Study On The Role Of CD30L/CD30 Signal Transduction In The Development And Immune Microenvironment Of Colitis-associated Colorectal Cancer

Objective:Colorectal cancer?CRC?is one of the most common gastrointestinal cancer.In recent years,CRC has gradually become the third malignancy incidence in the world.At present,there are more than one million new CRC patients in the world every year,and the number is still increasing year by year.Colitis-associated colorectal cancer?CAC?is a subtype of CRC.It is difficult to cure and has a high mortality rate,which brings great challenges to medicine.At present,the pathogenesis of CAC is not completely clear,so it is very important to explore the pathogenesis of CAC.CD30 and CD30 ligands?CD30 ligand,CD30L,CD153?belong to the members of tumor necrosis factor receptor superfamily?tnfrsf?and tumor necrosis factor superfamily?TNF?,respectively.They are type II membrane associated glycoproteins.In our previous work,CD30L or CD30 gene knockout mice were used to investigate the expression of CD30L and CD30 on intestinal mucosal immune cells and the effect of CD30L and CD30on the development of intestinal inflammation in mice with IBD.It is proved that CD30L/CD30 signal plays an important role in the formation mechanism of IBD intestinal inflammatory lesions.It is suggested that CD30L/CD30 signal may play a potential role in the transformation of"inflammation cancer"and the formation of tumor inflammatory microenvironment.At the same time,previous studies on CD30L and tumor were mostly limited in the field of lymphoid or hematogenous tumors,and the role of CD30L/CD30signal transduction in the pathogenesis of CAC has been reported.In order to study how CD30L/CD30 signal regulates tumor microenvironment and affects the occurrence and development of CAC,this study used CD30L gene knockout mice to establish colitis-related colorectal cancer CAC model,and preliminarily explored the effect of CD30L gene deletion on the formation of CAC immune microenvironment and tumor progression in mice,so as to further study the mechanism of CD30L in the occurrence and development of solid organ tumors provide important theoretical basis.Methods:1.Establish C57BL/6 mice CAC animal model by AOM and DSS:take colorectal and intestinal tumor tissues of mice,extract total protein,detect the expression changes of CD30L and CD30 protein in sample tissues by Western Blot,extract RNA,detect the mRNA expression changes of CD30L and CD30 in sample tissues by real time PCR.2.Establish CAC animal model with WT and CD30LKO mice:dynamically compare and observe the changes of weight,survival rate,intestinal length,number of tumors at different levels,tumor size,volume and weight of spleen during the formation of CAC lesions in mice caused by CD30L gene deletion.At the same time,the intestinal tumor changes of WT and CD30LKO mice before and after inducing CAC were detected by mice endoscope,and the histopathological changes of mice intestinal tract were detected by HE staining technology.3.The mRNA expression of tumor suppressor gene,oncogene,apoptosis suppressor gene,proinflammatory cytokine and chemokine in WT and CD30LKO mice intestinal tumor tissues were detected by real time PCR.4.Flow cytometry was used to detect the expression of PD-L1,MDSCs,TAMs and PD-L1 in the tumor immune microenvironment of WT and CD30LKO mice.The changes of cytokines secreted in tumor immune microenvironment were detected by Enzyme-linked immunosorbent assay.5.Flow cytometry was used to detect the expression of CD30 and CD30L of CD4⁺T and CD8⁺T cells in tumor immune microenvironment of mice in the control group and the induced CAC group after TCR stimulation for 0,16 and 32 hours;6.Flow cytometry was used to detect the expression of cytokines IFN-?,TNF-?,IL-17A,IL-2,differentiation related transcription factors ROR?T,Tbet,surface expression inhibitory molecule PD-1 and proliferation marker Ki-67 secreted by CD4⁺and CD8⁺T cells in the tumor immune microenvironment of WT and CD30LKO mice.The levels of IFN-?,TNF-?,IL-17A and IL-2 were detected by Enzyme-linked immunosorbent assay.Results:1.Compared with normal control group,the expression of CD30L and CD30protein in intestinal mucosa after CAC was induced to increase significantly.mRNA levels of CD30L and CD30 in intestinal tumor tissues and adjacent tissues of CAC mice were also significantly higher than those in distal intestinal tissues.That is,after inducing CAC intestinal tumor,the expression of CD30L and CD30 in mouse intestinal mucosa tissue increased abnormally,suggesting that CD30L/CD30 signal transduction may play a potentially important role in CAC lesion formation.2.Compared with WT mice,the weight and survival rate of CD30LKO mice are significantly reduced,and the number of intestinal tumors is significantly increased.Histopathological analysis shows that the muscularis in the intestinal mucosa of CD30LKO mice are significantly thickened,the gland structure is more incomplete,the number of severe atypical hyperplasia is more,and the inflammatory cell infiltration is significantly increased.The experimental results fully prove that CD30L gene deletion leads to significantly increased susceptibility to CAC and increased tumor burden in mice,suggesting that CD30L/CD30 signal transduction may play an important anti-tumor role in CAC lesion formation by regulating the formation of intestinal tumor immune microenvironment.3.Loss of CD30L leads to down-regulation of expression of tumor suppressor genes APC and p53,down-regulation of expression of apoptosis suppressor genes Bcl-xl and Bcl-2,and up-regulation of expression of vascular endothelial growth factor in intestinal tumor tissue.The expressions of cytokines IL-17A,Granzyme B,perforin,TNF-?,IFN-?and IL-2 mRNA,which are closely related to T cell activation and differentiation,are significantly down-regulated,while the expression levels of inflammatory cytokines mRNA such as IL-1?,IL-6 and IL-10 are significantly increased.The expression of chemokines CXCL1 and CXCL2 was up-regulated and CCL2 was significantly down-regulated,while the expression of CCL5 and CCL20 had no significant difference.In CAC intestinal mucosa tissue,CD30L deletion also resulted in significant down-regulation of IL-17A,Perforin,Granzyme B,TNF-?,IFN-?and IL-2 mRNA expression,while the expression of IL-10 and IL-6 were significantly up-regulated.The results suggest that the deletion of CD30L gene can lead to the transformation of intestinal tumor immune microenvironment in CAC mice to a direction favorable for tumor occurrence and development,thus promoting the formation of CAC lesions.4.After WT mice induced CAC,the proportion of MDSCs cell in intestinal mucosa lamina propria increased significantly,the expression of PD-L1 on its surface increased significantly,and the expression of CD30L also increased significantly.The results suggest that the deletion of CD30L may affect the differentiation of MDSCs.Compared with WT mice,after inducing CAC,the proportion of MDSCs in intestinal tissue of CD30LKO mice was significantly increased,and the proportion and quantity of G-MDSCs and M-MDSCs were both increased.PD-L1 expressed on MDSCs surface was significantly enhanced.Although CD30L deletion did not affect the proportion and quantity of TAM,it significantly increased the expression of MHCII⁺TAMs and MHCII^-TAMs cells PD-L1.Compared with WT mice,IL-1?,IL-6 and IL-10 in LPL culture supernatant of CD30LKO mice were significantly increased.The above results suggest that CD30L may promote the anti-tumor immune response mediated by CD30⁺effector T cells by inhibiting the accumulation of MDSCs in intestinal tumor immune microenvironment and the expression of MDSCs and TAMs cell surface immune checkpoint–PD-L1.5.Fresh CD4⁺and CD8⁺T-LPL cells can express low levels of CD30L and CD30.After 16 or 32 hours of culture in vitro,the expression of CD30L and CD30 on the surface of activated CD4⁺and CD8⁺T cells gradually increases.The expression of CD30L and CD30 on the surface of CD4⁺and CD8⁺T cells in intestinal mucosa lamina propria of CAC mice was significantly enhanced compared with that of normal mice.Moreover,TCR stimulation can significantly increase the intensity of CD30L and CD30 expression on CD4⁺and CD8⁺T cells.The results suggest that CD30L and CD30 regulate the differentiation and effect function of CD8⁺and CD4⁺T cells in intestinal mucosa lamina propria through intermolecular interaction,thus playing an important protective role in CAC intestinal tumor occurrence and tumor immune microenvironment formation.6.After CAC induction,CD4 TEM in intestinal mucosa lamina propria lymphocytes?LPL?of CD30LKO mice decreased significantly,and the expression intensity of PD-1 on CD4 T_EM surface in LPL and mesenteric lymph nodes?MLN?increased significantly.IFN-?⁺,TNF-?⁺,IL-17A⁺,IFN-?⁺TNF-?⁺,TNF-?⁺IL-17A⁺,IL-2⁺CD4⁺T cells decreased significantly.Moreover,the deletion of CD30L results in significant inhibition of the expression of nuclear transcription factors ROR?t and Tbet.The expression of IFN-?and Ki-67 in CD44⁺CD4⁺T cells also decreased significantly.ELISA results showed that the levels of IL-17A,IFN-?,TNF-?and IL-2 secreted by CD30LKO mice decreased significantly.In addition,CD8 T_EM in LPL of CD30LKO mice decreased significantly after CAC induction,and the expression intensity of PD-1 on CD8 T_EM surface in LPL and MLN increased significantly,but the expression of Ki-67 decreased significantly.Moreover,the expression of IFN-?,Perforin,Granzyme B and Ki-67 in effector CD44⁺CD8⁺CTL cells were significantly down-regulated.The results showed that CD30L gene deletion inhibited the activation,differentiation and proliferation of CD4⁺Th cells and CD8⁺CTL cells in the intestinal lamina propria of mice,further inhibited the anti-tumor immune response mediated by T cells in the intestinal tumor immune microenvironment,and finally led to significantly increased susceptibility of mice to CAC.Conclusion:1.The expression of CD30L and CD30 was high in mice after inducing CAC.2.CD30L gene deletion can increase the sensitivity of mice to CAC and aggravate the tumor of CD30LKO mice.3.CD30L gene deletion increased the accumulation of MDSCs,increased the expression of PD-L1,and transformed the immune microenvironment of intestinal tumor into a favorable direction for tumor development.4.CD30L/CD30 signal deletion can inhibit the activation,differentiation and proliferation of CD4⁺and CD8⁺T cells in intestinal lamina propria,inhibit the anti-tumor immune response mediated by T cells in intestinal tumor immune microenvironment,and promote the development of CAC.