负载抗原DC与CIK共培养对富集培养乳腺癌CTCs杀伤作用研究

Study on the Kill Activity of the Whole Tumor Cell Antigen Pulsed DC Co-Culture with CIK for Breast Cancer Circulating Tumor Cells

Abstract]ObjectiveTo study the kill activity of the whole tumor cell antigen (Ag) pulsed dendritic cell (DC) coculture with cytokine induced killer cell (CIK) for breast cancer circulating tumor cells (CTCs).Methods Peripheral blood mononuclear cells (PBMC) were isolated from breast cancer patients with CTCs using a blood cell separator instrument. The epidermal cell adhesion molecule (EpCAM ) (+) breast cancer CTCs enriched by MACS were cultured in vitro. The nested RT-PCR, cell immunofluorescence imaging (CK8/18), and immunohistochemistry (CK8/18and CK19) methods, were used for the detection and identification of the cells. EpCAM (-) cells were routinely induced to DC and CIK, which were grouped into Ag-DC-CIK group, DC-CIK group, DC group and CIK group. The cytotoxic activity of co-cultured DC-CIK against breast cancer CTCs was detected by flow cytometry and MTT assay. The cell morphology was observed by light microscopy and transmission electron microscopy.ResultsThe target band of CK19mRNA can be detected by nested RT-PCR. The expressions of CK8/18and CK19of EpCAM (+) cells in vitro were positive by immunofluorescence staining and immunohis?tochemical staining. The proliferative activity of co-cultured DC-CIK was higher than that of CIK (P＜0.001). The positive rates of CD1α+, CD83+CD86+, CD83+CD11C+, CD86+CD11C+DCs and CD3+CD8+, CD3+CD56+CIKs were significantly higher in Ag- DC- CIK group than those of DC- CIK group, DC group and CIK group (P＜0.05). The apoptosis of breast cancer CTCs was induced in Ag-DC-CIK, DC-CIK and CIK3groups, and apoptotic rates were (56.83±3.07)%,(31.43±1.77)% and (24.70±1.51)%, showing significant differences between them (P＜0.05). Transmission electron microscopy showed the typical micro-structure of breast cancer CTCs induced apoptosis.ConclusionMACS in combination with cell immunological methods can improve significantly the detective sensitivity of breast cancer CTCs. The co-cultured Ag-DC-CIK is highly effective immune cells,which shows a high er proliferation and cytoxicty against breast cancer CTCs. This may be used as a clinically immunotherapy means of anti-breast cancer recurrence and metastasis.