SRP492601 PRJNA1082303 GSE260587 Excess shed mesothelin disrupts pancreatic cancer cell clustering to impair peritoneal colonization Peritoneum is the second most common site of metastasis in patients with pancreatic ductal adenocarcinoma (PDAC). Peritoneal colonization is impaired in PDAC cells with knock-out (KO) of the cancer surface antigen mesothelin (MSLN) or by introducing Y318A mutation in MSLN to prevent binding to mucin-16 (MUC-16). MSLN has a membrane-bound form but is also shed to release soluble MSLN (sMSLN). Their individual roles in peritoneal metastasis are unknown. Here, a C-terminal truncated MSLN mutant (?591) incapable of cell membrane insertion but proficient in secretion was engineered. Expression of ?591 MSLN failed to rescue peritoneal metastasis in MSLN KO cells and inhibited peritoneal colonization when overexpressed in WT PDAC cells. Exposing PDAC cells to conditioned medium (CM) containing excess sMSLN impaired cancer cell clustering in vitro and in peritoneal fluid in vivo, while CM containing only Y318A sMSLN did not. These data demonstrate that interaction of membrane-bound MSLN with MUC-16 promotes cell clustering that is critical for efficient peritoneal metastasis. However, peritoneal colonization by MSLN KO cells was rescued by expression of ?591 mutant MSLN bearing Y318A mutation, suggesting that sMSLN also has a MUC-16-independent role in peritoneal spread. Alterations in inflammatory signaling pathways occurred following KO cell exposure to CM containing sMSLN, and CM from cancer cells with intact peritoneal metastasis provoked increased KO cell secretion of IL-1a. While excess sMSLN inhibits cell clustering and peritoneal colonization, sMSLN may also promote PDAC peritoneal metastasis independent of MUC-16. Overall design: Human pancreatic cancer cell lines KLM1 and T3M4 with MSLN knockout (KO) were cultured with the supernanat from MSLN KO KLM1 and T3M4 cell lines (Mock), engineering of KLM1 KO cells over-expressing full-length WT (WT) and Y318A (Y318A) mutant MSLN supernatant, as well as from the wild type KLM1 and T3M4 (WT) for 4 hours. total RNA was extracted for bulk RNAseq analysis. GSE260587 pubmed 39673668