Investigating the Biophysical and Biochemical Influences of Stromal Cells on Anti-Cancer Drug Resistance within Bioengineered Tumor Microenvironment Models

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Cancer is currently the second leading cause of death in the United States, claiming the lives of over half a million Americans annually. Although there has been a major thrust in cancer therapeutic research, metastatic cancers, i.e., cancers that have been formed by cancer cells that have been transmitted from an original growth at another site, are still deadly, largely due to their resistance to anti-cancer drugs. Many therapeutic strategies, developed using gold standard animal models and conventional cell culture methods, often fail due to not considering the complex cellular and molecular interactions of cancer cells within the surrounding tumor microenvironment. Despite significant advancements in the use of bioengineered tumor models to study different facets of cancer, none of the previous studies have accurately reconstructed the complex tumor microenvironment, composed of various types of stromal cells (cells in connective tissue) and the extracellular matrix (ECM) proteins that are crucial for determining the role of each microenvironmental component on anti-cancer drug resistance. The objective of this project is to directly addresses this knowledge gap by taking an innovative step forward to mechanistically study the role of stromal cells on anti-cancer drug resistance, utilizing bioengineered tumor microenvironment models that can accommodate key stromal cells and ECM components. The specific hypothesis of this study is that physical and chemical signaling cues, originating from tumor-stroma crosstalk, contribute to the drug resistance by a combinatorial mechanism of inhibiting drug penetration and activating compensatory drug resistance pathways in cancer cells. The research program brings a unique synergy between multiple scientific disciplines of tissue engineering, 3D imaging, and cancer cell biology. Insights gained will lead to more comprehensive understanding on the role of tumor-stroma crosstalk in anti-cancer drug resistance. The unique features of the tumor models will also overcome technical limitations of preexisting methods and thus allow better understanding of the effectiveness of anti-cancer therapeutics. With respect to the educational impact, the multidisciplinary nature of this study will engage and train a new generation of high school, undergraduate and graduate students in STEM fields and provide a unique opportunity to bring synergy and collaborations among bioengineers and cancer biologists. The proposed initiatives also promote dissemination of the generated fundamental knowledge to the scientific community through online modules and publications.This project focuses on mechanistically studying the role of tumor microenvironment and stromal cells on anti-cancer drug resistance using 3D bioengineered organotypic tumor microenvironmental models of breast cancer that are incorporated with key stromal cells, cancer associated fibroblasts (CAFs)and tumor associated macrophages (TAMs). The overarching hypothesis of the project is that the physiochemical signaling cues originating from tumor-stroma crosstalk lead to distinct mechanisms, which initially promote stromal remodeling and inhibit drug penetration and further results in emergence of compensatory drug-resistance pathways, which together lead to chemotherapeutic drug resistance. The research plan is organized under two objectives. The FIRST OBJECTIVE is to dissect the role of CAFs mediated stromal desmoplasia (growth of fibrous or connective tissue) on chemotherapeutic drug resistance. A high-density tumor-stroma model will be developed and characterized relative to ECM stiffness and remodeling during tumor growth. A panel of anti-fibrotic drugs will be used to inhibit stromal desmoplasia, and matrix remodeling and stiffness will be assessed in response to each drug. A 3D microfluidic platform will be used to test the penetration of chemotherapeutic drugs alone and in combination with the antfibrotic drugs, and the drug distribution profile will be correlated with stromal modeling and stiffness. The experiments are designed to test the hypothesis that the primary mechanism of failure of chemotherapeutic drugs is due to biophysical barriers and insufficient drug penetration within the stroma to reach the tumor core. The SECOND OBJECTIVE is to mechanistically study the synergistic influence of cellular signaling from stromal CAFs AND TAMs on drug resistance. The bioengineered 3D microfluidic platform will be used to construct a tumor-stroma model with a tri-culture of cancer cells, CAFs and TAMs. A panel of chemotherapeutic drugs that have been used in treating triple negative breast cancer will be screened to assess cancer cell invasion at the single cell level as well as tumor viability and growth in response to drug treatment. Then, in drug targets that have exhibited the lowest efficacy in inhibiting tumor growth and invasion, RNA sequencing will be utilized to assess alterations in cancer genomics and to identity compensatory pathways that lead to drug resistance. The experiments are designed to test the hypothesis that the integrated response within the stroma embedded with co-culture of CAFS and TAMs, due to cell-cell signaling crosstalk, is more pronounced than the sum of all the individual cues on drug resistance. The integrated studies and biological data from the project will enable the construction of a mechanistic framework on the comprehensive role of physiochemical signaling cues, in the context of tumor-stroma crosstalk, on anti-cancer drug resistance. Collectively, the research outcomes will advance fundamental science pertinent to cancer biology and anti-cancer drug resistance and will contribute to the broader biopharmaceutical tumor-on-chip communities.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.