Photodynamic therapy-mediated cancer vaccination enhances stem-like phenotype and immune escape, which can be blocked by thrombospondin-1 signaling through CD47 receptor protein

Abstract

Like most of the strategies for cancer immunotherapy, photodynamic therapy-mediated vaccination has shown poor clinical outcomes in application. The aim of this study is to offer a glimpse at the mechanisms that are responsible for the failure based on cancer immuno-editing theory and to search for a positive solution. In this study we found that tumor cells were able to adapt themselves to the immune pressure exerted by vaccination. The survived tumor cells exhibited enhanced tumorigenic and stem-like phenotypes as well as undermined immunogenicity. Viewed as a whole, immune-selected tumor cells showed more malignant characteristics and the ability of immune escape, which might contribute to the eventual relapse. Thrombospondin-1 signaling via CD47 helped prevent tumor cells from becoming stem-like and rendered them vulnerable to immune attack. These findings prove that the TSP-1/CD47/SIRP-α signal axis is important to the evolution of tumor cells in the microenvironment of immunotherapy and identify thrombospondin-1 as a key signal with therapeutic benefits in overcoming long term relapse, providing new evidence for the clinical promise of cancer vaccination.

Keywords: CD47; Cancer Stem Cells; Immunotherapy; Photodynamic Therapy; Tumor Immunology; Tumor Microenvironment.

FIGURE 1.

The growth rate, tumorigenicity, and stemness factor expression of tumor cells upon in vivo immune selection. A, tumor formation of T₀ and T₃ was monitored in the first 2 weeks post-re-challenge. Seven mice were included in each generation. The data are shown as the percentage of mice that formed tumors. B, tumor sizes were measured for 18 days post-re-challenge. Data are presented as the mean ± S.D. of seven mice in the T₀ and T₃ generations. C, representative images of immunohistochemistry for stemness factors in T₀–T₃ tumor tissues.

FIGURE 2.

The proliferation and stem-cell-like phenotype of immune-selected cancer cells. A, the cell proliferation of P₀-P₃ cells was measured by MTT assay. B, the cell-cycle distribution of P₀ and P₃ cells. C, Western blot analysis of cell-cycle proteins expression including cyclin A and p21 in cells of each generation. β-Actin was used as a loading control. The relative expression of each protein = (density of each band/density of the indicated β-actin band). Values are represented as -fold increases relative to P₀. D, flow cytometry analysis of stem cell markers including CD44, CD34, and CD133 for Lewis lung cancer in the P₀–P₃ population. Representative histograms are shown in D, left. The gray solid histogram represents isotype control. Results of quantitative analysis are shown in D, right. E, representative images of tumor spheres formation derived from P₀ and P₃ cells. The microscopic fields were photographed (upper), and the number of spheres ≥75 μm was counted (lower). F, expression of stemness factors in cancer cells undergone in vitro immune selection was analyzed by Western blot. G, the mRNA levels of stemness factors were measured by real-time PCR and normalized to levels of GADPH mRNA. Data mentioned above are represented as the mean ± S.D. (n ≥ 5 per group). *, p < 0.05; **, p < 0.01; ***, p < 0.001, versus P₀ cells.

FIGURE 3.

The immunogenicity and tumorigenicity of immune-selected cells. Spleen lymphocytes from vaccinated mice as effectors were fed with P₀-P₃ cells as targets. As effector cells, spleen lymphocytes were examined for the frequency of IFN-γ producing CD8+ T cells by flow cytometry. A, left, shows a summary, whereas A, right, shows representative dot plots. The frequency of apoptotic (cleaved-caspase-3 positive) tumor cells was measured by flow cytometry. Representative histograms of one of the experiments were shown in B, left). The gray solid histogram represents isotype control. B, right, shows quantitative analysis of the cleaved-caspase-3 expression. C, the cytotoxicity of CTL against P0-P3 cells was evaluated by a lactate dehydrogenase (LDH) release assay. D, the tumor formation of P₀ and P₃ cells in vivo was monitored for 18 days after tumor re-challenge at the indicated cell densities into nude mice. E, tumor weights were measured at the 18th day after re-challenge. Seven nude mice were included in each group. Values show a summary of >5 independent experiments. Data are represented as the mean ± S.D. *, p < 0.05; **, p < 0.01, compared with the P₀.

FIGURE 4.

The expression and the interaction of CD47 and its two ligands, TSP-1 and SIRP-α. A, upper, representative histograms of the CD47 expression. The gray solid histogram represents isotype control. A, lower, the mean fluorescence intensity (MFI) of CD47 was normalized by that of isotype control. The expression of CD47 in cells of each generation is shown as -fold change in the mean fluorescence intensity compared with P₀. Data show a summary of a total of five independent experiments. *, p < 0.05; **, p < 0.01, versus P₀. B, representative confocal microscopy images of P₀–P₃ cells stained for CD47. C, representative images of TSP-1 expression by immunohistochemistry in normal mice lungs and lung adenocarcinomas. D, co-immunoprecipitation of CD47 with SIRP-α from co-culture lysates of immature DC cells and LLC cells with or without TSP-1 pretreatment. Representative blots (IB) are shown in the upper graph, whereas quantification of three independent Western blots is shown in the lower graph.

FIGURE 5.

TSP-1 prevented the stem-like and immune-resistant phenotype of immune-selected tumor cells in the presence of CD47. A, Western blot analysis of the expression of cell-cycle proteins in P₀ and P₃ cells upon TSP-1/CD47 signal interference. B, cell proliferation of P₀-P₃ with or without TSP-1/CD47 signal interference. C, the impact of the TSP-1/CD47 signal on the cell-cycle distribution of P₀ and P₃ cells. D, the effect of the TSP-1/CD47 signal on the stemness factors expression in immune-selected cancer cells. β-Actin was used as a loading control. E, sphere-forming capacity of P₀ or P₃ cells treated with 2.2 nm TSP-1. F, P₀ and P₃ cells pretreated with 2.2 nm TSP-1 were incubated with CTLs for the indicated times. Representative dot plots (left) and a summary (right) of five experiments were shown. The population of IFN-γ-producing CD8+ T cells was measured by flow cytometry to detect CTL activation. G, P₀ and P₃ cells pretreated with 2.2 nm TSP-1 or not were co-incubated with tumor-specific CTLs, and the frequency of cleaved caspase-3+ cells was measured by flow cytometry. H, a lactate dehydrogenase release assay was also performed to evaluate the cytotoxicity of CTLs against P₀ and P₃ cells with TSP-1 treatment. Data above are the means of five independent experiments ± S.D. *, p < 0.05; **, p < 0.01, versus untreated; ns, not significant.

FIGURE 6.

TSP-1 also inhibited the cell proliferation and sphere-forming capacity of human cancer cells. The expression of CD47 on the surface of A549, HCT116, and HeLa cells was measured by flow cytometry. A, upper, representative histograms show the expression of CD47. The gray solid histogram represents isotype control. A, bottom, the expression of CD47 is shown as the percentage of FITC-positive cells. B, the inhibitory effect of TSP-1 on the growth rate of human tumor cells including A549, HCT116, and HeLa. A549, HCT116, and HeLa cells were treated with 2.2 nm TSP-1 for indicated times. C, the sphere-forming capacity of these cells was analyzed in suspension culture. A summary of five independent experiments is shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001.