Posts Tagged ‘angiogenesis’

Angiogenesis Requirement for Cancer Growth

Curator: Larry H. Bernstein, MD, FCAP



New Wrinkle in Cancer Growth Precept

David J. Kerr, CBE, MD, DSc, FRCP, FMedSci

Medscape  Oncology


Hello. I’m David Kerr, professor of cancer medicine at University of Oxford.

I’d like to discuss today a really interesting editorial that’s just recently been published in the Journal of the National Cancer Institute by a close friend and colleague of mine, Francesco Pezzella.[1] What he does is challenge one of the basic hallmarks of cancer. We know that Judah Folkman declared in the early 1970s[2] that cancer growth must be preceded by angiogenesis, that there can be no increase in cancer bulk or burden without new vessels dividing, sprouting, supporting, and oxygenating the growth of the cancer, these cancer nodules.

Indeed, angiogenesis has become one of the classic hallmarks of neoplasia, and there are by now a variety of drugs—bevacizumab, ramucirumab, and the tyrosine kinase inhibitors—that seek to prevent new angiogenesis. What Francesco talks about is a different model in which there are some tumors. This is first noted in non–small cell lung cancer[3] and pulmonary metastases, which are nonangiogenic. Rather than requiring the proliferation of new blood vessels, it seems that there are some tumor cells that are capable of co-opting existing blood vessels within organs such as the lungs and liver.

This is a new process, probably an active process. It’s probably cell surface adhesion molecule driven. But there are no clear ideas as yet as to what, mechanistically, the key events are. Of course, this poses a really huge number of really interesting questions. Is it possible that bevacizumab-resistant or ramucirumab-resistant tumors are those which switch from being angiogenic to nonangiogenic, thus devising and developing blood supply through co-option rather than new blood vessel formation?

If we understood more about the mechanism of action, it would lend itself to further drug targeting. If we combined an inhibitor of angiogenesis with an inhibitor of vascular co-option, would it prevent the outgrowth of resistance to the existing antiangiogenic drugs, which are used widely in clinic?

This is a fantastic new area of science, requiring much mechanistic work. It is not yet another hallmark of cancer but one for which I think, over the coming 5-10 years, we’ll see increasingly focused research, more mechanistic insights, and perhaps—if we’re lucky—an idea that can be used on development pathways.


  1. Pezzella F, Gatter KC. Evidence showing that tumors can grow without angiogenesis and can switch between angiogenic and nonangiogenic phenotypes. J Natl Cancer Inst. 2016; Apr 8. [Epub ahead of print]
  2. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971;285:1182-1186.Abstract
  3. Sardari NP, Colpaert C, Blyweert B, et al. Prognostic value of nonangiogenic and angiogenic growth patterns in non-small-cell lung cancer. Br J Cancer. 2004;91:1293-1300. Abstract


Evidence Showing That Tumors Can Grow Without Angiogenesis and Can Switch Between Angiogenic and Nonangiogenic Phenotypes

Francesco Pezzella and Kevin C. Gatter   JNCI J Natl Cancer Inst (2016) 108 (8): djw032

Although sorafenib is approved for the treatment of hepatocellular carcinoma (HCC), the ability of this anti-angiogenic tyrosine kinase inhibitor to extend survival in HCC patients is limited because of acquired drug resistance, whose mechanism is poorly understood. In this issue of the Journal, Kuczynski and colleagues (1) report a mechanism of acquired resistance to sorafenib using an orthotopic HCC xenograft model. The authors tell us how, although these HCC are initially angiogenic and respond to sorafenib, resistance occurs because the neoplastic cells switch to a non-angiogenic phenotype and actively co-opt the normal liver vasculature instead of inducing angiogenesis. These results have important implications for understanding this particular form of resistance to anti-angiogenic drugs and the biology of the tumors that can develop such a resistance.

The non-angiogenic growth of tumors has already been hypothesized to be one of the possible reasons for intrinsic and/or acquired cancer resistance to anti-angiogenic treatment (2). In this paper, it is now proven that an angiogenic malignancy can, following treatment with a drug inhibiting sprouting angiogenesis, switch to a non-angiogenic phenotype in order to keep growing.

This finding further supports the notion that neoplasia is not necessarily angiogenesis dependent as some tumors can be completely non-angiogenic while many more can present with a mixture of both angiogenic and non-angiogenic regions (3). Furthermore, it had been already described that the angiogenic status of a tumor is not an absolutely fixed characteristic but can change: eg, primary angiogenic breast carcinomas can relapse as non-angiogenic lung metastases (4) while non-angiogenic primary lung cancers can progress with angiogenic brain secondary (5). Now, the findings of Kuczynksi and coworkers show that a tumor can switch from angiogenic to non-angiogenic growth in response to treatment with an anti-angiogenic drug. Taken together, these observations demonstrate that the angiogenic status of a tumor is not an absolutely fixed characteristic, but can change.

This is in sharp contrast to the concept, first enunciated in 1971 by Judah Folkman (6) and subsequently included among the hallmarks of cancer (7), that tumor growth is always angiogenesis-dependent as “any increase in tumor population must be preceded by an increase in new capillaries converging on the tumor” (8).

Non-angiogenic tumors were first described in histopathology studies of primary non–small cell lung carcinomas (NSCLCs) and carcinoma metastases, from a variety of primaries, in the lung as these tumors fill the air spaces without destroying the parenchyma and co-opt the pre-existing alveolar vessels (9). Non-angiogenic human neoplasms were subsequently reported to occur as brain tumors (10), liver metastases growing by replacing the haepatocytes (11,12) or by colonizing the hepatic sinusoids (13) and lymph node metastases (14,15). Mouse models have now also been established for non-angiogenic malignant growths in brain (16,17) and lung (18).

The demonstration that some tumors do not induce angiogenesis is achieved on the grounds that in these lesions the only vessels present have the distribution and the phenotype of normal vessels and the overall architecture of the organ is preserved (10–12,19,20). This evidence now includes also a recent intravital microscopy study in an animal model (15). A second type of tumor growing without inducing sprouting angiogenesis and without co-opting vessels has also been discovered. This one exploits vascular mimicry, in which the neoplastic cells themselves assemble into channels that act like vessels (21).

The angiogenic NSCLC can also be told apart from non-angiogenic ones by gene transcription analyses. An investigation of differentially transcribed genes in these tumors suggested that metabolic reprogramming, with a switch toward oxidative phosphorylation under the control of selected heat shock proteins, oncogenes, and tumors suppressor genes, could be one of the key issues (22,23).

Another noteworthy piece of information is that the transcription and expression of genes associated with hypoxia and angiogenesis seems to be comparable in both angiogenic and non-angiogenic tumors (15,22,24). Therefore, the sensing of hypoxia and expression of VEGF protein by cancer cells do not invariably lead to new vessel formation.

Only a few studies so far have started to address the mechanisms of vessel co-option. Observations in animal models of brain malignancies indicate that vessel co-option is an active process that involves selected cell adhesion molecules and protection from apoptosis for the cell that is successfully co-opting a vessel (25–28).

All these results have therefore established the study of non-angiogenic tumors as a new field in cancer biology, but so far we have only just scratched the surface. So what now are the most pressing questions we face?

One is: what is the biology underlying the non-angiogenic phenotype, and what are the mechanisms that allow a cancer cell to switch between angiogenic and non-angiogenic phenotypes?

Second, how do the cancer cells interact with and co-opt the pre-existing vessels? Data published so far suggests that this is an active process (26,29). Moreover, how does this process change in different organs?

Third, does vessel co-option also facilitate resistance to other classes of anti-angiogenic drugs, such as the VEGF-neutralizing antibody bevacizumab?

Fourth, given that vessel co-option occurs in many human cancers, including some of the most prevalent (eg, malignancies from breast, colon, rectum, and lung), is vessel co-option also a mechanism of resistance to anti-angiogenic therapy in humans and not just in animal models?

Last, but not least, can vessel co-option be inhibited with drugs? The data of Kuczynski and colleagues shows that the response to sorafenib in HCC might be more durable if sorafenib were to be combined with a drug that targets vessel co-option. However, there are currently no drugs designed to target vessel co-option in humans. In our opinion, this represents a major deficiency in the current portfolio of oncology drugs and needs to be addressed urgently.

Hopefully, this new field of cancer biology will lead to novel therapeutic interventions designed according to the relationship observed between neoplastic cells and vessels in tumor lesions, in the knowledge that tumors can also grow without inducing angiogenesis.


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How cancer metastasis occurs

Larry H. Bernstein, MD, FCAP, Curator



How Cancer Cells Slide along Narrow Path to Metastasis

In tumors, abnormal protein-fiber environments and genetic perturbations conspire to give rise to metastatic behavior. In this looking-glass world, cells that bump into each other do not halt and reverse direction, as they ordinarily would. Instead, they slide around each other, enhancing migratory potential and bringing to mind the portmanteau “slithy,” which Lewis Carroll invented to describe the behavior of some of his imaginary creatures.

Slithy cancer cells do gyre and gimble in the tumor microenvironment, a looking-glass world in which abnormal protein-fiber scaffolds and genetic perturbations coincide, creating conditions that promote metastasis. Some cancer cells manage to circumnavigate or slide around other cells on protein fibers, and these cells can take relatively straightforward paths out of a primary tumor. Other cells, however, are more likely to turn back upon encountering other cells. They exit tumors less efficiently.

To understand how some cancer cells migrate more efficiently than others, researchers based at Northeastern University undertook a biophysical study. They developed a model environment that mimics protein fibers. First they stamped stripes of a protein called fibronectin on glass plates, making sure to represent various widths. Then they deposited the cells—alternately hundreds of breast cancer cells and hundreds of normal cells—on these fiber­like stripes and used a microscope with time-lapse capabilties to observe and quantify their behavior.

On fibers that were 6 or 9 microns wide—the typical size of fibers in tumors—half the breast cancer cells elongated and slid around the cells they collided with. Conversely, 99% of the normal breast cells did an about face.

To under­stand what gave the cancer cells this remarkable agility, the Northeastern researchers, led by Anand Asthagiri, explored the influence of fiber widths and genetic perturbations. They presented their results April 26 in the Biophysical Journal, in an article entitled, “Regulators of Metastasis Modulate the Migratory Response to Cell Contact under Spatial Confinement.”

“Downregulating the cell–cell adhesion protein, E-cadherin, enables MCF-10A cells to slide on narrower micropatterns; meanwhile, introducing exogenous E-cadherin in metastatic MDA-MB–231 cells increases the micropattern dimension at which they slide,” wrote the article’s authors.

This finding led the Northeastern team to consider the characteristic fibrillar dimension (CFD) at which effective sliding is achieved as a metric of sliding ability under spatial confinement.

“Using this metric, we show that metastasis-promoting genetic perturbations enhance cell sliding and reduce CFD,” the article’s authors continued. “Activation of ErbB2 combined with downregulation of the tumor suppressor and cell polarity regulator, PARD3, reduced the CFD, in agreement with their cooperative role in inducing metastasis in vivo. The CFD was further reduced by a combination of ErbB2 activation and transforming growth factor β stimulation, which is known to enhance invasive behavior.”

Asthagiri’s system is relatively easy to construct and suited for rapid imaging—two qualities that make it an excellent candidate for screening new cancer drugs. Pharmaceutical companies could input the drugs along with the cancer cells and mea­sure how effectively they inhibit sliding.

In the future, the system could also alert cancer patients and clinicians before metastasis starts. Studies with patients have shown that the structure of a tumor’s protein-fiber scaffolding can indicate how far the disease has progressed. The researchers found that certain aggressive genetic mutations enabled cells to slide on very narrow fibers, whereas cells with milder mutations would slide only when the fibers got much wider. Clinicians could biopsy the tumor and mea­sure the width of the fibers to see if that danger point were approaching. “We can start to say, ‘If these fibers are approaching X microns wide, it’s urgent that we hit certain path­ways with drugs,” said Asthagiri.

Questions, of course, remain. Do other types of cancer cells also have the ability to slide? What additional genes play a role?

Next steps, says Asthagiri, include expanding their fiber­like stripes into three-dimensional models that more closely represent the fibers in actual tumors and testing cancer and normal cells together. “There are so many types of cells in a tumor environment—immune cells, blood cells, and so on,” he noted. “We want to better emulate what’s hap­pening in the body rather than in isolated cells interacting on a platform.”


Regulators of Metastasis Modulate the Migratory Response to Cell Contact under Spatial Confinement.

The breast tumor microenvironment (TMEN) is a unique niche where protein fibers help to promote invasion and metastasis. Cells migrating along these fibers are constantly interacting with each other. How cells respond to these interactions has important implications. Cancer cells that circumnavigate or slide around other cells on protein fibers take a less tortuous path out of the primary tumor; conversely, cells that turn back upon encountering other cells invade less efficiently. The contact response of migrating cancer cells in a fibrillar TMEN is poorly understood. Here, using high-aspect ratio micropatterns as a model fibrillar platform, we show that metastatic cells overcome spatial constraints to slide effectively on narrow fiber-like dimensions, whereas nontransformed MCF-10A mammary epithelial cells require much wider micropatterns to achieve moderate levels of sliding. Downregulating the cell-cell adhesion protein, E-cadherin, enables MCF-10A cells to slide on narrower micropatterns; meanwhile, introducing exogenous E-cadherin in metastatic MDA-MB-231 cells increases the micropattern dimension at which they slide. We propose the characteristic fibrillar dimension (CFD) at which effective sliding is achieved as a metric of sliding ability under spatial confinement. Using this metric, we show that metastasis-promoting genetic perturbations enhance cell sliding and reduce CFD. Activation of ErbB2 combined with downregulation of the tumor suppressor and cell polarity regulator, PARD3, reduced the CFD, in agreement with their cooperative role in inducing metastasis in vivo. The CFD was further reduced by a combination of ErbB2 activation and transforming growth factor β stimulation, which is known to enhance invasive behavior. These findings demonstrate that sliding is a quantitative property and a decrease in CFD is an effective metric to understand how multiple genetic hits interact to change cell behavior in fibrillar environments. This quantitative framework sheds insights into how genetic perturbations conspire with fibrillar maturation in the TMEN to drive the invasive behavior of cancer cells.


There was a nice paper a few years ago by Dr. Edna Cukerman from Fox Chase showing how tumor cells slid down on fiber tracks generated from tumor stromal cells and how this pattern of movement is not as random as one would think. if extracellular matrix was generated from normal stromal cells you would not find athis type of coordinated movement.



Fatty acid oxidation disruption: a therapeutic alternative for triple negative breast cancer

Hormone therapy is ineffective against triple negative breast cancers (TNBC) as they lack HER2, Estrogen, and Progesterone receptors. Therefore new targetable pathways are needed to halt the cancer’s progression. Researchers at UCSF have outlined a means of treating TNBC through disruption of fatty acid oxidation (FAO). The pathway was first revealed as a potential target through metabolomics and gene signatures, identifying upregulated FAO intermediates in MYC-overexpressing TNBC samples. Considering the location, in the proximity of adipose-rich mammary glands, breast cancer FAO dependence pathway seemed to be a logical pathway. Subsequent inhibition of FAO with etomixir , an inhibitor of a major enzyme carnitine palmitoyltransferase 1 (CPT1) in the FAO pathway, lead to dramatic decreases in ATP production in MYC-overexpressing cell lines. Although a decrease in proliferation of cells in culture was observed viability remained unchanged. However, further testing of etomixir in vivo within patient derived xenograft models increased success of FAO disruption with a 4 to 6-fold decrease in relative tumor volume. The differential performance between in vitro and in vivo treatments indicates a need to recapitulate the actual tumor environment when studying metabolic manipulation regimens.

Camarda, et. al. Inhibition of fatty acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer.  Nature Medicine   

Inhibition of fatty acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer

Roman CamardaAlicia Y ZhouRebecca A Kohnz,….,Daniel K Nomura & Andrei Goga
Nature Medicine22,427–432(2016)

Expression of the oncogenic transcription factor MYC is disproportionately elevated in triple-negative breast cancer (TNBC), as compared to estrogen receptor–, progesterone receptor– or human epidermal growth factor 2 receptor–positive (RP) breast cancer1, 2. We and others have shown that MYC alters metabolism during tumorigenesis3, 4. However, the role of MYC in TNBC metabolism remains mostly unexplored. We hypothesized that MYC-dependent metabolic dysregulation is essential for the growth of MYC-overexpressing TNBC cells and may identify new therapeutic targets for this clinically challenging subset of breast cancer. Using a targeted metabolomics approach, we identified fatty acid oxidation (FAO) intermediates as being dramatically upregulated in a MYC-driven model of TNBC. We also identified a lipid metabolism gene signature in patients with TNBC that were identified from The Cancer Genome Atlas database and from multiple other clinical data sets, implicating FAO as a dysregulated pathway that is critical for TNBC cell metabolism. We found that pharmacologic inhibition of FAO catastrophically decreased energy metabolism in MYC-overexpressing TNBC cells and blocked tumor growth in a MYC-driven transgenic TNBC model and in a MYC-overexpressing TNBC patient–derived xenograft. These findings demonstrate that MYC-overexpressing TNBC shows an increased bioenergetic reliance on FAO and identify the inhibition of FAO as a potential therapeutic strategy for this subset of breast cancer.


3 Dimensional Ex-Vivo for In-situ Tumor Growth

Brain tumors are both difficult to treat and hard to study because of the organ they affect. The structure of the brain is extremely sensitive to alterations. Until recently the study of architectural alterations and their effects was mostly restricted to in vivo experiments. Typical culturing of brain tissue requires disaggregation and manipulation into a 2-dimensional format, losing any anatomically relevant structure. To study the in situ brain structure, a new technique has been described by researchers from the University of Erlangen-Nürnberg. By carefully sectioning the brains of 4 day-old mice and placing them on a 0.4 uM pore-size transwell membrane 6 well plate insert within required culture medium, they were able to study the endogenous structure under varying conditions. They injected astrocytes or glioma cells with a micropipette into the slices, and investigated the structural changes brain tumors effect in their environment. Termed the Vascular Organotypic Glioma Impact Model (VOGIM), it revealed all the characteristic pathological alterations normally associated with the disease in vivo such as tumor size and borders, vessel length, vessel junctions, and vessel branches, microglia, cell survival, and neuronal modifications. As this method allows for live cell fluorescent observation, they employed the technique to observe cultures treated with the chemotherapeutic Temozolamide (TMZ, Temodal/Temcad®). Indeed they found reduced tumor growth in treatment groups vs controls, but also revealed surprising reduction in microglial cells in the peritumoral region. Additionally, they were able to observe the lack of response TMZ elicited from microglial in healthy regions of the tissue, despite its overall reduction in vascularization towards normal levels. The VOGIM technique allows for ex vivo study of brain tissue requiring three dimensional measurements, but may also be extended to other tissues with unique morphology such as kidney, liver, and intestine.

Ghoochani, et al. (December, 2015) A versatile ex vivo technique for assaying tumor angiogenesis and microglia in the brain ONCOTARGET


A versatile ex vivo technique for assaying tumor angiogenesis and microglia in the brain

Ali Ghoochani1, Eduard Yakubov1, Tina Sehm1, Zheng Fan1, Stefan Hock1, Michael Buchfelder1, Ilker Y. Eyüpoglu1,*, Nicolai Savaskan1,*      PDF |  HTML

Primary brain tumors are hallmarked for their destructive activity on the microenvironment and vasculature. However, solely few experimental techniques exist to access the tumor microenvironment under anatomical intact conditions with remaining cellular and extracellular composition. Here, we detail an ex vivo vascular glioma impact method (VOGIM) to investigate the influence of gliomas and chemotherapeutics on the tumor microenvironment and angiogenesis under conditions that closely resemble the in vivo situation. We generated organotypic brain slice cultures from rats and transgenic mice and implanted glioma cells expressing fluorescent reporter proteins. In the VOGIM, tumor-induced vessels presented the whole range of vascular pathologies and tumor zones as found in human primary brain tumor specimens. In contrast, non-transformed cells such as primary astrocytes do not alter the vessel architecture. Vascular characteristics with vessel branching, junctions and vessel length are quantitatively assessable as well as the peritumoral zone. In particular, the VOGIM resembles the brain tumor microenvironment with alterations of neurons, microglia and cell survival. Hence, this method allows live cell monitoring of virtually any fluorescence-reporter expressing cell. We further analyzed the vasculature and microglia under the influence of tumor cells and chemotherapeutics such as Temozolamide (Temodal/Temcad®). Noteworthy, temozolomide normalized vasculare junctions and branches as well as microglial distribution in tumor-implanted brains. Moreover, VOGIM can be facilitated for implementing the 3Rs in experimentations. In summary, the VOGIM represents a versatile and robust technique which allows the assessment of the brain tumor microenvironment with parameters such as angiogenesis, neuronal cell death and microglial activity at the morphological and quantitative level.



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Lymph Node Metastases

Larry H. Bernstein, MD, FCAP, Curator


Investigation of the Lack of Angiogenesis in the Formation of Lymph Node Metastases

Journal of the National Cancer Institute

Han-Sin Jeong; Dennis Jones; Shan Liao; Daniel A. Wattson; Cheryl H. Cui; Dan G. Duda; Christopher G. Willett; Rakesh K. Jain; Timothy P. Padera

J Natl Cancer Inst. 2015; 107(9)

Background: To date, antiangiogenic therapy has failed to improve overall survival in cancer patients when used in the adjuvant setting (local-regional disease with no detectable systemic metastasis). The presence of lymph node metastases worsens prognosis, however their reliance on angiogenesis for growth has not been reported.

Methods: Here, we introduce a novel chronic lymph node window (CLNW) model to facilitate new discoveries in the growth and spread of lymph node metastases. We use the CLNW in multiple models of spontaneous lymphatic metastases in mice to study the vasculature of metastatic lymph nodes (n = 9–12). We further test our results in patient samples (n = 20 colon cancer patients; n = 20 head and neck cancer patients). Finally, we test the ability of antiangiogenic therapy to inhibit metastatic growth in the CLNW. All statistical tests were two-sided.

Results: Using the CLNW, we reveal the surprising lack of sprouting angiogenesis during metastatic growth, despite the presence of hypoxia in some lesions. Treatment with two different antiangiogenic therapies showed no effect on the growth or vascular density of lymph node metastases (day 10: untreated mean = 1.2%, 95% confidence interval [CI] = 0.7% to 1.7%; control mean = 0.7%, 95% CI = 0.1% to 1.3%; DC101 mean = 0.4%, 95% CI = 0.0% to 3.3%; sunitinib mean = 0.5%, 95% CI = 0.0% to 1.0%, analysis of variance P = .34). We confirmed these findings in clinical specimens, including the lack of reduction in blood vessel density in lymph node metastases in patients treated with bevacizumab (no bevacizumab group mean = 257 vessels/mm2, 95% CI = 149 to 365 vessels/mm2; bevacizumab group mean = 327 vessels/mm2, 95% CI = 140 to 514 vessels/mm2P = .78).

Conclusion: We provide preclinical and clinical evidence that sprouting angiogenesis does not occur during the growth of lymph node metastases, and thus reveals a new mechanism of treatment resistance to antiangiogenic therapy in adjuvant settings. The targets of clinically approved angiogenesis inhibitors are not active during early cancer progression in the lymph node, suggesting that inhibitors of sprouting angiogenesis as a class will not be effective in treating lymph node metastases.


Although antiangiogenic therapy is standard of care for several advanced (metastatic) cancers, all phase III clinical trials of antiangiogenic therapy to date have failed in the adjuvant setting.[1–4] The presence of lymph node metastases—the most common form of cancer dissemination—dictates treatment decisions,[5,6] however their reliance on angiogenesis for growth has not been reported. Furthermore, observations from preclinical and clinical studies suggest that lymph node metastases and primary tumors can respond differently to the same therapeutic regimen.[7–9] The clinical relevance of lymph node metastases has been the subject of debate for many years. Some argue that the presence of lymph node metastasis only demonstrates the ability of the cancer to metastasize and that disease in the lymph node is inconsequential.[10,11] The strong predictive power of lymph node metastases has led others to hypothesize that cancer cells in the lymph node can exit and spread to distant metastatic sites.[12,13] These advocates argue disease in lymph nodes needs to be treated in order to prevent distant metastasis and ultimately eradicate disease from the patient.[14,15] Likely the answer lies in between, depending where on the spectrum of progression to distant metastasis the cancer is diagnosed.[16]These issues highlight our fundamental lack of understanding of the biology of how metastatic cancer cells grow in a lymph node and affect the overall prognosis for the patient, limiting our ability to discover effective adjuvant therapy to treat lymph node metastases.

We and others have previously shown that antiangiogenic therapy did not stop the seeding or growth of lymph node metastases,[9,17,18] but no mechanism of failure has been determined. Nonsprouting angiogenesis mechanisms to sustain tumor growth, such as vessel co-option and intussusception, have been implicated in the growth of lung, liver, and brain metastases[19] and are thought to play a role in resistance to antiangiogenic therapy.[20] Based on these findings, we hypothesized that early growth of lymph node metastases is not dependent on sprouting angiogenesis.

Although reports show reduced vascular density in lymph node metastases compared with corresponding primary tumors and surrounding normal lymph node,[17,21,22] these data do not describe the degree of angiogenesis or whether the vessels are functional. Here, we introduce a novel model to longitudinally image the formation and growth of metastatic tumors in lymph nodes and reveal the surprising lack of sprouting angiogenesis, despite the presence of hypoxia in some lesions. Treatment with two different therapies designed to target sprouting angiogenesis showed no effect on the growth or vascular density of lymph node metastases in our models. These data are corroborated in clinical specimens and further add to mechanisms for the failure of antiangiogenic treatments in adjuvant settings.[1–4,20]


Intravital Multiphoton Microscopy

Intravital multiphoton microscopy was carried out as described previously on a custom-built multiphoton microscope.[25] Details of the imaging equipment, imaging protocols, and image analysis can be found in the Supplementary Methods (available online).


Longitudinal Imaging of the Formation of Spontaneous Lymph Node Metastases Using a Novel Chronic Lymph Node Window

Holding back our understanding of the biology of lymph node metastasis is our inability to longitudinally monitor spontaneous lymph node metastases. Inspired by pioneering intravital microscopy of the lymph node,[30–35] we developed a chronic lymph node window (CLNW)—a modification of the mammary fat pad chamber[23,24]—to create a CLNW that allows intravital imaging for up to 14 days with minimal morphological, cellular or biochemical changes in the inguinal lymph node (Figure 1, A and B; Supplementary Figure 1, available online).

Using multiphoton microscopy in the CLNW, we were able to serially image various stages of the growth of spontaneous metastasis in the lymph node from murine SCCVII squamous cell carcinoma[36,37]transduced with green fluorescence protein (SCCVII-GFP) (Figure 1C). Initially, cancer cells remain in or near the subcapsular sinus as individual cells (Figure 1C). Later, small aggregates of a few cancer cells form near the subcapsular sinus, which then grow into metastatic lesions that invade deeper into the lymph node (Figure 1C). This sequence was also observed in syngeneic MCa-P0008 breast cancer and B16F10 melanoma cells lines (Supplementary Figure 2, available online).

Recent genomic studies suggest that metastatic cells within lymph nodes consist of multiple clones.[38,39]To investigate this concept, we transduced SCCVII and SCCVII-GFP cells with a red fluorescence protein (DsRed), producing three different colors of cells (red, green, and red+green) that were mixed in equal proportions to form primary tumors. Single cells of multiple colors disseminated from the multicolor primary tumor and grew in the subcapsular sinus (Figure 1D). The metastatic lesions that subsequently formed contained all three colors with great spatial heterogeneity (Figure 1D), suggesting that lymph node metastases form from multiple cells. These findings were reproduced when using an equal mix of 4T1-DsRed and 4T1-GFP mammary carcinoma cells implanted in the mammary fat pad. In contrast, more than 80% of detected lung metastases from these 4T1 tumors were single color (Figure 1E).

The Role of the Existing Lymph Node Vascular Supply in Supporting the Growth of Lymph Node Metastases

Next, we directly measured for the first time whether angiogenesis is occurring in lymph node metastases by using intravital multiphoton microscopy to make longitudinal measurements in our CLNW. In early stages, metastatic cells resided in the lymph node sinus, away from blood vessels (Figure 2A). These metastatic tumor cells eventually invaded the lymph node cortex, growing closer to functional lymph node blood vessels and presumably utilizing the nutrient supply of these pre-existing vessels (Figure 2A). We found that the tumor cells started to access host lymph node blood vessels when they invaded approximately 50 to 100 μm into the cortex (Figure 2, B and C). Although the tumor invaded deeper into the node (day 6 mean depth = 43 μm, 95% CI = 24 to 61 μm; day 12 mean depth = 131 μm, 95% CI = 71 to 191 μm, P = .01), blood vessels did not invade toward the surface of the lymph node (day 6 mean depth = 52 μm, 95% CI = 49 to 55 μm; day 20 mean depth = 58 μm, 95% CI = 41 to 75 μm,P = .38), as would be expected for tumor-induced sprouting angiogenesis. These data provide the first direct evidence of the lack of sprouting angiogenesis during the growth of metastatic lesions in the lymph node.

Figure 2.

Intravital imaging of lymph node metastases and the native lymph node vasculature. A) Representative time course of images from a single metastatic lymph node, showing cancer cells (SCCVII, green) and blood vessels (TRITC-dextran, red) at three different depths in tissue. The image was created using multiphoton microscopy, and second harmonic generation was used to highlight fibrillar collagen (blue) in the lymph node capsule. The images are created from maximum intensity projections of 25 μm of tissue from inside the lymph node. In day 40 images, the red signal is background signal from the accumulation of TRITC-dextran as a result of the five intravenous injections over the course of the metastatic growth. Yellow arrows identify individual cancer cells. Yellow circles identify areas in which many cancer cells are found in the subcapsular sinus. White arrows identify blood vessels in the metastatic lesion. Purple, green and light blue arrows identify features in the lymph node vasculature that can be used to identify the same region in the mouse over the multiday experiment. White line marks edge of lymph node. Scale bars = 100 μm. B) A vertical image reconstruction showing the tumor cells (SCCVII, green) initially growing above the blood vessels (red). C) Measurements of the maximum depth of tumor cell invasion (SCCVII) and the minimum depth of blood vessels. Data are presented as mean ± 95% confidence interval.

Immunofluorescent staining for CD31 (Figure 3A) showed that the vessel density in lymph nodes with micrometastases from SCCVII tumors (Figure 3B) and macrometastases (lesions greater than 500 microns in one dimension) from 4T1 tumors (Figures 3E; Supplementary Figure 3A, available online) were not increased compared with those of control (from naïve mice with no tumor implantation) and contralateral nodes. The vessel density inside metastatic lesions was lower than the surrounding lymph node tissue (vessel density: SCCVII: metastatic lesion = 1.0%, 95% CI = 0.0% to 2.0%; nontumor area = 7.0%, 95% CI = 1.0% to 13.0%, P = .04; 4T1: metastatic lesion = 4.0%, 95% CI = 1.0% to 7.0%; nontumor area = 10.0%, 95% CI = 5.0% to 15.0%, P = .04) (Figure 3, C and F). To indicate sprouting angiogenesis, Ki67—a marker of cell proliferation—showed no difference in endothelial cell proliferation in micrometastatic lymph nodes (SCCVII) (Figure 3D; Supplementary Figure 4, available online) and a reduction in endothelial cell proliferation in macrometastatic lymph nodes (4T1) (Figure 3G; Supplementary Figure 3B, available online) in comparison with control and contralateral nodes. Vessel density in the metastatic lesions was not related to lesion size (Supplementary Figure 3C, available online). These data further indicate that sprouting angiogenesis is not induced in the lymph node at this stage of cancer progression.

Figure 3.

Immunohistochemical analysis of lymph node blood vessels and metastases. A) Representative sections of control (from non–tumor bearing mice), contralateral, and tumor-draining lymph nodes with micrometastases (SCCVII, green). Vessels were stained with CD31 (red) and nuclei with DAPI (blue).Scale bars = 300 μm. B) Quantification of CD31+ area per lymph node area in control, contralateral, and micrometastatic lymph nodes. C) In micrometastatic lymph nodes, quantification of CD31+ area per tissue area comparing tumor areas with nontumor areas. D) Costaining for CD105 and Ki67 measured blood vessel proliferation in micrometastatic lymph nodes. E) Using a different tumor model (4T1) that formed macrometastasis in the lymph node (greater than 500 μm in one direction), we measured CD31+ area in micrometastatic or macrometastatic lymph nodes, compared with control or contralateral nodes. F) The vascular area of macrometastatic lesions was measured in tumor areas and nontumor lymph node tissue. G) Costaining for CD31 and Ki67 measured blood vessel proliferation in macrometastatic lymph nodes. Data are presented as mean ± 95% confidence interval. Statistical significance was tested by one-way analysis of variance with Tukey’s Honestly Significant Difference post hoc test (B, D, E, G) or two-tailed paired Student’s t test (C, F).

In contrast, LYVE-1 staining for lymphatic vessels showed an increase in lymphatic vascular area (vessel density: SCCVII: control = 5.0%, 95% CI = 3.0% to 7.0%; contralateral = 8.0%, 95% CI = 6.0% to 10.0%; metastatic = 10.0%, 95% CI = 6.0% to 14.0%; control vs metastatic P = .03; 4T1: control = 5.0%, 95% CI = 2.0% to 8.0%; contralateral = 9.0%, 95% CI = 6.0% to 12.0%; nonmetastatic tumor draining = 22.0%, 95% CI = 18.0% to 26.0%; metastatic = 4.0%, 95% CI = 1.0% to 7.0%; control vs nonmetastatic tumor draining P < .001) and proliferating lymphatic endothelial cells in draining lymph nodes from SCCVII and 4T1 tumors (Supplementary Figures 5 and 6, available online), consistent with previous reports.[40–43] Interestingly, the lymphatic vascular area was greater in the contralateral and nonmetastatic tumor-draining lymph nodes of 4T1-bearing mice compared with lymph nodes with macrometastatic lesions (P < .001) (Supplementary Figure 6, available online), suggesting that the presence of cancer cells causes the lymphatic vasculature to regress. When compared with lymph nodes from tumor-naïve animals, contralateral lymph nodes show greater lymphatic vascular density (SCCVII: P = .04; 4T1: P < .001), suggesting that contralateral lymph nodes are also affected by the presence of the primary tumor, as others have reported.[44]

Although lesions growing in the subcapsular sinus of the lymph node showed markers for hypoxia (Figure 4, A–D), sprouting angiogenesis was not induced in these lesions and they remained avascular. Metastatic lesions that invaded the lymph node parenchyma where functional nodal blood vessels reside had only focally heterogeneous areas positive for hypoxia markers (Figure 4, A, C, and E). These data suggest that growing metastatic lesions can utilize the existing lymph node vasculature in order to meet their metabolic demand. Whether this demand or hypoxia drives cancer cell invasion of the lymph node remains unknown.

Figure 4.

Hypoxia in lymph node metastases. A) Representative images of pimonidazole staining for hypoxia (green) and perfused lectin staining for functional blood vessels (red) in lymph node metastases from 4T1 mammary carcinoma (cytokeratin, blue). The top panels show a lesion in the subcapsular sinus that is hypoxic and has no perfused blood vessels in the lesion. The bottom panels show a lesion in the parenchyma of the lymph node with perfused blood vessels and no hypoxia. Dashed line shows edge of the lymph node. Scale bars = 100 μm. B) Higher magnification of pimonidazole staining in metastatic lymph node showing colocalization of cytokeratin and pimonidazole. Contralateral lymph node is non–tumor bearing. Dashed line shows edge of the lymph node. Scale bars = 50 μm. C) Quantification of pimonidazole and perfused vessel staining in metastatic lesions in the subcapsular sinus and lymph node parenchyma. Data are presented as mean ± 95% confidence interval. Statistical significance was tested by two-tailed unpaired Student’s t test. D and E) Staining for CAIX, a marker of the cellular response to hypoxia, and CD31-positive blood vessels shows similar results to pimonidazole staining. Dashed line shows the outline of the metastatic lesions. Scale bars = 636 μm.

Hypoxia generally induces the production of vascular endothelial growth factor (VEGF). However, VEGF levels in control, contralateral, and metastatic lymph nodes were not different (4T1: control = 0.3 pg VEGF/mg protein, 95% CI = 0.2 to 0.4 pg VEGF/mg protein; contralateral = 0.4 pg VEGF/mg protein, 95% CI = 0.3 to 0.5 pg VEGF/mg protein; metastatic = 0.5 pg VEGF/mg protein, 95% CI = 0.2 to 0.8 pg VEGF/mg protein; Figure 5A; SCCVII: control = 0.4 pg VEGF/mg protein, 95% CI = 0.3 to 0.5 pg VEGF/mg protein; contralateral = 0.4 pg VEGF/mg protein, 95% CI = 0.3 to 0.5 pg VEGF/mg protein; metastatic = 0.4 pg VEGF/mg protein, 95% CI = 0.3 to 0.5 pg VEGF/mg protein; Figure 5B; and E0771: control = 0.3 pg VEGF/mg protein, 95% CI = 0.2 to 0.4 pg VEGF/mg protein; contralateral = 0.4 pg VEGF/mg protein, 95% CI = 0.3 to 0.5 pg VEGF/mg protein; metastatic = 0.4 pg VEGF/mg protein, 95% CI = 0.3 to 0.5 pg VEGF/mg protein; Figure 5C; all P values > .05 for each ANOVA containing these three lymph nodes types). Furthermore, levels of VEGF-C and VEGF-D were lower in metastatic and nonmetastatic tumor draining lymph nodes when compared with naïve lymph nodes (Supplementary Figure 6, C and D, available online). Next, we screened for transcriptional changes in sprouting angiogenesis-related genes in lymph nodes with metastasis when compared with naïve lymph nodes. No pro-angiogenesis related genes were upregulated in metastatic lymph nodes, but thrombospondin-1 (Thbs-1) and TIMP-1—both of which are antiangiogenic—were upregulated (Figure 5D). We confirmed no change in Vegf levels (control = 0.24 VEGF/GAPDH, 95% CI = 0.06 to 0.42 VEGF/GAPDH; metastatic = 0.16 VEGF/GAPDH, 95% CI = 0.04 to 0.28 VEGF/GAPDH, P = .37) and the elevation in Thbs-1 in lymph node metastasis by quantitative polymerase chain reaction (qPCR) (control = 0.10 THBS-1/GAPDH, 95% CI = 0.05 to 0.15 THBS-1/GAPDH; metastatic = 0.38 THBS-1/GAPDH, 95% CI = 0.23 to 0.53 THBS-1/GAPDH; P = .001) (Figure 5E). Thrombospondin-1 (TSP-1) was specifically located surrounding the blood vessels of control, contralateral, and metastatic lymph nodes (Figure 5F), further defining the nonangiogenic phenotype associated with these vessels. Taken together, these data describe an environment lacking prosprouting angiogenesis stimuli and abundant in antiangiogenesis molecules, suggesting metastatic lesions in the lymph node do not induce nor rely upon sprouting angiogenesis during their early growth.

Figure 5.

Molecular signature of quiescent lymph node vasculature. A-C) Levels of vascular endothelial growth factor (VEGF) protein were measured in metastatic lymph nodes containing 4T1 (A), SCCVII (B), or E0771 (C) and compared with control and contralateral lymph nodes. D) Quantitative polymerase chain reaction (qPCR) transcriptional array for angiogenesis-related genes compared the transcriptional profile of a diaeresis lymph node to a tumor-bearing lymph node. Differentially transcribed genes were defined as having more than a four-fold change and a P value under .01 when comparing metastatic lymph nodes to diaeresis lymph nodes. E) Confirmation of the qPCR transcriptional array for the Vegf and Thbs1 genes. *P < .05. F) Dual immunofluorescence staining for CD31 (red) and TSP-1 (green) showed distinctive TSP-1 staining surrounding the blood vessels in diaeresis, contralateral, and metastatic lymph nodes. Scale bars = 100μm. Data are presented as mean ± 95% confidence interval. Statistical significance was tested by one-way analysis of variance with Tukey’s Honestly Significant Difference post hoc test (A, B, and C) and two-tailed unpaired Student’s t test (E).

Blood Vessel Density in Metastatic Lymph Nodes From Colon Cancer and Head and Neck Cancer Patients

To confirm these findings in clinical specimens in a cancer where angiogenesis inhibitors have shown efficacy, we stained lymph nodes from 20 colon cancer patients with lymphatic metastasis for CD31 (Figure 6A; Supplementary Figure 7A, online). These patients did not have metastases on initial staging and went directly for surgical resection with no prior cancer-directed treatments (eg, chemotherapy, radiation therapy). We found that blood vessel densities in metastatic lymph nodes and large metastatic lesions where lymph node tissue was completely replaced with tumor cells were on average lower than those of tumor-negative lymph nodes (nonmetastatic- = 220 blood vessels/mm2, 95% CI = 172 to 268 blood vessels/mm2; metastatic = 135 blood vessels/mm2, 95% CI = 113 to 157 blood vessels/mm2; lymph node replaced by cancer = 104 blood vessels/mm2, 95% CI = 75 to 133 blood vessels/mm2; comparisons of either group of tumor-bearing to nonmetastatic lymph nodes: P < .001) (Figure 6, B and C). Furthermore, the vessel density inside metastatic lesions was statistically significantly lower than in the remaining lymph node tissue (metastatic lesion = 148 blood vessels/mm2, 95% CI = 124 to 172 blood vessels/mm2; nontumor area = 115 blood vessels/mm2, 95% CI = 95 to 135 blood vessels/mm2P = .03) (Figure 6, D and E). Accordingly, TSP-1 staining was also found to associate with lymph node blood vessels and to surround the gland-like structures formed by the cancer cells (Supplementary Figure 7B, available online), further suggesting that these vessels were not undergoing sprouting angiogenesis. Finally, the density of CD31-positive vessels was not dependent on the lesion size in the section, showing that vessel densities of macrometastases (clinically classified as lesions larger than 2mm in one direction[45]]) are the same as in micrometastases (Figure 6F). Blood vessel density and TSP-1 staining in specimens from head and neck cancer patients were similar to those from colon cancer patients (Supplementary Figure 7, C–G, available online). Taken together, these data from two different patient populations support the concept that the growth of metastatic lesions in the lymph nodes is not dependent upon sprouting angiogenesis.

Figure 6.

Vascular density in metastatic lymph nodes from colon cancer patients. A) Representative images of nonmetastatic (n = 19) and metastatic (n = 39) lymph nodes as well as lymph node tumors in which no normal lymph node tissue remained (n = 9). The sections were stained with CD31 (brown) to identify blood vessels. Scale bars = 200 μm. Images of whole lymph node sections can be found in Supplementary Figure 7 (available online). B) The number of vessels per area as determined by CD31 staining was measured in metastatic lymph nodes and in lymph node tumors in which no normal lymph node tissue remained and compared with nonmetastatic lymph nodes. C) The fraction of lymph node area composed of CD31-positive vessels was similarly measured in metastatic lymph nodes and in lymph node tumors in which no normal lymph node tissue remained and compared with nonmetastatic lymph nodes. *P value was determined by Tukey’s Honestly Significant Difference post hoc test of analysis of variance model. D and E) Within a metastatic lymph node, vascular density (D) and vessel area fraction (E) were measured in the tumor and the nontumor area. * P value was determined by paired Student’s t test.F) Vessel density was not dependent on the lesion size. Data are presented as mean ± 95% confidence interval throughout figure.

Growth of Lymph Node Metastases With Antiangiogenic Treatment

To directly measure the response of lymph node metastases to antiangiogenic therapy in the CLNW, we began treatment when micrometastases were between 100 and 125 μm in diameter (5–10×10–3 mm3)—the stage when we found blood vessels surrounding lymph node metastases—with either a monoclonal VEGF receptor (VEGFR)-2–blocking antibody (DC101, ImClone Systems) or the pan-VEGFR small-molecule tyrosine kinase inhibitor sunitinib. We chose agents with differential mechanisms of VEGF pathway inhibition—monoclonal antibody vs tyrosine kinase inhibitor (TKI)—to understand whether our findings were agent specific. Measuring lymph node blood vessels using the CLNW and longitudinal multiphoton microscopy, the growth of lymph node metastases (Figure 7, A–C) and functional blood vessel volume density remained at similar levels during treatment with either DC101 or sunitinib when compared with untreated controls (vessel density: day 10: untreated = 1.2%, 95% CI = 0.7% to 1.7%; control = 0.7%, 95% CI = 0.1% to 1.3%; DC101 = 0.4%, 95% CI = 0.0% to 3.3%; sunitinib = 0.5%, 95% CI = 0.0% to 1.0%; ANOVA P = .34) (Figure 7D). These direct measurements, supported by previous endpoint studies,[9,17] suggest that inhibitors of sprouting angiogenesis as a class of drugs will not be effective in inhibiting the early phase of lymph node metastasis. In contrast, sunitinib—a pan-VEGF receptor TKI—reduced the elevated lymphatic vessel density found in early metastatic lymph nodes compared with PBS control (Supplementary Figure 8, available online).

Figure 7.

Antiangiogenic therapy in the early growth of lymph node metastases. A) Representative intravital multiphoton microscopy images of spontaneous lymph node metastases treated with vehicle control, sunitinib, or the blocking monoclonal anti–VEGFR-2 antibody DC101. Tumor cells are shown in green and blood vessels in redScale bars = 200 μm. B) Primary tumors were of equal size at the time treatment began, when the lymph node micrometastases were 5–10×10–3 mm3C) The growth rate of the metastatic tumor in the lymph node was measured during antiangiogenesis therapy. D) The vessel density in metastatic lesions in the lymph node was measured during antiangiogenesis therapy. Biological replicates: untreated n = 15 (C), 12 (D), control (IgG = 2, PBS = 4) n = 6, sunitinib = 6, DC101 = 5. Data are presented as mean ± 95% confidence interval. Statistical significance was tested by one-way analysis of variance with Tukey’s Honestly Significant Difference post hoc test (B and C) and two-tailed unpaired Student’s t test (D and E).

Blood Vessel Density of Lymph Node Metastasis From Patients Treated With Bevacizumab

Finally, we identified rectal cancer patients that received neoadjuvant chemoradiation and bevacizumab and a comparator cohort of rectal cancer patients who received only neoadjuvant chemoradiation, as previously described.[46,47] Despite downstaging of the primary tumor after neoadjuvant therapy, lymph node metastases were often found at the time of surgery and pathological evaluation. Comparing lymph node metastases from 10 patients in each group, we found no difference in the vessel density in lymph node metastases (no bevacizumab group mean = 257 vessels/mm2, 95% CI = 149 to 365 vessels/mm2; bevacizumab group mean = 327 vessels/mm2, 95% CI = 140 to 514 vessels/mm2P = .78) (Figure 8, A and B). The vascular density in the tumor lesions specifically was also not different between the groups (no bevacizumab group mean = 307 blood vessels/mm2, 95% CI = 186 to 428 vessels/mm2; bevacizumab group mean = 318 blood vessels/mm2, 95% CI = 118 to 518 vessels/mm2P = .60) (Figure 8, C and D). Metastatic lymph nodes showed lower vascular density than nonmetastatic nodes after neoadjuvant therapy (Figure 8, A and B), independent of whether bevacizumab was used. Finally, lymphatic vessel density was not different in metastatic and nonmetastatic lymph nodes when comparing patients who received bevacizumab to those who did not (Supplementary Figure 8, D and E, available online). These data provide the first clinical evidence for the lack of response of lymph node metastasis to antiangiogenic therapy.

Figure 8.

Vascular density in lymph node metastases in rectal cancer patients treated with bevacizumab. The number of CD31+ vessels per area (A) and the fraction of lymph node area composed of CD31+ vessels(B) were measured in nonmetastatic and metastatic lymph nodes in colorectal cancer (CRC) patients that received neoadjuvant chemoradiation (No Bev.) or neoadjuvant chemoradiation with bevacizumab (Bev.).P value was determined by two-tailed unpaired Student’s t test. C and D) Within the tumor area of metastatic lymph nodes, we measured vascular density (C) and vessel area fraction (D) in rectal cancer patients that received neoadjuvant chemoradiation (No Bev.) or neoadjuvant chemoradiation with bevacizumab (Bev.). P value was determined by two-tailed unpaired Student’s t test. Data are presented as mean ± 95% confidence interval.


The main concept driving antiangiogenic therapy has been the hypothesis that tumors depend on new blood vessel growth. A critical observation made by longitudinal intravital microscopy in the CLNW is that metastatic lesions did not induce sprouting angiogenesis as they grew, in spite of the presence of hypoxia. Lesions that invaded into the blood vessel–rich lymph node parenchyma showed reduced hypoxia, suggesting that cancer cells survive in the lymph node by utilizing the existing lymph node vascular supply. The lack of VEGF, VEGF-C, and VEGF-D, along with the presence of TSP-1 surrounding lymph node blood vessels, provides a mechanism behind the lack of sprouting angiogenesis observed in lymph node metastases. A limitation of the use of longitudinal intravital microscopy is the limited imaging depth of 300 μm by multiphoton microscopy in the CLNW. To balance this, we used histological techniques, which allow full lymph node depth to be characterized but are limited in their ability to monitor the kinetic changes occurring as metastatic lesions grow in the lymph node. Using these complimentary techniques allowed better characterization of the growth of lymphatic metastases.

Our data show lymph node lymphangiogenesis is an early event in the natural history of cancer progression, in agreement with previous studies.[40,41,43] However, decreased lymphatic vessel density was found in macrometastatic lymph nodes, suggesting that the presence of the cancer cells in the lymph node causes lymphatic vessel regression. Furthermore, bevacizumab did not statistically significantly affect the lymphatic vasculature in patients. These data suggest that late intervention with antiangiogenic or antilymphangiogenic therapies after lymphatic vessel regression has begun in patients will show no effect on lymph node lymphatic vessels.

In patients, the observation that large metastatic lesions do not exhibit increased vascular density relative to those with micrometastases further suggests that sprouting angiogenesis is not required to sustain the growth of lymph node metastases. A limitation of our data is that we estimated lesion size based on the two-dimensional area available in the histological sections, so we are likely underestimating the size of the lesion. An additional limitation of our study is that we cannot rule out the contribution from different modes of new blood vessel formation in lymph node metastasis such as vasculogenesis, intussusception, vessel co-option, vascular mimicry, and tumor cell differentiation into endothelial cells.[20] The mechanisms of these alternative processes are not clearly defined, although VEGF and endothelial proliferation have been shown to contribute to these processes.[48–51] Our preclinical and clinical data, however, show that inhibitors targeting primarily sprouting angiogenesis will not inhibit the growth of metastases in the lymph node.

Predicted by recent genomic data,[38,39,52] we provide direct evidence that lymph node metastasis forms from multiple cells that disseminate from the primary tumor and suggest a fundamental difference in their formation compared with hematogenous metastases. Cancer cells that invade lymphatic vessels travel to the draining lymph node where they enter in locations defined by afferent lymphatic vessels. As such, lymph node metastasis can be reinforced by the continual arrival of new cells as they gain a foothold in their new microenvironment, leading to the spatially heterogeneous lesions imaged here and the genetically heterogeneous lesions documented previously.[38,39,52,53] In contrast, cells that metastasize through the blood spread out to different locations in an organ by the branching vasculature, leading to a higher probability of individually homogenous lesions. One can thus speculate that targeting a single genetic trait, unless ubiquitous in the primary tumor, may not be effective in eradicating lymph node metastases and any subsequent spread to distant sites.[39]

Using multiple spontaneous metastasis models, we show the first direct evidence that sprouting angiogenesis is not required in lymph node lesions during early metastatic growth. The lack of sprouting angiogenesis in lymph node metastases suggests an additional explanation for the poor outcomes of antiangiogenic therapy in adjuvant settings. As the lymph node is able to metabolically support rapid cellular expansion during an active immune response, it seems the existing vasculature of the lymph node is also able to support the growth of a nascent metastasis. Thus, the mechanisms of angiogenesis and the targets of clinically approved drugs are not active during this early step in cancer progression, suggesting that inhibitors of sprouting angiogenesis as a class will not be effective in treating lymph node metastases. Our novel preclinical models provide opportunities to uncover strategies to better control and eradicate disease in lymph nodes in metastatic cancer patients.

Gene Mutation Signals Poor Prognosis for Pancreatic Tumors

College of American Pathologists (CAP) 2015 Meeting

Neil Osterweil

NASHVILLE, Tennessee — For patients with pancreatic neuroendocrine tumors, the presence of recently identified mutations in two key genes is a prognostic factor for poor outcome, researchers report.

“We found loss of nuclear expression in about 23% of the tumors that we studied, and this loss of expression was associated with worse tumors from the outset,” lead investigator Michelle Heayn, MD, a second-year pathology resident at the University of Pittsburgh Medical Center, told Medscape Medical News.

Pancreatic tumors with neuroendocrine histology frequently respond to chemotherapy and have a more favorable prognosis than the more common pancreatic adenocarcinomas. However, the mutations are associated with worse disease-free and disease-specific survival.

The results of the study were presented here at the College of American Pathologists 2015 Meeting.

The mutations — in the alpha-thalassemia mental retardation syndrome X-linked gene (ATRX) and the death-domain-associated protein gene (DAXX) — cause loss of expression of the proteins coded by ATRX and DAXX, Dr Heayn explained.

We found loss of nuclear expression in about 23% of the tumors that we studied.

To test whether these mutations had any prognostic significance, Dr Heayn and her colleagues used immunolabeling in surgically resected pancreatic neuroendocrine tumors from 303 patients. They then correlated the findings with patient demographics, pathologic features, disease-free survival, and disease-specific survival. Follow-up ranged from 1.6 to 18.8 years.

Of the 303 tumors, 69 (23%) had mutations in one or both genes. Tumors with a gene mutation had a larger mean diameter than tumors with intact gene expression (5.0 vs 2.4 cm), as well as a significantly higher histologic grade, more lymphovascular and perineural invasion, a more advanced T stage, greater lymph node involvement, more synchronous metastases, and more frequent disease recurrence (P < .01 for all comparisons).

In addition, the mutations were associated with shorter mean disease-free survival (5.6 vs 17.2 years;< .01) and shorter mean disease-specific survival (12.5 vs 17.7 years; P = .01).

On multivariate analysis that controlled for patient and tumor factors, the mutations were a significant predictor of shorter disease-free survival (P < .01), independent of tumor size, stage, histology, lymphovascular or perineural invasion, and lymph node status.

Dr Heayn and her colleagues are currently exploring whether there is an association between metastatic pancreatic cancer and these genetic mutations.

Metastatic Pancreatic Cancer

Patients with these mutations in their tumors should be followed more closely for recurrence or disease progression, Dr Heayn said. And in this subset of patients, there is the possibility of new targeted therapies.

These findings are very important, said Safia Salaria, MD, from the Vanderbilt University Medical Center in Nashville.

“There is so much heterogeneity in these tumors, and currently we are just using clinicopathologic features and the WHO-recommended Ki-67 labelling and white count,” she told Medscape Medical News.

“If we have something that can be an adjunct to that — immunohistochemistry to determine the loss of these genes — it’s definitely going to be something that will help us, especially in low-grade tumors,” she explained.

Staining for the expression of the genes could also help pathologists identify patients who are at higher risk for disease recurrence or metastasis but don’t have metastases at the time of primary resection, Dr Salaria said.

Microbiome May Predict Colon Cancer Tumor Mutational Status

Neil Osterweil

BALTIMORE — Analysis of the microbiome surrounding colon cancer tumors could be used as a noninvasive screening test that is more sensitive and specific than fecal occult blood testing, according to the results of a new study.

“This is something that could be critical in colon cancer, because each tumor may have a different mutational landscape with different genes mutated, and that might have an effect on the microbiome,” said Ran Blekhman, PhD, from the University of Minnesota in Minneapolis.

The results of the study were presented here at the American Society of Human Genetics 2015.

Dr Blekhman and his colleagues looked at the genetic differences between healthy colon cells and tumor cells from adults with colorectal cancer, and found that specific tumor mutations are associated with the presence of specific bacteria in the gut.

For example, in people with an APC gene mutation, there is a strong association between familial adenomatous polyposis, a hereditary cancer syndrome, and an abundance of Fusobacterium, said Dr Blekhman.

He pointed out that his lab is the first to analyze the correlation between specific tumor mutations and the composition of the tumor microbiome.

More Mutations, More Diversity

The investigators used whole-exome sequencing to assess the protein-coding regions of tumors and microbiome profiling to characterize the microbiota in tumor biopsy specimens and normal colon tissue samples from 44 adults with colon cancer.

They found that the more mutations, the more varied the bacterial species in the tumor microbiome.

And for certain genes, there was a correlation between somatic mutations and changes in the abundance of specific microbes.

Other evidence of the correlation between bacteria and tumor was seen at the pathway level.

Loss-of-function mutations were detected in tumor glucose transport pathways and were strongly correlated with higher levels of energy utilization in the microbiome, said Dr Blekhman. This suggests that the tumor and the bacteria in its neighborhood are competing for bodily resources.

The investigators created a risk index that evaluated the correlation between microbes and each of several known tumor driver mutations. The index was able to accurately predict the presence of a loss-of-function mutation in ZFN717, a gene encoding for a zinc finger nuclease, part of a family of enzymes involved in DNA repair.

These findings suggest that it is possible to genetically classify tumors from fecal samples alone. Theoretically, this means that manipulation of the tumor microenvironment could be used to prevent or treat colon cancer, Dr Blekhman explained.

This study addresses, in part, the problem of “hidden heritability,” said Chris Gunter, PhD, from Emory University School of Medicine in Atlanta.

“If you look at cancer-sequencing studies now, they identify something like 10 possible driver mutations. We have not yet managed to predict what all the drivers and passengers will be,” she told Medscape Medical News.

“If this type of work can help us narrow down the list, that should add to our understanding of how cancer develops,” she said.

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Angiogenesis Inhibitors [9.5]

Writer and Curator: Larry H Bernstein, MD, FCAP

This article has the following structure:

9.5.1 Motesanib (AMG 706)

9.5.2 Drugs that block cancer blood vessel growth (anti angiogenics)

9.5.3 Recent Advances in Anti-Angiogenic Therapy of Cancer

9.5.4 Angiogenesis inhibitors in cancer therapy: mechanistic perspective on classification and treatment rationales

9.5.5 LUCITANIB a VEGFR/FGFR dual kinase inhibitor in Phase 2 trials

9.5.1 Motesanib (AMG 706)


Motesanib (AMG 706) is an experimental drug candidate originally developed by Amgen[1] but is now being investigated by theTakeda Pharmaceutical Company. It is an orally administered small molecule belonging to angiokinase inhibitor class which acts as an antagonist of VEGF receptorsplatelet-derived growth factor receptors, and stem cell factor receptors.[2] It is used as the phosphate salt motesanib diphosphate.

Motesanib, also known as AMG-706, is an orally administered multikinase inhibitor that selectively targets VEGF receptors, platelet-derived growth factor receptors, and Kit receptors.




9.5.2 Drugs that block cancer blood vessel growth (anti angiogenics)

When it has reached 1 to 2mm across, a tumor needs to grow its own blood vessels in order to continue to get bigger. Some cancer cells make a protein called vascular endothelial growth factor (VEGF). The VEGF protein attaches to receptors on cells that line the walls of blood vessels within the tumour.

Drugs that block blood vessel growth factor

Some drugs block vascular endothelial growth factor (VEGF) from attaching to the receptors on the cells that line the blood vessels. This stops the blood vessels from growing.

A drug that blocks VEGF is bevacizumab (Avastin). It is also a monoclonal antibody.

Drugs that block signalling within the cell

Some drugs stop the VEGF receptors from sending growth signals into the blood vessel cells. These treatments are also called cancer growth blockers or tyrosine kinase inhibitors (TKIs).

Sunitinib (Sutent) is a type of TKI that blocks the growth signals inside blood vessel cells. It is used to treat kidney cancer and a rare type of stomach cancer called gastrointestinal stromal tumour (GIST).

Drugs that affect signals between cells

Some drugs act on the chemicals that cells use to signal to each other to grow. This can block the formation of blood vessels. Drugs that works in this way include thalidomide and lenalidomide (Revlimid).

Each drug has different side effects. You can look up the name of your drug in our cancer drug section to find out about the side effects you may have.

To find trials using anti angiogenesis treatment go to our clinical trials database and type ‘angiogenesis’ into the search box.

Tumors can cause their blood supply to form by giving off chemical signals that stimulate angiogenesis. Tumors can also stimulate nearby normal cells to produce angiogenesis signaling molecules. The resulting new blood vessels “feed” growing tumors with oxygen and nutrients, allowing the cancer cells to invade nearby tissue, to move throughout the body, and to form colonies of cancer cells, called metastases. Because tumors cannot grow beyond a certain size or spread without a blood supply, scientists are trying to find ways to block tumor angiogenesis.

Angiogenesis requires the binding of signaling molecules, such as vascular endothelial growth factor (VEGF), to receptors on the surface of normal endothelial cells. When VEGF and other endothelial growth factors bind to their receptors on endothelial cells, signals within these cells are initiated that promote the growth and survival of new blood vessels.

Angiogenesis inhibitors interfere with various steps in this process. For example, bevacizumab (Avastin®) is a monoclonal antibody that specifically recognizes and binds to VEGF (1). When VEGF is attached to bevacizumab, it is unable to activate the VEGF receptor. Other angiogenesis inhibitors, including sorafenib and sunitinib, bind to receptors on the surface of endothelial cells or to other proteins in the downstream signaling pathways, blocking their activities (2).

The U.S. Food and Drug Administration (FDA) has approved bevacizumab to be used alone forglioblastoma that has not improved with other treatments and to be used in combination with other drugs to treat metastatic colorectal cancer, some non-small cell lung cancers, and metastatic renal cell cancer. Bevacizumab was the first angiogenesis inhibitor that was shown to slow tumor growth and, more important, to extend the lives of patients with some cancers.

The FDA has approved other drugs that have antiangiogenic activity, including sorafenib (Nexavar®), sunitinib(Sutent®), pazopanib (Votrient®), and everolimus (Afinitor®). Sorafenib is approved for hepatocellular carcinoma and kidney cancer, sunitinib and everolimus for both kidney cancer and neuroendocrine tumors, and pazopanib for kidney cancer.

Angiogenesis inhibitors are unique cancer-fighting agents because they tend to inhibit the growth of blood vessels rather than tumor cells. In some cancers, angiogenesis inhibitors are most effective when combined with additional therapies, especially chemotherapy. It has been hypothesized that these drugs help normalize the blood vessels that supply the tumor, facilitating the delivery of other anticancer agents, but this possibility is still being investigated.

Angiogenesis inhibitor therapy does not necessarily kill tumors but instead may prevent tumors from growing. Therefore, this type of therapy may need to be administered over a long period.

Initially, it was thought that angiogenesis inhibitors would have mild side effects, but more recent studies have revealed the potential for complications that reflect the importance of angiogenesis in many normal body processes, such as wound healing, heart and kidney function, fetal development, and reproduction. Side effects of treatment with angiogenesis inhibitors can include problems with bleeding, clots in the arteries (with resultant stroke or heart attack), hypertension, and protein in the urine (35). Gastrointestinal perforation and fistulas also appear to be rare side effects of some angiogenesis inhibitors.

In addition to the angiogenesis inhibitors that have already been approved by the FDA, others that target VEGF or other angiogenesis pathways are currently being tested in clinical trials (research studies involving patients). If these angiogenesis inhibitors prove to be both safe and effective in treating human cancer, they may be approved by the FDA and made available for widespread use.

In addition, phase I and II clinical trials are testing the possibility of combining angiogenesis inhibitor therapy with other treatments that target blood vessels, such as tumor-vascular disrupting agents, which damage existing tumor blood vessels (6).

9.5.3 Recent Advances in Anti-Angiogenic Therapy of Cancer

Rajeev S. Samant and Lalita A. Shevde
Oncotarget. 2011 Mar; 2(3): 122–134.

More than forty anti-angiogenic drugs are being tested in clinical trials all over the world. This review discusses agents that have approved by the FDA and are currently in use for treating patients either as single-agents or in combination with other chemotherapeutic agents.

Tumor angiogenesis is generation of a network of blood vessels within the cancerous growth. This process can occur two ways: The more accepted model involves the release of signaling molecules by the tumor cells; these molecules activate the surrounding tissue to promote growth of new blood vessels. This stimulates vascular endothelial cells to divide rapidly [910]. The other model proposes the generation of new vasculature by vasculogenic mimicry. This model argues that the tumor cells trans-differentiate in endothelial-like cells and create structures from inside of the tumor tapping into a nearby blood vessel [4].

Escape of the tumor cell from the confines of the primary tumor to distant body parts is the pre-requisite for hematogenous metastasis. This escape route is provided by the tumor vasculature. Thus, it was envisioned that inhibition of angiogenesis will also lead to inhibition of metastasis. This phenomenon was demonstrated by very elegant mouse model studies using angiostatin [1112]. Angiostatin was also demonstrated to be secreted by some primary tumors leading to restricted growth of the metastasis leading to “dormancy” of the metastasis. Mice deficient in angiogenesis (Id1 & Id3 deficient) showed significantly less tumor take rates [13]. Independent studies showed absence of metastasis in angiogenesis deficient mice [1415]. Defective angiogenesis was attributed to impaired VEGF-dependent recruitment of precursor endothelial cells from the bone marrow to the newly developing tumor vasculature [16].

Metastasis of malignant tumors to regional lymph nodes is one of the early signs of cancer spread in patients, and it occurs at least as frequently as hematogenous metastasis [17]. Particularly, in cancers, such as breast cancer, lymphatic metastasis is a predominant route for tumor spread. The contribution of lymphatic system to the tumor growth is an area that is relatively less studied. However, lymphatic vessels are speculated to contribute to tumor growth and metastasis in a variety of ways. The VEGF, FGF2 and PDGF produced by vascular endothelial cells are proposed to be involved in the activation of lymphatic endothelial cells, which in turn produce matrix metalloproteases and urokinase plasminogen activator (uPA) that can promote malignant tumor growth. Thus, there exists a synergistic crosstalk between the tumor and the lymphatic vessels and blood vessels.

Angiogenesis is a complex and intricately regulated process. Like all other regulated biological phenomena, angiogenesis has activators or pro-angiogenic factors and inhibitors or anti-angiogenic factors [9].

The Activators

Tumor cells activate signaling pathways that promote uncontrolled proliferation and survival. These include the PI3K/AKT/mTOR pathway, Hedgehog pathway and, Wnt pathway [1824] that produce pro-angiogenic signaling intermediates [2526]. Among the several reported activators of angiogenesis present in cells two proteins appear to be the most important for sustaining tumor growth: vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). VEGF and bFGF are secreted by the tumor into the surrounding tissue. They bind to their cognate receptors on endothelial cells. This activates a signaling cascade that transmits a nuclear signal prompting target genes to activate endothelial cell growth. Activated endothelial cells also produce matrix metalloproteinases (MMPs). These MMPs break down the extracellular matrix and allow the migration of endothelial cells. The division and migration of the endothelial cells leads to formation of new blood vessels [2728].

The Inhibitors

If angiogenesis is so critical for the tumor growth, then agents that inhibit angiogenesis would have great therapeutic value. With the discovery of endostatin, the concept of anti-angiogenic therapy was launched and popularized by Dr. Folkman [29]. Angiogenesis inhibitors have been discovered from a variety of sources. Some are naturally present in the human body e.g. specific fragments of structural proteins such as collagen or plasminogen (angiostatin, endostatin, tumstatin) [30]. Others are natural products in green tea, soy beans, fungi, mushrooms, tree bark, shark tissues, snake venom etc. [31]. A plethora of synthetic compounds are also characterized to have anti-angiogenic properties [32].


Since angiogenesis is an event critical to primary tumor growth as well as metastasis, anti-angiogenic treatment of tumors is a highly promising therapeutic avenue [33]. Thus, for over last couple of decades, there has been a robust activity aimed towards the discovery of angiogenesis inhibitors [3435]. More than forty anti-angiogenic drugs are being tested in human cancer patients in clinical trials all over the world. From the several anti-angiogenic agents reported, we have focused this review on discussing those agents that have received FDA approval in the United States and are currently in use for treating patients either as a single-agent or in combination with other chemotherapeutic agents (Figure ​(Figure1).1). Based on functionality, the anti-angiogenic drugs can be sub-divided into three main groups:

angiogenesis inhibitors oncotarget-02-122-g001

angiogenesis inhibitors oncotarget-02-122-g001

Figure 1

Targets of FDA-approved angiogenesis inhibitors: Angiogenesis inhibitors impact both, the tumor as well as the endothelial cells resulting in the disruption of the effects of the microenvironment in promoting tumor growth and angiogenesis

Drugs that inhibit growth of endothelial cells

e.g. Endostatin and combretastatin A4, cause apoptosis of the endothelial cells [36]. Thalidomide is also a potent inhibitor of endothelial cell growth [37].

Drugs that block angiogenesis signaling

e.g. anti-VEGF antibodies (Avastin, FDA approved for colorectal cancer), Interferon-alpha (inhibits the production of bFGF and VEGF) [36].

Drugs that block extracellular matrix breakdown

e.g. inhibitors of MMPs [38].


Conventional chemotherapy is usually a systemic therapy that tries to capture a narrow therapeutic window offered by rapid proliferation of tumor cells compared to the normal cells. Chemotherapy has significant side effects such as hair loss, diarrhea, mouth ulcer, infection, and low blood counts. Anti-angiogenic therapy has several advantages over chemotherapy as it is mostly not directed towards directly killing cells but stopping the blood vessel formation, an event that is rare in tissues other than growing tumor. Hence it is well tolerated by the patients and has fewer side effects [29]. There are currently seven approved anti-cancer therapies in two primary categories:

  1. Monoclonal antibodies directed against specific pro-angiogenic growth factors and/or their receptors
  2. Small molecule tyrosine kinase inhibitors (TKIs) of multiple pro-angiogenic growth factor receptors.

Besides these, inhibitors of mTOR (mammalian target of rapamycin), proteasome inhibitors and thalidomide have also been reported to indirectly inhibit angiogenesis through mechanisms that are not completely understood.


Four monoclonal antibody therapies are approved to treat several tumor types:

Bevacizumab (Avastin®)

The first FDA approved angiogenesis inhibitor, Avastin is a humanized monoclonal antibody that binds biologically active forms of vascular endothelial growth factor (VEGF) and prevents its interaction with VEGF receptors (VEGFR-1 and VEGFR-2), thereby inhibiting endothelial cell proliferation and angiogenesis. Bevacizumab has been tested in phase I studies in combination with chemotherapy with a good safety profile [39]. This treatment is approved for metastatic colorectal cancer and non-small cell lung cancer [4043]. Bevacizumab has also evolved as a first line of treatment in combination with paclitaxel in breast cancer patients by virtue of its ability to double median progression-free survival (PFS) [44]. In combination with chemoendocrine therapy (including capecitabine and vinorelbine, and letrozole) bevacizumab treatment significantly decreased the percentage of viable circulating endothelial cells and prevented the chemotherapy-induced mobilization of circulating progenitors [45]. In combination with irinotecan, bevacizumab significantly increased PFS in glioma patients [4647]. VEGF has emerged as a compelling therapeutic target for leukemias. Inhibition of angiogenesis in hematological malignancies interdicts the angiogenesis within the bone marrow ecosystem comprised of multiple cell types, including fibroblasts, endothelial progenitor cells, endothelial cells, dendritic cells and, malignant cells, blocking the availability of nutrients to cancer cells and disrupting crosstalk between the various cell types to curtail the malignant phenotype [48].

Cetuximab (Erbitux®)

This is a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), preventing ligand binding and activation of the receptor resulting in internalization and degradation of the receptor culminating in inhibition of cell proliferation and angiogenesis. Cetuximab downregulated VEGF expression in a dose-dependent manner in a human colorectal carcinoma (CRC) cell line and in human CRC mouse xenografts [49]. The xenografts also showed a significant reduction in blood vessel counts following several rounds of cetuximab treatment [49], indicating that the tumor-promoting effects of EGFR overexpression may be mediated through VEGF stimulation and tumor angiogenesis. This treatment is approved for metastatic CRC and head and neck cancer [50] in patients who are refractory to irinotecan-based chemotherapy. In combination with irinotecan (an inhibitor of topoisomerase I), cetuximab is the first monoclonal antibody that has been approved by the FDA as second-line treatment for metastatic colorectal cancer [5152]. In Phase I and Phase III trials [5354] cetuximab significantly improved the effects of radiotherapy in patients with unresectable (cannot be removed by surgery) squamous cell carcinoma of the head and neck (SCCHN). Cetuximab has also been shown to sensitize cells to radiation and chemotherapy, potentially through blocking EGFR nuclear import and the associated activation of DNA protein kinase enzymes necessary for repairing radiation- and chemotherapy-induced DNA damage [55]. Compared to radiation alone, cetuximab plus radiation therapy can nearly double the median survival in patients with a certain kind of head and neck cancer that has not spread to other parts of the body [54] making cetuximab the only drug achieving interesting response rate in second line treatment of advanced SCCHN [56]. Cetuximab was also found to be tolerated well in combination with cisplatin, or carboplatin, and fluorouracil [5758].

Panitumumab (Vectibix™)

It is a fully humanized anti-EGFR monoclonal antibody that binds specifically to the human EGFR. Panitumumab is a recombinant human monoclonal antibody [59]; therefore, the risk of an infusion reaction is minimized. Vectibix® is indicated as a single agent for the treatment of EGFR-expressing, metastatic colorectal carcinoma with disease progression on or following fluoropyrimidine-, oxaliplatin-, and irinotecan-containing chemotherapy regimens [6062]. The effectiveness of Vectibix® as a single agent for the treatment of EGFR-expressing, metastatic CRC is based on progression-free survival [6364]. Panitumumab is used in patients who are not responding to regimens containing fluorouracil, oxaliplatin, and irinotecan [60]. Patients often receive panitumumab after receiving bevacizumab or cetuximab. Panitumumab can be given with FOLFOX (oxaliplatin, leucovorin, and fluorouracil) or FOLFIRI (irinotecan, leucovorin, and fluorouracil) regimens, or as a single agent. Currently no data are available that demonstrate an improvement in disease-related symptoms or increased survival with Vectibix® in colon cancer [65]. This drug is also being tested for aerodigestive track and head and neck cancer [6667].

Trastuzumab (Herceptin®)

Is a humanized monoclonal antibody that binds the extracellular domain of HER-2, which is overexpressed in 25-30% of invasive breast cancer tumors [68]. HER2-positive breast cancer is highly aggressive disease with high recurrence rate, poorer prognosis with decreased survival compared with HER2-negative breast cancer [69]. Herceptin® is designed to target and block the function of HER2 protein overexpression. This is the first humanized antibody is approved for Breast cancer [70]. Herceptin® is approved by the FDA to treat HER2 positive breast cancer that has metastasized after treatment with other anticancer drugs [71]. It is also approved to be used with other drugs to treat HER2-positive breast cancer that has spread to the lymph nodes to be used after surgery. The FDA first approved Herceptin in September 1998 [7173]. In November 2006, the FDA approved Herceptin as part of a treatment regimen containing doxorubicin, cyclophosphamide and paclitaxel, for the adjuvant treatment of patients with HER2-positive, node-positive breast cancer ( In January 2008, the FDA approved Herceptin as a single agent for the adjuvant treatment of HER2-overexpressing node-negative (ER/PR-negative or with one high-risk feature) or node-positive breast cancer, following multi-modality anthracycline-based therapy ( Trastuzumab is also being studied in the treatment of other types of cancers such as pancreatic [74], endometrial [75], lung [76], cervical [77] and ovarian cancer [78]


Protein tyrosine kinases have emerged as crucial targets for therapeutic intervention in cancer especially because they play an important role in the modulation of growth factor signaling. As per (, there are 43 ongoing studies on tyrosine kinase inhibitors in angiogenesis. Since discussing all of them is beyond the scope of this article, we have focused our discussion on the three TKIs that are currently approved as anti-cancer therapies:

Erlotinib (Tarceva®)

Erlotinib hydrochloride (originally coded as OSI-774) is an orally available, potent, reversible, and selective inhibitor of the EGFR (ErbB1) tyrosine kinase activity. Erlotinib hydrochloride has been approved by FDA for treatment of patients with locally advanced or metastatic NSCLC after failure of at least one prior chemotherapy regimen [7980]. Interesting recent studies have demonstrated that since Erlotinib and Bevacizumab act on two different pathways critical to tumor growth and dissemination, administering these drugs concomitantly may confer additional clinical benefits to cancer patients with advanced disease. This combination therapy may prove to be a viable second-line alternative to chemotherapy in patients with NSCLC [81]. Also, for patients with locally advanced, unresectable or metastatic pancreatic carcinoma, Erlotinib has received FDA approval for the treatment in combination with gemcitabine [8283]. Erlotinib is also being studied in the treatment of other types of cancers. For example combination of Erlotinib with Bevacizumab has been evaluated in metastatic breast cancer [84], hepatocellular carcinoma [85] and in metastatic renal cancer [86] as phase II trials. Outcomes for prostate, cervical and colorectal cancers treated with Erlotinib are cautiously optimistic [8789].

Sorafenib (Nexavar®)

Sorafenib is an orally active inhibitor of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-β, and Raf-1 tyrosine kinase activity [90]. It has received the approval of FDA for the treatment of patients with unresectable hepatocellular carcinoma [91] and advanced renal cell carcinoma [92]. However, not all advanced hepatocellular carcinoma patients were able to tolerate sorafenib and some patients experienced tumor progression [91]. Sorafenib has shown improvements in PFS in patients with renal cell carcinoma [93]. It is one of the aggressively studied drugs. According to the NCI clinical trials search results, there are about 168 active clinical trials involving sorafenib in a variety of cancers.

Sunitinib (Sutent®)

Sunitinib targets activity of multiple tyrosine kinases such as VEGFR-1, VEGFR-2, VEGFR-3, PDGFR- β, and RET [94]. It is approved by FDA as Sunitinib malate for treating advanced (metastatic) renal cell carcinoma [95]. It is also approved by FDA for gastrointestinal stromal tumor (GIST) in patients whose disease has progressed or who are unable to tolerate treatment with imatinib (Gleevec), the current treatment for GIST patients [9596]. Sunitinib has shown early evidence of anti-tumor activity in Phase II trials in US, European and Asian patients with locally advanced, unresectable and metastatic hepatocellular carcinoma. A Phase III trial of sunitinib in hepatocellular carcinoma is ongoing [97]. According to the NCI clinical trials search results, Sunitinib is currently evaluated in about 150 active clinical trials. It is evaluated for ovarian [98], breast [99] and non small cell lung cancer [100] among others [101].

Inhibitors of mTOR

mTOR plays a part in the PI3 kinase/AKT pathway involved in tumor cell proliferation and angiogenesis [102]. Rapamycin and related mTOR inhibitors inhibit endothelial cell VEGF expression, as well as VEGF-induced endothelial cell proliferation [103]. Inhibitors of mTOR are an important class of anti-angiogenic agents. These include: deforolimus, everolimus, rapamycin (sirolimus), and temsirolimus [104105]. Temsirolimus (Toricel™) is a small molecule inhibitor of mTOR, approved for treating advanced renal cell carcinoma [106]. It is a type of rapamycin analog and a type of serine/threonine kinase inhibitor, it is also called CCI-779. In pre-clinical models combination therapy for treating breast cancer using anti-estrogen, ERA-923, and temsirolimus has been successfully tested [107]. It is found to be highly effective against human melanoma when tested in combination with cisplatin and DTIC (in independent studies) in a SCID mouse xenotranplantation model [108109]. There are over 41 active studies of Temsirolimus for a variety of solid tumors [110]. mTOR inhibition has also been strongly advocated in as a putative cancer therapeutic strategy for urologic malignancies [111]. In a pilot study (6 patients) with imatinib-resistant CML, rapamycin induced major and minor leukocyte responses, with an observed decrease in the mRNA levels of VEGFA in circulating leukaemic cells [112]. Combination treatments for breast cancer with aromatase inhibitor [113] and letrozol [114] are also being evaluated. Rapamycin treatment brought partial responses (>50% reduction in the absolute number of blood blasts) and stable disease in adult refractory/relapsed AML [115]. In a recent report, Deforolimus was studied in a Phase 2 trial in pretreated patients with various hematological malignancies, including ALL, AML, CLL, CML, MDS, agnogenic myeloid metaplasia, mantle cell lymphoma and T-cell leukemia/lymphoma [116]. Overall, 40% of deforolimus-treated patients experienced hematological improvement or stable disease.


Bortezomib (Velcade®)

Is a proteasome inhibitor that disrupts signaling of cancer cells, leading to cell death and tumor regression. It is the first compound in its class to be used in clinical practice. It has indirect anti-angiogenic properties [117]. While its exact mechanism is not understood, it induces the pro-apoptotic BH3-only family member NOXA in a p53 independent fashion triggering of a caspase cascade culminating in apoptosis in melanoma and myeloma cells [118]. It is FDA-approved for the treatment of myeloma that has relapsed after two prior treatments (or where resistance has developed following the last treatment). It was also found to induce high quality responses as third line salvage therapy with acceptable toxicity in a significant proportion of homogeneously pre-treated myeloma patients with progressive disease after autologous transplantation and thalidomide. [119]. In a Phase 3 trial involving 669 myeloma patients treated with at least one prior therapy, bortezomib increased median, improved overall survival, and increased response rate, compared with high-dose dexamethasone [120]. In combination with doxorubicin and gemcitabine, bortezomib was also found to be effective in heavily pretreated, advanced Cutaneous T cell Lymphomas (CTCL) [121]. Bortezomib was also reported to be active as a single agent for patients with relapsed/refractory CTCL and Peripheral T Cell Lymphoma (PTCL) with skin involvement [122]. On the contrary, the use of bortezomib was discouraged after a phase II study revealed that found in combination with dexamethasone, bortezomib is not active in heavily pre-treated patients with relapsed Hodgkin’s lymphoma [123124].

Thalidomide (Thalomid®)

Possesses immunomodulatory, anti-inflammatory, and anti-angiogenic properties, although the precise mechanisms of action are not fully understood. Thalidomide was the first angiogenesis inhibitor to demonstrate clinical efficacy in multiple myeloma [37125]. Specifically in myeloma, thalidomide down-regulated VEGF secretion from bone marrow endothelial cells obtained from patients with active disease. In a landmark Phase 2 clinical trial, 169 previously treated patients with refractory myeloma received thalidomide monotherapy [126]. Partial response, was achieved in 30% of patients, and 14% achieved a complete or nearly complete remission. The survival rate at 2 years was 48%. These results led to many subsequent clinical studies of thalidomide in myeloma, leading ultimately to FDA approval of the drug in 2006, for the treatment of newly diagnosed multiple myeloma, in combination with dexamethasone. In the pivotal Phase 3 trial, the response rate in patients receiving thalidomide plus dexamethasone was 63% compared to 41% with dexamethasone alone [127]. Long-term outcome measures, including time-to-progression (TTP) and PFS, were recently reported for a 470 patient randomized, placebo-controlled Phase 3 clinical trial of a similar protocol in newly diagnosed multiple myeloma, with comparable overall response rates [128]. Significant increases resulted in both median TTP and median PFS for the thalidomide plus dexamethasone group versus dexamethasone alone.

Thalidomide was found to be moderately tolerated and minimally effective in patients with histologically proven advanced hepatocellular carcinoma [129]. Thalidomide provided no survival benefit for patients with multiple, large, or midbrain metastases when combined with WBRT (whole-brain radiation therapy) [130]. On the contrary, thalidomide did not significantly add to the efficacy of the fludarabine, carboplatin, and topotecan (FCT) regimen in poor prognosis AML patients [131] and was also ineffective in improving prognosis or decreasing plasma VEGF levels in patients with persistent or recurrent leiomyosarcoma of the uterus [132].


While conventional anti-angiogenic therapy is based on Maximum Tolerated Doses (MTD), the cells involved in angiogenesis may regenerate during the three- to four-week interval between cycles of the chemotherapy. Taking advantage of the fact that endothelial cells are about 10–100 times more susceptible to chemotherapeutic agents than cancer cells, therapy based on daily, oral, low-dose chemotherapeutic drugs was designed. Metronomic chemotherapy refers to the close, rhythmic administration of low doses of cytotoxic drugs, with minimal or no drug-free breaks, over prolonged periods. Metronomic therapy appears promising mainly due to the fact that its anti-angiogenic and anti-tumorigenic effects are accompanied by low toxicity, limited side effects, no need for hospitalization and allowing for feasible combinations with selective inhibitors of angiogenesis. There are several foreseeable advantages and opportunities for metronomic chemotherapy: activity against the parenchymal and stromal components, pro-apoptotic activity, reduction of the likelihood of emergence of acquired resistance, feasibility of long term administration and acceptable systemic side effects [133]. In a pilot phase II study conducted by Correale et al [134] to investigate the toxicity and activity of the novel metronomic regimen of weekly cisplatin and oral etoposide in high-risk patients with NSCLC, the objective response rate was 45.2%, disease control was 58.1%, meantime to progression and survival were 9 and 13 months, respectively. Pharmacokinetic analysis showed that this regimen allowed a greater median monthly area under the curve of the drugs than conventional schedules. In a Phase I trial of metronomic dosing of docetaxel and thalidomide, of the 26 patients with advanced tumors enrolled, prolonged freedom from disease progression was observed in 44.4% of the evaluable patients [135].

Circulating endothelial progenitor cells (EPCs) also participate in tumor angiogenesis. In a study comparing the effects of metronomic chemotherapy over conventional dose-dense chemotherapy, it was found that the numbers of circulating EPCs and the plasma levels of VEGF increased sharply, doubling pre-therapeutic levels at day 21 after conventional chemotherapy, whereas under low-dose metronomic chemotherapy, the numbers of circulating EPCs decreased significantly and VEGF plasma concentrations remained unchanged. These observations provide evidence that conventional dose-dense chemotherapy leads to rebound EPC mobilization even when given with adjuvant intention, while low-dose metronomic scheduling of cytotoxic substances such as trofosfamide may sharply reduce EPC release into the circulation. [136].

Combined bevacizumab and metronomic oral cyclophosphamide was also discovered to be a safe and effective regimen for heavily pre-treated ovarian cancer patients [137]. Treatment with metronomic capecitabine and cyclophosphamide in combination with bevacizumab was shown to be effective in advanced breast cancer and additionally was minimally toxic [138]. Metronomic treatment with carboplatin and vincristine associated with fluvastatin and thalidomide significantly increased survival of pediatric brain stem tumor patients. Tumor volume showed a significant reduction accompanied by increased quality of life [139]. Thus, given the fact that the most evident effect of selective anti-angiogenic agents (i.e. bevacizumab) is the significant prolonging of the duration of response obtainable by chemotherapy alone, with minimal possible side effects of cytotoxic agents given in association metronomic chemotherapy should be considered both as novel up-front or maintenance treatment in patients with biologically poorly aggressive advanced cancer diseases [140].

Overall, metronomic chemotherapy was able to induce tumor stabilization and prolong the duration of clinical benefit, without much associated toxicity. Emerging evidence suggests that metronomic chemotherapy could also activate the host immune system and potentially induce tumor dormancy [141143].


While angiogenesis as a hallmark of tumor development and metastasis is now a validated target for cancer treatment, the overall benefits of anti-angiogenic drugs from the perspective of impacting survival have left much to desire, endorsing a need for developing more effective therapeutic regimens e.g., combining anti-angiogenic drugs with established chemotherapeutic drugs [144145]. There are now several agents that target the tumor vasculature through different pathways, either by inhibiting formation of the tumor neovasculature or by directly targeting the mature tumor vessels. The main body of evolving evidence suggests that their effects are compounded by their synergistic use with conventional chemotherapy rather than individual agents. Anti-angiogenic drugs such as bevacizumab can bring about a transient functional normalization of the tumor vasculature. This can have an additive effect when co-administered with chemo/radiotherapy. But long term inhibition of angiogenesis reduces tumor uptake of co-administered chemotherapeutic agents. This underscores the need for discovering new targets for anti-angiogenic therapy in order to effectively prohibit angiogenesis and circumvent mechanisms that contribute to resistance mechanisms that emerge with long term use of anti-angiogenic therapies. It also warrants a need to define reliable surrogate indicators of effectiveness of the anti-angiogenic therapy as well as dependable markers for identifying the patients who are most likely to benefit from the combination of anti-angiogenic therapy and conventional chemotherapy.

Several new frontiers are emerging. New advances in understanding endothelial cells, which constitute the tumor vasculature, towards developing antiangiogenic strategies are one of the important ones [146147]. Novel cellular targets such as integrins and microRNAs and novel treatment options such as possible use of pharmaconutrients to modulate angiogenic pathways need careful testing and evaluation [148151]. Finally, the administration of these drugs in a metronomic schedule is likely to improve the overall response to anti-angiogenic drugs making it feasible to administer them with conventionally toxic chemotherapeutic drugs, thus increasing the armamentarium of drug combinations that can be employed for treatment.

9.5.4 Angiogenesis inhibitors in cancer therapy: mechanistic perspective on classification and treatment rationales

El-Kenawi AE1, El-Remessy AB.
Br J Pharmacol. 2013 Oct; 170(4):712-29.

Angiogenesis, a process of new blood vessel formation, is a prerequisite for tumor growth to supply the proliferating tumor with oxygen and nutrients. The angiogenic process may contribute to tumour progression, invasion and metastasis, and is generally accepted as an indicator of tumor prognosis. Therefore, targeting tumor angiogenesis has become of high clinical relevance. The current review aimed to highlight mechanistic details of anti-angiogenic therapies and how they relate to classification and treatment rationales. Angiogenesis inhibitors are classified into either direct inhibitors that target endothelial cells in the growing vasculature or indirect inhibitors that prevent the expression or block the activity of angiogenesis inducers. The latter class extends to include targeted therapy against oncogenes, conventional chemotherapeutic agents and drugs targeting other cells of the tumor micro-environment. Angiogenesis inhibitors may be used as either monotherapy or in combination with other anticancer drugs. In this context, many preclinical and clinical studies revealed higher therapeutic effectiveness of the combined treatments compared with individual treatments. The proper understanding of synergistic treatment modalities of angiogenesis inhibitors as well as their wide range of cellular targets could provide effective tools for future therapies of many types of cancer.

Two major processes of blood vessel formation are implicated in the development of vascular system: vasculogenesis and angiogenesis. Vasculogenesis prevails in the embryo and refers to the formation ofde novo blood vessels by in situ differentiation of the mesoderm-derived angioblasts and endothelial precursors. Angiogenesis is the formation of new capillaries from pre-existing vessels and circulating endothelial precursors (Polverini, 2002; Chung et al., 2010; Ribatti and Djonov, 2012). Angiogenesis is a tightly controlled dynamic process that can occur physiologically in those tissues that undergo active remodeling in response to stress and hypoxia (Carmeliet, 2003; Folkman, 2007). However, it can be aberrantly activated during many pathological conditions such as cancer, diabetic retinopathy as well as numerous ischemic, inflammatory, infectious and immune disorders (Carmeliet, 2003; Ali and El-Remessy, 2009; Willis et al., 2011). Although the concept of proposing angiogenesis inhibitors as anticancer drugs received considerable skepticism when first presented by Dr. Folkman in the early 1970s (Folkman, 1971), active research in the field and subsequent clinical trials eventually resulted in US Food and Drug Administration (FDA) approval of bevacizumab for colorectal cancer in 2004 (Cohen et al., 2007). Since then, several angiogenic inhibitors have been identified. This review will provide an overview of the key mechanisms involved in tumor angiogenesis, classification of angiogenesis inhibitors as well as treatment rationales from the mechanistic point of view.

Sustained angiogenesis as a hallmark of cancer

Proliferating tumours tend to activate an angiogenic phenotype to fulfil their increased demand of oxygen and nutrients (Hanahan and Folkman, 1996; Carmeliet, 2005). Additionally, paracrine release of anti-apoptotic factors from activated endothelial cells in the newly formed vasculature supplies tumour cells with a survival privilege (Folkman, 2003). Consequently, in order to progress, tumors tend to activate an event called ‘angiogenic switch’ by shifting the balance of endogenous angiogenesis inducers and inhibitors towards a pro-angiogenic outcome. As a result, dormant lesion progresses into outgrowing vascularized tumor and eventually into a malignant phenotype (Hanahan and Folkman, 1996; Baeriswyl and Christofori, 2009). Hypoxia drives such imbalance through up-regulation of the transcription factor hypoxia inducible factor-1α (HIF-1α), which in turn increases the expression of many angiogenesis inducers as well as suppresses the expression of endogenous angiogenesis inhibitors (Pugh and Ratcliffe, 2003). In spite of that, accumulating evidence indicates that angiogenic cascade can be also driven by alternative HIF-1-independent pathways (Mizukami et al., 2007; Arany et al., 2008; Lee, 2013).

As summarized in Table 1, the angiogenesis inducers are a wide range of mediators that include many growth factors, a plethora of cytokines, bioactive lipids, matrix-degrading enzymes and a number of small molecules (Folkman, 1995; Folkman, 2003; Lopez-Lopez et al., 2004; Bouis et al., 2006; El-Remessy et al., 2007; Bid et al., 2011; MacLauchlan et al., 2011; Murakami, 2011; Fagiani and Christofori, 2013; Qin et al., 2013). Pro-angiogenic growth factors mostly activate a series of surface receptors in a series of paracrine and autocrine loops with the VEGF-A signaling representing the critical rate-limiting step, physiologically and pathologically. VEGF-A (traditionally known as VEGF) is the most potent VEGF isoform that acts mainly on VEGF receptor 2 (VEGFR2) to mediate vascular permeability, endothelial proliferation, migration and survival (Takahashi and Shibuya, 2005; Bouis et al., 2006). In spite of the well-established master roles of VEGF signaling in literature, those processes are probably accomplished through a highly regulated interplay between VEGF and other pro-angiogenic factors. In this context, basic fibroblast growth factor (bFGF) activation of the endothelium is required for maintenance of VEGFR2 expression and the ability to respond to VEGF stimulation (Murakami et al., 2011). Similarly, sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid that can directly contribute to tumor angiogenesis (reviewed in Sabbadini, 2011), is needed for VEGF-induced blood vessel formation, indicating the cooperation between S1P and VEGF in tumor angiogenesis (Visentin et al., 2006). As a net result, the pro-angiogenic interplay of those ligands and others dominates over the activities of two dozen endogenous angiogenesis inhibitors that can be either matrix-derived inhibitors or non–matrix-derived inhibitors (Nyberg et al., 2005).

Table 1. Pro-angiogenic mediators implicated in tumor angiogenesis

Category Examples References
Growth factors VEGFs Bouis et al., 2006
FGFs Ibid
TGFs Ibid
PDGFs Ibid
Insulin-like growth factors Lopez-Lopez et al., 2004; Bid et al., 2011
ANGs Fagiani and Christofori, 2013
Cytokines IL-8 Strieter et al., 2004
CSF-1 Lin et al., 2006
Bioactive lipids PGE2 Wang and Dubois, 2010
S1P Murakami, 2011
Matrix-degrading enzymes MMPs Bourboulia and Stetler-Stevenson, 2010
Heparanases Vlodavsky and Friedmann, 2001
Small mediators NO MacLauchlan et al., 2011
Peroxynitrite El-Remessy et al., 2007
Serotonin Qin et al., 2013
Histamine Qin et al., 2013

The multistep angiogenic process starts with vasodilation and increased permeability of existing vessels in response to tumor cell-secreted VEGF. This is accompanied by loosening of pericytes covering mediated by angiopoietin-2 (ANG2), a ligand of tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (TIE2) receptor (Bergers and Benjamin, 2003; Jain, 2003; Fagiani and Christofori, 2013). Meanwhile, many secreted matrix-degrading enzymes, such as MMPs and heparanases, function in concert to dissolve the basement membrane and to remodel the extracellular matrix (ECM) as well as to liberate more pro-angiogenic growth factors (bFGF and VEGF) from matrix heparan sulfate proteoglycans (HSPGs) respectively (Houck et al., 1992; Whitelock et al., 1996; Vlodavsky and Friedmann, 2001; Tang et al., 2005; van Hinsbergh and Koolwijk, 2008). The overall chemotactic angiogenic stimuli guide endothelial cells to migrate, to align into tube-like structures and to eventually form new blood vessels. However, such blood vessels are characterized by being disorganized, chaotic, hemorrhagic and poorly functioning (Bergers and Benjamin, 2003).

The angiogenic phenotype in tumor micro-environment can further be sustained and extravagated by the recruitment of other types of stromal cells. Stromal cells such as fibroblasts, mesenchymal stem cells and various bone marrow-derived myeloid cells including macrophages, TIE2-expressing monocytes, neutrophils and mast cells contribute to tumor angiogenesis through their production of growth factors, cytokines and proteases (Murdoch et al., 2008; Joyce and Pollard, 2009; Cirri and Chiarugi, 2011). For example, in response to cancer cell-derived TGF-β, PDGF or bFGF, fibroblasts are transformed to an activated phenotype with a higher proliferative activity and myofibroblastic characteristics (Kalluri and Zeisberg, 2006; Cirri and Chiarugi, 2011). Such carcinoma-associated fibroblasts (CAFs) were shown to promote angiogenesis and metastasis by secreting large amounts of MMP-2 and MMP-9 as well as by expressing many cytokines and chemokines that resulted in immune cell infiltration (Gerber et al., 2009; Giannoni et al., 2010). Furthermore, it has been shown that PDGF-C produced by CAFs is able to elicit VEGF production from tumor cells, thereby sustaining the angiogenic shift (Crawford et al., 2009). Similarly, tumor-associated macrophages (TAMs), one of the bone marrow myeloid-derived cells, are induced to develop into polarized type II (alternatively activated or M2 macrophages), upon exposure to tumor hypoxia and tumor cell-derived cytokines (Leek et al., 2002; Rogers and Holen, 2011). M2 macrophages tend to produce many pro-angiogenic growth factors, cytokines and matrix-degrading enzymes such as VEGF, PDGF, bFGF, TNF-α, COX-2, MMP-9, MMP-7 and MMP-12 (Lewis and Pollard, 2006).

From another perspective, angiogenesis may be dispensable for progression of some malignancies. For example, some tumours may co-opt pre-existent vessels as an alternative way to obtain blood supply. Vessel co-option was first described in the brain, one of the most densely vascularized organs, in which tumours may develop in earlier stages without the activation of angiogenic response (Holashet al., 1999; Leenders et al., 2002; Bergers and Benjamin, 2003; Hillen and Griffioen, 2007). In another example, hypovascularized tumors such as pancreatic ductal adenocarcinoma may involve certain adaptation to flourish in the absence of prominent angiogenesis (Bergers and Hanahan, 2008). Obviously, in both cases, tumors may be intrinsically indifferent to angiogenesis inhibitors. However, in most other cases, therapy directed towards the vasculature of solid tumors is being considered as an important direction in cancer treatment.

Classification of angiogenesis inhibitors

Growth of newly formed vessels in tumor micro-environment can be inhibited directly by targeting endothelial cells in the growing vasculature or indirectly by targeting either tumor cells or the other tumor-associated stromal cells. Therefore, angiogenesis inhibitors can be classified into direct and indirect inhibitors (Kerbel and Folkman, 2002; Folkman, 2007).

Direct endogenous inhibitors of angiogenesis

Direct endogenous inhibitors of angiogenesis, such as angiostatin, endostatin, arrestin, canstatin, tumstatin and others, are fragments released on proteolysis of distinct ECM molecules. Endogenous inhibitors prevent vascular endothelial cells from proliferating, migrating in response to a spectrum of angiogenesis inducers, including VEGF, bFGF, IL-8 and PDGF (Kerbel and Folkman, 2002; Abdollahi et al., 2004; Mundel and Kalluri, 2007; Ribatti, 2009). This direct anti-angiogenic effect may be mediated by interference with endothelial integrins along with several intracellular signaling pathways (Mundel and Kalluri, 2007). For example, the ability of tumstatin-derived active peptide to inhibit angiogenesis and tumour growth is associated with the expression of the adhesion receptor, αvβ3 integrin, on tumor endothelial cells (Eikesdal et al., 2008). Through binding αvβ3 integrin, full tumstatin was found to inhibit endothelial cell activation of focal adhesion kinase, PI3K, Akt, mammalian target of rapamycin (mTOR) and others (Maeshima et al., 2002). Direct targeting of those signaling pathways by endogenous inhibitors was thought to be the least likely to induce acquired drug resistance because they target endothelial cells with assumed genetic stability rather than unstable mutating tumour cells (Kerbel and Folkman, 2002). However, endostatin has not yet led to any documented benefit to patients in randomized phase III trials, or even modest activity in phase II trials (Ellis and Hicklin, 2008).

Indirect inhibitors of angiogenesis

Indirect inhibitors of angiogenesis classically prevent the expression or block the activity of pro-angiogenic proteins (Folkman, 2007). For example, Iressa, an EGF receptor (EGFR) TK inhibitor (TKI), blocks tumour expression of many pro-angiogenic factors; bevacizumab, a monoclonal antibody, neutralizes VEGF after its secretion from tumour cells whereas sunitinib, a multiple receptor TKI, blocks the endothelial cell receptors (VEGFR1, VEGFR2 and VEGFR3), preventing their response to the secreted VEGF (Folkman, 2007; Roskoski, 2007). In addition, this class extends to include conventional chemotherapeutic agents, targeted therapy against oncogenes and drugs targeting other cells of the tumor micro-environment (Kerbel et al., 2000; Ferrara and Kerbel, 2005).

Conventional chemotherapeutic agents

Conventional chemotherapeutic agents have been shown to have anti-angiogenic properties in addition to the ability to induce direct cancer cell death. Such chemotherapeutic agents can affect the endothelial cell population in the tumour bed during treatment cycles because they have significantly higher proliferation rates than resting endothelium outside a tumor, making them more susceptible to cytotoxic effect (Kerbel et al., 2000; Folkman, 2003). However, the cyclic treatment rationale of cytotoxic drugs allows the potential damage to the tumour vasculature to be repaired during the long breaks. Thus, continuous low doses of chemotherapeutic agents were suggested as a way to reduce side effects and drug resistance (Drevs et al., 2004). This modality is termed metronomic therapy, and clinically, it refers to the daily administration of 5–10% of the phase II-recommended dose of the chemotherapeutic agent (Penel et al., 2012). The extended use of such low doses of cytotoxic agents elicits an anti-angiogenic activity through induction of endothelial cell apoptosis and decreasing the level of circulating endothelial precursors (Hamano et al., 2004; Shahrzad et al., 2008). In clinical investigations, metronomic dosing of cyclophosphamide and others showed promising efficacy in patients with advanced, multiple metastasized and/or multiple pretreated solid tumours (Lord et al., 2007; Fontana et al., 2010; Nelius et al., 2011; Gebbia et al., 2012; Briasoulis et al., 2013; Navid et al., 2013).

VEGF-targeted therapy

VEGF-targeted therapy includes neutralizing antibodies to VEGF (e.g. bevacizumab) or VEGFRs (e.g. ramucirumab), soluble VEGFR/VEGFR hybrids (e.g. VEGF-Trap) and TKIs with selectivity for VEGFRs (e.g. sunitinib and sorafenib; Baka et al., 2006; Ellis and Hicklin, 2008; Hsu and Wakelee, 2009). Bevacizumab, a humanized monoclonal antibody against all isoforms of VEGF-A, has been approved for the treatment of colorectal, lung, glioblastoma and renal cell carcinoma (Hsu and Wakelee, 2009). Many other clinical trials with promising efficacy were also conducted in other cancers such as head and neck cancer, hepatocellular carcinoma, ovarian cancer, metastatic melanoma and gastric cancer (Argiris et al., 2011; 2013; Burger et al., 2011; Ohtsu et al., 2011; Fang et al., 2012; Minor, 2012; Schuster et al., 2012; Van Cutsem et al., 2012). However, for metastatic breast cancer, bevacizumab had been initially granted an accelerated FDA approval, which was later withdrawn due to lack of improvement evidence in disease-related symptoms or overall survival (Burstein, 2011; Montero et al., 2012). Similarly, clinical trials showed that the addition of bevacizumab to the treatment regimens of advanced pancreatic cancer did not extend overall survival (Chiu and Yau, 2012). The neutralization of VEGF-A can also be achieved by soluble receptor construct (VEGF-Trap) that monomerically ‘traps’ the different isoforms of VEGF-A, in addition to VEGF-B and placental growth factor (Rudge et al., 2007). VEGF-Trap showed clinical benefit in a phase III trial of oxaliplatin pretreated metastatic patients with colorectal cancer, and is currently being investigated in a prostate cancer phase III trial (Gaya and Tse, 2012). TKIs are small molecules with different chemical structures that have the ability to interact physically with the highly conserved kinase domain shared by different VEGFRs as well as PDGF receptors (PDGFRs), FGF receptors (FGFRs), EGFR, Raf kinases and c-Kit (a receptor of the pluripotent cell growth factor, stem cell factor). Such interaction directly inhibits tyrosine phosphorylation and the subsequent many downstream pro-angiogenic signaling networks (Baka et al., 2006; Ivy et al., 2009). Those multi-targeted TKIs demonstrated efficacy against various solid malignancies in different clinical trials, some of which have lead eventually to FDA approval of sunitinib and sorafenib. Sunitinib, known to inhibit several receptor TKs (RTKs) including VEGFR1–3, PDGFR-α, PDGFR-β, c-Kit, colony-stimulating factor-1 receptor (CSF-1R) and Flt-3, was approved for the treatment of renal cell carcinoma and gastrointestinal stromal cell tumours. Sorafenib that acts also by inhibiting VEGFR1–3 and PDGFR-β in addition to the serine–threonine kinases Raf-1, B-Raf, was approved for hepatocellular carcinoma in addition to renal cell carcinoma (Llovet et al., 2008; Ivy et al., 2009; Huang et al., 2010).

FGF-targeted therapies

FGF-targeted therapies were recently reconsidered as promising anti-angiogenic and anti-tumor agents after a long period of little attention for drug development, partly due to redundancy (Bono et al., 2013). The FGFR superfamily with its 18 ligands and four receptors has been involved in endothelial cell migration, proliferation and differentiation (Presta et al., 2005). Therapeutic targeting of FGF/FGFR signalling was accomplished by either monoclonal antibodies that inhibit FGFs binding, small molecules that inhibit FGFR TK activity or allosteric modulators that bind the extracellular FGFR domain. Monoclonal antibodies against bFGF displayed potent anti-tumor and anti-angiogenic effects in different preclinical cancer models, which warrant further clinical evaluation (Zhao et al., 2010; Wang et al., 2012). Pan inhibitors of the FGFR TKs such as AZD4547 (blocks the activity of FGFR1–3) and ponatinib (blocks all the FGFR isoforms) elicited potent anti-tumor activities in preclinical investigations so they are currently being evaluated in clinical trials. Those inhibitors displayed the greatest potency in FGFR-driven cancer models, which may be attributed to the interference with the oncogenic functions of either amplified or constitutively active FGFR (Dutt et al., 2011; Zhao et al., 2011; Gavine et al., 2012; Gozgit et al., 2012). Accordingly, further studies are needed to evaluate the relative contribution of angiogenic versus oncogenic inhibitory mechanisms towards the overall anti-tumor activity. The allosteric antagonist of the FGFR, SSR128129E, showed a strong anti-angiogenic activity in addition to tumour growth and metastasis inhibitory effects in animal models of arthritis and cancer respectively. Because allosteric modulators leave a residual level of baseline signalling, they have the ability to fine-tune target biological responses. As a result, allosteric multi-FGFR inhibitors may have an improved benefit/risk ratio that is not attainable with the other TKIs (Bonoet al., 2013; Herbert et al., 2013). However, preclinical findings suggest that long-term clinical outcomes may improve with blockade of additional pro-angiogenic RTKs that may also reduce the risk of drug resistance (Hilberg et al., 2008). For example, dual inhibition of VEGFRs and FGFRs using brivanib produced enduring tumour stasis and angiogenic blockade following the failure of VEGF-targeted therapies (Allen et al., 2011). Furthermore, triple inhibition of FGFRs, VEGFRs and PDGFR(s) using dovitinib (TKI258) or nintedanib (BIBF 1120) displayed broad-spectrum anti-tumour activities in several tumour xenograft models as well as promising data in clinical trials. Combined inhibition of FGFR/VEGFR/PDGFR targets not only tumour cells, but also endothelial cells, pericytes and smooth muscle cells, resulting in an effective inhibition of tumour growth, angiogenesis and metastasis even in advanced tumour stages (Hilberg et al., 2008; Ledermann et al., 2011; Taeger et al., 2011; Chenet al., 2012; Angevin et al., 2013).

Oncogene-targeted therapy

Oncogenes, genes that cause the transformation of normal cells into cancerous cells, are thought to up-regulate many pro-angiogenic proteins. Therefore, anticancer drugs that were developed for their capacity to block an oncogene also have an indirect anti-angiogenic activity (Kerbel et al., 2000; Bergers and Benjamin, 2003; Folkman, 2003). For example, dasatinib and other inhibitors of sarcoma (Src), an aberrantly activated non-RTK associated with many human malignancies, showed potent anti-angiogenic effects through the down-regulation of VEGF and IL-8 (Summy et al., 2005; Han et al., 2006; Haura et al., 2010). Another example is to target the oncogenic Ras using farnesyl transferase (FT) inhibitors, which inhibit post-translational farnesylation of Ras that governs the latter’s activity (Awada et al., 2002). FT inhibitors were found to inhibit tumor VEGF expression and block FTase-dependent Ras activation, which is critically involved in VEGF-elicited angiogenic signal transduction and angiogenesis (Han et al., 2005; Izbicka et al., 2005; Kim et al., 2010). In addition to classical oncogenes inhibition, interference with other tumor-deregulated signaling pathways would offer another approach in targeting angiogenesis. For example, inhibitors of heat shock protein 90 (HSP90), a chaperone molecule known to protect oncoproteins from misfolding and degradation in the protein-rich intracellular environment, were found to prevent VEGF production and to disrupt multiple pro-angiogenic signalling pathways in numerous cancer cells. They were also shown to inhibit tumour growth and vascularity of different human tumor xenografts (Sanderson et al., 2006; Langet al., 2007; Eccles et al., 2008; Trepel et al., 2010; Moser et al., 2012). Proteasome inhibitors, such as bortezomib (PS-341) or MG-132, were also shown to reduce tumour growth and vascularity of squamous cell carcinoma and pancreatic cancer xenograft probably through inhibition of NF–κB-dependent release of pro-angiogenic gene products, VEGF and IL-8 (Sunwoo et al., 2001; Nawrocki et al., 2002; Matsuo et al., 2009). Similarly, inhibition of B-cell lymphoma 2 (Bcl-2), a prosurvival protein that regulates apoptosis by preventing the mitochondrial release of pro-apoptogenic factors, was shown to prevent NF-κB-mediated release of the pro-angiogenic factors IL-8 and CXC chemokine ligand 1 (CXCL1) as well as VEGF in tumor-associated endothelial cells and pancreatic cell lines respectively (Karl et al., 2005; Wang et al., 2008). Moreover, (−)-gossypol, a natural BH3 mimetic that inhibits BH3 domain of Bcl-2 as well as related prosurvival proteins (Bcl-xL and Mcl-1), was shown to remarkably decrease microvessel density in human prostate tumour PC-3 xenografts through decrease of VEGF and IL-8 release as well as blocking multiple steps in VEGF-activated biological events (Karaca et al., 2008; Pang et al., 2011).

Matrix degrading and remodelling-targeted therapy

Matrix degrading and remodelling are activated by tumors to modify local micro-environment, which in turn promote their angiogenic potential (Bergers et al., 2000; Vlodavsky and Friedmann, 2001). Up-regulation of expression and activity of several endogenous MMPs including MMP-2, MMP-9 as well as MMP-3 and MMP-7 have been identified in invasive tumors (for a review, see Bourboulia and Stetler-Stevenson, 2010). Consequently, inhibitors of MMPs were extensively pursued as a therapeutic strategy for treating cancer. Unfortunately, MMPs intervention strategies had met with limited clinical success because of severe toxicities and associated metastasis-promoting effect (Coussens et al., 2002; Devy et al., 2009). Furthermore, the paradoxical roles of tissue inhibitors of metalloproteinases (TIMPs) may contribute to such failure depending on the net balance of TIMPs and MMPs in tumour stroma (Jiang et al., 2002). As a result, efforts were directed at therapies exploiting endogenous MMP inhibitors, TIMPs or monoclonal antibodies against individual MMPs (Martens et al., 2007; Jarvelainen et al., 2009). For example, DX-2400, a highly selective fully human MMP-14 inhibitory antibody, was found to block pro-MMP-2 processing on tumor and endothelial cells, inhibited angiogenesis, and slowed tumor progression and formation of metastatic lesions (Devy et al., 2009). Alternatively, in order to reduce toxicity and enhance drug delivery, polymeric nanoparticulate delivery systems could be used to target individual components of ECM. For example, targeted delivery of antisense inhibitors of laminin-8, a vascular basement membrane component, by conjugation to the natural drug carrier β-poly(L-malic acid) significantly reduced tumour microvessel density and increased animal survival in an experimental model of glioblastoma (Fujita et al., 2006). Similarly, a nano delivery system that incorporate peptides against proteolytically processed type IV collagen significantly accumulated in tumors and blocked angiogenesis in experimental models (Mueller et al., 2009). However, the highly sulfated oligosaccharides, Heparan (HS) mimetics highly sulfated oligosaccharides, were shown to have a heparanase-inhibiting effect sequestering, in turn, many heparan sulfate proteoglycan (HSPG)-binding factors (Johnstone et al., 2010; Dredge et al., 2011). In preclinical studies, HS mimetics have effectively targeted multiple HSPG-dependent functions and have resulted in decreased in vivo tumor growth, tumor invasion, tumor metastasis and angiogenesis (Johnstone et al., 2010; Dredge et al., 2011; Zhou et al., 2011). Clinically, the heparanase inhibitor PI-88 showed preliminary efficacy as an adjunct therapy for post-operative hepatocellular carcinoma (Liu et al., 2009).

Tumour-associated stromal cell-targeted therapy

Tumour-associated stromal cells crosstalk is a perquisite for the formation of a tumour vasculature, an essential step for tumour progression (Lorusso and Ruegg, 2008). Interference with those crosstalk circuits through intervention of cellular adhesion (highlighted in next paragraph) or tumor-induced recruitment of different stromal cells may be considered as an indirect way of anti-angiogenic therapy (Ferrara and Kerbel, 2005). The latter can be supported by studies in which inhibition of macrophage infiltration, for example, by either genetic ablation of the macrophage CSF-1 or liposomal clodronate-induced macrophage depletion, was shown to delay the angiogenic switch and malignant transition (Giraudo et al., 2004; Lin et al., 2006). Furthermore, CSF-1R kinase inhibitors were found to reduce tumor-associated vascularity in two different tumor mouse models (Kubota et al., 2009; Mantheyet al., 2009). In addition, clodronate and other related bisphosphonates, originally used to treat skeletal complications in patients with tumour-induced osteolysis, were shown to exert potent anti-tumour and anti-angiogenic effects in many other studies (Fournier et al., 2002; Santini et al., 2003; Stathopoulos et al., 2008). Zoledronic acid, a third-generation bisphosphonate, was also found to reduce a number of tumour-associated macrophages and shift their phenotype from M2 to M1, resulting in a reduction in TAM-associated production of VEGF in murine models of spontaneous mammary carcinogenesis and mesothelioma (Coscia et al., 2010; Veltman et al., 2010). Clinically, repeated low-dose therapy with zoledronic acid, which maintains active drug plasma concentration, was able to induce an early remarkable and long-lasting decrease of VEGF levels in patients with cancer (Santini et al., 2007). In another example, inhibition of mobilization of neutrophils, from bone marrow and their infiltration into tumour, using neutralizing anti–prokineticin-2, an antibody against a secreted protein known also as BV8, was shown to impair the initial angiogenic switch in a multistage pancreatic beta cell tumorigenesis model (Shojaei et al., 2008). Furthermore, the neutralizing anti-BV8 was found to prevent myeloid cell-dependent tumour angiogenesis in several xenograft models (Shojaei et al., 2007). Cancer-associated fibroblasts (CAF) can also be targeted with thapsigargin analogue coupled with peptides specific for fibroblast activation protein (FAP), a CAF membrane-bound protease whose catalytic site has access to the peritumoural fluid of the tumor micro-environment. This extracellular activation results in the death of CAFs as well as pericytes and endothelial cells within milieu of different human tumor xenografts (Brennen et al., 2012).

Cell adhesion molecules (CAMs)-targeted therapy

CAMs are cell surface proteins known to be involved in binding with other counter-receptors on adjacent cells or surrounding ECM macromolecules (Aplin et al., 1998). Many CAMs, such as αv-integrins, E-selectin, N-cadherin and VE-cadherin, have been implicated in tumour angiogenesis (Bischoff, 1997; Tei et al., 2002; Nakashima et al., 2003; Weis and Cheresh, 2011). For example, αv-integrins are expressed on surface of endothelial cells and can determine whether cells can adhere to and survive in a particular micro-environment. A number of matrix-derived fragments have the ability to act as endogenous angiogenesis inhibitors through binding to integrins on endothelial cells, disrupting physical connections and suppressing signalling events associated with cell survival, migration and proliferation (Nyberg et al., 2005). Consequently, integrins antagonism using peptidomimetics (e.g. cilengitide), monoclonal antibodies (e.g. volociximab) or oral small-molecule compounds have been investigated in a wide range of malignancies (Huveneers et al., 2007). Cilengitide is a cyclized pentapeptide peptidomimetic designed to compete for the arginine-glycine-aspartic acid (RGD) peptide sequence, thereby blocking the ligation of the αvβ3 and αvβ5 integrins to matrix proteins (Hariharan et al., 2007). Cilengitide is mainly under clinical development for glioblastoma; however, clinical trials of other malignancies such as head and neck cancer as well as lung cancer were also initiated (Reardon and Cheresh, 2011; Vermorken et al., 2012; Manegold et al., 2013). Alternatively, cyclic peptides containing RGD motif could guide nanoparticulate delivery system, which incorporates anti-angiogenic cytotoxic agents such as doxorubicin, paclitaxel or combretastatin A4, to accumulate specifically in tumor vasculature with no overt systemic toxicity (Murphy et al., 2008; Ruoslahti et al., 2010; Wang et al., 2011). Volociximab, a chimeric humanized monoclonal antibody that selectively inhibits the αvβ1 integrin interaction with fibronectin, has been evaluated also in clinical trials for solid tumours such as renal cell carcinoma, recurrent ovarian cancer, advanced non–small-cell lung cancer and metastatic pancreatic cancer (Figlin et al., 2006; Evans et al., 2007; Jarvelainen et al., 2009; Vergote et al., 2009; Besse et al., 2013). Cadherins constitute a superfamily of molecules that mediate calcium-dependent cell–cell adhesions. The intracellular domains of cadherins directly bind to β-catenin and link with cytoskeletal components, providing the molecular basis for stable cell–cell adhesion (Zhang et al., 2010). Targeting cadherin signalling may also represent another way for tumor angiogenesis intervention. For example, ADH-1, a cyclic pentapeptide containing the cell adhesion recognition site (His-Ala-Val) required for N-cadherin adhesion, was shown to possess anti-angiogenic and anti-tumour activity (Blaschuk et al., 2005; Blaschuk, 2012). Similarly, monoclonal antibody directed against specific region of VE-cadherin was able to inhibit tumor angiogenesis and growth with no side effects on normal vasculature (Corada et al., 2002; May et al., 2005).

Inflammatory angiogenesis-targeted therapy

Targeting inflammatory angiogenesis, responsible for a substantial part of tumour vascularization initiated by infiltrating leukocytes, may be considered as another indirect anti-angiogenic strategy (Albini et al., 2005). Moreover, as mentioned before, tumour-infiltrating leukocytes contribute into malignant progression through production of many pro-inflammatory cytokines, chemokines and enzymes that can mostly induce angiogenic cascade (Balkwill et al., 2005). Such vital roles have been supported by the early observation that nonsteroidal anti-inflammatory drugs can inhibit tumour angiogenesis and, in turn, tumor progression (Albini et al., 2005). For example, ibuprofen was found to decrease tumor growth and metastatic potential in mice models through modulation of angiogenesis (Yao et al., 2005). Moreover, selective inhibitors of COX-2, an inducible enzyme that catalyses the production of prostanoids from arachidonic acid, were also shown to inhibit angiogenesis (Tsujii et al., 1998; Wei et al., 2004). The anti-angiogenic effect of COX-2 inhibitors may be contributed, in part, by decreasing the COX-2 metabolic product PGE2, the predominant PG in solid tumors known to stimulate cancer cells to produce pro-angiogenic factors such as VEGF and bFGF as well as many other factors belonging to CXC chemokines family (Strieter et al., 2004; Wang et al., 2006; Wang and Dubois, 2010). Members of the CXC chemokine family are heparin-binding proteins that possess disparate regulative roles in angiogenesis. For example, the ELR+ CXC chemokines, characterized by highly conserved three amino acid motifs (Glu-Leu-Arg; ‘ELR’ motif), are potent promoters of angiogenesis, whereas the IFN-inducible (ELR−) CXC chemokines are inhibitors of angiogenesis (Strieter et al., 2004). The use of repertaxin, originally designed to target the ELR+ CXC chemokine receptors CXCR1 and CXCR2 on neutrophils to prevent their migration to sites of inflammation, was found to inhibit tumor angiogenesis, thereby suppressing tumour progression in a genetic model of pancreatic ductal adenocarcinoma (Ijichi et al., 2011). It would be beneficial to explore other small-molecule CXCR2 antagonists that have already been developed for the treatment of inflammatory diseases in different preclinical models of cancer, especially inflammation-associated cancers (refer to Chapman et al., 2009 for a list of newly developed CXCR2 antagonists used in the treatment of inflammatory diseases of the lung).

Mechanisms of enhanced therapeutic efficacy

  • Dual targeting of tumor vasculature
  • Targeting different cell types of tumor micro-environment
  • Normalization of tumor vasculature
  • Chemosensitization of tumor cells
  • Interference with the repair of cytotoxic drug-induced damage and resistance mechanisms

Consequences of anti-angiogenic therapy with other anticancer therapy

  • Contrary to initial expectations, treatment with angiogenesis inhibitors was associated with unexpected toxicities. The toxicity profiles of those inhibitors reflect the systemic disturbance of growth factor signalling pathways that mediate their anti-angiogenic activity (Elice and Rodeghiero, 20102012). In this context, disturbance of the tight endothelial cell-platelet interaction that maintains vascular integrity results in bleeding complications, gastrointestinal perforations, and disturbed wound and ulcer healing (Verheul and Pinedo, 2007). In general, the incidence of those adverse effects increases when anti-angiogenic agent is combined with chemotherapy. For example, bleeding complications have been observed in patients with colorectal cancer treated with chemotherapy in combination with bevacizumab (Kabbinavar et al., 2003; Giantonio et al., 2006). In non–small-cell lung cancer, some patients treated with bevacizumab in combination with carboplatin and paclitaxel experienced severe or fatal pulmonary haemorrhage (Johnson et al., 2004). Furthermore, a higher incidence of gastrointestinal perforation was observed in patients with colorectal cancer given bevacizumab in combination with chemotherapy compared with chemotherapy alone (Hurwitz et al., 2004). Similarly, thrombotic events have been observed in patients treated with angiogenesis inhibitors, especially when these agents are given in combination with chemotherapy (Verheul and Pinedo, 2007). Treatment of patients with cancer with angiogenesis inhibitors is frequently associated with hypertension, which may require the addition of regular anti-hypertensive agent (Izzedine et al., 2009).

Summary and future directions

  • Angiogenesis is a critical process that occurs pathologically in many malignancies due to changing balance of endogenous angiogenesis inducers and inhibitors, leading to the activation of nearby endothelial cells to form new vasculature. Consequently, angiogenesis can be targeted to restrict initiation, growth and progression of most of angiogenesis-dependent malignancies. Numerous angiogenic inhibitors have been identified, some of which are currently being investigated in clinical trials and some others were even approved for cancer therapies. These angiogenesis inhibitors were classified based on their target into two main classes: direct and indirect inhibitors. Indirect angiogenesis inhibitors can be further subclassified based on their interference mechanisms with the angiogenic cascade. A list of major categories and molecular targets for angiogenesis inhibitors is shown in Table 2.
  • Most angiogenesis inhibitors conferred clinical benefits mainly when combined with other chemotherapeutic/targeted therapies rather than being used as monotherapy. Unfortunately, many anti-angiogenic agents were shown to be associated with overt systemic toxicity as well as resistance emergence and disease recurrence. Drug resistance in anti-angiogenic therapy may result from a plethora of pro-angiogenic factors released by inappropriately functioning host cells in the tumor micro-environment as a compensatory mechanism. Therefore, the strategy of targeting endothelial cells alone may not be enough as explained in the previous texts, requiring the proposal of different rationales in which other cellular compartments of tumor micro-environment are targeted to attain proper anti-angiogenic and anti-tumor response. That highlights the importance of considering tumor micro-environment as a dynamic system, as depicted in Figure 1 in which interference with any of its components may be an approach to interfere with cancer hallmarks, including angiogenesis.

9.5.5 LUCITANIB a VEGFR/FGFR dual kinase inhibitor in Phase 2 trials

Dr.  Anthony Melvin Crasto


6-(7-((l-aminocyclopropyl)methoxy)-6-methoxyquinolin-4-yloxy)- N-methyl- 1 -naphthamide
1058137-23-7 (E-3810 free base); 1058137-84-0  (E-3810 HCl salt)
E-3810, E-3810 amine, UNII-PP449XA4BH, E3810, Lucitanib [INN], AL3810
Molecular Formula:C26H25N3O4
Molecular Weight:443.4944 g/mol
Spiro Substituted Compounds As Angiogenesis Inhibitors [US8163923] 2008-09-18 2012-04-24
A 4-(3-methoxypropoxy)-3-methylpyridinyl derivative of timoprazole that is used in the therapy of STOMACH ULCERS and ZOLLINGER-ELLISON SYNDROME. The drug inhibits H(+)-K(+)-EXCHANGING ATPASE which is found in GASTRIC PARIETAL CELLS.
For in advanced solid tumors.
Lucitanib (E-3810): Lucitanib, also known as E-3810,  is a novel dual inhibitor targeting human vascular endothelial growth factor receptors (VEGFRs) and fibroblast growth factor receptors (FGFRs) with antiangiogenic activity. VEGFR/FGFR dual kinase inhibitor E-3810 inhibits VEGFR-1, -2, -3 and FGFR-1, -2 kinases in the nM range, which may result in the inhibition of tumor angiogenesis and tumor cell proliferation, and the induction of tumor cell death. Both VEGFRs and FGFRs belong to the family of receptor tyrosine kinases that may be upregulated in various tumor cell type
Lucitanib (E-3810) Structure


Lucitanib is an oral, potent inhibitor of the tyrosine kinase activity of fibroblast growth factor receptors 1 through 3 (FGFR1-3), vascular endothelial growth factor receptors 1 through 3 (VEGFR1-3) and platelet-derived growth factor receptors alpha and beta (PDGFR α-ß). We own exclusive development and commercial rights to lucitanib on a global basis, excluding China. Lucitanib rights to markets outside of the U.S. and Japan have been sublicensed to Les Laboratoires Servier (Servier). We are collaborating with Servier on the global clinical development of lucitanib.

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 What is the key method to harness Inflammation to close the doors for many complex diseases?


Author and Curator: Larry H Bernstein, MD, FCAP


The main goal is to  have a quality of a healthy life.

When we look at the picture 90% of main fluid of life, blood, carried by cardiovascular system with two main pumping mechanisms, lung with gas exchange and systemic with complex scavenger actions, collection of waste, distribution of nutrition and clean gases etc.  Yet without lymphatic system body can’t make up the 100% fluid.  Therefore, 10% balance is completed by lymphatic system as a counter clockwise direction so that not only the fluid balance but also mass balance is  maintained. Finally, the immune system patches the  remaining mechanism by providing cellular support to protect the body because it contains 99% of white cells to fight against any kinds of invasion, attack, trauma.

These three musketeers, ccardiovascular, lyphatic and immune systems, create the core mechanism of survival during human life.

However, there is a cellular balance between immune and cardiovascular system since blood that made up off 99% red cells and 1% white blood cells that are used to scavenger hunt circulating foreign materials.   These three systems are acting with a harmony not only defend the body but provide basic needs of life.  Thus, controlling angiogenesis and working mechanisms in blood not only helps to develop new diagnostic tools but more importantly establishes long lasting treatments that can harness Immunomodulation.

The word inflammation comes from the Latin “inflammo”, meaning “I set alight, I ignite”.

Medical Dictionary description is:

“A fundamental pathologic process consisting of a dynamic complex of histologically apparent cytologic changes, cellular infiltration, and mediator release that occurs in the affected blood vessels and adjacent tissues in response to an injury or abnormal stimulation caused by a physical, chemical, or biologic agent, including the local reactions and resulting morphologic changes; the destruction or removal of the injurious material; and the responses that lead to repair and healing.”

The five elements makes up the signature of  inflammation:  rubor, redness; calor, heat (or warmth); tumor swelling; and dolor, pain; a fifth sign, functio laesa, inhibited or lost function.   However, these indications may not be present at once.

Please click on to the following link for genetic association of autoimmune diseases (Cho Et al selected major association signals in autoimmune diseases) from Cho JH, Gregersen PK. N Engl J Med 2011;365:1612-1623.

Inflammatory diseases grouped under two classification: the immune system related due to  inflammatory disorders, such as both allergic reactions  and some myopathies, with many immune system disorders.  The examples of inflammatory disorders  include Acne vulgaris, asthma, autoimmune disorders, celiac disease, chronic prostatitis, glomerulonepritis, hypersensitivities, inflammatory bowel diseases, pelvic inflammatory diseases, reperfusion diseases, rheumatoid arthritis, sarcoidosis, transplant rejection, vasculitis, interstitial cyctitis, The second kind of inflammation are related to  non-immune diseases such as cancer, atherosclerosis, and ischaemic heart disease.

This seems simple yet at molecular physiology and gene activation levels this is a complex response as an innate immune response from body.  There can be acute lasting few days after exposure to bacterial pathogens, injured tissues or chronic inflammation continuing few months to years after unresolved acute responses such as non-degradable pathogens, viral infection, antigens or any  foreignmaterials, or autoimmune responses.

As the system responses arise from plasma fluid, blood vessels, blood plasma through vasciular changes, differentiation in plasma cascade systems like coagulation system, fibrinolysis, complement system and kinin system.  Some of the various mediators include bradykinin produced by kinin system, C3, C5, membrane attack system (endothelial cell activation or endothelial coagulation activation mechanism) created by the complement system; factor XII that can activate kinin, fibrinolysys and coagulation systems at the same time produced in liver; plasmin from fibrinolysis system to inactivate factor Xii and C3 formation, and thrombin of coagulation system with a reaction through protein activated receptor 1 (PAR1), which is a seven spanning membrane protein-GPCR.   This system is quite fragile and well regulated.  For example activation of inactive Factor XII by collagen, platelets, trauma such as cut, wound, surgery that results in basement membrane changes since it usually circulate in inactive form in plasma automatically initiates and alerts kinin, fibrinolysis and coagulation systems.

Furthermore, the changes reflected through receptors and create gene activation by cellular mediators to establish system wide unified mechanisms. These factors (such as IFN-gamma, IL-1, IL-8, prostaglandins, leukotrene B4,  nitric oxide, histamines,TNFa) target immune cells and redesign their responses, mast cells, macrophages, granulocytes, leukocytes, B cells, T cells) platelets, some neuron cells and endothelial cells.  Therefore, immune system can react with non-specific or specific mechanisms either for a short or a long term.

As a result, controlling of mechanisms in blood and prevention of angiogenesis answer to cure/treat many diseases  Description of angiogenesis is simply formation of new blood vessels without using or changing pre-existing capillaries.  This involves serial numbers of events play a central role during physiologic and pathologic processes such as normal tissue growth, such as in embryonic development, wound healing, and the menstrual cycle.  However this system requires three main elements:  oxygen, nutrients and getting rid of waste or end products.

Genome Wide Gene Association Studies, Genomics and Metabolomics, on the other hand, development of new technologies for diagnostics and non-invasive technologies provided better targeting systems.

In this token recent genomewide association studies showed a clear view on a disease mechanism, or that suggest a new diagnostic or therapeutic approach particularly these disorders are related to  genes within the major histocompatibility complex (MHC) that predisposes the most significant genetic effect.  Presumably, these genes are reflecting the immunoregulatory effects of the HLA molecules themselves. As a result, the working mechanism of pathological conditions are revisited or created new assumptions to develop new targets for diagnosis and treatments.

Even though B and T cells are reactive to initiate responses there are several level of mechanisms control the cell differentiation for designing rules during health or diseases. These regulators are in check for both T and B cells.  For example, during Type 1 diabetes there are presence of more limited defects in selection against reactivity with self-antigens like insulin, thus, T cell differentiation is in jeopardy.  In addition, B cells have many active checkpoints to modulate the immune responses like  pre-B cells in the bone marrow are highly autoreactive yet they prefer to stay  in naïve-B cell forms in the periphery through tyrosine phosphatase nonreceptor type 22 (PTPN22) along with many genes play a role in autoimmunity.  In a nut shell this is just peeling the first layer of the onion at the level of Mendelian Genetics.

There is a great work to be done but if one can harness the blood and immune responses many complex diseases patients may have a big relief and have a quality of life.  When we look at the picture 90% of main fluid of life, blood, carried by cardiovascular system with two main pumping mechanisms, lung with gas exchange and systemic with complex scavenger actions, collection of waste, distribution of nutrition and clean gases.  Yet, without lymphatic system body can’t make up the 100% fluid.  Therefore, 10% balance is completed by lymphatic system as a counter clockwise direction so that not only the fluid balance but also mass balance is  maintained. Finally, the immune system patches the  remaining mechanism by providing cellular support to protect the body because it contains 99% of white cells to fight against any kinds of invasion, attack, trauma.


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Larry H. Bernstein, MD, FCAP, reviewer and curator,-lysine-methylation,-and-activation-of-receptor-tyrosine-kinase

Lysine Methylation Promotes VEGFR-2 Activation and Angiogenesis

 Edward J. Hartsough1*, Rosana D. Meyer1*, Vipul Chitalia2, Yan Jiang3, Victor E. Marquez4, Irina V. Zhdanova5, Janice Weinberg6, Catherine E. Costello3, and Nader Rahimi1{dagger}
 1 Departments of Pathology and Ophthalmology, School of Medicine, Boston University Medical Campus, Boston, MA 02118, USA.
2 Harvard-MIT Division of Health Science and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
3 Department of Biochemistry and Center for Biomedical Mass Spectrometry, School of Medicine, Boston University Medical Campus, Boston, MA 02118, USA.
4 Chemical Biology Laboratory, National Cancer Institute at Frederick, Frederick, MD 21702, USA.
5 Department of Anatomy and Neurobiology, Boston University Medical Campus, Boston, MA 02118, USA.
6 School of Public Health, Boston University Medical Campus, Boston, MA 02118, USA.
Activation of vascular endothelial growth factor receptor-2 (VEGFR-2), an endothelial cell receptor tyrosine kinase,
  • promotes tumor angiogenesis and ocular neovascularization.
We report the methylation of VEGFR-2 at multiple Lys and Arg residues, including Lys1041,
  • a residue that is proximal to the activation loop of the kinase domain.
Methylation of VEGFR-2 was
  • independent of ligand binding and
  • was not regulated by ligand stimulation.
Methylation of Lys1041 enhanced tyrosine phosphorylation and kinase activity in response to ligands. Additionally, interfering with the methylation of VEGFR-2 by pharmacological inhibition or by site-directed mutagenesis revealed that
  • methylation of Lys1041 was required for VEGFR-2–mediated angiogenesis
    • in zebrafish and
    • tumor growth in mice.
We propose that methylation of Lys1041 promotes the activation of VEGFR-2 and that
  • similar posttranslational modification could also regulate the activity of other receptor tyrosine kinases.
{dagger} Corresponding author. E-mail:
Citation: E. J. Hartsough, R. D. Meyer, V. Chitalia, Y. Jiang, V. E. Marquez, I. V. Zhdanova, J. Weinberg, C. E. Costello, N. Rahimi, Lysine Methylation Promotes VEGFR-2 Activation and Angiogenesis. Sci. Signal. 6, ra104 (2013).

Phosphoproteomic Analysis Implicates the mTORC2-FoxO1 Axis in VEGF Signaling and Feedback Activation of Receptor Tyrosine Kinases

Guanglei Zhuang, Kebing Yu, Zhaoshi Jiang, Alicia Chung, Jenny Yao, Connie Ha, Karen Toy, Robert Soriano, Benjamin Haley, Elizabeth Blackwood, Deepak Sampath, Carlos Bais, Jennie R. Lill, and Napoleone Ferrara (16 April 2013){dagger}
Sci. Signal. 16 April 2013;  6 (271), ra25.
Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
* These authors contributed equally to this work.{dagger}
{dagger} Present address: Department of Pathology and Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA.
The vascular endothelial growth factor (VEGF) signaling pathway plays a pivotal role in normal development and
  • also represents a major therapeutic target for tumors and intraocular neovascular disorders.
The VEGF receptor tyrosine kinases promote angiogenesis by phosphorylating downstream proteins in endothelial cells. We applied a large-scale proteomic approach to define
  1. the VEGF-regulated phosphoproteome and
  2. its temporal dynamics in human umbilical vein endothelial cells and then
  3. used siRNA (small interfering RNA) screens to investigate the function of a subset of these phosphorylated proteins in VEGF responses.
The PI3K (phosphatidylinositol 3-kinase)–mTORC2 (mammalian target of rapamycin complex 2) axis emerged as central
  1. in activating VEGF-regulated phosphorylation and
  2. increasing endothelial cell viability
    • by suppressing the activity of the transcription factor FoxO1 (forkhead box protein O1),
    • an effect that limited cellular apoptosis and feedback activation of receptor tyrosine kinases.
This FoxO1-mediated feedback loop not only reduced the effectiveness of mTOR inhibitors at decreasing protein phosphorylation and cell survival
  • but also rendered cells more susceptible to PI3K inhibition.
Collectively, our study provides a global and dynamic view of VEGF-regulated phosphorylation events and
  • implicates the mTORC2-FoxO1 axis in VEGF receptor signaling and
  • reprogramming of receptor tyrosine kinases in human endothelial cells.
{ddagger} Corresponding author. E-mail:
Citation: G. Zhuang, K. Yu, Z. Jiang, A. Chung, J. Yao, C. Ha, K. Toy, R. Soriano, B. Haley, E. Blackwood, D. Sampath, C. Bais, J. R. Lill, N. Ferrara, Phosphoproteomic Analysis Implicates the mTORC2-FoxO1 Axis in VEGF Signaling and Feedback Activation of Receptor Tyrosine Kinases. Sci. Signal. 6, ra25 (2013).

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Stem Cell Therapy for Coronary Artery Disease (CAD)

Author and Curator: Larry H. Bernstein, MD, FCAP


Curator: Aviva Lev-Ari, PhD, RN


There is great interest and future promise for stem cell therapy in ischemic heart disease.  This is another report for the active work in cardiology with stem cell therapy by MA Gaballa and associates at University of Arizona.

Stem Cell Therapy for Coronary Heart Disease

Julia N. E. Sunkomat and Mohamed A. Gaballa

The University ofArizona Sarver Heart Center, Section of Cardiology, Tucson, Ar
Cardiovascular Drug Reviews 2003: 21(4): 327–342

Keywords: Angiogenesis — Cardiac therapy — Coronary heart disease — Heart failure — Myoblasts — Myocardial ischemia — Myocardial regenera­tion — Stem cells


Coronary artery disease (CAD) remains the leading cause of death in the Western world. The high impact of its main sequelae, acute myocardial infarction and congestive heart failure (CHF), on the quality of life of patients and the cost of health care drives the search for new therapies. The recent finding that

stem cells contribute to neovascularization and possibly improve cardiac function after myocardial infarction makes stem cell therapy the most highly active research area in cardiology. Although the concept of stem cell therapy may revolutionize heart failure treatment, several obstacles need to be ad­dressed. To name a few:

  1.  Which patient population should be considered for stem cell therapy?
  2.  What type of stem cell should be used?
  3.  What is the best route for cell de­livery?
  4.  What is the optimum number of cells that should be used to achieve functional effects?
  5.  Is stem cell therapy safer and more effective than conventional therapies?

The published studies vary significantly in design, making it difficult to draw conclusions on the efficacy of this treatment. For example, different models of

  1. ischemia,
  2. species of donors and recipients,
  3. techniques of cell delivery,
  4. cell types,
  5. cell numbers and
  6. timing of the experiments

have been used. However, these studies highlight the landmark concept that stem cell therapy may play a major role in treating cardiovascular diseases in the near future. It should be noted that stem cell therapy is not limited to the treatment of ischemic cardiac disease.

  • Non-ischemic cardiomyopathy,
  • peripheral vascular disease, and
  • aging may be treated by stem cells.

Stem cells could be used as vehicle for gene therapy and eliminate the use of viral vectors. Finally, stem cell therapy may be combined with phar­macological, surgical, and interventional therapy to improve outcome. Here we attempt a systematic overview of the science of stem cells and their effects when transplanted into ischemic myocardium.



Congestive heart failure (CHF) is the leading discharge diagnosis in patients over the age of 65 with estimates of $24 billion spent on health care in the US (1,11). The number one cause of CHF is coronary artery diseases (CAD). Coronary care units, reperfusion therapy (lytic and percutaneous coronary intervention) and medical therapy with anti-pla­telet agents, statins, ACE-inhibitors and â-adrenoceptor antagonists all significantly reduce morbidity and mortality of CAD and CHF (9), but it is very difficult to regenerate new viable myocardium and new blood vessels.

Identification of circulating endothelial progenitor cells in peripheral blood that incor­porated into foci of neovascularization in hindlimb ischemia (4) and the successful engraftment of embryonic stem cells into myocardium of adult dystrophic mice (31) intro­duced a new therapeutic strategy to the field of cardiovascular diseases: tissue regeneration. This approach is supported by the discovery of primitive cells of extracardiac origin in cardiac tissues after sex-mismatched transplants suggesting that an endogenous repair mechanism may exist in the heart (35,45,54). The number of recruited cells varied significantly from 0 (19) to 18% (54), but the natural course of ischemic cardiomyopathy implies that cell recruitment for tissue repair in most cases is insufficient to prevent heart failure. Therefore, investigational efforts are geared towards

  • augmenting the number of multipotent stem cells and endothelial and myocardial progenitor cells at the site of ischemia to induce clinically significant angiogenesis and potentially myogenesis.

Stem and Progenitor Cells

Stem cells are defined by their ability to give rise to identical stem cells and progenitor cells that continue to differentiate into a specific tissue cell phenotype (23,33). The po­tential of mammalian stem cells varies with stage of development and age (Table 1).

In mammals, the fertilized oocyte and blastomere cells of embryos of the two to eight cell stage can generate a complete organism when implanted into the uterus; they are called totipotent stem cells. After the blastocyst stage, embryonic stem cells retain the ability to differentiate into all cell types, but

  • cannot generate a complete organism and thus are denoted pluripotent stem cells.

Other examples of pluripotent stem cells are embryo­nic germ cells that are derived from the gonadal ridge of aborted embryos and embryonic carcinoma cells that are found in gonadal tumors (teratocarcinomas) (23,33). Both these cell types can also differentiate into cells of all three germ layers, but are not as well inves­tigated as embryonic stem cells.

It is well established that embryonic stem cells can differentiate into cardiomyocytes (7,10,13,14,31,37,76), endothelial cells (55), and smooth muscle cells (5,22,78) in vitro, but it is unclear whether

  • pure populations of embryonic stem cell-derived cardiomyocytes can integrate and function appropriately in the heart after transplantation.
  • one study reported arrhythmogenic potential of embryonic stem cell-derived cardiomyocytes in vitro (80).

Adult somatic stem cells are cells that have already committed to one of the three germ layers: endoderm, ectoderm, or mesoderm (76). While embryonic stem cells are defined by their origin (the inner cell mass of the blastocyst), the origin of adult stem cells in mature tissues is still unknown. The primary role of adult stem cells in a living organism is thought to be maintaining and repairing the tissue in which they reside. They are the source of more identical stem cells and cells with a progressively more distinct phenotype of specialized tissue cells (progenitor and precursor cells) (Fig. 1). Until recently adult stem cells were thought to be lineage-specific, meaning that they can only differentiate into the cell-type of their original tissue. This concept has now been challenged with the discovery of multipotent stem and progenitor cells (26, 50, 51).

The presence of multipotent stem and progenitor cells in adult mammals has vast im­plications on the availability of stem cells to research and clinical medicine. Recent publi­cations, however, have questioned whether the adaptation of a phenotype in those dogma-challenging studies is really a result of trans-differentiation or rather a result of cell and nuclear fusion (60,68,75,79). Spontaneous fusion between mammalian cells was first re­ported in 1961 (8), but how frequently fusion occurs and whether it occurs in vivo is not clear.

The bone marrow is a known source of stem cells. Hematopoietic stem cells are fre­quently used in the field of hematology. Surface receptors are used to differentiate hematopoietic stem and progenitor cells from mature cells. For example, virtually all

  • hematopoietic stem and progenitor cells express the CD34+ glycoprotein antigen on their cell membrane (73),

though a small proportion of primitive cells have been shown to be CD34 negative (58).

The function of the CD34+ receptor is not yet fully understood. It has been suggested that it may act as a regulator of hematopoietic cell adhesion in the bone marrow microenvironment. It also appears to be involved in the maintenance of the hematopoietic stem/progenitor cell phenotype and function (16,21). The frequency of immature CD34+ cells in peripheral circulation diminishes with age.

  • It is the highest (up to 11%) in utero (69) and decreases to 1% of nucleated cells in term cord blood (63).
  • This equals the per­centage of CD34+ cells in adult bone marrow.
  • The number of circulating stem cells in adult peripheral blood is even lower at 0.1% of nucleated cells.

Since hematopoietic stem cells have been identified as endothelial progenitor cells (29,30,32) their low density in adult bone marrow and blood could explain the inadequacy of endogenous recruitment of cells to injured organs such as an ischemic heart. The bone marrow is also home to another stem cell population the so-called mesenchymal stem cells. These may constitute a subset of the bone marrow stromal cells (2,43). Bone marrow stromal cells are a mixed cell popu­lation that generates

  1. bone,
  2. cartilage,
  3. fat,
  4. connective tissue, and
  5. reticular network that sup­ports cell formation (23).

Mesenchymal stem cells have been described as multipotent (51,52) and as a source of myocardial progenitor cells (41,59). They are, however, much less defined than the hematopoietic stem cells and a characteristic antigen constellation has not yet been identified (44).

Another example of an adult tissue containing stem cells is the skeletal muscle. The cells responsible for renewal and growth of the skeletal muscle are called satellite cells or myoblasts and are located between the sarcolemma and the basal lamina of the muscle fiber (5). Since skeletal muscle and cardiac muscle share similar characteristics such as they both are striated muscle cells, satellite cells are considered good candidates for the repair of damaged myocardium and have been extensively studied (20,25,38–40,48,56, 64–67). Myoblasts are particularly attractive, because they can be autotransplanted, so that issues of donor availability, ethics, tumorigenesis and immunological compatibility can be avoided. They also have been shown to have a high growth potential in vitro and a strong resistance to ischemia in vivo (20). On the down side

  • they may have more arrhythmogenic potential when transplanted into myocardium than bone marrow or peripheral blood de­rived stem cells and progenitor cells (40).

Isolation of Cells Prior to Transplantation

Hematopoietic stem and progenitor cells are commonly identified by the expression of a profile of surface receptors (cell antigens). For example, human hematopoietic stem cells are defined as CD34+/CD59+/Thy-1+/CD38low//c-kit/low/lin, while mouse hema-topoietic stem cells are defined as CD34low//Sca-1+/Thy-1+/low/CD38+/c-kit+/lin (23). Additional cell surface receptors have been identified as markers for subgroups of hema-topoietic stem cells with the ability to differentiate into non-hematopoetic tissues, such as endothelial cells (57,78). These can be specifically targeted by isolation methods that use the receptors for cell selection (positive selection with antibody coated magnetic beads or fluorescence-activated cell sorting, FACS). Other stem cell populations are identified by their behavior in cell culture (mesenchymal stem cells) or dye exclusion (SP cells). Finally, embryonic stem cells are isolated from the inner cell mass of the blastocyst and skeletal myoblasts are mechanically and enzymatically dissociated from an easily acces­sible skeletal muscle and expanded in cell culture.

FIG. 1. Maturation process of adult stem cells: with acquisition of a certain phenotype the cell gradually loses its self-renewal capability.  (unable to transfer)


j.1527-3466.2003.tb00125.x  fig stem cell

FIG. 2. Intramyocardial injection:

the cells are injected directly into the myocardium through the epicardium. Usually a thoracotomy or sternotomy is required. Transendocardial injection: access can be gained from the ar­terial vasculature. Cells are injected through the endocardium into the myocardium, ideally after identifying the ischemic myocardium by perfusion studies and/or electromechanical mapping. Intracoronary injection: the coronary artery is accessed from the arterial vasculature. Stem cells are injected into the lumen of the coronary artery. Proximal washout is prevented by inflation of a balloon. Cells are then distributed through the capillary system. They eventually cross the endothelium and migrate towards ischemic areas.

The intracoronary delivery of stem cells (Fig. 2) and distribution through the coronary system has also been explored (6,62,74). This approach was pioneered by Robinson et al. (56), who demonstrated successful engraftment within the coronary distribution after intracoronary delivery of genetically labeled skeletal myoblasts. The risk of intracoronary injection is comparable to that of a coronary angiogram and percutaneous transluminal coronary angioplasty (PTCA) (62), which are safe and clinically well established.


Dif­ferentiation into cardiomyocytes was observed after transplantation of embryonic stem cells, mesenchymal stem cells, lin/c-kit+ and SP cells. The induction of angiogenesis was observed after transplantation of embryonic stem cells, mesenchymal stem cells, bone marrow-derived mononuclear cells, circulating endothelial progenitor cells, SP cells and lin/c-kit+ cells.

The use of embryonic stem cells in ischemia was examined in two studies (42,43). These studies demonstrated that mice embryonic stem cells transplanted into rat myo­cardium exhibited cardiomyocyte phenotype at 6 weeks after transplantation. In addition, generation of myocardium and angiogenesis were observed at 32 weeks after allogenic transplantation in rats. In these two studies no arrhythmias or cardiac tumors were reported.

Several studies have shown retardation of LV remodeling and improvement of cardiac function after administration of bone marrow-derived mononuclear cells. For example, decreases in infarct size, and increase in ejection fraction (EF), and left ventricular (LV) time rate change of pressure (dP/dtmax) were observed after direct injection of bone marrow-derived mononuclear cells 60 min after ischemia in swine (28). In humans, intra-coronary delivery and transendocardial injection of mononuclear cells leads to a decrease in LV dimensions and improvement of cardiac function and perfusion (49,62). A decrease in end systolic volume (ESV) and an increase in EF as well as regional wall motion were observed following intracoronary administration of CD34+/CD45+ human circulating en­dothelial cells (6). Injection of circulating human CD34+/CD117+ cells into infarcted rat myocardium induced neoangiogenesis and improved cardiac function (32). This study suggests that the improvement in LV remodeling after infarction appears to be in part me­diated by a decrease in apoptosis within the noninfarcted myocardium. Two other studies reported increased fractional shortening, improved regional wall motion and decreased left ventricular dimensions after transplantation of human CD34+ cells (29,30). Improved global left ventricular function and infarct perfusion was demonstrated after intramyo-cardial injection of autologous endothelial progenitor cells in humans (61).


The idea of replacing damaged myocardium by healthy cardiac tissue is exciting and has received much attention in the medical field and the media. Therefore, it is important for the scientist to know what is established and what is based on premature conclusions. Currently, there are data from animal studies and human trials (Table 2). However, some of these data are not very concrete. For example,

  • many animal studies do not report the level of achieved neoangiogenesis and/or regeneration of myocardium.
  • In studies where the numbers of neovessels and new cardiomyocytes are specified, these numbers are often very low.

While these experiments confirm the concept that bone marrow and peripheral blood-derived stem and progenitor cells can differentiate into cardiomyocytes and endo­thelial cells when transplanted into ischemic myocardium, they also raise the question how effective this treatment is.

The results of the clinical trials that have been conducted are encouraging, but they need to be interpreted with caution. The common endpoints of these studies include left ventricular dimensions, perfusion, wall motion and hemodynamic function. While all studies report improvement after mononuclear cell, myoblast or endothelial progenitor cell transplantation, it is difficult to separate the effects of stem cell transplantation from the effects of the state-of-the art medical care that the patients typically received.


While the majority of studies demonstrate neoangiogenesis and some studies also show regeneration of myocardium after stem/progenitor cell transplantation, it remains unclear whether the currently achieved level of tissue regeneration is sufficient to affect clinical outcome. Long-term follow-up of patients that received stem/progenitor cells in clinical trials will provide important information on the potential risks of neoplasm and arrhythmias and, therefore, safety of this treatment. Ultimately, postmortem histological confirmation of scar tissue repair by transplanted cells and randomized placebo control trials with long-term follow-up are required to prove efficacy of this treatment.


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6. Assmus B, Schaechinger V, Teupe C, et al. Transplantation of progenitor cells and regeneration en­hancement in acute myocardial infarction (TOPCARE-AMI). Circulation 2002;106:r53–r61.

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