Effect Of Anti-CD20 Monoclonal Antibody Therapy On Lung Adenocarcinoma In Mice

ObjectiveThis study aimed to clarify the anti-tumor effects of anti-CD20 monoclonal antibody treatment on LA795 cell line subcutaneous tumor bearing model T739 mice.Furthermore,the effects of anti-CD20 monoclonal antibody treatment in the different stages of tumor development,intestinal flora and related immune cells were explored.MethodsT739 mice aged 8-10 weeks were randomly divided into three groups:pre-tumor-treated anti-CD20 monoclonal antibody group;post-tumor-treated anti-CD20 monoclonal antibody group;control group.All animals were subcutaneously injected lung adenocarcinoma cell line LA795.Anti-CD20 monoclonal antibody demonstrated via tail vein before and after the tumor,the control group was treated with the same dose of PBS.Each group plotted tumor growth curves to record the volume and mass of tumors.HE staining and immunofluorescence technology were used to observe the structure of subcutaneous tumor tissue and the distribution of T cells.Meanwhile,flow cytometry assay was used to detect the ratio of peripheral blood to B cells in the spleen of mice in different subcutaneous tumor-bearing groups,and the proportion and activation status of B cells,T cells and NK cells in the drainage lymph nodes of subcutaneous tumor-bearing mice.Then,based on the sequencing technology while synthesizing,the Illumina platform was used to perform high-throughput sequencing of c DNA in tumor tissues,and the functional annotation and enrichment analysis of differentially expressed genes was carried out.Finally,based on the Illumina Novaseq sequencing platform,a small fragment library was constructed to analyze the differences of mouse intestinal flora using the method of double-terminal sequencing.Results1.Establishment of lung adenocarcinoma tumor-bearing mouse model:T739 mice were injected subcutaneously with 1x106 LA795 mouse lung adenocarcinoma cells,and tumor growth was visible subcutaneously;HE staining of the tumor tissue was observed,massive cells tightly arranged with large deep-stained nuclei were visible under the microscope.2.The effect of anti-CD20 monoclonal antibody on the growth of lung adenocarcinoma in mice injected at different times:(1)The tumor volume of mice demonstrsted with anti-CD20 monoclonal antibody before tumor formation was significantly smaller than that of mice in the control tumor formation group(P < 0.01),while the tumor volume of post-tumor-treated anti-CD20 monoclonal antibody group was not significantly different from that of the control group(P > 0.05).(2)The HE staining results showed that compared with the control group,the tumor cells in the pre-tumor-treated group were obviously necrotic,showing nuclear rupture and solidification,and the internal structure of the tumor was looser,while there was no obvious necrosis in the post-tumor-treated group.3.The immunofluorescence results showed that the infiltration of CD4+T cells in the pre-tumor-treated group before was significantly increased,and CD4+T and CD8+ T cells showed colocalization,which was conducive to the mutual activation of the two cell types.4.Effect of anti-CD20 monoclonal antibody injection on peripheral blood and spleen: T739 mice injected with anti-CD20 monoclonal antibody in the tail vein had significantly decresing B cells in peripheral blood and spleen than wild-type mice(P<0.001).5.Effect of anti-CD20 monoclonal antibody treatment on drainage lymph nodes in pre-tumor-bearing mice: lymphocyte types were detected by flow cytometry.The results demonstrated that B cells were significantly reduced in the drainage lymph nodes in the pre-tumor-treated anti-CD20 antibody group,compared with control group,furthermore,CD4+T cells and IFN-γ+CD8+T cells increased significantly(P<0.05),but there was no significant change in NK cells,these results indicated that pre-tumor-treated anti-CD20 monoclonal antibody could reduce the number of B cells and promoted the activation of T cells.6.Effect of pre-tumour injection of anti-CD20 monoclonal antibody on gene expression in tumour cells: transcriptome sequencing revealed that the gene expression profile of tumour cells in the pre-tumour injection group differed significantly(P<0.05)from that of tumour cells in control tumour-bearing mice.These differential genes were mostly enriched in molecular functions such as chemokine activity,metal chaperone activity and chemical rejection activity,and affected pathways related to diseases such as primary immunodeficiency and autoimmune diseases,the B-cell receptor signalling pathway and the NF-κB cell signalling pathway.7.Intestinal flora detection in Anti-CD20 monoclonal antibody treatment on in pre-tumor-treated mice: According to 16 Sr RNA gene detection,the intestinal flora of pre-tumor-treated antibody group was significantly different from that of control group.The proportion of Bacteroides decreased and the proportion of Proteobacteria and Actinomycetes increased.Furthermore,the abundance of metabolic pathways for in vivo replication and repair,nucleotide metabolism and resistance to antimicrobials decreases.The pathway abundance of specific cancers,biodegradation of exogenous substances and amino acid metabolism increased(P<0.05).ConclusionsThe tumor volume and mass of subcutaneous tumor-bearing mice was significantly reduced compared with the group that was treated with anti-CD20 monoclonal antibody before tumor charge compared with the group that gave monoclonal antibody treatment after tumor charge.In addition to the changes in the tumor itself,we also found that pre-tumor anti-CD20 monoclonal antibody therapy can deplete B cells in a short period of time and promote the activation of CD4+T and CD8+T cells,which can regulate primary immunodeficiency,autoimmune diseases and NF-κB pathways.At the same time,the abundance of intestinal flora was changed,that is,the proportion of Bacteroides decreased,and the proportion of Proteobacteria and Actinomycetes increased.This result shows that the administration of anti-CD20 monoclonal antibody therapy early in tumor development may be a synergistic anti-tumor effect through the immune system,intestinal flora and direct action of antibodies.