PROJECT SUMMARY Many aggressive cancers have a robust tumor microenvironment composed of heterogenous stromal and immune cells. Although the advent of immune checkpoint inhibitors has shifted therapeutic targets of cancer research to include tumor-cell extrinsic targets, the therapeutic potential of targeting the tumor stroma remains underexploited. Recently, cancer-associated fibroblasts (CAFs) have been implicated as drivers of disease progression. From preliminary attempts to harness the therapeutic potential of targeting CAFs, it has become clear that targeting CAFs as a bulk population of cells will not be sufficient. As such, there have been efforts in breast and pancreatic cancer to define the heterogeneity of CAFs. These efforts have yielded diverse subtypes commonly described by two overarching groups: myofibroblast CAFs (myCAFs) and inflammatory CAFs (iCAFs). In pancreatic cancer, myCAFs are observed to be tumor-adjacent and iCAFs are more distant from tumor cells. While defining subtypes of CAFs is a necessary first step, the development of novel therapeutic approaches will likely require the identification of specific functions of CAF subtypes. To this end, Wnt2 has been identified as upregulated in CAFs from pancreatic, breast, and colorectal cancer, yet the role of CAF-driven Wnt signaling on tumor progression remains largely unknown. Anaplastic thyroid carcinoma (ATC) is a lethal disease (~3-5 month median survival) with an abundant tumor stroma and no efficacious treatment options. The composition of the tumor stroma in ATC has been largely unexplored. My preliminary work identifies a prominent fibroblast population in ATC that expresses WNT2. As ATC is known to have upregulated Wnt signaling relative to other thyroid neoplasms, this provides a unique opportunity to study the dynamics of CAF-driven Wnt signaling. The goal of this proposal is to define the CAF subtypes present in thyroid carcinoma and determine the functional role of CAF-derived Wnt2 on tumor growth. I hypothesize that distinct CAF populations promote tumor growth and invasion in thyroid carcinoma through Wnt signaling and have unique spatial relationships. To test my hypothesis, I will perform experiments in ATC models and papillary thyroid carcinoma (PTC) models. PTC is a predominantly indolent thyroid carcinoma that can transform to ATC in vivo, making it ideal for examining the ability of CAFs to promote disease progression. In aim 1, I will elucidate subtypes of CAFs present in ATC and PTC and probe Wnt ligand-receptor interactions. Further, I will determine spatial resolution of myCAF and iCAF fibroblast populations in thyroid carcinoma. In aim 2, I will utilize >40 primary patient thyroid carcinoma CAF cultures that our lab has collected to demonstrate the role of CAF-derived Wnt2 signaling both paracrine on PTC and ATC tumor cells and autocrine to shape the phenotype of CAFs. In completing these studies, I will for the first time define the heterogeneity of...