Abstract 2626: Leveraging FFPE single-nucleus RNA-sequencing for Osteosarcoma

Abstract Osteosarcoma (OS), a heterogeneous class of bone sarcomas, can be subclassified based on predominant histological features. This molecular heterogeneity cannot fully be resolved with bulk sequencing. However, due to the rarity of several OS subtypes, including low-grade and small-cell, parosteal, periosteal, and high-grade surface, acquiring fresh specimens for single-cell omics is difficult. Logistical challenges exist when acquiring fresh specimens, such as transportation or adequate storage. Here, we leverage archival formalin-fixed paraffin-embedded (FFPE) samples of OS, which are routinely processed and preserved in pathology laboratories. We utilized 10x Flex assay to perform single-nucleus RNA-sequencing (snRNA-seq) from FFPE samples of OS, including osteoblastic OS, chondroblastic OS, low-grade, and periosteal. We compared fixed-frozen to FFPE samples to determine if both methods yielded concordant results. Two 25 μm FFPE scrolls and matched fixed-frozen tissue of approximately 60 mg were dissociated using the gentleMACs Dissociator. After confirming adequate cell count, we performed snRNA-seq on the fixed samples using the 10x Flex assay. Fixed-frozen (5201±3410 cells) had higher cell counts than FFPE (1185±823 cells), which may be attributed to tissue area in the FFPE scrolls. The confidentially mapped reads were similar for fixed-frozen (64%±13%) and FFPE (55%±17%). Higher UMIs and genes were detected in fixed-frozen (8566±110 UMIs and 2342±15 genes detected) than in FFPE (1464±25 UMIs and 835±9 genes detected). Regardless of specimen type, we detected the subtype-specific gene expression. Fixed-frozen and FFPE specimens of osteoblastic OS expressed osteoblastic markers, SPP1, CHD11, and IBSP. Chrondroblastic markers (SOX9 and ACAN) were expressed in both conditions for chondroblastic OS. Significantly, CDK4 and MDM2 were overexpressed in low-grade OS due to the amplification of CDK4 and MDM2 copy numbers, confirmed by CopyKAT and inferCNV. Overall, we found that the 10x Flex-based snRNA-seq adequately preserved each OS subtype's biology. FFPE had lower quality control metrics than fixed-frozen, possibly due to the smaller amount of tissue available from the scrolls. Future work will investigate the common theme of genetic drift from normal osteoblasts and their varied degrees of dedifferentiation observed along mesenchymal lineages among OS subtypes. Citation Format: Danh Truong, Clement Agyemang, Davis Ingram, Alexander Lazar, Valerae O. Lewis, Joseph Ludwig. Leveraging FFPE single-nucleus RNA-sequencing for Osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2626.