Paired single cell analysis reveals chemotherapy resistance in osteosarcoma

Abstract

Aim: Osteosarcoma remains aggressive with poor prognosis, particularly in chemotherapy-resistant cases. This study aimed to characterize transcriptional features of chemoresistant osteosarcoma cells, establish a prognostic resistance signature, and identify therapeutic vulnerabilities.

Methods: Single-cell RNA sequencing (scRNA-seq) was performed on paired pre- and post-neoadjuvant chemotherapy (NAC) specimens from three patients (6 samples; 16,272 cells). Resistance trajectories were reconstructed using Monocle 3 pseudotime analysis. A nine-gene resistance score was validated in the Peking University People’s Hospital (PKPH) bulk RNA-seq cohort (n = 70) and Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database (n = 87), with drug sensitivities predicted via oncoPredict.

Results: Chemotherapy reduced the malignant cell fraction but triggered expansion of cancer-associated fibroblasts and endothelial cells, creating a stromal-dominant, immune-sparse residual niche. Surviving tumor cells upregulated a nine-gene module along the resistance trajectory: KCNMA1, KIF21A, MIR181A1HG, RPS27, PDPN, ADIRF, PRELP, PHEX, and COL9A2. In an independent unpaired scRNA-seq cohort (two pre- and three post-chemotherapy samples), this signature remained associated with features of chemotherapy resistance. Higher scores correlated with poorer histopathologic response (r = -0.35, P = 0.006) and shorter progression-free survival [PKPH: hazard ratio (HR) = 2.4, 95% confidence interval (CI) 1.2-4.8, P = 0.01; TARGET: HR = 2.1, 95%CI 1.1-4.0, P = 0.02]. Of 198 compounds screened, only Pictilisib, a phosphoinositide 3-kinase (PI3K) inhibitor, showed lower predicted IC50 in the high-score subset across both datasets. However, the paired discovery cohort warrant further validation.

Conclusion: Our paired scRNA-seq approach identifies a nine-gene signature linking pre-treatment tumor biology to NAC response and outcome. The enhanced Pictilisib sensitivity in chemoresistant tumors positions PI3K blockade as a strategy meriting prospective testing in refractory osteosarcoma.

Keywords

Osteosarcoma / single-cell RNA sequencing / chemotherapy resistance / tumor microenvironment / prognostic signature / neoadjuvant chemotherapy

References

[1] Mirabello L,Savage SA.Osteosarcoma incidence and survival rates from 1973 to 2004: data from the Surveillance, Epidemiology, and End Results Program.Cancer2009;115:1531-43 PMCID:PMC2813207

[2] Bielack SS,Delling G.Prognostic factors in high-grade osteosarcoma of the extremities or trunk: an analysis of 1,702 patients treated on neoadjuvant cooperative osteosarcoma study group protocols.J Clin Oncol2002;20:776-90

[3] Kager L, Zoubek A, Pötschger U, et al.; Cooperative German-Austrian-Swiss Osteosarcoma Study Group. Primary metastatic osteosarcoma: presentation and outcome of patients treated on neoadjuvant Cooperative Osteosarcoma Study Group protocols.J Clin Oncol2003;21:2011-8

[4] Bacci G,Longhi A.Grade of chemotherapy-induced necrosis as a predictor of local and systemic control in 881 patients with non-metastatic osteosarcoma of the extremities treated with neoadjuvant chemotherapy in a single institution.Eur J Cancer2005;41:2079-85

[5] Whelan JS, Bielack SS, Marina N, et al.; EURAMOS collaborators. EURAMOS-1, an international randomised study for osteosarcoma: results from pre-randomisation treatment.Ann Oncol2015;26:407-14 PMCID:PMC4304379

[6] Garcia-Ortega DY,Caro-Sánchez HS.An overview of resistance to chemotherapy in osteosarcoma and future perspectives.Cancer Drug Resist2022;5:762-93 PMCID:PMC9511812

[7] Lilienthal I.Targeting molecular mechanisms underlying treatment efficacy and resistance in osteosarcoma: a review of current and future strategies.Int J Mol Sci2020;21:6885 PMCID:PMC7555161

[8] Du B.Targeting epithelial-mesenchymal transition (EMT) to overcome drug resistance in cancer.Molecules2016;21:965 PMCID:PMC6273543

[9] Sannino G,Kirchner T.Epithelial-to-mesenchymal and mesenchymal-to-epithelial transition in mesenchymal tumors: a paradox in sarcomas?.Cancer Res2017;77:4556-61

[10] Zeng Z,Zhang D.Development of a chemoresistant risk scoring model for prechemotherapy osteosarcoma using single-cell sequencing.Front Oncol2022;12:893282 PMCID:PMC9159767

[11] He L,Huang J.The tumor immune microenvironment and immune-related signature predict the chemotherapy response in patients with osteosarcoma.BMC Cancer2021;21:581 PMCID:PMC8138974

[12] Zhou Y,Yang Q.Single-cell RNA landscape of intratumoral heterogeneity and immunosuppressive microenvironment in advanced osteosarcoma.Nat Commun2020;11:6322 PMCID:PMC7730477

[13] Zheng X,Zhao Z,Yu S.Single-cell transcriptomic insights into chemotherapy-induced remodeling of the osteosarcoma tumor microenvironment.J Cancer Res Clin Oncol2024;150:356 PMCID:PMC11271355

[14] Liu T,Zhang Q.Targeting ABCB1 (MDR1) in multi-drug resistant osteosarcoma cells using the CRISPR-Cas9 system to reverse drug resistance.Oncotarget2016;7:83502-13 PMCID:PMC5347784

[15] Takemoto A,Ukaji T.Targeting podoplanin for the treatment of osteosarcoma.Clin Cancer Res2022;28:2633-45 PMCID:PMC9359727

[16] Maeser D,Huang RS.oncoPredict: an R package for predicting in vivo or cancer patient drug response and biomarkers from cell line screening data.Brief Bioinform2021;22:bbab260 PMCID:PMC8574972

[17] Feng B,Shen B,Feng J.Cancer-associated fibroblasts and resistance to anticancer therapies: status, mechanisms, and countermeasures.Cancer Cell Int2022;22:166 PMCID:PMC9052457

[18] Straussman R,Shee K.Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion.Nature2012;487:500-4 PMCID:PMC3711467

[19] Galon J.Approaches to treat immune hot, altered and cold tumours with combination immunotherapies.Nat Rev Drug Discov2019;18:197-218

[20] Sharma SV,Li B.A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations.Cell2010;141:69-80 PMCID:PMC2851638

[21] Kesh K,Durden B.Therapy resistance, cancer stem cells and ECM in cancer: the matrix reloaded.Cancers2020;12:3067 PMCID:PMC7589733

[22] Huang J,Wan D.Extracellular matrix and its therapeutic potential for cancer treatment.Signal Transduct Target Ther2021;6:153 PMCID:PMC8062524

[23] Piersma B,Weaver VM.Fibrosis and cancer: a strained relationship.Biochim Biophys Acta Rev Cancer2020;1873:188356 PMCID:PMC7733542

[24] Wilson TR,Yan Y.Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors.Nature2012;487:505-9 PMCID:PMC3724525

[25] Tzanakakis GN,Berdiaki A.The role of IGF/IGF-IR-signaling and extracellular matrix effectors in bone sarcoma pathogenesis.Cancers2021;13:2478 PMCID:PMC8160938

[26] Astarita JL,Turley SJ.Podoplanin: emerging functions in development, the immune system, and cancer.Front Immunol2012;3:283 PMCID:PMC3439854

[27] van der Vaart B,Doodhi H.CFEOM1-associated kinesin KIF21A is a cortical microtubule growth inhibitor.Dev Cell2013;27:145-60

[28] Xia C,Ren S,Zhang Q.Potassium channels, tumorigenesis and targeted drugs.Biomed Pharmacother2023;162:114673

[29] Groth-Pedersen L,Corcelle-Termeau E,Nylandsted J.Identification of cytoskeleton-associated proteins essential for lysosomal stability and survival of human cancer cells.PLoS One2012;7:e45381 PMCID:PMC3469574

[30] Warner JR.How common are extraribosomal functions of ribosomal proteins?.Mol Cell2009;34:3-11 PMCID:PMC2679180

[31] Jiao X,Ma M.MiR-181a enhances drug sensitivity in mitoxantone-resistant breast cancer cells by targeting breast cancer resistance protein (BCRP/ABCG2).Breast Cancer Res Treat2013;139:717-30

[32] Man TK,Visvanathan J.Expression profiles of osteosarcoma that can predict response to chemotherapy.Cancer Res2005;65:8142-50