Paired single cell analysis reveals chemotherapy resistance in osteosarcoma

<b>Aim:</b> 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. <b>Methods:</b> 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 (<i>n</i> = 70) and Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database (<i>n</i> = 87), with drug sensitivities predicted via oncoPredict. <b>Results:</b> 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: <i>KCNMA1</i>, <i>KIF21A</i>, <i>MIR181A1HG</i>, <i>RPS27</i>, <i>PDPN</i>, <i>ADIRF</i>, <i>PRELP</i>, <i>PHEX</i>, and <i>COL9A2</i>. 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, <i>P</i> = 0.006) and shorter progression-free survival [PKPH: hazard ratio (HR) = 2.4, 95% confidence interval (CI) 1.2-4.8, <i>P</i> = 0.01; TARGET: HR = 2.1, 95%CI 1.1-4.0, <i>P</i> = 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. <b>Conclusion:</b> 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.