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. 2012 Nov 1;1(8):1313-1322.
doi: 10.4161/onci.21680.

Studying the Role of the Immune System on the Antitumor Activity of a Hedgehog Inhibitor Against Murine Osteosarcoma

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Free PMC article

Studying the Role of the Immune System on the Antitumor Activity of a Hedgehog Inhibitor Against Murine Osteosarcoma

Christophe Paget et al. Oncoimmunology. .
Free PMC article

Abstract

Recent evidence demonstrates that the efficacy of conventional anticancer therapies including chemotherapy requires a functional immune system. Here, we addressed the possibility that the antitumor effect of a selective Smoothened antagonist and Hedgehog (Hh) pathway inhibitor (LDE225), a promising anticancer drug, might at least partially depend on the immune system. To this aim, we used tumor cell lines derived from a murine model of radiation-induced osteosarcoma. In vitro treatment of osteosarcoma cells with LDE225 resulted in a decreased ability of tumor cells to proliferate, but had no effect on their viability. Flow cytometry analysis demonstrated that LDE225-treatment did not detectably modulate the immunogenicity of tumor cells. Moreover, LDE225 did not display any pro-apoptotic properties on osteosarcoma cells, highlighting that its antitumor profile mainly derives from a cytostatic effect. Furthermore, calreticulin exposure, a key feature of immunogenic cell death, was not provoked by LDE225, neither alone nor combined with recognized immunogenic drugs. Finally, the oral administration of LDE225 to osteosarcoma-bearing mice did significantly delay the tumor growth even in an immunocompromised setting. These data suggest that inhibiting Hh signaling can control osteosarcoma cell proliferation but does not modulate the immunogenic profile of these cells.

Figures

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Figure 1. LDE225 reduced the proliferation ability of osteosarcoma cell lines. (A) Upper panel, CFSE-labeled (2.5 μΜ) 2 × 104 OS18 cells have been cultured for three days in complete media (α-MEM) in the absence (filled histograms) or in the presence (open histograms) of the indicated concentrations of LDE225. After three days, cells were monitored for CFSE dilution. Lower panel, OS18 cells were cultured in the absence or in the presence of LDE225 at the indicated doses. After 8 h, 0.5 μCu of 3H/well were added and radioactivity was measured after 48 h of culture using a liquid scintillation counter. Data represent means of 6 replicates per group ± SEM, from n = 2 independent experiments. Differences in means were analyzed using a one-way ANOVA test. *p < 0.05, ***p < 0.001. (B and C) LDE225-treated or untreated OS18 cells were cultured for 24 h in the presence of various concentrations of either doxorubicin (Dox) or etoposide (Eto) and then viability was assessed using the CellTiter-Blue® reagent. Data represent means of n = 3 independent experiments performed in triplicates. Differences in means were analyzed using a one-way ANOVA test. **p < 0.01, ***p < 0.001. Of note, in each experiment the amount of vehicle corresponds to the highest dose of LDE225 used.
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Figure 2. Phenotype of OS18 cells in the presence or absence of LDE225. Upper row, OS18 cells were monitored for the expression of markers including NK cell ligands (pan-Rae-1, CD155 and DR5), antigen presenting molecules (I-Ab, H2Kb and CD1d), immunosuppressive molecules (PD-1, PD-L1 and PD-L2) and co-stimulatory molecules (CD40, CD70 and CD86). All markers (open histograms) have been compared with their respective isotype controls (gray histograms). Lower row, marker expression has been assessed in the presence (open histograms) and in the absence (gray histograms) of LDE225 (5 μM).
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Figure 3. Effect of LDE225 on immunogenic cell death-induced apoptosis and calreticulin expression. (A) OS18 cells were cultured for 24 h with LDE225 (5 μM) alone or in combination with either doxorubicin (Dox, 1 μM) or etoposide (Eto, 500 nM) and apoptosis was assessed according to Annexin V/propidium iodide (PI) labeling. One representative experiment out of three is shown (left panel). Percentages ± SD of apoptotic cells (AnnexinV+ PI-) are represented in the right panel. (B) OS18 cells were treated for 24 h with LDE225 (5 μM), Dox (1 μM), Eto (500 nM) or the indicated combinations and monitored for calreticulin expression. The mean of fluorescence intensity is indicated. One representative experiment out of three is shown (upper panel). The average ± SEM of calreticulin expression on OS18 cells is shown in the lower panel (n = 9).
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Figure 4. In vitro activity of LDE225 on the development of immune responses. (A and B) One million vehicle- or LDE225-treated spleen cells were cultured in presence or not of lipopolysaccharide (LPS, 50 ng/mL) (A) or concanavalin A (Con A, 2.5 μg/mL) (B) and supernatants were collected after 48 h (A) or 72 h (B). Cytokines in supernatants were assessed using the CBA system. The average ± SD of one representative experiment out of two performed in triplicates is shown. Statistical analyses were performed using a one way ANOVA test followed by a Dunn’s Multiple Comparison post-test. *p < 0.05.
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Figure 5. In vivo activity of LDE225 in the context of host immunity. (A–C) Groups of 5 wild type (WT, A), Rag-1−/− (B), Rag-2−/−γc−/− (C) mice were inoculated s.c. with 1 × 106 OS18 cells. Mice then received either vehicle or LDE225 (80 mg/kg) daily on days 6–10 and 13–17 after tumor cell inoculation. Tumor size was measured as indicated. Data represent means of 5 mice per group ± standard errors. Statistical analyses were performed at the indicated time point using a Mann-Whitney test (*p < 0.05).
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Figure 6. Effect of immunotherapy alone and in combination with LDE225. (A–C) Groups of 5 wild type (WT) mice were inoculated s.c. with 1 × 106 OS18 cells. Tumours were harvested and tumor-infiltrating lymphocytes (TILs) were analyzed for Tim-3, PD-1 and CTLA-4 expression on CD4+ and CD8+ T cells and regulatory T cell (CD4+ FOXP3+) frequency (A). Mice received control immunoglobulins (cIg), anti-CTLA-4, anti-Tim-3, anti-PD-1, anti-CD137 or anti-PD-1/anti-CD137 combination (100 μg i.p each) on days 34, 38, 42 after tumor cell inoculation (B). Mice received as indicated either vehicle or LDE225 (80 mg/kg) daily on days 6–10 and 13–17 and/or cIg or anti-PD1/anti-CD137 on days 6, 10 and 14 after tumor cell inoculation (C). Tumor size was measured as indicated. Data represent means of 5 mice per group ± SEM. Statistical analyses were performed at the indicated time point a using Mann-Whitney test. *p < 0.05, as compared with cIg.

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