Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense desmoplastic stroma and profound spatial heterogeneity, yet the multicellular organizational principles governing its tumor microenvironment remain poorly understood. Here, we performed single-cell RNA sequencing (scRNA-seq; n = 9) and spatial transcriptomics (n = 4), integrating 61,234 single cells and 30,430 spots to identify an invasive-frontier myofibroblastic cancer-associated fibroblast (IF-myCAF), which is characterized by elevated expression of ACTA2, TAGLN, COL11A1, THBS2, CTHRC1, and POSTN. IF-myCAFs were validated in eight independent PDAC cohorts (159 samples with 67,959 CAFs), showing conserved transcriptional programs and representing a major CAF subpopulation across datasets. Spatial cellular community (CC) analysis identified five recurrent CCs that delineated the multicellular organization of PDAC. Notably, the invasive-desmoplastic CC (ID-CC), which localized to the tumor-stroma interface, was defined by a 5.8-fold enrichment of IF-myCAF; within this community, IF-myCAF colocalized with malignant ductal cells and macrophages. We also found that the ID-CC transcriptional signature was independently associated with worse outcome in TCGA-PAAD. Furthermore, ligand-receptor (LR) analysis of 4,009 IF-myCAF-associated LR pairs identified IF-myCAF as the predominant cell population mediating ECM-related intercellular signaling within ID-CC. Structure-based virtual screening against four IF-myCAF-derived ligand-receptor axes mediating intercellular communication within this community (THBS2-CD47, CTHRC1-FZD8, PPIA-BSG/CD147, MIF-CD74) identified five multi-target drug-repurposing candidates, including conivaptan, lonafarnib, rucaparib, desloratadine, and ketotifen. Collectively, this study first identified a novel CAF subpopulation, IF-myCAF, and established a CC-based organizational scheme for PDAC spatial architecture. Our findings advance our understanding of spatial ecosystem-level stromal heterogeneity in PDAC and suggest targeting IF-myCAF-driven signaling within ID-CC.
| [1] |
Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 2024, 630: 493-500.
|
| [2] |
Alver TN, Bergholtz H, Holm MB, Dorg LT, Skrede ML, Kure EH, et al. Spatial transcriptomics reveals cancer and stromal cell heterogeneity between center and invasive front of pancreatic cancer. Mod Pathol, 2025, 38. 100726
|
| [3] |
Cable DM, Murray E, Zou LS, Goeva A, Macosko EZ, Chen F, et al. Robust decomposition of cell type mixtures in spatial transcriptomics. Nat Biotechnol, 2022, 40: 517-526.
|
| [4] |
Chen T, Sun Y, Zhu S, Chen XT, Pu KJ, Huang HJ, et al. RBM15B recognizes H3K79me2 to guide selective m(6)A-modification of mRNA and enhance oncoprotein translation in MLL-r leukemia. EMBO J, 2026, 45: 1672-1696.
|
| [5] |
Elyada E, Bolisetty M, Laise P, Flynn WF, Courtois ET, Burkhart RA, et al. Cross-species single-cell analysis of pancreatic ductal adenocarcinoma reveals antigen-presenting cancer-associated fibroblasts. Cancer Discov, 2019, 9: 1102-1123.
|
| [6] |
Francescone R, Barbosa Vendramini-Costa D, Franco-Barraza J, Wagner J, Muir A, Lau AN, et al. Netrin G1 promotes pancreatic tumorigenesis through cancer-associated fibroblast–driven nutritional support and immunosuppression. Cancer Discov, 2021, 11: 446-479.
|
| [7] |
Gao R, Bai S, Henderson YC, Lin Y, Schalck A, Yan Y, et al. Delineating copy number and clonal substructure in human tumors from single-cell transcriptomes. Nat Biotechnol, 2021, 39: 599-608.
|
| [8] |
Greenwald AC, Darnell NG, Hoefflin R, Simkin D, Mount CW, Gonzalez Castro LN, et al. Integrative spatial analysis reveals a multi-layered organization of glioblastoma. Cell, 2024, 187: 2485-2501.
|
| [9] |
Hao Y, Hao S, Andersen-Nissen E, Mauck WMIIIZheng S, Butler A, et al. Integrated analysis of multimodal single-cell data. Cell, 2021, 184: 3573-3587.
|
| [10] |
Hosein AN, Brekken RA, Maitra A. Pancreatic cancer stroma: an update on therapeutic targeting strategies. Nat Rev Gastroenterol Hepatol, 2020, 17: 487-505.
|
| [11] |
Jin S, Guerrero-Juarez CF, Zhang L, Chang I, Ramos R, Kuan C-H, et al. Inference and analysis of cell-cell communication using Cell Chat. Nat Commun, 2021, 12: 1088.
|
| [12] |
Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. Nature, 2021, 596: 583-589.
|
| [13] |
Kang J, Lee JH, Cha H, An J, Kwon J, Lee S, et al. Systematic dissection of tumor-normal single-cell ecosystems across a thousand tumors of 30 cancer types. Nat Commun, 2024, 15: 4067.
|
| [14] |
Kim J, Bamlet WR, Oberg AL, Chaffee KG, Donahue G, Cao X-J, et al. Detection of early pancreatic ductal adenocarcinoma with thrombospondin-2 and CA19-9 blood markers. Sci Transl Med, 2017, 9. eaah5583
|
| [15] |
Kotliar D, Curtis M, Agnew R, Weinand K, Nathan A, Baglaenko Y, et al. Reproducible single-cell annotation of programs underlying T cell subsets, activation states and functions. Nat Methods, 2025, 22: 1964-1980.
|
| [16] |
Laklai H, Miroshnikova YA, Pickup MW, Collisson EA, Kim GE, Barrett AS, et al. Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression. Nat Med, 2016, 22: 497-505.
|
| [17] |
Liao X, Wang W, Yu B, Tan S. Thrombospondin-2 acts as a bridge between tumor extracellular matrix and immune infiltration in pancreatic and stomach adenocarcinomas: an integrative pan-cancer analysis. Cancer Cell Int, 2022, 22. 213
|
| [18] |
Lin J, Chen C, Li S, Chen L, Fang M, Fu S, et al. Spatial and single-cell characterization of human glioblastoma tumor microenvironment reveals malignant cellular communities. Nat Neurosci, 2026, 29: 1342-1354.
|
| [19] |
Ma L, Xiong B, Liu M, Tan K. Cellular neighborhoods in cancer. Nat Cancer, 2026, 7: 16-28.
|
| [20] |
Misra S, Hascall VC, Markwald RR, Ghatak S. Interactions between hyaluronan and its receptors (CD44, RHAMM) regulate the activities of inflammation and cancer. Front Immunol, 2015, 6. 201
|
| [21] |
Moses L, Pachter L. Museum of spatial transcriptomics. Nat Methods, 2022, 19: 534-546.
|
| [22] |
Nigro P, Pompilio G, Capogrossi MC. Cyclophilin A: a key player for human disease. Cell Death Dis, 2013, 4: e888.
|
| [23] |
Niu N, Shen X, Wang Z, Chen Y, Weng Y, Yu F, et al. Tumor cell-intrinsic epigenetic dysregulation shapes cancer-associated fibroblasts heterogeneity to metabolically support pancreatic cancer. Cancer Cell, 2024, 42: 869-884.
|
| [24] |
Öhlund D, Handly-Santana A, Biffi G, Elyada E, Almeida AS, Ponz-Sarvise M, et al. Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer. J Exp Med, 2017, 214: 579-596.
|
| [25] |
Özdemir BC, Pentcheva-Hoang T, Carstens JL, Zheng X, Wu C-C, Simpson TR, et al. Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival. Cancer Cell, 2014, 25: 719-734.
|
| [26] |
Pushpakom S, Iorio F, Eyers PA, Escott KJ, Hopper S, Wells A, et al. Drug repurposing: progress, challenges and recommendations. Nat Rev Drug Discov, 2019, 18: 41-58.
|
| [27] |
Pu K, Chen X, Zhu S, An Y, Feng XY, Huang HJ, et al. Chromatin-associated circRNA ciCRLF3(2) regulates cell differentiation blockage via activating non-homologous end joining-based DNA repair. Cell Death Differ, 2026, 33: 267-283.
|
| [28] |
Ravi VM, Will P, Kueckelhaus J, Sun N, Joseph K, Salié H, et al. Spatially resolved multi-omics deciphers bidirectional tumor-host interdependence in glioblastoma. Cancer Cell, 2022, 40: 639-655.
|
| [29] |
Rhim AD, Oberstein PE, Thomas DH, Mirek ET, Palermo CF, Sastra SA, et al. Stromal elements act to restrain, rather than support, pancreatic ductal adenocarcinoma. Cancer Cell, 2014, 25: 735-747.
|
| [30] |
Sahai E, Astsaturov I, Cukierman E, DeNardo DG, Egeblad M, Evans RM, et al. A framework for advancing our understanding of cancer-associated fibroblasts. Nat Rev Cancer, 2020, 20: 174-186.
|
| [31] |
Shiau C, Cao J, Gong D, Gregory MT, Caldwell NJ, Yin X, et al. Spatially resolved analysis of pancreatic cancer identifies therapy-associated remodeling of the tumor microenvironment. Nat Genet, 2024, 56: 2466-2478.
|
| [32] |
Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin, 2024, 74: 12-49
|
| [33] |
Sikic BI, Lakhani N, Patnaik A, Shah SA, Chandana SR, Rasco D, et al. First-in-human, first-in-class phase I trial of the anti-CD47 antibody Hu5F9-G4 in patients with advanced cancers. J Clin Oncol, 2019, 37: 946-953.
|
| [34] |
Street K, Risso D, Fletcher RB, Das D, Ngai J, Yosef N, et al. Slingshot: cell lineage and pseudotime inference for single-cell transcriptomics. BMC Genomics, 2018, 19. 477
|
| [35] |
Veghini L, Pasini D, Fang R, Delfino P, Filippini D, Neander C, et al. Differential activity of MAPK signalling defines fibroblast subtypes in pancreatic cancer. Nat Commun, 2024, 15: 10534.
|
| [36] |
Winkler J, Abisoye-Ogunniyan A, Metcalf KJ, Werb Z. Concepts of extracellular matrix remodelling in tumour progression and metastasis. Nat Commun, 2020, 11: 5120.
|
| [37] |
Wishart DS, Feunang YD, Guo AC, Lo EJ, Marcu A, Grant JR, et al. DrugBank 5.0: a major update to the DrugBank database for 2018. Nucleic Acids Res, 2018, 46: D1074D1082.
|
| [38] |
Xu H, Wang S, Fang M, Luo S, Chen C, Wan S, et al. SPACEL: deep learning-based characterization of spatial transcriptome architectures. Nat Commun, 2023, 14: 7603.
|
| [39] |
Yamamoto S, Nishimura O, Misaki K, Nishita M, Minami Y, Yonemura S, et al. Cthrc1 selectively activates the planar cell polarity pathway of Wnt signaling by stabilizing the Wnt-receptor complex. Dev Cell, 2008, 15: 23-36.
|
| [40] |
Zhang Q, Cao Z, Yan S, Chen B, Huang B, Liu Z, et al. Spatial multi-omics defines cancer-associated fibroblasts subtype gradients driving metabolic support and immune remodeling in pancreatic ductal adenocarcinoma. Cancer Lett, 2026, 653. 218585
|
| [41] |
Zhang W-M, Käpylä J, Puranen JS, Knight CG, Tiger C-F, Pentikäinen OT, et al. α11β1 integrin recognizes the GFOGER sequence in interstitial collagens. J Biol Chem, 2003, 278: 7270-7277.
|
| [42] |
Zhang W, Brown EL, Usmani A, Earland N, Kang M, Olelewe C, et al. Non-invasive profiling of the tumour microenvironment with spatial ecotypes. Nature, 2026, 654: 1076-1087.
|
| [43] |
Zhang Y, Ling L, Murad R, Maganti S, Manceau A, Hetrick HA, et al. Macropinocytosis maintains CAF subtype identity under metabolic stress in pancreatic cancer. Cancer Cell, 2025, 43: 1677-1696.
|
Funding
National Natural Science Foundation of China(32370594)
National Key R&D Program of China(2022YFA1303302)
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