Unveiling Cellular Traits of Osteosarcoma Cancer Stem Cells via Bulk and Single-Cell RNA-Sequencing Analysis

Osteosarcoma (OS) is a prototypical sarcoma, predominantly affecting adolescents. The hallmark of cancer stem cell (CSC) behavior pervades OS, invariably signifying an unfavorable prognosis. However, the intricacies underlying OS metastasis remain incompletely comprehended. As the frontiers of the scientific investigation push forward, encompassing both bulk sequencing and single-cell sequencing (scRNA), the domain of bioinformatics finds multifaceted utility. In this study, we combined scRNA and bulk sequencing with the clinical metadata to excavate the latent molecular substrates governing metastatic propensities within OS. Our scRNA analysis indicated that cell-stemness-related pathways might play vital roles in OS metastasis. Subsequently, an autonomous reservoir of bulk sequencing data set was subjected to weighted gene co-expression network analysis (WGCNA) to identify 10 gene clusters. After analyzing the clinical data, we were able to identify two hub genes, HDAC2 and HSPA4, which are strongly linked to the cancer cell stemness. Moreover, we performed transwell assays to validate the regulation of metastatic behaviors by miR-1-3p, HDAC2, and HSPA4 in OS cells. Significantly, our study was fortified by immunohistochemistry (IHC) analyses performed on tumor tissues acquired from OS patients, thereby accentuating the clinical import of our experimental endeavor. Notably, we unveiled the suppressive influence of miR-1-3p on both HDAC2 and HSPA4, with miRTarBase substantiating lower expression in tumor tissue relative to the normative cohort. Insights gleaned from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) program (https://ocg.cancer.gov/programs/target) further augmented the clinical significance of the identified hub genes. In conclusion, our findings highlight the significant role of cell junctions in governing OS stemness and metastasis, underscored by the integration of single-cell sequencing and bulk-sequencing analyses. Moreover, our foundational experiments identified three hub molecules closely associated with the metastatic behaviors. Our findings provide novel insights into the clinical treatment and fundamental understanding of OS.