Resolution of acinar dedifferentiation regulates tissue remodeling in pancreatic injury and cancer initiation.

Background and Aims: Acinar to ductal metaplasia (ADM) is crucial in the development of pancreatic ductal adenocarcinoma (PDAC). However, our understanding of the induction and resolution of ADM remains limited. We conducted comparative transcriptome analyses to identify conserved mechanisms of ADM in mouse and human. Methods: We identified Sox4 among the top upregulated genes. We validated the analysis by RNA in situ hybridization (ISH). We performed experiments in mice with acinar-specific deletion of Sox4 (Ptf1a: CreER; Rosa26-LSL-YFPLSL-YFP; Sox4fl/fl ) with and without an activating mutation in Kras (KrasLSL-G12D/+). Mice were given caerulein to induce pancreatitis. We performed phenotypic analysis by immunohistochemistry, tissue decellularization and single cell RNA sequencing. Results: We demonstrated that Sox4 is reactivated in ADM and PanINs. Contrary to findings in other tissues, Sox4 actually counteracts cellular dedifferentiation and helps maintain tissue homeostasis. Moreover, our investigations unveiled the indispensable role of Sox4 in the specification of mucin-producing cells and tuft-like cells from acinar cells. We identified Sox4-dependent non-cell-autonomous mechanisms regulating the stromal reaction during disease progression. Notably, Sox4-inferred targets are activated upon KRAS inactivation and tumor regression. Conclusions: Our results indicate that our transcriptome analysis can be used to investigate conserved mechanisms of tissue injury. We demonstrate that Sox4 acts as a switch to promote healing in pancreatic injury and cancer initiation and is activated upon Kras ablation and tumor regression in mice. By uncovering novel potential strategies to promote tissue homeostasis, our findings offer new avenues for preventing the development of PDAC. Overall design: To investigate the effect of Sox4 on acinar metaplasia and neoplastic development we knocked out Sox4 in the mouse acinar lineage by using the following alleles: LSL-KrasG12D, Ptf1aCreER, R26RYFP, and Sox4flox. Tissue injury was induced by repetitive caerulein injections over 2 days. The RNA was extracted from bulk pancreas at day 2 for study of acinar regeneration and at day 21 for the study of prenoeoplastic development. LSL-KrasG12D (MGI:2429948), Ptf1aCreER (MGI:3771322), R26RYFP (MGI:2449038), and Sox4flox (MGI:3773012) mouse strains were used for breeding. Mice were maintained on a genetically mixed background. Tamoxifen (T5648; Sigma-Aldrich) was administered through intraperitoneal injections every other day for 5 days (e.g., Monday, Wednesday, Friday) at a dose of 3mg per injection. Tamoxifen was dissolved in corn oil (C8267; Sigma-Aldrich) at a stock concentration of 30mg/ml (100?l/injection). A standard wash-out of 2 weeks was performed. To induce tissue injury in the pancreas, the cholecystokinin analogue caerulein (C9026; Sigma-Aldrich) was administered through intraperitoneal injections every hour for 7 hours (8 injections/day) on 2 consecutive days at a concentration of 125?g/kg body weight. Control mice were injected with equivalent volume of PBS following the same protocol. Pancreas tissue was finely chopped with scissors in digestion buffer (1mg/ml Collagenase (C9407-1G, Sigma-Aldrich), 1mg/ml DNase I (Sigma-Aldrich, 11284932001), 0,1mg/ml soybean trypsin inhibitor (STI, Fisher Scientific, 17-075-029) dissolved in Hank´s balanced salt solution (HBSS)). Digestion was performed in gentleMACS C Tubes using mTDK1 program on a gentleMACS™ Dissociator (Miltenyi Biotec). Reaction was stopped with STOP buffer (1mg/ml DNase I (Sigma-Aldrich, 11284932001), 0,1mg/ml STI and 5% Fetal bovine serum (FBS) dissolved in HBSS). Digested tissue was centrifuged 5 minutes at 2000 rpm and resuspended in 3 ml RBC lysis buffer (Thermo Scientific, 00-4333-57). After 2 minutes incubation cells were washed with STOP buffer twice and filtered through a 40 µm cell strainer. Cells were further processed for single cell sequencing. Nomenclature: CSox4WT: Ptf1CreER; -lslYFP. CSox4KO: Ptf1CreER; Sox4fl/fl; -lslYFP. KCSox4WT: Ptf1CreER; lsl-KrasG12D; -lslYFP. KCSox4KO: Ptf1CreER; Sox4fl/fl; lsl-KrasG12D; -lslYFP.