CXCR4 in human osteosarcoma malignant progression. The response of osteosarcoma cell lines to the fully human CXCR4 antibody MDX1338

Abstract

Osteosarcoma (OS) is the most frequent primary malignant tumour of bone and metastases occur in 30% of cases, the 5-year survival rate is 25-30%. Although pre- and post-operative chemotherapy has improved prognosis in osteosarcoma (OS), high toxicity and natural and acquired drug-resistance are the first cause of treatment failure. The identification of new predictive and therapeutic biomarkers may increase drug sensitivity and better control localized and metastatic disease. By the evidence that CXCR4 receptor by binding its ligand CXCL12 activates downstream critical endpoints for tumour malignancy, we first studied human OS progression correlating CXCR4 expression in OS biopsy with patient clinical data. By Real-time PCR and immunoistochemistry we found that high levels of CXCR4 gene and protein expression significantly correlated with OS progression, emphasizing the role of CXCR4/CXCL12 axis in tumour prognosis. This was supported by univariate analyses that showed a higher probability of local and/or systemic relapse in OS patients with a high CXCR4 gene expression and a significant increase of metastasis risk associated with an increasing score of CXCR4 protein staining intensity. Secondarily, to study the role of CXCR4 as a target for new therapeutic strategies, we evaluated the response of OS cells to the fully human CXCR4 antibody, MDX1338. In the study we also included AMD3100, the most studied CXCR4 antagonist. In CXCR4-positive OS cells cultured in CXCL12-rich BM-MCS-CM (bone marrow-derived mesenchymal stem conditioned medium), a decrease of cell proliferation up to 30%-40% of control was seen after drug exposure. However, an increase of apoptosis was seen in p53-positive U2OS and 143B after CXCR4 inhibitor incubation, while no changes were seen in treated SAOS-2 cells which also present a different labeling profile. The role of p53 in apoptotic response to CXCR4 inhibitors was confirmed by p53 silencing in U2OS cell line. Our data suggest that the response to anti-CXCR4 agents could be influenced by the genetic background and labeling profile which induces a different cross-talk between tumour cells and environment. The delay in cell cycle progression associated with increased apoptosis could sensitize p53-positive cells to conventional therapy and in vivo preclinical experiments are on going with the aim to suggest new combined target therapies in human OS.

Keywords: Biomarkers; CXCR4 antagonists; Metastasis; Prognosis; Sarcoma.

Fig. 1

Relative levels of CXCR4. mRNA expression. Mann–Whitney analysis revealed statistical significant differences between (A) primary OS and healthy bone tissue, (B) low and high grade OS, (C) disease-free and relapsed OS. (D) Kaplan–Meier analysis based on CXCR4 expression showed a higher probability of disease-free survival in patients with low CXCR4 mRNA levels. Cut-off rounded to the 50°percentile. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Fig. 2

Representative immunostaining of CXCR4 protein. CXCR4 was moderately to strongly expressed in cytoplasm and nucleus of high grade OS cells. In low grade OS CXCR4 was negative or week with a focal distribution. A week and diffuse distribution was seen for CXCL12 reactivity in all cases (IHC 20X).

Fig. 3

CXCR4 protein expression. (A) Mann–Whitney analysis showed higher CXCR4 protein levels in metastatic than in non metastatic OS. (B) Based on staining intensity score (range 1–5) metastasis-free survival was significantly higher in patients with CXCR4 low expression. (C) Higher CXCR4 staining levels were present in alive compared to deceased patients. (D) Overall survival probability was significantly higher in patients with no or low CXCR4 expression. Range 0–3 indicates a low or absent immunoreactivity; range 4–5 indicates a moderate to strong immunoreactivity. ** p ≤ 0.01; *** p ≤ 0.001.

Fig. 4

CXCR4 expression in OS cell lines. (A) CXCR4 mRNA levels in OS cell lines and osteoblasts by RT-PCR. (B) CXCR4 protein expression by FACS analysis in OS cells. Black= negative control; Grey=Ab anti-CXCR4; Each value indicates the average of three independent experiments, * p ≤ 0.05, ** p ≤ 0.01.

Fig. 5

Sensitivity of OS cells to CXCR4 antagonists. Cells were exposed to increasing doses of MDX1338 and AMD3100 for 48 h and 72 h. A proliferation decrease of 30–40% compared to non treated cells occurred at 48 h at the doses of 0.5 µg/ml and 30 µg/ml respectively by counting with trypan blue. Each point indicates the average of three independent experiments. C= non treated cells.

Fig. 6

Apoptosis and cell cycle. (A) By FACS analysis an increase of apoptosis was seen in U2OS and 143B after 48 h of 0.05 µg/ml MDX1338 and 30 µg/ml AMD3100 exposure. No changes in apoptotic fraction were seen in treated SAOS-2 cells. (B) Cell cycle analysis distribution of G1, S and G2/M phase at 48 h of 0.05 µg/ml MDX1338 and 30 µg/ml AMD3100 exposure shows a lengthening of G2/M and G1 in U2OS and 143B respectively. No differences were seen in cell cycle progression of treated SAOS-2 compared to control; Each value indicates the average of three independent experiments, *p ≤ 0.05.

Fig. 7

Wound healing assay of U20S. 30 µg/ml AMD3100 and 0.05 µg/ml MDX1338 exposure caused a relevant decrease in cell migration respectively up to 24 h and 48 h of treatment compared to control. At 48 h and 72 h migration rate shifted towards control values (100% of closure). Histograms show the percentage of wound closure. C= non treated cells.

Fig. 8

Wound healing assay of 143B Cells responded to the treatment with a relevant cell migration slowdown up to 12 h. At 24 h the percentage of wound closure approached the control value. Histograms show the percentage of wound closure. C= non treated cells.

Fig. 9

Wound healing assay of SAOS-2. MDX1338 reduced cell motility in a more marked and long-term way than AMD3100, delaying the wound closure. C= non treated cells.

Fig. 10

Response of U2OS to MDX1338 after p53 silencing. A) Western Blot analysis of p53 showed a marked reduction of protein expression after MDX1338 incubation. p53 levels were not affected by CTRL siRNA. B) CTRL siRNA transfected cells exposed to MDX1338 for 48 h responded by increasing apoptotic fraction, while no differences occurred in p53siRNA transfected cells. C) A slight delay in cell cycle progression was seen in both CTRL siRNA and p53 siRNA transfected cells after MDX1338 treatment. Actin = reference protein; C= control; CTRLsiRNA= Negative Control siRNA,.