Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
Cancer Lett. 2013 Oct 1;339(1):70-81. doi: 10.1016/j.canlet.2013.07.021. Epub 2013 Jul 20.
Liver cancer stem cells (LCSCs) can drive and maintain hepatocellular carcinoma (HCC) growth, metastasis, and recurrence. Therefore, they are potentially responsible for the poor prognosis of HCC. Oxygen and nutrient deficiencies are common characteristics of the tumor microenvironment. However, how LCSCs adapt to oxygen- and nutrient-deprived conditions is unclear. Here, we used immunofluorescent staining and flow cytometry analysis to show that CD133+ cells were significantly enriched after hypoxia and nutrient starvation (H/S) in the human HCC cell line Huh7. Sorted CD133+ cells showed higher survival, less apoptosis, and possess higher clonogenic ability under H/S compared to the CD133- population. Under H/S, electron microscopy revealed more advanced autophagic vesicles in CD133+ cells. Additionally, CD133+ cells had higher autophagy levels as measured by both RT-qPCR and Western blotting. CD133+ cells had more accumulated GFP-LC3 puncta, which can be detected by fluorescence microscopy. The autophagic inhibitor chloroquine (CQ) significantly increased apoptosis and decreased the clonogenic capacity of CD133+ cells under H/S. Pre-culturing in H/S enhanced the sphere-forming capacity of CD133+ cells. However, CQ significantly impaired this process. Therefore, autophagy is essential for LCSCs maintenance. CD133+ cells were also found to have a higher tumor-forming ability in vivo, which could be inhibited by CQ administration. Collectively, our results indicate that the involvement of autophagy in maintenance of CD133+ LCSCs under the oxygen- and nutrient-deprived conditions that are typical of the tumor microenvironment in HCC. Therefore, autophagy inhibitors may make LCSCs more sensitive to the tumor microenvironment and be useful in improving anti-cancer treatments.
肝癌干细胞 (LCSCs) 可以驱动和维持肝细胞癌 (HCC) 的生长、转移和复发。因此，它们可能是 HCC 预后不良的原因。氧气和营养物质的缺乏是肿瘤微环境的共同特征。然而，LCSCs 如何适应缺氧和营养饥饿的条件尚不清楚。在这里，我们使用免疫荧光染色和流式细胞术分析表明，在人肝癌细胞系 Huh7 中，缺氧和营养饥饿 (H/S) 后 CD133+细胞明显富集。分选的 CD133+细胞在 H/S 下表现出更高的存活率、更少的细胞凋亡和更高的克隆形成能力，而 CD133-群体则表现出更高的克隆形成能力。在 H/S 下，电子显微镜显示 CD133+细胞中晚期自噬小泡增多。此外，通过 RT-qPCR 和 Western blot 测量，CD133+细胞的自噬水平更高。CD133+细胞中 GFP-LC3 点状结构的积累更多，这可以通过荧光显微镜检测到。自噬抑制剂氯喹 (CQ) 显著增加了 H/S 下 CD133+细胞的凋亡并降低了其克隆形成能力。预先在 H/S 中培养增强了 CD133+细胞的球体形成能力。然而，CQ 显著削弱了这一过程。因此，自噬对于 LCSCs 的维持是必不可少的。体内实验也发现 CD133+细胞具有更高的肿瘤形成能力，CQ 的给药可显著抑制这一过程。总之，我们的研究结果表明，自噬参与了肝癌微环境中缺氧和营养物质缺乏条件下 CD133+ LCSCs 的维持。因此，自噬抑制剂可能使 LCSCs 对肿瘤微环境更加敏感，并有助于改善抗癌治疗。
