Spatial transcriptomic analysis across histological subtypes reveals molecular heterogeneity and prognostic markers in early-stage lung adenocarcinoma

Abstract

Background: The progression and prognosis of early-stage lung adenocarcinoma are closely associated with histologic subtypes, yet the presence of mixed histologic patterns often complicates prognostic assessment. Currently, the correlation between molecular and histologic features remains poorly understood.

Methods: Formalin-fixed paraffin-embedded (FFPE) samples were collected from patients with primary early-stage lung adenocarcinoma, encompassing three histologic subtypes: well-differentiated, moderately differentiated, and poorly differentiated. The GeoMx Digital Spatial Profiler platform was utilized to obtain spatial transcriptomic profiling. Regions of interest were carefully selected and further subdivided into three categories of areas of interest, specifically epithelial cell-enriched regions, macrophage-enriched regions, and other regions. Multiplex immunofluorescence (mIF) assays were employed to validate the obtained results.

Results: Distinct molecular characteristics were identified in tumor epithelial- and macrophage-enriched compartments spanning well-differentiated to poorly differentiated tumors. In poorly differentiated tumors, we observed enrichment of pathways related to humoral immune response, complement activation regulation, and extracellular matrix receptor interaction pathways, all of which are significantly associated with poorer prognosis. We integrated these pathways to develop a composite molecular signature that strongly correlate with adverse prognosis.

Conclusions: Our results provide new insights into the link between molecular and histologic subtypes in mixed-type lung adenocarcinomas. Specifically, the identified molecular signatures offer potential biomarkers for predicting disease progression and prognosis, thus facilitating more precise and personalized therapeutic approaches.

Keywords

digital spatial transcriptomic profiling / epithelial compartments / histologic subtypes / lung adenocarcinoma / macrophage compartments

References

[1] Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA Cancer J Clin. 2023; 73: 17-48. (1542-4863 (Electronic)).

[2] Nicholson AG, Tsao MS, Beasley MB, et al. The 2021 WHO classification of lung tumors: impact of advances since 2015. J Thorac Oncol. 2022; 17(3): 362-387.

[3] Moreira AL, Ocampo PSS, Xia Y, et al. A grading system for invasive pulmonary adenocarcinoma: a proposal from the international association for the study of lung cancer pathology committee. J Thorac Oncol. 2020; 15(10): 1599-1610.

[4] Nicholson AG. Grading in lung adenocarcinoma: another new normal. J Thorac Oncol. 2021; 16(10): 1601-1604.

[5] Tsuta K, Kawago M, Inoue E, et al. The utility of the proposed IASLC/ATS/ERS lung adenocarcinoma subtypes for disease prognosis and correlation of driver gene alterations. Lung Cancer. 2013; 81(3): 371-376. (1872-8332 (Electronic)).

[6] Yoshizawa A, Sumiyoshi S, Sonobe M, et al. Validation of the IASLC/ATS/ERS lung adenocarcinoma classification for prognosis and association with EGFR and KRAS gene mutations: analysis of 440 Japanese patients. J Thorac Oncol. 2013; 8(1): 52-61. (1556-1380 (Electronic)).

[7] von der Thüsen JH, Tham YS, Pattenden H, et al. Prognostic significance of predominant histologic pattern and nuclear grade in resected adenocarcinoma of the lung: potential parameters for a grading system. J Thorac Oncol. 2013; 8(1): 37-44. (1556-1380 (Electronic)).

[8] Li Z, Li F, Pan C, et al. Tumor cell proliferation (Ki-67) expression and its prognostic significance in histological subtypes of lung adenocarcinoma. Lung Cancer. 2021; 154: 69-75. (1872-8332 (Electronic)).

[9] Wright J, Churg A, Kitaichi M, et al. Reproducibility of visual estimation of lung adenocarcinoma subtype proportions. Modern Pathology. 2019; 32: 1587-1592. (1530-0285 (Electronic)).

[10] Wang Y, Liu B, Min Q, et al. Spatial transcriptomics delineates molecular features and cellular plasticity in lung adenocarcinoma progression. Cell Discov. 2023; 9(1): 96.

[11] Caso R, Sanchez-Vega F, Tan KS, et al. The underlying tumor genomics of predominant histologic subtypes in lung adenocarcinoma. J Thorac Oncol. 2020; 15(12): 1844-1856. (1556-1380 (Electronic)).

[12] Tavernari DA-O, Battistello E, Dheilly E, et al. Nongenetic evolution drives lung adenocarcinoma spatial heterogeneity and progression. Cancer Discov. 2021; 11(6): 1490-1507. (2159-8290 (Electronic)).

[13] Karasaki T, Moore DA, Veeriah S, et al. Evolutionary characterization of lung adenocarcinoma morphology in TRACERx. Nat Med. 2023; 29(4): 833-845.

[14] Akhave N, Zhang J, Bayley E, et al. Immunogenomic profiling of lung adenocarcinoma reveals poorly differentiated tumors are associated with an immunogenic tumor microenvironment. Lung Cancer. 2022; 172: 19-28. (1872-8332 (Electronic)).

[15] Zhang JT, Zhang J, Wang SR, et al. Spatial downregulation of CD74 signatures may drive invasive component development in part-solid lung adenocarcinoma. iScience. 2023; 26(10): 107699. (2589-0042 (Electronic)).

[16] Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma. Nature. 2014; 511(7511): 543-550.

[17] Martínez-Ruiz C, Black JRM, Puttick C, et al. Genomic-transcriptomic evolution in lung cancer and metastasis. Nature. 2023; 616(7957): 543-552.

[18] Roh W, Geffen Y, Cha H, et al. High-resolution profiling of lung adenocarcinoma identifies expression subtypes with specific biomarkers and clinically relevant vulnerabilities. Cancer Res. 2022; 82(21): 3917-3931.

[19] Merritt CR, Ong GT, Church SE, et al. Multiplex digital spatial profiling of proteins and RNA in fixed tissue. Nat Biotechnol. 2020; 38(5): 586-599. (1546-1696 (Electronic)).

[20] Chen B, Khodadoust MS, Liu CL, Newman AM, Alizadeh AA. Profiling tumor infiltrating immune cells with CIBERSORT. Methods Mol Biol. 2018; 1711: 243-259. (1940-6029 (Electronic)).

[21] Hu A, Liu Y, Zhang H, et al. BPIFB1 promotes metastasis of hormone receptor-positive breast cancer via inducing macrophage M2-like polarization. Cancer Sci. 2023; 114(11): 4157-4171.

[22] Cui M, Huang J, Zhang S, Liu Q, Liao Q, Qiu X. Immunoglobulin expression in cancer cells and its critical roles in tumorigenesis. Front Immunol. 2021; 12: 613530. (1664-3224 (Electronic)).

[23] Sha L, Lv Z, Liu Y, et al. Shikonin inhibits the Warburg effect, cell proliferation, invasion and migration by downregulating PFKFB2 expression in lung cancer. Mol Med Rep. 2021; 24(2): 560.

[24] Sui JSY, Martin P, Keogh A, et al. Altered expression of ACOX2 in non-small cell lung cancer. BMC Pulm Med. 2022; 22(1): 321. (1471-2466 (Electronic)).

[25] Kou W, Li B, Shi Y, et al. High complement protein C1q levels in pulmonary fibrosis and non-small cell lung cancer associated with poor prognosis. BMC Cancer. 2022; 22(1): 110.

[26] Chaudhary N, Jayaraman A, Reinhardt C, Campbell JD, Bosmann M. A single-cell lung atlas of complement genes identifies the mesothelium and epithelium as prominent sources of extrahepatic complement proteins. Mucosal Immunol. 2022; 15(5): 927-939. (1935-3456 (Electronic)).

[27] Zhang Y, Wu X, Kai Y, et al. Secretome profiling identifies neuron-derived neurotrophic factor as a tumor-suppressive factor in lung cancer. JCI Insight. 2019; 4(24): e129344.

[28] Hu X. HOXD1 inhibits lung adenocarcinoma progression and is regulated by DNA methylation. Oncol Rep. 2024; 52(6): 173.

[29] Xiubing C, Huazhen L, Xueyan W, et al. SERPINA1 promotes the invasion, metastasis, and proliferation of pancreatic ductal adenocarcinoma via the PI3K/Akt/NF-κB pathway. Biochem Pharmacol. 2024; 230(pt 2): 116580. (1873-2968 (Electronic)).

[30] Bischoff P, Trinks A, Obermayer B, et al. Single-cell RNA sequencing reveals distinct tumor microenvironmental patterns in lung adenocarcinoma. Oncogene. 2021; 40(50): 6748-6758.

[31] Wculek SK, Dunphy G, Heras-Murillo I, Mastrangelo A, Sancho D. Metabolism of tissue macrophages in homeostasis and pathology. Cell Mol Immunol. 2022; 19(3): 384-408.

[32] Yin W, Zhu H, Tan J, et al. Identification of collagen genes related to immune infiltration and epithelial‒mesenchymal transition in glioma. Cancer Cell Int. 2021; 21(1): 276. (1475-2867 (Print)).

[33] Gong Y, Hart E, Shchurin A, Hoover-Plow J. Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice. J Clin Invest. 2008; 118(9): 3012-3024. (0021-9738 (Print)).

[34] Balduit A, Agostinis C, Bulla R. Beyond the Norm: the emerging interplay of complement system and extracellular matrix in the tumor microenvironment. Semin Immunol. 2025; 77: 101929. (1096-3618 (Electronic)).

[35] Han L, Zhu W, Qi H, et al. The cuproptosis-related gene glutaminase promotes alveolar macrophage copper ion accumulation in chronic obstructive pulmonary disease. Int Immunopharmacol. 2024; 129: 111585. (1878-1705 (Electronic)).

[36] Magrini E, Di Marco S, Mapelli SN, et al. Complement activation promoted by the lectin pathway mediates C3aR-dependent sarcoma progression and immunosuppression. Nat Cancer. 2021; 2(2): 218-232. (2662-1347 (Electronic)).

[37] Medler TR, Murugan D, Horton W, et al. Complement C5a fosters squamous carcinogenesis and limits T cell response to chemotherapy. Cancer Cell. 2018; 34(4): 561-578.e6. (1878-3686 (Electronic)).

[38] Roumenina LA-O, Daugan MV, Petitprez F, Sautès-Fridman C, Fridman WH. Context-dependent roles of complement in cancer. Nat Rev Cancer. 2019; 19(12): 698-715. (1474-1768 (Electronic)).

[39] Ouyang X, Huang Y, Jin X, et al. Osteopontin promotes cancer cell drug resistance, invasion, and lactate production and is associated with poor outcome of patients with advanced non-small-cell lung cancer. Oncol Targets Ther. 2018; 11: 5933-5941.

[40] Su X. Deciphering tumor microenvironment: CXCL9 and SPP1 as crucial determinants of tumor-associated macrophage polarity and prognostic indicators. Mol Cancer. 2024; 23(1): 13.

[41] Viiklepp K, Nissinen L, Ojalill M. C1r upregulates production of matrix metalloproteinase-13 and promotes invasion of cutaneous squamous cell carcinoma. J Invest Dermatol. 2022; 142(5): 1478-1488.e9. (1523-1747 (Electronic)).

[42] Santos-Ribeiro D, Cunha C, Carvalho A. Humoral pathways of innate immune regulation in granuloma formation. Trends Immunol. 2024; 45(6): 419-427.

[43] Doni A, Parente R, Laface I, et al. Serum amyloid P component is an essential element of resistance against Aspergillus fumigatus. Nat Commun. 2021; 12(1): 3739. (2041-1723 (Electronic)).

[44] Doni A, Musso T, Morone D, et al. An acidic microenvironment sets the humoral pattern recognition molecule PTX3 in a tissue repair mode. J Exp Med. 2015; 212(6): 905-925. (1540-9538 (Electronic)).

[45] Dalakas MA-O, Alexopoulos H, Spaeth PA-O. Complement in neurological disorders and emerging complement-targeted therapeutics. Nat Rev Neurol. 2020; 16(11): 601-617. (1759-4766 (Electronic)).

[46] Sirois JP, Heinz A. Matrikines in the skin: origin, effects, and therapeutic potential. Pharmacol Ther. 2024; 260: 108682. (1879-016X (Electronic)).

[47] Sexauer D, Gray E, Zaenker P. Tumour-associated autoantibodies as prognostic cancer biomarkers—a review. Autoimmun Rev. 2022; 21(4): 103041.

[48] Janneh AH, Atkinson C, Tomlinson S, Ogretmen B. Sphingolipid metabolism and complement signaling in cancer progression. Trends Cancer. 2023; 9(10): 782-787. (2405-8025 (Electronic)).

[49] Qiu ZW, Zhong YT, Lu ZM, et al. Breaking physical barrier of fibrotic breast cancer for photodynamic immunotherapy by remodeling tumor extracellular matrix and reprogramming cancer-associated fibroblasts. ACS Nano. 2024; 18(13): 9713-9735. (1936-086X (Electronic)).

[50] Liu Q, Wang J, Sun H, et al. Targeting RORγ inhibits the growth and metastasis of hepatocellular carcinoma. Mol Ther. 2024; 32(3): 749-765. (1525-0024 (Electronic)).

[51] Mei J, Cai Y, Xu R, et al. Conserved immuno-collagenic subtypes predict response to immune checkpoint blockade. Cancer Commun. 2024; 44(5): 554-575.

[52] Puttock EA-O, Tyler EJ, Manni M, et al. Extracellular matrix educates an immunoregulatory tumor macrophage phenotype found in ovarian cancer metastasis. Nat Commun. 2023; 14(1): 2514. (2041-1723 (Electronic)).

[53] Revel M, Daugan MV, Sautés-Fridman C, Fridman WH, Roumenina LT. Complement system: promoter or suppressor of cancer progression? Antibodies. 2020; 9(4): 57.

[54] Zha H, Wang X, Zhu Y, et al. Intracellular activation of complement C3 leads to PD-L1 antibody treatment resistance by modulating tumor-associated macrophages. Cancer Immunol Res. 2019; 7(2): 193-207. (2326-6074 (Electronic)).

[55] Cho MS, Rupaimoole R, Choi HJ, et al. Complement component 3 is regulated by TWIST1 and mediates epithelial‒mesenchymal transition. J Immunol (Baltimore, Md: 1950). 2016; 196(3): 1412-1418.

[56] Talaat IM, Elemam NM, Saber-Ayad M. Complement system: an immunotherapy target in colorectal cancer. Front Immunol. 2022; 13: 810993. (1664-3224 (Electronic)).

[57] Kleczko EK. Targeting the complement pathway as a therapeutic strategy in lung cancer. Front Immunol. 2019; 10: 954.

[58] Chen Z, Long L, Wang J, et al. Enhanced tumor site accumulation and therapeutic efficacy of extracellular matrix‒drug conjugates targeting tumor cells. Small. 2024; 20(40): e2402040. (1613-6829 (Electronic)).

[59] Strader RL, Shmidov Y, Chilkoti A. Encoding structure in intrinsically disordered protein biomaterials. Acc Chem Res. 2024; 57(3): 302-311.

[60] Sleeboom JJF, van Tienderen GS, Schenke-Layland K, van der Laan LJW, Khalil AA, Verstegen MMA. The extracellular matrix as hallmark of cancer and metastasis: from biomechanics to therapeutic targets. Sci Transl Med. 2024; 16(728): eadg3840. (1946-6242 (Electronic)).

[61] Tavernari D, Battistello E, Dheilly E, et al. Nongenetic evolution drives lung adenocarcinoma spatial heterogeneity and progression. Cancer Discov. 2021; 11(6): 1490-1507.

[62] Lu Y, Yang A, Quan C, et al. A single-cell atlas of the multicellular ecosystem of primary and metastatic hepatocellular carcinoma. Nat Commun. 2022; 13(1): 4594. (2041-1723 (Electronic)).

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2025 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.