Antitumor Effect Of Macrophage Polarization And Immunotherapy Of Atezolizumab In Humanized Bladder Cancer Mouse Model

Tumor microenvironment is a complex system that enhances or evades the host immune surveillance and tumor killing.The core immune cells that make up the tumor immune environment include T cells,B cells,NK cells,macrophages,DC cells,and neutrophils.The location,number,functional phenotype,and the crosstalk of immune cells in the tumor microenvironment affect the tumor’s immune response,treatment,and prognosis outcomes.Therefore,remodeling the characteristics of immune cells and their inhibition in the tumor immune environment are crucial for the development of novel immunotherapy and chemotherapy resistance against cancer.Given that tumor growth is commonly accompanied with the accumulation of myeloid cells in the tumor and changes in immunophenotyping,immunotherapy for T-cell-targeted tumors is inhibited.This study aimed to investigate key security macrophages and specialist T lymphocytes in the tumor microenvironment,explore the substances that modulate macrophage-immune polarization phenotypes and their mechanisms,and construct humanized T-cell immunity.The mouse bladder cancer tumor evaluation model may provide insights into the key issues of remodeling tumor immune environment.1. Macrophages present two opposing phenotypes,namely,the classically activated M1 phenotype and the selectively activated M2 phenotype.M1 macrophages resist pathogen invasion,inhibit tumors,phagocytose microorganisms,promote inflammatory responses,and enhance immune surveillance.By contrast,M2 is a rest phenotype that suppresses immune responses,promotes tissue healing,reduces inflammation,and promotes tumor growth.Polyporus polysaccharides can exert immunomodulatory effects.These polysaccharides were obtained by water extraction and alcohol precipitation.Purification was performed by DEAE–cellulose ion exchange chromatography and Sephadex G-100filtration chromatography to obtain uniform Polyporus polysaccharides.M2 macrophages were induced through CD14 magnetic bead purification and M-CSF.A uniform polysaccharide form of P.umbellatus,named as PPS,was obtained.This polysaccharide affects macrophage adhesion,morphology changes,and M1 phenotype polarization;decreases the PD1 expression;increases the expression of MHC II molecules;promotes the secretion of cytokines,namely,interleukin（IL）-1β,IL-6,IL-8,IL-10,and tumor necrosis factor（TNF）-α（P<0.05）),and the tumor killing effects（P<0.05）;increases the expression levels of TLR2,TLR4,and NF-κB;and reduces the transcription of FN,COL III,and ICMA-1m RNA（P<0.05）.PPS also affects macrophage adhesion and pseudopod formation with significant immunopotentiation,and its mechanism may relate to the activation of the TLR2/4-NF-κB signaling pathway and the downregulation of adhesion proteins.Bacillus Calmette–Guérin（BCG）therapy is efficient for non-muscle-invasive bladder cancer.However,the underlying mechanisms of this therapy have not been completely clarified to date.The present study investigated the role and potential mechanism of BCG in the regulation of macrophage polarization and the significance of the signal regulatory protein-α（SIRPα）on macrophages in vitro.We differentiated the monocyte cell line THP-1into M2 macrophages(CD206^high and Th2 cytokine-secreting cells).Result indicated that BCG-induced M2 polarized into M1 phenotypes(CD86^high,CD197^high,and Th1cytokine-secreting cells),downregulated SIRPα（CD172α）,and promoted the expression of proinflammatory cytokines,such as IL-1β,IL-6,TNF-α,interferon-γ,and inducible nitric oxide synthase.The inhibition of T24 and RT4 human bladder cancer cell lines cocultured with BCG-treated M2 macrophages exhibited a strong effect belonging to CD47-high expression.In addition,BCG significantly increased the protein expression of Janus kinase2（JAK2）and p-signal transducer and the activator of transcription 1（STAT1）in a dose-dependent manner.Overall,these observations demonstrated that the JAK2/STAT1pathway mediates the M1 polarization and that SIRPαsuppression is an important mechanism of BCG on macrophages for combating bladder cancer.The role of macrophages in BCG therapy for bladder cancer has still not been determined in vivo.In this study,we investigated the role and potential mechanism of BCG（0.25,1.25,and 6.25μg/mouse,i.v.）immunotherapy for BCa in a NOD/scid ^IL2Rg-/-（NSI）mouse model by targeting macrophages in vivo.Notably,we observed that NSI mice with T24 bladdersignificant reduction in tumor volume after injection of live BCG into the tail vein.The levels of the inflammatory and macrophage maturation cytokines,such as TNF-α,IL-1β,IL-6,IL-12P70,RANKL,and MCP-1,were significantly increased in serum and the tumor supernatant compared with those in normal control subjects.Furthermore,BCG promoted the expression of the prodifferential genes PU-1,EGR-1,NF-κB,and C-Fos in the bone marrow.In conclusion,these observations suggested that the injection of live BCG can target macrophages against bladder tumor growth in vivo.The mechanism was related to promote macrophage maturation,immune activation,and increase in the number of macrophages infiltrating the bladder tumor.2. Humanized mice or mouse-human chimeras have become important models for the study of surrogate living organisms in vivo.In recent years,the humanized mouse model has been widely recognized and applied in studies of human AIDS,cancer,infectious diseases,hematological diseases,and degenerative diseases.Patient-derived xenograft tumors（PDX）retain the original tumor characteristics,such as histological heterogeneities,biomolecular characteristics,clinically malignant phenotypes,genotypes,tumor structures,and tumor vasculatures.These tumors can also provide relevant assessments,especially for novel cancer therapeutics and predictability of clinical outcomes,and a more reliable drug development platform than those of tumor cell lines.Thus,PDX have become recognized as preclinical drug evaluation models.Atezolizumab（MPDL3280A）,as an immunotherapeutic drug,is the first major drug for bladder cancer in the past 30 years.This drug enables the accurate targeted therapy for bladder cancer.A mouse model of PBMC-humanized subcutaneous tumor-bearing T24bladder cancer CDX was established.Atezolizumab（10 mg/kg,i.v.）was administered two times a week via the tail vein for 4 weeks.The immunotherapy effect and mechanism of atezolizumab were studied.Atezolizumab significantly reduced the tumor size compared with that in the control group（P<0.05）.Moreover,this drug increased the proportion of human T lymphocytes in the peripheral blood test（P<0.05）,but it exerted no effect on the T cell CD8⁺/CD4⁺ratio and decreased the T-cell PD1 expression（P<0.05）.In the tumor microenvironment,atezolizumab reduced the expression of PD1 and TIM3（P<0.05）and tumor PD-L1（P<0.01）,but it presented no effect on T-cell activation molecules CD25 and CD69.A humanized model of the PDX mouse model of bladder cancer was also constructed to observe the therapeutic effects of atezolizumab（5,10,20 mg/kg,i.v.）on patient-derived tumor samples.Atezolizumab significantly reduced the tumor size compared with that in the control group（P<0.05）.Additionally,atezolizumab increased the proportion of human T lymphocytes in the peripheral blood test（P<0.05）;significantly increase the number of CD4⁺subpopulations and decreased the expression of PD1 in T cells（P<0.05）,but it exerted no effect on CD25 and CD69 activations in peripheral T cells.In the tumor microenvironment,atezolizumab reduced the expression of PD1 and TIM3（P<0.05）and tumor PD-L1（P<0.01）,but it displayed no effect on T-cell activation molecules CD25 and CD69.The established PDX mouse model of humanized bladder cancer can well evaluate the effects of immunotherapeutic drugs and exhibit different characteristic phenotypes with CDX,especially the infiltration of T cells.Infiltrating T-cells are much larger than PDX in the CDX model.The experimentally established humanized bladder cancer CDX or PDX mouse model can well assess the effects of immunotherapeutic drugs.This model is a tumor immune evaluation model that shows the T lymphocyte characteristics.Results showed that atezolizumab not only exerted a significant inhibitory effect on tumor PD-L1 but also indirectly inhibited PD1 to increase the number of human T-cells in NSI mice.Moreover,this drug is crucial in remodeling the tumor immune environment.Differences between PDX and CDX models can provide preclinical evidence for patient tumor heterogeneity,different immune infiltration environments,and precise treatment.