Hedgehog signaling alters the balance of fibroblast subtypes in pancreatic cancer [bulk RNA-seq]

A better understanding of the roles of signaling pathways differentially activated in distinct fibroblast populations will shed light on the complexity of fibroblast heterogeneity in pancreatic cancer. Here, we show that Hedgehog pathway inhibition alters the fibroblast composition towards a more inflammatory microenvironment, opening the possibility for future combination therapies. Total RNA samples were isolated Trizol, according to manufacturer’s instructions. RNA concentration and quality were determined and ribosomal depleted mRNA libraries were prepared by the University of Michigan Advanced Genomics Core. Libraries were sequenced using 150 paired end NovaSeq 6000 (Illumina). Mouse tumors were treated with Vehicle or HH inhibitor for 14 days after formation of tumors. For sorting of fibroblasts from KPC tumors, cells were stained for 30 minutes with CD45-Alexa Fluor 488 (103122; BioLegend), CD326 (EpCAM)-Alexa Fluor 647 (118212; BioLegend), CD324 (E-cadherin)-Alexa Fluor 647 (147307; BioLegend), PDPN-APC/Cy7 (127418; BioLegend), and for 15 minutes with DAPI. DAPI- CD45- EpCAM- ECAD- PDPN+ cells were sorted on the FACSAria cell sorter (BD) and processed for RNA-sequencing. N=2 replicates for LDE225 group, N=3 for Vehicle group