Exploring Optimized Immunotherapy For Pancreatic Cancer By Tracing Dynamic Local Immunity And Gut Microbiome

Background and aimThe emergence of tumor immunotherapy and its success in a variety of tumor clinical trials are highly anticipated.Immunotherapy is considered as one of the important directions of tumor therapy in the future.However,pancreatic ductal adenocarcinoma(PDAC)has been recognized as an immunologic-cold tumor which is unresponsive to immunotherapies for reasons of different tumor-protective factors.A deeper understanding of the interaction and co-evolvement between the body and tumor is critical for the development of effective immunotherapeutic strategies.It has been recognized that the unique immunosuppressive microenvironment of pancreatic cancer is the biggest obstacle to immunotherapy,and the microbiome is also considered to be an important factor in shaping tumor immunity and impeding the success of immunotherapy.We explored the co-evolution between the body and the tumor in the development of pancreatic cancer from the perspectives of local immunity and gut microbiome,so as to provide a fundamental reference for the development of promising therapeutic strategies.MethodsIn this study,we explored the dynamic changes of tumor-infiltrating immune cells and gut microbiota using a genetic engineered mouse model,which spontaneously develop PDAC.Pancreas and PDAC samples were collected from KPC mice and analyzed by mass cytometry(Cy TOF)and immunohistochemistry.Human pancreas and PDAC samples were used to verify the immune characteristics of different developmental stages by immunohistochemistry.Feces samples in temporal order are collected and detected by metagenome sequencing.KPC syngeneic mouse model was used to determine the effects of gut microbiome on tumor growth by oral antibiotics and feces transplantation.Tumor infiltrating immune cells were evaluated by flow cytometry.ResultsWe observed two stages of immunosuppression,with significantly different characteristics during PDAC development.The early stage of immunosuppression occurs during acinar-to-ductal metaplasia,which presents with increased Treg abundance.The late stage of immunosuppression is initiated with metastasis,which features a large number of myeloid suppressive cells.Surprisingly,the early stage of PDAC continues to exhibit a prominent presence of T and B cells.A trajectory analysis of monocytes and macrophages showed that the differentiation/activation branch of Ly-6C~+monocytes changes from a BST2~+/MHC-II~+M1-like phenotype to an Arg-1+M2-like phenotype over time during PDAC development.Temporal immune characteristics were also confirmed in human specimens.In terms of microbiota,Akkermansia muciniphila maintained high abundance and Bacteroides vulgatus progressively increased specifically in KPC mice.In addition,Clostridium spiroforme was the only strain that rose in KPC mice and fell in healthy mice early in the disease.Metagenomic analysis of metabolic pathways showed that in KEGG level 2,pathways of carbohydrate metabolism,biosynthesis of other secondary metabolites,signal transduction and genetic information processing protein families were specifically altered in KPC mice.Specific metabolic changes of KPC mice were also screened at KEGG level 3,among which the arginine biosynthesis pathway was given special attention with almost all key genes in the pathway are upregulated in KPC mice.Depletion of gut microbiome significantly inhibited tumor growth,while transfer of feces from both wildtype and KPC mice could re-promote tumor progression.Interestingly,repopulation of the microbiome using feces from KPC mice significantly reduced the number of MHC-II~+and CD86~+antigen-presenting macrophages.ConclusionThe study demonstrates the coevolution of histopathology and immunology in the development of PDAC and highlights the necessity of selecting a specific immunotherapeutic strategy based on the corresponding tumor biology stage rather than the disease stage.Early intervention would be a better choice to the success of immunotherapy.In addition,it is a potential way to enable efficacy of immunotherapy by targeting gut microbiome.