Integrative analyses of bulk and single-cell transcriptomics reveals the infiltration and crosstalk of cancer-associated fibroblasts as a novel predictor for prognosis and microenvironment remodeling in intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is a highly malignant neoplasm and characterized by desmoplastic matrix. The heterogeneity and crosstalk of tumor microenvironment remain incompletely understood. METHODS: To address this gap, we performed Weighted Gene Co-expression Network Analysis (WGCNA) to identify and construct a cancer associated fibroblasts (CAFs) infiltration biomarker. We also depicted the intercellular communication network and important receptor-ligand complexes using the single-cell transcriptomics analysis of tumor and Adjacent normal tissue. RESULTS: Through the intersection of TCGA DEGs and WGCNA module genes, 784 differential genes related to CAFs infiltration were obtained. After a series of regression analyses, the CAFs score was generated by integrating the expressions of EVA1A, APBA2, LRRTM4, GOLGA8M, BPIFB2, and their corresponding coefficients. In the TCGA-CHOL, GSE89748, and 107,943 cohorts, the high CAFs score group showed unfavorable survival prognosis (p < 0.001, p = 0.0074, p = 0.028, respectively). Additionally, a series of drugs have been predicted to be more sensitive to the high-risk group (p < 0.05). Subsequent to dimension reduction and clustering, thirteen clusters were identified to construct the single-cell atlas. Cell-cell interaction analysis unveiled significant enhancement of signal transduction in tumor tissues, particularly from fibroblasts to malignant cells via diverse pathways. Moreover, SCENIC analysis indicated that HOXA5, WT1, and LHX2 are fibroblast specific motifs. CONCLUSIONS: This study reveals the key role of fibroblasts - oncocytes interaction in the remodeling of the immunosuppressive microenvironment in intrahepatic cholangiocarcinoma. Subsequently, it may trigger cascade activation of downstream signaling pathways such as PI3K-AKT and Notch in tumor, thus initiating tumorigenesis. Targeted drugs aimed at disrupting fibroblasts-tumor cell interaction, along with associated enrichment pathways, show potential in mitigating the immunosuppressive microenvironment that facilitates tumor progression.

Exosomal long non-coding RNA TRPM2-AS promotes angiogenesis in gallbladder cancer through interacting with PABPC1 to activate NOTCH1 signaling pathway.

BACKGROUND: Abnormal angiogenesis is crucial for gallbladder cancer (GBC) tumor growth and invasion, highlighting the importance of elucidating the mechanisms underlying this process. LncRNA (long non-coding RNA) is widely involved in the malignancy of GBC. However, conclusive evidence confirming the correlation between lncRNAs and angiogenesis in GBC is lacking. METHODS: LncRNA sequencing was performed to identify the differentially expressed lncRNAs. RT-qPCR, western blot, FISH, and immunofluorescence were used to measure TRPM2-AS and NOTCH1 signaling pathway expression in vitro. Mouse xenograft and lung metastasis models were used to evaluate the biological function of TRPM2-AS during angiogenesis in vivo. EDU, transwell, and tube formation assays were used to detect the angiogenic ability of HUVECs. RIP, RAP, RNA pull-down, dual-luciferase reporter system, and mass spectrometry were used to confirm the interaction between TRPM2-AS, IGF2BP2, NUMB, and PABPC1. RESULTS: TRPM2-AS was upregulated in GBC tissues and was closely related to angiogenesis and poor prognosis in patients with GBC. The high expression level and stability of TRPM2-AS benefited from m6A modification, which is recognized by IGF2BP2. In terms of exerting pro-angiogenic effects, TRPM2-AS loaded with exosomes transported from GBC cells to HUVECs enhanced PABPC1-mediated NUMB expression inhibition, ultimately promoting the activation of the NOTCH1 signaling pathway. PABPC1 inhibited NUMB mRNA expression through interacting with AGO2 and promoted miR-31-5p and miR-146a-5p-mediated the degradation of NUMB mRNA. The NOTCH signaling pathway inhibitor DAPT inhibited GBC tumor angiogenesis, and TRPM2-AS knockdown enhanced this effect. CONCLUSIONS: TRPM2-AS is a novel and promising biomarker for GBC angiogenesis that promotes angiogenesis by facilitating the activation of the NOTCH1 signaling pathway. Targeting TRPM2-AS opens further opportunities for future GBC treatments.

MarkovHC: Markov hierarchical clustering for the topological structure of high-dimensional single-cell omics data with transition pathway and critical point detection.

Clustering cells and depicting the lineage relationship among cell subpopulations are fundamental tasks in single-cell omics studies. However, existing analytical methods face challenges in stratifying cells, tracking cellular trajectories, and identifying critical points of cell transitions. To overcome these, we proposed a novel Markov hierarchical clustering algorithm (MarkovHC), a topological clustering method that leverages the metastability of exponentially perturbed Markov chains for systematically reconstructing the cellular landscape. Briefly, MarkovHC starts with local connectivity and density derived from the input and outputs a hierarchical structure for the data. We firstly benchmarked MarkovHC on five simulated datasets and ten public single-cell datasets with known labels. Then, we used MarkovHC to investigate the multi-level architectures and transition processes during human embryo preimplantation development and gastric cancer procession. MarkovHC found heterogeneous cell states and sub-cell types in lineage-specific progenitor cells and revealed the most possible transition paths and critical points in the cellular processes. These results demonstrated MarkovHC's effectiveness in facilitating the stratification of cells, identification of cell populations, and characterization of cellular trajectories and critical points.