Tumor-suppressive and tumor-promoting roles of cancer-associated fibroblast-released mediators in desmoplastic liver metastasis

Here we analyzed mouse derived samples to investigate origin, subtypes, functions and cell-cell interactions of cancer-associated fibroblasts in desmoplastic liver metastasis. Hepatic stellate cell-derived cancer-associated fibroblasts were isolated from three different models of liver metastasis induced by intra-splenic injection of cell lines Panc02, KPCY and CMT93 and analyzed by bulk RNA sequencing. In addition, CAF from Panc02- and CMT93-induced liver metastasis were analyzed by single cell sequencing. Overall design: Bulk RNA sequencing was performed in 9 samples: 1 isolate of quiescent mouse hepatic stellate cells (HSC); 3 isolates of HSC-derived CAF from Panc02; 3 isolates of HSC-derived CAF from KPCY and 2 isolates of HSC-derived CAF from CMT93. Mouse HSC were isolated by in situ liver perfusion as previously described, the cells were further purified by FACS using endogenous retinoid fluorescence and/or by LRAT-Cre-induced TdTomato fluorescence. CAFs from Lrat-Cre+ TdTom+ Col1a1-GFP+ mice were isolated from tumors following the HSC protocol with few modifications. Before FACS sorting, cells were subjected to a separation gradient using Nycodenz 34%. CAF were sorted for Col1a1-induced GFP; HSC-derived CAFs were sorted by Col1a1-induced GFP and TdTomato double positive signal. Single-cell RNA-sequencing was performed in 2 samples: one sample of CAF-enriched cells isolated from Panc02 and one sample of CAF-enriched cells isolated from CMT93. CAF were obtained by digesting livers as above with an addition trypsin digestion. Enriched CAF were obtained by sorting cells expressing high levels of Col1a1-induced GFP, followed by addition of a small fraction of unpurified cells. All cell sorting was performed on a BD Aria II Cell Sorter, followed by RNA sequencing or scRNA sequencing. For bulk RNA sequencing, purified HSC and HSC-CAF freshly isolated and never cultured, RNA was extracted using the RNeasy Micro or Mini Kit (Qiagen) with on-column DNase digestion accordingly to manufacturer instructions. RNA (RNA integrity number [RIN] >8, as determined by Bioanalyzer 2100, Agilent Technologies) was used to construct libraries using Illumina TruSeq RNA Preparation Kit according to the manufacturer's instructions. For single-cell RNA sequencing, CAF-enriched samples containing multiple cell populations were acquired by combining Col1a1-GFP+ cells (70%) with the entire live cell population (30%) after sorting on a BD Aria II Cell Sorter. Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 3' v3 Reagent Kit (10x Genomics) according to manufacturer's instructions. 12 cycles of cDNA amplification and 12 cycles of library amplification were performed, and samples were sequenced on an Illumina NovaSeq 6000 Sequencing System.