In vitro application of anti-CD47 monoclonal antibody for targeted therapy of ovarian cancer
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摘要:
目的 探讨抗CD47单克隆抗体在体外对巨噬细胞吞噬卵巢癌细胞作用的影响。 方法 收集天津医科大学附属肿瘤医院2011年6月至2011年12月手术切除的卵巢组织样本45例(包含卵巢癌组织样本39例, 正常卵巢组织样本6例), 获得的组织细胞通过流式细胞术分析CD47的表达水平, 通过细胞功能学实验观察抗CD47单克隆抗体在体外对小鼠巨噬细胞吞噬卵巢癌细胞的靶向治疗作用。 结果 流式细胞术分析显示, 卵巢癌细胞系SKOV-3细胞和卵巢癌组织细胞中CD47表达水平均明显高于正常人卵巢上皮细胞, 差异具有统计学意义(P < 0.01), 且与疾病的分化程度相关(P < 0.05)。细胞功能学实验结果显示, 抗CD47单克隆抗体在体外可促进巨噬细胞对卵巢癌细胞的吞噬作用, 各实验组(1μg/mL、5μg/mL、10μg/mL、20μg/mL)分别较空白对照组吞噬指数显著增加, 并且随抗体浓度的增加吞噬指数呈浓度依赖性, 差异均具有统计学意义(P < 0.01)。统计学分析显示, 10μg/mL浓度组吞噬指数达到峰值, 同20μg/mL浓度组吞噬指数无显著性差异(P > 0.05)。 结论 CD47高表达于卵巢癌组织细胞, 且与组织分化程度相关。抗CD47单克隆抗体在体外可促进巨噬细胞对卵巢癌肿瘤细胞的吞噬作用, 为卵巢癌的靶向治疗提供了实验依据。 Abstract:Objective CD47 is highly expressed on human ovarian cancer cell surface and is a ligand for signal-regulatory protein alpha(SIRPα).The CD47-SIRPα pathway has been found to negatively regulate macrophage phagocytosis.We hypothesize that this pathway enables ovarian cancer cells to escape during tumor immunosurveillance.CD47 is highly expressed in various malignant diseases.This study aimed to explore whether CD47 can serve as a potential therapeutic target for ovarian cancer. Methods Tissue samples were obtained from 39 epithelial ovarian cancer patients.Six patients underwent other operations during which normal ovarian tissue samples were obtained.The expression of CD47 in the ovary cancer cell line SKOV-3 in normal ovarian tissues and ovarian cancer cells were analyzed by quantitative fluorescence-activated cell sorting.Macrophages and CFSE-labeled SKOV-3 cells were co-cultured with the indicated antibodies through in vitro phagocytosis assay, and phagocytic index was calculated. Results Flow cytometry showed that the expression levels of CD47 in the ovarian cancer cell line SKOV-3 and ovarian cancer cells were significantly higher than in normal ovarian epithelial cells(P < 0.01).This result is associated with the grade of differentiation of the disease(P < 0.05).Blocking a monoclonal antibody against CD47 promotes in vitro phagocytosis of ovarian cancer cells by macrophages.The phagocytic index of the experimental groups(1, 5, 10, and 20 μg/ml) increased significantly(P < 0.01) compared with the control group.The occurrence was found to be concentration-dependent(P < 0.01).Statistical analysis showed that the maximum phagocytic index was found in the 10 μg/ml concentration group; the same was observed in the 20 μg/ml group.However, no significant difference was found between the two groups(P > 0.05). Conclusion CD47 is expressed in epithelial ovarian cancer cells at a higher level compared with normal ovarian cells.The level of expression is associated with tissue differentiation grade.Our study indicates that the anti-CD47 monoclonal antibody can significantly promote in vitro phagocytosis, indicating that the anti-CD47 monoclonal antibody might be a potential target in targeted cancer therapy. -
Key words:
- ovarian cancer /
- targeting therapy /
- macrophage /
- CD47 /
- monoclonal antibody
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图 1 正常卵巢组织与卵巢癌组织细胞中CD47的表达水平
Figure 1. Expression of CD47 in normal ovarian cells and ovarian cancer cells
图 2 不同浓度下抗CD47单克隆抗体对小鼠巨噬细胞吞噬作用的影响
Figure 2. Relationship between anti-CD47 monoclonal antibody concen-tration and the phagocytosis index
图 3 荧光显微镜观察小鼠巨噬 细胞对 CFSE 标记的卵巢癌肿瘤 细胞的吞噬作用（×200，红色箭头 所示）
Figure 3. Phagocytosis of CFSE-labeled ovarian cancer cells by mouse macrophages(×200, as indicated by the red arrowhead)
A：Anti-CD47，B：Anti-HLA；C： IgG1-Isotye
表 1 不同卵巢细胞中CD47表达水平的差异
Table 1. Expression of CD47 in different kinds of ovarian cells
表 2 卵巢癌组织细胞CD47表达水平与临床病理参数的关系
Table 2. Relationship of the expression of CD47 in ovarian cancer cells with clinicopathologic features
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