PROJECT SUMMARY/ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer death in the United States by 2030. One of the differentiating hallmarks of PDAC is an exceptionally high frequency of perineural invasion (PNI), a histopathologic manifestation of tumor-nerve crosstalk whereby cancer cells recruit, migrate towards and invade peripheral nerves. These tumor-nerve interactions may be biochemical, electrical, and metabolic in nature and involve both neoplastic and non-neoplastic cells. While it has become apparent that intra-tumoral nerves play an important role in cancer initiation, progression, recurrence, treatment resistance, metastasis, and survival, the diverse molecular mechanisms underlying tumor-nerve crosstalk remain largely unknown. To address this gap in knowledge, this proposal aims to perform a comprehensive spatially-resolved whole transcriptome screen in human PDAC to identify cell-type specific genes linked to PNI and dissect their functional roles by applying CRISPR activation (CRISPRa) in a novel orthotopic organoid transplant model. It is anticipated that this work will uncover a set of previously unknown mediators of PNI and provide a high-resolution molecular interconnectivity map of the tumor-nerve interactome, which will guide therapeutic development. Prior work studying molecular mediators of PNI using bulk or single-cell/nucleus RNA-sequencing lacked spatial context. The first aim applies a transcriptomic and proteomic digital spatial profiling (DSP; Nanostring) approach optimized for formalin-fixed paraffin-embedded sections of human PDAC to preserve the spatial relationships among different cell types/states. By performing DSP on tissue microarrays created from a custom cohort of PDAC patients with tumors spatially annotated for regions with and without PNI, the ability to extract gene expression differences that associate with PNI isolated from inter-patient genetic heterogeneity is greatly enhanced. The second aim will investigate the potential causal relationships between a shortlist of ~20 candidate genes and the development of PNI by performing an arrayed in vivo CRISPRa screen involving orthotopically transplanting pancreatic cancer organoids expressing single-guide RNAs (sgRNAs) targeting one or more candidate genes (KrasLSL-G12D/+;Trp53FL/FL;Rosa26LSL-dCas9-VPR-mNeonGreen;sgRNA) into syngeneic recipients. The density and properties of intra-tumoral nerves/PNI will be assessed using multiplexed imaging and machine learning approaches. The long-term goal of this research is the successful development of therapeutics that disrupt adaptive tumor-nerve crosstalk in the tumor microenvironment. The research proposal is part of an extensive career development plan that includes formal educational opportunities and seminar/conference presentations to enable the principal investigator to become an independent physician-scientist in translational cancer biology. A diverse and experienced team of mentors, scientific advisory committee members, and collaborators at MIT, MGH, DFCI, HMS, and Columbia University School of Medicine is committed to supporting and guiding the principal investigator toward achieving this goal.

PROJECT NARRATIVE Emerging evidence indicates that the active recruitment of nerve fibers into tumors and subsequent cancer cell invasion (perineural invasion) plays an important role in cancer development, metastasis, therapeutic resistance, recurrence, pain, and death across many malignancies including pancreatic cancer, but the diverse molecular mechanisms underlying this tumor-nerve dialogue remain largely unknown. To address this gap in knowledge, we will comprehensively identify cell-type specific genes linked to perineural invasion in human pancreatic cancer and then investigate their functional roles in a novel genetically-engineered mouse model. The proposed studies will provide a high-resolution roadmap of critical gene expression programs and intercellular interactions in the pancreatic cancer microenvironment that facilitate perineural invasion, which will guide the repurposing and development of therapeutics against this pervasive cause of morbidity and mortality in pancreatic cancer and many other malignancies.