The Role And Mechanism Of Adhesion Molecules In Promoting Enhanced CIK Cells To Resist Renal Cell Carcinoma

Objective:Renal cell carcinoma(RCC)is the most common malignant tumor of adult kidney,accounting for 2%of all malignant tumors.The incidence rate of RCC is third in the urologic malignancies after bladder cancer and prostate cancer,but the mortality rate is the highest.At present,surgical resection is the gold standard for the treatment ofrenal cell carcinoma without metastasis.However,the incidence of renal cell carcinoma is occult,and there are almost no clinical symptoms in the early and middle stages of RCC.About 30%of the RCC cases have been confirmed with local invasion or distant metastasis at the time of diagnosis.In addition,for those RCC patients undergoing surgery,the local recurrence rate can still reach 20%-30%within 3 years after surgery.Unlike most malignancies,renal cell carcinoma is not sensitive to radiotherapy and chemotherapy.On this account,the biological immunotherapy plays important role in adjuvant therapy for renal cell carcinoma.Cytokine induced killer cells(CIK cells)is a kind of cells with anti-tumor activity activated by cytokines IL-2,OKT3 and IFN-y.As an effective treatment of non-specific cellular immunotherapy for tumors,CIK cells have achieved exact therapeutic effect in experiments in vivo and in vitro or clinical researches.As yet,it is unclear how mononuclear cells acquire anti-tumor activity and the most important point is whether the standard preparation process of CIK cells is an efficient strategy,how to maximize the acquisition of CIK cells with high anti-tumor activity in the shortest time.Therefore,how to improve the proliferative activity and anti-tumor effect of CIK cells and figuring out its intrinsic mechanismis is still the crucial technical and scientific problem to be solved in the application of CIK cells.In the conventional preparation process,IFN-γ,IL-2 and CD3 are the main cytokines used to stimulate the PBMC to transform into CIK cells.However,in order to improve the CIK cells,some studies have tried to add different cytokines,such as IL-1,IL-17 and IL-12,in order to obtain more optimized and efficient CIK cells.IL-15 is a T cell activating factor homologous to IL-2 structure,which can effectively promote the activation and proliferation of T cells and NK cells,In addition,IL-15 can also convert activated T cells into memory T cells before reaching the effector cell stage,and promote the persistence of immune response by maintaining the long-term survival of memory T cells in vivo.Based on this special regulatory activity of IL-15 on T cells,we used IL-15 in the induction process of CIK cells additionally to obtain enhanced CIK cells(CIKIL-2+IL-15),and achieved good antitumor effect in the previous research.In this study,we will focus on the effect and mechanism of enhanced CIK cells on RCC,The proliferative efficiency and anti-tumor activity of CIKIL-2+IL-15 cells against RCC cells was tested by in vitro and in vivo experiments.Furthermore,the transcriptome sequencing was conducted to explore the key genes that regulating the CIKIL-2+IL-15 cells.Then,the interaction and signal transduction pathway of those key genes was systematically studied to further clarify the anti-tumor mechanism of CIKIL-2+IL-15 cells.Finally,the preliminary clinical study of CIKIL-2+IL-15 cells in the treatment of renal cell carcinoma was carried out to provide scientific basis and technical support for the clinical application.Methods:Part Ⅰ:Proliferative efficiency and anti-tumor activity of CIKIL-2+IL-15 cells against RCC:1.General CIK cells(CIKIL-2)and enhanced CIK cells(CIKIL-2+IL-15)were prepared by conventional and improved methods respectively.The proliferative efficiency of the two kinds of CIK cells were compared,and the clustering of CD3+,CD8+and CD56+cells of the two kinds of CIK cells were detected by flow cytometry.2.The antitumor activity and mechanism of CIKIL-2+IL-15 cells against RCC were studied in vitro and in vivo.In vitro experiments:CCK8 assay was used to test the cytotoxic activity of CIKIL-2 and CIKIL-2+IL-15 cells against RCC cells ACHN.The cytokines secreted by this two kinds of CIK cells were verified by ELISA.The ultra microstructure of enhanced cells killing ACHN cells was observed by electron microscope.In vivo experiments:The mouse model of xenograft tumor was established by subcutaneous injection of ACHN tumor cells.CIKIL-2+IL-15 cells were injected intratumorally and intravenously to verify its anti-tumor activity against RCC in vivo.The tumor tissues were stained with immunofluorescence(TUNEL,Ki-67 and CD3)to understand the local CIK cell infiltration and ACHN cell proliferation or apoptosis.Part II:Study on the intrinsic mechanism of CIKIL-2+IL-15 cells against RCC:1.The RNA of CIKIL-2+IL-15 cells and CIKIL-2 cells were extracted and sequenced.The key genes differentially expressed in CIKIL-2+IL-15 cells(adhesion molecules CD2,CD1la and CD56)were screened by GO(gene ontology)and KEGG enrichment analysis.2.siRNA interference was carried out to obtain enhanced CIK cells with low expression of CD2,CD11a and CD56(CIKCD2-low,CIKCD1la-low and CIKCD56-low).The RNA of CIKCD2-Iow,CIKCDC11a-Iow and CIKCD56-Iow cells were sequenced.The common differential genes of this three kinds of CIK cells were screened by Venn analysis from the sequencing data and analyzed by GO and KEGG function enrichment.Then the results indicated that the chemokines CXCL16,CXCR6,CCL5,CCR4 may be related to the immune regulation of CIKIL-2+IL-15 cells.The transcriptional level of the above chemokines in CIKCD2-Iow,CIKCD11a-low,CIKCD56-Iow and CIKIL-2+IL-15 cells were verified by qRT-PCR.And through PPI(protein protein interaction)analysis,it was finally revealed the signal pathways and key functional protein molecules that adhesion molecules CD2,CD11a and CD56 exert regulatory activities jointly in CIKIL-2+IL-15 cells.3.The difference of antitumor activities and cytokines level between CIKCD2-low,CIKCD11a-low,CIKCD56-low and enhanced CIK cells against ACHN cells was studied in vitro and in vivo to verify the regulatory activities of CD2,CD11a and CD56 in enhanced CIK cells.Part Ⅲ:The clinical study of CIKIL-2+IL-is cells in the treatment of renal cell carcinoma:11 patients with renal cell carcinoma were treated with CIKIL-2+IL-15 cells and compared with the other 9 patients without CIK cells treatment.The clinical anti-tumor efficacy of CIKIL-2+IL-15 cells was further observed by comparing the overall survival(OS),progression free survival(PFS)and CT imaging changes between the two groups.Results:Part Ⅰ:The proliferative efficiency of CIKIL-2+IL-15 cells was significantly higher than that of CIKIL-2 cells,and the proportion of CD3+CD56+cell subsets in CIKIL-2+IL-15 cells was also significantly higher.The CIKIL-2+IL-15 cells showed stronger anti-tumor activity against renal cell carcinoma in vitro and in vivo.In vitro,CIKIL-2+IL-15 cells exerted significantly stronger cytotoxic activity against renal cell carcinoma cells ACHN.The level of cytokine granzyme B,TNF-α and IFN-γ is also higher.Electron microscope scanning showed that the CIKIL-2+IL-15 cells wrapped ACHN cells through pseudopodia and adhered closely to them.In vivo,the anti-tumor activity of CIKIL-2+IL-15 cells in nude mouse models of RCC was enhanced.The decrease proliferation and increase apoptosis of RCC cells and infiltration of CIKIL-2+IL-15 cells were observed by immunofluorescence staining in site.Part Ⅱ:Transcriptome sequencing data showed that the transcriptional expression of adhesion molecules CD2,CDlla and CD56 was up-regulated in CIKIL-2+IL-15 cells.CIKCD2-low,CIKCD11a-low and CIKCD56-low cells were successfully obtained by siRNA interference.After Venn analysis of the transcriptome sequencing results of CIKCD2-low,CIKCD11a-low and CIKCD56-low cells,then through the functional enrichment analysis of Metascape,it was found that adhesion molecules CD56,CD1la and CD2 regulate the anti-tumor activity of CIKCD56-low cells by interacting with chemokines CXCL16,CXCR6,CCL5 and CCR4.PPI network analysis suggested that the above adhesion molecules and chemokines exert their biological activities through PI3K/Akt and MAPK signal transduction pathways.In vitro experiments confirmed that the anti-tumor activity of CIKCD2-low,CIKCD11a-low and CIKCD56-low cells against ACHN and six other common adult malignant tumor cells was significantly weakened,and the levels of IFN-γ and perforin decreased.At the same time,we verified that CIKCD2-low,CIKCD11a-low and CIKCD56-low cells had less inhibitory effect on the tumorigenesis of ACHN cells in nude mice.Part III:In the clinical study,11 patients with renal cell carcinoma treated with enhanced CIK cell adjuvant therapy after surgery were compared with the other 9 patients treated with surgery only.It was found that there was no significant difference in progression free survival(PFS)between the two groups,but enhanced CIK therapy can stabilize pulmonary metastasis,prolong the tumor bearing survival time of patients,and then prolong the overall survival time(OS).Patients receiving enhanced CIK were well tolerated and had no significant adverse reactions during treatment.Conclusions:1.Enhanced CIK cells(CIKIL-2+IL-15)showed higher proliferation efficiency and anti-RCC activity than CIKIL-2 cells It mediated tumor cell apoptosis by cytokines IFN-γ,TNF-α,Perforin and Granzyme,inhibited the survival and growth of RCC in vitro and in vivo.Meanwhile,we also observed that the cell adhesion between enhanced CIK cells and ACHN cells under electron microscope,this may be part of the anti-tumor activity of enhanced CIK cells.2.The adhesion molecules CD2,CD11a and CD56 enhanced the anti-tumor activity of CIKIL-2+IL-15 cells.Inhibiting the transcription and expression of CD2,CD11a and CD56 could weaken the anti-tumor activity of CIKI-2+IL-15 cells and reduce the secretion of cytokine IFN-y and Perforin.The regulatory activity of adhesion molecules CD2,CD1la and CD56 in CIKIL-2+IL-15 cells was based on the interaction with chemokines CCR4,CCL5,CXCL16 and CXCR6 through PI3K/Akt and MAPK signal transduction pathway to mediate the homing of CIK cells,drive CIK cells recognizing and adhering to tumor cells,then exerting anti-tumor immune activity.3.In the clinical study of renal cell carcinoma,CIKIL-2+IL-15 cells adjuvant therapy can delay the progression of tumor,stabilize pulmonary metastasis,prolong the survival time of patients with tumors,and then prolong the overall survival time(OS).Thus CIKIL-2+IL-15 cells treatment is a safe and effective choice for adjuvant therapy of RCC.