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Up to 50% of pancreatic ductal adenocarcinomas (PDA) harbor mutations in canonical TGFß pathway genes such as SMAD4 and TGFBR2. SMAD4 mutations have been shown to drive resistance to chemotherapy, metastasis, and margin positivity following surgical resection. To characterize the unique tumor microenvironment of SMAD4 mutant PDA, we developed isogenic, orthotopic mouse models using CRISPR mediated knockout of Smad4 in a cell line derived from KPfC (KrasLSL-G12D;Trp53fl/fl;Pdx1Cre) mice, a genetically engineered mouse model of pancreatic cancer. To characterize the alteration of the secretome caused by the loss of Smad4, we performed proteomic analysis with conditioned media collected from Smad4 wildtype and mutant cancer cell clones. Interestingly, the loss of Smad4 resulted in the production of neuroendocrine related factors. In addition, gene ontology analysis with the secretome suggested that these factors were associated with neuronal processes. These data indicate that loss of Smad4 in PDA cancer cells may lead to a neuroendocrine signature change. In particular, we found one of the most up-regulated secreted factors in the mutant cells was SPP1, which has been shown to promote tumor progression by affecting cancer cell stemness and macrophage polarization. To validate the proteomic results, we established orthotopic models with Smad4 wildtype and mutant clones. We found that loss of Smad4 resulted in larger tumor growth. Moreover, immunohistochemistry showed a significant up-regulation of SPP1 in the cancer cells of Smad4 mutant tumors. To further profile the alteration of immune landscape, we stained for markers of myeloid cells and lymphocytes. We found that Smad4 mutant tumors had an immunosuppressive microenvironment characterized by increased M2 macrophage, neutrophil and MDSC infiltration. Altogether, our study suggests that loss of SMAD4 in PDA may induce a neuroendocrine signature with increased SPP1 secretion in cancer cells. The production of this unique cancer cell secretome may result in a microenvironment marked by immunosuppressive myeloid cells. Citation Format: Gilbert Z. Murimwa, Francesca Rossi, Henry K. Fleming, Zeynep Yazgan, Dina Alzhanova, Huocong Huang, Rolf A. Brekken. Loss of Smad4 induces SPP1 secretion and immunosuppressive myeloid cell formation in pancreatic ductal adenocarcinoma. [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 3641. |
