Study Of Anti-tumor Effects And Mechanisms Of Colorectal Cancer Stem Cell Vaccines With High MUC1 Expression

Colorectal cancer(CRC)is the third most common cancer in the world accompanied with high morbidity,high mortality,and CRC is easy to relapse.Though conventional treatments include surgery dissection,radiotherapy,and chemotherapy reveal anti-tumor effects,the recurrence and low 5-year survival rate remain to resolve.Therefore,novel potent therapeutics including immunotherapy,which could promote the anti-CRC immune responses along with extending patient survival,are urgently required.Colorectal cancer stem cell(CCSC)hypothesis posits a small number of cancer cells which possess characteristics of sternness such as self-renew,unlimited proliferation,multidirectional differentiation,invasion,metastasis,drug resistance.On account of driving CRC development and recurrence,CCSC have been considered as"seed" of the CRC and become the potential target for CRC treatment in recent years.CD133 was widely considered as the most common surface marker to sort CCSC from tumor cells.In comparison with non-CCSC,CCSC reveal stronger immunogenicity as evidenced by overexpressing some dominant antigens on the surface,which may elicit robust target clearance of CCSC,and inhibit tumor growth.Epithelial cell molecule mucin 1(MUC1)has been reported that it involves in proliferation,invasion,metastasis,drug-resistance and angiogenesis of CRC cells,and is tightly related to the poor prognosis of CRC patients.MUC1 molecule consists of N-terminal subunit(MUC1-N)and C-terminal transmembrane subunit(MUC1-C).MUC1-N is composed of the variable number tandem repeat(VNTR)and T cell receptor(TCR)and B cell receptor(BCR)recognition sites,which make it a promising target for vaccine design.In the present study,we used magnetic-activated cell sorting system(MACS)to sort CD133 positive cells from human colorectal cancer cell line SW620 and mouse colorectal cancer cell line CT26.After identifying the CD133 positive cells as the CCSC,we prepared the CCSC vaccine via repeating freezing-thawing method and investigated the anti-tumor effects and mechanisms.AimBased on the investigation of anti-tumor efficacy and mechanisms of CCSC vaccines with high MUC1 expression,we would like to provide a novel immunoprophylaxis strategy for CRC patients.Methods1.Isolation and identification of CCSC:CD133 positive cells were isolated from human CRC cell line SW620 and mouse CRC cell line CT26 via MACS.A series of assays including colongenic assay,migration assay,invasion assay,drug-resistance assay and tummorigenicity assay were performed to investigate whether CD 133 positive cells possess the characteristics of CCSC.2.Screening surface dominant antigens of CCSC:Based on online tool R2,Genomics Analysis and Visualization Platform(http://r2.amc.n1)was used to analyze the dataset entitled with“Tumor Colon(Core-Exon)-333-rma-sketch-huex10p" which was uploaded by Sveen.The expression level of CD133,the CD133-related genes,and the relative genetic oncology were assessed using the 333 colon tumor samples.MUC1 was demonstrated as co-expressed gene with CD 133 and involved in CRC metastasis and poor prognosis of CRC patients.shMUC1 lentivirus,MUC1 knockin lentivirus and MUC1 knockdown lentivirus were used to established the stable expression of MUC1 knockdown in SW620 cell line,the stable expression of MUC1 knockin in CT26 cell line,and the stable expression of MUC1 knockdown in CT26 cell line,respectively.qRT-PCR and Western Blot assays were used to evaluate the mRNA and protein expression levels of MUC1.Cologenic assay,migration assay and invasion assay were performed to investigate whether MUC1 expression would affect cologenicity,migration and invasion of CRC cells.3.Anti-tumor effects and mechanisms of SW620 CCSC vaccines with downregulated MUC1 expression:in the vaccination and tumorigenesis assay,shMUC1 CCSC and CCSC were isolated from shMUCl SW620 cells and wild type SW620 cells.shMUC1 CCSC vaccine,CCSC vaccine,SW620 vaccine,and non-CCSC vaccine were prepared via a repeated freezing and thawing method(-80℃,30 min,4℃,30 min,repeating three times).Thirty mice were randomly divided into 5 groups of 6 mice each with comparable mean body weight.The mice received subcutaneous(s.c.)vaccination in the right flank with 2×105 cell lysates of CCSC,shMUC1 CCSC,SW620 cells,and non-CCSC for three times,at an interval of 10 days between adjacent immunizations.All the immunized mice were challenged s.c.with 2×106 SW620 cells in 0.1 mL PBS in the right flank at 10 days after their final vaccination.The experimental groups in BALB/c-nu mice included the PBS,shMUC1 CCSC,SW620 cell,CCSC,and non-CCSC vaccinated groups.Tumor growth was monitored with digital caliper,and tumor volume was recorded as length×width2/2 mm3.Tumor-free mice information was recorded as well.Measurable masses in diameter>2 mm were defined as tumors and thus mice with measurable masses of tumor volume in diameter<2 mm were labeled as tumor-free mice.Mice were also monitored for the general health indicators such as body weight,appearance of fur,etc.Ten days after the third immunization with the 2×105 different vaccines,the blood samples of immunized mice were collected.Serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),blood urea nitrogen(BUN),creatinine,cardiac troponin I(CTnI),and creatine kinase MB(CK-MB),were respectively evaluated by ELISA assays according to the manufacturer’s instructions.White blood cells(WBCs)and platelets(PLTs)were counted by the Chemray 240 Automated Chemistry Analyzer.The lungs,livers,intestines,kidneys and heart tissues of immunized mice were harvested.Hematoxylin and eosin(H&E)staining was performed to determine the toxicity of different vaccines in the immunized mice.The tumor tissues were harvested from the mice immunized with the different vaccines at the 40 days after the 2×106 wild type SW620 cell challenge and CD133+CCSC population,ALDH+population,tumor infiltrated CD3-CD49b+NK cell population were analyzed by Calibur flow cytometer(BD biosciences).IFN-γ production of tumor infiltrated CD3-CD49b+NK cells was determined by ELISPOT assay.The mRNA levels of perforin and granzyme B in the tumor infiltrated immune cells were assessed by qRT-PCR.CD3CD49b+NK cells were isolated from splenocytes and used in the NK cytotoxicity assay,and Antibody Dependent Cell mediated Cytotoxicity(ADCC)assay.Complement Dependent Cytotoxicity(CDC)assay was conducted using 200 μL serum from immunized mice and 200 μL serum from cavy mice in the presence of 1×106 SW620 cells.CD19+B population and B220+CD38+CD80+memory B cell population in the splenocytes were determined.The levels of IFN-y and anti-MUC1 IgG antibody were detected using the immunized serum from the different vaccine groups.4.Anti-tumor effects and mechanisms of CT26 CCSC vaccines with regulated MUC1 expression:(1)CCSC,MUC1 knockin CCSC and MUC1 knockdown CCSC were isolated from CT26 cell line,MUC1 knockin CT26 cell line,MUC1 knockdown CT26 cell line,respectively.Thirty-six BALB/c mice were randomly divided into six groups(six per group)including PBS,non-CCSC vaccine,CT26 cell vaccine,CCSC vaccine,MUC1 knockin CCSC vaccine,and MUC1 knockdown CCSC vaccine groups.The mice were immunized with 2×105 different cell lysates subcutaneously in the right flank for three times with an interval of 10 days,and then were challenged with 2×106 wild type CT26 cells subcutaneously in the right flank at 10 days after the third vaccination.Tumor volume of each mouse was monitored every five days by digital caliper with two tumor diameters(length ×width2/2 mm3),and tumor-free mice information was recorded as well.Measurable masses in diameter≥2 mm were defined as tumors,and thus mice with measurable masses in diameter were labeled as tumor-free mice.Mice were also monitored for the general health indicators such as body weight,appearance of fur,etc.Splenocytes were collected at 40 days after the 2×106 wild type of CT26 cell challenge.CD 16+NKG2D+NKp46+activated NK cell population,CD4+T and CD8+T cell populations,CD45R+CD19+B cell population,B220+CD38+CD80+memory B cell population were respectively defined by FCM.NK cell cytotoxicity,splenocyte cytotoxicity,ADCC activity,CDC activity,CTL cytotoxicity were evaluated using FCM.The levels of IFN-γ,TGF-β1 and anti-MUC1 IgG antibody in immunized serum were detected by ELISA.IFN-γ production of CD8+T cells was determined by ELISPOT assay.The perforin and granzyme B mRNA levels of tumor infiltrated immune cells were assessed by qRT-PCR.Tumor tissues were harvested from the immunized mice and CD133+CCSC,ALDH+cells,CD4+CD25+Tregs,MAC1+GR1+MDSCs were investigated,respectively.(2)The specific anti-MUC1 antibody was added to the cell lysate to neutralize the dominant antigen MUC1.Different cell lysates were prepared by repeating freezing-thawing method,including neutralized CCSC vaccine,CCSC vaccine,non-CCSC vaccine,PBS and IgG isotype neutralized CCSC as control.Thirty BALB/c mice were divided into five groups including PBS,non-CCSC vaccine,CCSC vaccine,anti-MUC1 antibody neutralized CCSC vaccine,and IgG isotype neutralized CCSC control groups.Anti-tumor effects of CCSC vaccines were analyzed.Results1.The percentages of both CD133+SW620 and CD133+CT26 were more than 93%after sorting.The results indicated that,in comparison with non-CD133+cells,CD133+cells revealed stronger abilities in proliferation,migration,invasion,and drug resistance in vitro,as well as the stronger ability of tumorigenesis in mice.The differences were statistically significant(P<0.05).2.Within 333 CRC cases,CD133 expression was observed in all samples with the average expression score 1476.2.Moreover,there were an expression of 19487 genes significantly correlated with CD133 expression,among which 10088 genes showed a positive correlation and 9399 genes showed a negative correlation.Several genes showed positive correlation and took part in the processes of protein glycosylation,protein O-linked glycosylation and negative regulation of cell adhesion,which is linked to the CRC cell metastasis.MUC1 expression levels of mRNA and protein were significantly higher in CD133+cells than non-CD133+cells.shMUC1 SW620 cell line,MUC1 knockdown and MUC1 knockin CT26 cell lines were established.Overexpressed MUC1 could enhance the abilities of clonogenicity,invasion and migration of CCSC.3.The results of vaccination and tumorigenesis assay suggested that,compared with non-CCSC vaccine,SW620 cell vaccine and shMUC1 CCSC vaccine,CCSC vaccine showed the strongest anti-tumor effects as evidenced by inhibited tumor growth.CCSC vaccine group showed the strongest anti-tumor effects as evidenced by decreased CD133+CCSC and ALDH+cell populations,and increased tumor infiltrated CD3-CD49b+NK cell population in tumor tissues of immunized mice.Elevated NK cytotoxicity,ADCC activity,CDC activity,CD19+B cell population,B220+CD38+CD80+memory B cell population were also observed in CCSC vaccine group.In addition,the enhanced levels of IFN-y and anti-MUC1 IgG antibody were detected in the serum from the CCSC immunized mice.The differences between the CCSC vaccine group and other vaccine groups were statistically significant(P<0.05).The serum levels of ALT and AST,BUN and creatinine that represent liver function markers,and the levels of CTnI and CKMB that represent heart function markers,WBCs,and PLTs were all in normal range.The H&E staining showed that the morphology of lungs,livers,intestines,kidneys and heart tissue were all normal state in each vaccine group,indicating these vaccines did not produce any toxicity to above tissues.4.The results of vaccination and tumorigenesis assays indicated that,in comparison with non-CCSC vaccine and CT26 cell vaccine,CCSC vaccine showed more potent anti-tumor effects as evidenced by delayed tumor growth,smaller tumor size.CCSC vaccine group showed that the increased CD16+NKG2D+NKp46+activated NK cell population,CD45R+CD19+B cell population,and B220+CD38+CD80+memory B cell population,NK cytotoxicity,splenocyte cytotoxicity,ADCC activity,CDC activity,and CTL cytotoxicity.Moreover,the increased serum levels of IFN-y and anti-MUC1 IgG antibody and the decreased level of TGF-β1 were simultaneously observed in the immunized mice.Furthermore,the decreased populations of CD133+CCSC,ALDH+cells,CD4+CD25+Treg,and MAC1+GR1+MDSC were respectively detected in the tumor tissues.MUC1 knockdown partly impaired the inhibition of tumor growth;whereas MUC1 knockin enhanced these effects compared with other control vaccines,which were statistically significant(P<0.05).As expected,neutralizing antibody against MUC1 significantly impaired the anti-tumor efficacy of CCSC vaccine.The differences were statistically significant(P<0.05).Conclusions1.In comparison with non-CCSC,CD133+CCSC highly expresses MUC1 and its expression is positively correlated with the immune responses induced by CD133+CCSC vaccines.Therefore,MUC1 is a dominant target antigen of SW620 CD133+CCSC and CT26 CD133+CCSC vaccines.2.The enriched dominance antigen of MUC1 in CD133+CCSC vaccines could activate immune system and induce the target clearance of CCSC,resulting in inhibiting the CRC growth.MUC1 might be an essential antigen for CCSC vaccine to elicit anti-tumor immune responses.MUC1 knockdown could impair the anti-tumor effects of SW620 CD133+CCSC and CT26 CD133+CCSC vaccines;whereas MUC1 knockin could enhance the anti-tumor effects of CT26 CD133+CCSC vaccine.3.In the present study,our developed CD133+CCSC vaccines could elicit the vaccinated mice to generate potent anti-CRC efficacy,and this investigation provided a scientific experiment data for immunoprophylaxis of CRC in clinic.