Cluster of differentiation 44 variant isoform 9 defines cancer stem cell reservoirs in pancreatic intraepithelial neoplastic spatial niches

Background: Pancreatic acinar-to-ductal metaplasia (ADM) is an adaptive process inwhich differentiated acinar cells acquire duct-like characteristics in response to chronicstress or injury. While ADM is a reversible cellular process allowing acinar cellregeneration, chronic inflammation or injury leads to the development of pancreaticintraepithelial neoplasia (PanIN) that are precancerous lesions often precedingpancreatic ductal adenocarcinoma (PDAC). Defining molecular targets of ADM canimpede the initiation and progression of disease leading to preventive strategies thatcould be translated to clinical practice. Our research team has identified a cancer stemcell (CSC) population that persists within the PDAC tumor microenvironment expressingCluster of Differentiation (CD) 44, variant 9 (CD44v9). A known marker of therapy-resistance, the role and detection of CD44v9 in early disease initiation remains undefined. Objective: To define CD44v9+ CSCs as reservoirs within ADM and PanIN precancerouslesions contributing to disease recurrence in patients with PDAC.Methods: Using CosMx ™ Whole Transcriptome Spatial Molecular Imaging (CosMx ™ WTx SMI) and Orion ™ Multiplex Immunofluorescence (MxIF), we analyzed PDAC tissuescollected after first line systemic chemotherapy and the Whipple surgical procedure frompatients with PDAC. To further investigate whether CD44v9 is expressed within ADM andPanIN lesions, we analyzed regions of interest in patient matched adjacent cancertissues. Results: MxIF staining identified cells co-expressing the acinar cell marker amylase andCD44v9, alongside CD44v9+ and ductal marker CK19+ cells lacking amylase. Consistentwith metaplastic transition cells co-expressing amylase and CK19 were also positive forCD44v9. Analysis of the CosMx ™ WTx SMI data showed the co-expression of CD44and ductal marker CK19 (KRT19) in the same cell populations identified as ADM. PanINlesions exhibited high co-expression of CD44, MUC6, MUC5AC and MUC1. Spatiallymatched by MxIF, the same CD44v9+ PanIN lesions also expressed senescent-associated secretory MIF and CEACAM genes. Analysis of cell-cell communication bythe CellChat computational tool revealed that CD44v9 CSCs expressed with PanINspatial niches had the highest number of interactions and strength with inflammatorycancer associated fibroblasts, myeloid derived suppressor cells and acinar and ductalcells. The highest expressing genes were associated with pathways regulatingextracellular matrix re-organization, senescence and immunosuppression. Conclusions: These findings support the hypothesis that CD44v9+ CSCs may arise inpre-malignant lesions, mirroring the persistent CD44 expression observed in primary andmetastatic sites. CD44v9+ CSCs may serve as reservoir for therapeutic resistance anddisease recurrence in patients with PDAC.