Jay George

Matrigel: From discovery and ECM mimicry to assays and models for cancer research☆

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.

In Vitro Microtumors Provide a Physiologically Predictive Tool for Breast Cancer Therapeutic Screening

Many anti-cancer drugs fail in human trials despite showing efficacy in preclinical models. It is clear that the in vitro assays involving 2D monoculture do not reflect the complex extracellular matrix, chemical, and cellular microenvironment of the tumor tissue, and this may explain the failure of 2D models to predict clinical efficacy. We first optimized an in vitro microtumor model using a tumor-aligned ECM, a tumor-aligned medium, MCF-7 and MDA-MB-231 breast cancer spheroids, human umbilical vein endothelial cells, and human stromal cells to recapitulate the tissue architecture, chemical environment, and cellular organization of a growing and invading tumor. We assayed the microtumor for cell proliferation and invasion in a tumor-aligned extracellular matrix, exhibiting collagen deposition, acidity, glucose deprivation, and hypoxia. We found maximal proliferation and invasion when the multicellular spheroids were cultured in a tumor-aligned medium, having low pH and low glucose, with 10% fetal bovine serum under hypoxic conditions. In a 7-day assay, varying doses of fluorouracil or paclitaxel had differential effects on proliferation for MCF-7 and MDA-MB-231 tumor spheroids in microtumor compared to 2D and 3D monoculture. The microtumors exhibited a tumor morphology and drug response similar to published xenograft data, thus demonstrating a more physiologically predictive in vitro model.

Abstract 4274: Porcine ovarian matrix supports maintenance of human intestinal organoid cultures

Currently, extracellular matrix hydrogels derived from the Engelbreth-Holmes-Swarm murine tumor (Cultrex BME, Matrigel) are used for stem cell maintenance and differentiation of both normal and tumor organoid culture models. While this matrix is suitable for many in vitro applications, there is increasing demand for a 3D organoid culture platform that is more suitable for clinical applications. Here, we have developed a soluble extracellular matrix derived from normal porcine ovaries that forms a hydrogel polymer at physiological temperature and that supports maintenance of human small intestine organoids in culture. This matrix supports expansion of LGR5+ human stem cell populations using Wnt, EGF, Noggin, and R-Spondin 1 cell culture medium. The matrix also supports differentiation of stem cells into mini organs by removing Wnt which is characterized by the formation of intestinal crypts which exhibit immunostaining for lineage specific markers. This new extracellular matrix hydrogel represents a non-tumor sourced, normal extracellular matrix for evaluating both development and cancer progression. Citation Format: Jay George, Sol De Gese, Gabriel Benton. Porcine ovarian matrix supports maintenance of human intestinal organoid cultures. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4274.

Abstract 324: High throughput triculture: A breast cancer spheroid model for drug screening

Many drugs that show promise in preclinical evaluation fail miserably during clinical trials, and the inability of current preclinical models to properly predict drug response may be attributed to oversimplified or non-existent tumor microenvironments. To provide a more physiologically predictive model for drug screening, we have developed an in vitro, 384 well, triculture system that presents key chemical (i.e. low pH, low glucose, low oxygen), extracellular matrix (ECM), and cellular (tumor cells, stromal cells and endothelial cells) attributes of tumor tissues. By using ECM proteins, we can promote the proper physiology for each of these cell types in triculture. To evaluate interactions between each cell type, they are fluorescently labeled with fluorophores with different excitation and emission spectra; MCF7 and MDA-MB-231 human breast cancer cell lines express a red fluorescence protein, while human umbilical vein endothelial cells (HUVECs) and human adipose-derived mesenchymal stem cells (hMSCs) are labeled with stable lipophilic membrane dyes. Breast tumor microtissues spontaneously assemble when breast cancer cells are co-cultured with hMSCs under low adhesion conditions. Once formed, these microtissues are deposited onto preformed tubule networks comprised of HUVECs and hMSCs and are embedded within a hydrogel composed of ECM proteins. Cellular interactions and dissemination are monitored via fluorescence microscopy, and cell proliferation of the breast cancer cells is quantified using a fluorescence plate reader. The breast cancer cells exhibit cell-cell interactions with endothelial tubules and stromal cells, forming microtissues. Furthermore, the response to anti-cancer drugs, paclitaxel and fluorouracil, in an in vitro triculture system is similar to the response for the same drugs in xenografts model, unlike 2D or 3D monoculture. This high throughput triculture cancer model will provide more insight into pathological mechanisms and can better predict response to anti-cancer drug treatment. Citation Format: Gabriel J. Benton, Gerald DeGray, Irina Arnaoutova, Hynda K. Kleinman, Jay George. High throughput triculture: A breast cancer spheroid model for drug screening. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 324. doi:10.1158/1538-7445.AM2015-324

Abstract 2379: 96-Well CometChip validation for simultaneous treatment and measurement of DNA damage in a single platform

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The 96-Well CometChip System is a high-throughput platform to simultaneously treat and measure DNA damage induced by different treatments, or among different cell types on a single slide using the comet assay. The CometChip is a consumable consisting of specifically sized micron pores patterned into agarose layered on a treated microscope slide. Ninety-six (96) separate wells are created by inserting the CometChip into a reusable 96-Well CometChip System, a magnetically-sealed cassette suitable for tissue culture incubators. Cells added to each well are captured by gravity into micropores and excess cells aspirated leaving an array of non-overlapping cells. Multiple experimental conditions are performed in parallel by the addition of different chemicals to respective wells. Once treatment is complete the CometChip is removed from the cassette and processed using standard alkaline comet conditions and imaging systems. A distributable CometChip requires precision manufacturing to ensure robust and reproducible performance. CometChip tolerances were investigated and compared to normal comet using cryopreserved comet control cells with known levels of DNA damage. After setting specifications, 96-Well CometChips with 30 micron pores were distributed to evaluate both intra and inter-chip variations between laboratories. To assure experimental consistency between labs, magnetically sealable cassettes, cryopreserved control cells and identical electrophoresis systems were provided to each lab. Data is presented to demonstrate the feasibility of manufacturing the CometChip for reproducible results based on the coefficients of variance (CVs) obtained between different wells and laboratories. In addition, data will be presented demonstrating the absence of cross talk between CometChip wells using the 96-Well CometChip System. (Work was supported by R44ES021116). Citation Format: Sandra R. Woodgate, Clare Whittaker, Jay George, Robert W. Sobol, Sandy Schamus-Haynes, Bevin P. Engelward, Jing Ge. 96-Well CometChip validation for simultaneous treatment and measurement of DNA damage in a single platform. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2379. doi:10.1158/1538-7445.AM2014-2379

Abstract 2423: Development of a validated high throughput ELISA assay for gamma H2AX as a pharmacodynamic marker

Histone H2AX is a 14 kDa ubiquitous member of the H2A histone family that becomes rapidly phosphorylated at Serine 139 by ATM and ATR kinases to yield a form known as γ-H2AX in response to double-strand DNA damage and apoptosis. γ-H2AX is an ideal Pharmacodynamic (PD) surrogate marker to measure molecular responses to a large number of drugs; however, methods such as western blots and immunohistochemistry are widely used but are difficult to validate to regulatory standards, and not suitable for high throughput screening applications. To address this need, we have developed a novel high throughput ELISA assay to measure γ-H2AX levels in cellular extracts and phosphorylation of H2AX in response to therapeutic intervention. This assay documents differences of γ-H2AX levels in PBMC, cultured cells, tissue biopsies, and will be useful in future clinical trials providing one of many needed tools to enable hypothesis-driven preclinical drug design strategies.p Background Histone H2AX is a 14 kDa ubiquitous member of the H2A histone family that contains an evolutionarily conserved SQ motif at the C-terminus in eukaryotes. Serine 139 within this motif becomes rapidly phosphorylated by ATM and ATR kinases to yield a form known as γ-H2AX in response to double-strand DNA damage and apoptosis (1). During the past year investigators have confirmed the value of γ-H2AX as an important Pharmacodynamic (PD) marker (2) and genotoxicity endpoint (3). There are over 21 anticancer drugs that are known to result in γ-H2AX formation. As a result, γ-H2AX is an ideal Pharmacodynamic PD surrogate marker to measure molecular responses to a large number of drugs (4,5,6). While many of these drugs have already garnered regulatory approval, and are currently being used to manage various types of cancers, they are the subject of ongoing clinical studies to evaluate their efficacy when used alone or in combination with molecularly targeted drugs. While methods such as western blots and immunohistochemistry are widely used but are difficult to validate to regulatory standards, the ELISA method is the most quantifiable and easiest to validate, and not suitable for high throughput screening applications. To address this need Trevigen9s quantitative pharmacodynamic HT γ-H2AX ELISA assay measures γ-H2AX levels in cellular extracts and phosphorylation of H2AX in response to therapeutic intervention. This assay documents differences of γ-H2AX levels in PBMC, cultured cells, tissue biopsies, and will be useful in future clinical trials providing one of many needed tools to enable hypothesis-driven preclinical drug design strategies. Citation Format: Jay George, Hai Xu. Development of a validated high throughput ELISA assay for gamma H2AX as a pharmacodynamic marker. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2423. doi:10.1158/1538-7445.AM2014-2423

Abstract 2033: A comprehensive 3D triple coculture model for evaluating breast cancer progression

Current models for evaluating breast cancer progression lack a comprehensive, physiological approach to modeling the complex tumor microenvironment. There has been much evidence supporting the use of tumor spheroids to mimic tumor physiology; they exhibit cell-cell bond formation, comparable morphology, elevated cell survival and proliferation in the outer cell layers, reduced proliferation rates in the intermediate layers, and a hypoxic core. While the tumor spheroid provides a physiological tumor model, other cell types within the surrounding tumor microenvironment are essential for tumor behavior and subsequent cancer progression. The tissue vasculature provides a critical component for tumor progression given the metabolic requirements of a growing tumor, and by incorporating endothelial tubules with the tumor spheroid, we are able to model interactions between vascular networks and growing tumors. At the same time, there is also an important stromal component involved in cancer progression where stromal cells have been shown to promote cell proliferation, dissemination, and drug resistance during cancer development. By using extracellular matrix proteins, we are able to promote the proper physiology for each of these cell types in triple cell coculture and compartmentalize their activities. To evaluate interactions between each cell type, they are fluorescently labeled with fluorophores with different excitation and emission spectra; MCF7 and MDA-MB-231 human breast cancer cell lines express a red fluorescence protein, while human umbilical vein endothelial cells (HUVECs) and human adipose-derived mesenchymal stem cells (hMSCs) are labeled with stable lipophilic membrane dyes. Tumor spheroids are formed using low adhesion microwells and deposited into fully formed HUVEC tubular networks, and the hMSCs are then added within a hydrogel overlay matrix. Cellular interactions and dissemination are monitored via fluorescence microscopy and cell proliferation of the breast cancer cells is monitored using a fluorescence plate reader. The breast cancer spheroids exhibit cell-cell interactions with endothelial tubules and stromal cells, and there is an increase in cell proliferation and invasion over standard spheroid monoculture model. Citation Format: Gabriel J. Benton, Jay George, Gerald DeGray, Irina Arnaoutova, Hynda K. Kleinman. A comprehensive 3D triple coculture model for evaluating breast cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2033. doi:10.1158/1538-7445.AM2014-2033

Abstract 3841: 3D spheroid culture model for studying cancer progression: fine tuning the extracellular environment.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC There is a need for more comprehensive and physiologically predictive cancer progression models. Tumor cell aggregates or spheroids are more demonstrative of tumors in vivo as they display several physiological traits including comparable morphology, reduced proliferation rates, elevated cell survival, cell-cell bond formation, tumor dormancy, and a hypoxic core. Embedding these spheroids in a physiological hydrogel composed of basement membrane proteins provides networks on which cells can travel out of the spheroid, mimicking early events during metastasis. Recent studies implicate the tumor microenvironment as a key component of the cancer progression process, and changes in tissue rigidity and collagen I content have been associated with this pathological process. The purpose of this study is to model the effects of differing extracellular matrix composition on morphology and invasiveness in different metastatic cell types during early metastatic events. Here, human cell lines representing different cancer types were grown in spheroid culture and subjected to embedding in hydrogels of differing extracellular matrix protein composition. The cell panel includes MDA-MB-231 for breast cancer, PC-3 for prostate cancer, U-87 MG for glioblastoma and HT-1080 for fibrosarcoma. Spheroids were photographed every 24 hours, and photographs were subjected to image analysis. The composition of the extracellular matrix hydrogel had a significant effect on the morphology of the invading cells as well as the rate of invasion. Increasing the collagen I content to model a more fibrotic environment resulted in fewer but longer process formation, representing a more motile cancer model; however, collagen I, alone, was insufficient to support robust invasion. In conclusion, the 3D spheroid invasion model suggests that the composition of the extracellular matrix proteins is important for cancer cell behavior during metastasis. Citation Format: Gabriel J. Benton, Irina Arnaoutova, Jay George. 3D spheroid culture model for studying cancer progression: fine tuning the extracellular environment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3841. doi:10.1158/1538-7445.AM2013-3841

The In Vitro Endothelial Cell Tube Formation Assay in 3D Culture on Gelled Basement Membrane Extract

In 1988, it was first observed that endothelial cells placed in 3D culture on a gelled basement membrane substratum, in vitro, form capillary-like structures with a lumen. Since then, the tube formation assay has been widely used to define angiogenic and anti-angiogenic factors as well as to identify genes and signaling pathways important in angiogenesis. More recently, it has been used to identify and characterize progenitor cells, and to better understand cell-cell organization, functional effects, and relation in 3D co-culture. The assay has been successful because it is quick, reliable, flexible, quantitative, easy to perform, and amenable for high throughput screening. The assay measures endothelial cell adhesion, migration, and tubule formation, and thus, it is more comprehensive than simple migration, invasion, or proliferation assays. Tubule formation requires protein synthesis; however, proliferation is not required. A variety of endothelial cells can be used as well, and modified gene expression may be used to define the function of certain genes in angiogenesis. There have been several reviews and methods papers recently published on this assay which detail the uses and methods.

Abstract 3884: A novel, in vitro tankyrase 1 trans-ribosylation assay

The poly (ADP-ribose) polymerase, tankyrase 1, is an encouraging pharmacological target for cancer therapy. First discovered by its association with the TRF1, tankyrase 1 plays an important role in the maintenance of telomere length, sister telomere association, and mitotic spindle organization. More recently, interactions between tankyrase 1 and axin were shown to stabilize β-catenin and modulate Wnt signaling, enabling selective targeting of the Wnt pathway with tankyrase inhibitors. In addition, tankyrase 1 has also demonstrated potential as a target for synthetic lethality for BRCA-deficient cancers. Given the effect on these cancer-associated pathways, there is a growing interest for identifying tankyrase-specific inhibitors. Historical methods to evaluate tankyrase activity are not amenable for high throughput screening. Limited availability of the full length enzyme required radioactivity to attain the needed sensitivity. Truncation of tankyrase 1 improved solubility and increased availability, but the physiological response may not be equivalent to the full length enzyme. Many of these assays have also used modified substrates where molecules such as fluorescein or biotin are linked to NAD without characterizing the effects on enzyme activity. Most historical assays have also relied on measuring autoribosylation of tankyrase 1; whereas, transribosylation may be more relevant for assessing the activity of tankyrase 1 on other molecules. To address these issues, we have developed a novel, in vitro, high throughput assay for evaluating the transribosylation activity of tankyrase 1. This is accomplished using an ELISA format which semi-quantitatively detects poly (ADP-ribose) (PAR) deposited onto immobilized histone proteins by tankyrase 1. An anti-PAR monoclonal antibody, goat anti-mouse IgG-HRP conjugate, and HRP substrate generate a colorimetric or chemiluminescent signal, and the conversion of substrate correlates with tankyrase 1 activity. This assay ensures physiological significance by utilizing the full length enzyme and unmodified substrate in a transribosylation format, providing sensitivity down to pmols of enzyme. This assay is utilized to demonstrate the specificity of the tankyrase 1 inhibitor, XAV939, for tankyrase 1 compared to PARP1 and to reveal differential effects of activated DNA for full-length tankyrase 1 compared to the truncated enzyme. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3884. doi:1538-7445.AM2012-3884