PROJECT SUMMARY/ABSTRACT Cutaneous squamous cell carcinoma (cSCC) is the second most common cancer overall in the U.S. and causes substantial morbidity, with a significant risk of metastatic spread and death. Treatment options are limited in unresectable cases, which are estimated at up to 40,000 per year. Advances in treatment have been hindered by incomplete knowledge of intratumoral heterogeneity (ITH) in cSCC, particularly regarding how different tumor subpopulations behave within a growing tumor. ITH can be driven by cell-intrinsic abnormalities within cancer cells, but increasing evidence suggests that cancer cells communicate with diverse cell types, such as immune or stromal cells, in the tumor microenvironment (TME) to coordinate malignant behavior such as invasion. Thus, a deeper understanding of how ITH arises in cSCC, from both TME and cell-intrinsic contributions, has the potential to yield new treatment strategies. Recent discovery of a novel subpopulation of cSCC tumor cells was facilitated through single-cell profiling of patient cSCC tumors. This tumor-specific keratinocyte (TSK) subpopulation was found to express a gene signature that was absent in normal skin, suggestive of invasive capacity, and associated with worse prognosis across several epithelial cancers. Spatial profiling demonstrated that TSKs resided at the tumor leading edge adjacent to cancer-associated fibroblasts (CAFs), suggesting that crosstalk among these cell types in the TME may control TSK cell state or function. Global ligand-receptor analyses integrating single-cell and spatial data nominated specific CAF-derived ligands matched with receptors expressed by TSKs, including several integrins. Integrin signaling genes mediating TME signals were required for tumor growth in CRISPR screens in vivo, including ITGB1, FERMT1, CD151, and ARPC2. Thus, these data support a model in which signals derived from TME cells proximal to TSKs promote tumorigenesis and potential invasion through ITGB1-mediated signaling. Through two specific aims, this work will determine if blocking cSCC-specific CAF-derived factors and disabling cancer subpopulation-intrinsic factors can impede TSK formation and subsequent tumor invasion and progression in preclinical models. Aim I will employ a human organoid model of cSCC to assess the contribution of CAF-derived ligands toward emergence of TSKs and invasion through genetic ablation and therapeutic blockade. Aim II will determine how TSK subpopulation-specific factors cooperate to facilitate invasion in organoids and tumorigenesis in xenograft mouse models through genetic manipulation of cancer cells to prevent factor interaction. This work will be performed under the mentorship of Dr. Paul Khavari, a physician-scientist and expert in skin cancer genetics, at Stanford University, a world-class research institution. These studies could aid the development of novel therapeutic strategies to treat unresectable cSCC and lead to a deeper mo...