To investigate the effect of endothelial cells in the perivascular niche on the stem cell-like characteristics of osteosarcoma cells and primarily explore the possible molecular mechanism.

A co-culture model was established in vitro using SV40T- human umbilical vein endothelial cells (HUVEC-T1) and osteosarcoma stem cells (OSCs) derived from the human osteosarcoma cell line 143B.Thus, there were 2 groups of cells, OSCs and co-cultured OSCs. The self-renewal capacity of OSCs between the 2 groups was assessed using a limiting dilution forming sphere assay. Flow cytometry and Western blotting were used to detect the expression of stem cell marker CD133 and stemness transcription factors SOX2 and NANOG. Fourteen female nude mice (4~6 weeks old, weighing 18~20 g) were randomly and equally divided into 2 groups of subcutaneous xenograft models: the control group (OSCs suspension) and the experimental group (OSCs+HUVEC-T1 mixed suspension). The tumor volume and mass were compared between the 2 xenograft groups. Immunofluorescence (IF) staining was used to verify the spatial proximity between endothelial cells and OSCs in vivo, while immunohistochemistry was employed to compare microvessel density (MVD) and CD133 expression level between the 2 groups. RNA-seq was performed to identify potential signaling pathways of endothelial cells affecting the stemness of OSCs. PCR and Western blotting were applied to confirm the RNA-seq findings. Exogenous protein treatment, IF staining, Western blotting and sphere formation assay were utilized to preliminarily validate the role of the identified pathway in regulating the stemness phenotype of OSCs.

The in vitro co-culture model of HUVEC-T1 and OSCs was successfully established. Compared with the control group, the co-culture group exhibited significantly enhanced self-renewal ability of OSCs, laeger proportion of the stemness marker CD133⁺ [(8.20±1.64)% vs (4.32±1.34)%, P<0.05], enhanced expression of CD117, SOX2 and NANOG (P<0.05), along with more sphere formation (P<0.05) and elevated SOX2/NANOG protein levels. The xenograft mice from the experimental group showed larger tumor volume (643.10±413.50 vs 247.90±93.66 mm³, P<0.05) and heavier tumor weight (0.52±0.27 vs 0.24±0.10 g, P<0.05) when compared the control group, correspondingly showing increased MVD (22.57±11.84 vs 11.43±5.38, P<0.05) and elevated CD133 expression (P<0.05). IF staining confirmed the adjacency of CD31-labeled endothelial cells and CD133-labeled OSCs in vivo. RNA-seq and functional experiments demonstrated that CXCR4 was highly expressed in co-cultured OSCs, CXCL12 was highly expressed in co-cultured endothelial cells, and exogenous CXCL12 promoted the sphere formation and expression levels of SOX2 and NANOG of OSCs (P<0.05).

Endothelial cells within the perivascular niche may promote the stemness phenotype of osteosarcoma cells via the CXCL12/CXCR4 pathway.