Cellular heterogeneity during mouse pancreatic ductal adenocarcinoma progression at single-cell resolution

Abstract

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...

Targeting TGF βR2‐mutant tumors exposes vulnerabilities to stromal TGF β blockade in pancreatic cancer

“…Furthermore, we found that CAFs are a major source of TGFβ‐induced IL‐6 expression. Several recent studies including ours demonstrate the existence of fibroblast heterogeneity within PDA (Ohlund et al , ; Bernard et al , ; Elyada et al , ; Hosein et al , ). Two major populations of CAFs have been identified, one is characterized by an inflammatory feature, while the other has been known as myofibroblast.…”

“…Recently, several studies, including ours, suggest the existence of two different subtypes of CAFs in PDA (Ohlund et al , ; Bernard et al , ; Elyada et al , ; Hosein et al , ). Of the two CAF populations, one appears to be inflammatory, characterized by PDGFRα expression and the secretion of a variety of inflammatory chemokines.…”

“…To identify the stromal cell type that secretes IL‐6 in a TGFβ‐dependent manner, we performed single‐cell RNA sequencing (scRNA‐seq) using whole tissue samples derived from normal mouse pancreas, early PDA, and late PDA from KIC mice (Hosein et al , ). We found that TGFβR1/R2 was mainly expressed by fibroblasts in the normal pancreas as well as early PDA (Fig A).…”

“…For scRNA‐seq (Hosein et al , ), normal mouse pancreas, 40‐day‐old KIC (early KIC ), and 60‐day‐old KIC pancreases (late KIC ) were freshly isolated and enzymatically digested. A library was generated with the single‐cell suspension using the 10× Chromium system (10× Genomics, Inc.).…”

Origin and functional heterogeneity of fibroblasts

“…In breast tumors, four subset of CAFs were captured in MMTV‐PyMT tumors, namely vascular CAFs (vCAF), matrix CAFs (mCAF), cycling CAFs (cCAF), and developmental CAFs (dCAF), representing a perivascular origin (vCAF and cCAF), a resident fibroblast origin (mCAF), and an EMT origin (dCAF) . In pancreatic cancer in contrast to normal pancreas, distinct CAFs subpopulations include a population associated with insulin‐like growth factor signaling (FB1), a population expressing Ly6a/c1 (lymphocyte antigen complex locus and locus c1) and Pi16 (peptidase inhibitor 16) (FB2), and a population with mesothelial markers, including Msln (mesothelin), and Cav1 , Cdh11 (cadherin 11), and Gas6 (growth arrest‐specific 6) (FB3) . Interestingly, only FB1 and FB3 persisted in pancreatic ductal adenocarcinoma (PDAC), with FB2 cells merging toward an FB1 transcriptome, and FB3 contain Acta2 + myofibroblasts as well as MHCII + cells, suggesting an immune modulatory function.…”