Abstract 2513: Distinct cancer-intrinsic mechanisms mediate nerve recruitment/outgrowth versus perineural invasion

Abstract Pancreatic ductal adenocarcinoma (PDAC) features abundant perineural invasion (PNI). Intra-tumoral nerves play critical roles in cancer initiation, progression, recurrence, treatment-resistance, metastasis, and mortality for many malignancies but the diverse molecular mechanisms underlying tumor-nerve crosstalk remain largely unknown—hindering the development of therapies targeting this key pathological process. To address this gap, we performed whole transcriptome digital spatial profiling on twelve custom tissue microarrays (n=288 cores) derived from intratumorally-matched regions with and without PNI in primary PDAC specimens (n=31 patients) and independently measured gene expression from cancer cells, fibroblasts, and nerves. We undertook a differential gene expression analysis comparing malignant cells in PNI-present and -absent regions. We quantified the growth kinetics of dorsal root ganglia (DRG) sensory neurons cultured with exogenous candidate proteins, which validated that some candidates augment (e.g., Lgals1) and others inhibit (e.g., Sema3b) neurite outgrowth. Next, we mapped our previously discovered malignant cell programs onto the epithelial segments and observed a significant enrichment of the mesenchymal, basal-like, and neural-like progenitor (NRP) programs versus depletion of the classical program in PNI-present regions. To determine the effects of malignant subtype on nerve outgrowth, we engineered isogenic KrasG12D/+;Trp53FL/FL;Rosa26-dCas9-VPR (KP;dCas9-VPR) organoids to overexpress the master transcription factors (TFs) for each malignant subtype (e.g., Gata6 for classical, Glis3 for NRP). We then performed the DRG neuronal outgrowth assay using conditioned media from each subtype-specific organoid line versus an off-target control and observed that the classical line suppressed neurite outgrowth, the mesenchymal and basal-like lines were neutral, and the NRP line enhanced neurite outgrowth dynamics comparable to the Ngf positive control. Taken together, our findings suggest that the mechanisms underlying nerve recruitment/outgrowth and perineural invasion may be partly decoupled. To further test this hypothesis, we are performing transwell invasion assays comparing KP;dCas9-VPR cancer cell lines that overexpress each of the candidate PNI-associated genes and malignant subtype TFs. We anticipate that this study will transform our understanding of how cancer cells and the peripheral nervous system collaborate and guide prioritization for therapeutic intervention in the burgeoning cancer neuroscience field. Citation Format: William L. Hwang, Jennifer Su, Carina Shiau, Peter L. Wang, Jimmy A. Guo, Nicole A. Lester, Jaimie L. Barth, Hannah I. Hoffman, Prajan Divakar, Jason W. Reeves, Eric Miller, Joseph M. Beechem, Andrew J. Aguirre, Lei Zheng, David T. Ting, Mari Mino-Kenudson, Tyler Jacks. Distinct cancer-intrinsic mechanisms mediate nerve recruitment/outgrowth versus perineural invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2513.