introduction: As the leading primary bone malignancy, osteosarcoma primarily affects pediatric and adolescent populations, occurring at a rate of about 3–5 cases per million worldwide each year1. Despite the use of multi-agent chemotherapy combined with surgical resection, the 5-year survival trend in localized osteosarcoma cases remains around 60–70%2, 3. However, for those presenting with metastases—most commonly to the lungs—the prognosis is significantly worse, with a 5-year survival rate dropping below 30%4, 5. Moreover, 30–40% of patients with initially localized disease eventually develop distant metastases or experience recurrence, highlighting the critical need to better understand the mechanisms driving tumor invasion and dissemination6. C-X-C chemokine receptor type 4 (CXCR4),a member of the GPCR superfamily that binds to its cognate ligand stromal-derived factor-1 (SDF-1, also known as CXCL12), has been implicated in multiple steps of the metastatic cascade. Elevated CXCR4 levels have been reported in over 20 forms of malignancies, including breast, lung, pancreatic, colorectal, and prostate cancers, and is frequently correlated with greater malignant potential, lymph node involvement, and distant metastasis7, 8. Functionally, CXCR4/CXCL12 signaling promotes tumor cell chemotaxis, invasion, angiogenesis, and survival through induction of downstream signaling mechanisms including PI3K/AKT, MAPK/ERK, and NF-κB9. In osteosarcoma, elevated CXCR4 expression has been correlated with poor clinical outcomes and enhanced metastatic potential, but its downstream effectors that mediate ECM degradation and invasion remain insufficiently characterized10. Matrix metalloproteinase 9 (MMP9), a zinc-dependent endopeptidase, plays a crucial role in extracellular matrix remodeling by degrading type IV collagen, a major component of basement membranes. MMP9 is upregulated in numerous malignancies and is closely associated with tumor cell invasion, angiogenesis, and metastasis11, 12. In osteosarcoma, clinical studies have reported a positive correlation between MMP9 expression and lung metastasis, as well as poor event-free survival13. Mechanistically, MMP9 can be induced by various oncogenic signals, including inflammatory cytokines, growth factors, and chemokine receptors such as CXCR4. For instance, the CXCR4/CXCL12 axis has been reported to transcriptionally regulate MMP9 via the stimulation of NF-κB and AP-1 in breast and prostate cancers14. However, whether such regulatory interplay occurs in osteosarcoma remains largely unexplored. We hypothesized that CXCR4 enhances osteosarcoma cell migration and matrix degradation through upregulation of MMP9. To test this, we conducted gain- and loss-of-function investigations in osteosarcoma cell lines, followed by comprehensive molecular and functional assays. Our findings aim to elucidate the functional involvement of the CXCR4/MMP9 axis in osteosarcoma advancement and suggest its promise as a novel intervention point in therapy. materials and methods: 1.1Cell Culture and Treatment Osteoblast cell line hFOB1.19 (ATCC, CRL-3602 ™) and osteosarcoma cell lines MG-63 (ATCC,CRL-1427™), U2OS (ATCC,HTB-96™), and 143B (ATCC, CRL-8303™) were purchased from the ATCC (Manassas, VA, USA). hFOB1.19 cells were grown in equal volumes of Ham’s F12 and DMEM supplemented with 10 results: 2.1Expression levels of CXCR4 and MMP9 in osteosarcoma cell lines To examine the abundance of CXCR4 and MMP9 in osteosarcoma, their mRNA and protein levels were assessed in hFOB1.19, MG-63, U2OS, and 143B cell lines. A significant upregulation of CXCR4 mRNA was detected in MG-63, U2OS, and 143B cells in comparison to hFOB1.19, according to qRT-PCR data, with the highest expression observed in 143B cells (P < 0.01)( Figure 1A). Western blot results showed that CXCR4 protein levels were also elevated in osteosarcoma cell lines, chiefly in U2OS and 143B cells (P < 0.01, Figure 1B).Similarly, MMP9 mRNA expression was markedly upregulated in U2OS, MG-63, and 143B cells relative to hFOB1.19 (P < 0.01)(Figure 1C). Western blot analysis verified higher MMP9 protein abundance in osteosarcoma cell lines, especially in 143B and U2OS cells (P < 0.01, Figure 1D).Overall, both CXCR4 and MMP9 exhibited higher expression in osteosarcoma cells than in normal osteoblasts. 2.2CXCR4 knockdown suppresses, while overexpression promotes, osteosarcoma cell migration and invasion To assess the biologically active role of CXCR4 in osteosarcoma, 143B cells were transfected with si-CXCR4 and MG-63 cells with OE-CXCR4. In 143B cells, both qRT-PCR and Western blot analysis showed no significant difference in CXCR4 expression between the control and si-NC groups, whereas a significant reduction in CXCR4 mRNA and protein levels was observed in the si-CXCR4 group compared with si-NC (P < 0.01, Figure 2A–B). In MG-63 cells, CXCR4 expression was comparable between the control and vector groups, while significantly elevated levels were detected in the OE-CXCR4 group relative to vector (P < 0.01, Figure 2C–D).Wound healing assays demonstrated that CXCR4 knockdown significantly reduced the migration rate of 143B cells versus the si-NC group, with no observable difference between control and si-NC (P < 0.01)(Figure 2E). In contrast, CXCR4 overexpression markedly enhanced the migratory capacity of MG-63 cells relative to the vector group, with no change between control and vector (P < 0.01, Figure 2F).Transwell invasion assays showed consistent results. The count of invading 143B cells was evidently decreased in the si-CXCR4 group compared with si-NC, with no difference between control and si-NC (P < 0.01, Figure 2G). In MG-63 cells, OE-CXCR4 significantly Led to a higher count of invasive cells relative to the vector group, while control and vector groups remained similar (P < 0.01)( Figure 2H). 2.3CXCR4 regulates MMP9 activity and expression in osteosarcoma cells To determine whether CXCR4 affects the matrix-degrading capacity of osteosarcoma cells, MMP9 activity and expression were examined in 143B and MG-63 cells following CXCR4 knockdown or overexpression. Consistently, ELISA analysis showed no difference in secreted MMP9 levels between the control and si-NC groups in 143B cells, whereas the si-CXCR4 group exhibited a significant reduction in MMP9 levels relative to si-NC (P < 0.01, Figure 3A). In MG-63 cells, MMP9 secretion was significantly increased in the OE-CXCR4 group relative to the vector group, with no difference between control and vector (P < 0.01)(Figure 3B). Western blot results additionally validated these findings. In 143B cells, MMP9 protein presence was significantly lower in the si-CXCR4 group versus si-NC, while control and si-NC groups showed similar levels (P < 0.01)(Figure 3C). In MG-63 cells, MMP9 protein was significantly upregulated in the OE-CXCR4 group compared to vector, with no difference between control and vector (P < 0.01)( Figure 3D). 2.4Co-localization of CXCR4 and MMP9 in osteosarcoma cells To assess the spatial relationship between CXCR4 and MMP9 in osteosarcoma cells, immunofluorescence staining was performed in 143B and MG-63 cells. In 143B cells, CXCR4 and MMP9 signals were both detected in the cytoplasm, with high degrees of overlap observed in the control and si-NC groups. In contrast, the si-CXCR4 group exhibited visibly reduced fluorescence intensity of both CXCR4 and MMP9, along with decreased co-localization (Figure 4A). Quantification of co-localization confirmed a significant reduction in the CXCR4-MMP9 overlap ratio in the si-CXCR4 group relative to si-NC (P < 0.01).In MG-63 cells, immunofluorescence signals for CXCR4 and MMP9 were similar between the control and vector groups, while cells in the OE-CXCR4 group showed increased staining intensity for both proteins and a higher degree of co-localization (Figure 4B). Quantitative analysis showed that the overlap ratio of CXCR4 and MMP9 was a notable elevation in the OE-CXCR4 group when compared to the vector group.(P < 0.01). 2.5CXCR4 promotes osteosarcoma cell migration and invasion through upregulation of MMP9 To determine whether MMP9 is engaged in CXCR4-mediated tuning of osteosarcoma cell motility, rescue and interference experiments were conducted. In 143B cells, qRT-PCR analysis showed that MMP9 mRNA expression was significantly increased in the OE-MMP9 group compared to the vector group (P < 0.01)(Figure 5A). In MG-63 cells, MMP9 expression was notably downregulated in the si-MMP9 group as opposed to si-NC(P < 0.01)(Figure 5B).Wound healing assays disclosed that CXCR4 knockdown significantly reduced the migration rate of 143B cells compared to si-NC, while co-transfection with OE-MMP9 partially rescued the migratory ability (P < 0.01, Figure 5C). In MG-63 cells, CXCR4 overexpression enhanced cell migration, which was significantly attenuated by co-transfection with si-MMP9 (P < 0.01)( Figure 5D).Consistently, Transwell assays showed that CXCR4 knockdown reduced the number of invading 143B cells relative to si-NC, and this reduction was partially reversed by MMP9 overremovedP < 0.01, Figure 5E). In MG-63 cells, increased invasion observed in the OE-CXCR4 group was significantly suppressed upon MMP9 silencing (P < 0.01, Figure 5F). 2.6CXCR4 enhances matrix degradation in osteosarcoma cells via MMP9 upregulation To determine whether CXCR4 regulates extracellular matrix degradation via MMP9, we assessed MMP9 activity and expression under both gain- and loss-of-function conditions, including M
Zhang et al. (Mon,) studied this question.