Promotion of Cholangiocarcinoma Growth by Diverse Cancer-associated Fibroblast Subpopulations

Here we analyzed mouse and human samples to characterize origin, subtypes, functions and cell-cell interactions of cancer-associated fibroblasts in cholangiocarcinoma, a highly desmoplastic tumor of the liver. Hepatic stellate cell-derived cancer-associated fibroblasts were isolated from two different models of murine intrahepatic cholangiocarcinoma, induced by overexpression of YAP+AKT or KRASG12D in combination with sg-p19, and compared by bulk RNA-sequencing to hepatic stellate cells from two models of liver fibrosis, induced by bile duct ligation or DDC diet. CAF-enriched fractions of from YAP+AKT or KRAS/sg-p19-induced intrahepatic cholangiocarcinoma, were analyzed by single-cell RNA sequencing. A cell suspension from human cholangiocarcinoma, containing all cell populations, was analyzed by single cell RNA-sequencing. Overall design: Bulk RNA sequencing was perfomed in 19 samples: 4 isolates of quiescent mouse hepatic stellate cells (HSC); 4 isolates of HSC from bile duct-ligated (BDL) mice, 4 HSC isolates from mice treated with DDC diet, 4 isolates fo HSC-derived CAF from YAP/AKT-induced intrahepatic cholangiocarcinoma (ICC); 3 isolateds of HSC-derived CAF from KRAS/sg-p19 induced ICC. Single cell RNA-sequencing was performed in 3 samples: one sample of CAF-enriched cells isolated from YAP/AKT-induced ICC; one sample of CAF-enriched cells isolated from KRAS/sg-p19-induced ICC; one sample containing all cell populations from a patient with hilar cholangiocarcinoma (CCA). Biliary liver fibrosis was induced in eight-week-old mice subjected to ligation of the common bile duct as previously described. Mice were euthanized 14 days after the surgery. As a second model of well-established cholestatic liver fibrosis, mice treated with diet containing 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) for three weeks. For induction of ICC, plasmids were injected into six to seven week old mice by hydrodynamic tail vein injection at 20µg:5µg ratio of transposon to transposase-encoding plasmid (pT3-EF1a-HA-myr-AKT (mouse), pT3-EF1a-YapS127A (human) for YAP/AKT-induced ICC model) or 25µg:5µg ratio of transposon to transposase-encoding plasmid (pCaggs- KRASG12D transposon plasmid) and 10µg CRISPR/Cas9 sgRNA-p19 plasmid (KRAS/P19 model). Mouse HSC were isolated by in situ liver perfusion as previously described, the cells were further purified by FACS using endogenous retinoid fluorescence 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. 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, freshly isolated cells from the YAP/AKT ICC sample were sorted for Col1a1-driven GFP. To obtain a CAF-enriched sample still containing multiple cell populations for CellPhoneDB analysis, for the KRAS/p19 ICC sample we combined Col1a1-GFP+ cells (60%) with the entire cell population (40%) after sorting on a BD Aria II Cell Sorter. Tumor specimen from 1 CCA patient was freshly dissociated, minced to 2-4 mm sized pieces and subsequently digested to single cell suspension using Multi Tissue Human Tumor Dissociation Kit 1 (Miltenyi Biotec) and a gentleMACS OctoDissociator (Miltenyi Biotec) according to the manufacturer's instructions. The dissociated samples were processed for scRNA sequencing. ***Please note that the AJ064 raw data was not provided due to IRB protocol as it contains identiable personal sequencing data of this patient currently undergoing treatment.