Research On The Mechnism Of ILC2s-derived IL-9 Regulating The Development Of Crc By Activating CD8~+ T Cells

Background:Colorectal cancer(CRC)is a kind of common gastrointestinal malignant tumor with no apparent early stage symptoms and serious systemic symptoms such as anemia and weight loss in the advanced stage.Surgical resection combined with chemotherapy and radiotherapy is the standard treatment for CRC.However,CRC patients frequently experience cancer recurrence,and the long-term survival rate is poor.Thus,in order to search the effective therapeutic strategies,in-depth study of the mechanism of development and progression of CRC is one of the important task of CRC treatment.In recent years,immunotherapy has been explored as a promising therapeutic option for CRC.Studies have shown that numerous immune cells modulate CRC progression by infiltrating the microenvironment of the CRC.Among these immune cells,GroupⅡinnate lymphoid cells(ILC2s)are abundant in the gut and play an essential role in regulating gut homeostasis.ILC2s,a subset of ILCs,is mainly present in the gut mucosa,lung,adipose tissue,and skin and is involved in the development of a great number of diseases including tumors by secreting typeⅡcytokines interleukin(IL)-4,IL-5,IL-9,and IL-13.but the role of ILC2s in CRC has not been elaborated.Objective:Detecting the regulatory role of ILC2s and ILC2s-derived IL-9 in the development of CRC,and further to investigate the regulatory mechanism of ILC2s in the development of CRC.Study on the effect and mechanism of IL-9 on the growth of CRC cells in vivo and in vitro.Exploring the method of sorting high-purity ILC2s,and investigating ILC2s chemokines in CRC microenvironment.Methods:1.The regulatory role and mechanism of ILC2s in the development of CRC(1)Constructing the AOM/DSS-induced CRC model in mice,and collecting tumor tissue specimens of CRC patients from hospital.Flow Cytometry(FCM)was used to analyze the levels of ILC2s in tumor tissue of CRC mice model and CRC patients.FCM was used to detect the level of IL-9 in mice CRC tumor tissue and the main IL-9-producing cell-subset in mice CRC tumor tissue.(2)Constructing the CT26 tumor-bearing mice model on nude mice,and anti-CD90.2 neutralizing antibody was used to knock out ILC2s in mice.Observing the growth of the tumors,FCM was used to detect the levels of ILC2s in tumor tissues,and RT-PCR was used to detect the expression of IL-9 in tumor tissues.(3)Mice spleen CD8~+T cells were sorted by immunomagnetic beads and transferred to untreated CT26 tumor-bearing nude mice and anti-CD90.2 pre-treated CT26 tumor-bearing nude mice.Observing the growth of the tumors in different groups,FCM was used to detect the levels of ILC2s in tumor tissues,RT-PCR was used to detect the expression of IL-9 in the tumor tissues of different groups.2.The effect and mechanism of IL-9 on the growth of CRC cells in vivo and in vitro(1)IL-9 protein was used to stimulate CRC cell line CT26 in vitro,FCM and Western blot were used to detect the apoptosis of CT26 cells,and MTT assay was used to detect the proliferation of CT26 cells.(2)CD8~+T cells in tumor tissue were sorted by immunomagnetic beads,and the purity was verified by FCM.IL-9 protein was used to stimulate the sorted CD8~+T cells,Enzyme-Linked Immunosorbent Assay(ELISA)was used to detect the level of Interferon-γ(IFN-γ)in the culture supernatant,and MTT assay was used to detect the proliferation of CD8~+T cells.(3)Sorted CD8~+T cells stimulated by IL-9 protein were co-cultured with CT26cells.The migration assay was used to analyze the migration ability of CT26 cells.The apoptosis of CT26 cells was analyzed by FCM.The proliferation of CT26 cells was observed by traditional counting methods.(4)Constructing the CT26 tumor-bearing mice model on BABL/c mice,IL-9was knocked out in mice with anti-IL-9 neutralizing antibody,FCM was used to detect the levels of ILC2s and CD8~+T cells in the tumor tissue,and observing the growth of the tumors.3.Isolation of ILC2s from mice spleen(1)Immunomagnetic beads and flow cytometry were used to sort the ILC2s in mice spleen.Firstly,the immunomagnetic beads was used to enrich the lineage-negative cells in mice spleen,and then flow cytometry was used to sort out the lineage~-CD90.2~+KLRG1~+cells,which was considered as mice ILC2s.FCM was used to detect the purity of the sorted ILC2s.(2)The sorted ILC2s were co-cultured with the sorted tumor-infiltrating CD8~+T cells,with or without anti-IL-9,the level of IFN-γin the supernatant was detected by ELISA and the proliferation of CD8~+T cells was detected by MTT assay.4.Investigating ILC2s chemokines in CRC tumor microenvironment.FCM was used to detect the levels of ILC2s in the spleen of CRC mice and the peripheral blood of CRC patients.Gene Expression Profiling Interactive Analysis(GEPIA)was used to explore the expression levels of ILC2s chemokines CCL25 and CXCL16 in the CRC tumor tissue.Reverse Transcription Polymerase Chain Reaction(q RT-PCR)was used to detect the expression levels of CCL25 and CXCL16 in tumor tissues and paracancer of CRC mice and CRC patients.Results:1.ILC2s is increased significantly in tumor tissue of CRC mice and CRC patients compared to paracancer.The expression level of IL-9 is also significantly up-regulated in CRC tumor tissue.ILC2s is the main IL-9-producing cell-subset(52%)in the CRC tumor microenvironment.2.IL-9 protein has no direct effect on the apoptosis and proliferation of CT26cells in vitro.IL-9 can promote the proliferation of sorted CD8~+T cells and activate CD8~+T cells up-regulating the secretion of IFN-γ.The migration and proliferation of CT26 cells can be inhibited when co-cultured with IL-9 activated CD8~+T cells,which can also promote the apoptosis of CT26 cells.3.FCM results show that we isolate a high purity ILC2s from mice spleen.The sorted ILC2s co-culture with tumor-infilitrating CD8~+T cells can obviously activate CD8~+T cells and up-regulate the secretion level of IFN-γ,and anti-IL-9 can reverse this effect.4.In vivo experiments show that IL-9 can significantly inhibit the tumor growth in CT26 tumor-bearing mice,while anti-IL-9 promotes the tumor growth.IL-9up-regulates the levels of ILC2s and CD8~+T cells in CT26 tumor-bearing mice tumor tissue,while anti-IL-9 down-regulates the levels.Anti-CD90.2 down-regulates the levels of ILC2s and IL-9 in CT26 tumor-bearing nude mice,but has no effect on tumor growth.After adoptive transfer of CD8~+T cells in anti-CD90.2 pre-treated CT26 tumor-bearing nude mice,the tumor growth is significantly increased compared with anti-CD90.2 untreated mice.5.The levels of ILC2s are significantly down-regulated in the spleen of CRC mice and the peripheral blood of CRC patients.GEPIA results show that the ILC2s chemokine CXCL16 significantly up-regulates in the CRC tumor tissue,while CCL25does not change.The q RT-PCR results also show that CXCL16 significantly up-regulates in the CRC mice and CRC patient tumor tissue,while CCL25 does not change.Conclusion:In CRC,up-regulated CXCL16 in CRC tumor microenvironment can recruit ILC2s to CRC tumor tissue and activate CD8~+T cells by secreting IL-9,thereby affecting the progression of CRC.