Transcriptome of senescent and non-senescent cells within PanIN pancreatic premalignant lesions in a KRas-driven mouse model

Cellular senescence is a central barrier to tumorigenesis, acting to block the proliferation of premalignant cells. However, senescent cells residing within tumor lesions can also exert paracrine effects influencing tumor growth and progression. Premalignant pancreatic intraepithelial neoplasia (PanIN) lesions contain senescent cells, yet whether these influence disease progression is unknown. Here we report that senescent cells in PanINs that develop in a Kras-driven mouse model express a pro-inflammatory gene signature, which includes high Cox2 levels. Pharmacologic Cox2 inhibition caused a dramatic reduction in PanIN growth. Senolytic treatment with the Bcl2-family inhibitor ABT-737 reduced the numbers of Cox2-expressing PanIN cells and blocked PanIN formation and progression to carcinoma. These findings indicate that senescent PanIN cells support tumor growth and progression through Cox2 activity, representing crosstalk between interspersed senescent and dividing premalignant cells. Targeted elimination of senescent cells may thus be effective in limiting progression of precancerous lesions. Overall design: For inducible Kras activation in pancreatic acinar cells, Ptf1a-CreER; Kras+/lsl-G12D; Rosalsl- tdTomato triple transgenic mice, and controls, were injected at 6 weeks of age subcutaneously with two doses of 400 mg/Kg tamoxifen. These mice developed premalignant PanIN lesions and were sacrificed at 3 months of age. We isolated tdTomato+SA-?Gal– and tdTomato+SA-?Gal high fractions from 3 mice. Total RNA from FACS-sorted cells was isolated by TRIzol (Invitrogen) extraction followed by RNeasy Plus Micro Kit (Qiagen), using >10,000 cells of each sample. Profiling was conducted using an adapted CEL-Seq2 protocol: 3' cDNA was synthesized and barcoded, followed by RNA synthesis, amplification by in vitro transcription and library generation for paired-end sequencing. Reads were demultiplexed, quality filtered and trimmed for adapters and poly-A tail using Cutadapt and aligned with the human genome (GRCh38) using Tophat2.