Single-nucleus RNA sequencing reveals the distinct heterogeneity of primary pulmonary sarcomas (PPS) and pulmonary sarcomatoid carcinoma (PSC)

Abstract

Background: Primary pulmonary sarcomas (PPS) and pulmonary sarcomatoid carcinoma (PSC) are rare and aggressive diseases that pose significant diagnostic challenges, requiring extensive sampling and comprehensive evaluation. To date, the single-cell characteristics and distinctions between these two conditions have not been thoroughly investigated.

Methods: In this study, we employed single-nucleus RNA sequencing (snRNA-seq) to characterise the cellular heterogeneity of PSC and PPS. Our analysis included 20 PSC samples, seven PPS samples and two non-malignant control samples obtained from adjacent normal tissue.

Results: Our results revealed that the majority of cells in PSC were of epithelial origin, while fibroblasts predominated in PPS. Specifically, AT2 cells, a major source of epithelial cells in PSC, underwent malignant transformation primarily through epithelial–mesenchymal transition, suggesting AT2 cells may serve as the origin of PSC. High Mobility Group AT-Hook 2 (HMGA2) expression was elevated in malignant AT2 cells of PSC and correlated with an unfavourable prognosis. Moreover, MET-mutated patients have a significantly higher expression level of HMGA2 (p < .001). In PPS, fibroblasts constituted the majority, only lipofibroblasts exhibited malignant features. A direct comparison between PSC and PPS lipofibroblasts revealed largely similar expression profiles, with the exception of an enrichment in DNA repair pathways specifically observed in PPS lipofibroblasts.

Conclusion: These findings provide novel insights of PSC and PPS at the single-cell level.

Keywords

primary pulmonary sarcomas / pulmonary sarcomatoid carcinoma / single-nucleus RNA sequencing / TME

References

[1] Li X, Wu D, Liu H, Chen J. Pulmonary sarcomatoid carcinoma: progress, treatment and expectations. Ther Adv Med Oncol. 2020; 12:1758835920950207.

[2] Rossi G, Cavazza A, Sturm N, et al. Pulmonary carcinomas with pleomorphic, sarcomatoid, or sarcomatous elements: a clinicopathologic and immunohistochemical study of 75 cases. Am J Surg Pathol. 2003; 27: 311-324.

[3] Pelosi G, Sonzogni A, De Pas T, et al. Review article: pulmonary sarcomatoid carcinomas: a practical overview. Int J Surg Pathol. 2010; 18: 103-120.

[4] Wei Y, Wang L, Jin Z, et al. Biological characteristics and clinical treatment of pulmonary sarcomatoid carcinoma: a narrative review. Transl Lung Cancer Res. 2024; 13: 635-653.

[5] Huang Q, Li W, He X, et al. Prognostic visualization model for primary pulmonary sarcoma: a SEER-based study. Sci Rep. 2023; 13:17774.

[6] Duran-Moreno J, Kokkali S, Ramfidis V, et al. Primary sarcoma of the lung—prognostic value of clinicopathological characteristics of 26 cases. Anticancer Res. 2020; 40: 1697-1703.

[7] Robinson LA, Babacan NA, Tanvetyanon T, Henderson-Jackson E, Bui MM, Druta M. Results of treating primary pulmonary sarcomas and pulmonary carcinosarcomas. J Thorac Cardiovasc Surg. 2021; 162: 274-284.

[8] Berzenji L, Van Schil PE. Commentary: primary pulmonary sarcomas and pulmonary carcinosarcomas, challenging and enigmatic, but treatable!. J Thorac Cardiovasc Surg. 2021; 162: 286-287.

[9] Huwer H, Kalweit G, Straub U, Feindt P, Volkmer I, Gams E. Pulmonary carcinosarcoma: diagnostic problems and determinants of the prognosis. Eur J Cardiothorac Surg. 1996; 10: 403-407.

[10] Gladish GW, Sabloff BM, Munden RF, Truong MT, Erasmus JJ, Chasen MH. Primary thoracic sarcomas. Radiographics. 2002; 22: 621-637.

[11] Yang Z, Xu J, Li L, et al. Integrated molecular characterization reveals potential therapeutic strategies for pulmonary sarcomatoid carcinoma. Nat Commun. 2020; 11: 4878.

[12] Zhou F, Huang Y, Cai W, et al. The genomic and immunologic profiles of pure pulmonary sarcomatoid carcinoma in Chinese patients. Lung Cancer. 2021; 153: 66-72.

[13] Yatabe Y, Dacic S, Borczuk AC, et al. Best practices recommendations for diagnostic immunohistochemistry in lung cancer. J Thorac Oncol. 2019; 14: 377-407.

[14] WHO Classification of Tumors Online (n.d.). Accessed December 1, 2024. https://tumourclassification.iarc.who.int/chapters/35

[15] Wiegleb G, Reinhardt S, Dahl A, Posnien N. Tissue dissociation for single-cell and single-nuclei RNA sequencing for low amounts of input material. Front Zool. 2022; 19: 27.

[16] Maitra M, Nagy C, Chawla A, et al. Extraction of nuclei from archived postmortem tissues for single-nucleus sequencing applications. Nat Protoc. 2021; 16: 2788-2801.

[17] Stuart T, Butler A, Hoffman P, et al. Comprehensive integration of single-cell data. Cell. 2019; 177: 1888-1902.e21.

[18] Hafemeister C, Satija R. Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Genome Biol. 2019; 20: 296.

[19] Aran D, Looney AP, Liu L, et al. Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage. Nat Immunol. 2019; 20: 163-172.

[20] Patel AP, Tirosh I, Trombetta JJ, et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science. 2014; 344: 1396-1401.

[21] Tirosh I, Izar B, Prakadan SM, et al. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science. 2016; 352: 189-196.

[22] Puram SV, Tirosh I, Parikh AS, et al. Single-cell transcriptomic analysis of primary and metastatic tumor ecosystems in head and neck cancer. Cell. 2017; 171: 1611-1624.e24.

[23] Wu T, Hu E, Xu S, et al. clusterProfiler 4.0: a universal enrichment tool for interpreting omics data. Innovation (Camb). 2021; 2:100141.

[24] Hänzelmann S, Castelo R, Guinney J. GSVA: gene set variation analysis for microarray and RNA-seq data. BMC Bioinformatics. 2013; 14: 7.

[25] Ritchie ME, Phipson B, Wu D, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015; 43:e47.

[26] Jin S, Guerrero-Juarez CF, Zhang L, et al. Inference and analysis of cell-cell communication using CellChat. Nat Commun. 2021; 12: 1088.

[27] Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018; 34: i884-i890.

[28] Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25: 1754-1760.

[29] Lai Z, Markovets A, Ahdesmaki M, et al. VarDict: a novel and versatile variant caller for next-generation sequencing in cancer research. Nucleic Acids Res. 2016; 44:e108.

[30] Li Q, Wang K. InterVar: clinical interpretation of genetic variants by the 2015 ACMG-AMP guidelines. Am J Hum Genet. 2017; 100: 267-280.

[31] Talevich E, Shain AH, Botton T, Bastian BC. CNVkit: genome-wide copy number detection and visualization from targeted DNA sequencing. PLoS Comput Biol. 2016; 12:e1004873.

[32] Ma Y, Li W, Li Z, et al. Immunophenotyping of pulmonary sarcomatoid carcinoma. Front Immunol. 2022; 13:976739.

[33] Gu H, Song R, Beeraka NM, et al. SEER-based survival nomogram (1998-2015) based on “stage, lymph node dissection, tumor size and degree of differentiation, and therapies” for prognosis of primary pulmonary sarcoma. Technol Cancer Res Treat. 2023; 22:15330338221150732.

[34] Chahal A, Manapragada PP, Singh SP, Winokur TS, Sonavane SK. Primary intrathoracic sarcomas: a review of cross-sectional imaging and pathology. J Comput Assist Tomogr. 2020; 44: 821-832.

[35] Maynard A, McCoach CE, Rotow JK, et al. Therapy-induced evolution of human lung cancer revealed by single-cell RNA sequencing. Cell. 2020; 182: 1232-1251.e22.

[36] Liberzon A, Birger C, Thorvaldsdóttir H, Ghandi M, Mesirov JP, Tamayo P. The molecular signatures database (MSigDB) hallmark gene set collection. Cell Syst. 2015; 1: 417-425.

[37] Wang Z, Li Z, Zhou K, et al. Deciphering cell lineage specification of human lung adenocarcinoma with single-cell RNA sequencing. Nat Commun. 2021; 12: 6500.

[38] Wu F, Fan J, He Y, et al. Single-cell profiling of tumor heterogeneity and the microenvironment in advanced non-small cell lung cancer. Nat Commun. 2021; 12: 2540.

[39] Juul NH, Yoon J-K, Martinez MC, et al. KRAS(G12D) drives lepidic adenocarcinoma through stem-cell reprogramming. Nature. 2023; 619: 860-867.

[40] Tian Y, Lv J, Su Z, et al. LRRK2 plays essential roles in maintaining lung homeostasis and preventing the development of pulmonary fibrosis. Proc Natl Acad Sci U S A. 2021; 118:e2106685118.

[41] Hashemi M, Rashidi M, Hushmandi K, et al. HMGA2 regulation by miRNAs in cancer: affecting cancer hallmarks and therapy response. Pharmacol Res. 2023; 190:106732.

[42] Gao Y, Zhang N, Zeng Z, et al. LncRNA PCAT1 activates SOX2 and suppresses radioimmune responses via regulating cGAS/STING signalling in non-small cell lung cancer. Clin Transl Med. 2022; 12:e792.

[43] Delhaye S, Bardoni B. Role of phosphodiesterases in the pathophysiology of neurodevelopmental disorders. Mol Psychiatry. 2021; 26: 4570-4582.

[44] Yu F-X, Zhao B, Guan K-L. Hippo pathway in organ size control, tissue homeostasis, and cancer. Cell. 2015; 163: 811-828.

[45] Sui Y, Liu F, Zheng S, et al. G-quadruplexes folding mediates downregulation of PBX1 expression in melanoma. Signal Transduct Target Ther. 2023; 8: 12.

[46] Zhang J, Pi S-B, Zhang N, et al. Translation regulatory factor BZW1 regulates preimplantation embryo development and compaction by restricting global non-AUG initiation. Nat Commun. 2022; 13: 6621.

[47] Kipps TJ. ROR1: an orphan becomes apparent. Blood. 2022; 140: 1583-1591.

[48] Gong C, Xiong H, Qin K, et al. MET alterations in advanced pulmonary sarcomatoid carcinoma. Front Oncol. 2022; 12:1017026.

[49] Marks JA, Gandhi N, Halmos B, et al. Molecular profiling METex14+ non-small cell lung cancer (NSCLC): impact of histology. Lung Cancer. 2024; 196:107935.

[50] Castiglione R, Alidousty C, Holz B, et al. Comparison of the genomic background of MET-altered carcinomas of the lung: biological differences and analogies. Mod Pathol. 2019; 32: 627-638.

[51] Zhang D-Y, Lei J-S, Sun W-L, Wang D-D, Lu Z. Follistatin like 5 (FSTL5) inhibits epithelial to mesenchymal transition in hepatocellular carcinoma. Chin Med J (Engl). 2020; 133: 1798-1804.

[52] Lucas LM, Dwivedi V, Senfeld JI, et al. The Yin and Yang of ERBB4: tumor suppressor and oncoprotein. Pharmacol Rev. 2022; 74: 18-47.

[53] Lugano R, Ramachandran M, Dimberg A. Tumor angiogenesis: causes, consequences, challenges and opportunities. Cell Mol Life Sci. 2020; 77: 1745-1770.

[54] de Visser KE, Joyce JA. The evolving tumor microenvironment: from cancer initiation to metastatic outgrowth. Cancer Cell. 2023; 41: 374-403.

[55] Pan X, Li X, Dong L, et al. Tumour vasculature at single-cell resolution. Nature. 2024; 632: 429-436.

[56] Baldovini C, Rossi G, Ciarrocchi A. Approaches to tumor classification in pulmonary sarcomatoid carcinoma. Lung Cancer (Auckl). 2019; 10: 131-149.

[57] Zepp JA, Morley MP, Loebel C, et al. Genomic, epigenomic, and biophysical cues controlling the emergence of the lung alveolus. Science. 2021; 371:eabc3172.

[58] Huang Y, Hong W, Wei X. The molecular mechanisms and therapeutic strategies of EMT in tumor progression and metastasis. J Hematol Oncol. 2022; 15: 129.

[59] Sadrkhanloo M, Entezari M, Orouei S, et al. STAT3-EMT axis in tumors: modulation of cancer metastasis, stemness and therapy response. Pharmacol Res. 2022; 182:106311.

[60] Johnson DE, O'Keefe RA, Grandis JR. Targeting the IL-6/JAK/STAT3 signalling axis in cancer. Nat Rev Clin Oncol. 2018; 15: 234-248.

[61] Wang X, Wang J, Zhao J, Wang H, Chen J, Wu J. HMGA2 facilitates colorectal cancer progression via STAT3-mediated tumor-associated macrophage recruitment. Theranostics. 2022; 12: 963-975.

[62] Wu J, Wei J-J. HMGA2 and high-grade serous ovarian carcinoma. J Mol Med (Berl). 2013; 91: 1155-1165.

[63] Meyer B, Loeschke S, Schultze A, et al. HMGA2 overexpression in non-small cell lung cancer. Mol Carcinog. 2007; 46: 503-511.

[64] Ding X, Wang Y, Ma X, et al. Expression of HMGA2 in bladder cancer and its association with epithelial-to-mesenchymal transition. Cell Prolif. 2014; 47: 146-151.

[65] Mansoori B, Mohammadi A, Ditzel HJ, et al. HMGA2 as a critical regulator in cancer development. Genes (Basel). 2021; 12: 269.

[66] Campos Gudiño R, McManus KJ, Hombach-Klonisch S. Aberrant HMGA2 expression sustains genome instability that promotes metastasis and therapeutic resistance in colorectal cancer. Cancers (Basel). 2023; 15: 1735.

[67] Chiou S-H, Dorsch M, Kusch E, et al. Hmga2 is dispensable for pancreatic cancer development, metastasis, and therapy resistance. Sci Rep. 2018; 8:14008.

[68] Hsu W-H, LaBella KA, Lin Y, et al. Oncogenic KRAS drives lipofibrogenesis to promote angiogenesis and colon cancer progression. Cancer Discov. 2023; 13: 2652-2673.

[69] Niu N, Shen X, Wang Z, et al. Tumor cell-intrinsic epigenetic dysregulation shapes cancer-associated fibroblasts heterogeneity to metabolically support pancreatic cancer. Cancer Cell. 2024; 42: 869-884.e9.

[70] Fuh KF, Shepherd RD, Withell JS, Kooistra BK, Rinker KD. Fluid flow exposure promotes epithelial-to-mesenchymal transition and adhesion of breast cancer cells to endothelial cells. Breast Cancer Res. 2021; 23: 97.

[71] Gong Z, Li Q, Shi J, et al. Lung fibroblasts facilitate pre-metastatic niche formation by remodeling the local immune microenvironment. Immunity. 2022; 55: 1483-1500.e9.

[72] Zhang H, Jiang H, Zhu L, Li J, Ma S. Cancer-associated fibroblasts in non-small cell lung cancer: recent advances and future perspectives. Cancer Lett. 2021; 514: 38-47.

[73] Huang Q, Liu L, Xiao D, et al. CD44+ lung cancer stem cell-derived pericyte-like cells cause brain metastases through GPR124-enhanced trans-endothelial migration. Cancer Cell. 2023; 41: 1621-1636.e8.

[74] Chiou G-Y, Chien C-S, Wang M-L, et al. Epigenetic regulation of the miR142-3p/interleukin-6 circuit in glioblastoma. Mol Cell. 2013; 52: 693-706.

[75] Sun J, Sun B, Zhu D, et al. HMGA2 regulates CD44 expression to promote gastric cancer cell motility and sphere formation. Am J Cancer Res. 2017; 7: 260-274.

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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.