IADR Abstract Archives
Mapping Immune Cell Infiltration in an HPV+ OPC Mouse Model Using Spatial Cell Profiling
Objectives: Oropharyngeal cancer (OPC) incidence is significantly increasing among men in the U.S. with most cases (~80%) caused by human papillomavirus type 16 (HPV16) infection. Despite presence of highly immunogenic viral antigens and robust immune infiltration, OPC tumors are characterized by immune evasion. OPC premalignancy and the mechanism by which anti-cancer immunity shapes early carcinogenesis remain poorly understood.
Methods: We developed a genetically engineered mouse model of OPC whereby expression of HPV16 E6 and E7 along with mutant PIK3CAE545K is regulated by a tamoxifen-inducible Cre recombinase with conditional expression directed to the murine oropharyngeal epithelium. We performed extensive molecular, histologic, immunohistochemical, and Lunaphore COMET-based spatial multiomics (multiplex Protein SeqIF + multiplex RNA in situ hybridization) of the tumor immune microenvironment (TIME) in normal epithelial mucosa, premalignant lesions, and OPC tumors.
Results: Oncogene induction leads to rapid development of premalignant lesions marked by immune cell accumulation, and a subset of these lesions progress to OPC. Macroscopic examination revealed that multifocal premalignant lesions develop with 100% penetrance and moderate burden and that, of these lesions, OPC tumors develop with ~40% penetrance. These tumors display significant leukocyte infiltration, namely neutrophils and CD3+ T cells, but also distinct spatial organization of antigen presenting cells.
Conclusions: Post-natal induction of HPV16 oncogenes and mutant PIK3CAE545K circumvents immune tolerance and permits analysis of mechanisms that regulate immune evasion during carcinogenesis. Anti-tumor immunity evolves as a continuum from preneoplasia, to preinvasive OPC, to frank invasive OPC with establishment of a pro-tumoral immune microenvironment. Spatial biology analyses and concurrent IHC highlighted NK cell (CD56+) foci, Collagen 1 associated fibroblasts (FAP+), DCs (CD11c+), neutrophil infiltrates (CD11b+), and imbalanced regulatory and cytotoxic T infiltrates within exophytic regions of the developing tumor. Collectively, immune cell recruitment and spatial organization is an early feature in disease progression, as evidenced by their presence in pre-malignant lesions which may guide future biomarker development of early disease.
Methods: We developed a genetically engineered mouse model of OPC whereby expression of HPV16 E6 and E7 along with mutant PIK3CAE545K is regulated by a tamoxifen-inducible Cre recombinase with conditional expression directed to the murine oropharyngeal epithelium. We performed extensive molecular, histologic, immunohistochemical, and Lunaphore COMET-based spatial multiomics (multiplex Protein SeqIF + multiplex RNA in situ hybridization) of the tumor immune microenvironment (TIME) in normal epithelial mucosa, premalignant lesions, and OPC tumors.
Results: Oncogene induction leads to rapid development of premalignant lesions marked by immune cell accumulation, and a subset of these lesions progress to OPC. Macroscopic examination revealed that multifocal premalignant lesions develop with 100% penetrance and moderate burden and that, of these lesions, OPC tumors develop with ~40% penetrance. These tumors display significant leukocyte infiltration, namely neutrophils and CD3+ T cells, but also distinct spatial organization of antigen presenting cells.
Conclusions: Post-natal induction of HPV16 oncogenes and mutant PIK3CAE545K circumvents immune tolerance and permits analysis of mechanisms that regulate immune evasion during carcinogenesis. Anti-tumor immunity evolves as a continuum from preneoplasia, to preinvasive OPC, to frank invasive OPC with establishment of a pro-tumoral immune microenvironment. Spatial biology analyses and concurrent IHC highlighted NK cell (CD56+) foci, Collagen 1 associated fibroblasts (FAP+), DCs (CD11c+), neutrophil infiltrates (CD11b+), and imbalanced regulatory and cytotoxic T infiltrates within exophytic regions of the developing tumor. Collectively, immune cell recruitment and spatial organization is an early feature in disease progression, as evidenced by their presence in pre-malignant lesions which may guide future biomarker development of early disease.