Spatial transcriptomics reveals a new cluster of fibroblasts to promote the progression of lung adenocarcinoma

Abstract

Methods: Our study applied spatio-temporal enhanced resolution omics-sequencing (Stereo-seq) to investigate the interactions between fibroblasts and lung cancer cells. A co-culture system of A549 lung cancer cells and HFL1 fibroblasts was established to validate the functional roles of identified fibroblast subtypes. Gene knockdown, proliferation, migration, invasion, and ELISA assays were conducted to investigate underlying mechanisms.

Results: Spatial transcriptomics revealed significant heterogeneity in LUAD tissues, with identification of a novel fibroblast cluster (C0) characterized by high expression of POSTN, THY1, BGN, and COL1A1, which co-localized with malignant epithelial cells (C5) in high-malignancy regions. Public dataset analysis confirmed C0 as CAFs, enriched in LUAD and associated with poor prognosis. Ligand-receptor analysis highlighted collagen I- and fibronectin 1-mediated interactions between C0 and C5. In vitro, co-culture with A549 induced differentiation of HFL1 into CAFs, with upregulation of COL1A1, COL1A2, and CAF markers. Knockdown of COL1A1 or COL1A2 in fibroblasts significantly reduced collagen I secretion and attenuated cancer cell proliferation, migration, and invasion, while exerting minimal effects on angiogenesis.

Conclusions: These findings not only provide a molecular map of lung cancer tissues for further study but also illustrate a novel co-location relationship between fibroblasts and cancer cells within the TME. Our results highlight the potential of CAFs as a promising therapeutic target, complementing existing approaches targeting genetic mutations and immune cells.

Keywords

cancer-associated fibroblasts / lung adenocarcinoma / spatial transcriptomics

References

[1] Thai AA, Solomon BJ, Sequist LV, Gainor JF, Heist RS. Lung cancer. Lancet. 2021;398:535-554.

[2] Melo Ferreira R, Sabo AR, Winfree S, et al. Integration of spatial and single-cell transcriptomics localises epithelial cell-immune cross-talk in kidney injury. JCI Insight. 2021;6:e147703.

[3] Moncada R, Barkley D, Wagner F, et al. Integrating microarray-based spatial transcriptomics and single-cell RNA-seq reveals tissue architecture in pancreatic ductal adenocarcinomas. Nat Biotechnol. 2020;38:333-342.

[4] Kuppe C, Ramirez Flores RO, Li Z, et al. Spatial multi-omic map of human myocardial infarction. Nature. 2022;608:766-777.

[5] Larroquette M, Guegan JP, Besse B, et al. Spatial transcriptomics of macrophage infiltration in non-small cell lung cancer reveals determinants of sensitivity and resistance to anti-PD1/PD-L1 antibodies. J Immunother Cancer. 2022;10:e003890.

[6] Zhang Qi, Abdo R, Iosef C, et al. The spatial transcriptomic landscape of non-small cell lung cancer brain metastasis. Nat Commun. 2022;13:5983.

[7] Tejada ML, Yu L, Dong J, et al. Tumour-driven paracrine platelet-derived growth factor receptor alpha signalling is a key determinant of stromal cell recruitment in a model of human lung carcinoma. Clin Cancer Res. 2006;12:2676-2688.

[8] Meng Q, Luo X, Chen J, et al. Unmasking carcinoma-associated fibroblasts: key transformation player within the tumour microenvironment. Biochim Biophys Acta Rev Cancer. 2020;1874:188443.

[9] Ershaid N, Sharon Y, Doron H, et al. NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis. Nat Commun. 2019;10:4375.

[10] Erez N, Glanz S, Raz Y, Avivi C, Barshack I. Cancer associated fibroblasts express pro-inflammatory factors in human breast and ovarian tumours. Biochem Biophys Res Commun. 2013;437:397-402.

[11] Hu H, Piotrowska Z, Hare PJ, et al. Three subtypes of lung cancer fibroblasts define distinct therapeutic paradigms. Cancer Cell. 2021;39:1531-1547.e1510.

[12] Navab R, Strumpf D, Bandarchi B, et al. Prognostic gene-expression signature of carcinoma-associated fibroblasts in non-small cell lung cancer. Proc Natl Acad Sci U S A. 2011;108:7160-7165.

[13] Albrengues J, Bertero T, Grasset E, et al. Epigenetic switch drives the conversion of fibroblasts into proinvasive cancer-associated fibroblasts. Nat Commun. 2015;6:10204.

[14] Rubinstein-Achiasaf L, Morein D, Ben-Yaakov H, et al. Persistent inflammatory stimulation drives the conversion of MSCs to inflammatory CAFs that promote pro-metastatic characteristics in breast cancer cells. Cancers. 2021;13:1472.

[15] Liu X, Jiang Y, Song D, et al, Clinical challenges of tissue preparation for spatial transcriptome. Clin Transl Med. 2022;12:e669.

[16] Kim N, Kim HK, Lee K, et al. Single-cell RNA sequencing demonstrates the molecular and cellular reprogramming of metastatic lung adenocarcinoma. Nat Commun. 2020;11:2285.

[17] Zhang X, Lan Y, Xu J, et al. CellMarker: a manually curated resource of cell markers in human and mouse. Nucleic Acids Res. 2019;47:D721-d728.

[18] Liu F, Liu X, Liu Y, et al. Deciphering the heterogeneity of neutrophil cells within circulation and the lung cancer microenvironment pre- and post-operation. Cell Biol Toxicol. 2024;40:11.

[19] Ding Y, Zhang H, Wen H, et al. Single-cell and spatial transcriptomics-based research reveals association between M2a macrophages and tumour spread through air spaces in lung adenocarcinoma. Am J Pathol. 2025;195:2477-2491.

[20] Zhang H, Zhang P, Lin X, et al. Integrative single-cell analysis of LUAD: elucidating immune cell dynamics and prognostic modelling based on exhausted CD8+ T cells. Front Immunol. 2024;15:1366096.

[21] Nicolini A, Ferrari P. Involvement of tumour immune microenvironment metabolic reprogramming in colorectal cancer progression, immune escape, and response to immunotherapy. Front Immunol. 2024;15:1353787.

[22] Bonnay Fç, Veloso A, Steinmann V, et al. Oxidative metabolism drives immortalisation of neural stem cells during tumorigenesis. Cell. 2020;182:1490-1507.e1419.

[23] Saksela O, Wahlström T, Meyer B, Vaheri A. Presence of alpha-2-macroglobulin in normal but not in malignant cervical epithelium. Cancer Res. 1984;44:2942-2946.

[24] Yan W, Hu W, Song Y, et al. Differential network analysis reveals the key role of the ECM-receptor pathway in α-particle-induced malignant transformation. Mol Ther Nucleic Acids. 2024;35:102260.

[25] Xu Y, Li J, Wang J, Deng F. A novel CAF-cancer cell crosstalk-related gene prognostic index based on machine learning: prognostic significance and prediction of therapeutic response in head and neck squamous cell carcinoma. J Transl Med. 2024;22:645.

[26] Wang K, He M, Fan X, et al. SEMA5A-PLXNB3 Axis Promotes PDAC Liver Metastasis Outgrowth through Enhancing the Warburg Effect. J Immunol Res. 2023;2023:3274467.

[27] Chen M-Z, Su Li-Yu, Ko P-H, et al. Extracellular domain of semaphorin 5A serves a tumour‑suppressing role by activating interferon signalling pathways in lung adenocarcinoma cells. Int J Oncol. 2022;60:21.

[28] Ko PH, Lenka G, Chen YA, et al. Semaphorin 5A suppresses the proliferation and migration of lung adenocarcinoma cells. Int J Oncol. 2020;56:165-177.

[29] Li K, Wang R, Liu G-W, et al. Refining the optimal CAF cluster marker for predicting TME-dependent survival expectancy and treatment benefits in NSCLC patients. Sci Rep. 2024;14:16766.

[30] Shi R, Zhang Z, Zhu A, et al. Targeting type I collagen for cancer treatment. Int J Cancer. 2022;151:665-683.

[31] Qin Z, Ma J, Chu Xu, Guo T, Feng P, Wang M. Delivery of miR-29a improves the permeability of cisplatin by downregulating collagen I expression. Biochem Biophys Res Commun. 2024;710:149884.

[32] Iwai M, Tulafu M, Togo S, et al. Cancer-associated fibroblast migration in non-small cell lung cancers is modulated by increased integrin α11 expression. Mol Oncol. 2021;15:1507-1527.

[33] van Kempen LC, Rijntjes J, Mamor-Cornelissen I, et al. Type I collagen expression contributes to angiogenesis and the development of deeply invasive cutaneous melanoma. Int J Cancer. 2008;122:1019-1029.

[34] Shintani Y, Maeda M, Chaika N, Johnson KR, Wheelock MJ. Collagen I promotes epithelial-to-mesenchymal transition in lung cancer cells via transforming growth factor-beta signalling. Am J Respir Cell Mol Biol. 2008;38:95-104.

[35] Yamazaki S, Higuchi Y, Ishibashi M, et al. Collagen type I induces EGFR-TKI resistance in EGFR-mutated cancer cells by mTOR activation through Akt-independent pathway. Cancer Sci. 2018;109:2063-2073.

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2026 The Author(s). Clinical and Translational Discovery published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.