(P 58) CHEMOTHERAPY-INDUCED EVOLUTION OF OSTEOSARCOMA REVEALED BY SINGLE-CELL RNA SEQUENCING

Objective: Osteosarcoma (OS) is the most common primary malignant bone tumor, particularly among children and adolescents. Neoadjuvant (preoperative) chemotherapy followed by surgery and adjuvant (postoperative) chemotherapy are the primary therapeutic strategy for OS. However, a considerable proportion of OS patients do not respond well to neoadjuvant chemotherapy and poor response stands as an obstacle to good prognosis. cancer cell intrinsic heterogeneity, cells within the tumor microenvironment (TME) and cellular plasticity were the major reasons of drug resistance. In this study, we performed scRNA-seq analyses on matched OS samples to reveal the heterogeneity of OS and cellular plasticity under drug resistance.

Methods: ScRNA-seq analyses were performed matched OS samples obtained from 5 patients before initiating systemic treatment (pre-treatment) and 3 paired surgical tumor samples after the standard neoadjuvant chemotherapy(post-treatment) which considered poor histological responders. Cell types and the corresponding marker genes were identified by Seurat and UMAP methods. Copy number Karyotyping of Tumors (copyKat) analysis was performed to identified the malignant cells. DEGs identification and GO enrichment analysis were used for differentially over-expressed genes. Tissue Microarray (TMA) and Immunohistochemical staining were used for protein validation.

Results: 13 cell clusters were identified. The proportions of endothelial cells and myofibrocytes in post-treatment samples were significantly increased compare with pre-treatment samples. While, B cells and neutrophil cells varied greatly among patients, the infiltration levels in pre-treatment samples are much higher than in post-treatment samples. Checkpoint genes (PDCD1, CTLA-4, TIGIT, LAG3 and VSIR) highly expressed in T cells of pre-non-respond samples compare with pre-respond samples, while chemotherapy did not change their expressions.
Chemotherapy-induced evolution in some types of cells of post-treatment tumors were observed. The subtypes of B cells have changed after chemotherapy. IGHG B cells became the predominant cells in post-treatment samples while the other two B cells decreased sharply. Cancer-related pathways were upregulated in the post-treatment tumor cells indicated evolution of tumor cells induced by chemotherapy. Patients with more MYC+ ANGPTL4+ CPE+ HSPA8+ IFITM5+ MRPL4+ tumor cells showed a worse prognosis, which also validated by IHC staining.

Conclusion: Our results focused on the unique aspects of the chemotherapy-induced evolution in OS, that might guide the development of optimized chemotherapy to benefit poor response of OS patient.