multiplexed-immunofluorescence-analysis-quantification-intratumoral

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 ...

Université Paris Descartes

Paroxysmal nocturnal hemoglobinuria (PNH) is associated with an increased risk of thrombosis through unknown mechanisms.

Evaluation of hemostasis and endothelial function in patients with paroxysmal nocturnal hemoglobinuria receiving eculizumab.

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.

Mechanistic study of the proangiogenic effect of osteoprotegerin.

Human adipose-derived stromal cells (hASCs) may hold potential for bone tissue engineering. Osteogenic differentiation of these cells is crucial to bone formation. Low-molecular-weight fucoidan (LMWF) is a sulfated polysaccharide that potentiates several growth factors, including pro-angiogenic growth factors. To investigate whether hASC preconditioning with LMWF promoted bone repair, we compared the effects of LMWF and low-molecular-weight heparin on hASC phenotype and osteogenic differentiation. LMWF did not modify the stem-cell phenotype of hASCs but enhanced their osteogenic differentiation (formation of calcium deposits, increased activity and expression of alkaline phosphatase, and increased expression of osteopontin and runt-related transcription factor 2). However, when hASCs were exposed to LMWF before their adhesion to biphasic calcium phosphate particles and implantation in a bone-growth mouse model, no bone formation was apparent after 5 or 8 weeks, probably due to cell death. In conclusion, LMWF may hold promise for enhancing the osteogenic differentiation of hASCs before their implantation. However, concomitant vascularization would be required to enhance bone formation.

The in vitro and in vivo effects of a low-molecular-weight fucoidan on the osteogenic capacity of human adipose-derived stromal cells.

Low-molecular-weight fucoidan (LMWF) is a sulfated polysaccharide extracted from brown seaweed that presents antithrombotic and pro-angiogenic properties. However, its mechanism of action is not well-characterized. Here, we studied the effects of LMWF on cell signaling and whole genome expression in human umbilical vein endothelial cells and endothelial colony forming cells. We observed that LMWF and vascular endothelial growth factor had synergistic effects on cell signaling, and more interestingly that LMWF by itself, in the absence of other growth factors, was able to trigger the activation of the PI3K/AKT pathway, which plays a crucial role in angiogenesis and vasculogenesis. We also observed that the effects of LMWF on cell migration were PI3K/AKT-dependent and that LMWF modulated the expression of genes involved at different levels of the neovessel formation process, such as cell migration and cytoskeleton organization, cell mobilization and homing. This provides a better understanding of LMWF's mechanism of action and confirms that it could be an interesting therapeutic approach for vascular repair.

Low-Molecular-Weight Fucoidan Induces Endothelial Cell Migration via the PI3K/AKT Pathway and Modulates the Transcription of Genes Involved in Angiogenesis.

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.

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

Regressing tumors are usually associated with a large immune infiltrate, but the molecular and cellular interactions that govern a successful anti-tumor immunity remain elusive. Here, we have triggered type I Interferon (IFN) signaling in a breast tumor model (MMTV-PyMT) using 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a ligand of the STimulator of Interferon Genes, STING. The 2 main events rapidly triggered by DMXAA in transplanted PyMT tumors are 1) the disruption of the tumor vasculature, followed by hypoxia and cell death; 2) the release of chemokines. Both events converged to trigger the recruitment of 2 waves of immune cells: a swift, massive recruitment of neutrophils, followed by a delayed rise in monocytes and CD8 T cells in the tumor mass. Depletion experiments in vivo revealed that myeloid cell subsets and T cells need to cooperate to achieve full-blown recruitment and activation at the tumor site and to induce effective secondary cell death leading to tumor regression (Illustration 1). Altogether, our study highlights that the tumor regression induced by the STING agonist DMXAA results from a cascade of events, with an initial vessel destruction followed by several infiltration waves of immune cells which have to cooperate to amplify and sustain the initial effect. We thus provide the first global and detailed kinetic analysis of the anti-tumoral effect of DMXAA and of its different articulated steps.

Isabelle Galy-Fauroux

INSERM U970

Université Paris Descartes

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

Isabelle Galy-Fauroux

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Université Paris Descartes

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

INSERM U970

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