The Roles And Mechanisms Of Tumor Microenvironment Mediated Immune Imbalance In Leukemia And Lymphoma

Chapter 1Study on the effect and mechanism of diffuse large B-cell lymphoma derived exosomes on macrophage polarizationBackgroundDiffuse large B-cell lymphoma(DLBCL)is the most common malignancy derived from lymphohematopoietic system and represents 25%-30%of non-Hodgkin lymphoma(NHL)patients.DLBCL includes a variety of subtypes,which are highly heterogeneous in terms of clinical manifestations,immunophenotyping,and molecular genetics.First-line chemotherapy regimens based on R-CHOP significantly improved treatment outcomes in patients with DBLCL,but about 40%of patients still had poor efficacy and even switched to refractory/relapsed DLBCL.Refractory/relapsed patients have limited effective treatment options and poor prognosis.Therefore,it is of great significance to explore the mechanism of tumorigenesis and progression of DLBCL and find new therapeutic targets for DLBCL.Tumor immune escape is tumor cells through their own or microenvironmental changes to evade the immune recognition and attack.Immune escape is closely related to the tumor progression and a poor prognosis of DLBCL.Tumor-associated macrophages(TAMs)are the main components of immune cells in the tumor microenvironment.Macrophages can differentiate into different subtypes under different stimuli,mainly including M1 type that exerts anti-tumor effects and M2 types that promote tumor effects.M2 macrophages secrete a variety of immunosuppressive cytokines,promote the proliferation of tumor cells,and inhibit the anti-tumor activity of T and NK cells.The infiltration abundance of TAM often predicts a patient’s adverse outcome.Therefore,M2 macrophage polarization-induced immune escape may be one of the pathogenesis of DLBCL.Exosomes are extracellular vesicles(EVs)with the size range of 30-150nm and carry a large amount of functional substances such as proteins,lipids,and non-coding RNA from maternal cells to mediate cell-to-cell signaling transfer.In various solid tumors and hematologic tumors,tumor-derived exosomes regulate the polarization and reprogramming of macrophages by delivering various signaling substances,resulting in tumor immune escape and progression.However,the regulation and mechanism of DLBCL-derived exosomes on macrophage polarization are poorly understood,and this topic aims to explore the influence of DLBCLderived exosomes on macrophage phenotype transformation and its role in tumor progression.ObjectivesTo explore the effects of DLBCL-derived exosomes on macrophage phenotype and function and provide a theoretical basis for the treatment of patients with DLBCL,which contributes to promote the treatment prognosis of patients.Methods1.Patient samples:peripheral blood samples from 36 newly diagnosed DLBCL patients and 40 healthy controls were obtained.We used Ficoll lymphocyte isolation solution to isolate PBMCs.Flow cytometry was performed to detect the proportion of CD163+cells in PBMCs in DLBCL patients and healthy controls.2.Cell line culture,exosome isolation and identification:human DLBCL cell line RC and human leukemia cell line THP-1 were cultured according to the manufacturer’s instructions.Exosome was isolated using differential centrifugation and identified by nanoparticle tracking analysis(NTA)and western blot(WB).3.Polarization of macrophages:Flow cytometry was used to study the effect of exosomes on the expression of CD 163 in macrophage.The expression levels of CD206,CD 163,interleukin 10(IL-10),TNFα and iNOS were determined by real time qPCR.4.Cell proliferation and apoptosis detection:DLBCL cells were co-cultured with macrophages treated by exosomes.CCK8 assay and flow cytometry were used to examine cell viability and apoptosis,respectively.5.Statistical analysis:Experimental data were statistically analyzed using GraphPad prism 9 software.Results are the average of three independent experiments,and the data was showed as mean ± sd.P value was calculated with Student t-test or the Mann-Whitney test and P<0.05 was statistically significant.Results1.Compared with healthy controls,the proportion of CD163+cells in PBMCs in newly diagnosed DLBCL patients were increased significantly.2.The pellets was isolated by differential centrifugation.Nanoparticle tracking analysis showed that the particle size range was 30-150nm;western blot demonstrated that CD63 and TSG-101 proteins,but not calnexin were observed in the pellets,confirming that the cell line culture supernatant extract was exosome without cellular components.3.Microscope observed that exosome-induced macrophages were long-spindle-shaped,adherent,and the cells stop proliferation and division.4.FCM analysis suggested that exosomes upregulated CD163 levels in macrophages.And real time qPCR analysis showed that exosomes can increase the expression of CD206,CD 163,IL-10 and decrease TNFa level in macrophages.However,exosome did not affect the expression level of iNOS.5.CCK8 assay showed that exosome-treated macrophages increased the proliferation of untreated DLBCL cells,and flow cytometry analysis also revealed that exosome-treated macrophages reduced the total apoptosis proportion of untreated DLBCL cells.ConclusionThis study showed that DLBCL-derived exosomes can up-regulate the expression of CD206,CD 163 and IL-10 in macrophages,promoting M2 macrophage polarization.And exosome-induced macrophages can promote proliferation and inhibit apoptosis of untreated DLBCL cells.Therefore,tumor-derived exosomes may serve as new therapeutic targets for DLBCL.Chapter 2Role of CDH23 as a prognostic biomarker and its relationship with immune infiltration in acute myeloid leukemiaBackgroundAcute myeloid leukemia(AML)is a highly aggressive malignancy characterized by clonal expansion of myeloid blast cells in peripheral blood,bone marrow,or other tissues,often accompanied by anemia,fatigue,and an increased risk of bleeding and infection.At present,the cause of AML is still unknown,and genetic mutations,chemical exposure,radiation,etc.are all possible risk factors.While there has been a clearer understanding of the biological processes of AML in recent years,it remains one of the most lethal malignancies.Cure rates are relatively low in AML patients,with a 5-year survival rate of less than 30%in adult AML patients.Therefore,exploring new therapeutic targets is of great significance for improving the prognosis of AML patients and reducing mortality.Cadherin superfamily have more than 100 members in humans involved in physiological processes such as signal transduction,self-recognition,and tumor suppression.Proteins of this superfamily member all have an extracellular cadherin domain of about 110 amino acids in size.There are tandem repeats in this domain,involved in mediating calcium binding and interaction with other cadherin molecules.Cadherin-23(CDH23)is an atypical form of cadherin in this family.Cadherin-23(CDH23),which plays an important role in intercellular adhesion,is involved in the progression of several types of cancer.Furthermore,the reduction of CDH23 expression was significantly association with poorer outcome in patients with DLBCL.However,the biological functions and the prognostic value of CDH23 expression in patients with AML are yet to be explored.Herein,we aim to characterize the role and molecular functions of CDH23 in AML.ObjectivesTo explore the expression level and prognostic value of CDH23 in AML and investigate the effect of CDH23 on immune cell infiltration and tumor microenvironment in AML and its role in AML disease progression,which may provide novel prognostic marker and therapeutic targets for AML.Methods1.Selection of study subjects:RNA expression profiles and clinical information of bone marrow tissue samples of 145 AML patients in TCGA database were extracted as training cohort,139 AML patients in Beat AML database were extracted as validation cohort.Patients’clinical information,genetic cytology and molecular biology information in the two cohorts were statistically analyzed.2.According to the median value of CDH23mRNA expression,patients were divided into high and low expression groups.Spearman rank correlation analyzed the correlation between CDH23 expression and AML clinical features.3.The expression level of CDH23 in bone marrow tissue samples of AML patients and healthy controls was compared by GEPIA database.Survival analysis curve was plotted by Kaplan-Meier tool.The relationship between CDH23 expression level and the overall survival of AML patients was explored.The prognostic value of CDH23 and patient clinical,genetic cytological and molecular biology information in AML was analyzed by univariate and multivariate Cox regression.4.LinkedOmics was used to analyze CDH23 expression-related genes and their prognostic value.The correlation between CDH23 and different immune cell infiltration and the tumor microenvironment was analyzed using the TIMER database.5.Using LinkedOmics,GO and KEGG enrichment analysis were used to explore the biological processes and molecular functions in which they may be involved,and further explore the possible signaling pathways of the gene in AML through GeneMANIA.6.Statistical analysis:Data analysis was used via R language(version 4.0.1).The data are expressed as mean ± standard deviation.Mann-Whitney U tests were performed to analyze group-to-group differences.P value less than 0.05 considered to be statistically significant.Results1.The expression value of CDH23 is related to age and genetic risk stratification,but not significantly related to other clinical pathological features.2.CDH23 was significantly highly expressed in bone marrow tissue samples of AML patients,while the high expression of CDH23 was inversely correlated with the overall survival of patients.3.Univariate and multivariate cox regression analysis showed that CDH23 expression levels,age,and cytogenetic risk stratification were independent prognostic factors affecting AML survival.4.The expression of CDH23 was positively correlated with the infiltration levels of monocytes,memory activated CD4+T cells,and regulatory T cells in the AML microenvironment,and was inverse with the infiltration ratios of eosinophils,resting mast cells,plasma cells and CD8+T cells.CDH23 expression levels were positively correlated with immune scores and stromal scores.5.CDH23 mediated tumorigenesis and development by mediating osteoclast differentiation,phagosome formation,lysosomal production,chemokine signaling pathway,endocytosis,NOD-like receptor signaling pathway,actin cytoskeleton regulation and other signaling pathways.ConclusionThis study found that CDH23 was upregulated in patients with AML and was correlated with adverse prognosis.The results showed that CDH23 was an independent prognostic factor predicting clinical outcomes in patients with AML.The expression of CDH23 affected the immune cell infiltration of AML microenvironment and was involved in the formation of the tumor immunosuppressive microenvironment.CDH23 regulated the tumor immune response as well as tumor metastasis by mediating the regulation of actin cytoskeletons.In summary,CDH23 may serve as a prognostic biomarker for AML and is expected to be a new target for the treatment of AML.