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fibroblasts (CAFs). These data will provide a resource for future studies aimed at further characterizing and targeting specific cell populations in PDA. ResultsCellular heterogeneity during PDA progression. We sought to determine the composition of single cells during the progression of PDA GEMMs. Normal mouse pancreas; 40-day-old Kras LSL−G12D/+ Ink4a fl/fl Ptf1a Cre/+ (KIC) (5) mouse pancreas, termed "early KIC" (with the early lesion initially confirmed by ultrasound; Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci. insight.129212DS1); and 60-day-old KIC pancreas, termed "late KIC" (Figure 1A) were freshly isolated and enzymatically digested followed by single-cell cDNA library generation using the 10× Genomics platform (6). Libraries were subsequently sequenced at a depth of more than 10 5 reads per cell. We performed stringent filtering, normalization, and graph-based clustering, which identified distinct cell populations in the normal pancreas and both stages of PDA.In the normal mouse pancreas, 2354 cells were sequenced and classified into appropriate cell types based on the gene expression of known markers: acinar cells, islet and ductal cells (Supplemental Figure 2), macrophages, T cells, and B cells, as well as 3 distinct populations of fibroblasts (Figure 1, B and E) were noted. In the early KIC lesion (3524 cells sequenced), the emergence of an expanded ductal population was observed (9.9% of cells), expressing known ductal markers, such as Krt18 and Sox9 (7), and displaying early neoplastic changes (Figure 1, A, C, and F, and Supplemental Figure 3). The acinar cell population was substantially reduced, while there was a marked increase in total macrophages and fibroblasts. Of note, the same 3 populations of fibroblasts seen in the normal pancreas were identified in the early KIC lesion. Additionally, endothelial cells were observed at this stage. This indicates that the expansion of fibroblasts and macrophages is an early event during PDA development. We next characterized the late KIC pancreas (804 cells sequenced) and noted the absence of normal exocrine (acinar) and endocrine (islet) cells (Figure 1, D and G). Instead, 2 distinct populations of cancer cells were present, suggesting phenotypic cancer cell heterogeneity as a late event in the course of the disease. We also observed the presence of only 2 distinct fibroblast populations, which had a similar percentage in relation to total cells. Noticeably, macrophages became a predominant cell population in the late KIC tumor. Moreover, we observed lymphocytes at this stage. The cellular heterogeneity in cancer cells and stromal cells in the early and late KIC lesions highlighted the dynamic cellular changes that occur during PDA progression.Cancer cells enriched with mesenchymal markers emerge in advanced PDA. Gene expression analysis of epithelial markers (Cdh1, Epcam, Gjb1, and Cldn3) and mesenchymal markers (Cdh2, Cd44, Axl, Vim, and S100a4) revealed that the early KIC neoplastic cell...

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Pancreatic ductal adenocarcinoma (PDA) is a highly metastatic disease that resists most current therapies. A defining characteristic of PDA is an intense fibrotic response that promotes tumor cell invasion and chemoresistance. Efforts to understand the complex relationship between the tumor and its extracellular network and to therapeutically perturb tumor-stroma interactions are ongoing. Fibronectin (FN), a provisional matrix protein abundant in PDA stroma but not normal tissues, supports metastatic spread and chemoresistance of this deadly disease. FN also supports angiogenesis, which is required for even hypovascular tumors such as PDA to develop and progress. Targeting components of the tumor stroma, such as FN, can effectively reduce tumor growth and spread while also enhancing delivery of chemotherapy. Here, we review the molecular mechanisms by which FN drives angiogenesis, metastasis and chemoresistance in PDA. In light of these new findings, we also discuss therapeutic strategies to inhibit FN signaling.