Benton G.,Trevigen, Inc. |
Arnaoutova I.,Trevigen, Inc. |
George J.,Trevigen, Inc. |
Kleinman H.K.,George Washington University |
Koblinski J.,Virginia Commonwealth University
Advanced Drug Delivery Reviews | Year: 2014
The basement membrane is an important extracellular matrix that is found in all epithelial and endothelial tissues. It maintains tissue integrity, serves as a barrier to cells and to molecules, separates different tissue types, transduces mechanical signals, and has many biological functions that help to maintain tissue specificity. A well-defined soluble basement membrane extract, termed BME/Matrigel, prepared from an epithelial tumor is similar in content to authentic basement membrane, and forms a hydrogel at 24-37. °C. It is used in vitro as a substrate for 3D cell culture, in suspension for spheroid culture, and for various assays, such as angiogenesis, invasion, and dormancy. In vivo, BME/Matrigel is used for angiogenesis assays and to promote xenograft and patient-derived biopsy take and growth. Studies have shown that both the stiffness of the BME/Matrigel and its components (i.e. chemical signals) are responsible for its activity with so many different cell types. BME/Matrigel has widespread use in assays and in models that improve our understanding of tumor biology and help define therapeutic approaches. © 2014 Elsevier B.V.
Arnaoutova I.,Trevigen, Inc. |
Kleinman H.K.,U.S. National Institutes of Health
Nature Protocols | Year: 2010
A protocol is presented here for a rapid, quantitative and reliable in vitro angiogenesis assay that can be adapted for high throughput use. Endothelial cells are plated on a gelled basement matrix, their natural substrate, and form capillary-like structures with a lumen. The assay can be used to identify inhibitors or stimulators of angiogenesis, as well as genes and signaling pathways involved in angiogenesis. It has also been used to identify endothelial progenitor cells. This assay involves endothelial cell adhesion, migration, protease activity and tubule formation. This tube formation assay is preferred, as other in vitro assays for angiogenesis, such as cell adhesion, migration and invasion, measure limited steps in the angiogenesis process. The tube formation assay on basement membrane can be completed in a day because transformed endothelial cells form tubes within 3 h, whereas non-transformed endothelial cells form tubes within 6 h. © 2010 Nature Publlshing Group.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 216.88K | Year: 2012
DESCRIPTION (provided by applicant): Apoptosis is an evolutionarily conserved cell death process that involves over 100 gene products. In response to cellular stress or to maintain tissue homeostasis, the apoptotic machinery initiates and carries out a series of biochemical events leading to cell death in the absence of inflammation characteristic of necrosis. Apoptosis is essential to remove damaged or dangerous cells, and defects in apoptosis contribute both to tumorigenesis and resistance to anti-cancerchemotherapeutic regimens. The complexity of the apoptotic response to chemotherapy coupled with functional crosstalk between apoptosis and the cell survival process of autophagy presents a significant challenge in our understanding of the cellular resistance to chemotherapy. To help characterize the cellular response to different classes of chemotherapeutic agents, particularly in tumor cells with defects in apoptosis, we propose to develop a set of isogenic human cell lines as discovery tools for characterizing the apoptosis genes involved in chemotherapy resistance. In this Phase I feasibility project, we will prepare and characterize shRNA expressing lentiviruses specific for six human proteins that are key nodes in either the extrinsic or intrinsic apoptotic pathways (DR4, Caspase-8, PUMA, BAX, Caspase-9 and Caspase-3). These lentiviruses will be used for the development of stable cell lines with specific gene knockdown in both the glioma cell line LN428 and the colon cancer cell line HCT-116, followed by mRNA expression (qRT-PCR) characterization of each of the knockdown cells and single-cell clones. This will be coupled with analysis to validate apoptosis deficiency via protein expression loss, functional analysis of multiple apoptotic and autophagy endpoints and selective response to apoptosis inducing agents (Temozolomide, Camptothecin, staurosporine and Sulindac). The optimum shRNA for each will then inform for the development of cell lines with the specific gene knockdown together with (i) a far-redfluorescent reporter (FP635) for selection, (ii) a luciferase reporter amenable to real-time imaging of apoptosis and (iii) expression of LC3-EGFP for a direct analysis of autophagy induction, linked via T2A sequences in a single gene cassette. These cellswill function as valuable tools for the identification of key apoptotic targets in chemoresistance and the discovery of agents designed to overcome gene-specific defects in apoptosis. In addition, these novel cell lines are designed to be amenable to high-throughput drug testing or analysis using cell-based and xenograft models. The development of such isogenic human cells specific for an additional 100 genes coding for apoptosis proteins will be the topic of the second phase of this proposal. PUBLIC HEALTH RELEVANCE: We describe the creation of isogenic human cell lines as discovery tools for the identification of key apoptotic targets in chemoresistance and the discovery of agents designed to overcome gene-specific defects in apoptosis. In this Phase I project, we will demonstrate the feasibility of this approach by developing isogenic LN428 and HCT-116 cell lines functionally deficient in one of six human proteins that are key nodes in either the extrinsic or intrinsic apoptotic pathways. Finally,these cell lines will be modified by co-expression of fluorescent markers for utility as valuable tools for discovery of agents designed to be amenable to high-throughput drug testing or analysis using cell-based and xenograft models.
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 1.50M | Year: 2013
DESCRIPTION provided by applicant Preserving genomic integrity is essential in order to suppress cancer neurodegeneration aging and other diseases At odds with genomic preservation is DNA damage which can drive mutations sequence rearrangements and cellular toxicity DNA damage is unavoidable as DNA damaging agents are present in our environment and in our cells To counteract the deleterious effects of DNA damage we have evolved sophisticated DNA repair systems It is now known that every major DNA repair pathway suppresses cancer Furthermore since cancer is often treated using DNA damaging agents it is not surprising that the DNA repair capacity of tumors modulates sensitivity to chemotherapy Despite its importance measurements of DNA damage and repair are far from routine primarily due to the lack of reliable and rapid DNA damage assays Here by bringing together convergent expertise among engineers biologists and computer programmers we propose to meet this need by developing a platform for rapid semi automated single cell DNA damage quantification that can be broadly distributed and readily applied by researchers in public health academia industry and medicine As defined in the Phase I submission we created and tested a prototype for a well CometChip platform and have optimized the engineering design and a production apparatus to produce spatially encoded and well demonstrated that supplementation of the Microwell Comet gels with extracellular matrix proteins EMPs supports the growth of human cells for up to two weeks and the EMPs do not impact the formation of comets To enable characterization of the genotoxicity of chemicals used commercially those found in the environment or newly developed pharmaceuticals and to quantify DNA repair capacity without the need to identify specific DNA Repair technology This proposal to develop the andapos DNA Repair on a Chipandapos technology combines the use of agarose based Microwell arrays spatially encoded cellular recognition automated data processing and extra cellular matrix proteins to optimize validate and commercialize a series of Spatially Encoded Microwell Arrays We will demonstrate that we have significantly advanced the manufacturing process Aim have developed a macrowell former to produce well and welll CometChips Aim and propose the implementation of a graphical user interface for data analysis Aim Finally we will rigorously validate this new technology by analyzing the genotoxic effects of a range of compounds from the NTP library for their impact on DNA damage and repair responses and to reveal inter individual and inter cell type variation in DNA damage responses Aim Through the integration of traditional methods in biology and engineering the DNA Repair on a Chip platform described here represents a significant technological advance providing high throughput objective and quantitative measurements that have the potential to become a new standard in DNA damage analysis PUBLIC HEALTH RELEVANCE We describe a new methodology that provides for robust high throughput DNA damage and repair analysis by exploiting gravity capture of single cells into a Microwell array DNA damage levels are revealed morphologically by single cell gel electrophoresis The Microwell array enables fully automated DNA damage and DNA repair measurement of multiple experimental conditions simultaneously This technological advance opens the door to new strategies for drug discovery genotoxicity testing and environmental health research through objective quantitative analyses
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 252.27K | Year: 2013
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 2.53M | Year: 2010
DESCRIPTION (provided by applicant): Successful completion of Phase I led to the development a panel of human cell lines, each deficient in one of the eleven DNA glycosylase enzymes. Depletion of target mRNA was as high as 95%, with corresponding depletion of target protein levels and enzymatic activity. To expand background diversity, the same shRNA lentiviruses were also used to develop parallel cell line panels in diferent tumor backgrounds, including glioma and breast cancer cell lines, demonstrating similar target mRNA depletion across different tumor cell backgrounds. Gene expression knockdown of the DNA glycosylases exemplify the impact of DNA repair defects on the human transcriptome. As an example of the far reaching potential for a panel of DNA repair deficient cell lines, we show that DNA glycosylase deficiency modulated both the transcriptome and epigenome, implicating some DNA glycoylases in methylation maintenance and genome expression diversity. Further, by combining both DNA glycosylase and BRCA1 knockdown, we have begun to investigate the requirement for DNA glycosylases in the effectiveness of PARP inhibitors in a BRCA1 knockdown tumor line. Phase II of the project wil utilize the successful work-flow paradigm optimized in Phase I for the development, functional characterization, cell banking and transcriptome analysis of isogenic human cel lines deficient in all known DNA repair genes. These include genes involved in Base Excision Repair, Direct Reversal of Damage, Mismatch Excision Repair, Nucleotide Excision Repair, Homologous Recombination, Non- homologous End-Joining, the modulation of nucleotide pools, DNA polymerases, editing and processing nucleases, the Rad6 pathway, Chromatin Structure, DNA Repair genes defective in diseases and conserved DNA Damage Response genes. The studies described in Aim 1 involve the preparation of the shRNA expressing lentiviruses, transduction and generation of three different human tumor cell knockdown panels for all known DNA repair genes (gt150), followed by the mRNA expression characterization (qRT-PCR) of the knockdown cell lines and optimized scale-up and step-wise characterization to prepare for cell line distribution (Cell Banking). In aim 2, the cell lines will be validated for the expected DNA repair functional deficiency by protein expression profiling and genotoxin challenge. Finally (Aim 3), whole-genome transcriptional profiles will be conducted to quantitate transcriptional reprogramming mediated by changes in endogenous DNA repair capacity and where appropriate, following specific genotoxic stress. With the expectation that DNA repair capacity impacts basic cellular functions both spontaneously and in response to genotoxic stress, alters the transcriptional and epigenetic landscape and dictates the cellular response to stress, the development of a complete panel of isogenic DNA repair deficient cell lines across multiple backgrounds will be a valuable platform for gene and drug discovery, validation of inhibitor specificity and the identification of response biomarkers and novel targets for gene/drug synthetic-lethality combinations. The ready availability of this panel of cell lines will permit both academic and pharmaceutical scientists to study the molecular etiology of tumor genomic instability and to exploit it in oncology research. We envision robust market demand for the cell lines and information that relates to the global transcriptome. PUBLIC HEALTH RELEVANCE: In this Phase II proposal we plan to utilize the successful work-flow paradigm optimized in Phase I for the cell-line development and transcriptome analysis of isogenic human cells lines deficient in all known DNA repair genes. These highly characterized and annotated isogenic cell lines will form the basis for a platform for gene and drug discovery, validation of inhibitor specificity and the identification of response biomarkers and novel targets for gene/drug synthetic-lethality combinations.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 202.05K | Year: 2014
Project Summary / Abstract DNA repair pathways maintain the integrity of the genome and thereby help prevent the onset of cancer, disease and aging phenotypes. Further, many cancer treatments function by inducing genomic DNA damage. As such, the critical requirement for DNA repair proteins and pathways in response to radiation and genotoxic chemotherapeutics implicates DNA repair proteins as prime targets for improving response to currently available anti-cancer regimens. Essential to the development of specific DNA repair inhibitors is the availability of robust, highly sensitive assays to measure DNA repair capacity. In addition, defects in critical DNA repair pathways or proteins can predispose to cancer onset and may also provide an option for therapeutic selectivity. Many of these defects in the 150 or more DNA repair proteins can be detected using current omics technologies. However, there are many defects that can only be detected using functional assays such as those described herein. To effectivel
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 318.78K | Year: 2011
DESCRIPTION (provided by applicant): Human exposure to dangerous genotoxins is unavoidable, as DNA damaging agents are ubiquitous both in our environment and within our cells. DNA damaging agents and other genotoxins that arise from cellular metabolism, environmental sources or disease-related cellular defects contribute to cell death (e.g., neurodegeneration), gene mutations, gene rearrangements and in many cases, the onset of cancer, disease and aging phenotypes. In addition, many exogenous exposures suchas chemotherapy and radiation treatment rely on the induction of tumor cell genotoxicity to mediate therapeutic response. Further, the ability to effectively and accurately repair spontaneous or induced DNA damage depends on the cellular DNA repair capacity. Therefore, the ability to quantify DNA damage and the rate of repair of the damage to the nuclear genome directly in human cells is critical in applications ranging from epidemiology to drug development. To address this technological need in the research community, to be better positioned to characterize the genotoxicity of newly developed pharmaceuticals, and to quantify DNA repair capacity without the need to identify specific DNA Repair gene defects, we propose the development of the next generationin DNA damage detection and quantification technology. This proposal, to develop the 'DNA Repair on a Chip' technology, combines the use of agarose-based Microwell arrays, spatially- encoded cellular recognition, human tumor cell lines with genetically-defined DNA repair status and extra-cellular matrix proteins to optimize, validate and commercialize a series of Spatially Encoded Microwell Arrays that will function as a tool to quantify DNA damage and measure cellular DNA Repair capacity at baseline and following genotoxin exposure on a single array or chip (DNA Repair on a Chip). The studies described in Aim 1 involve the development of a series of 24-well Spatially Encoded Microwell Arrays, with Microwells ranging from 10-50 5M in diameter and 20-50 5M indepth, suitable for gravity capture of a single cell of various sizes. Efficacy of the Microwell Arrays will be validated using radiation and small molecule inhibitors. Further, the sensitivity of the Microwell Arrays for analysis of cellular DNA Repair capacity will be evaluated using an isogenic panel of human tumor cell lines with defined defects in DNA Repair gene expression and following genotoxic stress. Iterative analysis and Microwell characterization will inform to finalize a set of 24-well Microwell Arrays for production and distribution. The studies described in Aim 2 involve additives to the Microwell Arrays that will enhance cell growth and attachment, providing optimal analysis of baseline DNA damage and most importantly, critical data on cellular capacity for in vivo repair post-damage. This technological advance opens the door to new strategies for drug discovery, genotoxicity testing, and environmental health research through objective, quantitative analyses. Phase II of the project will beexpanded to offer 96-well capability, end-user software for spatial recognition and quantitation plus micro-well additive options for specialized cell growth and attachment. PUBLIC HEALTH RELEVANCE: We describe a new methodology that provides for robust, high-throughput DNA damage and repair analysis by exploiting gravity capture of single cells into a Microwell array. DNA damage levels are revealed morphologically by single-cell gel electrophoresis. The Microwell array enables fully automated DNA damage and DNA repair measurement of multiple experimental conditions simultaneously. This technological advance opens the door to new strategies for drug discovery, genotoxicity testing, and environmental health research through objective, quantitative analyses.
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: STTR | Phase: Phase I | Award Amount: 153.00K | Year: 2016
DESCRIPTION provided by applicant The objective of this Phase STTR research is to quantify genotoxicity in basal cell keratinocytes from organotypic cultures Epiderm in response to commonly used chemical agents The proposed product is a Comet Chip assay that measures DNA damage in basal cells derived from a reconstructed human epidermis Technical questions that will be addressed are Can we modify our currently used Comet Chip assay to incorporate extracellular matrix proteins or antibodies Can we isolate individual basal keratinocytes from a D organotypic skin culture on the basis of their preferential adhesion to these matrix proteins including collagen I and IV or by using immobilized antibodies to integrin Can we confirm their identity by quantum dot coupled antibodies specific for or collagen ligand integrins Can we use the isolated antibody labeled epidermal basal cells to detect and quantify levels of DNA damage in response to known environmental genotoxic agents Can we use our Immuno CometChip assay to screen large numbers of agents currently or proposed to be marketed The impact of the proposed research will be to reduce animal model use for toxic agent screening since human organotypic culture has been shown to be almost identical to human skin with respect to its cytokine profile in response to corrosive or irritating agents The market for screening skin genotoxic agents is immense since the current screening procedures cannot keep pace with the number of new agents currently being introduced Aim will be to develop a new method for the isolation of basal keratinocytes and an immunostaining method for simultaneous visualization of specific antigens including integrin and DNA damage Aim will be to validate the Immuno CometChip assay using known DNA damaging agents including H O This adds three new parameters to the Comet assay The first is obtaining treatment groups of single basal epidermal keratinocytes from an organotypic culture on a single chip the second is verifying their identity with surface markers and the third is the simultaneous reproducible assay for DNA damage PUBLIC HEALTH RELEVANCE The Comet assay has appeal for many reasons The assay is rapid simple sensitive reliable and fairly inexpensive Its use for testing for skin toxicity hs been hampered by a number of factors including background DNA damage incurred during the normal process of differentiation in organotypic skin models which mimic human skin We therefore propose to develop a Comet Chip assay a high throughput reproducible and quantitative method to isolate basal keratinocytes and measure genotoxicity in response to commonly used chemical agents
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 225.00K | Year: 2016
DESCRIPTION provided by applicant The objective of this application is to develop an accurate high throughput genotoxicity screening system with high specificity and accuracy using our established and well validated toxicogenomic biomarker in cultured human cells Genotoxicity represented by chromosome damage and mutations in DNA is considered to be the hallmark of carcinogenic risk The standard genotoxicity assays especially in the case of in vitro chromosome aberration assays have a high false positive rate which results in costly and time consuming follow up assays that increase the cost of drug development and chemical safety assessment Hence gaining insight into genotoxic mechanisms and distinguishing those andquot falseandquot positive genotoxicity findings caused by nongenotoxic mechanisms is of great value so a simple reliable technology proposed here would be sought after by pharmaceutical and chemical companies Our biomarker TGx is capable of recognizing incorrectly identified compounds The specificity of genotoxicity prediction by TGx the intra and inter laboratory In the Phase I feasibility project reproducibility and the reproducibility on differen technical platforms have been carefully validated by us and by a second laboratory in follow up studies Our TGx biomarker recently was incorporated into the andquot Genesandquot panel for the Tox Phase III high throughput transcriptomics project we propose to develop a commercially viable and efficient high throughput genotoxicity screening system using TGx which has shown remarkable specificity and robustness for genotoxicity prediction Our technical approach will employ direct digital counting technology to achieve high levels of precision linearity and reproducibility in measuring the expression levels of genes in TGx simultaneously The proposed approach will provide significant benefits in comparison to the current genotoxicity battery and is poised to be commercially successful PUBLIC HEALTH RELEVANCE Genotoxicity testing is an essential component of the safety assessment paradigm required by regulatory agencies world wide for drug candidates industrial chemicals and environmental pollutants However the current genotoxicity testing battery features high incidence of false positive finding for in vitro chromosome damage assays that provides a challenge to both industry and regulatory agencies This proposal addresses the high Incidence of false positive findings by applying the genomic biomarker TGx which was identified by the Fornace laboratory and that is capable of identifying relevant genotoxic responses The Phase I proposal will primarily be a feasibility and proof of principle project for developing a high throughput TGx based screening service and later a genotoxicity kit The successful completion of this project will enable a broad application of the first toxicogenomics assay for genotoxicity i e DNA damage assessment in the pharmaceutical and chemical industry Furthermore the automated screening system is expected to improve drug discovery and risk assessment of industrial chemicals