| Colorectal cancer(CRC)is the third most common cancer in the world.The number of new cases per year in the world is about 1.4 million.The incidence rate is the third highest in solid tumors in both men and women,while the causes of death by cancer have risen to the 2nd and 3rd places,respectively [1].In recent years,the incidence of colorectal cancer has risen steadily,while China's rise rate is twice faster than the international average growth rate(2%),especially in developed cities such as Shanghai,where the incidence rate reaches the second highest level of solid tumors.It is threatening human health and quality of life seriously [2,3].Due to the occult occurrence of colorectal cancer,a considerable number of patients have had distant metastasis when they are firstly diagnosed [4].At present,for patients with advanced colorectal cancer,palliative surgery,neo-adjuvant chemotherapy and targeted drugs are the main treatment methods,but the efficacy and prognosis are not ideal because of the heterogeneity between patients and the emergence of drug resistance [5].In the past decade,cancer immunotherapy has attracted more and more attention.In 2012,it was evaluated by Science as one of the six areas of concern in the future [6];in 2013,it was named “Top one of the ten breakthroughs of the year” by Science [7];it also has continuously become the hotspot and focus of the American Society of Clinical Oncology(ASCO)meeting in 2015 and 2016,which represents the top level of the globle [8,9].Dr.Topalian was awarded as the "David A.Karnofsky Memorial Award of 2015" ——the most important award of ASCO,for her outstanding contributions to the study of PD-1 and PDL1 immunotherapy.Currently,a large number of basic and clinical studies have focused on cancer immunotherapy.Patients with CRC also catch hope from cancer immunotherapy,especially those with mismatch repair deficiency.They are much more sensitive to the PD-1 blockades than those with MSS [10].As we know,microsatellite instability(MSI)is one of the important causes of CRC,which refers to the addition or deletion of DNA repeats(microsatellites)caused by the mutations of mismatch repair genes such as MSH2,MSH6,MLH1,PMS1,and PMS2,which then lead to carcinogenesis [11,12].Due to the accumulation of mutations in the microsatellite sequence,frameshift mutations during protein translation,tumor cells produce a large number of abnormal polypeptide fragments that are more easily recognized by the immune system and stimulate the antitumor immune response of the host [13].That's why the patients with MSI benefit much more than those with MSS from PD-1 blockades.However,the frequency of MSI in sporadic CRC is only about 15% [14].For most CRC patients with MSS,it is difficult to have objective clinical response of PD-1 blockades.So,how to improve the efficacy in MSS has become an important problem.It has become the consensus of scientists worldwide that the combination of tumor immunotherapy and comprehensive therapy are still the ideal treatment mode for cancer.In the hundreds of years of fighting with cancer,people have realized that one therapy alone cannot overcome this complex disease.Only synergistic therapy targeting the different mechanisms,such as traditional radiotherapy,chemotherapy combined with immunotherapy,vaccine combined with immune checkpoint inhibitors,CAR-T cell therapy combined with immune checkpoint inhibitors,targeted drugs and immunotherapy,as well as the combination of tumor immunotherapy and gene therapy may we win the battle [15-19].Traditional surgery,radiotherapy and chemotherapy reduce the burden of the patients significantly.After that,suitable tumor immunotherapy would lead to the elimination of tiny residual lesions,inhibition of cancer cell proliferation,improvement of the prognosis and quality of life,prolong of the survival time and even cure the cancer.In-depth understanding of the interaction between tumors and immunity,taking advantages of various immunotherapy,and optimization of treatment programs have become the focus of the current research.Decitabine is an analog of adenosine that inhibits the methylation of DNA by inhibiting DNA methyltransferase,thereby inhibiting the proliferation of tumor cells,which has been widely used in chemotherapy for myelodysplastic syndrome(MDS)and acute leukemia.As a methylation inhibitor,it has been widely studied in solid tumors.Studies have shown that low dose DAC not only can significantly induce and increase the expression of tumor antigens,such as NY-ESO-1,MAGE-A3/6,but also can increase the expression of immune markers such as CD80,MHC-I molecules [20,21],and then increase the infiltration of immune cells into tumor site,which provide with a more appropriate immune microenvironment for immune checkpoint inhibitors,therapeutic vaccines,adoptive cell therapy and other immunotherapy.This study aims to explore the effects of low dose DAC on tumor biological characteristics and immunogenicity,as well as the re-modulation and mechanisms of tumor microenvironment,and discuss the relationship and synergistic effects with PD-1 blockades,NY-ESO-1 specific TCR-T cell therapy,providing with new strategies and scientific basis for the clinical treatment for MSS colorectal cancer.This study is divided into three parts:Part One: Effect of low dose decitabine on the tumor cellDNA methylation is a reversible gene modification that does not change the gene base sequence,and its methylation level is closely related to the "open" state of the gene.Based on the methylation role of low dose DAC,we conducted the DAC treatment of microsatellite stable(MSS)murine colon cancer cell line CT26 [22],and extracted the genomic DNA and total RNA.The characteristics of the tumor cell were detected and analyzed by whole genome methylation sequencing(WGBS)and whole transcription group sequencing(RNAseq).The results showed that the genomic methylation level of CT26 cells was significantly decreased after DAC treatment.The methylation levels of promoter regions of antigen processing and presenting genes,cytokine-related genes,chemokine and STAT gene,immune response related gene,MAPK signaling pathway gene and PI3K-AKT signaling pathway gene were significantly decreased.The results of the RNA-seq showed that the expression profile of CT26 cells changed markedly and found a large number of differentially expressed functional-related genes,including 180 up-regulated genes and 139 down-regulated genes.We focused on the m RNA expression of the genes indicated by the change of methylation level in WGBS results.The results showed that the expression of antigen-related genes,cytokine-related genes,chemokine-related genes,STAT genes,immune response-related genes,MAPK signaling pathway genes and PI3K-AKT signaling pathway genes changed significantly.It is noteworthy that the antigen processing and presenting genes were significantly increased.This indicates that the high methylation level in the gene promoter region could inhibit gene expression,and when the level of methylation is decreased,the gene expression is reactivated and the expression level increases.So,the expression level of the gene is related to the methylation level of promoter region.After DAC treatment,the level of genome methylation decreased significantly,the cell expression profile changed markedly,and the immunogenicity of tumor cells was improved.Further,we collected the fresh tumor tissue of the patients,and established the CRC PDX model on the severe immunodeficiency NCG mice,and identified the microsatellite status according to standards of the National Cancer Institute of America [23].We selected the MSS PDX model for DAC treatment,extracted the genome DNA and total RNA from the tumor tissue,and also carried out WGBS and RNA-seq detection and analysis.We found a similar phenomenon with CT26 cells after DAC treatment: The methylation level of PDX tumor cells was significantly decreased after DAC treatment.The methylation levels in the promoter regions of the immune-related gene and protein-modified gene were downregulated.The expression profile also changed markedly,with 89 genes significantly upregulated and 50 genes significantly down-regulated.The most important genes were antigen processing and presenting genes,immune-related genes and protein-modified genes,which were consistent with the low methylation level shown by WGBS results.This suggests that the low dose DAC has altered the characteristics of PDX tumors and increased its immunogenicity.In order to explore the effect of DAC on CRC cells with different microsatellite states,we selected and treated two MSI cell lines HCT116,LOVO and two MSS cells lines HT29,SW480,with different concentrations of DAC at 0 ?m,0.1 ?m,1 ?m,10 ?m.The total RNA,DNA and protein of the tumor cells were collected at different time after DAC exposure(1st day,3rd day,7th Day),and the expression levels of NY-ESO-1 antigen m RNA and protein of tumor cells were detected by q PCR,Western blot.Methylation of NY-ESO-1 promoter region in genomic DNA was detected by methylation sequencing.The results showed that low dose DAC could induce or improve the expression of NY-ESO-1 gene in CRC cell lines.In the MSS cell line,the NY-ESO-1 expression of HT29 and SW480 needs to be induced by a lower concentration DAC and shorter induction time.The NY-ESO-1 expression level of HT29 and SW480 is 20-30 folds higher than that of the untreated cells,significantly higher than the 5-10 times of the MSI cell.The level of methylation in NY-ESO-1 promoter region is closely related to the gene expression level.In the first part of the study,we confirmed that the genome methylation level of CT26 cells was significantly decreased after low-dose DAC treatment,and a large number of functional genes were remarkably up-regulated,which changed the immunological characteristics of the tumor and improved its immunogenicity.In the PDX tumor model,this phenomenon is verified again.The results of this part provide a theoretical basis for the next study.Part Two: Effect of low dose decitabine on tumor microenvironmentThe main problem that affects the efficacy of immunotherapy in solid tumors is the inhibitory immune microenvironment of tumor.How to break and re-modulate the tumor microenvironment and elite the anti-tumor immune response is the key to the breakthrough of solid tumor therapy.Immune microenvironment of the tumor microenvironment determines the effectiveness of immunotherapy [24-27],while the expression of immunerelated molecules and immune cell infiltration are the key components of tumor microenvironment.In the first part,we have confirmed that low dose DAC can change the characteristics of the tumor and improve its immunogenicity.Here,we have established a CT26 tumorbearing mice model.Low dose DAC was given by intraperitoneal injection,while PBS and Cytidine(cytosine analogues)were as control.The tumor tissues were collected after DAC treatment at different time(before,DAC-1 day,DAC-3 day,DAC-7 day,DAC-14 day).The effects of low dose DAC on tumor microenvironment were further explored by immunohistochemistry.We found that more and more T lymphocytes infiltrated to the tumor site.To further detection,more T lymphocyte infiltration in DAC group than control group,including CD4+ T lymphocytes and CD8+ T lymphocytes,and the number of IFN-? secreting cells was significantly higher than that in PBS and cytidine groups.Notably,we found that most of the infiltrating T lymphocytes were PD-1 positive,suggesting that the effect of DAC on TME may be inhibited by PD-1/PD-L1 pathways.In the second part of the study,we found that low-dose DAC can re-modulate the tumor microenvironment by recruiting more T lymphocytes,including CD4+ T cells,CD8+ T cells to infiltrate the tumor and IFN-? secreting cells,but most of these T cells are PD-1 positive,and their function may be suppressed through PD-1/PD-L1 pathway.Part Three: Synergistic Immunotherapy Strategies Based on DecitabineI.Synergistic strategy of decitabine and PD-1 blockadeStudies have shown that PD-1 monoclonal antagonists are essentially ineffective for MSS tumors [10].In the previous results of the study,we found that low dose DAC can change the immunological characteristics of tumor,improve its immunogenicity,and can recruit a large number of T lymphocytes infiltration to the tumor part,which may remodulate the tumor microenvironment.However,most of the T lymphocytes infiltrating into the tumor were PD-1 positive,so we followed up with a joint application of PD-1 blockade to explore the possibility of synergistic effects to improve efficacy.In this section,we inoculate the CT-26 cells with MSS to establish the tumor-bearing mice model.Separately,PBS,DAC alone,PD-1 blockade alone,and DAC+PD-1 blockade treatment were given.The results showed that the DAC+PD-1 blockade group had obvious inhibitory effect on tumor growth compared with PD-1 blockade group and DAC group,and the effects of prolonging the survival time of mice were also more significant.The results indicated that the pretreatment of DAC combined with PD-1 treatment could significantly inhibit tumor growth and prolong its survival in CT26-bearing mice.Considered the results of Part One,the mechanism may be that tumor cells modified by low dose DAC recruited more T lymphocytes into the local area of the tumor.The PD-1-positive T lymphocytes suppressed by PD-1 were reactivated by PD-1 blockade and then played an anti-tumor effect role.II.Synergistic strategy of decitabine and NY-ESO-1 specific TCR-T cellsIn the first part,we confirmed that low dose DAC could induce and improve the expression of NY-ESO-1 antigen in MSS tumor cells,providing with target for specific cellular immunotherapy.So,we explored the synergistic effect of DAC and NY-ESO-1 specific TCR-T cells.Firstly,we obtained the NY-ESO-1157-165(SLLMWITQC)specific TCR ?/? gene sequence from the literature and the database,and changed the TS of ? chain 95-96 bits to LY to improve the specific lysis function [28,29].The mutation of the ?-chain Thr48 and ?-chain Ser57 into Cys is beneficial to the formation of disulfide bonds and the success rate of their matching [30].TCR ?/? chain genes are linked by P2 A with "selfcleavege" functions,so that the translated products could be cut into ?/? chains for better match [30,31].The optimized sequence was cloned into a lentiviral vector.Subsequently,we isolated lymphocytes from human peripheral blood and detect the HLA-A2 phenotype due the MHC restriction of TCR.HLA-A2 strong positive lymphocytes were selected and activated by CD3+/CD28+ Dynabeads.These T lymphocytes were transduced with lentivirus after activation.The TCR expression was detected by anti-TCR V?13.1 antibody,and the specific binding affinity was detected by MHC-tetramer.The results showed that the transduction efficiency of specific TCR was 56%,and the positive rate of specific binding was 15%,which indicated that we had successfully prepared NY-ESO-1 specific TCR-T cells.In order to verify the specific recognition to the NY-ESO-1 epitope and cytokines secretion in vitro of the prepared TCR-T cells,the HLA-A2-restrcted NY-ESO-1157-165(SLLMWITQC)peptide and unrelated cognate peptide OVA257-264(SIINFEKL)were loaded into the T2 cells as target cells,and the TCR-T cells and the T2 cell loaded with peptides were co-cultured.The release of IFN-? in the supernatant was detected by ELISPOT.The results showed that TCR-T cells co-cultured with T2 cells loaded with NY-ESO-1157-165 secreted significantly higher IFN-? than those of T2 cells with OVA257-264.The results showed that the prepared TCR-T cells could recognize the NY-ESO-1 epitope of T2 cells and induce the secretion of cytokines IFN-?.Subsequently,we further validated the lysis effect of the prepared TCR-T cells in vitro.Tumor cells with different phenotype of HLA-A2 and NY-ESO-1 were selected as target cells: LOVO(NY-ESO-1-/HLA-A2-),MGC 803(NY-ESO-1+/ HLA-A2-)SW480(NY-ESO-1±/ HLA-A2+),A375 cells(NY-ESO-1++/ HLA-A2+)were as positive control,and according to the WB results in the first part,HCT116(NY-ESO-1+/ HLA-A2+)was induced by the 1?m DAC.The target cells were co-cultured with the TCR-T cells,and LDH assay was detected to evaluate the effect of TCR-T and ELISPOT for IFN-? secretion.The results showed that a large number of spots were formed in the wells of A375,HCT116,and the quantity was much higher than that of LOVO,MGC 803,and SW480.Accordingly,a large amount of LDH was detected in the wells of A375,HCT116,and its release level was much higher than that of LOVO,MGC 803,and SW480.So,TCR-T cells had a good killing effect on A375 and HCT116 cells.Experiments in vitro showed that TCR-T cells have a specific killing effect on NY-ESO-1+/HLA-A2+ cells,but there is no killing effect for NY-ESO-1 or HLA-A2 single positive or double negative cells.Then,we prepared the A375 tumor-bearing mice model,and administrated the TCR-T cells by tail vein,while the PBS and the empty virus transducted T cell(ctrl-t cell)as the control.We found that the prepared TCR-T cells could inhibit the tumor growth in mice significantly,and the survival time of mice was significantly prolonged.The results above confirmed that the NY-ESO-1 specific TCR-T cells could secreted IFN-? in vivo and in vitro specifically,and have anti-tumor effect on HLA-A2 restrictive NY-ESO-1 specific tumor.After the successful acquisition of NY-ESO-1 specific TCR-T cells,we further studied whether DAC and TCR-T cells had synergistic therapeutic effects.We treated the SW480 cells(NY-ESO-1±/HLA-A2+)with 0?m,0.1?m,1?m DAC,and co-cultured with the TCRT cells according to the different target ratios 10:1,20:1 and 40:1.The results showed that,compared with the SW480 group,the LDH release level in the DAC treated SW480 cells was much higher.ELISPOT results also showed that the level of IFN-? in supernatant increased markedly in DAC-treated SW480 compared with the SW480 group.Further,we have inoculated the SW480 cells treated with different concentrations DAC at 0?m,0.1?m,1?m on the severe immunodeficiency NPG mice,establishing the MSS SW480 tumor mice model.Then the TCR-T cells infusion therapy was performed once a week for totally three times,while the PBS and the virus transduced T cells(ctrl-t cells)were given at the same condition as control.The results showed that the DAC-untreated SW480 cells and the 0.1?m DAC-treated SW480 cells had a certain inhibition of tumor growth after the treatment of TCR-T cells,but the survival time was not prolonged.The inhibition effect of tumor growth and survival of mice of 1?m DAC-treated SW480 group were significantly inhibited and prolonged than other groups after the infusion of TCR-T cells.The above results showed that MSS tumor cells treated with appropriate concentration of DAC(1?m)could significantly improve their cytotoxicity to TCR-T cells,inhibit the growth of MSS tumors and improve survival,which confirmed that the combination of appropriate DAC and TCRT cells has synergistic anti-tumor effect.In this part,we confirmed that low dose DAC can improve the therapeutic effect of PD-1 blockade and NY-ESO-1 specific TCR-T cells in vitro and in vivo.The synergistic effect on the MSS tumor has been validated,which provides a novel strategy and theory basis for the MSS patients.In addition,DAC induced or improved the expression NY-ESO-1 and other tumor antigens specifically,which provided more targets for therapeutic vaccines,TCR-T cells and other antigen-based immunotherapy.This indicated that the DAC-based synergistic immunotherapy is expected to significantly improve the clinical efficacy of solid tumor.Results and Conclusions:1.Low dose DAC could significantly reduce the genome methylation level of CT26 cells and PDX model tumor cells with MSS,and improve the expression of antigen processing and presenting genes and cytokines genes of colorectal cancer cells.Low-dose DAC could induce and improve the expression of NY-ESO-1 in MSI-H and MSS CRC cells through promoter region demethylation of target genes.This shows that the low dose DAC changes the immunological characteristics of CRC cells and improves the immunogenicity.2.After the low dose DAC treatment,CRC tumor could recruit more CD4+ and CD8+ T lymphocytes infiltrated into the tumor local,larger number of IFN-? secreting cells.Notably,most of these infiltrating T lymphocytes showed PD-1 positive.This indicated that low dose DAC changed the CRC tumor microenvironment,but its state may be inhibited by the PD/PD-L1 pathway.3.The combination of low-dose DAC and PD-1 blockade could significantly inhibit the growth of CRC tumor CT26 in mice and prolong the survival time of mice.The results show that low dose DAC provides a good tumor microenvironment for the PD-1 blockade.And the PD-1 blockade activated the T lymphocytes inhibited by PD-1/PD-L1 pathway in the tumor microenvironment,and showed an anti-tumor effect.So,DAC and PD-1 blockade could play a synergistic role in improving anti-tumor efficacy.4.NY-ESO-1-specific TCR-T cells were successfully prepared by lentivirus transduction of human peripheral blood T lymphocytes,and functional verification was performed in vivo and in vitro.The prepared TCR-T cells have good specificity and anti-tumor effect on HLAA2 restrictive NY-ESO-1-positive tumors.5.The pretreatment of low dose DAC could improve the expression level of NY-ESO-1 in MSS colorectal cancer cells,and the combined application of TCR-T cells could obviously inhibit tumor growth and prolong the survival time of mice.The results showed that low dose DAC could provide more targets for TCR-T cells to play anti-tumor effect,and improve its therapeutic effect,which showed the synergistic effect. |
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