Dissecting the Tumor Microenvironment: The Role of Cancer-Associated Fibroblast-Tumor Crosstalk in Solid Malignancies

Abstract

Solid tumors are the leading cause of cancer-related mortality worldwide that accounts for approximately 10 million deaths annually. Effective treatments often fail due to the dense, desmoplastic stroma characterized by the excessive deposition of extracellular matrix (ECM) proteins. This extensive desmoplasia is strongly associated with worse patient outcomes and is a prominent feature in pancreatic ductal adenocarcinoma (PDAC) and breast cancer, where the stroma can comprise up to 60-90% of the tumor mass. Cancer-associated fibroblasts (CAFs), the primary stromal cell type in desmoplastic tumors, dynamically remodel the tumor microenvironment (TME) by secreting ECM proteins, growth factors, cytokines and other factors that not only generate a physical barrier to treatment, but also establish a supportive environment that promotes tumor growth and immune evasion. Therefore, elucidating mechanisms by which CAF-secreted factors promote drug resistance and tumor progression is critical to identify more effective treatments in desmoplastic tumors. PDAC is a hallmark desmoplastic tumor that is extremely resistant to chemotherapy partially due to the high stromal content that is largely comprised of CAF-secreted ECM proteins. Chapter 2 investigates the mechanism by which CAF-secreted ECM proteins promote chemoresistance to identify a previously unrecognized protein, N-myc downstream regulated gene 1 (NDRG1), that becomes phosphorylated in cancer cells in response to CAF-secreted ECM proteins. Upon activation, NDRG1 localizes to replication forks where it stabilizes and resolves chemotherapy-induced stalled forks. NDRG1 additionally reduces replication stress by resolving R-loops, RNA-DNA hybrids that occur during transcription-replication conflicts (TRCs) or in response to chemotherapy. Our data show that loss of NDRG1 sensitizes pancreatic cancer cells to chemotherapy by enhancing DNA damage and R-loop accumulation. Collectively, these findings identify a novel DNA repair protein that directly links CAF-secreted ECM proteins to chemotherapy resistance in PDAC. In addition to CAF-secreted ECM proteins, this work investigates CAF-secreted cytokines as additional drivers of PDAC progression. Chapter 3 profiles the secretome of patient-derived CAFs to identify Chitinase-3-Like 1 (CHI3L1/YKL-40) protein as a highly abundant cytokine. Importantly, high expression of YKL-40 is significantly correlated with worse patient survival in PDAC. We show that YKL-40 exerts dual pro-tumorigenic effects by directly promoting tumor cell proliferation and by promoting immune evasion through the recruitment of regulatory (Tregs) and inhibition of CD8+ T cell anti-tumor activity. Importantly, therapeutic inhibition of YKL-40 in vivo attenuates tumor growth and reprograms the TME toward an immunostimulatory state. Together, these findings identify YKL-40 as a key mediator of CAF-driven tumor progression and immune suppression that highlight its potential as a promising therapeutic target in PDAC. Finally, this work extends the role of CAF-derived cytokines as mediators of tumor progression to hormone-receptor positive (HR+) breast cancer, a disease in which patients typically respond successfully to endocrine therapy but can relapse due to extrinsic factors such as the desmoplastic TME. Chapter 4 interrogates CAF-secreted cytokines in HR+ breast cancer using a patient-derived organoid platform that incorporates matching primary patient-derived CAFs to identify cytokines, such as Growth Regulated Oncogene α (GROα), as drivers of resistance to endocrine therapy. These findings highlight the conserved role of CAF-derived factors in therapy resistance across tumor types. Collectively, this work underscores the stroma’s pivotal role in tumor progression to provide the foundation for the development of stroma-directed therapies in desmoplastic tumors.