EPCAM Antigen Expressing Dendritic Cells Induce Specific Immune Killing Effect In Adenocarcinoma

Objective: As a promising new treatment approach, immunotherapy isan entirely new class of cancer therapeutic method besides surgery,chemotherapy, and radiotherapy. By stimulating the body’s natural immunesystem and inducing, enhancing, or suppressing an immune response,immunotherapy mobilizes the body’s own powerful anticancer mechanisms tohelp achieve a durable response. Adoptive cellular immunotherapy is a highlyindividual systemic cancer treatment method through killing tumor cells byinfusing autologous immune cells which expanded largely in vitro. Comparedto other anti-tumor methods, adoptive cellular immunotherapy has thefollowing advantages: slight side effects, higher efficiency and killing bothstationary phase cells and tumor stem cells without immune suppression. Theimmune cells applied in clinical are mainly cytokine-induced killer cells (CIK)and mixed immune cells load of holoantigen, but these can not meet higherexpectations of tumor-killing effect.By the comparative study of dendriticcells (DC) expressing specific antigen EPCAM induced mixed immune cells（EP-effector）and CIK with DC expressing tumor holoantigen induced mixedimmune cells（EP-effector）,the experiment is to investigative whether thespecific immune destruction efficiency that dendritic cells (DC) loaded withspecific antigen EPCAM induced adenocarcinoma is higher.Methods:1Culture two adenocarcinoma cell lines in vitro, applyingflow cytometry technology to detect EPCAM expressing of Colo205andLS-174adenocarcinoma cell lines on the surface;2Collect mononuclear cells(PBMCs) from healthy adult peripheral blood by density gradientcentrifugation and detach lymphocytes and imDCs by using adherent. Andinduce T lymphocytes cell to transform into CIK cells and amplified in vitro.3the preparation of Colo205lysates.Put the Colo205lysates and specific antigen EPCAM into DC cells in d5,respectively,load imDCs and promotetheir maturation to prepare mature DC (Co-mDCs) loaded of Colo205celllysates and mature DC (EP-mDCs) loaded of specific antigen EPCAM.applying flow cytometry to detect the difference of DC cells’immunephenotype before and after adding antigen and make statistical credits.4Co-mDCs,EP-mDCs and CIK cells were co-cultured in vitro, the twoDCswere co-cultured with CIK by d7, by DC cell CIK cells can obtain relativeinformation to tumor holoantigen and specific antigen EPCAM, to makesome CIK cells transform into CTL with specific killing activity,therebyproducing a DC-CIK-CTL mixed immune cells Co-effector and EP-effector.5culture adenocarcinoma cells Colo205of Logarithmic phase, and add100μlColo205adenocarcinoma cells into96-well plates according to104/well.CIK,Co-effector and EP-effector were added to co-cultured with tumor cellsaccording to effector-target ratio E: T5:1,10:1,20:1. Simultaneously set up asingle set of target cells, a single set of effector cells and the control group, setup three auxiliary wells for each sample,37℃,5%CO2moisturizingincubator for18hours and added in CCK-820ul/holes in the incubator for4hours, measure the absorbance of each group with a microplate reader at450nm wavelength.According to the formula: killing rate (%)=1-(experimental group A value-effector cells alone group A value)/target cellsalone A value×100%, calculate the killing effect rate of effector cells ontumor cells.Results:1EPCAM antigen expression n the surface of Colo205and LST-174cells was99.73%,99.61%respectively.2After five days of cell culture, DC`s surface marker CD80, CD83,CD86, HLA-DR) expression levels were11.9%,16.1,16.1%,28.1%,significantly low, which suggested that the DCs are in the immature state;After pulsing with whole tumor antigens and antigen EPCAM for another twodays from D5, the expression levels of the cell surface (CD80, CD83, CD86,HLA-DR) were41.1%,66.3%,79.3%,83%and41.7%,69.6%,78.9%, 85.7%respectively, And immature DC cell surface molecule expression wasstatistically significant P <0.05;DC cells before and after adding antigenexpression of cell surface molecules from low to high expression, indicatingthat the cell has matured DC with antigen presentation capabilities.3CIK、Co-effector and EP-effector all can kill tumor cells.When E: Tequal5:1,10:1,20:1,the killing rate (%) of CIK were40.54±2.71,49.27±2.57,67.04±3.20correspondingly, the killing rate (%)of Co-effector were:54.13±3.83、66.40±4.58、75.65±5.37.the killing rate (%)of EP-effector were:60.51±4.54、75.17±6.10、92.53±8.87. It revealed that the killing rate increasedas effector-target ratio elevated in these three groups above. Under theconditions of E:T20:1, the killing rate of three groups were statisticallysignificant,CIK killing rate than the Co-effector, EP-effector low statisticalsignificance between each other, P <0.05;EP-effector ratio of Co-effectorkilling rate, was statistically significant between the two, P <0.05.Conclusion: The three group of immune cells all had killing effect onColo205tumor cell, But EPCAM load DC-induced antigen-specific immunekilling rate of adenocarcinoma is more efficient and specific.