Osteoprotegerin regulates cancer cell migration through SDF-1/CXCR4 axis and promotes tumour development by increasing neovascularization

We previously reported that OPG is involved in ischemic tissue neovascularization through the secretion of SDF-1 by pretreated-OPG endothelial colony-forming cells (ECFCs). As the vascularization is one of the key factor influencing the tumour growth and cancer cell dissemination, we investigated whether OPG was able to modulate the invasion of human MNNG-HOS osteosarcoma and DU145 prostate cancer cell lines in vitro and in vivo. Cell motility was analysed in vitro by using Boyden chambers. Human GFP-labelled MMNG-HOS cells were inoculated in immunodeficient mice and the tumour nodules formed were then injected with OPG and/or FGF-2, AMD3100 or 0.9% NaCl (control group). Tumour growth was manually followed and angiogenesis was assessed by immunohistochemistry. In vitro, SDF-1 released by OPG-pretreated ECFCs markedly attracted both MNNG-HOS and DU145 cells and induced spontaneous migration of cancer cells. In vivo, tumour volumes were significantly increased in OPG-treated group compared to the control group and OPG potentiated the effect of FGF-2. Concomitantly, OPG alone or combined with FGF-2 increased the number of new vasculature compared to the control group. Interestingly AMD3100, an inhibitor of SDF-1, prevented the in vivo effects of OPG induced by SDF-1 This study provides experimental evidence that OPG promotes tumour development trough SDF-1/CXCR4 axis

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it is now well established that the immune infiltrate in human tumors can correlate with prognosis and response to therapy. The analysis of the immune infiltrate in the tumor microenvironment has become a major challenge for the classification of patients and the response to treatment. The co-expression of inhibitory receptors such as Program Cell Death Protein 1 (PD1; also known as CD279), Cytotoxic T Lymphocyte Associated Protein 4 (CTLA-4), T-Cell Immunoglobulin and Mucin Containing Protein-3 (Tim-3; also known as CD366), and Lymphocyte Activation Gene 3 (Lag-3; also known as CD223), is a hallmark of T cell exhaustion. We developed a multiparametric in situ immunofluorescence staining to identify and quantify at the cellular level the co-expression of these inhibitory receptors. On a retrospective series of frozen tissue of renal cell carcinomas (RCC), using a fluorescence multispectral imaging technology coupled with an image analysis software, it was found that co-expression of PD-1 and Tim-3 on tumor infiltrating CD8 T cells is correlated with a poor prognosis in RCC. To our knowledge, this represents the first study demonstrating that this automated multiplex in situ technology may have some clinical relevance

Cxcr6-deficiency impairs cancer vaccine efficacy and resident memory CD8+ T-cells recruitment in tumor.

Immunization route is directly correlated with cancer vaccine efficacy. Our team previously showed that mucosal (intranasal, i.n.) and systemic (intramuscular) vaccinations are both able to induce systemic specific CD8+ T-cells but only i.n. immunization allows an efficient control of mucosal tumor growth. Indeed, the i.n. vaccination favors the tumor infiltration of specific CD8+ T-cells and especially tissue-resident memory T-cells (Trm), which are crucial for a potent antitumor activity. Based on a transcriptomic analysis, we have identified a chemokine receptor, CXCR6, highly expressed by these specific Trm CD8+ T-cells, induced by the i.n. vaccination. To understand the role of CXCR6 in vaccination efficacy, we have set up a body of experiments using heterozygous Cxcr6gfp/+ mice, where GFP reflects CXCR6 expression and homozygous Cxcr6-deficient mice Cxcr6gfp/gfp.We have then confirmed CXCR6 expression on specific Trm CD8+ T-cells induced by i.n. vaccination in lung mucosa. Using an orthotopic head and neck tumor model, CXCR6 expression on tumor-infiltrating Trm induced by i.n. immunization has also been reported. In addition, we showed that Cxcr6-deficiency impairs mice survival in a prophylactic and therapeutic i.n. vaccination settings in various mucosal tumor models. In this Cxcr6-deficient mouse model, the loss of vaccine-induced protection against tumor graft correlates with a clear reduction of Trm infiltration in tumor. Finally, in a cohort of human lung cancer, we have observed endogenous CXCR6+ Trm infiltrating these tumors in situ. To conclude, we have identified CXCR6 as a required parameter to recruit the crucial anti-tumor Trm in mucosal tumor. Our results finally indicate that CXCR6 might be a new surrogate biomarker to evaluate antitumor vaccine efficacy

Mechanistic study of the proangiogenic effect of osteoprotegerin

Osteoprotegerin (OPG), a soluble tumour necrosis factor receptor superfamily member, inhibits RANKL-mediated osteoclastogenesis. We have previously reported that OPG enhances the proangiogenic properties of endothelial colony-forming cells (ECFCs) in vitro, and promotes vasculogenesis in vivo. Here we investigated how OPG promotes neovascularisation. Proteomic experiments showed that OPG pretreatment affected ECFCs protein expression in two ways, 23 spots being down-regulated and 6 upregulated. These spots corresponded to proteins involved in cell motility, adhesion, signal transduction and apoptosis. In keeping with these proteomic results, we found that OPG induced ECFCs adhesion to activated endothelium in shear stress conditions, promoting intermediate but not focal adhesion to fibronectin and collagen. Treatment with OPG induced a reorganization of the ECFCs cytoskeleton, with the emergence of cell protrusions characteristic of a migratory phenotype. These effects correlated with decreased FAK phosphorylation and enhanced integrin αVβ3 expression. OPG drastically reduced caspase-3/7 activities and maintained ECFCs viability after 48 h of treatment. All these effects were significantly attenuated by ECFCs incubation with the CXCR4 antagonist AMD-3100, and by prior heparan sulphate proteoglycan disruption. The proangiogenic properties of OPG appeared to be mediated by the proteoglycan syndecan-1, although OPG 1-194 lacking its heparin-binding domain still had pro-vasculogenic effects in vitro and in vivo. These results suggest that OPG may interact with ECFCs by binding to HSPGs/syndecan-1, thereby induce an anti-adhesive effect and promoting ECFCs migration through a SDF-1/CXCR4 dependent pathway