Tumor microenvironment heterogeneity and progression mechanisms in intrahepatic cholangiocarcinoma: A study based on single-cell and spatial transcriptomic sequencing

Background and Aims: Intrahepatic cholangiocarcinoma (ICC) is characterized by high malignancy, and its global incidence is predicted to continue to increase over the past decades. However, the mechanisms underlying ICC pathogenesis and progression remain unclear. Approach and Results: The training cohort consisted of single-cell sequencing of 12 treatment-naïve ICC samples and spatial transcriptomics of four ICC samples. The validation cohort comprised of RNA-seq data from 87 ICC tumor samples. Finally, we validated our findings via multiplex immunofluorescence, organoids, and mice models both in vivo and in vitro . We found significant heterogeneity within the tumor microenvironment (TME) of ICC patients. ICC cells were classified into five molecular subtypes, and we revealed that aspartate beta-hydroxylase (ASPH) was a marker gene for invasion subtypes. We then selected cepharanthine (CEP) as an ASPH inhibitor that effectively suppressed tumor progression. Regarding the ICC lymphatic metastasis mechanism, we found that tumor cells in N1 lymph nodes highly expressed tumor-specific MHC-II molecules but lacked co-stimulatory factors CD80/CD86, inducing a state of anergy in CD4+ T cells, which might facilitate ICC immune evasion. Conclusions: The TME of ICC was heterogeneous. ASPH markedly enhanced ICC invasion The ASPH inhibitor CEP significantly inhibits ICC progression and may serve as a targeted therapeutic drug for ICC. Tumor cells in N1 lymph nodes demonstrate high expression of tumor-specific MHC-II molecules, but silencing of co-stimulatory factors such as CD80/CD86 induces CD4+ T cells into an anergic state. Our study indicated that ASPH and MHC-II may serve as novel therapeutic targets for ICC.