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. 2022 Oct;148(10):2743-2757.
doi: 10.1007/s00432-022-04036-8. Epub 2022 May 12.

Attenuated Toxoplasma gondii enhances the antitumor efficacy of anti-PD1 antibody by altering the tumor microenvironment in a pancreatic cancer mouse model

Affiliations

Attenuated Toxoplasma gondii enhances the antitumor efficacy of anti-PD1 antibody by altering the tumor microenvironment in a pancreatic cancer mouse model

Said Ahmed Bahwal et al. J Cancer Res Clin Oncol. 2022 Oct.

Abstract

Purpose: To investigate whether attenuated Toxoplasma is efficacious against solid tumors of pancreatic cancer and whether attenuated Toxoplasma improves the antitumor activity of αPD-1 antibody on pancreatic cancer.

Methods: The therapeutic effects of attenuated Toxoplasma NRTUA strain monotherapy and combination therapy of NRTUA with anti-PD-1 antibody on PDAC tumor volume and tumor weight of Pan02 tumor-bearing mice were investigated. We characterized the effects of combination therapy of NRTUA with anti-PD-1 antibody on tumor-infiltrating lymphocytes and tumor-specific IFN-γ by using immunohistochemistry, flow cytometry and ELISA. The antitumor mechanisms of combination therapy of NRTUA with anti-PD-1 antibody were investigated via depletion of CD8+ T cells and IL-12.

Results: NRTUA strain treatment inhibited tumor growth in a subcutaneous mouse model of PDAC through activating dendritic cells and increasing CD8+ T cell infiltration in the tumor microenvironment. More importantly, combination therapy of NRTUA with anti-PD-1 antibody elicited a significant antitumor immune response and synergistically controlled tumor growth in Pan02 tumor-bearing mice. Specifically, the combination treatment led to elevation of CD8+ T cell infiltration mediated by dendritic cell-secreted IL-12 and to tumor-specific IFN-γ production in the PDAC tumor microenvironment. Also, the combination treatment markedly reduced the immunosuppressive myeloid-derived suppressor cell population in PDAC mice.

Conclusion: These findings could provide a novel immunotherapy approach to treating solid tumors of PDAC and overcoming resistance to anti-PD-1 agents in PDAC tumors.

Keywords: Dendritic cells; Immunotherapy; Programmed cell death 1 receptor; Toxoplasma gondii; Vaccination.

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Conflict of interest statement

The author(s) declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
NRTUA vaccination effectively controls tumor growth in a Pan02 subcutaneous tumor model. A Six to 8-week-old female C57BL/6 J mice were subcutaneously inoculated with 1 ×ばつ 106 Pan02 cells. Mice were randomly divided into 4 groups when tumor volume reached 50 mm3. Each group of mice was treated with a relevant treatment according to the schematic diagram above. Tumor growth was measured with digital calipers every 3 days. B Left: representative images of tumors separated from each group at the experimental endpoint. Middle: change in tumor volume over time in each administration group. Right: corresponding tumor weight at the experimental endpoint (n = 4). C CD8+ tumor-infiltrating T cells were evaluated through IHC. Representative CD8 immunohistochemically stained sections and semiquantitative density results are shown, scale bar: 50 μm. Tumor growth curve (B, middle panel) was analyzed using a two-way ANOVA with a Bonferroni multiple comparison test. Tumor weight (B, right panel) and CD8+ T cell infiltration (C) were analyzed using one-way ANOVA. Data represented as mean ± SD. *P < 0.05; ***P < 0.001; ****P < 0.0001; n.s. not significant
Fig. 2
Fig. 2
NRTUA activates invaded DCs in the tumor microenvironment and increases PD-1 expression on tumor-infiltrating CD8+ T cells. A Dendritic cells from NRTUA treated and untreated Pan02 tumor–bearing mice were analyzed 1 day after treatment for expression of CD80 and CD86 (mean fluorescence index, MFI). NRTUA-invaded cells were tracked using CFSE-labeled NRTUA parasites. B Bone marrow cells were isolated from 4- to 6-week-old mice and differentiated into DCs by GM-CSF in vitro. BMDCs were cocultured with NRTUA (MOI = 1:1) for 24 h. Total mRNA was extracted from DCs, and reverse transcribed into cDNA. The levels of IL12 gene expression were detected by RT-qPCR. Results were normalized to the level of GAPDH mRNA and presented as fold increase in mRNA level over the untreated control that was arbitrarily set as 1. C Protein expression in immature DCs (iDCs) and NRTUA-infected DCs (NRTUA-DC) was analyzed by immunoblotting using antibodies against MYD88, NF-kB and GAPDH (loading control). D Supernatants collected from iDCs and DCs were cocultured with NRTUA for 24 h, washed, and added to the lower chambers of transwell plates. Medium only was used as a control. Anti-IL-12 or corresponding isotype (rabbit-IgG) control antibody was added as indicated. CD8+ T cells were added to the upper chamber and cells were incubated for 24 h. Cells in the lower chambers were harvested and stained with PE-conjugated anti-CD8 and analyzed by flow cytometry. E The intra-tumoral CD8+ T cells were gated and tested for expression of PD-1 by flow cytometry. Data in (A, B and E) were analyzed by student’s t test. Data in (D) was analyzed using one-way ANOVA tests. Data represented as mean ± SD. **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s. not significant
Fig. 3
Fig. 3
Combination treatment with NRTUA and αPD-1 antibody enhances the inhibition of subcutaneous Pan02 tumor growth via increasing infiltration of CD8+ T cells in the TME. A Each group of mice was treated with a relevant treatment according to the schematic diagram above. B Left: representative images of tumors separated from each group at the experimental endpoint. Middle: change in tumor volume over time in each administration group. Right: corresponding tumor weight at the experimental endpoint. C CD8a tumor-infiltrating T cells in the TME were evaluated through IHC. Representative CD8α immunohistochemically stained sections and semi-quantitative density results are shown, scale bar: 50 μm. D Schematic representation of the treatment schedule for the CD8+ T cell depletion experiment. C Left: representative images of tumors separated from each group at the experimental endpoint. Middle: change in tumor volume over time in each administration group. Right: corresponding tumor weight at the experimental endpoint, (n = 4). F CD8a tumor-infiltrating T cells in the TME were evaluated through IHC. Representative CD8α immunohistochemically stained sections and semi-quantitative density results are shown, scale bar: 50 μm. Tumor growth curves were analyzed using a two-way ANOVA with a Bonferroni multiple comparison test. Tumor weight and CD8+ T cell infiltration data were analyzed by using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 4
Fig. 4
NRTUA combined with PD-1 blockade enhances tumor-specific IFN-γ production in CD8+ T cells within the PDAC TME. A CD8+ T cells were isolated and purified from tumors of Pan02 tumor-bearing mice. Tumor-bearing mice were treated with IgG, NRTUA, αPD-1, or NRTUA + αPD-1 therapy as indicated. The percentage of IFNγ producing CD8+ T cells amongst all CD8+ T cells within the tumor-infiltrating lymphocytes is shown. ELISA assays were performed using autologous irradiated Pan02 tumor cells as antigenic targets for CD8+ T cells isolated from (B) tumor-infiltrating lymphocytes and (C) the spleen. Each experimental group consisted of 5 mice, pooled, and analyzed individually in triplicates. Data were analyzed using one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s. not significant
Fig. 5
Fig. 5
Antitumor efficacy of combined treatment requires IL-12 production for CD8+ T cell activation. A The IL-12 secreting DC subpopulation in the TME was quantified by flow cytometry analysis. B Schematic representation of the treatment schedule for the IL12p75 depletion experiment. C Left: representative images of tumors separated from each group at the experimental endpoint. Middle: change in tumor volume over time in each administration group. Right: corresponding tumor weight at the experimental endpoint, (n = 4). D CD8a tumor-infiltrating T cells were evaluated through IHC. Representative CD8α immunohistochemically stained sections and semiquantitative density results are shown, scale bar: 50 μm. Tumor growth curve (B) was analyzed by two-way ANOVA with a Bonferroni multiple comparison test. Data in (A, D) were analyzed by one-way ANOVA test. Data represented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s. not significant
Fig. 6
Fig. 6
Combination therapy with NRTUA and PD-1 blockade reduces the MDSC population in the PDAC TME. MDSC subpopulations PMN-MDSCs (B) M-MDSCs (C) and in the TME were determined by flow cytometry (n = 4). Polymorphonuclear MDSCs are CD11b+Ly6CloLy6Ghi, whereas monocytic MDSCs are CD11b+Ly6ChiLy6Glo. Data were analyzed by one-way ANOVA. n.s. non significance, *P < 0.05, ***P < 0.001. PMN-MDSCs polymorphonuclear myeloid-derived suppressor cells, M-MDSCs monocytic myeloid-derived suppressor cells

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