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Natural Killer Cell Response: Treatment of Cancer

Posted in Gene Regulation, Human Immune System in Health and in Disease, Immunology, Inflammasome, Proteomics, Signaling & Cell Circuits, Stress Disorders, Translational Science, tagged Apoptosis, CD155 DNAM-1 ligand, cellular stress; NK cells; therapy; cancer; immunogenic, chemotherapy and radiotherapy, DNA-damage response, heat shock proteins, immunogenic cell death, major histocompatibility complex (MHC) class I, natural killer cells, NK cell-activating ligand expression, NKG2D ligands, oncogenes, p53, senescence, tumor suppressors on April 13, 2016| Leave a Comment »

Natural Killer Cell Response: Treatment of Cancer

Curator: Larry H. Bernstein, MD, FCAP

Molecular mechanisms of natural killer cell activation in response to cellular stress

C J Chan^1,2,3, M J Smyth^1,2,3,4,5 and L Martinet^1,2,4,5 Edited by M Piacentini

Cell Death and Differentiation (2014) 21, 5–14; http://www.nature.com/cdd/journal/v21/n1/full/cdd201326a.htm

Protection against cellular stress from various sources, such as nutritional, physical, pathogenic, or oncogenic, results in the induction of both intrinsic and extrinsic cellular protection mechanisms that collectively limit the damage these insults inflict on the host. The major extrinsic protection mechanism against cellular stress is the immune system. Indeed, it has been well described that cells that are stressed due to association with viral infection or early malignant transformation can be directly sensed by the immune system, particularly natural killer (NK) cells. Although the ability of NK cells to directly recognize and respond to stressed cells is well appreciated, the mechanisms and the breadth of cell-intrinsic responses that are intimately linked with their activation are only beginning to be uncovered. This review will provide a brief introduction to NK cells and the relevant receptors and ligands involved in direct responses to cellular stress. This will be followed by an in-depth discussion surrounding the various intrinsic responses to stress that can naturally engage NK cells, and how therapeutic agents may induce specific activation of NK cells and other innate immune cells by activating cellular responses to stress.

Stress induces specific intrinsic and extrinsic physiological mechanisms within cells that lead to their identification as functionally abnormal
Sources of cellular stress can be nutritional, physical, pathogenic, or oncogenic
Intrinsic responses to cellular stress include activation of the DNA-damage response, tumor-suppressor genes, and senescence
The extrinsic response to cellular stress is activation of the immune system, such as natural killer cells
Intrinsic responses to cellular stress can directly upregulate factors that can activate the immune system, and the immune system been shown to be indispensable for the efficacy of some chemotherapy

Further critical determinants of intrinsic responses to stress and cell death that can activate the immune system must be identified

Identification of the different cellular pathways and molecular determinants controlling the immunogenicity of different cancer therapies is required
How can we harness the ability of therapeutic agents to activate both the intrinsic and extrinsic responses to cellular stress to achieve more specific and safer approaches to cancer treatment?

Any insult to a cell that leads to its abnormal behavior or premature death can be defined as a source of stress. As the turnover and maintenance of cells in all multi-cellular organisms is tightly regulated, it is essential that stressed cells be rapidly identified to avoid widespread tissue damage and to maintain tissue homeostasis. Various intrinsic cellular mechanisms exist within cells that become activated when they are exposed to stress. These include activation of DNA-damage response proteins, senescence programs, and tumor-suppressor genes.¹ Extrinsic mechanisms also exist that combat cellular stress, through the upregulation of mediators that can activate different components of the immune system.² Although frequently discussed separately, much recent evidence has indicated that intrinsic and extrinsic responses to cellular stress are intimately linked.³

As the link between cell intrinsic and extrinsic responses to stress have been uncovered, these observations are now being harnessed therapeutically, particularly in the context of cancer.⁴ Indeed, various chemotherapeutic agents and radiotherapy are critically dependent on the immune system to elicit their full therapeutic benefit.^{5, 6} The mechanisms by which this occurs may be twofold: (i) the induction of intrinsic cellular stress mechanisms activates innate immunity and (ii) the release and presentation of tumor-specific antigens engages an inflammatory adaptive immune response.

NK cells are the major effector lymphocyte of innate immunity found in all the primary and secondary immune compartments as well as various mucosal tissues.⁷ Through their ability to induce direct cytotoxicity of target cells and produce pro-inflammatory cytokines such as interferon-gamma, NK cells are critically involved in the immune surveillance of tumors^{8, 9, 10} and microbial infections.^{11, 12} The major mechanism that regulates NK cell contact-dependent functions (such as cytotoxicity and recognition of targets) is the relative contribution of inhibitory and activating receptors that bind to cognate ligands.

Under normal physiological conditions, NK cell activity is inhibited through the interaction of their inhibitory receptors with major histocompatibility complex (MHC) class I.^{13, 14} However, upon instances of cellular stress that are frequently associated with viral infection and malignant transformation, ligands for activating receptors are often upregulated and MHC class I expression may be downregulated. The upregulation of these activating ligands and downregulation of MHC class I thus provides a signal for NK cells to become activated and display effector functions. Activating receptors are able to provide NK cells with a strong stimulus in the absence of co-stimulation due to the presence of adaptor molecules such as DAP10, DAP12, FcRγ, and CD3ζ that contain immunoreceptor tyrosine-based activating motifs (ITAMs).^{15, 16,17} By contrast, inhibitory receptors contain inhibitory motifs (ITIMs) within their cytoplasmic tails that can activate downstream targets such as SHP-1 and SHP-2 and directly antagonize those signaling pathways activated through ITAMs.^{18, 19, 20} The specific details of individual classes of inhibitory and activating receptors and their ligands are summarized in Figure 1 and have been extensively reviewed elsewhere.^{14, 21} Instead, this review will more focus on the relevant activating receptors that are primarily involved in the direct regulation of NK cell-mediated recognition of cellular stress: natural killer group 2D (NKG2D) and DNAX accessory molecule-1 (DNAM-1).

Figure 1.

NK cell receptors and their cognate ligands. Major inhibitory and activating receptors on NK cells and their cognate ligands on targets are depicted. BAT3, human leukocyte antigen (HLA)-B-associated transcript 3; CRTAM, class I-restricted T-cell-associated molecule; HA, hemagglutinin; HLA-E, HLA class I histocompatibility antigen, alpha chain E; IgG, immunoglobulin G; LFA-1, leukocyte function-associated antigen-1; LLT1, lectin-like transcript 1; TIGIT, T cell immunoglobulin and ITIM domain

Full figure and legend (185K)

NK Cell-Mediated Recognition of Cellular Stress by NKG2D and DNAM-1

NKG2D is a lectin-like type 2 transmembrane receptor expressed as a homodimer in both mice and humans by virtually all NK cells.^{22, 23} Upon interaction with its ligands, NKG2D can trigger NK cell-mediated cytotoxicity against their targets. The ligands for NKG2D are self proteins related to MHC class I molecules.²⁴ In humans, these ligands consist of the MHC class I chain-related protein (MIC) family (e.g., MICA and MICB) and the UL16-binding protein (ULBP1-6) family.^{25, 26} In mice, ligands for NKG2D include the retinoic acid early inducible (Rae) gene family, the H60 family, and mouse ULBP-like transcript-1 (MULT-1).^{27, 28, 29} NKG2D ligands are generally absent on the cell surface of healthy cells but are frequently upregulated upon cellular stress associated with viral infection and malignant transformation.^{3, 30} Indeed, NKG2D ligand expression has been found on many transformed cell lines, and NKG2D-dependent elimination of tumor cells expressing NKG2D ligands has been well documented in vitro and in tumor transplant experiments.^{25, 30, 31, 32, 33} In humans, NKG2D ligands have been described on different primary tumors^{34, 35} and specific NKG2D gene polymorphisms are associated with susceptibility to cancer.³⁶ Finally, blocking NKG2D through gene inactivation or monoclonal antibodies leads to an increased susceptibility to tumor development in mouse models,^{37, 38}demonstrating the key role played by NKG2D in immune surveillance of tumors. NKG2D can also contribute to shape tumor immunogenicity, a process called immunoediting, as demonstrated by the frequent ability of tumor cells to avoid NKG2D-mediated recognition through NKG2D ligand shedding, as discussed later in this review.^{38, 39, 40}

DNAM-1 is a transmembrane adhesion molecule constitutively expressed on T cells, NK cells, macrophages, and a small subset of B cells in mice and humans.^{41, 42, 43} DNAM-1 contains an extracellular region with two IgV-like domains, a transmembrane region and a cytoplasmic region containing tyrosine- and serine-phosphorylated sites that is able to initiate downstream activation cascades.^{41, 44} There is accumulating evidence showing that DNAM-1 not only promotes adhesion of NK cells and CTLs but also greatly enhances their cytotoxicity toward ligand-expressing targets.^{41, 45, 46, 47, 48, 49, 50} The ligands for DNAM-1 are the nectin/nectin-like family members CD155 (PVR, necl-5) and CD112 (PVRL2, nectin-2).^{45, 46} Like NKG2D ligands, DNAM-1 ligands are frequently expressed on virus-infected and transformed cells.^{51, 52}DNAM-1 ligands, especially CD155, are overexpressed by many types of solid and hematological malignancies and blocking DNAM-1 interactions with its ligands reduces the ability of NK cells to kill tumor cells in vitro.^{41, 49, 53, 54, 55, 56, 57} Further evidence of the role of DNAM-1 in tumor immune surveillance is provided by studies using experimental and spontaneous models of cancer in vivo showing enhanced tumor spread in the absence of DNAM-1.^{47, 48, 49, 50, 58}

As NKG2D and DNAM-1 ligands are frequently expressed on stressed cells, many studies have sought to determine the mechanisms that underpin these observations. The guiding hypothesis for these studies is that cell-intrinsic responses to stress are directly linked to cell-extrinsic responses that can trigger rapid NK cell surveillance and elimination of stressed cells. Indeed, major cell-intrinsic responses to cellular stress can directly lead to NK cell-activating ligand upregulation and are outlined in the following sections.

The DNA-Damage Response

Cellular stress caused by the activation of the DNA-damage response leads to downstream apoptosis or cell-cycle arrest. The activation of DNA-damage checkpoints occurs when there are excessive DNA strand breaks and replication errors, thereby representing an important tumorigenesis barrier that can slow or inhibit the progression of malignant transformation.^{59, 60} Two major transducers of the DNA-damage response are the PI3-kinase-related protein kinases ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). ATM and ATR can modulate numerous signaling pathways such as checkpoint kinases (Chk1 and Chk2, which inhibit cell-cycle progression and promote DNA repair) and p53 (which mediates cell-cycle arrest and apoptosis).⁶¹

In addition to the induction of cell-cycle arrest and apoptosis, activation of the DNA-damage response has been shown to promote the expression of several activating ligands that are specific for NK cell receptors, primarily those of the NKG2D receptor. These findings have shown a critical direct link between cellular transformation, apoptosis, and surveillance by the immune system.⁶² The first evidence of this link between DNA damage and immune cell activation was provided by Raulet and colleagues who showed that NKG2D ligands were upregulated by genotoxic stress and stalled DNA replication conditions known to activate either ATM or ATR.⁶³ These observations have now been extended by several other studies that have defined further DNA-damaging conditions (e.g., genotoxic drugs/chemotherapy, deregulated proliferation, or oxidative stress) that can promote NKG2D ligand upregulation.^{64, 65, 66, 67}

The role of the DNA-damage response in controlling NKG2D ligand expression and subsequent NK cell activation has also been demonstrated in the context of anti-viral immunity, specifically in Abelson murine leukemia virus infection.⁶⁸ This pathogen was shown to induce activation-induced cytidine deaminase (AID) expression outside the germinal center, resulting in generalized hypermutation, DNA-damage checkpoint activation, and Chk1 phosphorylation. The genotoxic activity of virally induced AID not only restricted the proliferation of infected cells but also induced the expression of NKG2D ligands. More recently, another member of APOBEC-AID family of cytidine deaminases, A3G, has been shown to promote the recognition of HIV-infected cells by NK cells after DNA-damage response activation.⁶⁹ In this study, viral protein Vpr-mediated repair processes, which generate nicks, gaps, and breaks of DNA, activate an ATM/ATR DNA-damage response that leads to NKG2D ligand expression.

The DNA-damage sensors ATM and ATR have also been shown to regulate other key NK cell-activating ligands such as the DNAM-1 ligand, CD155.^{58, 65, 70} For example, in the Eμ-myc spontaneous B-cell lymphoma model, activation of the DNA-damage response leads to the upregulation of CD155 in the early-stage transformed B cells, subsequently activating spontaneous tumor regression in an NK cell- and T-cell-dependent manner.⁵⁸ The DNA-damage response can also regulate the expression of the death receptor DR5.⁷¹ The engagement of DR5 by the effector molecule TRAIL, which is expressed by NK cells and T cells, can induce apoptosis of target cells and has been shown to have a key role in immune surveillance against tumors.⁷² Collectively, these results suggest that the detection of DNA damage, primarily through ATM and ATR, may represent a conserved protection mechanism governing the immunogenicity of infected or transformed cells, leading to direct recognition by NK cells (Figure 2).

Figure 2.

Overview of the molecular pathways leading to NK cell recognition of intrinsic cellular stress. Oncogenic transformation and viral infection can activate intrinsic cellular responses to stress. These responses include activation of the DNA-damage response, senescence, tumor suppressors, and the presentation and/or release of HSPs that, in turn, can activate NK cells through various receptor–ligand interactions. Senescent cells can also release pro-inflammatory cytokines that can recruit NK cells and other innate immunity, such as macrophages. CCL2, C-C motif chemokine ligand 2; CXCL11, C-X-C motif chemokine ligand 11; DR, death receptor 5; IFN, interferon; IL, interleukin; LFA-1, leukocyte function-associated antigen-1; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand

Full figure and legend (146K)

As a result of these studies, many therapeutic agents known to induce DNA damage have been evaluated for their ability to increase the immunogenicity of cancer cells for a more targeted therapeutic approach using NK cells.^{64, 65} For example, treatment of multiple myeloma cells with doxorubicin, melphalan, or bortezomib can lead to DNAM-1 and NKG2D ligand upregulation.⁶⁵Indeed, many chemotherapeutic agents commonly used, especially in hematological malignancies, can trigger the DNA-damage pathway. Therefore, it is reasonable to speculate that there is a general role of ATM and ATR in the induction of NK cell activation as a therapeutic effect of these agents.

Senescence

Cellular senescence is generally defined as a growth-arrest program in mammalian cells that limits their lifespan.⁷³ The major type of cellular senescence is replicative senescence that occurs due to telomere shortening. However, it is now generally accepted that premature senescence can also occur due to oncogene activation (oncogene-induced senescence) and/or the loss/gain of tumor-suppressor gene function, in the absence of telomere shortening.⁷⁴ Thus, premature senescence is an important barrier against malignant transformation.⁵⁹ Upon engagement of the senescence program, although cells are in growth arrest, they remain metabolically active and can produce many pro-inflammatory cytokines, as well as upregulate adhesion molecules and activating ligands to alert the immune system.^{75, 76, 77}Activation of the immune system, in particular innate immunity, has a critical role in the clearance of senescent cells.^{78, 79, 80, 81}More specifically, in a model of hepatocellular carcinoma, it has been shown that reactivation of p53 can induce a senescence program, resulting in tumor regression through the activation of NK cells, macrophages, and neutrophils. Of note, intercellular adhesion molecule (ICAM)-1, which can trigger both adhesion and cytotoxicity of NK cells,⁸² and interleukin-15, a cytokine that can promote NK cell effector function,⁸³ were both upregulated in senescent tumors. More recently, the potential contribution of NK cells was also shown in the clearance of senescent hepatic stellate cells, a mechanism important in limiting liver fibrosis in response to a fibrogenic agent.⁸⁰ ICAM-1, NKG2D ligands (MICA and ULPB2), and DNAM-1 ligands (CD155) were all upregulated on senescent hepatic stellate cells.

The specific mechanisms linking the senescence program to immune activation are not yet fully understood. However, the intracellular molecular mechanisms that govern induction of senescence may provide possible indications. Both replicative senescence and premature senescence (e.g., oncogene-induced senescence) have been shown to have common molecular determinants, such as the activation of the DNA-damage response pathway (e.g., ATM and ATR) and downstream activation of p53 and p16^INK4A.^{1, 59, 84, 85, 86} Activation of the DNA-damage response would presumably initiate the upregulation of NK cell-activating ligands as previously discussed. However, how senescence may be linked to the induction of pro-inflammatory cytokine release is a more compelling question and requires further investigation (Figure 2). Nevertheless, induction of pro-inflammatory cytokines is an important protective mechanism in order to recruit immune cells that can rapidly recognize and remove senescent cells. Interestingly, activation of NK cells by senescent cells has been observed in a clinical context when multiple myeloma cells were treated with chemotherapy and genotoxic agents.⁶⁵ In this setting, NKG2D and DNAM-1 ligands were both upregulated through a mechanism that required activation of the DNA-damage pathway initiated by ATM and ATR.⁶⁵

Tumor Suppressors: p53

p53 is a potent tumor suppressor and central regulator of apoptosis, DNA repair, and cell proliferation, that is activated in response to DNA damage, oncogene activation, and other cellular stress.⁸⁷ The number of identified cellular functions that p53 regulates has greatly increased over the past few years, and there is now a vast array of evidence that shows that p53 can be induced by viral infection⁸⁸ to limit pathogen spread by inducing apoptosis.^{89, 90} Furthermore, p53 not only acts as an intrinsic barrier against tumorigenesis or pathogenic spread but can also lead to increased cellular immunogenicity. For example, p53 reactivation in a hepatocellular carcinoma can promote tumor regression mediated by innate immunity.⁷⁸ A direct link between p53 expression and immune cell recognition was recently provided by Textor et al.⁹¹ where expression of p53 in lung cancer cell lines strongly upregulated the NKG2D ligands ULBP1 and 2, resulting in NK cell activation. Subsequently, p53-responsive elements were found to directly regulate ULBP1 and 2 expression, the deletion of which abolished the capacity of p53 to mediate ULBP1 and 2 upregulation. Another recent report that used a pharmacological activator of p53 confirmed the ability of p53 to directly induce ULBP2 expression that was independent of ATM/ATR.⁹² However, it has also been shown that miR34a and miR34C microRNAs (miRNAs) induced by p53 can target ULBP2 mRNA and reduce its cell-surface expression, suggesting that p53 may have a dual role in regulating ULBP2 expression.⁹³ Finally, early work showed that NKG2D ligands can be upregulated by ATR/ATM in the total absence of p53 in tumor cell lines,^{62, 63} suggesting the existence of ATM/ATR-dependent and p53-independent pathways that regulate NKG2D ligand expression in response to cellular stress.

In addition to regulating NK cell ligand expression, genetic reactivation of p53 in tumors can also induce a wide array of pro-inflammatory mediators ranging from adhesion receptor (ICAM-1) expression to the production of various chemokines (CXCL11 and monocyte chemoattractant protein-1) and cytokines (interleukin-15).⁷⁸ Furthermore, recent studies in anti-viral immunity indicate that several interferon-inducible genes and Toll-like receptor-3 expression are direct transcriptional targets of p53 and that p53 contributes to production of type I interferon by virally infected cells.^{94, 95, 96} All together, these studies suggest that p53 accumulation could represent a key determinant of the immunogenicity of stressed cells that are infected or undergoing malignant transformation through its ability to regulate innate immune activation.

Oncogenes

Malignant transformation is a complex process that frequently involves the activation of one or more oncogenes in addition to the inactivation or mutation of tumor-suppressor genes (e.g., p53). Oncogene activation is a powerful inducer of cellular stress that is able to activate intrinsic cellular programs that lead to cell apoptosis or senescence (e.g., activation of the DNA-damage response and p53).¹ In addition, many recent reports have also shown that major oncogenes can activate extrinsic responses to cellular stress through inducing the upregulation of NK cell-activating ligands.^{63, 97, 98} This suggests that oncogene activation can represent a key cellular event in alerting the immune system to ongoing cellular transformation (Figure 3).

Figure 3.

Molecular mechanisms that regulate the cell surface expression of NKG2D ligands. The major group of NK cell-activating ligands that are upregulated by intrinsic cellular responses to stress are those that bind the NKG2D receptor. Activation of the DNA-damage response, senescence, oncogenes, tumor suppressors, or sensing of deregulated proliferation can induce NKG2D ligand gene transcription and increase mRNA translation, leading to extracellular protein expression. MMP, matrix metalloproteases

Full figure and legend (183K)

The enhanced expression of the proto-oncogene Myc has been described as a critical event leading to cellular transformation and is a frequently found genetic alteration in cancer.⁹⁹ In a recent study, again using the Eμ-myc model, Medzhitov and colleagues demonstrated the ability of c-Myc to alert NK cells to early oncogenic transformation through the upregulation of Rae-1.⁹⁷ In this study, the induction of Rae-1 was dependent on the direct regulation of Rae-1 transcription by Myc through its interaction with the Raet1 epsilon gene. Collectively, these results provide a possible direct molecular mechanism to explain the increased susceptibility of NKG2D gene-targeted mice to lymphoma development in the Eμ-myc model.³⁸

Recent evidence suggests that several oncogenic mutations of Ras (H-Ras, N-Ras, and K-Ras) can also regulate NKG2D ligand expression in both mice and humans.⁹⁸ Interestingly, in this case, NKG2D ligands were regulated through MAPK/MEK and PI3K pathways downstream of oncogenic H-RasV12. The activation of PI3K pathways, and more particularly the p110α subunits by virus-encoded proteins, has also been shown to induce the Rae-1 family of ligands.¹⁰⁰ As many viruses can manipulate the PI3K pathway¹⁰¹ and tumors often bear Ras and p110α oncogene mutations,¹⁰² collectively, this data suggests that there is the existence of a common molecular mechanism by which NK cells sense cellular stress mediated by PI3K-dependent regulation of NKG2D ligands.

Interestingly, whereas Myc was involved in the transcriptional regulation of NKG2D ligands, PI3K can increase NKG2D ligand expression by increasing the translation of Rae-1 mRNA.⁹⁸ This involved the induction of eIF4E, a protein that enhances the translation of mRNA.¹⁰³ As number of tumors and viruses can upregulate host translation initiation machinery through the overexpression of eIF4E,^{104, 105} this may represent an important means by which NK cells can discriminate tumor- and virus-infected cells from normal cells.

Heat-Shock Proteins (HSPs)

HSPs are highly conserved intracellular chaperone molecules that are present in most prokaryotic and eukaryotic cells that mediate protection against cellular damage under conditions of stress. HSPs are distributed in most intracellular compartments of cells where they support the correct folding of nascent polypeptides, prevent protein aggregation, and assist in protein transport across membranes.¹⁰⁶ Many tumors display overexpression of HSPs as a response to cellular stress induced by oncogenic transformation.^{107, 108} HSPs can also be mobilized to the plasma membrane, or even released from cells, under conditions of stress.¹⁰⁹

Although intracellular HSPs can promote cell survival by interfering with different apoptosis components, many studies have reported that membrane-bound or soluble HSPs can directly stimulate innate immunity.¹¹⁰ A major immunostimulatory function of HSPs is to promote the presentation of tumor-specific antigens by MHC class I to CD8 T cells.^{111, 112, 113} Soluble and membrane-bound HSPs can also induce antigen-presenting cell maturation and the resultant secretion of pro-inflammatory cytokines.^{114, 115, 116}Finally, HSPs may directly activate NK cells as HSP70, when overexpressed on tumor cells, can induce a selective dose-dependent increase in NK cell-mediated cytotoxicity in vitro.¹¹⁷ NK cells may directly recognize HSP70 through a 14-amino-acid oligomer (TKD) that is localized in the C-terminal domain of the protein through CD94.^{118, 119} Tumor-specific HSP70 that is either presented at the cell surface or secreted on exosomes can also enhance NK cell activity against diverse types of cancer in vivo.^{120, 121} Most importantly, hepatocellular carcinoma cells that are treated with various chemotherapeutic agents can become more susceptible to NK cell-mediated cytotoxicity through their release of HSP-containing exosomes, giving the aforementioned findings a therapeutic context.¹²² Collectively, these results suggest that HSP translocation to the plasma membrane or secretion during cellular stress may represent a potent danger signal that can stimulate NK cell activity, particularly in the context of cancer.

Emerging Links Between Cellular Stress and NK Cell Activation

Deregulated proliferation

Deregulated proliferation is a critical determinant of cellular transformation and pathogenic infection.^{123, 124} Interestingly, recent results indicate that the Rae-1 family of ligands is a transcriptional target for E2F transcription factors, which control cell-cycle progression.¹²⁵ Cellular stress induced by deregulated proliferation may therefore activate NKG2D-bearing lymphocytes, including NK cells, as an early barrier to tumorigenesis. Future studies will have to address whether E2F transcription factors or indeed other cell-cycle regulators can also modulate other NK cell-activating ligands and whether these mechanisms can promote NK cell-tumor immunosurveillance in vivo.

MiRNAs

MiRNAs are an abundant class of small non-coding RNAs that are approximately 22 nucleotides in length. The functions of miRNAs are to directly regulate protein-encoding mRNAs to suppress gene expression.¹²⁶ Owing to their ability to regulate gene expression, miRNAs have been implicated in many biological and pathological processes.¹²⁷ Recent studies demonstrate that several miRNAs can regulate the cell-surface expression of NKG2D ligands (MICA, MICB, and ULBP2), therefore implicating miRNAs as a link between intrinsic and extrinsic responses to oncogenic transformation.^{93,128, 129} Indeed, in the context of cancer, many miRNAs (designated oncomiRs) are activated and are involved in the initiation and progression of disease.¹³⁰ As such, several oncomiRs have been implicated in the suppression of NKG2D ligand upregulation in various types of cancer, leading to their ability to facilitate tumor escape from NK cell-mediated immunosurveillance.^{93, 129}

Regulation of cell-surface expression

In addition to transcriptional and post-transcriptional regulation of NK cell receptor ligand expression by intrinsic cellular pathways, recent evidence suggest that stress-induced mechanisms can affect cell-surface expression of such ligands. For example, the expression of MULT-1 NKG2D ligand is repressed at the protein level on normal cells by polyubiquitination of its cytoplasmic tail leading to its degradation. By contrast, upon cellular stress induced by UV irradiation or heat shock, MULT-1 is less ubiquitinated resulting in an increase in cell-surface expression.¹³¹

NKG2D ligand shedding, secretion, or excretion in vesicles such as exosomes also seems to regulate their level of expression at the cell surface. For example, cleavage of the extracellular domain of NKG2D ligands by matrix metalloproteases results in their shedding from the cell surface, a common escape mechanism used by tumor cells to avoid NK cell recognition.^{39, 40, 132, 133} Indeed, soluble NKG2D ligands have been described in many cancer types in humans and usually correlates with a negative prognosis.^{40, 133,134, 135} Therefore, a better understanding of the intrinsic and extrinsic molecular pathways implicated in NKG2D ligand shedding could have direct therapeutic implications.

Cellular hyperploidy

Cellular hyperploidy occurs when there is an acquisition of more than two sets of chromosomes. For example, tetraploidy (or tetraploidization), where there is the presence of four sets of chromosomes, has been associated with early tumorigenesis due the acquisition of extra genetic material inherently making cells more susceptible to genetic abnormalities. As a barrier to potential oncogenic transformation from cellular hyperploidy, this event can be intrinsically sensed, leading to the inhibition of cell-cycle progression.¹³⁶ In a recent report, it has also been shown that cellular hyperploidy can be extrinsically sensed by the immune system.¹³⁷ Hyperploid cells were shown to become immunogenic as a result of a constitutive endoplasmic reticulum stress response that leads to the cell-surface exposure of calreticulin, a potent phagocytic signal for antigen-presenting cells, such as macrophages.¹³⁸ Interestingly, in this study, it was also shown that ex vivo tumors from Eμ-myc mice crossed to a DNAM-1^−/−background exhibited a larger nuclear diameter and P-eIF2αphosphorylation (as surrogate markers for cellular hyperploidy) than tumors from a wild-type background. This suggests that DNAM-1 on NK cells (and T cells) may participate in the immunoselection of hyperploid cells.

Immunogenic cell death

There is now clear evidence that demonstrates that the full clinical efficacy of some conventional chemo- and radiotherapy requires the activation of several immune components.^{139, 140} A common determinant of this requirement is the ability of these therapies to induce immunogenic cancer cell death. More specifically, it has been shown that cell-surface exposure of calreticulin and the release of high mobility group box-1 and adenosine triphosphate can activate dendritic cells leading to engulfment of dying cells, tumor-specific antigen presentation, and production of pro-inflammatory cytokines such as interleukin-1β^{6, 141, 142} (Figure 4). Interestingly, although CD8 T cells were the main effectors responsible for the therapeutic effects of chemotherapy in these settings, the efficient priming of these cells required the presence of upstream innate immunity, such as γδ T cells.¹⁴³ As the phenotype and function of NK cells are modulated in humans during chemotherapy^{144, 145} and many cellular stress pathways activated by chemotherapy can increase tumor cell sensitivity to NK cell-mediated cytotoxicity and recognition (as discussed in the previous sections), it will be important to determine whether NK cells can also contribute to the efficacy of chemotherapy.

Figure 4.

The role of NK cells during immunogenic cell death induced by cancer therapy. (1) Immunogenic cell death induced by various cancer therapies such as chemotherapy and radiotherapy involves the cell surface exposure of calreticulin (CRT), and the release of high mobility group box (HMGB)-1 and adenosine triphosphate (ATP) that triggers dendritic cell engulfment of dying cells, antigen presentation, and production of interleukin (IL)-1β, ultimately leading to activation of CD8 T cells. The efficient priming of tumor-specific CD8 T cells may require the presence of innate immunity such as γδ T cells that produce IL-17 in response to IL-1β. (2) Various cancer therapies can also activate intrinsic cellular responses to stress in tumor cells that can result in the upregulation of NKG2D ligands and the DNAM-1 ligand, CD155. This subsequently leads to the recognition and destruction of tumors by NK cells and CD8 T cells. HDAC, histone deacetylases; IFN, interferon

Full figure and legend (148K)

Concluding Remarks

NK cells provide an important primary defense mechanism against stressed cells. This is due to their ability to respond rapidly to the upregulation of multiple activating ligands. Although frequently documented on stressed cells, the mechanisms that govern the regulation of NK cell-activating ligand expression have only recently become apparent, particularly in the context of cancer. These mechanisms include multiple intrinsic cellular responses, such as

the activation of the DNA-damage response, senescence,
the activation of tumor suppressors and
oncogenes, and
the sensing of deregulated cell proliferation.

Indeed, many forms of chemo- and radiotherapy that are currently used in the clinic that aim to engage these intrinsic cellular responses have been shown to require the immune system, including NK cells for their full efficacy. Thus, formulating specific therapies that can concurrently engage intrinsic and extrinsic responses to cellular stress will hopefully lead to more targeted, effective, and safer approaches for the treatment of cancer.

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Insight on Cell Senescence

Posted in Cancer - General, Curation, Disease Biology, Human aging, Human Immune System in Health and in Disease, Neurodegenerative Diseases, tagged Aging, granular exostosis, immune surveillance, mediation, senescence on April 13, 2016| Leave a Comment »

Insight on Cell Senescence

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Granule exocytosis mediates immune surveillance of senescent cells

A Sagiv1 , A Biran1 , M Yon2,3, J Simon2,4, SW Lowe2,4 and V Krizhanovsky1,2
Oncogene (2013) 32, 1971–1977 http://www.nature.com/onc/journal/v32/n15/pdf/onc2012206a.pdf

Senescence is a stable cell cycle arrest program that contributes to tumor suppression, organismal aging and certain wound healing responses. During liver fibrosis, for example, hepatic stellate cells initially proliferate and secrete extracellular matrix components that produce fibrosis; however, these cells eventually senesce and are cleared by immune cells, including natural killer (NK) cells. Here, we examine how NK cells target senescent cells and assess the impact of this process on liver fibrosis. We show that granule exocytosis, but not death-receptor-mediated apoptosis, is required for NK-cell-mediated killing of senescent cells. This pathway bias is due to upregulation of the decoy death receptor, Dcr2, an established senescence marker that attenuates NK-mediated cell death. Accordingly, mice with defects in granule exocytosis accumulate senescent stellate cells and display more liver fibrosis in response to a fibrogenic agent. Our results thus provide new insights into the immune surveillance of senescent cells and reveal how granule exocytosis has a protective role against liver fibrosis. Oncogene (2013) 32, 1971–1977; http://dx.doi.org:/10.1038/onc.2012.206

Senescence is accompanied by phenotypic and transcriptional changes that identify senescent cells in vitro and in vivo. For example, senescent cells display a large and flat morphology in vitro and upregulate a senescence-associated b-galactosidase (SA-b-gal).9 Senescent cells often display global changes in chromatin structure10 that are associated with downregulation of cell cycle genes and components of the extracellular matrix and upregulation of immune modulators and matrix degrading enzymes.4 Comparative analyses of gene expression data have produced some markers that appear specific for senescence,11 including the p15ink4b cyclin-dependent kinase inhibitor and the decoy receptor 2 (Dcr2, formally TNFRSF10D). Although p15ink4b likely contributes to the senescence-associated cell cycle arrest,12 whether decoy receptors or some other senescence markers actively participate in the program remains unknown.

Senescence acts through a coordinated program involving cell autonomous and cell nonautonomous components.13 In a cell autonomous manner, the Rb and p53 tumor suppressor pathways act to produce the stable cell cycle arrest that is the hallmark of senescence.1 These proteins are activated by, or activate, cyclindependent kinase inhibitors, such as p15ink4b, p16ink4a and p21, which lead to stable suppression of E2F target genes.10,14 Secreted proteins, regulated at least partially by NF-kB, enhance cell cycle arrest and are largely responsible for mediating the impact of senescent cells on tissue biology.15–17 These factors can attract immune cells, including natural killer (NK) cells, triggering the recognition and ultimate clearance of the senescent cells from tumors or tissue.4,18 Such mechanisms may be necessary to prevent the long-term damage that might be produced by senescent cells, and to facilitate tissue repair and homeostasis.

The mechanisms whereby NK cells eliminate senescent cells from tissues are not known. NK cells rely on two independent mechanisms to eliminate a variety of external and internal threats, including tumor cells.19,20 The ligands on the surface of NK cells, TRAIL and FAS ligand (FasL) bind corresponding receptors on target cells leading to caspase activation and cell death—a process that can be exquisitely controlled though the expression of various positive and negative regulators.21,22 NK cells can also eliminate target cells through granule exocytosis, a process involving the production of perforin and granzyme (A, B) containing granules, which are secreted from the NK cell upon interaction with the target cell.21,23 Perforin is responsible for perforating the cell membrane and thus enabling granzyme release into the target cells where it can induce cell death by both caspase-dependent and independent pathways.24 B

Here, we set out to understand how NK cells eliminate senescent cells from tissues and the implications of such mechanisms on liver fibrosis. Our results indicate that the granule exocytosis, and not death-receptor-mediated apoptosis, is essential for the NK-mediated surveillance of the senescent cells and that disruption of this pathway leads to the accumulation of senescent cells in damaged livers and increased fibrosis. Our study thus provides the key biological and mechanistic insights into the immune surveillance of senescent cells.

Figure 1. NK cells preferentially recognize senescent cells in a wide range of target:effector cell ratios. Senescent or growing IMR-90 fibroblasts were co-incubated with YT cells for 12 h at the indicated ratios and cytotoxicity was determined. The graphs represent the average and the s.e. of triplicate measurements from at least three independent experiments. *Po0.005.

Figure 2. Caspases are dispensable for NK-mediated cell killing of senescent cells. Senescent or growing IMR-90 fibroblasts were incubated for 12 h with either 2 or 10 nM FasL (a). Caspase inhibitors Z-VAD-FMK or Z-IEDT-FMK were added at the concentration of 10 mM as indicated. Cytotoxicity was determined at the end of the coincubation period. Senescent or growing IMR-90 fibroblasts were coincubated with YT cells for 12 h in the presence of 10 mM of caspase inhibitors Z-VAD-FMK or Z-IEDT-FMK and then the cytotoxicity was determined (b). The graphs represent the average and the s.e. of triplicate measurements from at least three independent experiments. *Po0.05, ***Po0.001.

Figure 3. Granule exocytosis pathway is required for NK-cell-mediated killing of senescent cells. Senescent and growing IMR-90 fibroblasts (a, c) or HSCs (b) were co-incubated with YT cells for 12 h (a, b) or with primary NK cells for 2 h (c). Cytotoxicity assays were performed either in the presence of 100 nM granule exocytosis inhibitor, CMA or following pre-incubation of the YT or primary NK cells with 25 mM Granzyme B inhibitor 3,4-DCI. The graphs represent the average and the the s.e. of triplicate measurements from at least three independent experiments. *Po0.01, **Po0.001, ***Po0.0001.

Figure 4. Dcr2 attenuates killing of senescent cells through the death receptor pathway. Dcr2 expression level in senescent and growing IMR- 90 fibroblasts (a, b) and human HSCs (c, d) were evaluated by quantitative RT–PCR analysis (a, c) and immunoblotting (b, d). Dcr2-deficient senescent IMR-90 cells were incubated with either 10 or 100 ng/ml TRAIL and cytotoxicity was determined (e), and Dcr2 knockdown confirmed (f). Senescent IMR-90 cells with siDcr2 or siControl were incubated with YT cells for 12 h and cytotoxicity was determined (g). In the parallel approach IMR-90 cells were infected with short hairpin RNA (shRNA) targeting Dcr2 (shDcr2) or control shRNA targeting luciferase (shLuci) and induced to senescence by etoposide treatment. Dcr2 protein level was assessed by immunoblot (h). The cells were co-incubated for 12 h with YT cells and cytotoxicity was determined (i). The graphs represent the average and the s.e. of triplicate measurements from at least four independent experiments *Po0.05, **Po0.001, ***Po0.0001.

Figure 5. Perforin promotes senescent cell clearance and limits liver fibrosis. Perforin knockout (Prf / ) and wt mice were treated with CCl4 to induce fibrosis. H&E and Sirius red staining show liver morphology and accumulation of fibrotic scar following the treatment (a). Morphometric analysis of Sirius red stained, entire liver sections (b). Expression of markers of activated HSCs, aSMA and Colagen1a, and senescence marker p15ink4b were tested by immunoblotting of whole-liver extracts (c). Four mice of each genotype are shown. SA-b-gal staining identified accumulation of senescent cells along the fibrotic scar areas in the livers (d). The presence of SA-b-gal-positive cells was quantified in the entire liver sections (e). At least five mice of each genotype were used for the analysis in B and E; **Po0.001, ***Po0.0001.

…..

NK-cell-mediated clearance of senescent cells is one component of the coordinated process whereby cellular senescence limits the extent of liver fibrosis and facilitates wound repair.4,18 Recent studies also suggest that senescent cell clearance by immune cells promotes tumor regression in established tumors.18 Our results demonstrate that the granule exocytosis pathway, but not the death receptor pathway, is necessary for the specific killing of senescent fibroblasts and stellate cells by NK cells and participates in the clearance of senescent activated HSCs to limit liver fibrosis. Therefore, NK-cell-mediated cytotoxicity through granule exocytosis contributes to immune surveillance of senescent cells in vitro and in vivo.

In addition to the granule exocytosis pathway, most cytotoxic lymphocytes engage the death receptor pathway to eliminate target cells. This pathway is widely used by NK cells in the liver.21 NK cell express high levels of the death receptor ligand TRAIL upon activation with IL-2,26 are suggested to participate in the surveillance of the HSCs,35 and protect against tumor development following chemical carcinogenesis.36 Given this, we were surprised that death-receptor-mediated cytotoxicity was dispensable for the immune surveillance of senescent cells. Consistent with these findings, an anti-TRAIL antibody failed to inhibit immune system-mediated tumor clearance following p53 restoration in a liver carcinoma model18 (W Xue and SWL, unpublished data). Of course, we cannot rule out the possibility that death receptor pathways contribute to senescent cell clearance in other settings.

Why does granule exocytosis, and not the death-receptor signaling, mediate NK-cell surveillance of senescent cells? Mechanistically, this appears partly because of the accumulation of Dcr2 during senescence, which occurs in fibroblasts, certain epithelial cells11,18 and, as shown here, also senescent activated HSCs. Dcr2 can bind death-receptor ligands, with higher affinity to TRAIL, but as it lacks the activation domain it prevents downstream signaling through the death receptor pathway31,37 and, therefore, can protect senescent cells from death-receptorligand-mediated killing. Another decoy receptor, Dcr3, has higher affinity to FASL.38 However, in contrast to Dcr2, Dcr3 is a secreted receptor and is much less likely to have a role in direct interaction between senescent and NK cells. Although previously considered merely a senescence marker, our results establish a functional role for Dcr2 in protecting senescent cells from cytotoxicity through the death receptor pathway induced by NK cells and possibly other cells as well. The biological rationale for this regulation remains unclear, but may serve to prevent autoimmunity following short-term tissue damage.

In addition to blocking the death receptor pathway, senescent cells may also stimulate NK cells to induce the perforin-mediated killing. Senescent cells upregulate expression of several ligands of NK-cell receptor NKG2D4,39 and ICAM-1, the ligand of NK-cell receptor LFA-1.40 Studies suggest that activation of the NKG2D receptor induces granule exocytosis to eliminate cancer cells, a process that might be reinforced by signaling from LFA-1.41 In this manner, ligands upregulated in senescent cells might activate multiple NK-cell receptors to trigger granule exocytosis.

The role of granule exocytosis in the surveillance of senescent cells has important ramifications for understanding and treating wound healing and cancer. Indeed, we show that the immune clearance of senescent activated HSCs has a significant impact on the pathophysiology of liver fibrosis in which the granule exocytosis pathway has been previously implicated.42,43 Beyond the liver, immune surveillance of senescent cells might have a significant role in other fibrosis-related pathological conditions.

Still, the most prevalent conditions where senescence has been studied to date involve cancer and aging.3,9 Senescent cells accumulate with age and contribute to functional decline of multiple tissues7,9 while perforin-mediated granule exocytosis diminishes at that time.47,48 Separate studies suggest that the integrity of the granule exocytosis pathway can modulate a variety of cancer phenotypes.49,50 Though definitive proof will require further testing, we speculate that the granule exocytosis pathway contributes to immune surveillance of senescent cells in each of these conditions. In principle, pharmacological modulation of this pathway, as has been recently described using IL21,51 might increase the clearance of senescent cells from premalignant, damaged or aged tissues to limit carcinogenesis and the decline in tissue function accompanying the accumulation of senescent cells.

REFERENCES 1 Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 1997; 88: 593–602.

2 Schmitt CA, Fridman JS, Yang M, Lee S, Baranov E, Hoffman RM et al. A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy. Cell 2002; 109: 335–346.

3 Narita M, Lowe SW. Senescence comes of age. Nat Med 2005; 11: 920–922.

4 Krizhanovsky V, Yon M, Dickins RA, Hearn S, Simon J, Miething C et al. Senescence of activated stellate cells limits liver fibrosis. Cell 2008; 134: 657–667.

5 Jun JI, Lau LF. The matricellular protein CCN1 induces fibroblast senescence and restricts fibrosis in cutaneous wound healing. Nat Cell Biol 2010; 12: 676–685.

6 Pitiyage GN, Slijepcevic P, Gabrani A, Chianea YG, Lim KP, Prime SS et al. Senescent mesenchymal cells accumulate in human fibrosis by a telomereindependent mechanism and ameliorate fibrosis through matrix metalloproteinases. J Pathol 2011; 223: 604–617.

7 Baker DJ, Wijshake T, Tchkonia T, LeBrasseur NK, Childs BG, van de Sluis B et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 2011; 479: 232–236.

8 Kang TW, Yevsa T, Woller N, Hoenicke L, Wuestefeld T, Dauch D et al. Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature 2011; 479: 547–551.

9 Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA. 1995; 92: 9363–9367.

10 Narita M, Nunez S, Heard E, Narita M, Lin AW, Hearn SA et al. Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. Cell 2003; 113: 703–716

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Conduction, graphene, elements and light

Posted in Alzheimer's Disease, Bioengineering & reverse engineering design, Curation, Developmental biology, Hematopoiesis, Human Immune System in Health and in Disease, Immunotherapy, Innovations, Lasers and photonics, Materials Science & Engineering, Medical Imaging Technology, Microbiology, Nanotechnology for Drug Delivery, Neurodegenerative Diseases, tagged biomedical, Electronics, embryonic stem cells, light, MRI, Nanotech/Materials Science, Surface plasmon resonance on March 27, 2016| Leave a Comment »

Conduction, graphene, elements and light

Larry H. Bernstein, MD, FCAP, Curator

LPBI

New 2D material could upstage graphene Mar 25, 2016

Can function as a conductor or semiconductor, is extremely stable, and uses light, inexpensive earth-abundant elements
http://www.kurzweilai.net/new-2d-material-could-upstage-graphene

The atoms in the new structure are arranged in a hexagonal pattern as in graphene, but that is where the similarity ends. The three elements forming the new material all have different sizes; the bonds connecting the atoms are also different. As a result, the sides of the hexagons formed by these atoms are unequal, unlike in graphene. (credit: Madhu Menon)

A new one-atom-thick flat material made up of silicon, boron, and nitrogen can function as a conductor or semiconductor (unlike graphene) and could upstage graphene and advance digital technology, say scientists at the University of Kentucky, Daimler in Germany, and the Institute for Electronic Structure and Laser (IESL) in Greece.

Reported in Physical Review B, Rapid Communications, the new Si₂BN material was discovered in theory (not yet made in the lab). It uses light, inexpensive earth-abundant elements and is extremely stable, a property many other graphene alternatives lack, says University of Kentucky Center for Computational Sciences physicist Madhu Menon, PhD.

Limitations of other 2D semiconducting materials

A search for new 2D semiconducting materials has led researchers to a new class of three-layer materials called transition-metal dichalcogenides (TMDCs). TMDCs are mostly semiconductors and can be made into digital processors with greater efficiency than anything possible with silicon. However, these are much bulkier than graphene and made of materials that are not necessarily earth-abundant and inexpensive.

Other graphene-like materials have been proposed but lack the strengths of the new material. Silicene, for example, does not have a flat surface and eventually forms a 3D surface. Other materials are highly unstable, some only for a few hours at most.

The new Si₂BN material is metallic, but by attaching other elements on top of the silicon atoms, its band gap can be changed (from conductor to semiconductor, for example) — a key advantage over graphene for electronics applications and solar-energy conversion.

The presence of silicon also suggests possible seamless integration with current silicon-based technology, allowing the industry to slowly move away from silicon, rather than precipitously, notes Menon.

https://youtu.be/lKc_PbTD5go

Abstract of Prediction of a new graphenelike Si₂BN solid

While the possibility to create a single-atom-thick two-dimensional layer from any material remains, only a few such structures have been obtained other than graphene and a monolayer of boron nitride. Here, based upon ab initiotheoretical simulations, we propose a new stable graphenelike single-atomic-layer Si₂BN structure that has all of its atoms with sp² bonding with no out-of-plane buckling. The structure is found to be metallic with a finite density of states at the Fermi level. This structure can be rolled into nanotubes in a manner similar to graphene. Combining first- and second-row elements in the Periodic Table to form a one-atom-thick material that is also flat opens up the possibility for studying new physics beyond graphene. The presence of Si will make the surface more reactive and therefore a promising candidate for hydrogen storage.

references:

Antonis N. Andriotis, Ernst Richter, and Madhu Menon. Prediction of a new graphenelike Si2BN solid. Phys. Rev. B 93, 081413(R) – Published 26 February 2016; DOI: 10.1103/PhysRevB.93.081413

Nano-enhanced textiles clean themselves with light

Catalytic uses for industrial-scale chemical processes in agrochemicals, pharmaceuticals, and natural products also seen
http://www.kurzweilai.net/nano-enhanced-textiles-clean-themselves-with-light

Close-up of nanostructures grown on cotton textiles. Image magnified 150,000 times. (credit: RMIT University)

Researchers at at RMIT University in Australia have developed a cheap, efficient way to grow special copper- and silver-based nanostructures on textiles that can degrade organic matter when exposed to light.

Don’t throw out your washing machine yet, but the work paves the way toward nano-enhanced textiles that can spontaneously clean themselves of stains and grime simply by being put under a light or worn out in the sun.

The nanostructures absorb visible light (via localized surface plasmon resonance — collective electron-charge oscillations in metallic nanoparticles that are excited by light), generating high-energy (“hot”) electrons that cause the nanostructures to act as catalysts for chemical reactions that degrade organic matter.

Steps involved in fabricating copper- and silver-based cotton fabrics: 1. Sensitize the fabric with tin. 2. Form palladium seeds that act as nucleation (clustering) sites. 3. Grow metallic copper and silver nanoparticles on the surface of the cotton fabric. (credit: Samuel R. Anderson et al./Advanced Materials Interfaces)

The challenge for researchers has been to bring the concept out of the lab by working out how to build these nanostructures on an industrial scale and permanently attach them to textiles. The RMIT team’s novel approach was to grow the nanostructures directly onto the textiles by dipping them into specific solutions, resulting in development of stable nanostructures within 30 minutes.

When exposed to light, it took less than six minutes for some of the nano-enhanced textiles to spontaneously clean themselves.

The research was described in the journal Advanced Materials Interfaces.

Scaling up to industrial levels

Rajesh Ramanathan, a RMIT postdoctoral fellow and co-senior author, said the process also had a variety of applications for catalysis-based industries such as agrochemicals, pharmaceuticals, and natural products, and could be easily scaled up to industrial levels. “The advantage of textiles is they already have a 3D structure, so they are great at absorbing light, which in turn speeds up the process of degrading organic matter,” he said.

Cotton textile fabric with copper-based nanostructures. The image is magnified 200 times. (credit: RMIT University)

“Our next step will be to test our nano-enhanced textiles with organic compounds that could be more relevant to consumers, to see how quickly they can handle common stains like tomato sauce or wine,” Ramanathan said.

“There’s more work to do to before we can start throwing out our washing machines, but this advance lays a strong foundation for the future development of fully self-cleaning textiles.”

Abstract of Robust Nanostructured Silver and Copper Fabrics with Localized Surface Plasmon Resonance Property for Effective Visible Light Induced Reductive Catalysis

Inspired by high porosity, absorbency, wettability, and hierarchical ordering on the micrometer and nanometer scale of cotton fabrics, a facile strategy is developed to coat visible light active metal nanostructures of copper and silver on cotton fabric substrates. The fabrication of nanostructured Ag and Cu onto interwoven threads of a cotton fabric by electroless deposition creates metal nanostructures that show a localized surface plasmon resonance (LSPR) effect. The micro/nanoscale hierarchical ordering of the cotton fabrics allows access to catalytically active sites to participate in heterogeneous catalysis with high efficiency. The ability of metals to absorb visible light through LSPR further enhances the catalytic reaction rates under photoexcitation conditions. Understanding the modes of electron transfer during visible light illumination in Ag@Cotton and Cu@Cotton through electrochemical measurements provides mechanistic evidence on the influence of light in promoting electron transfer during heterogeneous catalysis for the first time. The outcomes presented in this work will be helpful in designing new multifunctional fabrics with the ability to absorb visible light and thereby enhance light-activated catalytic processes.

references:

Samuel R. Anderson, Mahsa Mohammadtaheri, Dipesh Kumar, Anthony P. O’Mullane, Matthew R. Field, Rajesh Ramanathan, Vipul Bansal. Robust Nanostructured Silver and Copper Fabrics with Localized Surface Plasmon Resonance Property for Effective Visible Light Induced Reductive Catalysis. Advanced Materials Interfaces, 2016; DOI: 10.1002/admi.201500632

New type of molecular tag makes MRI 10,000 times more sensitive

Could detect biochemical processes in opaque tissue without requiring PET radiation or CT x-rays
http://www.kurzweilai.net/new-type-of-molecular-tag-makes-mri-10000-times-more-sensitive

Duke scientists have discovered a new class of inexpensive, long-lived molecular tags that enhance MRI signals by 10,000 times. To activate the tags, the researchers mix them with a newly developed catalyst (center) and a special form of hydrogen (gray), converting them into long-lived magnetic resonance “lightbulbs” that might be used to track disease metabolism in real time. (credit: Thomas Theis, Duke University)

Duke University researchers have discovered a new form of MRI that’s 10,000 times more sensitive and could record actual biochemical reactions, such as those involved in cancer and heart disease, and in real time.

Let’s review how MRI (magnetic resonance imaging) works: MRI takes advantage of a property called spin, which makes the nuclei in hydrogen atoms act like tiny magnets. By generating a strong magnetic field (such as 3 Tesla) and a series of radio-frequency waves, MRI induces these hydrogen magnets in atoms to broadcast their locations. Since most of the hydrogen atoms in the body are bound up in water, the technique is used in clinical settings to create detailed images of soft tissues like organs (such as the brain), blood vessels, and tumors inside the body.

MRI’s ability to track chemical transformations in the body has been limited by the low sensitivity of the technique. That makes it impossible to detect small numbers of molecules (without using unattainably more massive magnetic fields).

So to take MRI a giant step further in sensitivity, the Duke researchers created a new class of molecular “tags” that can track disease metabolism in real time, and can last for more than an hour, using a technique called hyperpolarization.* These tags are biocompatible and inexpensive to produce, allowing for using existing MRI machines.

“This represents a completely new class of molecules that doesn’t look anything at all like what people thought could be made into MRI tags,” said Warren S. Warren, James B. Duke Professor and Chair of Physics at Duke, and senior author on the study. “We envision it could provide a whole new way to use MRI to learn about the biochemistry of disease.”

Sensitive tissue detection without radiation

The new molecular tags open up a new world for medicine and research by making it possible to detect what’s happening in optically opaque tissue instead of requiring expensive positron emission tomography (PET), which uses a radioactive tracer chemical to look at organs in the body and only works for (typically) about 20 minutes, or CT x-rays, according to the researchers.

This research was reported in the March 25 issue of Science Advances. It was supported by the National Science Foundation, the National Institutes of Health, the Department of Defense Congressionally Directed Medical Research Programs Breast Cancer grant, the Pratt School of Engineering Research Innovation Seed Fund, the Burroughs Wellcome Fellowship, and the Donors of the American Chemical Society Petroleum Research Fund.

* For the past decade, researchers have been developing methods to “hyperpolarize” biologically important molecules. “Hyperpolarization gives them 10,000 times more signal than they would normally have if they had just been magnetized in an ordinary magnetic field,” Warren said. But while promising, Warren says these hyperpolarization techniques face two fundamental problems: incredibly expensive equipment — around 3 million dollars for one machine — and most of these molecular “lightbulbs” burn out in a matter of seconds.

“It’s hard to take an image with an agent that is only visible for seconds, and there are a lot of biological processes you could never hope to see,” said Warren. “We wanted to try to figure out what molecules could give extremely long-lived signals so that you could look at slower processes.”

So the researchers synthesized a series of molecules containing diazarines — a chemical structure composed of two nitrogen atoms bound together in a ring. Diazirines were a promising target for screening because their geometry traps hyperpolarization in a “hidden state” where it cannot relax quickly. Using a simple and inexpensive approach to hyperpolarization called SABRE-SHEATH, in which the molecular tags are mixed with a spin-polarized form of hydrogen and a catalyst, the researchers were able to rapidly hyperpolarize one of the diazirine-containing molecules, greatly enhancing its magnetic resonance signals for over an hour.

The scientists believe their SABRE-SHEATH catalyst could be used to hyperpolarize a wide variety of chemical structures at a fraction of the cost of other methods.

Abstract of Direct and cost-efficient hyperpolarization of long-lived nuclear spin states on universal ¹⁵N₂-diazirine molecular tags

Conventional magnetic resonance (MR) faces serious sensitivity limitations, which can be overcome by hyperpolarization methods, but the most common method (dynamic nuclear polarization) is complex and expensive, and applications are limited by short spin lifetimes (typically seconds) of biologically relevant molecules. We use a recently developed method, SABRE-SHEATH, to directly hyperpolarize ¹⁵N₂ magnetization and long-lived ¹⁵N₂singlet spin order, with signal decay time constants of 5.8 and 23 min, respectively. We find >10,000-fold enhancements generating detectable nuclear MR signals that last for more than an hour. ¹⁵N₂-diazirines represent a class of particularly promising and versatile molecular tags, and can be incorporated into a wide range of biomolecules without significantly altering molecular function.

references:

Thomas Theis, Gerardo X. Ortiz Jr., Angus W. J. Logan, Kevin E. Claytor, Yesu Feng, William P. Huhn, Volker Blum, Steven J. Malcolmson, Eduard Y. Chekmenev, Qiu Wang and Warren S. Warren. Direct and cost-efficient hyperpolarization of long-lived nuclear spin states on universal ¹⁵N₂-diazirine molecular tags. Science Advances, March 25, 2016. DOI: 10.1126/sciadv.1501438

[Seems like they have a great idea, now all they need to do is confirm very specific uses or types of cancers/diseases or other processes they can track or target. Will be interesting to see if they can do more than just see things, maybe they can use this to target and destroy bad things in the body also. Keep up the good work….. this sounds like a game changer.]

Scientists time-reverse developed stem cells to make them ‘embryonic’ again

May help avoid ethically controversial use of human embryos for research and support other research goals
http://www.kurzweilai.net/scientists-time-reverse-developed-stem-cells-to-make-them-embryonic-again

Researchers have reversed “primed” (developed) “epiblast” stem cells (top) from early mouse embryos using the drug MM-401, causing the treated cells (bottom) to revert to the original form of the stem cells. (credit: University of Michigan)

University of Michigan Medical School researchers have discovered a way to convert mouse stem cells (taken from an embryo) that have become “primed” (reached the stage where they can differentiate, or develop into every specialized cell in the body) to a “naïve” (unspecialized) state by simply adding a drug.

This breakthrough has the potential to one day allow researchers to avoid the ethically controversial use of human embryos left over from infertility treatments. To achieve this breakthrough, the researchers treated the primedembryonic stem cells (“EpiSC”) with a drug called MM-401* (a leukemia drug) for a short period of time.

Embryonic stem cells are able to develop into any type of cell, except those of the placenta (credit: Mike Jones/CC)

…..

* The drug, MM-401, specifically targets epigenetic chemical markers on histones, the protein “spools” that DNA coils around to create structures called chromatin. These epigenetic changes signal the cell’s DNA-reading machinery and tell it where to start uncoiling the chromatin in order to read it.

A gene called Mll1 is responsible for the addition of these epigenetic changes, which are like small chemical tags called methyl groups. Mll1 plays a key role in the uncontrolled explosion of white blood cells in leukemia, which is why researchers developed the drug MM-401 to interfere with this process. But Mll1 also plays a role in cell development and the formation of blood cells and other cells in later-stage embryos.

Stem cells do not turn on the Mll1 gene until they are more developed. The MM-401 drug blocks Mll1’s normal activity in developing cells so the epigenetic chemical markers are missing. These cells are then unable to continue to develop into different types of specialized cells but are still able to revert to healthy naive pluripotent stem cells.

Abstract of MLL1 Inhibition Reprograms Epiblast Stem Cells to Naive Pluripotency

The interconversion between naive and primed pluripotent states is accompanied by drastic epigenetic rearrangements. However, it is unclear whether intrinsic epigenetic events can drive reprogramming to naive pluripotency or if distinct chromatin states are instead simply a reflection of discrete pluripotent states. Here, we show that blocking histone H3K4 methyltransferase MLL1 activity with the small-molecule inhibitor MM-401 reprograms mouse epiblast stem cells (EpiSCs) to naive pluripotency. This reversion is highly efficient and synchronized, with more than 50% of treated EpiSCs exhibiting features of naive embryonic stem cells (ESCs) within 3 days. Reverted ESCs reactivate the silenced X chromosome and contribute to embryos following blastocyst injection, generating germline-competent chimeras. Importantly, blocking MLL1 leads to global redistribution of H3K4me1 at enhancers and represses lineage determinant factors and EpiSC markers, which indirectly regulate ESC transcription circuitry. These findings show that discrete perturbation of H3K4 methylation is sufficient to drive reprogramming to naive pluripotency.

Abstract of Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass

Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals.

How to kill bacteria in seconds using gold nanoparticles and light

March 24, 2016

Could treat bacterial infections without using antibiotics, which could help reduce the risk of spreading antibiotics resistance

Researchers at the University of Houston have developed a new technique for killing bacteria in 5 to 25 seconds using highly porous gold nanodisks and light, according to a study published today in Optical Materials Express. The method could one day help hospitals treat some common infections without using antibiotics

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Selye’s Riddle solved

Posted in Biochemical pathways, Curation, Developmental biology, Gene Regulation and Evolution, Human aging, Human Immune System in Health and in Disease, Human Sensation and Cellular Transduction: Physiology and Therapeutics, Inflammasome, Metabolism, Microbiology, Micronutrients, Nephrology, Stress Disorders, Systemic Inflammatory Response Related Disorders, tagged adaptation, Evolution, Fitness, General adaptation syndrome, Optimality, Physiology on March 26, 2016| Leave a Comment »

Selye’s Riddle solved

Larry H. Bernstein, mD, FCAP, Curator

LPBI

Mathematicians Solve 78-year-old Mystery

3/23/2016 University of Leicester http://www.scientificcomputing.com/news/2016/03/mathematicians-solve-78-year-old-mystery

Mathematicians developed a solution to Selye’s riddle which has puzzled scientists for almost 80 years.

In previous research, it was suggested that adaptation of an animal to different factors looks like spending of one resource, and that the animal dies when this resource is exhausted. In 1938, Hans Selye introduced “adaptation energy” and found strong experimental arguments in favor of this hypothesis. However, this term has caused much debate because, as it cannot be measured as a physical quantity, adaptation energy is not strictly energy.

Evolution of adaptation mechanisms: Adaptation energy, stress, and oscillating death

Alexander N. Gorban ^a^,^,, Tatiana A. Tyukina ^a^,, Elena V. Smirnova ^b^,, Lyudmila I. Pokidysheva ^b^,

Journal of Theoretical Biology 19 Jan 2016 http://www.sciencedirect.com/science/article/pii/S0022519316000205 http://dx.doi.org:/10.1016/j.jtbi.2015.12.017

Highlights

• We formalize Selye׳s ideas about adaptation energy and dynamics of adaptation.
• A hierarchy of dynamic models of adaptation is developed.
• Adaptation energy is considered as an internal coordinate on the ‘dominant path’ in the model of adaptation.
• The optimal distribution of resources for neutralization of harmful factors is studied.
• The phenomena of ‘oscillating death’ and ‘oscillating remission’ are predicted.

In previous research, it was suggested that adaptation of an animal to different factors looks like spending of one resource, and that the animal dies when this resource is exhausted.

In 1938, Selye proposed the notion of adaptation energy and published ‘Experimental evidence supporting the conception of adaptation energy.’ Adaptation of an animal to different factors appears as the spending of one resource. Adaptation energy is a hypothetical extensive quantity spent for adaptation. This term causes much debate when one takes it literally, as a physical quantity, i.e. a sort of energy. The controversial points of view impede the systematic use of the notion of adaptation energy despite experimental evidence. Nevertheless, the response to many harmful factors often has general non-specific form and we suggest that the mechanisms of physiological adaptation admit a very general and nonspecific description.

We aim to demonstrate that Selye׳s adaptation energy is the cornerstone of the top-down approach to modelling of non-specific adaptation processes. We analyze Selye׳s axioms of adaptation energy together with Goldstone׳s modifications and propose a series of models for interpretation of these axioms. Adaptation energy is considered as an internal coordinate on the ‘dominant path’ in the model of adaptation. The phenomena of ‘oscillating death’ and ‘oscillating remission’ are predicted on the base of the dynamical models of adaptation. Natural selection plays a key role in the evolution of mechanisms of physiological adaptation. We use the fitness optimization approach to study of the distribution of resources for neutralization of harmful factors, during adaptation to a multifactor environment, and analyze the optimal strategies for different systems of factors.

In this work, an international team of researchers, led by Professor Alexander N. Gorban from the University of Leicester, have developed a solution to Selye’s riddle, which has puzzled scientists for almost 80 years.

Alexander N. Gorban, Professor of Applied Mathematics in the Department of Mathematics at the University of Leicester, said: “Nobody can measure adaptation energy directly, indeed, but it can be understood by its place already in simple models. In this work, we develop a hierarchy of top-down models following Selye’s findings and further developments. We trust Selye’s intuition and experiments and use the notion of adaptation energy as a cornerstone in a system of models. We provide a ‘thermodynamic-like’ theory of organism resilience that, just like classical thermodynamics, allows for economics metaphors, such as cost and bankruptcy and, more importantly, is largely independent of a detailed mechanistic explanation of what is ‘going on underneath’.”

Adaptation energy is considered as an internal coordinate on the “dominant path” in the model of adaptation. The phenomena of “oscillating death” and “oscillating remission,” which have been observed in clinic for a long time, are predicted on the basis of the dynamical models of adaptation. The models, based on Selye’s idea of adaptation energy, demonstrate that the oscillating remission and oscillating death do not need exogenous reasons. The developed theory of adaptation to various factors gives the instrument for the early anticipation of crises.

Professor Alessandro Giuliani from Istituto Superiore di Sanità in Rome commented on the work, saying: “Gorban and his colleagues dare to make science adopting the thermodynamics style: they look for powerful principles endowed with predictive ability in the real world before knowing the microscopic details. This is, in my opinion, the only possible way out from the actual repeatability crisis of mainstream biology, where a fantastic knowledge of the details totally fails to predict anything outside the test tube.¹”

Citation: Alexander N. Gorban, Tatiana A. Tyukina, Elena V. Smirnova, Lyudmila I. Pokidysheva. Evolution of adaptation mechanisms: Adaptation energy, stress, and oscillating death. Journal of Theoretical Biology, 2016; DOI:10.1016/j.jtbi.2015.12.017. Voosen P. (2015) Amid a Sea of False Findings NIH tries Reform, The Chronicle of Higher Education.

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Success in Psoriasis Treatment

Posted in Curation, FDA Regulatory Affairs, FDA, CE Mark & Global Regulatory Affairs: process management and strategic planning - GCP, GLP, ISO 14155, Genetics & Pharmaceutical, Human Immune System in Health and in Disease, Inflammasome, Pharmaceutical Analytics, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, tagged autoimmune diseases, IL-17A inhibitor, ixekizumab, monoclonal antibody drugs, nitric oxide pathway, pro-inflammatory cytokine, psoriasis on March 8, 2016| Leave a Comment »

Success in Psoriasis Treatment

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Anti-IL17A Tx Clears Most Cases of Psoriasis

Durable long-term responses with ixekizumab, secukinumab

http://www.medpagetoday.com/MeetingCoverage/AAD/56597?xid=nl_mpt_guptaguide_2016-03-07

by Charles Bankhead Staff Writer, MedPage Today

Note that these studies were published as abstracts and presented at a conference.
These data and conclusions should be considered to be preliminary until published in a peer-reviewed journal.
More than half of patients with moderate-to-severe plaque psoriasis remained clear of lesions after a year of treatment with the interleukin-17A inhibitor ixekizumab.
Note that in another study, comparing two monoclonal antibodies for secukinumab (Cosentyx) or ustekinumab (Stelara) in patients with moderate to severe plaque psoriasis. showed sustained superiority for secukinumab (Cosentyx) over ustekinumab (Stelara).

More than half of patients with moderate-to-severe plaque psoriasis remained clear of lesions after a year of treatment with the interleukin-17A inhibitor ixekzumab, according to data reported here.

The 60-week follow-up data showed that 54% of patients treated with either of two doses of ixekizumab had 100% improvement in the Psoriasis Area and Severity Index (PASI 100). More than 70% achieved PASI 90, and more than 80% met criteria for PASI 75 response.

In general, the monoclonal antibody demonstrated good tolerability, associated with a discontinuation rate of about 5%, Andrew Blauvelt, MD, of Oregon Medical Research Center in Portland, reported at the American Academy of Dermatology meeting.

“Izekizumab treatment led to high clinical response rates and sustained efficacy in a majority of patients,” Blauvelt said. “More than half of ixekizumab-treated patients achieved complete resolution of psoriatic plaques at week 60. The safety profile for ixekizumab was similar to what was observed during the 12-week induction period.”

Ixekizumab is a specific inhibitor of the IL-17A receptor. The antibody was compared against placebo and etanercept (Enbrel) in two phase III trials that evaluated two ixekizumab dosing schedules (administration every 2 or 4 weeks). More than 1,200 patients were randomized 1:2:2:2 to placebo, etanercept (Enbrel) or one of the ixekizumab schedules.

As previously reported, the antibody demonstrated superior efficacy after a 12-week induction period. PASI 75 response rates were 7.3% with placebo, 53.4% with etanercept, and 84.2% and 87.3% with the two ixekizumab regimens. PASI 90 rates were 3.1%, 25.7%, 65.3%, and 68.1%. PASI 100 responses were attained by 0%, 73%, 35%, and 37.7%.

Upon completion of the induction phase, all patients transitioned to open-label ixekizumab, administered every 4 weeks. Blauvelt reported findings for patients who received only ixekizumab for the entire 60-week follow-up period.

The ixekizumab trial was supported by Eli Lilly.

Blauvelt disclosed relevant relationships with AbbVie, Amgen, Boehringer Ingelheim, Celgene, Dermira, Genentech, Janssen Ortho Biotech, Eli Lilly, Merck, Novartis, Pfizer, Regeneron, and Sandoz.

The secukinumab trial was supported by Novartis.

Thaci disclosed relevant relationships with AbbVie, Almiral, Amgen, Astellas, Biogen-Idec, Boehringer Ingelheim, Celgene, Dignity, Eli Lilly, Forward Pharma, GlaxoSmithKline, LEO Pharma, Janssen-Cilag, Maruho, Merck Sharp & Dohme, Mitsubishi Pharema, Novartis, Pfizer, Roche, Sandoz, Galapagos, Xenoport, Roche, and Mundipharma.

Lancet. 2015 Aug 8;386(9993):541-51. http://dx.doi.org:/10.1016/S0140-6736(15)60125-8. Epub 2015 Jun 10.

Comparison of ixekizumab with etanercept or placebo in moderate-to-severe psoriasis (UNCOVER-2 and UNCOVER-3): results from two phase 3 randomised trials.

Griffiths CE, Reich K, Lebwohl M, van de Kerkhof P, Paul C, Menter A, Cameron GS, Erickson J, Zhang L, Secrest RJ, Ball S, Braun DK, Osuntokun OO, Heffernan MP, Nickoloff BJ, Papp K; UNCOVER-2 and UNCOVER-3 investigators.

BACKGROUND:

Ixekizumab is a humanised monoclonal antibody against the proinflammatory cytokine interleukin 17A. We report two studies of ixekizumab compared with placebo or etanercept to assess the safety and efficacy of specifically targeting interleukin 17A in patients with widespread moderate-to-severe psoriasis.

METHODS:

In two prospective, double-blind, multicentre, phase 3 studies (UNCOVER-2 and UNCOVER-3), eligible patients were aged 18 years or older, had a confirmed diagnosis of chronic plaque psoriasis at least 6 months before baseline (randomisation), 10% or greater body-surface area involvement at both screening and baseline visits, at least a moderate clinical severity as measured by a static physician global assessment (sPGA) score of 3 or more, and a psoriasis area and severity index (PASI) score of 12. Participants were randomly assigned (1:2:2:2) by computer-generated random sequence with an interactive voice response system to receive subcutaneous placebo, etanercept (50 mg twice weekly), or one injection of 80 mg ixekizumab every 2 weeks, or every 4 weeks after a 160 mg starting dose. Blinding was maintained with a double-dummy design. Coprimary efficacy endpoints were proportions of patients achieving sPGA score 0 or 1 and 75% or greater improvement in PASI at week 12. Analysis was by intention to treat. These trials are registered with ClinicalTrials.gov, numbers NCT01597245 and NCT01646177.

FINDINGS:

Between May 30, 2012, and Dec 30, 2013, 1224 patients in UNCOVER-2 were randomly assigned to receive subcutaneous placebo (n=168), etanercept (n=358), or ixekizumab every 2 weeks (n=351) or every 4 weeks (n=347); between Aug 11, 2012, and Feb 27, 2014, 1346 patients in UNCOVER-3 were randomly assigned to receive placebo (n=193), etanercept (n=382), ixekizumab every 2 weeks (n=385), or ixekizumab every 4 weeks (n=386). At week 12, both primary endpoints were met in both studies. For UNCOVER-2 and UNCOVER-3 respectively, in the ixekizumab every 2 weeks group, PASI 75 was achieved by 315 (response rate 89·7%; [effect size 87·4% (97·5% CI 82·9-91·8) vs placebo; 48·1% (41·2-55·0) vs etanercept]) and 336 (87·3%; [80·0% (74·4-85·7) vs placebo; 33·9% (27·0-40·7) vs etanercept]) patients; in the ixekizumab every 4 weeks group, by 269 (77·5%; [75·1% (69·5-80·8) vs placebo; 35·9% (28·2-43·6) vs etanercept]) and 325 (84·2%; [76·9% (71·0-82·8) vs placebo; 30·8% (23·7-37·9) vs etanercept]) patients; in the placebo group, by four (2·4%) and 14 (7·3%) patients; and in the etanercept group by 149 (41·6%) and 204 (53·4%) patients (all p<0·0001 vs placebo or etanercept). In the ixekizumab every 2 weeks group, sPGA 0/1 was achieved by 292 (response rate 83·2%; [effect size 80·8% (97·5% CI 75·6-86·0) vs placebo; 47·2% (39·9-54·4) vs etanercept]) and 310 (80·5%; [73·8% (67·7-79·9) vs placebo; 38·9% (31·7-46·1) vs etanercept]) patients; in the ixekizumab every 4 weeks group by 253 (72·9%; [70·5% (64·6-76·5) vs placebo; 36·9% (29·1-44·7) vs etanercept]) and 291 (75·4%; [68·7% (62·3-75·0) vs placebo; 33·8% (26·3-41·3) vs etanercept]) patients; in the placebo group by four (2·4%) and 13 (6·7%) patients; and in the etanercept group by 129 (36·0%) and 159 (41·6%) patients (all p<0·0001 vs placebo or etanercept). In combined studies, serious adverse events were reported in 14 (1·9%) of 734 patients given ixekizumab every 2 weeks, 14 (1·9%) of 729 given ixekizumab every 4 weeks, seven (1·9%) of 360 given placebo, and 14 (1·9%) of 739 given etanercept; no deaths were noted.

INTERPRETATION:

Both ixekizumab dose regimens had greater efficacy than placebo and etanercept over 12 weeks in two independent studies. These studies show that selectively neutralising interleukin 17A with a high affinity antibody potentially gives patients with psoriasis a new and effective biological therapy option.

FUNDING:

Eli Lilly and Co.

Comment in

Ixekizumab for psoriasis–Authors’ reply. [Lancet. 2016]
Ixekizumab for psoriasis. [Lancet. 2016]
Interleukin-17 inhibition: a route to psoriasis clearance? [Lancet. 2015]

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Brain Cancer Vaccine in Development and other considerations

Posted in CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Clinical & Translational, Clinical Diagnostics, Curation, Diagnostic Immunology, Diagnostics and Lab Tests, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Human Immune System in Health and in Disease, Image Processing/Computing, Imaging-based Cancer Patient Management, Immuno-Oncology & Genomics, Immunodiagnostics, Immunology, Lasers and photonics, Neurological Diseases, Neuroscience, Nuclear Medicine, tagged "inhibiting" RNAs, antitumor immunity, Brain tumors, dendritic cell vaccines, glioblastoma multiforme, Glioma, high resolution microscopy, immunogenic cell death, immunogenicity, Immunotherapy, metabolic labelled radioimaging, tumor lysate on March 5, 2016| Leave a Comment »

Brain Cancer Vaccine in Development and other considerations

Larry H. Bernstein, MD, FCAP, Curator

LPBI

GEN News Highlights Mar 3, 2016

Advanced Immunotherapeutic Method Shows Promise against Brain Cancer

http://www.genengnews.com/gen-news-highlights/advanced-immunotherapeutic-method-shows-promise-against-brain-cancer/81252433/

http://www.genengnews.com/Media/images/GENHighlight/Mar3_2016_LeuvenLab_CellDeathMouseBrain6232214015.jpg

The researchers induced a specific type of cell death in brain cancer cells from mice. The dying cancer cells were then incubated together with dendritic cells, which play a vital role in the immune system. The researchers discovered that this type of cancer cell killing releases “danger signals” that fully activate the dendritic cells. “We re-injected the activated dendritic cells into the mice as a therapeutic vaccine,” Professor Patrizia Agostinis explains. “That vaccine alerted the immune system to the presence of dangerous cancer cells in the body. As a result, the immune system could recognize them and start attacking the brain tumor.” [©KU Leuven Laboratory of Cell Death Research & Therapy, Dr. Abhishek D. Garg]

Scientists from KU Leuven in Belgium say they have shown that next-generation cell-based immunotherapy may offer new hope in the fight against brain cancer.

Cell-based immunotherapy involves the injection of a therapeutic anticancer vaccine that stimulates the patient’s immune system to attack the tumor. Thus far, the results of this type of immunotherapy have been mildly promising. However, Abhishek D. Garg and Professor Patrizia Agostinis from the KU Leuven department of cellular and molecular medicine believe they have found a novel way to produce more effective cell-based anticancer vaccines.

“We re-injected the activated dendritic cells into the mice as a therapeutic vaccine,” explains Prof. Agostinis. “That vaccine alerted the immune system to the presence of dangerous cancer cells in the body. As a result, the immune system could recognize them and start attacking the brain tumor.”

Combined with chemotherapy, this novel cell-based immunotherapy drastically increased the survival rates of mice afflicted with brain tumors. Almost 50% of the mice were completely cured. None of the mice treated with chemotherapy alone became long-term survivors.

“The major goal of any anticancer treatment is to kill all cancer cells and prevent any remaining malignant cells from growing or spreading again,” says Professor Agostinis. “This goal, however, is rarely achieved with current chemotherapies, and many patients relapse. That’s why the co-stimulation of the immune system is so important for cancer treatments. Scientists have to look for ways to kill cancer cells in a manner that stimulates the immune system. With an eye on clinical studies, our findings offer a feasible way to improve the production of vaccines against brain tumors.”

The team published its study (“Dendritic Cell Vaccines Based on Immunogenic Cell Death Elicit Danger Signals and T Cell–Driven Rejection of High-Grade Glioma”) in Science Translational Medicine.

Dendritic cell vaccines based on immunogenic cell death elicit danger signals and T cell–driven rejection of high-grade glioma

Abhishek D. Garg¹,*, Lien Vandenberk²,*, Carolien Koks², Tina Verschuere³, Louis Boon⁴, Stefaan W. Van Gool²,†,‡ and Patrizia Agostinis¹,†,‡
Science Translational Medicine 02 Mar 2016: Vol. 8, Issue 328, pp. 328ra27 DOI: 10.1126/scitranslmed.aae0105

DAMPening Glioma

Dendritic cell (DC)–based vaccines have shown promise for treating high-grade glioma (HGG), but efficacy has been limited by antigenic heterogeneity of the tumors. Now, Garg et al. combine DC vaccines with hypericin-based photodynamic therapy–induced immunogenic cell death (ICD) to treat HGG in an animal model. They found that ICD-based DC vaccines improved survival, and that this effect was dependent on the cell-associated reactive oxygen species and release of damage-associated molecular patterns (DAMPs) acting as danger signals. These ICD-based DC vaccines synergized with standard-of-care therapy to further improve survival in HGG-bearing mice and shifted the tumor immune signature from regulatory to T_H1/T_H17, which is associated with positive outcome in patients.

The promise of dendritic cell (DC)–based immunotherapy has been established by two decades of translational research. Of the four malignancies most targeted with clinical DC immunotherapy, high-grade glioma (HGG) has shown the highest susceptibility. HGG-induced immunosuppression is a roadblock to immunotherapy, but may be overcome by the application of T helper 1 (T_H1) immunity–biased, next-generation, DC immunotherapy. To this end, we combined DC immunotherapy with immunogenic cell death (ICD; a modality shown to induce T_H1 immunity) induced by hypericin-based photodynamic therapy. In an orthotopic HGG mouse model involving prophylactic/curative setups, both biologically and clinically relevant versions of ICD-based DC vaccines provided strong anti-HGG survival benefit. We found that the ability of DC vaccines to elicit HGG rejection was significantly blunted if cancer cell–associated reactive oxygen species and emanating danger signals were blocked either singly or concomitantly, showing hierarchical effect on immunogenicity, or if DCs, DC-associated MyD88 signal, or the adaptive immune system (especially CD8⁺ T cells) were depleted. In a curative setting, ICD-based DC vaccines synergized with standard-of-care chemotherapy (temozolomide) to increase survival of HGG-bearing mice by ~300%, resulting in ~50% long-term survivors. Additionally, DC vaccines also induced an immunostimulatory shift in the brain immune contexture from regulatory T cells to T_H1/cytotoxic T lymphocyte/T_H17 cells. Analysis of the The Cancer Genome Atlas glioblastoma cohort confirmed that increased intratumor prevalence of T_H1/cytotoxic T lymphocyte/T_H17 cells linked genetic signatures was associated with good patient prognosis. Therefore, pending final preclinical checks, ICD-based vaccines can be clinically translated for glioma treatment.

SLC7A11 expression is associated with seizures and predicts poor survival in patients with malignant glioma

Stephanie M. Robert¹, Susan C. Buckingham¹, Susan L. Campbell¹, Stefanie Robel¹, ….., Adrienne C. Lahti³, Louis B. Nabors² and Harald Sontheimer¹,*
Science Translational Medicine 27 May 2015: Vol. 7, Issue 289, pp. 289ra86 DOI: 10.1126/scitranslmed.aaa8103

Glioma is the most common malignant primary brain tumor. Its rapid growth is aided by tumor-mediated glutamate release, creating peritumoral excitotoxic cell death and vacating space for tumor expansion. Glioma glutamate release may also be responsible for seizures, which complicate the clinical course for many patients and are often the presenting symptom. A hypothesized glutamate release pathway is the cystine/glutamate transporter System x_c⁻ (SXC), responsible for the cellular synthesis of glutathione (GSH). However, the relationship of SXC-mediated glutamate release, seizures, and tumor growth remains unclear. Probing expression of SLC7A11/xCT, the catalytic subunit of SXC, in patient and mouse-propagated tissues, we found that ~50% of patient tumors have elevated SLC7A11 expression. Compared with tumors lacking this transporter, in vivo propagated and intracranially implanted SLC7A11-expressing tumors grew faster, produced pronounced peritumoral glutamate excitotoxicity, induced seizures, and shortened overall survival. In agreement with animal data, increased SLC7A11 expression predicted shorter patient survival according to genomic data in the REMBRANDT (National Institutes of Health Repository for Molecular Brain Neoplasia Data) database. In a clinical pilot study, we used magnetic resonance spectroscopy to determine SXC-mediated glutamate release by measuring acute changes in glutamate after administration of the U.S. Food and Drug Administration–approved SXC inhibitor, sulfasalazine (SAS). In nine glioma patients with biopsy-confirmed SXC expression, we found that expression positively correlates with glutamate release, which is acutely inhibited with oral SAS. These data suggest that SXC is the major pathway for glutamate release from gliomas and that SLC7A11 expression predicts accelerated growth and tumor-associated seizures.

Cortical GABAergic excitation contributes to epileptic activities around human glioma

Johan Pallud¹,²,³,⁴, Michel Le Van Quyen¹,²,*, Franck Bielle¹,⁵,*, Christophe Pellegrino⁶,⁷,*, …., Richard Miles¹,², Laurent Capelle¹,¹¹ and Gilles Huberfeld¹,²,⁹,¹²,¹³,†
Science Translational Medicine 09 Jul 2014: Vol. 6, Issue 244, pp. 244ra89 DOI: 10.1126/scitranslmed.3008065

Brain gliomas are highly epileptogenic. Excitatory glutamatergic mechanisms are involved in the generation of epileptic activities in the neocortex surrounding gliomas. However, chloride homeostasis is known to be perturbed in glioma cells. Thus, the contribution of γ-aminobutyric acidergic (GABAergic) mechanisms that depend on intracellular chloride merits closer study. We studied the occurrence, networks, cells, and signaling basis of epileptic activities in neocortical slices from the peritumoral surgical margin resected around human brain gliomas. Postoperative glioma tissue from 69% of patients spontaneously generated interictal-like discharges, synchronized, with a high-frequency oscillation signature, in superficial layers of neocortex around areas of glioma infiltration. Interictal-like events depended both on glutamatergic AMPA receptor–mediated transmission and on depolarizing GABAergic signaling. GABA released by interneurons depolarized 65% of pyramidal cells, in which chloride homeostasis was perturbed because of changes in expression of neuronal chloride cotransporters: KCC2 (K-Cl cotransporter 2) was reduced by 42% and expression of NKCC1 (Na-K-2Cl cotransporter 1) increased by 144%. Ictal-like activities were initiated by convulsant stimuli exclusively in these epileptogenic areas. This study shows that epileptic activities are sustained by excitatory effects of GABA in human peritumoral neocortex, as reported in temporal lobe epilepsies, suggesting that both glutamate and GABA signaling and cellular chloride regulation processes, all also involved in oncogenesis as already shown, induce an imbalance between synaptic excitation and inhibition underlying epileptic discharges in glioma patients. Thus, the control of chloride in neurons and glioma cells may provide a therapeutic target for patients with epileptogenic gliomas.

Spherical Nucleic Acid Nanoparticle Conjugates as an RNAi-Based Therapy for Glioblastoma

Samuel A. Jensen¹,*, Emily S. Day²,³,*, Caroline H. Ko²,³,*, Lisa A. Hurley¹, …, David A. Giljohann²,³, Chad A. Mirkin²,³,† and Alexander H. Stegh¹,³,†
Science Translational Medicine 30 Oct 2013: Vol. 5, Issue 209, pp. 209ra152 DOI: 10.1126/scitranslmed.3006839

Glioblastoma multiforme (GBM) is a neurologically debilitating disease that culminates in death 14 to 16 months after diagnosis. An incomplete understanding of how cataloged genetic aberrations promote therapy resistance, combined with ineffective drug delivery to the central nervous system, has rendered GBM incurable. Functional genomics efforts have implicated several oncogenes in GBM pathogenesis but have rarely led to the implementation of targeted therapies. This is partly because many “undruggable” oncogenes cannot be targeted by small molecules or antibodies. We preclinically evaluate an RNA interference (RNAi)–based nanomedicine platform, based on spherical nucleic acid (SNA) nanoparticle conjugates, to neutralize oncogene expression in GBM. SNAs consist of gold nanoparticles covalently functionalized with densely packed, highly oriented small interfering RNA duplexes. In the absence of auxiliary transfection strategies or chemical modifications, SNAs efficiently entered primary and transformed glial cells in vitro. In vivo, the SNAs penetrated the blood-brain barrier and blood-tumor barrier to disseminate throughout xenogeneic glioma explants. SNAs targeting the oncoprotein Bcl2Like12 (Bcl2L12)—an effector caspase and p53 inhibitor overexpressed in GBM relative to normal brain and low-grade astrocytomas—were effective in knocking down endogenous Bcl2L12 mRNA and protein levels, and sensitized glioma cells toward therapy-induced apoptosis by enhancing effector caspase and p53 activity. Further, systemically delivered SNAs reduced Bcl2L12 expression in intracerebral GBM, increased intratumoral apoptosis, and reduced tumor burden and progression in xenografted mice, without adverse side effects. Thus, silencing antiapoptotic signaling using SNAs represents a new approach for systemic RNAi therapy for GBM and possibly other lethal malignancies.

Rapid, Label-Free Detection of Brain Tumors with Stimulated Raman Scattering Microscopy

Minbiao Ji¹,*, Daniel A. Orringer²,*, Christian W. Freudiger¹, Shakti Ramkissoon³,⁴, Xiaohui Liu⁵, …, Sandra Camelo-Piragua⁸, Keith L. Ligon³,⁴,⁹, Oren Sagher² and X. Sunney Xie¹,†
Science Translational Medicine 04 Sep 2013: Vol. 5, Issue 201, pp. 201ra119 DOI: 10.1126/scitranslmed.3005954

Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (κ = 0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct.

Neural Stem Cell–Mediated Enzyme/Prodrug Therapy for Glioma: Preclinical Studies

Karen S. Aboody¹,²,*, Joseph Najbauer¹, Marianne Z. Metz¹, Massimo D’Apuzzo³,…., Christine E. Brown⁸, Seung U. Kim⁹, Behnam Badie² and Jana Portnow^{10

Science Translational Medicine 08 May 2013: Vol. 5, Issue 184, pp. 184ra59 DOI: 10.1126/scitranslmed.3005365}

High-grade gliomas are extremely difficult to treat because they are invasive and therefore not curable by surgical resection; the toxicity of current chemo- and radiation therapies limits the doses that can be used. Neural stem cells (NSCs) have inherent tumor-tropic properties that enable their use as delivery vehicles to target enzyme/prodrug therapy selectively to tumors. We used a cytosine deaminase (CD)–expressing clonal human NSC line, HB1.F3.CD, to home to gliomas in mice and locally convert the prodrug 5-fluorocytosine to the active chemotherapeutic 5-fluorouracil. In vitro studies confirmed that the NSCs have normal karyotype, tumor tropism, and CD expression, and are genetically and functionally stable. In vivo biodistribution studies demonstrated NSC retention of tumor tropism, even in mice pretreated with radiation or dexamethasone to mimic clinically relevant adjuvant therapies. We evaluated safety and toxicity after intracerebral administration of the NSCs in non–tumor-bearing and orthotopic glioma–bearing immunocompetent and immunodeficient mice. We detected no difference in toxicity associated with conversion of 5-fluorocytosine to 5-fluorouracil, no NSCs outside the brain, and no histological evidence of pathology or tumorigenesis attributable to the NSCs. The average tumor volume in mice that received HB1.F3.CD NSCs and 5-fluorocytosine was about one-third that of the average volume in control mice. On the basis of these results, we conclude that combination therapy with HB1.F3.CD NSCs and 5-fluorocytosine is safe, nontoxic, and effective in mice. These data have led to approval of a first-in-human study of an allogeneic NSC-mediated enzyme/prodrug-targeted cancer therapy in patients with recurrent high-grade glioma.

Magnetic Resonance Metabolic Imaging of Glioma

Philippe Metellus¹,²,^* and Dominique Figarella-Branger²,^{3

Science Translational Medicine 11 Jan 2012: Vol. 4, Issue 116, pp. 116ps1 DOI: 10.1126/scitranslmed.3003591}

2-Hydroxyglutarate (2-HG) is a potential oncometabolite involved in gliomagenesis that has been identified as an aberrant product of isocitrate dehydrogenase (IDH)–mutated glial tumors. Recent genomics studies have shown that heterozygous mutation of IDH genes 1 and 2, present in up to 86% of grade II gliomas, is associated with a favorable outcome. Two reports in this issue describe both ex vivo and in vivo methods that could noninvasively detect the presence of 2-HG in glioma patients. This approach could have valuable implications for diagnosis, prognosis, and stratification of brain tumors, as well as for monitoring of treatment in glioma patients.

Exploiting the Immunogenic Potential of Cancer Cells for Improved Dendritic Cell Vaccines

Lien Vandenberk,^1,^*Jochen Belmans,¹Matthias Van Woensel,^2,³Matteo Riva,^1,⁴ andStefaan W. Van Gool^1,^5,^6,^*

Front Immunol. 2015; 6: 663. doi: 10.3389/fimmu.2015.00663

Cancer immunotherapy is currently the hottest topic in the oncology field, owing predominantly to the discovery of immune checkpoint blockers. These promising antibodies and their attractive combinatorial features have initiated the revival of other effective immunotherapies, such as dendritic cell (DC) vaccinations. Although DC-based immunotherapy can induce objective clinical and immunological responses in several tumor types, the immunogenic potential of this monotherapy is still considered suboptimal. Hence, focus should be directed on potentiating its immunogenicity by making step-by-step protocol innovations to obtain next-generation Th1-driving DC vaccines. We review some of the latest developments in the DC vaccination field, with a special emphasis on strategies that are applied to obtain a highly immunogenic tumor cell cargo to load and to activate the DCs. To this end, we discuss the effects of three immunogenic treatment modalities (ultraviolet light, oxidizing treatments, and heat shock) and five potent inducers of immunogenic cell death [radiotherapy, shikonin, high-hydrostatic pressure, oncolytic viruses, and (hypericin-based) photodynamic therapy] on DC biology and their application in DC-based immunotherapy in preclinical as well as clinical settings.

Cancer immunotherapy has gained considerable momentum over the past 5 years, owing predominantly to the discovery of immune checkpoint inhibitors. These inhibitors are designed to release the brakes of the immune system that under physiological conditions prevent auto-immunity by negatively regulating cytotoxic T lymphocyte (CTL) function. Following the FDA approval of the anti-cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) monoclonal antibody (mAb) ipilimumab (Yervoy) in 2011 for the treatment of metastatic melanoma patients (1), two mAbs targeting programed death (PD)-1 receptor signaling (nivolumab and pembrolizumab) have very recently joined the list of FDA-approved checkpoint blockers (respectively, for the treatment of metastatic squamous non-small cell lung cancer and relapsed/refractory melanoma patients) (2, 3).

However, the primary goal of cancer immunotherapy is to activate the immune system in cancer patients. This requires the induction of tumor-specific T-cell-mediated antitumor immunity. Checkpoint blockers are only able to abrogate the brakes of a functioning antitumoral immune response, implying that only patients who have pre-existing tumor-specific T cells will benefit most from checkpoint blockade. This is evidenced by the observation that ipilimumab may be more effective in patients who have pre-existing, albeit ineffective, antitumor immune responses (4). Hence, combining immune checkpoint blockade with immunotherapeutic strategies that prime tumor-specific T cell responses might be an attractive and even synergistic approach. This relatively new paradigm has lead to the revival of existing, and to date disappointing (as monotherapies), active immunotherapeutic treatment modalities. One promising strategy to induce priming of tumor-specific T cells is dendritic cell (DC)-based immunotherapy.

Dendritic cells are positioned at the crucial interface between the innate and adaptive immune system as powerful antigen-presenting cells capable of inducing antigen-specific T cell responses (5). Therefore, they are the most frequently used cellular adjuvant in clinical trials. Since the publication of the first DC vaccination trial in melanoma patients in 1995, the promise of DC immunotherapy is underlined by numerous clinical trials, frequently showing survival benefit in comparison to non-DC control groups (6–8). Despite the fact that most DC vaccination trials differ in several vaccine parameters (i.e., site and frequency of injection, nature of the DCs, choice of antigen), DC vaccination as a monotherapy is considered safe and rarely associates with immune-related toxicity. This is in sharp contrast with the use of mAbs or cytokine therapies. Ipilumumab has, for instance, been shown to induce immune-related serious adverse events in up to one-third of treated melanoma patients (1). The FDA approval of Sipuleucel-T (Provenge), an autologous DC-enriched vaccine for hormone-resistant metastatic prostate cancer, in 2010 is really considered as a milestone in the vaccination community (9). After 15 years of extensive clinical research, Sipileucel-T became the first cellular immunotherapy ever that received FDA approval, providing compelling evidence for the substantial socio-economic impact of DC-based immunotherapy. DC vaccinations have most often been applied in patients with melanoma, prostate cancer, high-grade glioma, and renal cell cancer. Although promising objective responses and tumor-specific T cell responses have been observed in all these cancer-types (providing proof-of-principle for DC-based immunotherapy), the clinical success of this treatment is still considered suboptimal (6). This poor clinical efficacy can in part be attributed to the severe tumor-induced immune suppression and the selection of patients with advanced disease status and poor survival prognostics (6, 10–12).

There is a consensus in the field that step-by-step optimization and standardization of the production process of DC vaccines, to obtain a Th1-driven immune response, might enhance their clinical efficacy (13). In this review, we address some recent DC vaccine adaptations that impact DC biology. Combining these novel insights might bring us closer to an ideal DC vaccine product that can trigger potent CTL- and Th1-driven antitumor immunity.

One factor requiring more attention in this production process is the immunogenicity of the dying or dead cancer cells used to load the DCs. It has been shown in multiple preclinical cancer models that the methodology used to prepare the tumor cell cargo can influence the in vivo immunogenic potential of loaded DC vaccines (14–19). Different treatment modalities have been described to enhance the immunogenicity of cancer cells in the context of DC vaccines. These treatments can potentiate antitumor immunity by inducing immune responses against tumor neo-antigens and/or by selectively increasing the exposure/release of particular damage-associated molecular patterns (DAMPs) that can trigger the innate immune system (14, 17–19). The emergence of the concept of immunogenic cell death (ICD) might even further improve the immunogenic potential of DC vaccines. Cancer cells undergoing ICD have been shown to exhibit excellent immunostimulatory capacity owing to the spatiotemporally defined emission of a series of critical DAMPs acting as potent danger signals (20, 21). Thus far, three DAMPs have been attributed a crucial role in the immunogenic potential of nearly all ICD inducers: the surface-exposed “eat me” signal calreticulin (ecto-CRT), the “find me” signal ATP and passively released high-mobility group box 1 (HMGB1) (21). Moreover, ICD-experiencing cancer cells have been shown in various mouse models to act as very potent Th1-driving anticancer vaccines, already in the absence of any adjuvants (21, 22). The ability to reject tumors in syngeneic mice after vaccination with cancer cells (of the same type) undergoing ICD is a crucial hallmark of ICD, in addition to the molecular DAMP signature (21).

Here, we review the effects of three frequently used immunogenic modalities and four potent ICD inducers on DC biology and their application in DC vaccines in preclinical as well as clinical settings (Tables (Tables11 and and2).2). Moreover, we discuss the rationale for combining different cell death-inducing regimens to enhance the immunogenic potential of DC vaccines and to ensure the clinical relevance of the vaccine product.

Table 1

A list of prominent enhancers of immunogenicity and ICD inducers applied in DC vaccine setups and their associations with DAMPs and DC biology.

Table 2

A list of preclinical tumor models and clinical studies for evaluation of the in vivo potency of DC vaccines loaded with immunogenically killed tumor cells.

The Impact of DC Biology on the Efficacy of DC Vaccines

Over the past years, different DC vaccine parameters have been shown to impact the clinical effectiveness of DC vaccinations. In the next section, we will elaborate on some promising adaptations of the DC preparation protocol.

Given the labor-intensive ex vivo culturing protocol of monocyte-derived DCs and inspired by the results of the Provenge study, several groups are currently exploiting the use of blood-isolated naturally circulating DCs (76–78). In this context, De Vries et al. evaluated the use of antigen-loaded purified plasmacytoid DCs for intranodal injection in melanoma patients (79). This strategy was feasible and induced only very mild side effects. In addition, the overall survival of vaccinated patients was greatly enhanced as compared to historical control patients. However, it still remains to be determined whether this strategy is more efficacious than monocyte-derived DC vaccine approaches (78). By contrast, experiments in the preclinical GL261 high-grade glioma model recently showed that vaccination with tumor antigen-loaded myeloid DCs resulted in more robust Th1 responses and a stronger survival benefit as compared to mice vaccinated with their plasmacytoid counterparts (80).

In view of their strong potential to stimulate cytotoxic T cell responses, several groups are currently exploring the use of Langerhans cell-like DCs as sources for DC vaccines (81–83). These so-called IL-15 DCs can be derived from CD14⁺monocytes by culturing them with IL-15 (instead of the standard IL-4). Recently, it has been shown that in comparison to IL-4 DCs, these cells have an increased capacity to stimulate antitumor natural killer (NK) cell cytotoxicity in a contact- and IL-15-dependent manner (84). NK cells are increasingly being recognized as crucial contributors to antitumor immunity, especially in DC vaccination setups (85, 86). Three clinical trials are currently evaluating these Langerhans cell-type DCs in melanoma patients (NCT00700167, NCT 01456104, and NCT01189383).

Targeting cancer stem cells is another promising development, particularly in the setting of glioma (87). Glioma stem cells can foster tumor growth, radio- and chemotherapy-resistance, and local immunosuppression in the tumor microenvironment (87, 88). Furthermore, glioma stem cells may express higher levels of tumor-associated antigens and MHC complex molecules as compared to non-stem cells (89, 90). A preclinical study in a rodent orthotopic glioblastoma model has shown that DC vaccines loaded with neuropsheres enriched in cancer stem cells could induce more immunoreactivity and survival benefit as compared to DCs loaded with GL261 cells grown under standard conditions (91). Currently there are four clinical trials ongoing in high-grade glioma patients evaluating this approach (NCT00890032, NCT00846456, NCT01171469, and NCT01567202).

With regard to the DC maturation status of the vaccine product, a phase I/II clinical trial in metastatic melanoma patients has confirmed the superiority of mature antigen-loaded DCs to elicit immunological responses as compared to their immature counterparts (92). This finding was further substantiated in patients diagnosed with prostate cancer and recurrent high-grade glioma (93, 94). Hence, DCs need to express potent costimulatory molecules and lymph node homing receptors in order to generate a strong T cell response. In view of this finding, the route of administration is another vaccine parameter that can influence the homing of the injected DCs to the lymph nodes. In the context of prostate cancer and renal cell carcinoma it has been shown that vaccination routes with access to the draining lymph nodes (intradermal/intranodal/intralymphatic/subcutaneous) resulted in better clinical response rates as compared to intravenous injection (93). In melanoma patients, a direct comparison between intradermal vaccination and intranodal vaccination concluded that, although more DCs reached the lymph nodes after intranodal vaccination, the melanoma-specific T cells induced by intradermal vaccination were more functional (95). Furthermore, the frequency of vaccination can also influence the vaccine’s immunogenicity. Our group has shown in a cohort-comparison trial involving relapsed high-grade glioma patients that shortening the interval between the four inducer DC vaccines improved the progression-free survival curves (58, 96).

Another variable that has been systematically studied is the cytokine cocktail that is applied to mature the DCs. The current gold standard cocktail for DC maturation contains TNF-α, IL-1β, IL-6, and PGE₂ (97, 98). Although this cocktail upregulates DC maturation markers and the lymph node homing receptor CCR7, IL-12 production by DCs could not be evoked (97, 98). Nevertheless, IL-12 is a critical Th1-driving cytokine and DC-derived IL-12 has been shown to associate with improved survival in DC vaccinated high-grade glioma and melanoma patients (99, 100). Recently, a novel cytokine cocktail, including TNF-α, IL-1β, poly-I:C, IFN-α, and IFN-γ, was introduced (101, 102). The type 1-polarized DCs obtained with this cocktail produced high levels of IL-12 and could induce strong tumor-antigen-specific CTL responses through enhanced induction of CXCL10 (99). In addition, CD40-ligand (CD40L) stimulation of DCs has been used to mature DCs in clinical trials (100, 103). Binding of CD40 on DCs to CD40L on CD4⁺ helper T cells licenses DCs and enables them to prime CD8⁺ effector T cells.

A final major determinant of the vaccine immunogenicity is the choice of antigen to load the DCs. Two main approaches can be applied: loading with selected tumor antigens (tumor-associated antigens or tumor-specific antigens) and loading with whole tumor cell preparations (13). The former strategy enables easier immune monitoring, has a lower risk of inducing auto-immunity, and can provide “off-the-shelf” availability of the antigenic cargo. Whole tumor cell-based DC vaccines, on the other hand, are not HLA-type dependent, have a reduced risk of inducing immune-escape variants, and can elicit immunity against multiple tumor antigens. Meta-analytical data provided by Neller et al. have demonstrated enhanced clinical efficacy in several tumor types of DCs loaded with whole tumor lysate as compared to DCs pulsed with defined tumor antigens (104). This finding was recently also substantiated in high-grade glioma patients, although this study was not set-up to compare survival parameters (105).

Toward a More Immunogenic Tumor Cell Cargo

The majority of clinical trials that apply autologous whole tumor lysate to load DC vaccines report the straightforward use of multiple freeze–thaw cycles to induce primary necrosis of cancer cells (8, 93). Freeze–thaw induced necrosis is, however, considered non-immunogenic and has even been shown to inhibit toll-like receptor (TLR)-induced maturation and function of DCs (16). To this end, many research groups have focused on tackling this roadblock by applying immunogenic modalities to induce cell death.

Immunogenic Treatment Modalities

Tables Tables11 and and22 list some frequently applied treatment methods to enhance the immunogenic potential of the tumor cell cargo that is used to load DC vaccines in an ICD-independent manner (i.e., these treatments do not meet the molecular and/or cellular determinants of ICD). Immunogenic treatment modalities can positively impact DC biology by inducing particular DAMPs in the dying cancer cells (Table (Table1).1). Table Table22 lists the preclinical and clinical studies that investigated their in vivo potential. Figure Figure11 schematically represents the application and the putative modes of action of these immunogenic enhancers in the setting of DC vaccines.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712296/bin/fimmu-06-00663-g001.jpg

A schematic representation of immunogenic DC vaccines. Cancer cells show enhanced immunogenicity upon treatment with UV irradiation, oxidizing treaments, and heat shock, characterized by the release of particular danger signals and the (increased) production of tumor (neo-)antigens. Upon loading onto DCs, DCs undergo enhanced phagocytosis and antigen uptake and show phenotypic and partial functional maturation. Upon in vivo immunization, these DC vaccines elicit Th1- and cytotoxic T lymphocyte (CTL)-driven tumor rejection.

Ultraviolet Irradiation ….

Oxidation-Inducing Modalities

In recent years, an increasing number of data were published concerning the ability of oxidative stress to induce oxidation-associate molecular patterns (OAMPs), such as reactive protein carbonyls and peroxidized phospholipids, which can act as DAMPs (28, 29) (Table (Table1).1). Protein carbonylation, a surrogate indicator of irreversible protein oxidation, has for instance been shown to improve cancer cell immunogenicity and to facilitate the formation of immunogenic neo-antigens (30, 31).

One prototypical enhancer of oxidation-based immunogenicity is radiotherapy (21,23). In certain tumor types, such as high-grade glioma and melanoma, clinical trials that apply autologous whole tumor lysate to load DC vaccines report the random use of freeze–thaw cycles (to induce necrosis of cancer cells) or a combination of freeze–thaw cycles and subsequent high-dose γ-irradiation (8, 18) (Table (Table2).2). However, from the available clinical evidence, it is unclear which of both methodologies has superior immunogenic potential. In light of the oxidation-based immunogenicity that is associated with radiotherapy, we recently demonstrated the superiority of DC vaccines loaded with irradiated freeze–thaw lysate (in comparison to freeze–thaw lysate) in terms of survival advantage in a preclinical high-grade glioma model (18) (Table (Table2).2). ….

Heat Shock Treatment

Heat shock is a term that is applied when a cell is subjected to a temperature that is higher than that of the ideal body temperature of the organisms of which the cell is derived. Heat shock can induce apoptosis (41–43°C) or necrosis (>43°C) depending on the temperature that is applied (110). The immunogenicity of heat shock treated cancer cells largely resides within their ability to produce HSPs, such as HSP60, HSP70, and HSP90 (17, 32) (Table (Table1).1). …

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Figure 2

A schematic representation of immunogenic cell death (ICD)-based DC vaccines. ICD causes cancer cells to emit a spatiotemporally defined pattern of danger signals. Upon loading of these ICD-undergoing cancer cells onto DCs, they induce extensive phagocytosis and antigen uptake. Loaded DCs show enhanced phenotypic and functional maturation and immunization with these ICD-based DC vaccines instigates Th1-, Th17-, and cytotoxic T lymphocyte (CTL)-driven antitumor immunity in vivo.

Inducers of Immunogenic Cell Death

Immunogenic cell death is a cell death regimen that is associated with the spatiotemporally defined emission of immunogenic DAMPs that can trigger the immune system (20, 21, 113). ICD has been found to depend on the concomitant induction of reactive oxygen species (ROS) and activation of endoplasmatic reticulum (ER) stress (111). Besides the three DAMPs that are most crucial for ICD (ecto-CRT, ATP, and HMGB1), other DAMPs such as surface-exposed or released HSPs (notably HSP70 and HSP90) have also been shown to contribute to the immunogenic capacity of ICD inducers (20, 21). The binding of these DAMPs to their respective immune receptors (CD91 for HSPs/CRT, P2RX7/P2RY2 for ATP, and TLR2/4 for HMGB1/HSP70) leads to the recruitment and/or activation of innate immune cells and facilitates the uptake of tumor antigens by antigen-presenting cells and their cross-presentation to T cells eventually leading to IL-1β-, IL-17-, and IFN-γ-dependent tumor eradiation (22). This in vivo tumor rejecting capacity induced by dying cancer cells in the absence of any adjuvant, is considered as a prerequisite for an agent to be termed an ICD inducer. …

Although the list of ICD inducers is constantly growing (113), only few of these immunogenic modalities have been tested in order to generate an immunogenic tumor cell cargo to load DC vaccines (Tables (Tables11 and and2).2). Figure Figure22 schematically represents the preparation of ICD-based DC vaccines and their putative modes of action.

Radiotherapy

Ionizing X-ray or γ-ray irradiation exerts its anticancer effect predominantly via its capacity to induce DNA double-strand breaks leading to intrinsic cancer cell apoptosis (114). The idea that radiotherapy could also impact the immune system was derived from the observation that radiotherapy could induce T-cell-mediated delay of tumor growth in a non-irradiated lesion (115). This abscopal (ab-scopus, away from the target) effect of radiotherapy was later explained by the ICD-inducing capacity (116). Together with anthracyclines, γ-irradiation was one of the first treatment modalities identified to induce ICD. …

Shikonin

The phytochemical shikonin, a major component of Chinese herbal medicine, is known to inhibit proteasome activity. It serves multiple biological roles and can be applied as an antibacterial, antiviral, anti-inflammatory, and anticancer treatment. …

High-hydrostatic pressure

High-hydrostatic pressure (HHP) is an established method to sterilize pharmaceuticals, human transplants, and food. HHP between 100 and 250 megapascal (MPa) has been shown to induce apoptosis of murine and human (cancer) cells (121–123). While DNA damage does not seem to be induced by HHP <1000 MPa, HHP can inhibit enzymatic functions and the synthesis of cellular proteins (122). Increased ROS production was detected in HHP-treated cancer cell lines and ER stress was evidenced by the rapid phosphorylation of eIF2α (42). …

Oncolytic Viruses

Oncolytic viruses are self-replicating, tumor selective virus strains that can directly lyse tumor cells. Over the past few years, a new oncolytic paradigm has risen; entailing that, rather than utilizing oncolytic viruses solely for direct tumor eradication, the cell death they induce should be accompanied by the elicitation of antitumor immune responses to maximize their therapeutic efficacy (128). One way in which these oncolytic viruses can fulfill this oncolytic paradigm is by inducing ICD (128).

Thus far, three oncolytic virus strains can meet the molecular requirements of ICD; coxsackievirus B3 (CVB3), oncolytic adenovirus and Newcastle disease virus (NDV) (Table (Table1)1) (113). Infection of tumor cells with these viruses causes the production of viral envelop proteins that induce ER stress by overloading the ER. Hence, all three virus strains can be considered type II ICD inducers (113). …

Photodynamic therapy

Photodynamic therapy (PDT) is an established, minimally invasive anticancer treatment modality. It has a two-step mode of action involving the selective uptake of a photosensitizer by the tumor tissue, followed by its activation by light of a specific wavelength. This activation results in the photochemical production of ROS in the presence of oxygen (129–131). One attractive feature of PDT is that the ROS-based oxidative stress originates in the particular subcellular location where the photosensitizer tends to accumulate, ultimately leading to the destruction of the tumor cell (132). …

Combinatorial Regimens

In DC vaccine settings, cancer cells are often not killed by a single treatment strategy but rather by a combination of treatments. In some cases, the underlying rationale lies within the additive or even synergistic value of combining several moderately immunogenic modalities. The combination of radiotherapy and heat shock has, for instance, been shown to induce higher levels of HSP70 in B16 melanoma cells than either therapy alone (16). In addition, a combination therapy consisting of heat shock, γ-irradiation, and UV irradiation has been shown to induce higher levels of ecto-CRT, ecto-HSP90, HMGB1, and ATP in comparison to either therapy alone or doxorubicin, a well-recognized inducer of ICD (57). ….

Triggering antitumor immune responses is an absolute requirement to tackle metastatic and diffusely infiltrating cancer cells that are resistant to standard-of-care therapeutic regimens. ICD-inducing modalities, such as PDT and radiotherapy, have been shown to be able to act as in situ vaccines capable of inducing immune responses that caused regression of distal untreated tumors. Exploiting these ICD inducers and other immunogenic modalities to obtain a highly immunogenic antigenic tumor cell cargo for loading DC vaccines is a highly promising application. In case of the two prominent ICD inducers, Hyp-PDT and HHP, preclinical studies evaluating this relatively new approach are underway and HHP-based DC vaccines are already undergoing clinical testing. In the preclinical testing phase, more attention should be paid to some clinically driven considerations. First, one should consider the requirement of 100% mortality of the tumor cells before in vivo application. A second consideration from clinical practice (especially in multi-center clinical trials) is the fact that most tumor specimens arrive in the lab in a frozen state. This implies that a significant number of cells have already undergone non-immunogenic necrosis before the experimental cell killing strategies are applied. ….

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Retroviruses and Immunity

Posted in Cell Biology, Clinical Genomics, Curation, Human Immune System in Health and in Disease, Immunology, Virology, tagged evolution and endogenous retrovirus, Evolutionary Biology, human evolution, immunity, Immunology, innate immunity, retrovirus on March 4, 2016| Leave a Comment »

Retroviruses and Immunity

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Viral Remnants Help Regulate Human Immunity

Endogenous retroviruses in the human genome can regulate genes involved in innate immune responses.

By Jyoti Madhusoodanan | March 3, 2016
http://www.the-scientist.com//?articles.view/articleNo/45503/title/Viral-Remnants-Help-Regulate-Human-Immunity/

http://www.the-scientist.com/images/News/March2016/310ERVs.png

Dendrogram of various classes of endogenous retroviruses WIKIMEDIA, FGRAMMEN

Remnants of retroviruses that entered the human genome millions of years ago can regulate some innate immune responses. These viral sequences have previously been linked to controlling early mammalian development and formation of the placenta, among other things. A study published today (March 3) in Science establishes that one such endogenous retrovirus in human cells can also regulate the interferon response, which helps organisms quickly respond to infections. The work is one of the first reports to show that human cells could have adopted retroviral sequences to regulate their genes.

“Before we started this project . . . we knew our genomes were full of these elements and many of them are activated during normal development in cells,” said study coauthor Edward Chuong, a postdoc at the University of Utah in Salt Lake City. “Our motivation was: How can we take the next step and figure out their potential biological consequences?”

Chuong and his University of Utah mentors Nels Eldeand Cédric Feschotte began by scanning the sequences around interferon-induced genes, finding at least 27 transposable elements that likely originated from the long repeats at the ends of retroviral sequences. One such element, known as MER41, comes from a virus that invaded the genome approximately 45 million to 60 million years ago; the team found that its sequence in present-day human cells contained interferon-inducible binding sites.

The group then focused on a MER41 sequence that occurs 220 base pairs upstream of an interferon-induced gene called AIM2, which activates an inflammatory response in cells. When the researchers deleted this MER41 element in a cell line using CRISPR/Cas9 gene editing, interferon treatment could not trigger the AIM2 gene. Without the interferon-mediated response, these cells were more susceptible to viral infections, the team found.

“This is a really strong paper,” said Dixie Mager of the University of British Columbia who was not involved with the study. Although previous studies have considered the regulatory functions of endogenous retroviruses, most have been genome-wide correlations, Mager added. “[Here] they go in and delete the specific endogenous retroviruses and show an effect. That’s one of the things that sets this study apart.”

In addition to AIM2, the group found MER41 elements helped regulate at least three other interferon-inducible genes involved in human immunity. Looking across the genomes of other mammals, the researchers also found MER41-like regulatory elements in lemurs, bats, and other species.

The work is “simple and elegant,” said Todd Macfarlan of the Eunice Kennedy Shriver National Institute of Child Health and Human Development who was not involved with the study. “The novelty here is that it extends this idea that retroviruses are continually being coopted for things—not just for placental or early development, but also for other types of gene regulatory pathways. In the future the question might be: Are there any pathways where retroviruses don’t play a role?”

Whether host cells coopted the viral sequences for their regulatory needs or if ancient viruses used their regulatory abilities to control host immunity during invasion is still unknown, according to Feschotte. “We can only speculate why ancient viruses might have carried these regulatory switches to begin with, but data suggest they had these systems built into their sequence already,” he told The Scientist.

Endogenous retroviral elements make up about 8 percent of the human genome, and similar regulatory effects might be found on other mammalian gene functions, said Mager. “What’s cool about endogenous retroviruses is that their ends, known as LTRs, are optimized to have all these regulatory sequences in just 300 to 400 base pairs of DNA,” she said. “These units are powerhouses of regulatory potential.”

Future studies are needed to establish that these regulatory mechanisms are functional in animals, said Macfarlan. In subsequent work, Feschotte and his colleagues aim to extend their studies to a mouse model and immune cell lines.

To Feschotte’s mind, understanding how these sequences regulate human genes could shed light on previously unknown mechanisms of many diseases. While studies of cancer, autoimmune diseases, and other conditions have reported that endogenous retroviruses are reactivated in disease, the reasons for reactivation— and its consequences—are still unclear.

“What has plagued this field is that we don’t the consequences or molecular mechanisms by which these endogenous retroviruses contribute to disease,” he said.

E.B. Chuong et al., “Regulatory evolution of innate immunity through co-option of endogenous retroviruses,” Science, doi:10.1126/science.aad5497, 2016.

Regulatory evolution of innate immunity through co-option of endogenous retroviruses

Edward B. Chuong, Nels C. Elde*,†, Cédric Feschotte*,†
Science 2016;Vol. 351, Issue 6277, pp. 1083-1087 http://dx.doi.org:/10.1126/science.aad5497

Regulatory use of endogenous retroviruses

Mammalian genomes contain many endogenous retroviruses (ERVs), which have a range of evolutionary ages. The propagation and maintenance of these genetic elements have been attributed to their ability to contribute to gene regulation. Chuong et al. demonstrate that some ERV families are enriched in regulatory elements, so that they act as independently evolved enhancers for immune genes in both humans and mice (see the Perspective by Lynch). The analysis revealed a primate-specific element that orchestrates the transcriptional response to interferons. Selection can therefore act on selfish genetic elements to generate novel gene networks.

Science, this issue p. 1083 see also p. 1029

Endogenous retroviruses (ERVs) are abundant in mammalian genomes and contain sequences modulating transcription. The impact of ERV propagation on the evolution of gene regulation remains poorly understood. We found that ERVs have shaped the evolution of a transcriptional network underlying the interferon (IFN) response, a major branch of innate immunity, and that lineage-specific ERVs have dispersed numerous IFN-inducible enhancers independently in diverse mammalian genomes. CRISPR-Cas9 deletion of a subset of these ERV elements in the human genome impaired expression of adjacent IFN-induced genes and revealed their involvement in the regulation of essential immune functions, including activation of the AIM2 inflammasome. Although these regulatory sequences likely arose in ancient viruses, they now constitute a dynamic reservoir of IFN-inducible enhancers fueling genetic innovation in mammalian immune defenses.

Researchers Trace Spread of Ancient Viruses

Wed, 03/09/2016 Greg Watry, Digital Reporter http://www.dddmag.com/articles/2016/03/researchers-trace-spread-ancient-viruses

Viruses have been present for billions of years, affecting the gamut of life from single celled to multicellular organisms. But these diminutive infectious agents don’t leave behind fossils. Therefore, understanding their origin and evolution has proven difficult.

However, researchers from Boston College have traced the spread of an ancient group of retroviruses—known as ERV-Fc—that affected 28 of 50 studied mammalian ancestors between 15 and 30 million years ago.

“Over the course of millions of years, genetic sequences from the viruses accumulate in the DNA genomes of living organisms (including humans),” the researchers wrote in their paper appearing in eLife. “These sequences can serve as molecular ‘fossils’ for exploring the natural history of viruses and their hosts.”

Retroviruses affect various populations, and included in that group are immunodeficiency viruses, such as HIV-1 and HIV-2, and T-cell leukemia viruses.

The ancient viruses studied “affected a diverse range of hosts, including carnivores, rodents and primates,” the researchers wrote. “The distribution of ERV-Fc among different mammals indicates that the viruses spread to every continent except Antarctica and Australia, and that they jumped between species more than 20 times.”

The ERV-Fc virus was traced to the beginning of the Oligocene Epoch, which was marked by the first appearance of elephants with trunks, early horses, and extensive grasslands, according to the Univ. of California Museum of Paleontology.

In order to trace the virus group, the researchers searched mammalian genome sequence databases for ERV-Fc loci, and then “reconstructed the sequences of proteins representing the virus that colonized the ancestors of that particular species,” according to eLife.

The researchers also followed the changing patterns in the ERV-Fc viruses’ genes as it adapted to various hosts.

“As part of this process, the viruses often exchanged genes with each other and with other types of viruses,” the researchers wrote. “Such genetic recombination is likely to have played a significant role in the evolutionary success of the ERV-Fc viruses.”

According to study co-author William E. Diehl, the research may help humanity predict the long-term effects of viral infections, and the future evolution of such organisms.

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Inflammatory Disorders: Articles published @ pharmaceuticalintelligence.com

Posted in Alzheimer's Disease, Autoimmune Inflammatory DIseases, Autophagy, Bacterial Resistance, Cancer and Current Therapeutics, Crohn's disease, Cytokines, Diabetes Mellitus, Disease Biology, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Enzymes and isoenzymes, Etiology, Gastroenterology, Hematology, Human Immune System in Health and in Disease, Inflammasome, Lung and pulmonology, Lymphoma, Metabolism, Metabolomics, Microbiology, Micronutrients, Nephrology, Neurodegenerative Diseases, Neurological Diseases, Nitric Oxide in Health and Disease, Nutrition, Obesity, Protein-energy malnutrition, Proteolysis, Proteomics, Ulcerative Colitis, Viral diseases, Virology, tagged autoimmunity, systemic inflammatory response, T cells on February 28, 2016| Leave a Comment »

Inflammatory Disorders: Articles published @ pharmaceuticalintelligence.com

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

This is a compilation of articles on Inflammatory Disorders that were published

@ pharmaceuticalintelligence.com, since 4/2012 to date

There are published works that have not been included. However, there is a substantial amount of material in the following categories:

Proteomics, metabolomics and diabetes

http://pharmaceuticalintelligence.com/2015/11/16/reducing-obesity-related-inflammation/

http://pharmaceuticalintelligence.com/2015/10/25/the-relationship-of-stress-hypermetabolism-to-essential-protein-needs/

http://pharmaceuticalintelligence.com/2015/10/24/the-relationship-of-s-amino-acids-to-marasmic-and-kwashiorkor-pem/

http://pharmaceuticalintelligence.com/2015/10/24/the-significant-burden-of-childhood-malnutrition-and-stunting/

http://pharmaceuticalintelligence.com/2015/04/14/protein-binding-protein-protein-interactions-therapeutic-implications-7-3/

http://pharmaceuticalintelligence.com/2015/03/07/transthyretin-and-the-stressful-condition/

http://pharmaceuticalintelligence.com/2015/02/13/neural-activity-regulating-endocrine-response/

http://pharmaceuticalintelligence.com/2015/01/31/proteomics/

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Breast Cancer Extratumor Microenvironment has Effect on Progression

Posted in Biochemical pathways, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Informatics, Cell Biology, Signaling & Cell Circuits, Clinical & Translational, Clinical Diagnostics, Computational Biology/Systems and Bioinformatics, Curation, Cytoskeleton, Diagnostics and Lab Tests, Disease Biology, Gene Regulation, Genome Biology, Genomic Endocrinology, Human Immune System in Health and in Disease, Lipid metabolism, Metabolism, Metabolomics, Micronutrients, Mutagenesis, tagged breast cancer, extratunot microenvironment, metastasis on February 20, 2016| Leave a Comment »

Breast Cancer Extratumor Microenvironment has Effect on Progression

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Tumor Microenvironment Diversity Predicts Breast Cancer Outcomes

GEN News Highlights Feb 17, 2016 http://www.genengnews.com/gen-news-highlights/tumor-microenvironment-diversity-predicts-breast-cancer-outcomes/81252378/

Intratumor heterogeneity, it is known, can complicate cancer treatments. Now it appears the same may be true of tumor microenvironment heterogeneity. According to a new study from the Institute of Cancer Research (ICR), London, breast cancers that develop within an “ecologically diverse” breast cancer microenvironment are particularly likely to progress and lead to death.

The study took an unusual approach: It combined ecological scoring methods with genome-wide profiling data. This approach, besides showing clinical utility in the evaluation of breast cancer outcomes, demonstrated that even so contextual a discipline as genomics can benefit from being placed within a larger context. In this case, the context is essentially Darwinian, albeit at a small scale.

Natural selection is typically studied at the level of ecosystems consisting of animals and plants. In the current study, however, it was assessed at the level of the tumor microenvironment, which consists of cancer cells, immune system lymphocytes, and stromal cells.

The ICR scientists, led by Yinyin Yuan, Ph.D., presented their work February 16 in the journal PLoS Medicine, in an article entitled “Microenvironmental Heterogeneity Parallels Breast Cancer Progression: A Histology–Genomic Integration Analysis.” The article describes how the scientists developed a tumor ecosystem diversity index (EDI), a scoring system that indicates the degree of microenvironmental heterogeneity along three spatial dimensions in solid tumors. EDI scores take account of “fully automated histology image analysis coupled with statistical measures commonly used in ecology.”

“[EDI] was compared with disease-specific survival, key mutations, genome-wide copy number, and expression profiling data in a retrospective study of 510 breast cancer patients as a test set and 516 breast cancer patients as an independent validation set,” wrote the authors. “In high-grade (grade 3) breast cancers, we uncovered a striking link between high microenvironmental heterogeneity measured by EDI and a poor prognosis that cannot be explained by tumor size, genomics, or any other data types.”

By using the EDI, the ICR team was able to identify several particularly aggressive subgroups of breast cancer. In fact, the EDI was a stronger predictor of survival than many established markers for the disease.

The ICR researchers also looked at the EDI in addition to genetic factors. For example, the researchers found that the prognostic value of EDI was enhanced with the addition of TP53 mutation status. By integrating EDI data and genome-wide profiling data, the researchers identified losses of specific genes on 4p14 and 5q13 that were enriched in grade 3 tumors. These tumors, which showed high microenvironmental diversity, substratified patients into poor prognostic groups.

“Our findings show that mathematical models of ecological diversity can spot more aggressive cancers,” said Dr. Yuan. “By analyzing images of the environment around a tumor based on Darwinian natural selection principles, we can predict survival in some breast cancer types even more effectively than many of the measures used now in the clinic.

“In the future, we hope that by combining cell diversity scores with other factors that influence cancer survival, such as genetics and tumor size, we will be able to tell apart patients with more or less aggressive disease so we can identify those who might need different types of treatment.”

“This ingenious study…teaches us a valuable lesson,” added Paul Workman, Ph.D., chief executive of the ICR. “[We] should always remember that cancer cells are not developing and growing in isolation, but are part of a complex ecosystem that involves normal human cells, too. By better understanding these ecosystems, we aim to create new ways to diagnose, monitor and treat cancer.”

Microenvironmental Heterogeneity Parallels Breast Cancer Progression: A Histology–Genomic Integration Analysis

Rachael Natrajan, Heba Sailem, Faraz K. Mardakheh, Mar Arias Garcia, Christopher J. Tape, Mitch Dowsett, Chris Bakal, Yinyin Yuan

Feb 16, 2016 http://dx.doi.org:/10.1371/journal.pmed.1001961

Background

The intra-tumor diversity of cancer cells is under intense investigation; however, little is known about the heterogeneity of the tumor microenvironment that is key to cancer progression and evolution. We aimed to assess the degree of microenvironmental heterogeneity in breast cancer and correlate this with genomic and clinical parameters.

Methods and Findings

We developed a quantitative measure of microenvironmental heterogeneity along three spatial dimensions (3-D) in solid tumors, termed the tumor ecosystem diversity index (EDI), using fully automated histology image analysis coupled with statistical measures commonly used in ecology. This measure was compared with disease-specific survival, key mutations, genome-wide copy number, and expression profiling data in a retrospective study of 510 breast cancer patients as a test set and 516 breast cancer patients as an independent validation set. In high-grade (grade 3) breast cancers, we uncovered a striking link between high microenvironmental heterogeneity measured by EDI and a poor prognosis that cannot be explained by tumor size, genomics, or any other data types. However, this association was not observed in low-grade (grade 1 and 2) breast cancers. The prognostic value of EDI was superior to known prognostic factors and was enhanced with the addition of TP53 mutation status (multivariate analysis test set, p = 9 × 10⁻⁴, hazard ratio = 1.47, 95% CI 1.17–1.84; validation set, p = 0.0011, hazard ratio = 1.78, 95% CI 1.26–2.52). Integration with genome-wide profiling data identified losses of specific genes on 4p14 and 5q13 that were enriched in grade 3 tumors with high microenvironmental diversity that also substratified patients into poor prognostic groups. Limitations of this study include the number of cell types included in the model, that EDI has prognostic value only in grade 3 tumors, and that our spatial heterogeneity measure was dependent on spatial scale and tumor size.

Conclusions

To our knowledge, this is the first study to couple unbiased measures of microenvironmental heterogeneity with genomic alterations to predict breast cancer clinical outcome. We propose a clinically relevant role of microenvironmental heterogeneity for advanced breast tumors, and highlight that ecological statistics can be translated into medical advances for identifying a new type of biomarker and, furthermore, for understanding the synergistic interplay of microenvironmental heterogeneity with genomic alterations in cancer cells.

Background

The human body contains millions of cells, all of which grow, divide, and die in an orderly fashion to build tissues during early life and to replace worn-out or dying cells and repair injuries during adult life. Sometimes, however, normal cells acquire genetic changes (mutations) that allow them to divide uncontrollably and to move around the body (metastasize), resulting in cancer. Because any cell in the body can acquire the mutations needed for cancer development, there are many types of cancer. For example, breast cancer, the most common cancer in women, begins when the cells in the breast that normally make milk become altered. Moreover, different types of cancer progress and evolve differently—some cancers grow quickly and kill their “host” soon after diagnosis, whereas others can be successfully treated with drugs, surgery, or radiotherapy. The behavior of individual cancers depends both on the characteristics of the cancer cells within the tumor and on the interactions between the cancer cells and the normal stromal cells (the connective tissue cells of organs) and other cells (for example, immune cells) that surround and feed cancer cells (the tumor microenvironment).

Why Was This Study Done?

Although recent studies have highlighted the importance of the tumor microenvironment for disease-related outcomes, little is known about how the heterogeneity of the tumor microenvironment—the diversity of non-cancer cells within the tumor—affects outcomes. Mathematical modeling suggests that tumors with heterogeneous and homogeneous microenvironments have different growth patterns and that heterogeneous microenvironments are more likely to be associated with aggressive cancers than homogenous microenvironments. However, the lack of methods to quantify the spatial variability and cellular composition across solid tumors has prevented confirmation of these predictions. Here, the researchers develop a computational system for quantifying microenvironmental heterogeneity in breast cancer based on tumor morphology (shape and form) in histological sections (tissue samples taken from tumors that are examined microscopically). They then use this system to analyze the associations between clinical outcomes, molecular changes, and microenvironmental heterogeneity in breast cancer.

What Did the Researchers Do and Find?

The researchers used automated image analysis and statistical analysis to develop the ecosystem diversity index (EDI), a numerical measure of microenvironmental heterogeneity in solid tumors. They compared the EDI with prognosis (likely outcome), key mutations, genome-wide copy number (tumor cells often contain abnormal numbers of copies of specific genes), and expression profiling data (the expression of several key proteins is altered in tumors) in a test set of 510 samples from patients with breast cancer and in a validation set of 516 additional samples. Among high-grade breast cancers (grade 3 cancers; the grade of a cancer indicates what the cells look like; high-grade breast cancers have a poor prognosis), but not among low-grade breast cancers (grades 1 and 2), a high EDI (high microenvironmental heterogeneity) was associated with a poor prognosis. Specifically, patients with grade 3 tumors and a high EDI had a ten-year disease-specific survival rate of 51%, whereas the remaining patients with grade 3 tumors had a ten-year survival rate of 70%. Notably, the combination of a high EDI with specific DNA alterations—mutations in a gene called TP53 and loss of genes on Chromosomes 4p14 and 5q13—improved the accuracy of prognosis among patients with grade 3 breast cancer and stratified them into subgroups with disease-specific five-year survival rates of 35%, 9%, and 32%, respectively.

What Do These Findings Mean?

These findings establish a method for measuring the spatial heterogeneity of the microenvironment of solid tumors and suggest that the measurement of tumor microenvironmental heterogeneity can be coupled with information about genomic alterations to provide an accurate way to predict outcomes among patients with high-grade breast cancer. The association between EDI, specific genomic alterations, and outcomes needs to be confirmed in additional patients. However, these findings suggest that microenvironmental heterogeneity might provide an additional biomarker to help clinicians identify those patients with advanced breast cancer who have a particularly bad prognosis. The ability to identify these patients is important because it will help clinicians target aggressive treatments to individuals with a poor prognosis and avoid the overtreatment of patients whose prognosis is more favorable. Finally, and more generally, these findings describe a new way to investigate the interactions between the tumor microenvironment and genomic alterations in cancer cells.

Additional Information

This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at http://dx.doi.org/10.1371/journal.pmed.1001961.

The US National Cancer Institute provides comprehensive information about cancer and its development (in English and Spanish), including detailed information about breast cancer and an online booklet for patients
Cancer Research UK, a not-for-profit organization, provides information about cancer, including detailed information about breast cancer and a science blog on the tumor microenvironment
Breast Cancer Now is a not-for-profit organization that provides up-to-date information about breast cancer (in English and Spanish)
The UK National Health Service Choices website has information and personal stories about breast cancer; the not-for-profit organization Healthtalkonline also provides personal stories about dealing with breast cancer
Wikipedia has a page about the tumor microenvironment (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)

Citation: Natrajan R, Sailem H, Mardakheh FK, Arias Garcia M, Tape CJ, Dowsett M, et al. (2016) Microenvironmental Heterogeneity Parallels Breast Cancer Progression: A Histology–Genomic Integration Analysis.
PLoS Med 13(2): e1001961. http://dx.doi.org:/10.1371/journal.pmed.1001961

http://journals.plos.org/plosmedicine/article/figure/image?size=large&id=info:doi/10.1371/journal.pmed.1001961.g001

Fig 1. In silico tumor dissection pipeline for quantifying spatial diversity in the tumor ecosystem. (A) Flow diagram depicting the overall study design. (B) Schematic of our pipeline for quantifying spatial diversity in pathological samples. H&E sections are morphologically classified and divided into regions to be spatially scored. The number of clusters k in the regional scores is indicative of the number of sub-populations of cell types in the tumor regions. (C) Examples of tumor regions with low and high diversity scores using the Shannon diversity index, accounting for cancer cells (outlined in green), lymphocytes (blue), and stromal cells (red). Cell classification is automated by image analysis. (D) The 3-D landscape of cell diversity scores on an example H&E section; the x- and y-axes are the geometric axes of the image, and the z-axis is cell diversity computed on a region-by-region basis. (E) The distribution of regional scores in a tumor from the METABRIC study with two regional clusters identified using Gaussian mixture clustering (grey shading: histogram; dashed black line: density; solid black lines: mixture components/clusters).

http://journals.plos.org/plosmedicine/article/figure/image?size=medium&id=info:doi/10.1371/journal.pmed.1001961.g002

Fig 2. Application of EDI to 1,026 breast tumors from the METABRIC study. (A) The frequencies of EDI scores in breast tumors. (B) H&E staining, distribution of classified cells (green: cancer; blue: lymphocyte; red: stromal cells), and the heatmap of regional diversity scores for a tumor with the highest EDI score (EDI = 5). (C) Representative regions from each of the clusters k1–k5 are shown in a tumor with EDI = 5, with cluster k1 having the lowest diversity score and k5 the highest. By mapping regional clusters to the H&E image, we can begin to interpret these clusters with different cell diversity. We observed predominantly cancer cells in k1, increasingly more stromal cells and ductal in situ carcinoma cells (DCIS) in k2, and a vessel in k3. Cluster k4 features extensive stromal lymphocytes between ductal in situ carcinoma components, while k5 shows tumor-infiltrating lymphocytes (TIL) associated with invasive carcinoma cells.

Fig 3. Reproducibility, stability, and independence of the EDI-high group in 507 grade 3 breast tumors.

http://journals.plos.org/plosmedicine/article/figure/image?size=large&id=info:doi/10.1371/journal.pmed.1001961.g003

Fig 3. Reproducibility, stability, and independence of the EDI-high group in 507 grade 3 breast tumors. (A) Kaplan–Meier curves of disease-specific survival to illustrate the prognosis of EDI-high samples compared to other grade 3 samples in two independent patient cohorts. Shown below the graph are the number of patients (the number of disease-specific events) per group for EDI-low (grey) and EDI-high (red). (B) Agreement of the EDI subtyping between 100% data and resampling with progressively fewer tumor regions in 200 repeats. (C) Distribution of known subtypes in grade 3 tumors stratified by EDI; asterisks mark subtypes enriched in the EDI-high group. (D) Kaplan–Meier curves illustrating the duration of disease-specific survival according to tumor size (left) and improvement of stratification with the addition of EDI information (right).

Accumulating evidence suggests that the interactions of cancer cells and stromal cells within their microenvironment govern disease progression, metastasis, and, ultimately, the evolution of therapeutic resistance [1–3]. Recent reports have highlighted the significance of the contribution of stromal gene expression and morphological structure as powerful prognostic determinants for a number of tumor types, emphasizing the importance of the tumor microenvironment in disease-related outcomes [4–7]. In breast cancer, a number of studies have demonstrated the prognostic correlation of individual cell types, including the immune cell infiltrate that predicts response to therapy [8–10], and the high percentage of tumor stroma that predicts poor prognosis in triple-negative disease but good prognosis in estrogen receptor (ER)–positive disease [11,12]. Nevertheless, different types of cells coexist with varying degrees of heterogeneity within a tumor. This fundamental feature of human tumors and the combinatorial effects of cell types have been largely ignored, and the collective implications for clinical outcome remain elusive. Consistent observations from mathematical models have highlighted that tumors with diverse microenvironments show growth patterns dramatically different from those of tumors with homogeneous environments [13] and are more likely to be associated with aggressive cancer phenotypes [2] that select for cell migration and eventual metastasis by allowing cancer cells to evolve more rapidly [14]. These observations highlight the need to understand the collective physiological characteristics and heterogeneity of tumor microenvironments. However, there is a lack of methods to quantify the high spatial variability and diverse cellular composition across different solid tumors. Moreover, the interplay of genomic alterations in cancer cells and microenvironmental heterogeneity and its subsequent role in treatment response have not been explored. Our aims were (i) to develop a computational system for quantifying microenvironmental heterogeneity based on tumor morphology in routine histological sections, (ii) to define the clinical implications of microenvironmental heterogeneity, and (iii) to integrate this histologybased index with RNA gene expression and DNA copy number profiling data to identify molecular changes associated with microenvironmental heterogeneity.

The Ecosystem Diversity Index To characterize the tumor ecosystem based on cell compositions, we developed a new index to be used in conjunction with our image analysis tool [16]. First, we used our automated morphological classification method [16] to identify and classify cells into cancer, lymphocyte, or stromal cell classes in H&E sections (Fig 1B). We next divided sections into smaller spatial regions and quantified the diversity of the tumor ecosystem in a tumor region j using the Shannon diversity index: dj ¼ Sm i pi logpi ; ð1Þ where m is the number of cell types and pi is the proportion of the ith cell type (Fig 1B and 1C). A high value of the Shannon diversity index dj reports a heterogeneous environment populated by many cell types, whilst a low value indicates a homogeneous environment (Fig 1C). Compared to other methods such as the Simpson index, the Shannon diversity index accounts for rare species and, hence, is less dominated by main species [17]. Subsequently, we derived the ecosystem diversity index (EDI) by applying unsupervised clustering that identifies the optimum number of clusters in the dataset in an unbiased manner, in order to group tumor regions and quantify the degree of spatial heterogeneity. Let D = d1,d2,…,dn be the Shannon index for n regions in a tumor. We used Gaussian mixture models to fit data D: D SK k¼1okNðmk; s2 kÞ: ð2Þ where μk, ,s2 k, and ωk are the mean, variance, and weight of a Gaussian distribution k, and K is the number of clusters. The Bayesian information criterion was then used to select the best number of clusters K [18]. We used K = 1–5 as the range of K to avoid small EDI groups (S1 Text). The final value of K thus is a measurement of heterogeneity and the score of EDI for a tumor.

Fig 5. The relationship between ecological heterogeneity and cancer genomic aberrations in 507 grade 3 tumors. (A) Genome-wide copy number aberrations in grade 3 breast tumors and genomic coordinates of genes with copy number aberrations enriched in the EDI-high group. Lengths of black lines denote level of enrichment significance with copy number gains (above the horizontal line) or losses (below the horizontal line). (B) Kaplan–Meier curves illustrating the duration of disease-specific survival in grade 3 breast cancer patients according to copy number loss of the 4p14 region (left) and the EDI-high group with additional information of 4p14 copy number loss (right). (C) Kaplan–Meier curves illustrating the duration of disease-specific survival according to copy number loss of the 5q13 region (left) and the EDI-high group with additional information of 5q13 copy number loss (right).

http://dx.doi.org:/10.1371/journal.pmed.1001961.g005

This study has a number of limitations. The motivation for our computational development was to use a data-driven model and measure the degree of spatial heterogeneity in tumor pathological specimens. In this model, only three major cell types in breast tumors were considered. Further sub-classification of the different types of stromal and immune cells by immunohistochemistry may add additional discriminatory value to our model. For dissecting spatial heterogeneity, we chose to use square regions with equal sizes. We found that EDI was correlated with the size of the region chosen for calculation of the Shannon diversity index, and as such the spatial heterogeneity is scale dependent. This phenomenon has been well described in a number of studies in ecology that show that a scale needs to be chosen that is appropriate for the ecological process under study [38,39], further highlighting the analogy between tumor studies and ecology. Similar to the recent observation that breast cancer subclonal heterogeneity is correlated with tumor size [35], we also found an association between microenvironmental heterogeneity and tumor size; hence, EDI may have more limited value in smaller tumors. However, small tumors were present in the EDI-high group, and addition of EDI within tumors grouped by size further stratified their prognosis. We found that EDI was prognostic only in grade 3 tumors in our study, which could be a limitation, given the possible discordance in grading between pathologists.

The identification of additional biomarkers in subgroups of patients that identify them as high risk is important for patient management and to avoid overtreatment for low-risk patients. We envision that the use of our measure of microenvironment heterogeneity, together with key genomic alterations, will enable the diagnosis of patients at very high risk of relapse and facilitate the enrollment of these patients into additional clinical trials for novel therapies or treatment intensification. Our novel computational approach provides a fully automated tool that is relatively easy to implement. Integration of this measure with genomic profiling provides additional prognostic information independent of known clinical parameters. The results of this study highlight the possibility of a grade-3-specific prognostic tool that may aid in further classification of high-grade breast cancer patients beyond standard assays such as ER and HER2 status.

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Checkpoint inhibitors for gastrointestinal cancers

Posted in Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Curation, Disease Biology, Drug Toxicity, Human Immune System in Health and in Disease, Immuno-Oncology & Genomics, Monoclonal Immunotherapy, tagged gastrointestinal carcinomas, Immunotherapy, Monoclonal antibodies on February 14, 2016| Leave a Comment »

Checkpoint inhibitors for gastrointestinal cancers

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Updated 5/03/2019

Modern Immunotherapy for the Treatment of Advanced Gastrointestinal Cancers –

Oncology Journal, Gastrointestinal Cancer January 15, 2016

By Danny N. Khalil, MD, PhD and Neil H. Segal, MD, PhD

http://www.cancernetwork.com/oncology-journal/modern-immunotherapy-treatment-advanced-gastrointestinal-cancers?GUID=08B7ACA4-07B7-4253-8ACC-0C9AAFF0371A&XGUID=&rememberme=1&ts=11022016#sthash.NdRaifcd.dpuf

Since the first immune checkpoint–blocking monoclonal antibody was approved in the United States in 2011 for the treatment of advanced cancer, the rate of progress in the field of cancer immunotherapy has only accelerated. This mode of cancer treatment has yielded durable complete responses in a subset of patients with metastatic cancer for whom no other treatment was effective. It is a class of therapy that is not inherently cancer type–specific, and investigators are only beginning to understand why some cancers, such as melanoma, are more sensitive to immunotherapy than others. Although immunotherapy is not yet approved for the treatment of gastrointestinal cancers, it is already clear that many gastrointestinal cancers can be sensitive to it. We will review recent clinical trial results demonstrating this, and offer our perspective on the role that immunotherapy might play in the treatment of advanced gastrointestinal malignancies in the years ahead.

Introduction Immunotherapy can be defined as a therapeutic intervention that is focused on the immune system, as opposed to the cancer itself. Thus, it becomes the patient’s own immune response, rather than an exogenous drug, that acts directly against the disease. This approach to the treatment of cancer is viewed by many as a modern paradigm shift in oncology, in part because of recent successes of immune checkpoint blockade in diverse cancers.[1-3] It is important to keep in mind, however, that attempts to recruit the immune system in the effort against cancer are not new, and there is much to learn from early experiences in the field.

Immunotherapy has long been part of the standard treatment for early-stage cancers. For example, the intravesical Bacillus Calmette-Guérin vaccine and topical imiquimod are used to treat non–muscle-invasive bladder cancer and superficial basal cell carcinoma, respectively. Both of these agents are immunostimulants that function by activating immune cells in an antigen-nonspecific manner.[4,5] Their efficacy suggests that directing the immune response to a specific target is unnecessary in some cases, presaging disappointing efforts in therapeutic cancer vaccination designed to direct the immune system to targets associated with malignant cells.[6,7]

The experience with systemic immunotherapy for cancer in prior decades has been more controversial. High-dose interleukin (IL)-2 treatment for renal cell carcinoma and melanoma has led to extremely durable responses for a minority of patients, but has also led to excessive toxicity for others.[8] Without evidence of improved overall survival (OS) in a large randomized clinical trial, the precise setting for this therapy in patient care has been disputed. Nevertheless, IL-2 allowed the oncology community to glimpse both the potential efficacy and the potential harms of using the immune system to treat metastatic cancer.

Immune Checkpoint Blockade

Immune checkpoint blockade represents a class of anticancer agents that function by blocking inhibitory immune cell receptors. Among the most important members of this category are monoclonal antibodies (mAbs) that block cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) and programmed death 1 (PD-1) or its ligand PD-L1. After an antigen-presenting cell (APC) captures a tumor-associated antigen, it presents a portion of the antigen as a peptide to naive T cells in the context of a so-called immunologic synapse. Both stimulatory and inhibitory signaling between the T cell and the APC occur at this synapse. One inhibitory T-cell receptor that functions in this context is CTLA-4; therapeutically blocking CTLA-4 strengthens the immunogenic signal that the APC transmits to the T cell. Once the T cell is activated by the APC, it can then encounter a malignant cell presenting a cognate peptide and mediate its lysis. It is at this phase that the T cell encounters another set of inhibitory signals, including PD-L1 and PD-L2, which are both recognized by PD-1 on T cells. Anti–PD-1 mAbs block this interaction and thus enhance the ability of the activated T cell to lyse its target cell.

Immune checkpoint blockade as a means of treating cancer rose to prominence in 2010 when the anti–CTLA-4 mAb ipilimumab was found to improve median OS for patients with metastatic melanoma from 6.4 to 10 months.[7] This result was important for a number of reasons. First, ipilimumab was the first therapy to improve OS in this patient population in a phase III clinical trial. Second, since an independent study arm incorporated a therapeutic vaccine, it showed that such antigen-directed therapy did not add benefit in this context. Finally, it demonstrated that anti–CTLA-4 therapy can result in durable remissions.[9]

Following the unprecedented activity of CTLA-4 blockade, PD-1 blockade quickly rose to prominence. In fact, anti–PD-1 axis (ie, anti–PD-1 or anti–PD-L1) therapy showed response rates of over 40% in some melanoma studies,[1,10] and it has shown activity in a host of other malignancies, including non–small-cell lung cancer (NSCLC; response rate of 20%),[11,12] bladder cancer (response rate of over 40% in select patients),[3] and gastrointestinal malignancies, as discussed below.

The marked, but non-uniform, responses to checkpoint blockade triggered an international effort to identify biomarkers of response. PD-L1 expression in the tumor, whether on malignant cells or tumor-associated cells, was found to correlate with response to PD-1 axis blockade across a range of malignancies.[3,13,14] It should be noted, however, that a subset of tumors found to be PD-L1–negative did benefit from anti–PD-1 axis therapy, highlighting the fact that PD-L1 should not necessarily be used as a binary biomarker to predict response to therapy.

Although baseline PD-L1 expression correlates with response to PD-1 axis blockade, there is now evidence that genomic alterations may predict for response to checkpoint blockade more broadly. Whole-exome sequencing has demonstrated that mutation burden correlates with response to CTLA-4 blockade in melanoma,[15] and similar work revealed that mutation burden also correlates with response to PD-1 blockade in NSCLC.[16] It is not yet clear, however, that specific mutated sequences (so-called neoepitopes) reliably predict for response to any form of immunotherapy.[17] Such a finding, if prospectively validated, would enable clinicians to administer immunotherapy in much the same way that modern targeted therapies are used—based on the presence of discreet and predefined genetic lesions.

In addition, tumors that were responsive to checkpoint blockade were found to be more inflamed at baseline. For example, tumors rich in infiltrating T cells, and T helper 1 (Th1)-associated cytokines, were found to be particularly responsive.[18,19]

These findings do not only further our understanding of why immunotherapy is effective for some patients, but they also impact how immunotherapy will be used in the future. Therefore, they are of major significance as the field of immunotherapy begins to expand into gastrointestinal malignancies.

Pancreatic Cancer

Despite its historic intransigence, there are multiple lines of evidence indicating that pancreatic cancer can be responsive to immunotherapy. Pancreatic tumors have been found to exclude T cells at baseline in a manner that can be reversed.[20] Combination regimens designed to stimulate T cells with PD-L1 blockade and overcome T-cell exclusion via inhibition of the chemokine C-X-C ligand 12 (CXCL12) mediated tumor regression in an autochthonous animal model of pancreatic ductal adenocarcinoma.[21]

Based on clinical data, considering the paucity of responses to date, it is unlikely that anti–CTLA-4 therapy alone will have a role in the care of pancreatic cancer patients in the future. Nevertheless, there is instructive anecdotal evidence that even single-agent ipilimumab has activity among patients with pancreatic cancer. ….

Gastric Cancer

As with pancreatic cancer, responses to anti–CTLA-4 monotherapy in gastric carcinoma are rare and can be quite delayed. For example, in a phase II study of the anti–CTLA-4 mAb tremelimumab, 1 of 18 gastric cancer patients achieved a PR after 25 months on treatment.[30]

Consistent with other cancers, responses to PD-1 axis blockade in gastric cancer appear to be more frequent than responses to CTLA-4 blockade. Such results were anticipated by preclinical data showing that PD-L1 expression on gastric carcinoma cells, but not healthy gastric tissue or gastric adenomas, could induce T-cell apoptosis in a manner that was reversible with PD-L1–blocking mAbs.[31]

The anti–PD-1 mAb pembrolizumab is currently being tested in an ongoing phase I study of patients with adenocarcinoma of the stomach or gastroesophageal junction.[32] Preliminary results were presented at the European Society for Medical Oncology 2014 Congress. ….

Colorectal Cancer

There is extensive circumstantial data suggesting that colorectal cancer can respond to immune modulation. For example, colorectal cancer is generally associated with a relatively high mutation burden similar to other immune-responsive cancers, such as gastric and head and neck cancers.[33] In addition, there are reports associating immune signatures (eg, increased lymphocytes, especially cytotoxic and Th1 T cells, within the tumor or at the invasive margin) with improved prognosis.[34-36]

It is now apparent that two distinct immunologic subtypes of colorectal cancer exist, according to their mismatch repair (MMR) status. MMR deficiency occurs in approximately 4% of patients with metastatic colorectal cancer.[37] Tumors with MMR deficiency are rich in mutations that may be recognized as neoepitopes when presented to the adaptive immune system.[38,39] As would therefore be expected, MMR-deficient colorectal cancers are enriched for tumor-infiltrating lymphocytes.[40] This immunologic subtype of colorectal cancer represents an inherently sensitive population for T-cell stimulatory therapy. In a recently published phase II study of pembrolizumab,[41] 4 of 10 MMR-deficient patients had an immune-related objective response[23] vs 0 of 18 MMR-proficient patients. In an update presented at the 2015 American Society of Clinical Oncology Annual Meeting, which reported on 13 MMR-deficient and 25 MMR-proficient patients,[42] objective response rates were 62% and 0%, respectively. It is against this background that patients with MMR-deficient colorectal cancer will be evaluated for their response to pembrolizumab in phase II (Clinicaltrials.gov identifier: NCT02460198) and phase III (Clinicaltrials.gov identifier: NCT02563002) clinical trials; as well as for their response to durvalumab in an ongoing phase II study (Clinicaltrials.gov identifier: NCT02227667) we are currently conducting.

The Future of Immunotherapy in Gastrointestinal Cancers

We are optimistic that immunotherapy will become standard of care in at least a subset of gastrointestinal malignancies. In the near term, we anticipate that PD-1 axis blockade will be incorporated into the care of patients with gastroesophageal cancer and MMR-deficient colorectal cancer, and perhaps others, as it has been for patients with NSCLC and melanoma.

CTLA-4 and PD-1 are only two receptors among over a dozen known inhibitory and stimulatory T-cell receptors that can be targeted to augment antitumor T-cell activity.[45] There are thus innumerable combination regimens that can be designed to boost the already notable activity of checkpoint blockade. Furthermore, receptors on other immune cell populations can be activated or blocked to synergize with T-cell stimulatory therapy.[46] For example, current clinical trials are coupling the blockade of an inhibitory killer-cell immunoglobulin-like receptor on natural killer (NK) cells with anti–CTLA-4 (Clinicaltrials.gov identifier: NCT01750580) and anti–PD-1 (Clinicaltrials.gov identifier: NCT01714739) mAbs.

Given that tumor antigen–targeting mAbs (eg, cetuximab, trastuzumab) are approved or in clinical development for several types of gastrointestinal cancers,[47-49] there is interest in enhancing their efficacy through stimulation of immune cells. NK cells represent an attractive target for such a strategy, as they can mediate antibody-dependent cell-mediated cytotoxicity of malignant cells bound by tumor-targeting mAbs. In one such study that includes colorectal cancer patients, cetuximab is being combined with the anti-CD137 agonist mAb urelumab, which is designed to stimulate NK cells, in addition to T cells (Clinicaltrials.gov identifier: NCT02110082). …..

Although adoptive T-cell therapy is not yet ready for widespread clinical application, it has immense potential significance. Tran et al have effectively treated a patient with metastatic cholangiocarcinoma using CD4 T cells selected to recognize the product of a mutation specific to the patient’s tumor.[54] This type of adoptive transfer of selected, but unmodified, T cells has the notable limitation of being restricted to cancer-specific epitopes presented within patient-specific major histocompatibility complex (MHC) molecules. ….

The need for ex vivo manipulation to direct T cells to malignant cells in an MHC-independent manner can be circumvented using so-called bispecific T-cell engager (BiTE) technology. With this approach a therapeutic protein is constructed using mAb fragments specific to CD3 (present on the surface of T cells) and a molecule on the surface of the malignant cell. As with CAR technology, BiTEs have been studied primarily for the treatment of hematologic malignancies.[57] However, BiTEs that recognize the colorectal cancer–associated carcinoembryonic antigen have been developed,[58] and they will soon undergo clinical testing.

Most modern cancer immunotherapy is not inherently disease-specific. Furthermore, such treatments offer patients a chance at durable remissions, something not typically associated with cytotoxic chemotherapy or so-called targeted therapies. For these two reasons it is clear that, despite the remarkable successes to date, we are only at the start of an era in which the patient’s own immune system—with its unique combination of potency, specificity, and memory—begins to take the place of therapies that are designed to be directly toxic to malignant cells.

– See more at: http://www.cancernetwork.com/oncology-journal/modern-immunotherapy-treatment-advanced-gastrointestinal-cancers/page/0/2?GUID=08B7ACA4-07B7-4253-8ACC-0C9AAFF0371A&XGUID=&rememberme=1&ts=11022016#sthash.EnRTDdFt.dpuf

-see also

Immune-Oncology Molecules In Development & Articles on Topic in @pharmaceuticalintelligence.com

Curators: Stephen J Williams, PhD and Aviva Lev-Ari, PhD, RN

http://pharmaceuticalintelligence.com/2016/01/11/articles-on-immune-oncology-molecules-in-development-pharmaceuticalintelligence-com/

Updated 5/02/2019

Lack of microsatellite instability in colon cancer dooms a Combination MEK/PD-L1 Inhibitor Trial

IMblaze370 a ‘great disappointment’ following promise in preclinical models

by Ian Ingram, Deputy Managing Editor, MedPage Today April 24, 2019

An immunotherapy and targeted therapy combination failed to improve survival over standard third-line therapy for patients with chemorefractory metastatic colorectal cancer (CRC) and microsatellite-stable disease, a phase III trial found.

Median overall survival with the PD-L1 inhibitor atezolizumab (Tecentriq) plus MEK inhibitor cobimetinib (Cotellic) was no better than treatment with regorafenib (Stivarga) for these patients (8.9 vs 8.5 months; HR 1.00, 95% Cl 0.73-1.38, P=0.99), reported Fortunato Ciardiello, MD, PhD, of Università degli Studi della Campania Luigi Vanvitelli in Naples, Italy, and colleagues.

And with a median overall survival of 7.1 months, atezolizumab alone was numerically worse than regorafenib (HR 1.19, 95% Cl 0.83-1.71, P=0.34), the researchers wrote in Lancet Oncology.

Median progression-free survival was 1.9 months in each of the atezolizumab arms versus 2.0 months in the regorafenib arm, and objective responses occurred in 3% of patients treated with atezolizumab-cobimetinib and in 2% of patients treated with each of the single agents.

“Although many patients with metastatic colorectal cancer who have tumors with high microsatellite instability benefit from clinical improvement after immune checkpoint inhibitor therapy, patients with microsatellite-stable tumors do not,” Ciardiello’s group wrote.

Only about 3% to 5% of CRC patients have microsatellite instability, a genetic marker for immunotherapy response that led to the FDA approval of the anti-PD-1 agents pembrolizumab (Keytruda) and nivolumab (Opdivo) and the anti–CTLA-4/PD-1 combination of ipilimumab (Yervoy) plus nivolumab for all solid tumor patients who harbor this genetic abnormality and have previously been treated with chemotherapy.

Mouse models of cobimetinib showed anti-tumor activity “while promoting the effector phenotype and longevity of tumor-infiltrating CD8+ T cells,” and an anti-MEK/PD-L1 combination had a synergistic effect that led to durable treatment responses and complete regression in some cases. A phase Ib trial that reported objective responses in 8% of CRC patients with microsatellite stable disease led to development of the phase III IMblaze370 trial.

“Despite the rationale supported by preclinical data, our results suggest that dual inhibition of the PD-L1 immune checkpoint and MAPK-mediated immune suppression is insufficient to generate anti-tumor immune responses in immune-excluded tumors, such as microsatellite-stable metastatic colorectal cancer,” the authors wrote. “This failure to generate a response could be because of alternative mechanisms to bypass the inhibition of the MAPK pathway by a MEK inhibitor.”

In an editorial that accompanied the study, Francesco Sclafani, MD, of the Institut Jules in Brussels, said the findings appear to put an end to the suggestion that MEK inhibition can overcome immune resistance in CRC patients with microsatellite-stable disease.

“There is great disappointment for the negative results of the IMblaze370 trial because of the scientific interest and general enthusiasm for the underlying biological rationale and supportive preliminary clinical findings,” he wrote. “Dwelling on potential reasons for such an unexpected failure is therefore imperative.”

Sclafani noted that the immunomodulatory effects of MEK inhibition are not actually a settled matter, with some data reporting “suppression of T lymphocyte proliferative response and antigen-specific expansion and impairment of antigen processing by dendritic cells,” which could account for the trial’s negative findings.

He also questioned the trial’s lack of a biomarker strategy and said that heterogeneous tumor characteristics in microsatellite-stable CRC may require “distinct immunomodulatory strategies” to restore immunogenicity and generate anti-tumor immune responses.

The investigators noted that a limitation of the study was that it was not designed to examine patient subgroups that may have been more likely to respond to the combination therapy.

From 2016 to 2017, the IMblaze370 study randomized 363 adult CRC patients 2:1:1 to the combination of 840-mg atezolizumab (IV every 2 weeks) plus 60-mg oral cobimetinib daily (days 1-21 of 28-day cycles), 1200-mg atezolizumab monotherapy (IV every 3 weeks), or 160-mg regorafenib monotherapy (days 1-21 of 28-day cycles). Patients were eligible if they had an Eastern Cooperative Oncology Group performance status of 0-1 and had progressed or were intolerant of ≥2 prior lines of systemic therapy. Enrollment of patients with microsatellite instability–high CRC was allowed, but capped at 5%.

Grade 3/4 adverse events (AEs) in the combination arm were twice as frequent as in the atezolizumab monotherapy arm (61% vs 31%, respectively), but similar to the regorafenib arm (58%). Common grade 3/4 AEs (>5%) in the combination arm included diarrhea (11%), increased blood creatine phosphokinase (7%), and anemia (6%).

Serious AEs occurred in 40% of patients in the combination arm versus 23% with regorafenib and 17% with atezolizumab alone. There were two therapy-related deaths with the combination arm due to sepsis and one in the regorafenib arm due to intestinal perforation.

The study was funded by Roche/Genentech.

Ciardiello disclosed financial relationships with Roche/Genentech, Merck Serono, Pfizer, Amgen, Servier, Lilly, Bayer, Bristol-Myers Squibb, and Celgene. Co-authors reported relationships with Roche/Genentech and various other industry entities.

Other posts on the correlation of Microsatellite Instability with PDL1 efficacy on this Open Access Journal include:

Collaboration With Bristol Myers Squib Led to Successful Launch of Ono Pharmaceutical’s Cancer Immune Therapy (Opdivo®)

Immunotherapy Resistance Rears Its Ugly Head: PD-1 Resistant Metastatic Melanoma and More

First Drug in Checkpoint Inhibitor Class of Cancer Immunotherapies has demonstrated Superiority over Standard of care in the treatment of First-line Lung Cancer Patients: Merck’s Keytryda

Read Full Post »

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Archive for the ‘Human Immune System in Health and in Disease’ Category

Natural Killer Cell Response: Treatment of Cancer

Molecular mechanisms of natural killer cell activation in response to cellular stress

NK Cell-Mediated Recognition of Cellular Stress by NKG2D and DNAM-1

The DNA-Damage Response

Senescence

Tumor Suppressors: p53

Oncogenes

Heat-Shock Proteins (HSPs)

Emerging Links Between Cellular Stress and NK Cell Activation

Deregulated proliferation

MiRNAs

Regulation of cell-surface expression

Cellular hyperploidy

Immunogenic cell death

Concluding Remarks

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