Expression of GPX4 by oncolytic vaccinia virus can significantly enhance CD8+T cell function and its impact against pancreatic ductal adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is currently difficult to treat, even when therapies are combined with immune checkpoint blockade (ICB). A novel strategy for immunotherapy would be to maximize the therapeutic potential of oncolytic viruses (OVs), which have been proven to engage the regulation of tumor microenvironment (TME) and cause-specific T-cell responses. To boost tumor sensitivity to ICB therapy, this study aimed to investigate how glutathione peroxide 4 (GPX4)-loaded OVs affect CD8⁺ T cells and repair the immunosuppressive environment. Here, we successfully constructed a novel recombinant oncolytic vaccinia virus (OVV) encoding the mouse GPX4 gene. We found the OVV-GPX4 effectively replicated in tumor cells and prompted the expression of GPX4 in T cells. Our research indicated that OVV-GPX4 could reshape the TME, rectify the depletion of CD8⁺T cells, and enhance the antitumor effects of ICB therapy.

Keywords: Glutathione peroxide 4; oncolytic vaccinia virus; pancreatic ductal adenocarcinoma; tumor microenvironment.

Figure 1.

Generation of a GPX4-expressing oncolytic vaccinia virus and its oncolytic properties in vitro (a) Schematic representation of OVV-GPX4 structure. The GPX4 gene under the control of the promoter Pse/l was inserted into the TK gene of the vaccinia virus. TKR, right flank sequences of thymidine kinase gene; TKL, left flank sequences of thymidine kinase gene; gpt, guanine phosphoribosyl transferase; EGFP, enhanced green fluorescent protein; P7.5, vaccinia virus P7.5 early/late promoter; Pse/l, synthesized vaccinia virus early/late promoter. (b) Detection of GPX4 expression and secretion by western blot. Vero cells were infected with OVV or OVV-GPX4 at MOI 1. At 48 h post-infection, cell lysates and supernatants were collected, and GPX4 was detected with anti-GPX4 antibody. (c) Comparative cytotoxicity of OVV and OVV-GPX4. Panc02, CT26, MC38, and Hepa 1–6 cells infected with serial dilutions of OVV or OVV-GPX4. Cell viability was measured 48 h post-infection by CCK8. (d) Viral infectivity of OVV-GPX4. Panc02, CT26, MC38, and Hepa 1–6 cells infected with OVV or OVV-GPX4 at MOI of 0.1. At indicated time points, cells were harvested and detected by flow cytometry. Data represent the mean ± standard deviation (SD) of ≥three independent experiments.

Figure 2.

Effect of OVV-GPX4 on T cells in vitro (a) After staining T cells with CFSE and co-incubating them with tumor cells at a ratio of 6:1 (CD3⁺T: Panc02 cells), and adding OVV or OVV-GPX4 viral supernatant, T cell proliferation was measured by flow cytometry. In a parallel experiment, Co-culture of T cells with OVV or OVV-GPX4 (MOI = 10). After 48 h, the expression of CD36 and lipid peroxidation levels in CD8⁺T cells was evaluated by flow cytometry. (b) Representative diagram of flow cytometric analysis of T cell proliferation. (c) Bar chart showing expression of GPX4 in T cells. MOI for OVV or OVV-GPX4 infection of T cells was 10. Cells were collected 48 h after infection, RNA was extracted, and cDNA was reverse-transcribed. qPCR was utilized to detect GPX4 expression in cells. (d) Representative diagram of flow cytometric analysis of the activation indicator CD25 in CD8⁺ T cells. (e) Representative diagram of flow cytometric analysis of the expression of CD36 in CD8⁺ T cells. (f) Representative diagram of flow cytometric analysis of lipid peroxidation levels in CD8⁺T cells. Data represent the mean ± standard deviation (SD) of ≥three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 3.

Intratumoral injection of OVV-GPX4 enhanced antitumor efficacy in subcutaneous Panc02 tumor models (a) C57BL/6 mice bearing subcutaneous Panc02 tumors were treated with PBS, OVV, OVV-GPX4 (2 × 10 pfu/tumor, I.T.), or GPX4 (1 µg/tumor, I.T.) for five times. (b) Plot of mouse tumor progression. (c) Mouse survival curves. (d) Tumor growth curve for each mouse. (e) Bar chart showing the abundance of OVV and OVV-GPX4 in important organs and tumors of mice. Data represent the mean ± standard deviation (SD) of ≥three independent experiments. *p < 0.05.

Figure 4.

Intratumoral injection of OVV-GPX4 improved immune microenvironment in Panc02 tumor model (a) C57BL/6 mice bearing subcutaneous Panc02 tumors were treated with PBS, OVV, OVV-GPX4 (2 × 10 pfu/tumor, I.T.), or GPX4 (1 µg/tumor, I.T.). On day 2 after virus injection, tumors were harvested, and a cell suspension was prepared. Percentages of CD8⁺T cells were analyzed by flow cytometry. In other parallel experiments, CD8⁺T cell proliferation indicator Ki67, percentages of MDSCs and the expression of PD-1 on the surface of CD8⁺T cells in tumors was analyzed by flow cytometry. (b) Representative diagram of flow cytometric analysis of percentages of CD8⁺T cells in tumors. (c) Representative diagram of flow cytometric analysis of CD8⁺T cell proliferation indicator Ki67 in tumors. (d) Representative diagram of flow cytometric analysis showing percentages of MDSCs in tumors. (e) Representative diagram of flow cytometric analysis of IFN-γ producing in CD8⁺T cells, which exert effector functions in tumors. (f) Representative diagram of flow cytometric analysis of producing perforin-1 in CD8⁺T cell in tumors. (g) Representative diagram of flow cytometric analysis of PD-1 on the surface of CD8⁺T cells in tumors. (h,i) Representative diagram of flow cytometric analysis of depletion indexes tim-3 and LAG-3 on the surface of CD8⁺T cell in tumors. (j) Representative diagram of flow cytometric analysis of percentage of Foxp3⁺ CD4⁺ Treg cells in tumors. (k) Detection of CD8⁺T infiltration in tumors by immunohistochemistry. Scale bar,100 µm. (n = 3 per group). Data represent the mean ± standard deviation (SD) of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.0001.

Figure 5.

Intratumoral injection of OVV-GPX4 improved systemic immunity in Panc02 tumor model (a) C57BL/6 mice bearing subcutaneous Panc02 tumors were treated with PBS, OVV, OVV-GPX4 (2 × 10 pfu/tumor, I.T.), or GPX4 (1 µg/tumor, I.T.). On day 2 after virus injection, spleens were harvested, and a cell suspension was prepared. Percentages of CD8⁺T cells, proliferation indicator Ki67 and percentages of MDSCs in spleens were analyzed by flow cytometry. (b) Representative diagram of flow cytometric analysis of percentages of CD8⁺T cells in spleens. (c) Representative diagram of flow cytometric analysis of CD8⁺T cell proliferation indicator Ki67 in spleens. (d) Representative diagram of flow cytometric analysis showing percentages of MDSCs in spleens. (e) Representative diagram of flow cytometric analysis of IFN-γ producing in CD8⁺T cells in spleens. (f) Representative diagram of flow cytometric analysis of perforin-1 producing in CD8⁺T cells in spleens. Data represent the mean ± standard deviation (SD) of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6.

CD8⁺ T cells mediated the antitumor immunity of OVV-GPX4 (a) The depletion of CD8⁺T and CD4⁺T cells in the tumor of mice was analyzed by flow cytometry. (b) Flow cytometric analysis of the proportion of CD8⁺T cells and CD4⁺T cells. (c) Treatment scheme of Panc02 S.C. tumor models. C57BL/6 mice bearing subcutaneous Panc02 tumors were injected with PBS, OVV-GPX4 (2 × 10⁷ pfu/mouse, I.T.), OVV-GPX4 (2 × 10⁷ pfu/mouse, I.T.) combined with anti-CD4 or anti-CD8 antibody (100 µg/mouse, I.P.). Tumor volume was measured every 2 days. (d) Plot of mouse tumor progression. (e) Individual tumor growth curve. Data represent the mean ± standard deviation (SD) of ≥ three independent experiments. ns > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 7.

Intratumoral injection of OVV-GPX4 combined with anti-PD-1 antibody enhanced antitumor efficacy in subcutaneous Panc02 tumor model (a) Illustration of mouse injection procedure. C57BL/6 mice bearing subcutaneous Panc02 tumors were injected with PBS, OVV-GPX4 (2 × 10 pfu/mouse, I.T.), anti-PD-1 antibody (200 µg/mouse, I.P.), or OVV-GPX4 combined with anti-PD-1 antibody for five times. Tumor volume was measured every 2 days. (b) Plot of mouse tumor progression. (c) Mouse survival curves. (d) Tumor growth curve for each mouse. Data represent the mean ± standard deviation (SD) of five independent experiments. *p < 0.05; **p < 0.01.

Figure 8.

Immune mediation mechanism of OVV-GPX4 (a) C57BL/6 mice bearing subcutaneous Panc02 tumors were treated with PBS, OVV, or OVV-GPX4 (2 × 10 pfu/tumor, I.T.) for three times. On day 2 after virus injection, RNA from fresh tumor tissues was sequenced. Bubble plot of KEGG analysis of signaling pathway gene expression enrichment. (b) Heat map of differentially expressed genes in the chemokine signaling pathway. (c) Heat map of differentially expressed genes in the cytokine-cytokine receptor interaction pathways. (d) Detection of fold changes in CCL4, CXCL9, CXCL10, CXCL13, ICOS, CD28, and GZMB at mRNA level by qPCR. Data represent the mean ± standard deviation (SD) of ≥three independent experiments. *p < 0.05.