Book Description

Cancer is the second leading cause of mortality in the United States. The National Cancer Institute estimated 1.7 million new cases of cancer and 0.6 million cancer deaths in the United States in 2019. Cancer is a heterogeneous disease that involves not only cancer cells, but also different cells and proteins in the tumor stroma. Various cells such as fibroblasts, infiltrating immune cells, and endothelial cells, the extracellular matrix (ECM) proteins, growth factors, chemokines, cytokines, and other bioactive agents constitute the tumor microenvironment (TME). Traditional cancer treatments only target cancer cells but growing evidence has established pivotal roles for the TME in driving tumor progression and chemoresistance. As such, targeting the TME and its interactions with cancer cells (known as tumor-stromal interactions) is now being pursued as a new approach to improve treatment outcomes for patients. However, preclinical models such as standard in vitro cell cultures and animal models routinely used in cancer drug discovery fail to recapitulate tumor-stromal interactions of human tumors. To overcome limitations of existing tumor models, we developed a three-dimensional (3D) organotypic tumor model that enables mechanistic studies of tumor-stromal interactions to enable drug discovery efforts against the TME. The organotypic model incorporates three key components of the TME: a mass of cancer cells, fibroblasts, and collagen as the ECM. The resulting model resembles the architecture of solid tumors and spatial distribution of cells within the TME. We used a novel cell and protein micropatterning approach based on an aqueous two-phase system (ATPS) to first generate a cancer cell spheroid and then overlay it with a collagen solution containing fibroblasts. We automated this technology and adapted it to a high throughput 384-well plate format to enable both mechanistic and phenotypic studies of tumor-stromal interactions and testing arrays of drugs. We focused on triple negative breast cancer (TNBC) as a disease model because TNBC is the most aggressive subtype of breast cancer with very limited targeted therapy options and poor patient outcomes from cytotoxic chemotherapies, underscoring an unmet need for new treatment strategies. We leveraged our organotypic tumor model to demonstrate the feasibility of mechanistic studies of tumor-stromal interactions in TNBC, focusing on cancer-associated fibroblasts (CAFs) as the most abundant stromal cells in breast tumors. To establish the validity of our model, we used a well-known chemokine-receptor interaction mechanism in TNBC. Specifically, we showed that fibroblasts-secreted CXCL12 chemokine promotes the ECM invasion of CXCR4+ TNBC cells by activating oncogenic mitogen-activated protein kinase (MAPK) pathway. Additionally, the fibroblast cells remodeled the ECM through the RhoA/ROCK/myosin light chain-2 pathway. Following the validation step, we incorporated patient-derived CAFs in our model and studied their dynamic interactions with TNBC cells to explore whether CAFs-TNBC interactions would present therapy targets. Our mechanistic studies showed that hepatocyte growth factor (HGF) secreted by CAFs predominantly activates MET receptor tyrosine kinase on TNBC cells to promote proliferation, invasiveness, and epithelial-to-mesenchymal transition (EMT) of TNBC cells. This interaction axis led to activation of oncogenic pathways such as MAPK, phosphatidylinositol 3-kinase-Akt (PI3K/Akt), and signal transducer and activator of transcription (STAT) in TNBC cells. Importantly, we found that TNBC cells become resistant to single-agent treatment with a potent MAPK pathway inhibitor (trametinib) and demonstrated a design-driven approach to select drug combinations that effectively inhibit pro-metastatic functions of TNBC cells. We also demonstrated that the HGF-MET axis is implicated in lung metastasis of TNBC and that blocking this signaling axis is a potential approach against both primary TNBC tumorigenesis and metastases formation in the lung. Future studies are needed to study long-term effectiveness of drug combinations in our organotypic tumor model. Overall, this work established the utility of our 3D organotypic tumor model to elucidate the role of tumor stroma in promoting pro-metastatic functions of TNBC cells, thereby facilitating the design and development of novel therapeutic approaches against tumor-stromal interactions. This technology will facilitate future studies to incorporate other components of tumor stroma and patient-derived cancer and stromal cells to expedite the translation of the findings.