Spatial profiling of neuro-immune interactions in gastroenteropancreatic NETs

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are heterogeneous malignancies that arise from complex cellular interactions within the tissue microenvironment. Here, we sought to decipher tumor-derived signals from the surrounding microenvironment by applying Nanostring Digital Spatial Profiling (DSP) to hormone-secreting and non-functional GEP-NETs. DSP was used to evaluate the expression of 40 neural and immune-related proteins in surgically resected duodenal and pancreatic NETs (n=20) primarily comprised of gastrinomas (18/20). A total of 279 regions of interest were examined between tumors, adjacent normal and abnormal-appearing epithelium, and the surrounding stroma. The results were stratified by tissue type and Multiple Endocrine Neoplasia I (MEN1) status and protein expression was validated by immunohistochemical (IHC) staining. A tumor immune cell autonomous inflammatory signature was further evaluated by IHC and RNAscope, while functional pro-inflammatory signaling was confirmed using patient-derived duodenal organoids. Gastrin-secreting and non-functional pancreatic NETs showed a higher abundance of immune cell markers and immune infiltrate compared to duodenal gastrinomas. Tumors displayed strong intra-tissue variation in the expression of neural- and immune-related proteins. Compared to non-MEN1 tumors, MEN1 gastrinomas showed reduced expression of immune cell markers and upregulated expression of neuropathological proteins. Duodenal gastrinomas showed strong expression of the pro- inflammatory and pro-neural factor IL-17B. Treatment of human duodenal organoids with IL- 17B activated NF-kB and STAT3 signaling and induced the expression of neuroendocrine markers. In conclusion, multiplexed spatial protein analysis identified tissue-specific neuro- immune signatures in GEP-NETs. Moreover, duodenal gastrinomas cell autonomously express immune and pro-inflammatory factors, including tumor-derived IL-17B, that stimulate the neuroendocrine phenotype.