Imunoterapia direcionada à anidrase carbônica IX em carcinoma renal: impactos no checkpoint imunológico e tumorigênese
Data
2024-06-14
Autores
Pivetta, Renata Schmieder 
Tipo
Dissertação de mestrado
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ISSN da Revista
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Resumo
Introdução: A expressão das enzimas pró-tumorigênicas anidrase carbônica IX (CAIX) e ciclooxigenase-2 (COX-2) ocorre em cerca de 95% e 50% dos casos de carcinoma renal de células claras (ccRCC), respectivamente. O ccRCC também é caracterizado por um microambiente imunossupressor, e aproximadamente 24% dos casos superexpressam o ligante de morte celular programada-1 (PD-L1), aumentando o risco de morte em 53%. Objetivos: Avaliar a existência de um mecanismo regulatório de expressão de PD-L1 e COX-2 mediado pela inibição imunoterapêutica da CAIX em linhagens celulares de ccRCC positivas para CAIX, COX-2 e PD-L1. Verificar possível correlação in silico entre a expressão de CAIX e PD-L1 e/ou CAIX e COX2 em 534 tumores e 9 linhagens celulares de ccRCC. Analisar o efeito da imunoterapia com linfócitos T (LT) expressando receptor quimérico de antígeno (CAR) anti-CAIX no checkpoint imunológico in vivo. Metodologia: Realizamos a separação (“sorting”) por citometria de fluxo de subpopulações de células SKRC52 baseadas na expressão de CAIX e PD-L1. Promovemos o bloqueio da CAIX com dois anticorpos monoclonais em duas linhagens de ccRCC, SKRC52 e SKRC59. Avaliamos a viabilidade celular e a expressão de CAIX, PD-L1, COX-2 por imunofluorescência e a expressão de proteínas relacionadas a possíveis vias de sinalização envolvidas por Western Blotting. No ensaio in vivo, avaliamos PD-L1, Ki67, CD3 e Granzima B, assim como marcadores de exaustão de LT. Resultados: A subpopulação de SKRC52 selecionada como negativa para CAIX e positiva para PD-L1, tornou-se espontaneamente negativa para a expressão de ambas as moléculas. Por bioinformática, foi observada uma correlação positiva entre CAIX e PD-L1 e CAIX e COX-2 em células de ccRCC. O bloqueio de CAIX com anticorpo monoclonal anti-CAIX, gerou uma diminuição nos níveis de expressão de PD-L1 e COX-2 em ccRCC. LT CAR Anti-CAIX in vivo reduziu PD-L1 e reverteu a exaustão dos LT infiltrados em ccRCC. Conclusões: Terapias com inibidores de CAIX podem indiretamente inibir o checkpoint imunológico via PD-L1/PD-1 e a tumorigênese via COX-2.
Introduction: The carbonic anhydrase IX (CAIX) and cyclooxygenase-2 (COX-2) enzymes are expressed in about 95% and 50% of the clear cell renal cell carcinoma (ccRCC) cases, respectively. An immunosuppressive microenvironment characterizes this tumor, and approximately 24% of cases overexpress the programmed cell death ligand-1 (PD-L1), increasing the risk of death by 53%. Objectives: Herein, we evaluated a regulatory mechanism for PD-L1 and COX2 expression control mediated by immunotherapeutic inhibition of CAIX in ccRCC cell lines positive for CAIX, COX-2, and PD-L1. We have also correlated in silico the expression of CAIX and PD-L1 or CAIX and COX2 in 534 tumors and 9 ccRCC cell lines, and we analyzed the effect of immunotherapy of CAIX-targeted chimeric antigen receptor (CAR) T cells on the immunological checkpoint in vivo. Methods: By flow cytometry, we sorted SKRC52 cell subpopulations based on the expression of CAIX and PD-L1. The CAIX blockade was performed with two monoclonal antibodies in two ccRCC cell lines, SKRC52 and SKRC59. Cell viability and the expression of CAIX, PD-L1, and COX-2 were evaluated by immunofluorescence. The expression of proteins related to possible signaling pathways involved in this modulation was determined by western blotting. PD-L1, Ki67, CD3, and Granzyme B expression were evaluated in the in vivo assay by immunohistochemistry and the expression of T cell exhaustion markers by flow cytometry. Results: The subpopulation of SKRC52 was selected as negative for CAIX and positive for PD-L1, and it became spontaneously negative for the expression of both molecules. A positive correlation was observed between CAIX and PD-L1 and CAIX and COX-2 in a panel of ccRCC cells. Blocking CAIX with anti-CAIX monoclonal antibodies decreased the expression levels of PD-L1 and COX-2 in ccRCC. CAIX-targeted CAR T cells in vivo reduced PD-L1 and reversed the exhaustion of T cells infiltrated in the tumor. Conclusions: CAIX inhibitor therapies may indirectly inhibit immune checkpoint via PD-L1/PD-1 and tumorigenesis via COX-2.
Introduction: The carbonic anhydrase IX (CAIX) and cyclooxygenase-2 (COX-2) enzymes are expressed in about 95% and 50% of the clear cell renal cell carcinoma (ccRCC) cases, respectively. An immunosuppressive microenvironment characterizes this tumor, and approximately 24% of cases overexpress the programmed cell death ligand-1 (PD-L1), increasing the risk of death by 53%. Objectives: Herein, we evaluated a regulatory mechanism for PD-L1 and COX2 expression control mediated by immunotherapeutic inhibition of CAIX in ccRCC cell lines positive for CAIX, COX-2, and PD-L1. We have also correlated in silico the expression of CAIX and PD-L1 or CAIX and COX2 in 534 tumors and 9 ccRCC cell lines, and we analyzed the effect of immunotherapy of CAIX-targeted chimeric antigen receptor (CAR) T cells on the immunological checkpoint in vivo. Methods: By flow cytometry, we sorted SKRC52 cell subpopulations based on the expression of CAIX and PD-L1. The CAIX blockade was performed with two monoclonal antibodies in two ccRCC cell lines, SKRC52 and SKRC59. Cell viability and the expression of CAIX, PD-L1, and COX-2 were evaluated by immunofluorescence. The expression of proteins related to possible signaling pathways involved in this modulation was determined by western blotting. PD-L1, Ki67, CD3, and Granzyme B expression were evaluated in the in vivo assay by immunohistochemistry and the expression of T cell exhaustion markers by flow cytometry. Results: The subpopulation of SKRC52 was selected as negative for CAIX and positive for PD-L1, and it became spontaneously negative for the expression of both molecules. A positive correlation was observed between CAIX and PD-L1 and CAIX and COX-2 in a panel of ccRCC cells. Blocking CAIX with anti-CAIX monoclonal antibodies decreased the expression levels of PD-L1 and COX-2 in ccRCC. CAIX-targeted CAR T cells in vivo reduced PD-L1 and reversed the exhaustion of T cells infiltrated in the tumor. Conclusions: CAIX inhibitor therapies may indirectly inhibit immune checkpoint via PD-L1/PD-1 and tumorigenesis via COX-2.
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Citação
PIVETTA, Renata Schmieder. Imunoterapia direcionada à anidrase carbônica IX em carcinoma renal: impactos no checkpoint imunológico e tumorigênese. 2024. 120 f. Dissertação (Mestrado em Hematologia e Oncologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2024.