Abstract C052: Identifying mediators of perineural invasion in pancreatic cancer using spatial transcriptomics

Abstract Intratumoral nerves play important and versatile roles in cancer initiation, progression, recurrence, treatment-resistance, metastasis, morbidity, and mortality for many malignancies but the diverse molecular mechanisms underlying tumor-nerve crosstalk remain largely unknown. One of the differentiating hallmarks of pancreatic ductal adenocarcinoma (PDAC) is an exceptionally high frequency of perineural invasion (PNI), a histopathologic manifestation of tumor-nerve crosstalk whereby cancer cells recruit, migrate towards, and envelop or invade peripheral nerves. Evidence for some neurochemicals/neurotrophins involved in PNI have been uncovered, but most of the underlying work was limited by a lack of cell-type specificity, spatial context, and fragmented focus on individual pathways. To address these shortcomings, we set out to comprehensively identify cell-type specific genes spatially linked to PNI in patient tumors and then dissect the functional roles of these genes through live imaging of dorsal root ganglia (DRG) sensory neurons incubated in conditioned media from cancer cell organoids overexpressing candidate genes via CRISPR activation (CRISPRa). First, we performed whole transcriptome digital spatial profiling (NanoString GeoMx) on twelve custom tissue microarrays (n=288 cores) derived from intratumorally-matched malignant regions with and without PNI in primary resected PDAC specimens (n=31 patients). Differential gene expression (DE) analysis (FDR < 0.001) for PNI demonstrated that for malignant cells there were 271 enriched and 65 depleted genes, and for fibroblasts there were 16 enriched and 27 depleted genes. We further evaluated associations between PNI and expression of malignant subtypes previously identified from single-nucleus RNA-seq applied to 43 primary resected PDAC specimens. We found that malignant cells engaged in PNI were enriched in the mesenchymal, basaloid and neural-like progenitor (NRP) subtypes and depleted in the classical subtype. To test these associations functionally, we generated isogenic murine organoid lines (KrasG12D/+;Trp53FL/FL;R26-dCas9-VPR) overexpressing subtype-driving transcription factors and collected conditioned media. DRG sensory neurons demonstrate enhanced and suppressed growth kinetics when grown in NRP and classical conditioned media, respectively; mesenchymal and basal-like conditioned media do not appear to influence growth kinetics. These results suggest that while mesenchymal, basaloid, and NRP cells likely all play a role in cancer cell invasion of nerves, NRP cells may have an additional role in tumor-nerve tropism. Additional experiments exploring the functional effects of the top enriched and depleted genes from the DE analysis are ongoing. We anticipate that this study will provide a high-resolution understanding of critical intercellular interactions in the PDAC tumor microenvironment that facilitate PNI and tumor-nerve crosstalk more broadly to guide novel therapeutic strategies. Citation Format: William L. Hwang, Jennifer Su, Jimmy A. Guo, Carina Shiau, Jaimie L. Barth, Hannah I. Hoffman, Prajan Divakar, Jason W. Reeves, Eric Miller, Grissel Cervantes-Jaramillo, William Freed-Pastor, Vanessa Funes, Jennifer Y. Wo, Theodore S. Hong, Carlos Fernandez-del Castillo, Lei Zheng, Andrew J. Aguirre, David T. Ting, Mari Mino-Kenudson, Tyler Jacks. Identifying mediators of perineural invasion in pancreatic cancer using spatial transcriptomics [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C052.