Local Anti-PD-1 Delivery Prevents Progression of Premalignant Lesions in a 4NQO-Oral Carcinogenesis Mouse Model

Abstract

Although the principle of systemic treatment to prevent the progression of oral premalignant lesions (OPL) has been demonstrated, there remains a lack of consensus about an optimal approach that balances clinical efficacy with toxicity concerns. Recent advances in cancer therapy using approaches targeting the tumor immune microenvironment (TIME) including immune-checkpoint inhibitors indicate that these agents have significant clinically activity against different types of cancers, including oral cancer, and therefore they may provide an effective oral cancer prevention strategy for patients with OPLs. Our past work showed that systemic delivery of a monoclonal antibody to the programmed death receptor 1 (PD-1) immune checkpoint can inhibit the progression of OPLs to oral cancer in a syngeneic murine oral carcinogenesis model. Here we report a novel approach of local delivery of a PD-1 immune-checkpoint inhibitor loaded using a hydrogel, which significantly reduces the progression of OPLs to carcinomas. In addition, we detected a significant infiltration of regulatory T cells associated with oral lesions with p53 mutation, and a severe loss of expression of STING, which correlated with a decreased infiltration of dendritic cells in the oral lesions. However, a single local dose of PD-1 inhibitor was found to restore stimulator of interferon response cGAMP interactor 1 (STING) and CD11c expression and increase the infiltration of CD8⁺ T cells into the TIME irrespective of the p53 mutational status. Overall, we provide evidence for the potential clinical value of local delivery of biomaterials loaded with anti-PD-1 antibodies to prevent malignant progression of OPLs. PREVENTION RELEVANCE: Oral cancer is an aggressive disease, with an overall survival rate of 50%. Preinvasive histologic abnormalities such as tongue dysplasia represent an early stage of oral cancer; however, there are no treatments to prevent oral carcinoma progression. Here, we combined biomaterials loaded with an immunotherapeutic agent preventing oral cancer progression.

Figure 1.

Experimental carcinogen-induced mouse model and local immunotherapy delivery reduces oral lesion incidence. A, Study timeline and end points for immunotherapy studies. B, Representative lesions and histopathology of 4NQO-induced oral lesions in the mouse tongue, scale ruler (mm). Photomicrographs show the histopathologic progression in this model system, mild, moderate, severe dysplasia, and carcinoma in situ. Scale bars, 200 and 50 μm, top and bottom panels, respectively. C, The square shows a magnification area of each histologic stage, and the table below the graph represents the incidence of normal, low-, and high-grade incidence of lesions after treatment in each mouse group (n = 5–7).

Figure 2.

Increased infiltration of CD4⁺ and CD8⁺ T cells in oral lesions after anti–PD-1 treatment. A, Representative images of low- and high-grade oral lesions and IHC quantification signal of CD4⁺ T cells (right side graphs). B, Representative IHC images of infiltrating CD8⁺ T cells in oral lesions. A significant difference of CD4⁺ and CD8⁺ T cells was detected between untreated and PD-1 treated mice groups. Immunostaining signal was defined as the number of positive cells per mm². Right graphs: *, P < 0.05; **, P <0.01; ***, P <0.001; ****, P < 0.0001. Scale bar, 50 μm.

Figure 3.

Local immunotherapy delivery reduced the expression of PD-1 levels in oral lesions. Immunotherapy significantly reduced the PD-1 levels in oral lesions of wild-type and mutant p53 mice. Immunostaining signal was defined as the number of positive cells per mm². Right graphs: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Scale bar, 50 μm.

Figure 4.

PD1 blockage reduced the infiltration of Foxp3⁺ T cells in low- and high-grade oral lesions. Mutant p53 mice showed a higher Foxp3⁺ T-cell infiltration compared with wild-type mice. PD-1 hydrogel reduced the numbers of positive Foxp3⁺ T cells in low- and high-grade oral lesions. Immunostaining signal was defined as the number of positive cells per mm². Right graphs: *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Scale bar, 50 μm.

Figure 5.

Local delivery of anti–PD-1 increases STING protein levels in low- and high-grade lesions. Representative IHC images of STING expression in oral lesions. A, significant difference of STING expression was detected between untreated and PD-1 treated mice groups. Immunostaining signal was defined as the number of positive cells per mm². Right graphs: **, P <0.01; ***, P < 0.001; ****, P < 0.0001. Scale bar, 50 μm.

Figure 6.

PD-1 blockage promoted recruitment of CD11c⁺ DCs into oral lesions of wild-type and mutant p53 mice. Representative IHC images of CD11c DCs in oral lesions. A significant difference of CD11⁺ cells was detected between untreated and PD1-treated mice groups. Immunostaining signal was defined as the number of positive cells per mm². Right graphs: **, P <0.01; ***, P < 0.001; ****, P < 0.0001. Scale bar, 50 μm.