| ObjectiveThe incidences and mortalities of cancer are still rising. Emerging as a potential treatment option for various tumors, adoptive immunotherapy has attracted tremendous attentions. The CIK cells derived from peripheral blood have been applied in clinically. However, the application of CIK is limited owing to obstacles, such as low proliferation rates, short survival period in vivo, lower ability of anti-apoptosis. The purpose of this study is to analyze the proliferation, immunophenotype, immunogenicity, differential expressions of activated or inhibitory cell surface markers, and drug-resistance gene ABCG2of cytokine-induced killer (CIK) cells derived from umbilical cord blood or peripheral blood in cancer patients. Those CIK cells were pretreated with chemotherapy drug cisplatin, and then the apoptosis of CIK cells were evaluated. We also detected the intracellular cytokines and the cytotoxic activity on KYSE70in vitro of CIK cells derived from cord blood and peripheral blood. Furthermore, the esophagus tumor-xenografted nude mice model was established, and the tumor volumes were compared after different CIK cells were infused separately.MethodsCord blood mononuclear cells (CBMCs) and peripheral blood mononuclear cells (PBMCs) in cancer patients were isolated using Ficoll-Plaque density gradients centrifuge. The CBMC and PBMC were re-suspended in RPMI1640containing10%fetal bovine serum, and cultured in the presence of Interferon-y, anti-CD3antibody and interlukin-2to induce CIK cells expansion and activation. The proliferation, immunophenotypes, immunogenicity, activated cell surface markers (CD28, CD27) and inhibitory cell surface marker (PD-1) were analyzed by flow cytometry assay, and the apoptosis of CIK cells after treatment with cisplatin was determined using Annexin V/PI staining. The expression of drug-resistance gene ABCG2was detected by qRT-PCR. The secretion of intracellular cytokines including interferon-y, interlukin-2, and tumor necrosis factor-a were detected by ELISA. The expression of CD107a were detected by flow cytometry. Five-weeks old Balb/c nude mice were randomly divided into three groups,(5for each group). Mice were injected i.p. with1×107KYSE70cells. When the tumors were visible, PBS, cord blood CIK. When the tumors were visible, PBS, cord blood CIK and peripheral blood CIK cells were infused via trail vein, respectively, and then the growth of tumors was observed.ResultsThe proliferation of index (PI) of CIK cells derived from cord blood was statistically significantly higher than that from peripheral blood on day10in vitro culture [(251.52±16.76) vs (158.00±43.19), P＜0.05]. Thirteen days after incubation, the expression of HLAI and HLAⅡ were significantly lower in CIK cells derived from cord blood [(18.86±9.76)%vs (65.63±10.02),(14.53±3.34)%vs (67.20±4.50)%, P<0.01], the proportions of CD8HLAII, CD4HLAII and CD8HLAI were lower in CIK cells derived from cord blood than its counterpart [(5.86±0.53)%vs (51.20±4.85)%,(2.32±0.34)%vs (12.42±3.65)%,(17.56±1.85)%vs (52.10±5.72)%, P＜0.05]. However, the proportions of CD4HLAI had no significant differences (P>0.05). The proportion of CD3+CD56+cells was higher in CIK cells derived from cord blood than that from peripheral blood [(21.20±4.82)%vs (10.06±3.46)]%, P＜0.05). When detecting the status of CIK cells, the proportions of activated CD4+CD28+, CD4+CD27+and CD8+CD27+T cells were higher in cord blood derived-CIK than peripheral blood derived-CIK [(32.40±16.81)%vs (18.65±9.23)%,(27.48±13.53)%vs (0.98±0.55)%,(41.76±13.98)%vs (2.58±2.10)%, P<0.05or P<0.01], while the proportion of inhibitory CD8+PD-1+T cells was significantly lower in the cord blood-derived CIK cells [(3.25±2.13)%vs (8.05±9.23)%, P<0.01). However, the proportions of CD8+CD28+and CD4+PD-1+T cells showed no significant differences between CIK cells from the cord blood and those from the peripheral blood (P>0.05). ABCG2, a drug-resistance gene, was not expressed in the CIK cells from the peripheral blood but in those from the umbilical cord blood. The apoptosis of cisplatin-treated CIK cells derived from cord blood was less than CIK cells derived from peripheral blood [(10.10±4.20)%vs (27.12±12.18)%, P<0.05]. The secretion level of IFN-γ and IL-2was higher in CIK derived from cord blood (P<0.01), while there was no difference in the secretion level of TNF-a [(4.70±0.44)pg/mL vs (4.82±0.51)pg/mL, P>0.05].The expression of CD107a in CIK derived from cord blood is higher [(9.16±1.58)%vs (2.36±0.86)%, P<0.01]. In xenograft models, the CIK derived from cord blood showed more potential of therapy, resulting in a significant decline in tumor volumes.ConclusionCompared with the peripheral blood-derived CIK cells, the counterparts derived from the umbilical cord blood demonstrate increased proliferation rates, higher activity of CIK cells. The anti-apoptosis of cord blood-derived CIK is higher, furthermore, the cytokine and cytotoxicity is higher than CIK derived from peripheral blood, suggesting a potential clinically use. |
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