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.