Single-Cell Transcriptomic Profiling Reveals Cellular Heterogeneity and Identifies Novel Therapeutic Targets in...

Osteosarcoma is the most prevalent primary malignant bone tumor predominantly affecting children and adolescents, yet prognosis for metastatic disease remains dismal. Understanding the cellular complexity within the tumor microenvironment is essential for developing targeted therapeutic strategies.

We performed comprehensive single-cell RNA sequencing analysis on an osteosarcoma tissue sample (GSM4952363) using the Seurat pipeline (v4.3.0). Following rigorous quality control (200-6000 genes per cell, < 15% mitochondrial reads), cells were filtered for downstream analysis. Dimensionality reduction (PCA and UMAP) and unsupervised clustering (Louvain algorithm, resolution = 0.8) identified seven distinct cellular clusters. Differential expression analysis (Wilcoxon rank-sum test, |log₂FC| > 0.25, adjusted p < 0.05) identified cluster-specific markers, while Gene Ontology and KEGG pathway enrichment analyses (clusterProfiler, adjusted p < 0.05) revealed functional programs. Four candidate genes (F11, ACRP2, LEPR, and POSTN) were selected for validation by quantitative real-time PCR (mRNA level) and ELISA (protein level) in MG-63 osteosarcoma cells compared to hFOB 1.19 normal osteoblasts.

Single-cell transcriptomic profiling identified seven distinct cellular clusters within the osteosarcoma microenvironment, including macrophages (Cluster 0, 28.0%), osteoblasts (Cluster 1, 24.2%), fibroblasts (Cluster 2, Fibro_COMP, 14.7%), proliferating cells (Cluster 3, 12.3%), osteoclasts (Cluster 4, 11.6%), monocytes (Cluster 5, 6.3%), and T cells (Cluster 6, 2.9%). Functional enrichment analysis highlighted activation of PI3K-Akt signaling, focal adhesion, and extracellular matrix organization as core pathways. qRT-PCR validation (mRNA level) demonstrated that F11 was significantly downregulated (0.31 ± 0.04 vs. 1.00 ± 0.07, 69% reduction, p < 0.001), whereas ACRP2 (2.87 ± 0.33-fold), LEPR (4.52 ± 0.48-fold), and POSTN (6.23 ± 0.57-fold) were significantly upregulated (all p < 0.001). ELISA validation (protein level) confirmed consistent trends: F11 protein decreased by 65% (0.35 ± 0.05 vs. 1.00 ± 0.08, p < 0.001), whereas ACRP2 (2.64 ± 0.29-fold), LEPR (4.18 ± 0.44-fold), and POSTN (5.89 ± 0.53-fold) protein levels were elevated (all p < 0.001). Among the four candidates, POSTN exhibited the most pronounced changes at both mRNA and protein levels, suggesting its involvement in osteosarcoma matrix remodeling.

This study provides a single-cell transcriptomic atlas of the osteosarcoma microenvironment, revealing substantial cellular heterogeneity. The differentially expressed genes F11, ACRP2, LEPR, and POSTN represent candidate biomarkers that warrant further investigation for their potential roles in osteosarcoma biology and as putative therapeutic targets.