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Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014). This article is part of the Topical Collection: Special Issue on Big Data and Smart Computing in Network Systems H.W. conceived the project; H.W., J.F. and Y.J.Z. designed the experiments; X.W., C.L.L., J.A.C., L.C., M.H., Y.J., E.L.,Y.L.Z. and X.Z. performed the experiments and bioinformatics analysis; X.W., A.D., X.F., W.Y. and Z.Y. collected the human samples; H.W., J.F., X.R. and X.C. analyzed the data; J.F., X.W. and C.L.L. wrote the manuscript. Corresponding authors RNA velocity was performed to investigate the potential inter-relationship of mesenchymal cell lineage using velocyto 89. BAM file containing all the mesenchymal cells was used in this pipeline, spliced/unspliced reads were annotated. The calculation of RNA velocity values for each gene in each cell and embedding RNA velocity vector to low-dimension space was done by following the velocyto pipeline. All the parameters were set as default. The result was visualized into UMAP plot. Tissue distribution preference of clusters

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Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014). Sun, L., Cai, J. & Gonzalez, F. J. The role of farnesoid X receptor in metabolic diseases, and gastrointestinal and liver cancer. Nat. Rev. Gastroenterol. Hepatol. 18, 335–347 (2021). Based on accumulated evidence in a century, GBC typifies chronic inflammation-associated cancers (CIACs) 4. The majority of GBCs reside in a harsh environment besieged by infectious (e.g., pathogens), chemical (e.g., pancreatic juice reflux), mechanical (e.g., gallstone irritation), metabolic (e.g., bile cholesterol supersaturation), and hydrodynamic (e.g., biliary sludge) stresses which, along with heightened cell division stress with aging, contribute to a multitude of microbial, cytotoxic, metabolic, genotoxic, and senescence-related inflammation processes 5, many of which have initiated and evolved from inflamed gallbladders for ~15 years or more 6.Schewe, M. et al. Secreted phospholipases A2 are intestinal stem cell niche factors with distinct roles in homeostasis, inflammation, and cancer. Cell Stem Cell 19, 38–51 (2016). Veglia, F., Sanseviero, E. & Gabrilovich, D. I. Myeloid-derived suppressor cells in the era of increasing myeloid cell diversity. Nat. Rev. Immunol. 21, 485–498 (2021). Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, i884–i890 (2018). Following resection, digestion, quality filtering, and doublet removal, we obtained a total of 140,870 cells. We identified 8 cell subpopulations using the t-distributed stochastic neighbor embedding ( t-SNE) method, including T cells ( n = 50,871; CD3D, CD3E), B cells ( n = 5,925; CD79A, MS4A1), plasma B cells ( n = 8,490; CD79A, MZB1), myeloid cells ( n = 14,958; CD68, CD14, CD163), mast cells ( n = 1,971; TPSA B1, KIT, CPA3), mesenchymal cells ( n = 24,680; COL1A1, COL14A1, LUM), endothelial cells ( n = 8,897; CD34, PECAM1, VWF), and epithelial cells (EPCs, n = 25,078; EPCAM, KRT19) (Fig. 1b–d). EPCs primarily clustered by sample, indicating marked inter-patient heterogeneity, but the stromal and immune cells mainly clustered by cell type with mixed biological origins (Fig. 1e, f). These cell types dispersed among benign and tumor samples to varying extents, and this variation remained for paired lesions from the same case, implying intra- and inter-patient heterogeneity (Fig. 1g; Supplementary Table S2). Generally, compared with CCs, PTs were enriched with more EPCs and mesenchymal cells but with fewer immune cells, especially T cells behaving as tumor-infiltrating lymphocytes (TILs) (Supplementary Fig. S2a, b). These discrepancies implied dynamic cellular adaptation and competition for space and survival amidst different ecosystems 13. Classification of malignant and non-malignant epithelial cells

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Pandey, A. et al. Integrated genomic analysis reveals mutated ELF3 as a potential gallbladder cancer vaccine candidate. Nat. Commun. 11, 4225 (2020). Newman, A. M. et al. Determining cell type abundance and expression from bulk tissues with digital cytometry. Nat. Biotechnol. 37, 773–782 (2019). Efremova, M., Vento-Tormo, M., Teichmann, S. A. & Vento-Tormo, R. CellPhoneDB: inferring cell-cell communication from combined expression of multi-subunit ligand-receptor complexes. Nat. Protoc. 15, 1484–1506 (2020).

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Muthuswamy, S. K. Self-organization in cancer: Implications for histopathology, cancer cell biology, and metastasis. Cancer Cell 39, 443–446 (2021). Cheng, S. et al. A pan-cancer single-cell transcriptional atlas of tumor infiltrating myeloid cells. Cell 184, 792–809.e23 (2021).

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Biswas J, Wang E, Gaowei M, Liu W, Rahman O, Sadowski JT (2021) High quantum effic Department of General Surgery, The Second Xiangya Hospital, Central South University, Changsha 410011, People's Republic of China. State Key Laboratory of Powder Metallurgy, Central South University, Changsha 410083, People's Republic of China. Dorhoi, A. & Du Plessis, N. Monocytic myeloid-derived suppressor cells in chronic infections. Front. Immunol. 8, 1895 (2017).Chen S, Hu M, Liu L, Pan Y, Li P, He J, Ding J. Chen S, et al. iScience. 2022 Nov 13;25(12):105563. doi: 10.1016/j.isci.2022.105563. eCollection 2022 Dec 22. iScience. 2022. PMID: 36444307 Free PMC article. Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015). The primers, primary and secondary antibodies were listed in Supplementary Table S14, and were used at the concentrations indicated by manufacturer’s instructions. Statistics International Cooperation Laboratory on Signal Transduction, National Center for Liver Cancer, Ministry of Education Key Laboratory on Signaling Regulation and Targeting Therapy of Liver Cancer, Shanghai Key Laboratory of Hepato-biliary Tumor Biology, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China

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In terms of GBO lentivirus infection, the organoids were resuspended in a 500 μL growth medium after trypsinization for 5 min at 37 °C. Then cells were seeded into 48-well plates at 80%–90% confluence and were infected with lentivirus of NC labeling GFP and KRAS G12D labeling GFP according to standard procedures 107. Three days after infection, the growth medium was exchanged with medium containing puromycin at a concentration of 5 μg/mL for selection for two weeks. Co-culture experiment and senescence-associated β-galactosidase (SA-β-gal) assay Zhang Y, Liu W, Fang Y, Wu D (2006) Secure localization and authentication in ultra-wideband sensor networks. IEEE J Sel Areas Commun 24(4):829–835Vassiliadis, D. & Dawson, M. A. Mutation alters chromatin changes during injury response to drive cancer. Nature 590, 557–558 (2021).