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New Mutant KRAS Inhibitors Are Showing Promise in Cancer Clinical Trials: Hope For the Once ‘Undruggable’ Target

Curator: Stephen J. Williams, Ph.D.

The November 1st issue of Science highlights a series of findings which give cancer researchers some hope in finally winning a thirty year war with the discovery of drugs that target KRAS, one of the most commonly mutated oncogenes  (25% of cancers), and thought to be a major driver of tumorigenesis. Once considered an undruggable target, mainly because of the smooth surface with no obvious pockets to fit a drug in, as well as the plethora of failed attempts to develop such an inhibitor, new findings with recently developed candidates, highlighted in this article and other curated within, are finally giving hope to researchers and oncologists who have been hoping for a clinically successful inhibitor of this once considered elusive target.

 

For a great review on development of G12C KRas inhibitors please see Dr. Hobb’s and Channing Der’s review in Cell Selective Targeting of the KRAS G12C Mutant: Kicking KRAS When It’s Down

Figure 1Mechanism of Action of ARS853 showing that the inhibitors may not need bind to the active conformation of KRAS for efficacy

Abstract: Two recent studies evaluated a small molecule that specifically binds to and inactivates the KRAS G12C mutant. The new findings argue that the perception that mutant KRAS is persistently frozen in its active GTP-bound form may not be accurate.

 

Although the development of the KRASG12C-specific inhibitor, compound 12 (Ostrem et al., 2013), was groundbreaking, subsequent studies found that the potency of compound 12 in cellular assays was limited (Lito et al., 2016, Patricelli et al., 2016). A search for more-effective analogs led to the development of ARS853 (Patricelli et al., 2016), which exhibited a 600-fold increase of its reaction rate in vitro over compound 12 and cellular activities in the low micromolar range.

 

A Summary and more in-depth curation of the Science article is given below:

After decades, progress against an ‘undruggable’ cancer target

Summary

Cancer researchers are making progress toward a goal that has eluded them for more than 30 years: shrinking tumors by shutting off a protein called KRAS that drives growth in many cancer types. A new type of drug aimed at KRAS made tumors disappear in mice and shrank tumors in lung cancer patients, two companies report in papers published this week. It’s not yet clear whether the drugs will extend patients’ lives, but the results are generating a wave of excitement. And one company, Amgen, reports an unexpected bonus: Its drug also appears to stimulate the immune system to attack tumors, suggesting it could be even more powerful if paired with widely available immunotherapy treatments.

Jocelyn Kaiser. After decades, progress against an ‘undruggable’ cancer target. Science  01 Nov 2019: Vol. 366, Issue 6465, pp. 561 DOI: 10.1126/science.366.6465.561

The article highlights the development of three inhibitors: by Wellspring Biosciences, Amgen, and Mirati Therapeutics.

Wellspring BioSciences

 

In 2013, Dr. Kevan Shokat’s lab at UCSF discovered a small molecule that could fit in the groove of the KRAS mutant G12C.  The G12C as well as the G12D is a common mutation found in KRAS in cancers. KRAS p.G12C mutations predominate in NSCLC comprising 11%–16% of lung adenocarcinomas (45%–50% of mutant KRAS is p.G12C) (Campbell et al., 2016; Jordan et al., 2017), as well as 1%–4% of pancreatic and colorectal adenocarcinomas, respectively (Bailey et al., 2016; Giannakis et al., 2016).  This inhibitor was effective in shrinking, in mouse studies conducted by Wellspring Biosciences,  implanted tumors containing this mutant KRAS.

 

See Wellspring’s news releases below:

March, 2016 – Publication – Selective Inhibition of Oncogenic KRAS Output with Small Molecules Targeting the Inactive State

February, 2016 – Publication – Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism

Amgen

 

Amgen press release on AMG510 Clinical Trial at ASCO 2019

 

THOUSAND OAKS, Calif., June 3, 2019 /PRNewswire/ — Amgen (NASDAQ: AMGN) today announced the first clinical results from a Phase 1 study evaluating investigational AMG 510, the first KRASG12C inhibitor to reach the clinical stage. In the trial, there were no dose-limiting toxicities at tested dose levels. AMG 510 showed anti-tumor activity when administered as a monotherapy in patients with locally-advanced or metastatic KRASG12C mutant solid tumors. These data are being presented during an oral session at the 55th Annual Meeting of the American Society of Clinical Oncology (ASCO) in Chicago.

“KRAS has been a target of active exploration in cancer research since it was identified as one of the first oncogenes more than 30 years ago, but it remained undruggable due to a lack of traditional small molecule binding pockets on the protein. AMG 510 seeks to crack the KRAS code by exploiting a previously hidden groove on the protein surface,” said David M. Reese, M.D., executive vice president of Research and Development at Amgen. “By irreversibly binding to cysteine 12 on the mutated KRAS protein, AMG 510 is designed to lock it into an inactive state. With high selectivity for KRASG12C, we believe investigational AMG 510 has high potential as both a monotherapy and in combination with other targeted and immune therapies.”

The Phase 1, first-in-human, open-label multicenter study enrolled 35 patients with various tumor types (14 non-small cell lung cancer [NSCLC], 19 colorectal cancer [CRC] and two other). Eligible patients were heavily pretreated with at least two or more prior lines of treatment, consistent with their tumor type and stage of disease. 

Canon, J., Rex, K., Saiki, A.Y. et al. The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity. Nature 575, 217–223 (2019) doi:10.1038/s41586-019-1694-1

Besides blocking tumor growth, AMG510 appears to stimulate T cells to attack the tumor, thus potentially supplying a two pronged attack to the tumor, inhibiting oncogenic RAS and stimulating anti-tumor immunity.

 

Mirati Therapeutics

 

Mirati’s G12C KRAS inhibitor (MRTX849) is being investigated in a variety of solid malignancies containing the KRAS mutation.

 

For recent publication on results in lung cancer see Patricelli M.P., et al. Cancer Discov. 2016; (Published online January 6, 2016)

For more information on Mirati’s KRAS G12C inhibitor see https://www.mirati.com/pipeline/kras-g12c/

 

KRAS G12C Inhibitor (MRTX849)

Study 849-001 – Phase 1b/2 of single agent MRTX849 for solid tumors with KRAS G12C mutation

Phase 1b/2 clinical trial of single agent MRTX849 in patients with advanced solid tumors that have a KRAS G12C mutation.

See details for this study at clinicaltrials.gov

 

Additional References:

Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism.

Lito P et al. Science. (2016)

Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor.

Janes MR et al. Cell. (2018)

Potent and Selective Covalent Quinazoline Inhibitors of KRAS G12C.

Zeng M et al. Cell Chem Biol. (2017)

Campbell, J.D., Alexandrov, A., Kim, J., Wala, J., Berger, A.H., Pedamallu, C.S., Shukla, S.A., Guo, G., Brooks, A.N., Murray, B.A., et al.; Cancer Genome Atlas Research Network (2016). Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas. Nat. Genet.48, 607–616

Jordan, E.J., Kim, H.R., Arcila, M.E., Barron, D., Chakravarty, D., Gao, J., Chang, M.T., Ni, A., Kundra, R., Jonsson, P., et al. (2017). Prospective comprehensive molecular characterization of lung adenocarcinomas for efficient patient matching to approved and emerging therapies. Cancer Discov. 7, 596–609.

Bailey, P., Chang, D.K., Nones, K., Johns, A.L., Patch, A.M., Gingras, M.C., Miller, D.K., Christ, A.N., Bruxner, T.J., Quinn, M.C., et al.; Australian Pancreatic Cancer Genome Initiative (2016). Genomic analyses identify molecular subtypes of pancreatic cancer. Nature 531, 47–52.

Giannakis, M., Mu, X.J., Shukla, S.A., Qian, Z.R., Cohen, O., Nishihara, R., Bahl, S., Cao, Y., Amin-Mansour, A., Yamauchi, M., et al. (2016). Genomic correlates of immune-cell infiltrates in colorectal carcinoma. Cell Rep. 15, 857–865.

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

One of the most contagious diseases known to humankind, measles killed an average of 2.6 million people each year before a vaccine was developed, according to the World Health Organization. Widespread vaccination has slashed the death toll. However, lack of access to vaccination and refusal to get vaccinated means measles still infects more than 7 million people and kills more than 100,000 each year worldwide as reported by WHO. The cases are on the rise, tripling in early 2019 and some experience well-known long-term consequences, including brain damage and vision and hearing loss. Previous epidemiological research into immune amnesia suggests that death rates attributed to measles could be even higher, accounting for as much as 50 percent of all childhood mortality.

 

Over the last decade, evidence has mounted that the measles vaccine protects in two ways. It prevents the well-known acute illness with spots and fever and also appears to protect from other infections over the long term by giving general boost to the immune system. The measles virus can impair the body’s immune memory, causing so-called immune amnesia. By protecting against measles infection, the vaccine prevents the body from losing or “forgetting” its immune memory and preserves its resistance to other infections. Researchers showed that the measles virus wipes out 11% to 73% of the different antibodies that protect against viral and bacterial strains a person was previously immune to like from influenza to herpes virus to bacteria that cause pneumonia and skin infections.

 

This study at Harvard Medical School and their collaborators is the first to measure the immune damage caused by the virus and underscores the value of preventing measles infection through vaccination. The discovery that measles depletes people’s antibody repertoires, partially obliterating immune memory to most previously encountered pathogens, supports the immune amnesia hypothesis. It was found that those who survive measles gradually regain their previous immunity to other viruses and bacteria as they get re-exposed to them. But because this process may take months to years, people remain vulnerable in the meantime to serious complications of those infections and thus booster shots of routine vaccines may be required.

 

VirScan detects antiviral and antibacterial antibodies in the blood that result from current or past encounters with viruses and bacteria, giving an overall snapshot of the immune system. Researchers gathered blood samples from unvaccinated children during a 2013 measles outbreak in the Netherlands and used VirScan to measure antibodies before and two months after infection in 77 children who’d contracted the disease. The researchers also compared the measurements to those of 115 uninfected children and adults. Researchers found a striking drop in antibodies from other pathogens in the measles-infected children that clearly suggested a direct effect on the immune system resembling measles-induced immune amnesia.

 

Further tests revealed that severe measles infection reduced people’s overall immunity more than mild infection. This could be particularly problematic for certain categories of children and adults, the researchers said. The present study observed the effects in previously healthy children only. But, measles is known to hit malnourished children much harder, the degree of immune amnesia and its effects could be even more severe in less healthy populations. Inoculation with the MMR (measles, mumps, rubella) vaccine did not impair children’s overall immunity. The results align with decades of research. Ensuring widespread vaccination against measles would not only help prevent the expected 120,000 deaths that will be directly attributed to measles this year alone, but could also avert potentially hundreds of thousands of additional deaths attributable to the lasting damage to the immune system.

 

References:

 

https://hms.harvard.edu/news/inside-immune-amnesia?utm_source=Silverpop

 

https://science.sciencemag.org/content/366/6465/599

 

www.who.int/immunization/newsroom/measles-data-2019/en/

 

https://www.ncbi.nlm.nih.gov/pubmed/20636817

 

https://www.ncbi.nlm.nih.gov/pubmed/27157064

 

https://www.ncbi.nlm.nih.gov/pubmed/30797735

 

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Nano-guided cell networks as conveyors of molecular communication

Nature Communications
6,
Article number:
8500
doi:10.1038/ncomms9500
Received
07 March 2015
Accepted
28 August 2015
Published
12 October 2015

Abstract

Advances in nanotechnology have provided unprecedented physical means to sample molecular space. Living cells provide additional capability in that they identify molecules within complex environments and actuate function. We have merged cells with nanotechnology for an integrated molecular processing network. Here we show that an engineered cell consortium autonomously generates feedback to chemical cues. Moreover, abiotic components are readily assembled onto cells, enabling amplified and ‘binned’ responses. Specifically, engineered cell populations are triggered by a quorum sensing (QS) signal molecule, autoinducer-2, to express surface-displayed fusions consisting of a fluorescent marker and an affinity peptide. The latter provides means for attaching magnetic nanoparticles to fluorescently activated subpopulations for coalescence into colour-indexed output. The resultant nano-guided cell network assesses QS activity and conveys molecular information as a ‘bio-litmus’ in a manner read by simple optical means.

At a glance

Figures

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left

  1. Nano-guided cell networks for processing molecular information.
    Figure 1
  2. Cells express functional, interchangeable protein components indicating both fluorescence and ability for streptavidin-linked surface coupling.
    Figure 2
  3. Cells equipped with magnetic nanoparticles (mNPs) via streptavidin-mediated interaction with surface-expressed proteins.
    Figure 3
  4. Affinity-based probing for functional analysis of AI-2-induced protein expression.
    Figure 4
  5. Single and multi-population cell responses to autoinducer-2.
    Figure 5
  6. Binning molecular information through cell-based parallel processing and magnetically focusing fluorescence into collective consensus output.
    Figure 6
  7. Extension of nano-guided cell networks for hypothetical regulatory structures.
    Figure 7

right

Introduction

It has become increasingly apparent that a wealth of molecular information exists, which, when appropriately accessed, can provide feedback on biological systems, their componentry and their function. Thus, there is a developing niche that transcends length scales to concurrently recognize molecular detail and at the same time provide understanding of the overall system1, 2. An emerging scheme is to develop nano- to micro-scaled tools that intimately engage with biological systems through monitoring and interacting at the molecular level, with synthetic biology being one such tool3, 4, 5, 6, 7.

While synthetic biology is often viewed as an innovative means for ‘green’ product synthesis through the genetic rearrangement of cells, their biosynthetic capabilities and their regulatory networks can instead be tuned for executive function8, 9, 10. That is, cells can be rewired to survey molecular space3, 11, 12 as they have sophisticated capabilities to recognize, amplify and transduce chemical information13. Further, they provide a means to connect biological systems with traditional microelectronic devices and in doing so present a potential interface between chemically based biomolecular processing and conventional vectors of information flow, such as electrons and photons14, 15, 16. Specifically, through engineered design, cell-based molecular processing can be further coupled to enable external abiotic responses. Cells, then, represent a versatile means for mediating the molecular ‘signatures’ common in complex environments, or in other words, they are conveyors of molecular communication17, 18, 19.

Further, beyond clonal cell-based sensors, there is an emerging concept of population engineering to establish microorganisms in deliberate networks that enable enriched system identification through a combination of distinctive yet coexistent behaviours, including, perhaps, competitive or cooperative features8, 20, 21, 22, 23, 24, 25. We posit the use of cell populations assembled in parallel¸ where multiple microbes with distinct molecular recognition capabilities work congruently. An advantage is that populations, as opposed to few cells, can facilitate thorough sampling since the presence of many cells increases their spatial breadth and per-cell data contributions (Fig. 1a). Each cellular unit undergoes independent decision-making and contributes a datum to its entire constituency. The prevalence of data provided within the population, then, substantiates a collective output by the system based on the molecular landscape. As follows in a multi-population system, molecular input thus influences the outcomes of each population, and elicits plural responses when the molecular input ranges overlap the ranges of the sensing populations21, which can define classification boundaries (Fig. 1a). Cell-mediated classification was posited in silico by Didovyk et al.21, where reporter libraries with randomized sensitivities to a molecular cue elicit concentration-dependent fluorescent patterns and these are elucidated by population screening . In the present construct, multiple populations enable multiplexed analysis, resulting, here, in a response gradation that is designed to index the molecular input ‘signature’. Consequently, the fed-back information becomes transfigured beyond a dose-dependent cell-by-cell analysis. That is, the output is predicated by the comparison between the populations rather than accumulation of response within a total population.

Figure 1: Nano-guided cell networks for processing molecular information.

Nano-guided cell networks for processing molecular information.

(a) Biotic (multicellular) processing is facilitated by cell recognition, signal transduction and genetic response. The genetically encoded response reflects the identity and prevalence of the target molecule(s). Biotic processing includes both increased cell number of responders and their genetically tuned response patterns. (b) Abiotic processing, used in conjunction with biotic processing, adds dimensionality to cell-based output by modifying through a physical stimulus (in our example, magnetic focusing). (c) Schematic of a cell population and nanomaterial-based network comprising both biotic (green/red axis) and abiotic (black axis) processing mechanisms. This conceptual system interprets molecular information by intercepting diverse molecular inputs, processes them autonomously through independent cell units within the system and refines output to include positive responders that are viewed via orthogonal means (visual classification). The system’s hierarchical structure allows molecular information to be refined into categorized collective outputs.

With population engineering as a premise for enriched molecular information processing, we engineered cell species, each to achieve an appropriate output through genetic means. There is conceptual basis for incorporation into networks, such as through mobile surveillance and position-based information relay26, 27. Hence, it is conceivable that, in addition to autonomous molecular recognition and processing afforded by synthetic biology, the use of physical stimuli to enable cell response could confer similar networking properties28, 29. For example, the complete information-processing ‘repertoire’ can be expanded beyond specific cell responses by the integration of external stimuli that serve to collate cell populations30. Specifically, we envision integration of nanomaterials that enable co-responses to molecular inputs, such that cell populations employ traditional reporting functions, that is, fluorescence marker expression, as well as responses that enable additional processing via the integration of stimuli-responsive abiotic materials (Fig. 1b).

In our example, cells are engineered to respond by permitting the attachment of magnetic nanoparticles (mNPs), such that each fluorescent cell becomes receptive to a magnetic field. Thus, the combination of cell-nanoparticle structures provides further dimensionality for the conveyance of molecular information (via magnetic stimulation). That is, without magnetic collation the fully distributed system would harbour diffuse responses; a magnetically stimulated system results in acute output due to a filtering and focusing effect (Fig. 1b)31, 32, allowing binned information to be readily, and fluorescently, conveyed.

The detection and interpretation of signalling molecules in our example is based on a microbial communication process known as quorum sensing (QS). The molecules, autoinducers (AIs), are secreted and perceived within a microbial community; once accumulated, the AI level indicates that the population size has reached a ‘quorum’33, 34. By surpassing a threshold concentration, the AI signalling coordinates population-wide phenotypic changes35. We have designed a QS information processor that utilizes two cell populations to independently interrogate natural microbial communities and generate information about QS activity by accessing AI-2 (ref. 36). Each cell population becomes ‘activated’ in response to a characteristic AI-2 level by expressing a fluorescent marker and a streptavidin-binding peptide (SBP) on the outer membrane38. SBP provides a means for collating data by binding mNPs that are introduced into the community. Using a post-processing magnetic sweep, the system as a whole interprets a molecular landscape and refines output into colour-categorized, or ‘binned,’ states (no fluorescence, red, or red and green) through (1) parallel population processing and (2) acute focusing (Fig. 1c).

The use of engineered cells as data-acquiring units and selectively equipping each with functional nanomaterials to form a redistributable processing system merges two paradigms: decentralized, active probing at a molecular scale and self-organization of units through structured dependencies on stimuli42. The population-based system overall contributes categorized feedback about a biological environment.

Results

Surface expression of SBP and fluorescent protein fusions

First, we established expression of a fusion protein consisting of a fluorescent marker (enhanced green fluorescent protein (eGFP) and variants) and SBP. Importantly, for SBP to function as a coupling agent between cells and mNPs, we used AIDAc (kindly shared by J. Larssen)40 to export the chimeric protein to Escherichia coli’s outer surface. Translocation to a cell’s surface utilizes a signal peptide (for inner membrane translocation) and AIDAc as an outer membrane autotransporter pore38, 39, 40, 41, with the passenger protein linked to each. In Fig. 2a, we depict expression of three different constructs using Venus, eGFP and mCherry for optical transmission, and the AIDAc translocator domain for surface localization. These constructs are mapped inSupplementary Fig. 1. After induction with isopropyl B-D-1-thiogalactopyranoside (IPTG), cultures were probed for surface expression of the SBP portion of the tagged fluorescent protein. Cells were incubated with fluorescently labelled streptavidin; the fluorophore of the streptavidin probe was orthogonal to the expressed fluorescent protein. The multiple fluorescence emissions were analysed by confocal microscopy without spectral overlap. The fraction of cells (fc) that exhibit colocalized fluorescent protein and the fluorescently-labeled streptavidin is reported in Fig. 2b, showing that SBP–Venus cells bound streptavidin at a slightly lower frequency than SBP–mCherry and SBP–eGFP, which exhibited statistically similar fractions (fc=0.7).

Figure 2: Cells express functional, interchangeable protein components indicating both fluorescence and ability for streptavidin-linked surface coupling.

Cells express functional, interchangeable protein components indicating both fluorescence and ability for streptavidin-linked surface coupling.

(a) A T7 cassette was used to express chimeric proteins consisting of a membrane autotransporter domain (AIDAc), one of several fluorescent proteins and a streptavidin-binding peptide (SBP). Fluorophore-tagged streptavidin (SA) was used to bind SBP. (b) Of cells expressing fluorescent proteins (FP), those also marked by SBP coupling are represented as a ‘colocalized fraction (fc),’ plotted with image analysis-based s.d. of at least five replicates. The asterisk ‘*’ denotes fc that +SBP–eGFP and+SBP–mCherry are statistically equivalent (fc~0.7) by t-test and greater than +SBP–Venus cells. (c) Composite images show cell fluorescence (Column I) from the fluorescent protein (FP); labelled streptavidin using orthogonal filter sets (Column II); and an overlay of both (Column III). Arrows indicate representative cells with strong colocalization. Plotted in Column IV are the fluorescence mean grey values (y-axis) from a representative horizontal slice of the composite image (x-axis). Vertical bars displayed between Columns III and IV identify the position of each analysed slice. Arrows indicate peaks that match the highlighted cells in Column III. fc values are noted. Fluorophores with non-overlapping spectra were paired. Row 1, Venus expression (yellow-green) was paired with Dylight405-labelled SA (blue). Row 2, eGFP expression (green) was paired with Alexafluor594-labeled SA (red). Row 3, mCherry expression (red) was paired with Alexafluor488-labeled SA (green). Scale bar in lower left, 50μm.

That is, microscopy results related to the colocalization analysis are depicted for pairings of Venus and blue-streptavidin (SA), eGFP and red-SA, and mCherry and green-SA (Fig. 2c). Strong signals were observed in both filter sets (the fluorescent protein (Column I) and the labelled streptavidin (Column II)). Overlaying each image reveals colocalization, as indicated in Column III, where arrows point to examples of strong colocalization. In addition, Column IV plots fluorescence intensities across horizontal sections of the images, where cells that exhibit colocalized fluorescence are indicated by superimposed peaks. For +pSBP–Venus cells, those with both a blue and yellow signal are observed as pale blue–violet in the overlaid image. Cells with +pSBP–eGFP and +pSBP–mCherry and labelled streptavidin emit both green and red signals; their colocalization appears yellow. Controls shown in Supplementary Fig. 2, verify that fluorescent streptavidin (all colours) has specificity for only SBP-expressing cells over negative controls. Colocalization indicates that not only are both components of the fusion, SBP and the fluorescent protein, expressed, but that SBP is accessible to bind streptavidin on the cell’s surface. This is the first use of AIDAc for cell surface anchoring of fluorescent proteins, each having been functionalized with an affinity peptide.

Cell hybridization via mNPs

Given that expression of a fluorescent protein tagged with SBP enabled external binding of streptavidin, we employed this interaction for fastening streptavidin-functionalized materials directly to the cell surface. We chose streptavidin-conjugated mNPs, 100nm in diameter (an order of magnitude smaller than a cell), for binding to a cell surface (Fig. 3a) to impart the abiotic magnetic properties. Scanning electron microscopy (SEM) was used to observe surface interaction between cell surface-expressing SBP and streptavidin-functionalized mNPs. Supplementary Fig. 3a,bshows electron micrographs of E. coli cells (dimensions 1.5–2μm in length) and the mNPs (~100nm in diameter). The SEM image in Fig. 3b, shows a magnetically isolated SBP-expressing cell with streptavidin-mNPs. The sample was prepared by mixing SBP-expressing cells with streptavidin-mNPs, then collecting or ‘focusing’ into a magnetized pellet via magnetic field, then separating from unbound cells in the supernatant. The cells were then washed and resuspended. In Fig. 3b, clusters of surface-bound mNPs are observed. In addition, the elemental composition was analysed with energy-dispersive X-ray spectroscopy, shown in Fig. 3c by an element map superimposed with carbon (red) and iron (green). While the cell appears to be of a uniform carbon composition, the particles localized at the cell surface (highlighted with arrows) were found having a strong iron composition; thus, elemental analysis confirmed particle identity as iron oxide mNPs. Additional characterization of magnetic functionality, including detailed SEM and fluorescent microscopic analysis prior to and after application of magnetic fields, is described in theSupplementary Information (Supplementary Fig. 3).

Figure 3: Cells equipped with magnetic nanoparticles (mNPs) via streptavidin-mediated interaction with surface-expressed proteins.

Cells equipped with magnetic nanoparticles (mNPs) via streptavidin-mediated interaction with surface-expressed proteins.

(a) Cell surface binding of streptavidin-conjugated magnetic nanoparticles occurs via surface-anchored streptavidin-binding peptide (SBP). The fusion of T7-expressed SBP-fluorescent protein (FP)-AIDAc enables the cell surface accessibility. (b) Scanning electron micrograph of an E. coli cell with surface-bound particles. (c) Element map of carbon (red) and iron (green) through energy-dispersive spectroscopy.

In sum, the well-known affinity interaction between streptavidin and the peptide SBP is harnessed to endow cells with non-natural abiotic properties. Here coupling a functionalized nanomaterial to the surface-displayed peptide physically extends the fusion protein and also adds physical (magnetic) functionality to the cell.

Linking expression to AI-2 recognition

The expression system for pSBP–Venus was then put under AI-2 control so that the protein is expressed in the presence of AI-2 instead of IPTG. That is, we coupled the native QS signal transduction circuitry to the reporter cassette. To ensure ample expression (as the native operon is fairly weak), we placed expression of T7 RNA polymerase under control of the natural QS circuitry43. Phosphorylated AI-2 activates the system through derepression of the regulator LsrR, naturally upregulating AI-2 import and phosphorylation44, and, by design, the T7 RNA polymerase on a sensor plasmid43. When sbp–Venus is included downstream of a T7 promoter region on a second plasmid, expression is then triggered by AI-2 uptake (Supplementary Fig. 4a). Then, we used two host sensor strains engineered to provide varied AI-2 sensitivity (denoted responders ‘A’ and ‘B’). In ‘A’, lsrFG, genes required for internally phosphorylated AI-2 degradation45, 46 are deleted. Also, both strains lack the terminal AI-2 synthase, luxS, so they cannot produce AI-2 and, instead, must ‘receive’ AI-2 from an external source (Supplementary Fig. 4a). The phenotypic difference between A and B is the threshold level of AI-2 that activates the genetic response47, 48. Fully constructed, these cells are designed to take up and process AI-2 to generate fluorescence output (that co-functions with streptavidin binding).

We next evaluated the kinetics of surface-fusion protein expression and effects on cell growth. The AI-2-induced expression for AIDAc-linked and SBP-tagged fluorescent proteins did not alter growth kinetics for either cell type (Supplementary Fig. 4b,c). Expression efficacy was also evaluated via immunoassay of the outer membrane, probing for AI-2-induced surface display. After induction with 20μM AI-2, extracts from cell types A and B were size-separated and blotted using alkaline phosphatase-conjugated streptavidin to probe for the SBP-tagged protein fusion (Supplementary Fig. 5). The 88kDa AIDAc–Venus–SBP protein was only found in the membrane-containing pellet fraction (Fig. 4a). Analogously, protein orientation was assessed by immunolabeling the fluorescent protein. Cell type B transformed with pSBP–eGFP was induced with 20μM AI-2 overnight; cell surfaces were then probed for eGFP using a mouse anti-GFP primary antibody and red-labelled secondary anti-mouse IgG. Simultaneously, cells were observed using phase contrast and fluorescence confocal microscopy. We noted a punctate pattern for eGFP, which was in one-to-one correspondence with red immunostaining of the surface-expressed protein. The positive staining of eGFP-expressing cells for red fluorescence, contrasted by the absence of negative control immunostaining indicated surface exposure of the fusion (Supplementary Fig. 6). Confocal microscopy confirmed precise colocalization of the eGFP and red-labelled antibodies within the confines of individual cells (Fig. 4b). Therefore, efficient transport of this functionality to the membrane under AI-2 induction was demonstrated in each host.

Figure 4: Affinity-based probing for functional analysis of AI-2-induced protein expression.

Affinity-based probing for functional analysis of AI-2-induced protein expression.

(a) 64–82kDa region of western blot for pelleted (P) and supernatant (S) protein fractions isolated from Type A and B cells. Alkaline phosphatase-conjugated streptavidin was used to target AIDAc–Venus–SBP at expression timepoints. Arrows indicate the expected position of the full fusion protein. (b) Immunostaining for assessment of the fluorescent protein surface accessibility. The external surfaces of cells expressing AIDAc–eGFP–SBP were probed with an anti–eGFP and Alexafluor594-labelled antibody pair. A representative overlaid fluorescence and phase contrast image is shown along with fluorescence images of the green (G) and red (R) filters for the boxed-in region. Scale bar, 2μM.

Establishing molecular ranges for cell interrogation

Importantly, the engineered cells each provide a characteristic response to the level of AI-2. Recently, we showed that AI-2 level influences the quorum size of responding engineered populations but does not alter the expression level within each quorum47. Here we evaluated our engineered AI-2 responders, again for quorum size (or in other words, percentage of AI-2-responsive cells in the population), this time varying the compositions of molecular input and the configuration of responders (Fig. 5a). First, we added AI-2, synthesized in vitro, to each of the two responder populations (Fig. 5b). We also added conditioned medium (CM), the spent medium from an AI-2 producer culture containing metabolic byproducts, as well as AI-2 (refs 36, 49; Fig. 5c). We also mixed the responder populations and added AI-2 to gauge responses in complex cultures (Fig. 5d).

Figure 5: Single and multi-population cell responses to autoinducer-2.

Single and multi-population cell responses to autoinducer-2.

(a) Fluorescence output is linked to small molecule input, derived from purified or crude sources. Fluorescence from Responders A and B was analysed after exposure to autoinducer-2 (AI-2) in mono and mixed culture environments. (b) Venus expression from in vitro-synthesized AI-2 added to monocultures of A and B. (c) Venus expression from conditioned media (CM) added to monocultures of A and B. CM was isolated from WT W3110 E. coli cultures sampled at indicated OD. Data are averages from triplicate cultures with s.d. indicated. (d) Red and green fluorescence responses to AI-2 during co-incubation of Responders A (pSBP–mCherry+, red) and B (pSBP–eGFP+, green). Representative fluorescence images show colocalization of red and green cells. Scale bar, 10μm. The average cell count per responder cell is plotted against AI-2 concentration, as determined by image analysis in quadruplicate. All data are plotted as averages of at least triplicate samples with s.d.

Specifically, in Fig. 5b, A and B populations were incubated at mid-exponential phase with in vitro-synthesized AI-2 (refs 50, 51) at concentrations: 0, 2, 10, 28 and 75μM. After 12h, samples were observed for fluorescence by confocal microscopy and then quantified by fluorescence-activated cell sorting (FACS; Supplementary Fig. 4c). We found that SBP–Venus expression for responder A cells occurred at the lowest tested level (2μM AI-2), where 56% of the population expressed SBP–Venus and this fraction increased with AI-2 reaching a maximum of 90% at 28μM. For type B, a more gradual trend was found; only ~1% was fluorescent from 0-2μM, and this increased from 9 to 46% as AI-2 was increased to 28μM. Finally, the highest fraction of fluorescing cells was found at the highest concentration tested, 75μM.

We next isolated CM, which contains a dynamic composition of unfiltered metabolites and media components, from W3110 E. coli cultures at intervals during their exponential growth, throughout which AI-2 accumulates (AI-2 levels for the samples are indicated in Supplementary Fig. 7). CM aliquots were mixed with either A or B cells and cultured in triplicate for 12h. Through FACS analysis it was found, again, that a larger subpopulation of A expressed Venus compared with population B at any concentration (Fig. 5c). Statistically relevant expression from B was not apparent until incubated with CM from cultures at an optical density (OD) of 0.23. In all cases, population A recognized AI-2 presence, including from media isolated at a W3110 OD of 0.05, the minimum cell density tested in this study.

The sensitivities of both strains to AI-2-mediated induction corroborate previous literature10, 47. These trends demonstrate that strains engineered for altered sensitivity to molecular cues provide discrimination of concentration level. That is, the identical plasmid expression system was transformed into different hosts, providing robust and distinct levels of expression.

Having developed cell types A and B with differential ability to detect AI-2, we next altered the reporters so that each cell type expressed a unique SBP-fluorescence fusion for colour-coded designation. Cell type A was engineered with pSBP–mCherry and type B with pSBP–eGFP, resulting in red and green fluorescence, respectively. These populations were mixed together in equal proportion at mid-exponential phase, introduced to a range of AI-2 concentrations, and incubated overnight. Populations A and B exhibited equal growth rates when cultured alone and together (Supplementary Fig. 8c); it followed that the cocultures should comprise a 1:1 ratio of each constituent. Fluorescence output is shown by representative images in Fig. 5d. Also in Fig. 5d, the green and red cell count is plotted from a quadruplicate analysis for each input concentration.

Coculturing enables parallel processing as the molecule-rich environment is perceived by each cell, and is processed uniquely per cell type. Yet, since each sensing mechanism is a living and proliferating population, we tested whether the potentially altered dynamics of coculturing would permit the same sensitivities as isolated culturing. We evaluated the Monod-type saturation constant for each population independently and in cocultures. We found, in Fig. 5d, the general trends in response to an increasing AI-2 level were as predicted by modelled response curves (Supplementary Table 4), which were also well-correlated to Fig. 5b data (Supplementary Fig. 8a,b). That is, the saturation constants that describe dependence on AI-2 were unchanged when measured in cocultures. Phenomenologically, as expected, an initial accumulation of red type A responders was found. Then, at higher AI-2 levels, we found an emergence of a green subpopulation (type B). Above 28μM, there was no longer an apparent differential response that would otherwise enable discrimination of AI-2 concentration; based on the consistency with modelled behaviour, coculturing contributed to dampen the response as the maximum percentage of responding cells in cocultures is 50% instead of 100%. However, the overall fluorescence output is enriched by the combination of multiple populations since the ranges of sensitivity overlap and effectively expand that of the master population (Supplementary Fig. 8d). Specifically, because the fluorescence of B is described by a larger saturation constant, its fluorescence continually increases at higher AI-2 concentrations, while the fluorescence of A remains unchanged. Thus, coculturing between A and B enables resolvable output that is lower than the detection limit of B (due to A) yet surpasses the upper limit at which A saturates by the inclusion of B. The choice to fluorescently differentiate A and B was important because the output would otherwise be biased by extracellular components including the existence of non-sensing cells. Due to colour designation of A and B, a colour ‘pattern’ emerges as a feature of the parallel response, which we recognize is independent of the absolute fluorescence of the population.

Consensus feedback through multidimensional processing

We hypothesized that the value of cell-based sensing would be enhanced if the cell output could be collated in an unbiased manner that in turn were easily ‘read’ using optical means. We engaged magnetic processing, which represents an abiotic processing step that enhances the signal by focusing the collective response. Hence, cells were equipped with streptavidin-conjugated mNPs (Fig. 3). The ability of a magnetic field to refine fluorescence output through filtering and focusing is described in the Supplementary Information (Supplementary Fig. 11). Thus, in our combinatorial approach, fluorescence feedback about molecular information within a microbial community entails biotic processing through constituencies of two independent cell types in conjunction with magnetic post-processing that is enabled by guidance at the nanoscale (Fig. 6c). Moreover, since the fluorescence feedback data is provided through two constituencies, consensus from each independently provides an aggregate output; in our example, the output becomes relayed as a distinctive ‘binned’ category due to finite colour-combinations generated from constituencies A and B (Fig. 6c).

Figure 6: Binning molecular information through cell-based parallel processing and magnetically focusing fluorescence into collective consensus output.

Binning molecular information through cell-based parallel processing and magnetically focusing fluorescence into collective consensus output.

(a) A and B cell types were co-incubated with AI-2 levels ranging from 0 to 55μM AI-2 (left axis), then imaged after magnetic nanoparticle coupling and magnetic collation. Fluorescence results (centred directly over the magnet) are shown from high to low input (top left to bottom right). (b) Quantification of red and green fluorescence cell densities per AI-2 level. (c) The process of accessing molecular information begins by distributing Responders A and B within the environment of an AI-2 producer, P. A and B independently express fluorophore fusions and are linked with magnetic nanoparticles on processing autoinducer-2. Magnetic focusing translocates fluorescing responders. Image analysis of the magnetically collated cell aggregate reveals classified fluorescence output, representing the AI-2 composition of the interrogated environment. (d) Bright field (left) and fluorescence (right, red and green filters) images positioned over the edge of a magnet, as indicated by the inset. The sample in the bottom image pair was isolated from an environment of low AI-2 accumulation. The sample in the top image pair was isolated from a high AI-2 environment. (e) Quantification of visual space occupied by collated cells (eGFP and mCherry expressers) while distributed (- magnet) and magnetically focused (+). Scale bars, 50μm.

Again, type A transmits red output (SBP–mCherry+) and type B transmits green (SBP–eGFP+). These were first co-incubated with titred concentrations of AI-2, to obtain results similar to those ofFig. 5d. By coupling mNPs to the responsive parallel populations, we tested for aggregate two-colour output to provide informative feedback within a set of outcomes ranging from no colour, red-only to red+green. After overnight co-incubation and a magnetic sweep with streptavidin-mNPs, fluorescence results are shown in Fig. 6a, where the recovered cells are displayed above a magnet’s center in order from highest to lowest AI-2 level (top left to bottom right). The processing output generated by the range of conditions was quantitatively assessed for contributions from A and B responders. The spatial density of each fluorophore, or the area occupied by fluorescent responders as a percentage of total visible area, was quantified and plotted in Fig. 6b. Here the trend of increasing fluorescence with AI-2 is followed by both A and B cell types; however, red A cells accumulate at a higher rate than green B cells. This relationship between A and B processing is not only consistent with their previous characterizations (Fig. 5) but indicates that the aggregate output is unbiased regardless of assembly with mNPs and magnetic-stimulated redistribution (Supplementary Information, Supplementary Fig. 14a).

Next, A and B cells were added together to probe the QS environment of Listeria innocua, an AI-2-producing cell type that is genetically and ecologically similar to the pathogenic strain L. monocytogenes52. The environment was biased towards low and high cell density conditions by altering nutrient levels to develop contrasting scenarios of AI-2 level. Preliminary characterization in the Supplementary Information indicated that L. innocua proliferation is unperturbed by the presence of E. coli responders (Supplementary Fig. 12) and that type A cells detect AI-2 at lowListeria densities limited by sparse nutrients; then with rich nutrient availability, cell proliferation permits a higher AI-2 level that can be detected by type B (Supplementary Fig. 13). Replicating these conditions, we expected red fluorescence to be observed at low culture density and for green fluorescence to be reported when high (Fig. 6c). Two conditions were tested: L. innocua was proportioned to responder cells at 20:1 in dilute media to establish a low culture density condition or, alternatively, a ratio of 200:1 in rich media for a high culture density condition. After overnight co-incubation and a magnetic sweep (applied directly to the triple strain cultures) with streptavidin-mNPs, the recovered cells are displayed above a magnet’s edge (shown in Fig. 6d). Acute focusing of the fluorescence signals, contributed by each subset population of the processor (A and B), is visually apparent. The magnetic field had a physical effect of repositioning the ‘on’ subsets to be tightly confined within the magnetic field.

The processing output generated by the contrasting culture conditions was again assessed for the respective contributions of A and B, and for changes in spatial signal density due to the magnetic sweep (Fig. 6e). The analysis was based on images provided in Supplementary Fig. 14b. Data inFig. 6e indicate that red type A cells are prevalent regardless of culture condition (except negative controls). However, compared with the low AI-2 condition, the abundance of green cells is 100-fold higher in the high AI-2 condition. In addition, the ratio of green to red was consistent prior to and after magnetic concentration, substantiating observations in the distributed system. Further, data show that magnetic refining increased per-area fluorescence 100-fold or 10-fold in low and high cell culture studies, respectively.

Based on the thresholds established for responder populations A and B, we found colour-coded binning corresponded to AI-2 level, where ‘red-only’ represented less AI-2 than ‘red+green’ (Fig. 5d). Thus, we found a binned output was established via this multidimensional molecular information-processing system and that this matched the expectations. Red feedback (from responder A) indicated dilute AI-2 accumulation occurred in the low density culture. In the dense cultures, high AI-2 accumulation turned on both A and B for combined red and green feedback.

System response patterns defined by parallel populations

Our example demonstrates the concept of an amorphous processing system that utilizes several biotic and abiotic components for multidimensional information processing. Interestingly, a binning effect was enabled: our system yields an index of colour-categorized feedback that characterizes the sampled environment. In Fig. 7, we present a means to extend our approach to multidimensional systems, those with more than one molecule-of-interest and at different concentrations. That is, by appropriate design of the cell responders, we can further enrich the methodology, its depth and breadth of applicability. We depict 10 hypothetical pairs of responses (with defining equations located in Supplementary Table 5)—those that can be driven by appropriately engineering cells to portend altered genetic responses. For example, rows 1 and 3 provide genetic outcomes as a function of analyte (AI-2) concentration. The hypothetical depictions are feasible as ‘designer’ signal transduction and marker expression processes enabled by synthetic biology21, 53, 54. Rows 2 and 4 demonstrate the corresponding visual planes, where red cell numbers (x-axis) are plotted against green (y-axis), illustrated by the first example. If one divides the two-dimensional space into quadrants (no colour, majority red, majority green, and equivalent ratios of red and green), it becomes apparent that the relationship between cell types influences the ‘visual’ or optical output. Thus, the 10 arbitrary response sets yield a variety of pairings that can provide unique visual patterns for categorizing molecular information. We have simplified the analysis by placing dot marker symbols at the various coincident datapoints, revealing visual patterns. In this way, the ability to incorporate unique responses to a multitude of molecular cues, all within a single pair of cells, or through further multiplexing with additional cell populations becomes apparent.

Figure 7: Extension of nano-guided cell networks for hypothetical regulatory structures.

Extension of nano-guided cell networks for hypothetical regulatory structures.

(a) Rows 1 and 3 depict 10 hypothetical genetic responses to molecular inputs for pairs of fluorescence-reporting cell populations (red, R and green, G). Rows 2 and 4 depict genetic responses as phase-plane plots yielding distinct patterns. This establishes a visual field, showing the extent of any population–population bias (illustrated in example case 1). (b) Left panel: a two-population pairing (shown in case 10) defines visual output that inherently bins into three quadrants: Q1, negligible colour; Q2, red bias due to majority red cell output; and Q4, combined red and green output. Right panel: data from Figs 5d and 6bare plotted analogously, where each data point represents an autoinducer-2 input (labelled, μM). As expected, red and green outputs were binned into Q1, Q2 and Q4 as indicated by coloured outlines.

Our AI-2-conveying cell network is similar to example 7 in Fig. 7a and the AI-2 response curves inFig. 5 (characterized by Supplementary Table 4 equations). Example 7 establishes output into three basic quadrants, including Q1 (negligible colour), Q2 (majority red) and Q4 (roughly equal red and green) (Fig. 7b). We recast the data from Figs 5d and 6b as a phase-plane portrait in Fig. 7c. This reveals the mechanisms by which the output is binned and how the originating cell response curves lead to this pattern, which in turn, was unchanged due to magnetic refinement. InSupplementary Fig. 15, we demonstrate a parameterization of the red and green response curves that suggest the methodology is robust, that when cells are appropriately engineered one could ‘tune’ system characteristics to enhance or diminish a binning effect. We suggest that the utility of subcellular genetic tuning extends well beyond per-cell performance. Rather, we suggest such a strategy may be used to guide the dynamics of population architecture for actuation of by-design response patterns at a systems level.

Discussion

While cell-based sensors work well in well-defined assay conditions, extension to complex environments remains a challenge. They grow, they move, they perturb their environs, they report in a time and concentration-dependent manner, small numbers of sensor cells may require signal amplification and so on. Also, increasingly, bacterial cells are engineered for user specified ‘executive’ functions in complex environments55, 56, 57. Their performance depends on their ability to filter out extraneous noise while surveying the molecular landscape, and providing informed actuation.

Our system interrogates the molecular space by focusing on bacterial QS and a widely distributed signal molecule, AI-2. In addition to genetic attributes of the AI-2-responding sensor cells, AI-2 is a chemoattractant for E. coli, and hence E. coli engineered to sense and respond to AI-2 will naturally move towards its sources, enabling full sampling of the prevailing state10, 37. Each strain evaluates AI-2 with a distinct sensitivity. When ‘activated’ in response to a characteristic level, the cells simultaneously expressed a fluorescent marker and a SBP on the outer membrane via AIDAc translocation. SBP provides a means for cell hybridization through its strong affinity to streptavidin, and here, aids in binding mNPs. This enables the non-genetically coded property of cell translocation within a magnetic field through physically stimulated focusing and binning.

By making use of a diversity of biotic and abiotic features, our multidimensional system of ‘responder’ populations exemplifies several key metrics that promote executive performance in such environments: active molecule capture, post-capture refining of the detection output and finally the utilization of multiple feedback thresholds58, 59, 60. Here cells facilitate AI-2 recognition autonomously and actively because, as a distributed network they reside planktonically, chemotaxing to and continually processing signals over time. When AI-2 is detected, a processor cell’s cognate machinery responds by upregulation of the native QS operon, leading to rapid signal uptake and thereby creating an active-capture signal-processing mechanism. To maximize information acquisition and account for a potentially heterogeneous molecular landscape, cells serve as molecular sampling units among a distributed population, which leads to data fed back as a consensus of fluorescent ‘datapoints’. Then, distributed data collection can be selectively reversed via the incorporated abiotic feature: mNPs, fastened externally on the cell through affinity-guided self-assembly. As such, responding cells obtained this extendable feature, thereby becomes sensitized to repositioning within a magnetic field.

The layered nature of the processor here, from the subcellular to multicellular scale, permits a series of selective steps: it commences with the AI-2-triggered expression cascade which releases a tight repressor, surface localization of both the fluorescent protein and SBP tag, and finally nanoparticle binding for recovery. In addition, multiple layers of amplification result in orthogonal fluorescence feedback. The AI-2 detection event leads to whole-cell fluorescence through expression of many protein copies47. Then their physical collection further amplifies the signal, yielding a macroscopic composite of many individual cell units. When utilized as a network of multiple constituencies, responder cell types A and B contribute individual recognition results (off, red or green) to a single consensus output. Finally, due to their overlapping thresholds for recognition of the same molecule, in this case, AI-2, parallel processing by A and B responders can contribute to visual interpretation of information about the molecule. Outcomes are classified into a finite number of states: here output to no fluorescence, red, or red and green, with each addition of colour as a metric of a higher interval of AI-2. In many respects, the elucidation of layered information networks as demonstrated here is analogous to computer information processing via information theory61, 62, 63.

Here, however, interrogation of biological systems requires a reliable means for accessing molecular information—that which is communicated between biological species and that which can be relayed to the end user. The responder cells need not be present in high concentration, nor must they all be collected in the present format. We suggest that engineered biological mechanisms are well-poised to serve at this critical interface between information acquisition and user interaction. Thus, the functional design of components for autonomous self-assembly, decision-making and networking is requisite in the field of micro- and nano-scaled machines. Our combinatorial approach allows for cells to independently assess, yet collectively report, on molecular information. Its processing is enabled through appropriate integration of synthetic biology and nanomaterials design. We suggest this approach provides a rich opportunity to direct many formats of multi-population response through genetic tuning and systems-level engineering. Further development of cellular networks and incorporation of alternate abiotic attributes can expand the depth and breadth of molecular communication for user specified actuation.

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Cancer Genomics: Multiomic Analysis of Single Cells and Tumor Heterogeneity

Curator: Stephen J. Williams, PhD

 

scTrio-seq identifies colon cancer lineages

Single-cell multiomics sequencing and analyses of human colorectal cancer. Shuhui Bian et al. Science  30 Nov 2018:Vol. 362, Issue 6418, pp. 1060-1063

To better design treatments for cancer, it is important to understand the heterogeneity in tumors and how this contributes to metastasis. To examine this process, Bian et al. used a single-cell triple omics sequencing (scTrio-seq) technique to examine the mutations, transcriptome, and methylome within colorectal cancer tumors and metastases from 10 individual patients. The analysis provided insights into tumor evolution, linked DNA methylation to genetic lineages, and showed that DNA methylation levels are consistent within lineages but can differ substantially among clones.

Science, this issue p. 1060

Abstract

Although genomic instability, epigenetic abnormality, and gene expression dysregulation are hallmarks of colorectal cancer, these features have not been simultaneously analyzed at single-cell resolution. Using optimized single-cell multiomics sequencing together with multiregional sampling of the primary tumor and lymphatic and distant metastases, we developed insights beyond intratumoral heterogeneity. Genome-wide DNA methylation levels were relatively consistent within a single genetic sublineage. The genome-wide DNA demethylation patterns of cancer cells were consistent in all 10 patients whose DNA we sequenced. The cancer cells’ DNA demethylation degrees clearly correlated with the densities of the heterochromatin-associated histone modification H3K9me3 of normal tissue and those of repetitive element long interspersed nuclear element 1. Our work demonstrates the feasibility of reconstructing genetic lineages and tracing their epigenomic and transcriptomic dynamics with single-cell multiomics sequencing.

Fig. 1 Reconstruction of genetic lineages with scTrio-seq2.

Global SCNA patterns (250-kb resolution) of CRC01. Each row represents an individual cell. The subclonal SCNAs used for identifying genetic sublineages were marked and indexed; for details, see fig. S6B. On the top of the heatmap, the amplification or deletion frequency of each genomic bin (250 kb) of the non-hypermutated CRC samples from the TCGA Project and patient CRC01’s cancer cells are shown.

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Fig. 1 Reconstruction of genetic lineages with scTrio-seq2.

Global SCNA patterns (250-kb resolution) of CRC01. Each row represents an individual cell. The subclonal SCNAs used for identifying genetic sublineages were marked and indexed; for details, see fig. S6B. On the top of the heatmap, the amplification or deletion frequency of each genomic bin (250 kb) of the non-hypermutated CRC samples

 

 

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from The American Association for Cancer Research aacr.org:

 

AACR Congratulates Dr. William G. Kaelin Jr., Sir Peter J. Ratcliffe, and Dr. Gregg L. Semenza on 2019 Nobel Prize in Physiology or Medicine

10/7/2019

PHILADELPHIA — The American Association for Cancer Research (AACR) congratulates Fellow of the AACR Academy William G. Kaelin Jr., MDSir Peter J. Ratcliffe, MD, FRS, and AACR member Gregg L. Semenza, MD, PhD, on receiving the 2019 Nobel Prize in Physiology or Medicine for their discoveries of how cells sense and adapt to oxygen availability.

Kaelin, professor of medicine at the Dana-Farber Cancer Institute and Harvard Medical School in Boston; Ratcliffe, director of Clinical Research at the Francis Crick Institute in London; and Semenza, director of the Vascular Program at the Institute for Cell Engineering at Johns Hopkins University School of Medicine in Baltimore, are being recognized by the Nobel Assembly at the Karolinska Institute for identifying the molecular machinery that regulates the activity of genes in response to varying levels of oxygen, which is one of life’s most essential adaptive processes. Their work has provided basic understanding of several diseases, including many types of cancer, and has laid the foundation for the development of promising new approaches to treating cancer and other diseases.

Kaelin, Ratcliffe, and Semenza were previously recognized for this work with the 2016 Lasker-DeBakey Clinical Medical Research Award.

Kaelin’s research focuses on understanding how mutations affecting tumor-suppressor genes cause cancer. As part of this work, he discovered that a tumor-suppressor gene called von Hippel–Lindau (VHL) is involved in controlling the cellular response to low levels of oxygen. Kaelin’s studies showed that the VHL protein binds to hypoxia-inducible factor (HIF) when oxygen is present and targets it for destruction. When the VHL protein is mutated, it is unable to bind to HIF, resulting in inappropriate HIF accumulation and the transcription of genes that promote blood vessel formation, such as vascular endothelial growth factor (VEGF). VEGF is directly linked to the development of renal cell carcinoma and therapeutics that target VEGF are used in the clinic to treat this and several other types of cancer.

Kaelin has been previously recognized with numerous other awards and honors, including the 2006 AACR-Richard and Hinda Rosenthal Award.

Ratcliffe independently discovered that the VHL protein binds to HIF. Since then, his research has focused on the molecular interactions underpinning the binding of VHL to HIF and the molecular events that occur in low levels of oxygen, a condition known as hypoxia. Prior to his work on VHL, Ratcliffe’s research contributed to elucidating the mechanisms by which hypoxia increases levels of the hormone erythropoietin (EPO), which leads to increased production of red blood cells.

Semenza’s research, which was independent of Ratcliffe’s, identified in exquisite detail the molecular events by which the EPO gene is regulated by varying levels of oxygen. He discovered HIF and identified this protein complex as the oxygen-dependent regulator of the EPO gene. Semenza followed up this work by identifying additional genes activated by HIF, including showing that the protein complex activates the VEGF gene that is pivotal to the development of renal cell carcinoma.

The recognition of Kaelin and Semenza increases the number of AACR members to have been awarded a Nobel Prize to 70, 44 of whom are still living.

The Nobel Prize in Physiology or Medicine is awarded by the Nobel Assembly at the Karolinska Institute for discoveries of major importance in life science or medicine that have changed the scientific paradigm and are of great benefit for mankind. Each laureate receives a gold medal, a diploma, and a sum of money that is decided by the Nobel Foundation.

The Nobel Prize Award Ceremony will be Dec. 10, 2019, in Stockholm.

Please find following articles on the Nobel Prize and Hypoxia in Cancer on this Open Access Journal:

2018 Nobel Prize in Physiology or Medicine for contributions to Cancer Immunotherapy to James P. Allison, Ph.D., of the University of Texas, M.D. Anderson Cancer Center, Houston, Texas. Dr. Allison shares the prize with Tasuku Honjo, M.D., Ph.D., of Kyoto University Institute, Japan

The History, Uses, and Future of the Nobel Prize, 1:00pm – 6:00pm, Thursday, October 4, 2018, Harvard Medical School

2017 Nobel prize in chemistry given to Jacques Dubochet, Joachim Frank, and Richard Henderson  for developing cryo-electron microscopy

Tumor Ammonia Recycling: How Cancer Cells Use Glutamate Dehydrogenase to Recycle Tumor Microenvironment Waste Products for Biosynthesis

Hypoxia Inducible Factor 1 (HIF-1)[7.9]

 

 

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Disentangling molecular alterations from water-content changes in the aging human brain using quantitative MRI

Reporter: Dror Nir, PhD

 

Authors’ list: Shir Filo, Oshrat Shtangel, Noga Salamon, Adi Kol, Batsheva Weisinger, Sagiv Shifman & Aviv A. Mezer
Published in: Nature Communications volume 10, Article number: 3403 (2019)

Abstract

It is an open question whether aging-related changes throughout the brain are driven by a common factor or result from several distinct molecular mechanisms. Quantitative magnetic resonance imaging (qMRI) provides biophysical parametric measurements allowing for non-invasive mapping of the aging human brain. However, qMRI measurements change in response to both molecular composition and water content. Here, we present a tissue relaxivity approach that disentangles these two tissue components and decodes molecular information from the MRI signal. Our approach enables us to reveal the molecular composition of lipid samples and predict lipidomics measurements of the brain. It produces unique molecular signatures across the brain, which are correlated with specific gene-expression profiles. We uncover region-specific molecular changes associated with brain aging. These changes are independent from other MRI aging markers. Our approach opens the door to a quantitative characterization of the biological sources for aging, that until now was possible only post-mortem.

 

Introduction

The biology of the aging process is complex, and involves various physiological changes throughout cells and tissues1. One of the major changes is atrophy, which can be monitored by measuring macroscale brain volume reduction1,2. In some cases, atrophy can also be detected as localized microscale tissue loss reflected by increased water content3. This process is selective for specific brain regions and is thought to be correlated with cognitive decline in Alzheimer’s disease2,4,5. In addition to atrophy, there are molecular changes associated with the aging of both the normal and pathological brain5,6. Specifically, lipidome changes are observed with age, and are associated with several neurological diseases7,8,9,10,11.

It is an open question as to whether there are general principles that govern the aging process, or whether each system, tissue, or cell deteriorates with age for different reasons12,13. On one hand, the common-cause hypothesis proposes that different biological aging-related changes are the result of a single underlying factor14,15. This implies that various biomarkers of aging will be highly correlated16. On the other hand, the mosaic theory of aging suggests that there are several distinct aging mechanisms that have a heterogenous effect throughout the brain12,13. According to this latter view, combining different measurements of brain tissue is crucial in order to fully describe the state of the aging brain. To test these two competing hypotheses in the context of volumetric and molecular aging-related changes, it is essential to measure different biological aspects of brain tissue. Unfortunately, the molecular correlates of aging are not readily accessible by current in vivo imaging methods.

The main technique used for non-invasive mapping of the aging process in the human brain is magnetic resonance imaging (MRI)2,17,18,19. Advances in the field have led to the development of quantitative MRI (qMRI). This technique provides biophysical parametric measurements that are useful in the investigation and diagnosis of normal and abnormal aging20,21,22,23,24,25,26,27. qMRI parameters have been shown to be sensitive to the microenvironment of brain tissue and are therefore named in vivo histology28,29,30. Nevertheless, an important challenge in applying qMRI measurements is increasing their biological interpretability. It is common to assume that qMRI parameters are sensitive to the myelin fraction20,23,30,31,32,33, yet any brain tissue including myelin is a mixture of multiple lipids and proteins. Moreover, since water protons serve as the source of the MRI signal, the sensitivity of qMRI parameters to different molecular microenvironments may be confounded by their sensitivity to the water content of the tissue34,35. We hypothesized that the changes observed with aging in MRI measurements20,23,30,31,32,33,36 such as R1, R2, mean diffusivity (MD), and magnetization transfer saturation (MTsat)37, could be due to a combination of an increase in water content at the expense of tissue loss, and molecular alterations in the tissue.

Here, we present a qMRI analysis that separately addresses the contribution of changes in molecular composition and water content to brain aging. Disentangling these two factors goes beyond the widely accepted “myelin hypothesis” by increasing the biological specificity of qMRI measurements to the molecular composition of the brain. For this purpose, we generalize the concept of relaxivity, which is defined as the dependency of MR relaxation parameters on the concentration of a contrast agent38. Instead of a contrast agent, our approach exploits the qMRI measurement of the local non-water fraction39 to assess the relaxivity of the brain tissue itself. This approach allows us to decode the molecular composition from the MRI signal. In samples of known composition, our approach provides unique signatures for different brain lipids. In the live human brain, it produces unique molecular signatures for different brain regions. Moreover, these MRI signatures agree with post-mortem measurements of the brain lipid and macromolecular composition, as well as with specific gene-expression profiles. To further validate the sensitivity of the relaxivity signatures to molecular composition, we perform direct comparison of MRI and lipidomics on post-mortem brains. We exploit our approach for multidimensional characterization of aging-related changes that are associated with alterations in the molecular composition of the brain. Finally, we evaluate the spatial pattern of these changes throughout the brain, in order to compare the common-cause and the mosaic theories of aging in vivo.

 

Results

Different brain lipids have unique relaxivity signatures
The aging process in the brain is accompanied by changes in the chemophysical composition, as well as by regional alterations in water content. In order to examine the separate pattern of these changes, we developed a model system. This system was based on lipid samples comprising common brain lipids (phosphatidylcholine, sphingomyelin, phosphatidylserine, phosphatidylcholine-cholesterol, and phosphatidylinositol-phosphatidylcholine)7. Using the model system, we tested whether accounting for the effect of the water content on qMRI parameters provides sensitivity to fine molecular details such as the head groups that distinguish different membrane phospholipids. The non-water fraction of the lipid samples can be estimated by the qMRI measurement of lipid and macromolecular tissue volume (MTV, for full glossary of terms see Supplementary Table 1)39. By varying the concentration of the lipid samples, we could alter their MTV and then examine the effect of this manipulation on qMRI parameters. The parameters we estimated for the lipid samples were R1, R2, and MTsat. The potential ambiguity in the biological interpretation of qMRI parameters is demonstrated in Fig. 1a. On one hand, samples with similar lipid composition can present different R1 measurements (Fig. 1a, points 1 & 2). On the other hand, scanning samples with different lipid compositions may result in similar R1 measurements (Fig. 1a, points 2 & 3). This ambiguity stems from the confounding effect of the water content on the MR relaxation properties.

Screenshot 2019-08-01 at 14.36.20

We evaluated the dependency of different qMRI parameters on the non-water fraction estimated by MTV. This analysis revealed strong linear dependencies (median R2 = 0.74, Fig. 1a, b and Supplementary Fig. 1a, b). These linear MTV dependencies change as a function of the lipid composition, reflecting the inherent relaxivity of the different lipids. We could therefore use the MTV derivatives of qMRI parameters (dqMRIdMTV, i.e., the slope of the linear relationship between each qMRI parameter and MTV) as a measure that is sensitive to molecular composition. By accounting for the Multidimensional Dependency on MTV (“MDM”) of several qMRI parameters, a unique MRI relaxivity signature was revealed for each lipid (Fig. 1c). This implies that the water-related ambiguity demonstrated in the inset of Fig. 1a can be removed by measuring the MTV dependencies (Fig. 1c). Creating mixtures of several lipids provided supportive evidence for the generality of our framework. Figure 1d and Supplementary Fig. 1c show that the qMRI measurements of a mixture can be predicted by summing the MTV dependencies of pure lipids (for further details see Supplementary Note 1 and Supplementary Fig. 2). Furthermore, we used this biophysical model to predict the lipid composition of a mixture from its MDM measurements (Fig. 1e). This model provided a good estimation of the sphingomyelin (Spg) and phosphatidylserine (PS) content (R2 > 0.64) but failed to predict phosphatidylcholine (PtdCho) content (for further details see Supplementary Note 2). While lipids are considered to be a major source of the MRI signal in the brain 40,41,42,43,44,45, our approach can be applied to other compounds to reveal differences in the MRI signal between different proteins, sugars, and ions (Supplementary Fig. 1d). Hence, the relationships between qMRI parameters and MTV account for the effect of water on MRI measurements and could be of use in quantifying the biological and molecular contributions to the MRI signal of water protons.

The tissue relaxivity of the human brain is region-specific.
In order to target age-related changes in molecular composition, we applied the same approach for the human brain (Fig. 2a).

Screenshot 2019-08-01 at 14.41.35

We found that the linear dependency of qMRI parameters on MTV is not limited to in vitro samples and a similar relationship was also evident in the human brain (Fig. 2b and Supplementary Figs. 3–5). Importantly, different brain regions displayed a distinct dependency on MTV. Therefore, the relaxivity of brain tissue is region-specific. Figure 2b provides an example for the regional linear trends of R1 and MTsat in a single subject. Remarkably, while the thalamus and the pallidum presented relatively similar R1 dependencies on MTV, their MTsat dependencies were different (p < 0.001, two-sample t-test). Compared to these two brain regions, frontal white-matter demonstrated different dependencies on MTV (p < 0.001, two-sample t-test). A better separation between brain regions can therefore be achieved by combining the MTV dependencies of several qMRI parameters (MTsat, MD, R1 and R2). The MTV derivatives of qMRI parameters are consistent across subjects (Fig. 2c and Supplementary Fig. 6), with good agreement between hemispheres (Supplementary Fig. 5). Moreover, they provide a novel pattern of differentiation between brain regions, which is not captured by conventional qMRI methods (Supplementary Fig. 7). In our lipid sample experiments, the MDM approach revealed unique relaxivity signatures of different lipids (Fig. 1c). Therefore, we attribute the observed diversity in the MTV derivatives of qMRI parameters across brain regions to the intrinsic heterogeneity in the chemophysical microenvironment of these regions. The multidimensional dependency of various qMRI parameters on MTV can be represented by the space of MTV derivatives to reveal a unique chemophysical MDM signature for different brain regions (Fig. 2d, see explanatory scheme of the MDM method in Supplementary Fig. 8). Fig. 2 figure2 The MDM method provides region-specific signatures in the in vivo human brain. a Representative MTV, MTsat, and R1 maps. b Calculating the MDM signatures. The dependency of R1 (left) and MTsat (right) on MTV in three brain regions of a single subject. For each region, MTV values were pooled into bins (dots are the median of each bin; shaded area is the median absolute deviation), and a linear fit was calculated (colored lines). The slopes of the linear fit represent the MTV derivatives of R1 and MTsat and vary across brain regions. c The reliability of the MDM method across subjects. Variation in the MTV derivatives of R1 (left) and MTsat (right) in young subjects (N = 23). Different colors represent 14 brain regions (see legend). Edges of each box represent the 25th, and 75th percentiles, median is in black, and whiskers extends to extreme data points. Different brain regions show distinct MTV derivatives. d Unique MDM signatures for different brain regions (in different colors). Each axis is the MTV derivative (“MDM measurements”) of a different qMRI parameter (R1, MTsat, R2, and MD). The range of each axis is in the legend. Colored traces extend between the MDM measurements, shaded areas represent the variation across subjects (N = 23). An overlay of all MDM signatures is marked with dashed lines Full size image The in vivo MDM approach captures ex vivo molecular profiles To validate that the MDM signatures relate to the chemophysical composition of brain tissue, we compared them to a previous study that reported the phospholipid composition of the human brain7. First, we established the comparability between the in vivo MRI measurements and the reported post-mortem data. MTV measures the non-water fraction of the tissue, a quantity that is directly related to the total phospholipid content. Indeed, we found good agreement between the in vivo measurement of MTV and the total phospholipid content across brain regions (R2 = 0.95, Fig. 3a). Söderberg et al.7 identified a unique phospholipid composition for different brain regions along with diverse ratios of phospholipids to proteins and cholesterol. We compared this regional molecular variability to the regional variability in the MDM signatures. To capture the main axes of variation, we performed principal component analysis (PCA) on both the molecular composition of the different brain regions and on their MDM signatures. For each of these two analyses, the first principal component (PC) explained >45% of the variance. The regional projection on the first PC of ex vivo molecular composition was highly correlated (R2 = 0.84, Fig. 3b) with the regional projection on the first PC of in vivo MDM signatures. This confirms that brain regions with a similar molecular composition have similar MDM. Supplementary Fig. 9a provides the correlations of individual lipids with MDM. Importantly, neither MTV nor the first PC of standard qMRI parameters was as strongly correlated with the ex vivo molecular composition as the MDM (Supplementary Fig. 9b, c). We next used the MDM measurements as predictors for molecular properties of different brain regions. Following our content predictions for lipids samples (Fig. 1e), we constructed a weighted linear model for human data (for further details see Supplementary Note 3). To avoid over fitting, we reduced the number of fitted parameters by including only the MDM and the molecular features that accounted for most of the regional variability. The MTV derivatives of R1 and MTsat accounted for most of the variance in MDM. Thus, we used these parameters as inputs to the linear model, while adjusting their weights through cross validation. We tested the performance of this model in predicting the three molecular features that account for most of the variance in the ex vivo molecular composition. Remarkably, MRI-driven MDM measurements provided good predictions for the regional sphingomyelin composition (R2 = 0.56, p < 0.05 for the F-test, Fig. 3c) and the regional ratio of phospholipids to proteins (R2 = 0.56, p < 0.05 for the F-test, Fig. 3c).

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Last, we compared the cortical MDM signatures to a gene co-expression network based on a widespread survey of gene expression in the human brain46. Nineteen modules were derived from the gene network, each comprised of a group of genes that co-varies in space. Six out of the nineteen gene modules were significantly correlated with the first PC of MDM. Interestingly, the first PC of MDM across the cortex was correlated most strongly with the two gene modules associated with membranes and synapses (Fig. 4, for further details see Supplementary Note 4 and Supplementary Figs. 10 and 11).

Screenshot 2019-08-01 at 14.47.04

Post-mortem validation for the lipidomic sensitivity of MDM.
The aforementioned analyses demonstrate strong agreement between in vivo MDM measurements and ex vivo molecular composition based on a group-level comparison of two different datasets. Strikingly, we were able to replicate this result at the level of the single brain. To achieve this we performed MRI scans (R1, MTsat, R2, MD, and MTV mapping) followed by histology of two fresh post-mortem porcine brains (Fig. 5a, b). First, we validated the qMRI estimation of MTV using dehydration techniques. MTV values estimated using MRI were in agreement with the non-water fraction found histologically (adjusted R2 = 0.64, p < 0.001 for the F-test, Fig. 5c).

Screenshot 2019-08-01 at 14.50.12
Next, we estimated the lipid composition of different brain regions. Thin-layer chromatography (TLC) was employed to quantify seven neutral and polar lipids (Supplementary Table 2 and Supplementary Fig. 12a). In accordance with the analysis in Fig. 3, we performed PCA to capture the main axes of variation in lipidomics, standard qMRI parameters, and MDM. Figure 5d shows that MTV did not correlate with the molecular variability across the brain, estimated by the 1st PC of lipidomics. Likewise, the molecular variability did not agree with the 1st PC of standard qMRI parameters (Fig. 5e).

Last, we applied the MDM approach to the post-mortem porcine brain. Similar to the human brain, different porcine brain regions have unique MDM signatures (Fig. 5f, g and Supplementary Fig. 12b). Remarkably, we found that agreement between lipid composition and MRI measurements emerges at the level of the MDM signatures. The molecular variability across brain regions significantly correlated with the regional variability in the MDM signatures (adjusted R2 = 0.3, p < 0.01 for the F-test, Fig. 5h). Excluding from the linear regression five outlier brain regions where the histological lipidomics results were 1.5 standard deviations away from the center yielded an even stronger correlation between MDM signatures and lipid composition (adjusted R2 = 0.55, p < 0.001 for the F-test, Supplementary Fig. 12c). This post-mortem analysis validates that the MDM approach allows us to capture molecular information using MRI at the level of the individual brain.

Disentangling water and molecular aging-related changes.
After establishing the sensitivity of the MDM signatures to the molecular composition of the brain, we used them to evaluate the chemophysical changes of the aging process. To assess aging-related changes across the brain, we scanned younger and older subjects (18 older adults aged 67 ± 6 years and 23 younger adults aged 27 ± 2 years). First, we identified significant molecular aging-related changes in the MDM signatures of different brain regions (Figs. 6 and 7, right column; Supplementary Fig. 13). Next, we tested whether the changes in MRI measurements, observed with aging, result from a combination of changes in the molecular composition of the tissue and its water content. We found that although it is common to attribute age-related changes in R1 and MTsat to myelin28,30,36, these qMRI parameters combine several physiological aging aspects. For example, using R1 and MTsat we identified significant aging-related changes in the parietal cortex, the thalamus, the parietal white-matter and the temporal white-matter (Figs. 6 and 7, left column). However, the MDM approach revealed that these changes have different biological sources (Figs. 6 and 7, middle columns; see Supplementary Figs. 14–17 for more brain regions).

Screenshot 2019-08-01 at 14.51.53

Screenshot 2019-08-01 at 14.54.44

Screenshot 2019-08-01 at 14.56.06

In agreement with the mosaic hypothesis, we identified distinct aging patterns for different brain regions. For example, in the hippocampus we found a change in R2* values related to a higher iron concentration with age, along with significant reduction in the total hippocampal volume (Fig. 8a). This age-related shrinkage was not accompanied by lower MTV values, indicating conserved tissue density (Fig. 7b). In addition, there was no significant difference in the hippocampal MDM signature with age (Fig. 7b). Cortical gray-matter areas also exhibited similar trends of volume reduction without major loss in tissue density (Fig. 8a). Unlike the gray matter, in the white matter we did not find volume reduction or large iron accumulation with age (Fig. 8a). However, we did find microscale changes with age in tissue composition, as captured by the MDM signature (Figs. 6a and 7c, and Supplementary Fig. 13), accompanied by a significant density-related decline in MTV (Fig. 8a). These findings are consistent with previous histological studies49,50,51 (see Discussion), and provide the ability to monitor in vivo the different components of the aging mosaic.

Last, to test whether the different biological aging trajectories presented in Fig. 8a share a common cause, we evaluated the correlations between them (Fig. 8b). Importantly, the chemophysical trajectory did not correlate significantly with the iron or volume aging patterns. The spatial distribution of water-related changes was found to correlate with iron content alterations (R2 = 0.27) and chemophysical alterations (R2 = 0.25). However, the strongest correlation between aging-related changes was found in volume and iron content (R2 = 0.77). As shown previously, this correlation may be explained to some extent by a systematic bias in automated tissue classification23. Additional analysis revealed that the different dimensions of the MDM signature capture distinct patterns of aging-related changes (Supplementary Fig. 30). Hence, complementary information regarding the various chemophysical mechanisms underlying brain aging could be gained by combining them.

 

Discussion

Normal brain aging involves multiple changes, at both the microscale and macroscale level. MRI is the main tool for in vivo evaluation of such age-related changes in the human brain. Here, we propose to improve the interpretation of MRI findings by accounting for the fundamental effect of the water content on the imaging parameters. This approach allows for non-invasive mapping of the molecular composition in the aging human brain.

Our work is part of a major paradigm shift in the field of MRI toward in vivo histology30,36,52. The MDM approach contributes to this important change by providing a hypothesis-driven biophysical framework that was rigorously developed. We demonstrated the power of our framework, starting from simple pure lipid phantoms to more complicated lipid mixtures, and from there, to the full complexity of the brain. In the brain, we show both in vivo and post-mortem validations for the molecular sensitivity of the MDM signatures. Early observations relate different qMRI parameters to changes in the fraction of myelin20,23,30,31,32,33,36. The current approach enriches this view and provides better sensitivity to the molecular composition and fraction of myelin and other cellular tissues.

We developed a unique phantom system of lipid samples to validate our method. While the phantom system is clearly far from the complexity of brain tissue, its simplicity allowed us to verify the specificity of our method to the chemophysical environment. Remarkably, our approach revealed unique signatures for different lipids, and is therefore sensitive even to relatively subtle details that distinguish one lipid from another. We chose to validate our approach using membrane lipids based on previous experiments40,41,42,43,44,45. Nevertheless, we do acknowledge the fact that brain tissue comprises many other compounds beside lipids, such as proteins, sugars, and ions. As we have shown, these other compounds also exhibit unique dependency on MTV. The effect of such compounds, along with other factors such as microstructure, and multi-compartment organization28 is probably captured when we apply the MDM approach to the in vivo human brain. Therefore, the phantoms were made to examine the MRI sensitivity for the chemophysical environment, and the human brain data was used to measure the true biological effects in a complex in vivo environment.

Our relaxivity approach captures the molecular signatures of the tissue, but is limited in its abilities to describe the full complexity of the chemophysical environment of the human brain. For example, R1 and R2, which are used to generate the MDM signatures, are also sensitive to the iron content23,48,52. However, we found that most of our findings cannot be attributed to alterations in iron content as measured with R2* (for more details see Supplementary Note 5). While there is great importance in further isolating different molecular components, we argue that accounting for the major effect of water on qMRI parameters (for R2 distributions see Supplementary Fig. 5) is a crucial step towards more specific qMRI interpretation.

We provide evidence from lipids samples and post-mortem data for the sensitivity of the MDM signatures to the molecular environment (Figs. 1e, 3b, and 5h). The variability of MDM values between human brain regions also correlated with specific gene-expression profiles (Fig. 4). While the comparison of in vivo human brain measurements to previously published ex vivo findings is based on two different datasets, these measurements are highly stable across normal subjects and the intersubject variabilities are much smaller than the regional variability. The agreement between the modalities provides strong evidence for the ability of our method to capture molecular information.

Remarkably, we were able to demonstrate the sensitivity of MDM signatures to lipid composition using direct comparison on post-mortem porcine brains. Even though there are many challenges in scanning post-mortem tissue, segmenting it, and comparing it to anatomically relevant histological results, we were able to replicate our in vivo findings. We provide histological validation for the MRI estimation of MTV. Moreover, we find that while standard qMRI parameters and MTV do not explain the lipidomic variability across the brain, the MDM signatures are in agreement with histological results. Lipids constitute the majority of the brain’s dry weight and are known to be important for maintaining neural conduction and chemical balance53,54. The brain lipidome was shown to have a great deal of structural and functional diversity and was found to vary according to age, gender, brain region, and cell type55. Disruptions of the brain lipid metabolism have been linked to different disorders, including Alzheimer’s disease, Parkinson’s disease, depression, and anxiety7,8,11,54,55,56,57. Our results indicate that the MDM approach enhances the consistency between MRI-driven measurements and lipidomics, compared with standard qMRI parameters.

The simplicity of our model, which is based on a first-order approximation of qMRI dependencies, has great advantages in the modeling of complex environments. Importantly, we used lipids samples to show that the contributions of different mixture-components can be summed linearly (Fig. 1d). For contrast agents, the relaxivity is used to characterize the efficiency of different agents. Here, we treated the tissue itself, rather than a contrast material, as an agent to compute the relaxivity of the tissue. While relaxivity is usually calculated for R1 and R2, we extended this concept to other qMRI parameters. Our results showed that the tissue relaxivity changes as a function of the molecular composition. This suggests that the relaxivity of the tissue relates to the surface interaction between the water and the chemophysical environment. A theoretical formulation for the effect of the surface interaction on proton relaxation has been proposed before58,59. Specifically, a biophysical model for the linear relationship between R1 and R2 to the inverse of the water content (1/WC = 1/(1 – MTV)) was suggested by Fullerton et al.43. Interestingly, 1/WC varies almost linearly with MTV in the physiological range of MTV values. Applying our approach with 1/WC instead of MTV produces relatively similar results (Supplementary Fig. 28). However, using MTV as a measure of tissue relaxivity allowed us to generalize the linear model to multiple qMRI parameters, thus producing multidimensional MDM signatures.

We show that the MDM signatures allow for better understanding of the biological sources for the aging-related changes observe with MRI. Normal brain aging involves multiple changes, at both the microscale and macroscale levels. Measurements of macroscale brain volume have been widely used to characterize aging-associated atrophy. Our method of analysis can complement such findings and provide a deeper understanding of microscale processes co-occurring with atrophy. Moreover, it allows us to test whether these various microscale and macroscale processes are caused by a common factor or represent the aging mosaic. Notably, we discovered that different brain regions undergo different biological aging processes. Therefore, combining several measurements of brain tissue is crucial in order to fully describe the state of the aged brain. For example, the macroscale aging-related volume reduction in cortical gray areas was accompanied by conserved tissue density, as estimated by MTV, and region-specific chemophysical changes, as estimated by the MDM. In contrast, in white-matter areas both MDM and MTV changed with age. These microscale alterations were not accompanied by macroscale volume reduction. Our in vivo results were validated by previous histological studies, which reported that the cortex shrinks with age, while the neural density remains relatively constant49,50. In contrast, white matter was found to undergo significant loss of myelinated nerve fibers during aging51. In addition, we found that the shrinkage of the hippocampus with age is accompanied with conserved tissue density and chemophysical composition. This is in agreement with histological findings, which predict drastic changes in hippocampal tissue composition in neurological diseases such as Alzheimer, but not in normal aging49,50,60,61. In contrast, hippocampal macroscale volume reduction was observed in both normal and pathological aging2.

It should be noted that most of the human subjects recruited for this study were from the academic community. However, the different age groups were not matched for variables such as IQ and socioeconomic status. In addition, the sample size in our study was quite small. Therefore, the comparison we made between the two age groups may be affected by variables other than age. Our approach may benefit from validation based on larger quantitative MRI datasets27,62. Yet, we believe we have demonstrated the potential of our method to reveal molecular alterations in the brain. Moreover, the agreement of our findings with previous histological aging studies supports the association between the group differences we measured and brain aging. Our results suggest that the MDM approach may be very useful in differentiating the effects of normal aging from those of neurodegenerative diseases. There is also great potential for applications in other brain research fields besides aging. For example, our approach may be used to advance the study and diagnosis of brain cancer, in which the lipidomic environment undergoes considerable changes63,64,65.

To conclude, we have presented here a quantitative MRI approach that decodes the molecular composition of the aging brain. While common MRI measurements are primarily affected by the water content of the tissue, our method employed the tissue relaxivity to expose the sensitivity of MRI to the molecular microenvironment. We presented evidence from lipid samples, post-mortem porcine brains and in vivo human brains for the sensitivity of the tissue relaxivity to molecular composition. Results obtained by this method in vivo disentangled different biological processes occurring in the human brain during aging. We identified region-specific patterns of microscale aging-related changes that are associated with the molecular composition of the human brain. Moreover, we showed that, in agreement with the mosaic theory of aging, different biological age-related processes measured in vivo have unique spatial patterns throughout the brain. The ability to identify and localize different age-derived processes in vivo may further advance human brain research.

Methods

Phantom construction
The full protocol of lipids phantom preparation is described in Shtangel et al.66.

In short, we prepared liposomes from one of the following lipids: phosphatidylserine (PS), phosphatidylcholine (PtdCho), phosphatidylcholine-cholesterol (PtdCho-Chol), Phosphatidylinositol-phosphatidylcholine (PI-PtdCho), or sphingomyelin (Spg). These phantoms were designed to model biological membranes and were prepared from lipids by the hydration–dehydration dry film technique67. The lipids were dissolved over a hot plate and vortexed. Next, the solvent was removed to create a dry film by vacuum-rotational evaporation. The samples were then stirred on a hot plate at 65 °C for 2.5 h to allow the lipids to achieve their final conformation as liposomes. Liposomes were diluted with Dulbecco’s phosphate buffered saline (PBS), without calcium and magnesium (Biological Industries), to maintain physiological conditions in terms of osmolarity, ion concentrations and pH. To change the MTV of the liposome samples we varied the PBS to lipid volume ratios66. Samples were then transferred to the phantom box for scanning in a 4 mL squared polystyrene cuvettes glued to a polystyrene box, which was then filled with ~1% SeaKem Agarose (Ornat Biochemical) and ~0.0005 M Gd (Gadotetrate Melumine, (Dotarem, Guerbet)) dissolved in double distilled water (ddw). The purpose of the agar with Gd (Agar-Gd) was to stabilize the cuvettes, and to create a smooth area in the space surrounding the cuvettes that minimalized air–cuvette interfaces. In some of our experiments we used lipid mixtures composed of several lipids. We prepared nine mixtures containing different combinations of two out of three lipids (PtdChol, Spg and PS) in varying volume ratios (1:1,1:2,2:1). For each mixture, we prepared samples in which the ratio between the different lipid components remained constant while the water-to-lipid volume fraction varied.

For the bovine serum albumin (BSA) phantoms, samples were prepared by dissolving lyophilized BSA powder (Sigma Aldrich) in PBS. To change the MTV of these phantoms, we changed the BSA concentration. For the BSA + Iron phantoms, BSA was additionally mixed with a fixed concentration of 50 µg/mL ferrous sulfate heptahydrate (FeSO4*7H2O). Samples were prepared in their designated concentrations at room temperature. Prepared samples were allowed to sit overnight at 4 ℃ to ensure BSA had fully dissolved, without the need for significant agitation, which is known to cause protein cross-linking. Samples were then transferred to the phantom box for scanning.

For Glucose and Sucrose phantoms, different concentrations of D-( + )-Sucrose (Bio-Lab) and D-( + )-Glucose (Sigma) were dissolved in PBS at 40 ℃. Samples were allowed to reach room temperature before the scan.

MRI acquisition for phantoms

Data was collected on a 3 T Siemens MAGNETOM Skyra scanner equipped with a 32-channel head receive-only coil at the ELSC neuroimaging unit at the Hebrew University.

For quantitative R1 & MTV mapping, three-dimensional (3D) Spoiled gradient (SPGR) echo images were acquired with different flip angles (α = 4°, 8°, 16°, and 30°). The TE/TR was 3.91/18 ms. The scan resolution was 1.1 × 1.1 × 0.9 mm. The same sequence was repeated with a higher resolution of 0.6 × 0.6 × 0.5 mm. The TE/TR was 4.45/18 ms. For calibration, we acquired an additional spin-echo inversion recovery (SEIR) scan. This scan was done on a single slice, with adiabatic inversion pulse and inversion times of TI = 2000, 1200, 800, 400, and 50. The TE/TR was 73/2540 ms. The scan resolution was 1.2 mm isotropic.

For quantitative T2 mapping, images were acquired with a multi spin-echo sequence with 15 equally spaced spin echoes between 10.5 ms and 157.5 ms. The TR was 4.94 s. The scan resolution was 1.2 mm isotropic. For quantitative MTsat mapping, images were acquired with the FLASH Siemens WIP 805 sequence. The TR was 23 ms for all samples except PI:PtdCho for which the TR was 72 ms. Six echoes were equally spaced between 1.93 ms to 14.58 ms. The on-resonance flip angle was 6°, the MT flip angle was 220°, and the RF offset was 700. We used 1.1-mm in-plane resolution with a slice thickness of 0.9 mm. For samples of sucrose and glucose, MTsat mapping was done similar to the human subjects, based on 3D Spoiled gradient (SPGR) echo image with an additional MT pulse. The flip angle was 10°, the TE/TR was 3.91/28 ms. The scan resolution was 1 mm isotropic.

Estimation of qMRI parameters for phantoms

MTV and R1 estimations for the lipids samples were computed based on a the mrQ39 (https://github.com/mezera/mrQ) and Vista Lab (https://github.com/vistalab/vistasoft/wiki) software. The mrQ software was modified to suit the phantom system66. The modification utilizes the fact that the Agar-Gd filling the box around the samples is homogeneous and can, therefore, be assumed to have a constant T1 value. We used this gold standard T1 value generated from the SEIR scan to correct for the excite bias in the spoiled gradient echo scans. While the data was acquired in two different resolutions (see “MRI acquisition”), in our analysis we use the median R1 and MTV of each lipid sample and these are invariant to the resolution of acquisition (Supplementary Fig. 1e). Thus, we were able to use scans with different resolutions without damaging our results. T2 maps were computed by implementing the echo‐modulation curve (EMC) algorithm68.

For quantitative MTsat mapping see the “MTsat estimation” section for human subjects.

MDM computation for phantoms

We computed the dependency of each qMRI parameter (R1, MTsat, and R2) on MTV in different lipids samples. This process was implemented in MATLAB (MathWorks, Natwick, MI, USA). To manipulate the MTV values, we scanned samples of the same lipid in varying concentrations. We computed the median MTV of each sample, along with the median of qMRI parameters. We used these data points to fit a linear model across all samples of the same lipid. The slope of this linear model represents the MTV derivative of the linear equation. We used this derivative estimate of three qMRI parameters (R1, R2, and MTsat) to compute the MDM signatures. The same procedure was used for the MDM computation of lipid mixtures.

 

MDM modeling of lipid mixtures

We tested the ability of MDM to predict the composition of lipid mixtures. For this analysis we used nine mixture phantoms (see “Phantom construction”), along with the three phantoms of the pure lipid constituents of the mixtures (PS, Spg, and Ptd-Cho).

In order to predict the qMRI parameters of a lipid mixture (Fig. 1d) we used Supplementary Eq. 1 (Supplementary Note 1). To further predict the composition of the mixtures (Fig. 1e) we used Supplementary Eq. 5 (Supplementary Note 2). We solved this equation using the QR factorization algorithm.

Ethics

Human experiments complied with all relevant ethical regations. The Helsinki Ethics Committee of Hadassah Hospital, Jerusalem, Israel approved the experimental procedure. Written informed consent was obtained from each participant prior to the procedure.

Human subjects

Human measurements were performed on 23 young adults (aged 27 ± 2 years, 11 females), and 18 older adults (aged 67 ± 6 years, five females). Healthy volunteers were recruited from the community surrounding the Hebrew University of Jerusalem.

MRI acquisition for human subjects

Data was collected on a 3 T Siemens MAGNETOM Skyra scanner equipped with a 32-channel head receive-only coil at the ELSC neuroimaging unit at the Hebrew University.

For quantitative R1, R2*, & MTV mapping, 3D Spoiled gradient (SPGR) echo images were acquired with different flip angles (α = 4°, 10°, 20°, and 30°). Each image included five equally spaced echoes (TE = 3.34–14.02 ms) and the TR was 19 ms (except for six young subjects for which the scan included only one TE = 3.34 ms). The scan resolution was 1 mm isotropic. For calibration, we acquired additional spin-echo inversion recovery scan with an echo-planar imaging (EPI) read-out (SEIR-epi). This scan was done with a slab-inversion pulse and spatial-spectral fat suppression. For SEIR-epi, the TE/TR was 49/2920 ms. TI were 200, 400, 1,200, and 2400 ms. We used 2-mm in-plane resolution with a slice thickness of 3 mm. The EPI read-out was performed using 2 × acceleration.

For quantitative T2 mapping, multi‐SE images were acquired with ten equally spaced spin echoes between 12 ms and 120 ms. The TR was 4.21 s. The scan resolution was 2 mm isotropic. T2 scans of four subjects (one young, three old) were excluded from the analysis due to motion.

For quantitative MTsat mapping, 3D Spoiled gradient (SPGR) echo image were acquired with an additional MT pulse. The flip angle was 10°, the TE/TR was 3.34/27 ms. The scan resolution was 1 mm isotropic.

Whole-brain DTI measurements were performed using a diffusion-weighted spin-echo EPI sequence with isotropic 1.5-mm resolution. Diffusion weighting gradients were applied at 64 directions and the strength of the diffusion weighting was set to b = 2000 s/mm2 (TE/TR = 95.80/6000 ms, G = 45mT/m, δ = 32.25 ms, Δ = 52.02 ms). The data includes eight non-diffusion-weighted images (b = 0). In addition, we collected non-diffusion-weighted images with reversed phase-encode blips. For five subjects (four young, one old) we failed to acquire this correction data and they were excluded from the diffusion analysis.

Anatomical images were acquired with 3D magnetization prepared rapid gradient echo (MP-RAGE) scans for 24 of the subjects (14 from the younger subjects, 10 from the older subjects). The scan resolution was 1 mm isotropic, the TE/TR was 2.98/2300 ms. Magnetization Prepared 2 Rapid Acquisition Gradient Echoes (MP2RAGE) scans were acquired for the rest of the subjects. The scan resolution was 1 mm isotropic, the TE/TR was 2.98/5000 ms.

Estimation of qMRI parameters for human subjects

Whole-brain MTV and R1 maps, together with bias correction maps of B1 + and B1-, were computed using the mrQ software39,69 (https://github.com/mezera/mrQ). Voxels in which the B1 + inhomogeneities were extrapolated and not interpolated were removed from the MTV and R1 maps. While we did not correct our MTV estimates for R2*, we showed that employing such a correction does not significantly change our results (see Supplementary Note 6, Supplementary Figs. 20–27). MTV maps of four subjects had bias in the lower part of the brain and they were therefore excluded from the analysis presented in Fig. 3, which includes ROIs in the brainstem.

Whole-brain T2 maps were computed by implementing the echo‐modulation curve (EMC) algorithm68. To combine the MTV and T2 we co-registered the quantitative MTV map to the T2 map. We used the ANTS software package70 to calculate the transformation and to warp the MTV map and the segmentation. The registration was computed to match the T1 map to the T2 map. Next, we applied the calculated transformation to MTV map (since MTV and T1 are in the same imaging space) and resampled the MTV map to match the resolution of the T2 map. The same transformation was also applied to the segmentation. R2 maps were calculated as 1/T2.

Whole-brain MTsat maps were computed as described in Helms et al.37. The MTsat measurement was extracted from Eq. (1):

MTsat=𝑀0𝐵1𝛼𝑅1TR𝑆MT−(𝐵1𝛼)22−𝑅1TR
(1)
Where SMT is the signal of the SPGR scan with additional MT pulse, α is the flip angle and TR is the repetition time. Mo (the equilibrium magnetization parameter), B1 (the transmit inhomogeneity), and R1 estimations were computed from the non-MT weighted SPGR scans, during the pipeline described under “MTV & R1 estimation”. Registration of the SMT image to the imaging space of the MTV map was done using a rigid-body alignment (R1, B1, and MO are all in the same space as MTV).

Diffusion analysis was done using the FDT toolbox in FSL71,72. Susceptibility and eddy current induced distortions were corrected using the reverse phase-encode data, with the eddy and topup commands73,74. MD maps were calculated using vistasoft (https://github.com/vistalab/vistasoft/wiki). We used a rigid-body alignment to register the corrected dMRI data to the imaging space of the MTV map (Flirt, FSL). In order to calculate the MD-MTV derivatives, we resampled the MTV map and the segmentation to match the dMRI resolution.

We used the SPGR scans with multiple echoes to estimate R2*. Fitting was done through the MPM toolbox75. As we had four SPGR scans with variable flip angles, we averaged the R2* maps acquired from each of these scans for increased SNR.

Human brain segmentation

Whole-brain segmentation was computed automatically using the FreeSurfer segmentation algorithm76. For subjects who had an MP-RAGE scan, we used it as a reference. For the other subjects the MP2RAGE scan was used as a reference. These anatomical images were registered to the MTV space prior to the segmentation process, using a rigid-body alignment. Sub-cortical gray-matter structures were segmented with FSL’s FIRST tool77. To avoid partial volume effects, we removed the outer shell of each ROI and left only the core.

MDM computation in the human brain

We computed the dependency of each qMRI parameter (R1, MTsat, MD, and R2) on MTV in different brain areas. This process was implemented in MATLAB (MathWorks, Natwick, MI, USA). For each ROI, we extracted the MTV values from all voxels and pooled them into 36 bins spaced equally between 0.05 and 0.40. This was done so that the linear fit would not be heavily affected by the density of the voxels in different MTV values. We removed any bins in which the number of voxels was smaller than 4% of the total voxel count in the ROI. The median MTV of each bin was computed, along with the median of the qMRI parameter. We used these data points to fit the linear model across bins using Eq. (2):

qMRIparameters=𝑎∗MTV+𝑏
(2)
The slope of this linear model (“a”) represents the MTV derivative of the linear equation. We used this derivative estimate to compute the MDM signatures.

For each subject, ROIs in which the total voxel count was smaller than a set threshold of 500 voxels for the MTsat and R1 maps, 150 voxels for the MD map, and 50 voxels for the R2 map were excluded.

Principal component analysis (PCA) in the human brain

To estimate the variability in the MDM signatures across the brain, we computed the first principal component (PC) of MDM. For each MDM dimension (MTV derivatives of R1, MTsat, MD, and R2), we evaluated the median of the different brain areas across the young subjects. As each MDM dimension has different units, we then computed the z-score of each dimension across the different brain area. Finally, we performed PCA. The variables in this analysis were the different MDM dimensions, and the observations were the different brain areas. From this analysis, we derived the first PC that accounts for most of the variability in MDM signatures across the brain. To estimate the median absolute deviations (MAD) across subjects of each MDM measurement in the PC basis, we applied the z-score transformation to the original MAD and then projected them onto the PC basis.

To compute the first PC of standard qMRI parameters we followed the same procedure, but used R1, MTsat, MD, and R2 instead of their MTV derivatives.

For the first PC of molecular composition, we followed the same procedure, but used the phospholipid composition and the ratio between phospholipids to proteins and cholesterol as variables. The data was taken from eight post-mortem human brains7. Brains were obtained from individuals between 54 and 57 years of age, which were autopsied within 24 h after death.

Linear model for prediction of human molecular composition

We used MDM measurements in order to predict the molecular composition of different brain areas (Fig. 3c). For this analysis we used Supplementary Eq. 5 in the Supplementary Note 2. We solved this equation using QR factorization algorithm (for more details see Supplementary Note 3).

Gene-expression dataset

For the gene-expression analysis we followed the work of Ben-David and Shifman46. Microarray data was acquired from the Allen Brain Atlas (http://human.brain-map.org/well_data_files) and included a total of 1340 microarray profiles from donors H0351.2001 and H0351.2002, encompassing the different regions of the human brain. The donors were 24 and 39 years old, respectively, at the time of their death, with no known psychopathologies. We used the statistical analysis described by Ben-David and Shifman46. They constructed a gene network using a weighted gene co-expression network analysis. The gene network included 19 modules of varying sizes, from 38 to 7385 genes. The module eigengenes were derived by taking the first PC of the expression values in each module. In addition, we used the gene ontology enrichment analysis described by Ben-David and Shifman to define the name of each module. The colors of the different modules in the Fig. 4 and Supplementary Fig. 10 are the same as in the original paper.

Next, we matched between the gene-expression data and the MRI measurements. This analysis was done on 35 cortical regions extracted from FreeSurfer cortical parcellation. We downloaded the T1-weighted images of the two donors provided by the Allen Brain Atlas (http://human.brain-map.org/mri_viewers/data) and used them as a reference for FreeSurfer segmentation. We then found the FreeSurfer label of each gene-expression sample using the sample’s coordinates in brain space. We removed samples for which the FreeSurfer label and the label provided in the microarray dataset did not agree (there were 72 such samples out of 697 cortical samples). For each gene module, we averaged over the eigengenes of all samples from the same cortical area across the two donors.

Last, we compared the cortical eigengene of each module to the projection of cortical areas on the first PC of MDM. In addition, we compared the modules’ eigengenes to the MTV values of the cortical areas and to the projection of cortical areas on the first PC of standard qMRI parameters (Supplementary Fig. 10). These 57 correlations were corrected for multiple comparisons using the FDR method.

Brain region’s volume computation

To estimate the volume of different brain regions, we calculated the number of voxels in the FreeSurfer segmentation of each region (see “Brain segmentation”).

R2* correction for MTV
To correct the MTV estimates for R2* we used Eq. (3):

MTV𝐶=1−(1−MTV)⋅exp(TE⋅R2∗)
(3)
Where MTVC is the corrected MTV.

Statistical analysis

The statistical significance of the differences between the age groups was computed using an independent-sample t-test (alpha = 0.05, both right and left tail) and was corrected for multiple comparisons using the false-discovery rate (FDR) method. For this analysis, MRI measurements of both hemispheres of bilateral brain regions were joined together. R2 measurements were adjusted for the number of data points. All statistical tests were two-sided.

Post-mortem tissue acquisition

Two post-mortem porcine brains were purchased from BIOTECH FARM.

Post-mortem MRI acquisition

Brains were scanned fresh (without fixation) in water within 6 h after death. Data was collected on a 3 T Siemens MAGNETOM Skyra scanner equipped with a 32-channel head receive-only coil at the ELSC neuroimaging unit at the Hebrew University.

For quantitative R1, R2*, & MTV mapping, 3D Spoiled gradient (SPGR) echo images were acquired with different flip angles (α = 4°, 10°, 20°, and 30°). Each image included five equally spaced echoes (TE = 4.01 – 16.51 ms) and the TR was 22 ms. The scan resolution was 0.8 mm isotropic. For calibration, we acquired additional spin-echo inversion recovery scan with an echo-planar imaging (EPI) read-out (SEIR-epi). This scan was done with a slab-inversion pulse and spatial-spectral fat suppression. For SEIR-epi, the TE/TR was 49/2920 ms. TI were 50, 200, 400, 1200 ms. The scan resolution was 2 mm isotropic. The EPI read-out was performed using 2 × acceleration.

For quantitative T2 mapping, multi‐SE images were acquired with ten equally spaced spin echoes between 12 and 120 ms. The TR was 4.21 s. The scan resolution was 2 mm isotropic.

For quantitative MTsat mapping, 3D Spoiled gradient (SPGR) echo image were acquired with an additional MT pulse. The flip angle was 10°, the TE/TR was 4.01/40 ms. The scan resolution was 0.8 mm isotropic.

Whole-brain DTI measurements were performed using a diffusion-weighted spin-echo EPI sequence with isotropic 1.5-mm resolution. Diffusion weighting gradients were applied at 64 directions and the strength of the diffusion weighting was set to b = 2000 s/mm2 (TE/TR = 95.80/6000 ms, G = 45mT/m, δ = 32.25 ms, Δ = 52.02 ms). The data includes eight non-diffusion-weighted images (b = 0).

For anatomical images, 3D magnetization prepared rapid gradient echo (MP-RAGE) scans were acquired. The scan resolution was 1 mm isotropic, the TE/TR was 2.98/2300 ms.

Histological analysis

Following the MRI scans the brains were dissected. Total of 42 brain regions were identified. Four samples were excluded as we were not able to properly separate the WM from the GM. One sample was excluded as we could not properly identify its anatomical origin. Additional two samples were too small for TLC analysis.

The non-water fraction (MTV) was determined by desiccation, also known as the dry-wet method. A small fraction of each brain sample (~0.25 g) was weighed. In order to completely dehydrate the fresh tissues, they were left for several days in a vacuum dessicator over silica gel at 4 °C. The experiment ended when no further weight loss occurred. The MTV of each brain sample was calculated based on the difference between the wet (Wwet) and dry (Wdry) weights of the tissue (Eq. 4):

MTV=𝑊wet−𝑊dry𝑊wet
(4)
For lipid extraction and lipidomics analysis78, Brain samples were weighted and homogenized with saline in plastic tubes on ice at concentration of 1 mg/12.5 µL. Two-hundred fifty microliters from each homogenate were utilized for lipid extraction and analysis with thin-layer chromatography (TLC). The lipid species distribution was analyzed by TLC applying 150 µg aliquots. Samples were reconstituted in 10 µL of Folch mixture and spotted on Silica-G TLC plates. Standards for each fraction were purchased from Sigma Aldrich (Rehovot, Israel) and were spotted in separate TLC lanes, i.e., 50 µg of triacylglycerides (TG), cholesterol (Chol), cholesteryl esters (CE), free fatty acids (FFA), lysophospholipids (Lyso), sphingomyelin (Spg), phosphatidylcholine (PtdCho), phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidylethanolamine (PE). Plates were then placed in a 20 × 20 cm TLC chamber containing petroleum ether, ethyl ether, and acetic acid (80:20:1, v/v/v) for quantification of neutral lipids or chloroform, methanol, acetic acid, and water (65:25:4:2, v:v:v:v) for quantification of polar lipids and run for 45 min. TG, Chol, CE, FFA, phospholipids (PL), Lyso, Spg, PtdCho, PI, PS, and PE bands were visualized with Iodine, scanned and quantified by Optiquant after scanning (Epson V700). Lyso, CE, TG, and PI were excluded from further analysis as their quantification was noisy and demonstrated high variability across TLC plates. This analysis was conducted under the guidance of Prof. Alicia Leikin-Frenkel in the Bert Strassburger Lipid Center, Sheba, Tel Hashomer.

Estimation of qMRI parameters in the post-mortem brain

Similar to human subjects.

Brain segmentation of post-mortem brain

Brain segmentation was done manually. Five tissue samples were excluded as we could not identify their origin location in the MRI scans.

MDM computation in the post-mortem brain

We computed the dependency of each qMRI parameter (R1, MTsat, MD, and R2) on MTV in different brain areas similarly to the analysis of the human subjects.

Principal component analysis (PCA) in the post-mortem brain

To estimate the variability in the MDM signatures across the brain, we computed the first principal component (PC) of MDM. PCA analysis was performed with four variables corresponding to the MDM dimensions (MTV derivatives of R1, MTsat, MD, and R2), and 30 observations corresponding to the different brain regions. As each MDM dimension has different units, we first computed the z-score of each dimension across the different brain areas prior to the PCA. From this analysis we derived the first PC that accounts for most of the variability in MDM signatures across the brain.

To compute the first PC of standard qMRI parameters we followed the same procedure, but used R1, MTsat, MD, and R2 instead of their MTV derivatives.

To estimate the variability in the lipid composition across the brain, we computed the first principal component (PC) of lipidomics. PCA analysis was performed with seven variables corresponding to the different polar and neutral lipids (Chol, FFA, PL, Spg, PtdCho, PS, PE), and 30 observations corresponding to the different brain regions. From this analysis, we derived the first PC that accounts for most of the variability in lipid composition across the brain.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Code availability

A toolbox for computing MDM signatures is available at [https://github.com/MezerLab/MDM_toolbox].

The code generating the figures of in the paper is available at [https://github.com/MezerLab/MDM_Gen_Figs].

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Acknowledgements

This work was supported by the ISF grant 0399306, awarded to A.A.M. We acknowledge Ady Zelman for the assistance in collecting the human MRI data. We thank Assaf Friedler for assigning research lab space and advising on the lipid sample experiments. We thank Inbal Goshen for assigning research lab space and advising on the protein and ion samples as well as the porcine brain experiments. We thank Magnus Soderberg for advising on histological data interpretation. We are grateful to Brian A. Wandell, Jason Yeatman, Hermona Soreq, Ami Citri, Mark Does, Yaniv Ziv, Ofer Yizhar, Shai Berman, Roey Schurr, Jonathan Bain, Asier Erramuzpe Aliaga, Menachem Gutman, and Esther Nachliel for their critical reading of the manuscript and very useful comments. We thank Prof. Alicia Leikin-Frenkel for her guidance with the TLC analysis. We thank Rona Shaharabani for guidance and support in the post-mortem experiments.

Affiliations

The Edmond and Lily Safra Center for Brain Sciences, The Hebrew University of Jerusalem, Jerusalem, 9190401, Israel
Shir Filo, Oshrat Shtangel, Noga Salamon, Adi Kol, Batsheva Weisinger & Aviv A. Mezer
Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, 9190401, Israel
Sagiv Shifman
Contributions
S.F., O.S., and A.A.M. conceived of the presented idea. S.F. and A.A.M. wrote the manuscript and designed the figures. S.F. collected the human and non-human brain datasets and analyzed them. O.S. performed the phantom experiments and analyzed them. B.W. performed the phantom experiments for non-lipid compounds. N.S. performed the gene-expression analysis. S.S. assisted and instructed with the gene-expression analysis. A.K. performed the porcine brain dissection.

Corresponding author

Correspondence to Aviv A. Mezer.

Ethics declarations & Competing interests

A.A.M, S.F., O.S. and the Hebrew University of Jerusalem have filed a patent application describing the technology used to measure MDM in this work. The other authors declare no competing interests.

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An Intelligent DNA Nanorobot to Fight Cancer by Targeting HER2 Expression

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

HER2 is an important prognostic biomarker for 20–30% of breast cancers, which is the most common cancer in women. Overexpression of the HER2 receptor stimulates breast cells to proliferate and differentiate uncontrollably, thereby enhancing the malignancy of breast cancer and resulting in a poor prognosis for affected individuals. Current therapies to suppress the overexpression of HER2 in breast cancer mainly involve treatment with HER2-specific monoclonal antibodies. However, these monoclonal anti-HER2 antibodies have severe side effects in clinical trials, such as diarrhea, abnormal liver function, and drug resistance. Removing HER2 from the plasma membrane or inhibiting the gene expression of HER2 is a promising alternative that could limit the malignancy of HER2-positive cancer cells.

 

DNA origami is an emerging field of DNA-based nanotechnology and intelligent DNA nanorobots show great promise in working as a drug delivery system in healthcare. Different DNA-based nanorobots have been developed as affordable and facile therapeutic drugs. In particular, many studies reported that a tetrahedral framework nucleic acid (tFNA) could serve as a promising DNA nanocarrier for many antitumor drugs, owing to its high biocompatibility and biosecurity. For example, tFNA was reported to effectively deliver paclitaxel or doxorubicin to cancer cells for reversing drug resistance, small interfering RNAs (siRNAs) have been modified into tFNA for targeted drug delivery. Moreover, the production and storage of tFNA are not complicated, and they can be quickly degraded in lysosomes by cells. Since both free HApt and tFNA can be diverted into lysosomes, so,  combining the HApt and tFNA as a novel DNA nanorobot (namely, HApt-tFNA) can be an effective strategy to improve its delivery and therapeutic efficacy in treating HER2-positive breast cancer.

 

Researchers reported that a DNA framework-based intelligent DNA nanorobot for selective lysosomal degradation of tumor-specific proteins on cancer cells. An anti-HER2 aptamer (HApt) was site-specifically anchored on a tetrahedral framework nucleic acid (tFNA). This DNA nanorobot (HApt-tFNA) could target HER2-positive breast cancer cells and specifically induce the lysosomal degradation of the membrane protein HER2. An injection of the DNA nanorobot into a mouse model revealed that the presence of tFNA enhanced the stability and prolonged the blood circulation time of HApt, and HApt-tFNA could therefore drive HER2 into lysosomal degradation with a higher efficiency. The formation of the HER2-HApt-tFNA complexes resulted in the HER2-mediated endocytosis and digestion in lysosomes, which effectively reduced the amount of HER2 on the cell surfaces. An increased HER2 digestion through HApt-tFNA further induced cell apoptosis and arrested cell growth. Hence, this novel DNA nanorobot sheds new light on targeted protein degradation for precision breast cancer therapy.

 

It was previously reported that tFNA was degraded by lysosomes and could enhance cell autophagy. Results indicated that free Cy5-HApt and Cy5-HApt-tFNA could enter the lysosomes; thus, tFNA can be regarded as an efficient nanocarrier to transmit HApt into the target organelle. The DNA nanorobot composed of HApt and tFNA showed a higher stability and a more effective performance than free HApt against HER2-positive breast cancer cells. The PI3K/AKT pathway was inhibited when membrane-bound HER2 decreased in SK-BR-3 cells under the action of HApt-tFNA. The research findings suggest that tFNA can enhance the anticancer effects of HApt on SK-BR-3 cells; while HApt-tFNA can bind to HER2 specifically, the compounded HER2-HApt-tFNA complexes can then be transferred and degraded in lysosomes. After these processes, the accumulation of HER2 in the plasma membrane would decrease, which could also influence the downstream PI3K/AKT signaling pathway that is associated with cell growth and death.

 

However, some limitations need to be noted when interpreting the findings: (i) the cytotoxicity of the nanorobot on HER2-positive cancer cells was weak, and the anticancer effects between conventional monoclonal antibodies and HApt-tFNA was not compared; (ii) the differences in delivery efficiency between tFNA and other nanocarriers need to be confirmed; and (iii) the confirmation of anticancer effects of HApt-tFNA on tumors within animals remains challenging. Despite these limitations, the present study provided novel evidence of the biological effects of tFNA when combined with HApt. Although the stability and the anticancer effects of HApt-tFNA may require further improvement before clinical application, this study initiates a promising step toward the development of nanomedicines with novel and intelligent DNA nanorobots for tumor treatment.

 

References:

 

https://pubs.acs.org/doi/10.1021/acs.nanolett.9b01320

 

https://www.ncbi.nlm.nih.gov/pubmed/27939064

 

https://www.ncbi.nlm.nih.gov/pubmed/11694782

 

https://www.ncbi.nlm.nih.gov/pubmed/27082923

 

https://www.ncbi.nlm.nih.gov/pubmed/25365825

 

https://www.ncbi.nlm.nih.gov/pubmed/26840503

 

https://www.ncbi.nlm.nih.gov/pubmed/29802035

 

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