Molecular Mechanisms of Intratumoral Nerve Recruitment and Perineural Invasion Elucidated with Spatial Transcriptomics and CRISPR Activation.

International journal of radiation oncology, biology, physics

PURPOSE/OBJECTIVE(S): Perineural invasion (PNI) is an aggressive manifestation of tumor-nerve interactions associated with postoperative recurrence, metastasis, pain, and decreased survival. Hence, PNI is included in the staging criteria of several malignancies and often an indication for treatment intensification using adjuvant radiotherapy. However, the diverse molecular mechanisms underlying tumor-nerve crosstalk remain largely unknown-hindering the development of new therapies targeting this key pathological process. Moreover, prior studies were limited by a lack of cell-type information, spatial context, and/or a fragmented focus on a small number of pathways.MATERIALS/METHODS: Using pancreatic ductal adenocarcinoma (PDAC) as an exemplar given the exceptionally high frequency of PNI in this malignancy, we performed the first comprehensive, cell-type specific, and spatially resolved whole-transcriptome analysis of human PDAC to identify molecular mediators of tumor-nerve crosstalk and PNI. We constructed 12 custom tissue microarrays (TMAs) derived from matched malignant regions with and without tumor-nerve proximity (n = 288 cores). We performed whole-transcriptome digital spatial profiling (DSP) to independently determine mRNA abundance from the malignant, fibroblast, and nerve compartments through optical sectioning.RESULTS: We mapped malignant subtypes we previously identified onto the spatial data and found strong (p<0.0001) positive nerve associations with the mesenchymal, basaloid, and neural-like progenitor subtypes and a negative nerve association with the classical subtype. Numerous genes expressed by malignant cells were enriched (e.g., MMP2, PLXND1, NRP1) or depleted (e.g., SEMA3B) in association with radial distance from nerves, including recapitulation of prior literature. To functionally explore these candidate mediators of tumor-nerve crosstalk, we derived genetically-engineered murine organoids (Kras; Trp53; Rosa26-dCas9-VPR) and transduced them with guide RNAs to overexpress subtype-specific transcription factors or candidate genes from the spatial analysis. We quantified (1) cancer cell invasion through extracellular matrix using cultured dorsal root ganglia (DRG) sensory neurons as the chemoattractant, and (2) the role of cancer-intrinsic signaling on nerve recruitment/outgrowth by applying conditioned media or exogenous proteins to cultured DRG sensory neurons and tracking their growth with live imaging.CONCLUSION: Our results suggest that the mechanisms enabling cancer cells to recruit nerves into the tumor microenvironment are distinct from those facilitating perineural invasion. This study has transformed our understanding of how cancer cells and the peripheral nervous system collaborate to promote tumor growth, survival, and dissemination, and is now guiding prioritization of therapeutic strategies that synergize with adjuvant radiotherapy in the burgeoning field of cancer neuroscience.

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International journal of radiation oncology, biology, physics
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