Drug-loaded tumor exosomes combined with radiotherapy inhibit the proliferation of breast cancer stem cells

doi:10.3969/j.issn.2095-4344.0502

Abstract

Abstract:

BACKGROUND: In recent years, studies have found that drug-loaded tumor exosomes have a high affinity with tumor cells from corresponding origins, but have a low binding force to other cells, thus becoming an efficient targeted drug carrier.
OBJECTIVE: To observe the effect of drug-loaded tumor exosomes combined with radiotherapy on the proliferation of breast cancer stem cells.
METHODS: Tumor exosomes were isolated from ZR-75-30 human breast ductal carcinoma cells by ultracentrifugation and used to wrap methotrexate and prepare drug-loaded tumor exosomes. CD133⁺ ZR-75-30 was isolated from human breast cancer cells in breast cancer stem cells by flow cytometry. The CD133⁺ cells were cultured in routine medium as blank control group, in RPMI 1640 medium containing drug-loaded tumor exosomes for 24 hours as exosome group, and in RPMI 1640 medium containing drug-loaded tumor exosomes for 24 hours followed by 2, 4, 6 Gy X-ray irradiation as low-, middle- and high-dose groups. After 7 days of culture, tumor spheres formed under inverted microscope, and sphere number and volume were recorded. Expression of Nanog, Sox-2, and Oct-4 was detected by western blot assay.
RESULTS AND CONCLUSION: Compared with the blank control group, the tumor exosome group had significantly decreased number and size of tumor spheres (P < 0.05); compared with the tumor exosome group, low-, middle- and high-dose radiotherapy groups had significantly decreased number and size of tumor spheres (P < 0.05), and the reduction was increased with the increasing of radiation dose. Compared with the blank control group, lower expression of Nanog, Sox-2, and Oct-4 was found in the tumor exosome group (P < 0.05); compared with the tumor exosome group, there was also a significant reduction in the expression of Nanog Sox-2, Oct-4 in the three radiotherapy groups (P < 0.05), and the reduction was increased with the increasing of radiation dose. To conclude, the combined use of radiotherapy and drug-loaded tumor exosomes inhibits the proliferation activity of CD133⁺ breast cancer stem cells in a radiation dose-dependent manner.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Breast Neoplasms, Neoplastic Stem Cells, Exosomes, Radiotherapy, Cell Proliferation, Tissue Engineering

CLC Number:

R394.2

Luo Qiong, Gao Guo-xiang, Qin Shi-yun. Drug-loaded tumor exosomes combined with radiotherapy inhibit the proliferation of breast cancer stem cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(21): 3286-3291.

Figures/Tables 1

2.1 乳腺癌肿瘤干细胞的分离与鉴定利用流式细胞仪成功收集到CD133+和CD133-细胞，分选前后CD133阳性表达率分别为(5.20±1.68)%和(86.36±1.64)%(图1)。 2.2 CCK-8增殖实验检测CD133+细胞增殖能力 CCK-8检测结果显示，两组细胞吸光度值随着培养时间的延长逐渐升高，CD133+细胞培养3，5，7 d的吸光度值明显高于CD133-细胞(P < 0.05)，见图2，说明CD133+细胞具有更强的增殖能力。 2.3 Transwell小室检测CD133+细胞侵袭能力 Transwell小室实验结果显示，CD133+细胞的侵袭能力明显高于CD133-细胞(P < 0.05)，见图3，说明CD133+细胞具有更强的侵袭能力。 2.4 CCK-8检测筛选最佳肿瘤细胞外泌体稀释倍数培养72 h后CCK-8检测发现，CD133+肿瘤干细胞增殖活性受到不同程度的影响，随着稀释比例的增加，细胞存活率逐渐降低，当载药肿瘤细胞外泌体稀释比例>1︰1 000时，细胞存活率下降明显(图4)，因此实验选择稀释倍数1 000进行后续实验。 2.5 CD133+乳腺癌肿瘤干细胞肿瘤球形成实验结果 2.5.1 肿瘤球数量与空白组比较，肿瘤细胞外泌体组肿瘤球数量明显减少(P < 0.05)；与肿瘤细胞外泌体组比较，低、中、高剂量放疗组肿瘤球数量明显减少(P < 0.05)；与低剂量放疗组比较，中、高剂量放疗组肿瘤球数量明显减少(P < 0.05)；与中剂量放疗组比较，高剂量放疗组肿瘤球数量明显减少(P < 0.05)，见表1。 2.5.2 肿瘤球体积与空白组比较，肿瘤细胞外泌体组肿瘤球体积明显减小(P < 0.05)；与肿瘤细胞外泌体组比较，低、中、高剂量放疗组肿瘤球体积明显减小(P < 0.05)；与低剂量放疗组比较，中、高剂量放疗组肿瘤球体积明显减少(P < 0.05)；与中剂量放疗组比较，高剂量放疗组肿瘤球体积明显减小(P < 0.05)，见表1。 2.6 CD133+肿瘤干细胞表面标志物表达与空白组比较，肿瘤细胞外泌体组Nanog、Sox-2、Oct-4表达降低(P < 0.05)；与肿瘤细胞外泌体组比较，低、中、高剂量放疗组Nanog、Sox-2、Oct-4表达进一步降低(P < 0.05)；与低剂量放疗组比较，中、高剂量放疗组Nanog、Sox-2、Oct-4表达明显降低(P <0.05)；与中剂量放疗组比较，高剂量放疗组Nanog、Sox-2、Oct-4表达明显降低(P < 0.05)，见图5。"

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