RESUMO
Senescence is a mechanism associated with aging that alters tissue regeneration by depleting the stem cell pool. Chronic obstructive pulmonary disease (COPD) displays hallmarks of senescence, including a diminished stem cell population. DNA damage from cigarette smoke (CS) induces senescence via the p16 pathway. This study evaluated the contribution of p16 to CS-associated lung pathologies. p16 expression was prominent in human COPD lungs compared with normal subjects. CS induces impaired pulmonary function, emphysema, and increased alveolar epithelial cell (AECII) senescence in wild-type mice, whereas CS-exposed p16-/- mice exhibit normal pulmonary function, reduced emphysema, diminished AECII senescence, and increased pro-growth IGF1 signaling, suggesting that improved lung function in p16-/- mice was due to increased alveolar progenitor cell proliferation. In conclusion, our study suggests that targeting senescence may facilitate alveolar regeneration in COPD emphysema by promoting IGF1 proliferative signaling.
Assuntos
Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Enfisema/etiologia , Enfisema/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fumar/efeitos adversos , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/genética , Citocinas/metabolismo , Enfisema/fisiopatologia , Fator de Crescimento Insulin-Like I/genética , Pulmão/metabolismo , Pulmão/patologia , Camundongos Endogâmicos C57BL , Modelos Biológicos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4',6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.
