Role Of Immune Microenvironment On The Progression Of Castration-resistant Prostate Cancer

Background:Prostate Cancer(PCa)has become the second most common cancer and the fifth most deadly cancer in the world.It is a serious threat to the health of middle-aged and elderly men.In the United States,prostate cancer is the first ranking male morbidity and the mortality rate is second only behind to lung cancer.The incidence of prostate cancer has obvious geographical differences.In China,although the incidence of prostate cancer is much lower than that of developed countries in Europe and America,the growth rate is increasing due to the great material life of people,changes in dietary structure,etc.From 1998 to 2008,the annual increase rate of prostate cancer incidence in China was about 12.07%,which is the fastest growing male tumor.Androgen deprivation therapy(ADT)is currently the most effective treatment for patients with advanced prostate cancer,but these patients will eventually progress to the castration-resistant prostate cancer(CRPC)phase.Although there are currently many treatments to try to solve this problem,such as chemotherapy(docetaxel,cabazitaxel),radiotherapy,new development of androgen receptor(AR)pathway inhibitors(enzarumide,abiraterone),Ra-223,and the PARP inhibitor Olapani,which is still in the experimental stage,etc.Without exception,it is difficult to completely reverse the progress of CRPC,so more research is needed to clarify the resistanct mechanism of these drugs.And There is an urgent need for more emerging drug developed to treat patients with CRPC.Notably,most of the current research focuses on the tumor cells themselves,but less on the surrounding environment in which they depend.However,we know that the development of tumors is inseparable from the environment around them.The "seed soil" theory points out that although "seed" is important,the "soil" around is also indispensable.Therefore,for tumors,the process of its development and metastasis is inseparable from its surrounding environment.We should study the tumor and its microenvironment as a whole,which may solve the current dilemma.The tumor microenvironment includes microvessels,fibroblasts,mesenchymal cells,immune cells,etc.It also includes cytokines,chemokines,chemical factors,etc.secreted by various cells,which together form a complex network that regulates the development and metastasis of tumor cells.Among them,immune cells play a crucial role in the progression of tumors.Antibodies against CTLA4 or PD1/PDL1 have been shown to be effective in a number of tumors including kidney cancer,bladder cancer,melanoma,lung cancer.This suggests that strategies for targeted therapy of the tumor microenvironment are feasible.However,in prostate cancer,traditional immunotherapy seems to have no effect on the two immunological checkpoint inhibitors.Therefore,we need to figure out the resistant mechanisms and further explore new immunotherapy methods.Purpose:This study aims to explore the relationship between the mechanism of drug resistance of advanced prostate cancer and the surrounding immune microenvironment,which is mainly divided into the following three parts:First,we explored the relationship between tumor-associated macrophages(TAMs)and tumor cells from patients with advanced prostate cancer with enzalutamide resistance.Secondly,explore the specific mechanism of interaction between tumor-associated macrophages(TAMs)and tumor cells.Finally,the patients with advanced prostate cancer and their surrounding immune microenvironment were studied as a whole.The overall distribution of immune cells in the microenvironment of prostate cancer tissue during CRPC progression was compared and the immunological map was drawn.Single-cell Bulk sequencing analysis of CRPC tissue immune cells revealed changes in molecular levels within the cells,for the purpose of finding suitable intervention targets,designing small molecule drugs and monoclonal antibodies,and exploring the possibility of clinical treatment.Methods:The first two parts of the study discovered the relationship between TAMs and patients with advanced drug-resistant prostate cancer by immunohistochemistry,and verified the basic data of 140 clinical patients,followed by establishing co-cultured cell lines in vitro.A co-culture system between nuclear macrophage cell lines and prostate cancer resistant cell lines was built to study the relationship and mechanism between TAM and prostate tumor cells.Molecular changes between the transcriptional and expressional levels were also determined by Western Blot,qPCR,coimmunoprecipitation(IP),chromatin immunoprecipitation(CHIP).In addition,we used antibody microarrays,ELISA and other experiments to determine the mechanism of TAMs affecting the resistance of tumor cells.In addition,after identifying key targets of action mechanisms,we constructed a prostate cancer resistance model with immunodeficient mice,and tested drugs and treatments for several key targets in this model.In the third part of the study,we applied CyTOF technology to the early prostate cancer and advanced prostate cancer tissue and explore changes in various immune cells in the immune microenvironment which are classified according to certain functional molecules.For the T cells of their main immune response,we isolated and purified them,and carried out Bulk sequencing.We found the detailed changes in the transcriptome,thus discovering the key immune checkpoint of NRP1.We also found its role in the ligand,that is,SEMA3 A secreted by tumor cells through a series of T cell proliferation and secretion experiments in vitro level to verify its ability to inhibit T cell immune response through NRP1.Results:First,we found that TAMs around the tumor were positively correlated with ADTinduced neuroendocrine differentiation of prostate cancer by immunohistochemistry and in vivo animal experiments.The relationship between the two cell types and the prognosis and biochemical recurrence of the patient’s clinical data are also statistically relevant.Then,by establishing a co-culture system,we found that drug resistant prostate cancer cells can recruit and polarize TAMs.More interestingly,in turn the recruitment and polarization of TAMs can further lead to drug resistance and neuroendocrine differentiation of prostate cancer cells.Then,through the antibody microchip experiment,we found that TAMs affect the drug resistance of tumor cells by secreting IL6/STAT3 pathway.Prostate cancer cells with enzalutamide-resistant properties affect TAMs by amplifying and secreting HMGB1.HMGB1 not only stabilizes β-catenin in tumor cells but also degrades them,allowing them to smoothly enter the nucleus to activate downstream neuroendocrinerelated genes.Also,HMGB1 is transcribed and secreted into the intercellular space,which acts on monocyte macrophages in a paracrine manner,recruiting and polarizing monocytes into TAMs.Then,TAMs secrete more IL6 to affect the tumor cells,thus forming a Positive feedback loops leading to drug resistance in prostate cancer cells.Next,we constructed a prostate cancer resistance model for immunodeficient mice.The drugs targeting several key targets,that is HMGB1 and IL6 R,were applied to the mouse model.The drug,compared with the placebo group,was able to significantly improve its resistance characteristics,and the combination of the two was better.Finally,we extended the research focus to the entire immune microenvironment of advanced prostate cancer.CyTOF technology found that the immune microenvironment of patients with advanced castration-resistant prostate cancer CRPC changed compared to the early stage of prostate cancer and the immunological difference map between the two was drawn.Differences in changes in various immune cells include CD4 T cells,CD8 T cells,Treg cells,monocytes/macrophages,B cells,NK cells,MDSC cells,DC cells.It was found that the changes in T cells in the immune microenvironment accounted for the vast majority.Therefore,we performed Bulk sequencing of T cells in tumor tissues.Interestingly,we identify that NRP1 is a key molecule for T cell function changes.Then we found its functional ligand,SEMA3 A,through a series of in vitro experiments.It was found that SEMA3 A inhibits NRP1 on the surface of T cells,ultimately leading to T cell proliferation inability,the secretion function decreased,the ability to exert immune function decreased.Thus,the mechanism of immune escape is generated.Conclusion:In this study,we investigated the interaction between TAMs and advanced drugresistant tumor cells.We found the effect of immune microenvironment around tumor on tumor cell progression and drug resistance,and found that in advanced prostate cancer,the resistant cell line secreted HMGB1.The autocrine and paracrine functions recruit and polarize monocyte or macrophages,and TAMs in turn promotes the resistance of tumor cells through IL6/STATA3 pathway.In addition,a clear map of the entire immune microenvironment of advanced prostate cancer and early prostate cancer is drawn.What’s more,a significant pathway for suppressing the immune response is found after comparative analysis,providing new immunotherapeutic strategy,which is a solid foundation for providing new hope for patients with advanced drug-resistant prostate cancer.