Distal cholangiocarcinoma (dCCA) are a group of epithelial cell malignancies that occurs at the distal common bile duct, and account for approximately 40% of all cholangiocarcinoma cases. dCCA remains a highly lethal disease as it typically features remarkable cellular heterogeneity. A comprehensive exploration of cellular diversity and the tumor microenvironment is essential to depict the mechanisms driving dCCA progression. Single-cell RNA sequencing was used here to dissect the heterogeneity landscape and tumor microenvironment composition of human dCCAs. Seven human dCCAs and adjacent normal bile duct samples were included in the current study for single-cell RNA sequencing and subsequent validation approaches. Additionally, the results of the analyses were compared with bulk transcriptomic datasets from extrahepatic cholangiocarcinoma and single-cell RNA data from intrahepatic cholangiocarcinoma. We sequenced a total of 49,717 single cells derived from human dCCAs and adjacent tissues, identifying 11 distinct cell types. Malignant cells displayed remarkable inter- and intra-tumor heterogeneity with 5 distinct subsets were defined in tumor samples. The malignant cells displayed variable degree of aneuploidy, which can be classified into low- and high-copy number variation groups based on either amplification or deletion of chr17q12 - chr17q21.2. Additionally, we identified 4 distinct T lymphocytes subsets, of which cytotoxic CD8+ T cells predominated as effectors in tumor tissues, whereas tumor infiltrating FOXP3+ CD4+ regulatory T cells exhibited highly immunosuppressive characteristics. Our single-cell transcriptomic dataset depicts the inter- and intra-tumor heterogeneity of human dCCAs at the expression level.

Single-cell RNA-sequencing (scRNA-seq) is one of the most used single-cell omics in recent decades. The exponential growth of single-cell data has immense potential for large-scale integration and in-depth explorations that are more representative of the study population. Efforts have been made to consolidate published data, yet extensive characterization is still lacking. Many focused on raw-data database constructions while others concentrate mainly on gene expression queries. Hereby, we present HTCA (www.htcatlas.org), an interactive database constructed based on ∼2.3 million high-quality cells from ∼3000 scRNA-seq samples and comprised in-depth phenotype profiles of 19 healthy adult and matching fetal tissues. HTCA provides a one-stop interactive query to gene signatures, transcription factor (TF) activities, TF motifs, receptor–ligand interactions, enriched gene ontology (GO) terms, etc. across cell types in adult and fetal tissues. At the same time, HTCA encompasses single-cell splicing variant profiles of 16 adult and fetal tissues, spatial transcriptomics profiles of 11 adult and fetal tissues, and single-cell ATAC-sequencing (scATAC-seq) profiles of 27 adult and fetal tissues. Besides, HTCA provides online analysis tools to perform major steps in a typical scRNA-seq analysis. Altogether, HTCA allows real-time explorations of multi-omics adult and fetal phenotypic profiles and provides tools for a flexible scRNA-seq analysis.

Background and Aims: Ambiguous understanding of tumors and the tumor microenvironments (TMEs) hinders accurate diagnosis and available treatment for multifocal hepatocellular carcinoma (HCC) covering intrahepatic metastasis (IM) and multicentric occurrence (MO). Here, we characterized the diverse TMEs of IM and MO identified by whole-exome sequencing (WES) at single-cell resolution. Approach and Results: We performed parallel WES and single-cell RNA sequencing (scRNA-seq) on twenty-three samples from seven patients to profile their TMEs when major results were validated by immunohistochemistry in the additional cohort. Integrative analysis of WES and scRNA-seq found that malignant cells in IM showed higher intra-tumor heterogeneity, stemness and more activated metabolism than those in MO. Tumors from IM shared similar TMEs while distinct TMEs were noticed in those from MO. Furthermore, CD20+ B cells, plasma cells and conventional type II dendritic cells (cDC2s) were decreased in IM relative to MO while T cells in IM exhibited a more terminally exhausted capacity with a higher proportion of proliferative/exhausted T cells than that in MO. Both CD20 and CD1C correlated with better prognosis in multifocal HCC. Additionally, MMP9+ tumor-associated macrophages were enriched across IM and MO which formed cellular niches with regulatory T cells (Tregs) and proliferative/exhausted T cells. Conclusions: Our findings deeply decipher the heterogeneous TMEs between IM and MO, which provide a comprehensive landscape of multifocal HCC. Export

Extrahepatic cholangiocarcinoma (eCCA) contains perihilar cholangiocarcinoma and distal cholangiocarcinoma both of which can arise at any point of the biliary tree and originate from disparate anatomical sites. Generally, the incidence of eCCA is increasing globally. Though surgical resection is the principal treatment of choice for the early stages of eCCA, optimal survival remains restricted by the high risk of recurrence when most patients are present with unresectable disease or distant metastasis. Furthermore, both intra- and intertumoral heterogeneity make it laborious to determine molecularly targeted therapies. In this review, we mainly focused on current findings in the field of eCCA, mostly including epidemiology, genomic abnormalities, molecular pathogenesis, tumor microenvironment, and other details while a summary of the biological mechanisms driving eCCA may shed light on intricate tumorigenesis and feasible treatment strategies.

Background: Multiple studies have shown that tumor-associated macrophages (TAMs) promote cancer initiation and progression. However, the reprogramming of macrophages in the tumor microenvironment (TME) and the cross-talk between TAMs and malignant subclones in intrahepatic cholangiocarcinoma (iCCA) has not been fully characterized, especially in a spatially resolved manner. Deciphering the spatial architecture of variable tissue cellular components in iCCA could contribute to the positional context of gene expression containing information pathological changes and cellular variability. Methods: Here, we applied spatial transcriptomics (ST) and digital spatial profiler (DSP) technologies with tumor sections from patients with iCCA. Results: The results reveal that spatial inter- and intra-tumor heterogeneities feature iCCA malignancy, and tumor subclones are mainly driven by physical proximity. Tumor cells with TME components shaped the intra-sectional heterogenetic spatial architecture. Macrophages are the most infiltrated TME component in iCCA. The protein trefoil factor 3 (TFF3) secreted by the malignant subclone can induce macrophages to reprogram to a tumor-promoting state, which in turn contributes to an immune-suppressive environment and boosts tumor progression. Conclusions: In conclusion, our description of the iCCA ecosystem in a spatially resolved manner provides novel insights into the spatial features and the immune suppressive landscapes of TME for iCCA.

Immune checkpoint blockade (ICB) therapy has transformed cancer treatment, yet many patients fail to respond. Employing single-cell multiomics, we unveil T cell dynamics influencing ICB response across 480 pan-cancer and 27 normal tissue samples. We identify four immunotherapy response-associated T cells (IRATs) linked to responsiveness or resistance and analyze their pseudotemporal patterns, regulatory mechanisms, and T cell receptor clonal expansion profiles specific to each response. Notably, transforming growth factor β1 (TGF-β1)+ CD4⁺ and Temra CD8⁺ T cells negatively correlate with therapy response, in stark contrast to the positive response associated with CXCL13+ CD4⁺ and CD8⁺ T cells. Validation with a cohort of 23 colorectal cancer (CRC) samples confirms the significant impact of TGF-β1+ CD4⁺ and CXCL13+ CD4⁺ and CD8⁺ T cells on ICB efficacy. Our study highlights the effectiveness of single-cell multiomics in pinpointing immune markers predictive of immunotherapy outcomes, providing an important resource for crafting targeted immunotherapies for successful ICB treatment across cancers.