Immune Effect Of Mucin 1 Gene Transfected Dendritic Cells On Breast Cancer And Evaluation Of Curative Effect By Optical Molecular Imaging

The incidence rate of breast cancer worldwide has been increasing since the end of 1970s.It has become the leading malignancy of women in China.It shows a younger trend.Moreover,young women have a high local invasion,a poor pathological grade and a poor prognosis.Surgery,chemotherapy,radiotherapy and endocrine therapy are the four traditional treatment methods for breast cancer.Since the beginning of this century,biological therapy,the fifth major treatment of breast cancer,has been paid more and more attention.Immunotherapy is an important part of biotherapy.However,the development of immunotherapy for breast cancer has been relatively slow.Therefore,how to reasonably select the population and treatment mode,so that more breast cancer patients benefit from immunotherapy,is the key development direction in this field in the future.DC cells(DC)are the most powerful antigen-presenting cells(APC).As one of the main effector cells of APC,DC closely link innate immunity with adaptive immunity,and play a unique immune effect in specific immune response.According to the different biological characteristics of DC in different stages,some scholars propose to enhance its anti-tumor effect by improving the function of DC.Mucin 1(MUC1)is a type I transmembrane glycoprotein,which can be expressed in epithelial cells of various tissues and organs near the lumen or glandular surface under normal conditions,and can also be seen on the surface of adenocarcinoma epithelial cells,and has a high expression trend.Related studies have shown that the higher the expression of MUC1,the more aggressive the tumor is,the worse the prognosis is,and it is more prone to local invasion,lymph node metastasis and hematogenous metastasis.Combined with the new discovery of MUC1 in tumor biotherapy and the maturity and promotion of gene modification technology in recent years,we take MUC1 as the starting point and gene modified DC as the main technical route to systematically explore the immune effect of MUC1 gene transfected DC on breast cancer in the following three parts,in order to explore new ways and methods of tumor immunotherapy for breast cancer.1.Preparation of DC Vaccine Transfected with MUC1 GeneObjective:To verify the over expression of MUC1 in human breast cancer,and construct and package MUC1 recombinant lentivirus to infect DC,and obtain DC vaccine over-expressing MUC1.Materials and methods:(1)The expression of MUC1 protein in 90 cases of breast cancer tissues,30 cases of adjacent normal tissues and three kinds of cells(neuroblastoma cell line SH-SY5Y,two kinds of breast cancer cell lines MCF7and MDA-MB-231)was detected by immunohistochemistry.The expression of MUC1 protein in 26 cases of fresh breast cancer tissues,adjacent normal tissues and three kinds of cells was detected by q PCR to verify the expression of MUC1in breast cancer tissues and cell lines;(2)The recombinant MUC1 lentivirus was obtained by using gv657 vector,amplification of target gene,exchange of PCR products with vector,sequencing,plasmid extraction and purity,and virus packaging;(3)Human peripheral blood mononuclear cells(PBMC)were isolated by density gradient centrifugation in vitro.Mature DCs were induced by rh GM CSF,rh IL-4 and TNF-?in serum-free medium.The expression of DC surface markers was detected by flow cytometry;(4)DC infected with MUC1 lentivirus were divided into MUC1 gene infected DC group(MUC1-DC group),empty vector infected DC group(GFP-DC group)and control group DC group);(5)The expression of MUC1 m RNA was detected by RT-PCR;(6)The expression of MUC1 protein was detected by Western blotting.Results:(1)The expression of MUC1 in 90 cases of breast cancer tissues was significantly higher than that in adjacent normal tissues.MUC1 was highly expressed in 92.2%(83/90)cases of breast cancer tissues.MUC1 was highly expressed in 5 cases(5/30)of adjacent tissues(16.7%),and the rest 25 cases(83.3%)negative or weakly expressed it in cytoplasm.In 26 cases of breast cancer,92.3%(24/26)of MUC1 m RNA level was significantly higher than that of matched adjacent tissues(P<0.01),and only 2 cases of breast cancer had the similar level of MUC1 m RNA expression as that of adjacent tissues.Compared with neuroblastoma cell line SH-SY5Y,the expression of MUC1 m RNA in MCF7and MDA-MB-231 was significantly higher(P<0.05);(2)The sequencing results of MUC1 plasmid were consistent with the target gene,and the recombinant MUC1 lentivirus with viral titer of 1×10~9TU/ml was obtained;(3)The morphological changes of DC from peripheral blood were observed by phase contrast microscope,and cultured to 5-7 d most of the cells were detached and suspended,and the volume of the cells increased significantly with different sizes and irregular shapes.There were many burr like processes with different thickness,uneven shape on the surface showing typical DC morphology.After 7d of culture,the expression rates of CD1a,CD80,CD83 and CD86 were(21.6±2.4)%,(22.7±1.6)%,(24.9±2.1)%and(95.4±4.3)%,respectively,indicating that mature DC were obtained;(4)The specific GFP expression was observed at 24 h after transfection with GFP lentivirus vector pc DNA3.1(+)-MUC1,and the transfection efficiency was(11.7±1.0)%,(36.8±1.6)%and(12.9±0.9)%,respectively;(5)Compared with the empty vector group and the control group,a 346 bp amplification band was detected in MUC1-DC group by RT-PCR;(6)Western blotting showed that there were obvious MUC1 protein bands in MUC1-DC transfection group.Conclusion:(1)MUC1 was over-expressed in human breast cancer tissue and two human breast cancer cell lines MCF7 and MDA-MB-231;(2)MUC1recombinant plasmid was successfully constructed and packaged to obtain recombinant lentivirus carrying MUC1 gene;(3)MUC1-DC vaccine was successfully obtained by infecting DC with MUC1 recombinant lentivirus.2.Research on Immune Function of MUC1-DC in vitroObjective:To explore the immune effect of MUC1-DC vaccine in vitro and its killing activity on human breast cancer cell line MCF7.Materials and methods:(1)Specific cytotoxic T lymphocytes(CTL)were induced and cultured.The IL-12 and TNF-?production of CTL induced by MUC1-DC at different transfection time was detected by immunofluorescence;(2)The levels of IL-12 and TNF-?in each group(MUC1-DC-CTL,GFP-DC-CTL,DC-CTL)were detected by ELISA;(3)The killing activity of CTL was detected by LDH release method with MCF7 as target cells and MUD1-DC-CTL,DC-CTL and Ct-CTL as effector cells.The specific killing rate of CTL was(%)=(test hole value-effector cell control hole value)/(maximum killing control value-minimum killing control value)×100%.Results:(1)CTL induced by MUC1-DC began to express IL-12 and TNF-?after 2d detected by immunofluorescence,but the amount of expression was small and the fluorescence intensity was weak.The expression level of 4d was significantly higher than that of 3d and 2d(P<0.05,P<0.05).The fluorescence intensity was the strongest on the 5d,and decreased gradually on the 6d of culture;(2)ELISA results showed that the secretion of IL-12 and TNF-?by CTL induced by GFP-DC was(101.83±5.79)ng/m L and(119.26±11.52)ng/m L,respectively.There was no significant difference between GFP-DC group and DC group(P>0.05).Compared with DC group and GFP-DC group,the ability of MUC1-DC to induce CTL to secrete these two factors was significantly increased(202.52±17.10)ng/m L and(349.07±79.42)ng/m L,respectively(P<0.01);(3)LDH release assay showed the killing activity of the three groups increased with the increase of effect target ratio.At the same effect target ratio,compared with DC-CTL group or GFP-DC-CTL group,MUC1-DC-CTL group had more obvious killing activity on MCF7(P<0.01).Conclusion:(1)The ability of secreting IL-12 and TNF-?of MUC1-DC-CTL was the highest in 5d,which could be used in the follow-up experiment of the study;(2)DC vaccine transfected with MUC1 gene(MUC1-DC)had stronger immune ability to secrete IL-12 and TNF-?cytokines than the CTL induced by DC alone;(3)The killing activity of MUC1-DC-CTL against human breast cancer MCF7 cells was significantly higher than that of DC-CTL alone,and the killing activity of CTL gradually was increased with the increase of the ratio of effector cells to target cells.3.Evaluation of Immune Effect of MUC1-DC on Breast Cancer by Monitoring Tumor Growth with Optical Molecular Imaging in vivoObjective:To evaluate the immune effect of MUC1-DC vaccine on breast cancer and its possible mechanism by monitoring the growth of MCF7 tumor in nude mice.Materials and methods:(1)GFP lentivirus transfected MCF7 cells(GFP-MCF7);(2)BALB/c nude mice were subcutaneously implanted with GFP-MCF7(1×10~7cells/mouse).After tumorigenesis,the mice in each group were randomly divided into three groups.First,CIK cells activated in vitro(1×10~8cells/mouse)were injected via tail vein.In the treatment group,MUC1-DC(MUC1-DC group)or DC(DC group)were subcutaneously injected with 1×10~7cells/mouse,with a volume of 0.2 m L/mouse.In the control group,normal saline was injected with 0.2m L/mouse,once a day for 5 consecutive days;(3)The fluorescence imaging of transplanted tumor was observed before and 35 days after treatment with small animal in vivo optical imaging system,fluorescence intensity and fluorescence area were analyzed;(4)The expression of Caspase 3 was detected by immunohistochemistry;(5)Apoptosis was detected by TUNEL.Results:(1)The tumor formation rate of nude mice was 100%at 7 days after GFP-MCF7 was implanted.The results of optical molecular imaging showed that there was no significant difference in fluorescence signal intensity among MUC1-DC group,DC group and Ct group before treatment(F=0.4341,P>0.05);At 35d after treatment,the fluorescence signal intensity of MUC1-DC group was significantly lower than that of Ct group(F=7.864,P<0.05);there was no significant difference between DC and Ct group,MUC1-DC and DC group(P>0.05),but the fluorescence signal of MUC1-DC group was lower than that of DC group.There was no significant difference in the distribution area of fluorescence signal among MUC1-DC group,DC group and Ct group before treatment(F=1.084,P>0.05).At the 35th day after the beginning of treatment,the fluorescence signal in Ct group was scattered in many places.The area of fluorescence signal in MUC1-DC group and DC group was significantly lower than that in Ct group(P<0.01),but there was no significant difference between MUC1-DC group and DC group(P>0.05);(2)Caspase3 expression was the least in Ct group,followed by DC group,and it was the highest in MUC1-DC group.There were significant differences among the three groups(P<0.05);(3)TUNEL results showed that the apoptosis rates of three groups were as following:Ct group(4.11±2.61%),DC group(9.63±2.27)%,MUC1-DC group(25.30±8.24)%,and there were significant differences among the three groups(P<0.05).Conclusion:(1)GFP-labeled human breast cancer cells can continuously express fluorescence signal in nude mice,and can detect the optical density by optical molecular imaging system to observe the location and growth of tumor.It is an effective method to dynamically observe the growth and metastasis of tumor in vivo and evaluate the anti-tumor effect;(2)MUC1-DC vaccine can inhibit tumor growth more effectively than DC alone;(3)Compared to DC,MUC1-DC plays a better role in promoting tumor cell apoptosis.