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Development of Human Tissue Culture Systems that Mimic the Tumor Microenvironment


There is a critical need to improve the accuracy of preclinical drug efficacy screening and testing through the development of in vitro culture systems that more effectively mimic the in vivo environment. Currently, two-dimensional (2D) in vitro culture systems or in vivo animal models are the primary tools used to test cancer cell responses to drugs. However, drug sensitivity data obtained via 2D culture systems can be misrepresentative, while animal models are expensive, time-consuming, and not always predictive of the effects on human tumors in their native environment. Three-dimensional (3D) culture systems that mimic the tumor microenvironment using human tissue could be a better tool for drug screening by providing a more accurate, in vivo-like structure and organization than 2D culture systems, without the cost and time associated with using animal models. In addition, culture systems using human tissue may produce responses more predictive of humans than animal models. Advances in bioengineering and 3D cell culture models have led to in vitro systems that better replicate the structure, physiology, and function of tissues seen in vivo. 3D models more accurately mimic the in vivo milieu than current 2D in vitro culture systems by recreating the morphology and arrangement of individual cells, concentration gradients of signaling molecules and therapeutic agents, and the composition, structure, and mechanical forces of extracellular matrix around cells. The use of 3D systems that recreate the human tumor microenvironment could improve drug development in at least two ways: 1) speed decision-making for whether a particular therapeutic agent is worth pursuing in an animal model, reducing the time and cost of development; 2) lead to fewer clinical trial failures because of earlier, more relevant results from human tissue. Properly representing the tumor microenvironment is particularly critical for testing the effectiveness of anti-cancer therapeutic agents. For example, extravascular transport in solid tumors is a fundamental determinant of the efficacy of both locally and systemically administered cancer agents. Large diffusion distances in tumor tissues, elevated interstitial fluid pressure, and interactions between anti-cancer drugs, tumor tissue, and normal tissue are factors that significantly limit drug diffusion in the extravascular compartment. Additionally, due to rapid proliferation and poor blood supply to tumor cells, the tumor microenvironment is often acidic and hypoxic, which can lead to the resistance of tumor cells to both drug and radiation therapy. Thus, systems to properly recreate the tumor microenvironment are essential to advance the discovery and development of effective anti-cancer agents. Project Goals The focus of this topic is the development of 3D human tissue model culture systems that accurately mimic the tumor microenvironment, including factors affecting tumor cell responses such as vascularization and interactions with heterogeneous cell types. The project goal is to produce a system that is validated against known effective anti-cancer agents to demonstrate the system’s utility as a predictive tool and screening assay. It is anticipated that the development of 3D systems representative of human tumor microenvironments will lead to an increase in the quality of and reduction in the timelines and costs associated with screening drugs, and enhancement in efficacy information for regulatory decisions. Essential characteristics of an in vitro tumor microsystem should include all or some of the following features: 1) multicellular architecture that represents physiologically relevant characteristics of the tumor and tissue of origin; 2) reproducible and viable operation with simple and clear protocols; 3) ability to examine multiple aspects of cancer, such as tumor growth, angiogenesis, cell proliferation, migration, and/or invasion; and 4) compatibility with high content screening platforms that include multiple molecular read-outs, such as genomic, proteomic, metabolomic, or epigenomic analyses. System development should permit scale-up production such that the system can be reliably reproduced at a cost with reasonable expectation for market success. An eventual goal for such systems may include the ability to incorporate individual patient tumor biopsies to test patient-specific responses to available agents. It is important to note that full 3D tumor microenvironment systems will consist of more than just an extracellular matrix containing tumor cells and will facilitate the inclusion of various cell types to mimic tumor cell interaction with surrounding normal cells and their effects on cancer aggressiveness and response to anti-cancer drugs. Examples include stromal cells that can induce chemoresistance and encourage metastasis, as well as endothelial cells that can carry therapeutics to the cancer. This topic is not intended to fund microphysiological organ systems for the study of toxicity, though tumor culture systems developed under this topic may be combined as a module with systems such as those being developed through the collaborative program between NIH, FDA, and DARPA: Phase I Activities and Expected Deliverables • Develop 3D culture system prototype that incorporates human tumor cells o System should include: • Co-culture with multiple cell types, such as stromal cells, endothelial cells, etc. • Components to address cell-cell or cell-extracellular matrix (ECM) adhesion • Method to deliver and control necessary growth factors o Use a tumor cell line or biopsy tissue that is readily available and well characterized o Model should be developed using or easily adapted for use with high content screening platforms for sample analysis o Develop standardized protocol to enable reproducible culture of tumor cells in 3D microenvironment o Recapitulate tissue-tissue interfaces, spatiotemporal chemical gradients (e.g. oxygen, nutrients, and/or growth factors), and mechanical context of tumor microenvironment • Submit a statement to NCI that specifies metrics used and criteria for prediction of clinical efficacy prior to demonstration of accurate prediction of clinical efficacy o Identify specific biomarkers (e.g. gene expression patterns, cell surface proteins) that characterize cell types and tumors used o Specify criteria for assessing whether the tumor microenvironment is representative of human physiological environment o Specify markers of tumor activity o Specify metrics that will be used to evaluate efficacy and milestones for desired efficacy • Demonstrate accurate prediction of clinical efficacy in the developed prototype o Test at least one anti-cancer agent with a known clinical profile using the developed prototype (e.g., agent used may be from the NCI Developmental Therapeutics Program [DTP] Approved Oncology Drugs Set) ( o Benchmark performance in developed system against 2D (e.g., NCI-60 Human Tumor Cell Line), and currently available 3D culture systems (e.g., tumor spheroids, hollow-fiber bioreactors) Phase II Activities and Expected Deliverables • Benchmark performance in developed system against applicable in vivo animal model(s) and known clinical performance o Test multiple agents with known clinical profiles in the developed prototype • Test at least one agent that has proven efficacious in animal trials but not in clinical trials o Assess genomic, proteomic, metabolomic, and epigenomic profile of the tumor system • Use validated markers and/or evaluative criteria from in vivo histologic analysis • Genomic data may be compared to The Cancer Genome Atlas ( o Compare dose-response relationships of known anti-cancer agents • Demonstrate the ability to scale-up the system for use in high-throughput therapeutic agent screening assays o Demonstrate the ability to perform high-throughput quantitative analysis on samples, such as simple harvesting and/or automated imaging.
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