Cellular heterogeneity and transcriptomic profiles during intrahepatic cholangiocarcinoma initiation and progression

Abstract

Background and Aims Intrahepatic cholangiocarcinoma (ICC) is not fully investigated, and how stromal cells contribute to ICC formation is poorly understood. We aimed to uncover ICC origin, cellular heterogeneity, and critical modulators during ICC initiation/progression, and to decipher how fibroblast and endothelial cells in the stromal compartment favor ICC progression. Approach and Results We performed single‐cell RNA sequencing (scRNA‐seq) using AKT/Notch intracellular domain–induced mouse ICC tissues at early, middle, and late stages. We analyzed the transcriptomic landscape, cellular classification and evolution, and intercellular communication during ICC initiation/progression. We confirmed the findings using quantitative real‐time PCR, western blotting, immunohistochemistry or immunofluorescence, and gene knockout/knockdown analysis. We identified stress‐responding and proliferating subpopulations in late‐stage mouse ICC tissues and validated them using human scRNA‐seq data sets. By integrating weighted correlation network analysis and protein–protein interaction through least absolute shrinkage and selection operator regression, we identified zinc finger, MIZ‐type containing 1 (Zmiz1) and Y box protein 1 (Ybx1) as core transcription factors required by stress‐responding and proliferating ICC cells, respectively. Knockout of either one led to the blockade of ICC initiation/progression. Using two other ICC mouse models (YAP/AKT, KRAS/p19) and human ICC scRNA‐seq data sets, we confirmed the orchestrating roles of Zmiz1 and Ybx1 in ICC occurrence and development. In addition, hes family bHLH transcription factor 1, cofilin 1, and inhibitor of DNA binding 1 were identified as driver genes for ICC. Moreover, periportal liver sinusoidal endothelial cells could differentiate into tip endothelial cells to promote ICC development, and this was Dll4‐Notch4‐Efnb2 signaling–dependent. Conclusions Stress‐responding and ICC proliferating subtypes were identified, and Zmiz1 and Ybx1 were revealed as core transcription factors in these subtypes. Fibroblast–endothelial cell interaction promotes ICC development.

Single-cell profiling reveals the sustained immune infiltration, surveillance, and tumor heterogeneity of infiltrative BCC

“…Antigen-presenting fibroblasts were recently identified in malignancies and adjacent normal tissues and could activate CD4 + T cells[38–40]. Here, we found that apFib expressed both fibroblast (LUM, COL1A1, and COL1A2) and antigen presentation-associated genes (CD74, HLA-DRB1, and HLA-DRA) (Fig.…”

“…It was newly reported that hepatic stellate cells could deliver MHC-I molecules with target antigens to hepatic sinusoidal endothelial cells, which, in turn, activated CD8 + T cells[50]. Multiple subtypes of fibroblasts with different phenotypes have been identified at the single-cell level, including antigen-presenting fibroblasts that carry high levels of MHC-2 molecules[38–40]. Whether the upregulated MHC-I molecules in fibroblasts play a role in activating CD8 + T/NK cells and the associated activation mechanisms need to be further explored (Fig.…”

Stromal yin‐yang of myofibroblasts and endothelial cells in the progression of intrahepatic cholangiocarcinoma

“…Taking advantage of the well‐characterized murine model of AKT–Notch intracellular domain (NICD) hydrodynamic tail vein injection–induced iCCA tumors combined with scRNAseq profiling, [ 13 ] to the best of our knowledge, Wang et al are the first to study how cells in the stromal compartment contribute to iCCA formation (Figure 1). Their study exploits the possibility in a model to tightly define the tumor stages, investigate the initiation of tumor formation, and homogenize sample inclusion.…”

“…[ 14 ] At 5 weeks, tumors already show evidence of necrosis, high mitotic activity, and invasion of the normal liver parenchyma. Therefore, to investigate the intratumoral heterogeneity in the initiating phases of the establishing tumor, Wang et al [ 13 ] sampled murine livers starting at day 10, at a point in which tumor budding appeared, and this model typically only shows microscopic single or clusters of cytologically malignant cells. At day 17, small lesions with ductular structures are formed, whereas day 31 shows fully progressed cytokeration 19‐positive iCCA with bile‐like fluid and a glandular histomorphology.…”

“…Although the consensus is that AKT–NICD gives rise to 100% iCCA‐like tumors, we do not know if this model develops through the small or large duct type morphology. Indeed, this question may have been resolved if Wang et al, [ 13 ] during the effort to determine how consistent their murine single‐cell types resemble those found in human iCCA, had incorporated their data together with the recently suggested iCCA subtype markers S100 calcium binding protein P and SPP1. [ 17 ] In this study, the authors concluded that the peripheral‐type iCCA comprises tumor cells at different differentiation states, including albumin‐positive hepatocytic and inhibitor of DNA‐binding 3–positive cells, which suggests a hepatoblast status.…”