title: Autophagy contributes to the survival of CD133+ liver cancer stem cells in the hypoxic and nutrient-deprived tumor microenvironment.
abstract: 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.
authors.affiliation: Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
authors.forename: Yu-Jiao
authors.initials: YJ
authors.lastname: Song
authors.forename: Shan-Shan
authors.initials: SS
authors.lastname: Zhang
authors.forename: Xian-Ling
authors.initials: XL
authors.lastname: Guo
authors.forename: Kai
authors.initials: K
authors.lastname: Sun
authors.forename: Zhi-Peng
authors.initials: ZP
authors.lastname: Han
authors.forename: Rong
authors.initials: R
authors.lastname: Li
authors.forename: Qiu-Dong
authors.initials: QD
authors.lastname: Zhao
authors.forename: Wei-Jie
authors.initials: WJ
authors.lastname: Deng
authors.forename: Xu-Qin
authors.initials: XQ
authors.lastname: Xie
authors.forename: Jian-Wei
authors.initials: JW
authors.lastname: Zhang
authors.forename: Meng-Chao
authors.initials: MC
authors.lastname: Wu
authors.forename: Li-Xin
authors.initials: LX
authors.lastname: Wei
mesh_terms: D000071916:AC133 Antigen; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D001343:Autophagy / Q000187:drug effects; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002738:Chloroquine / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism*; D008297:Male; D051379:Mice; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D013217:Starvation; D014183:Transplantation, Heterologous; D059016:Tumor Microenvironment; D014410:Tumor Stem Cell Assay
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D006023:Glycoproteins; C000606023:PROM1 protein, human; D010455:Peptides; C000606024:Prom1 protein, mouse; D002738:Chloroquine
keywords: 4′,6′-diamidino-2-phenylindole; ATG; Autophagy; CCK8; CD133; CQ; Cell Counting Kit-8; DAPI; FITC; GFP-LC3; H/S; HCC; Hypoxia and nutrient starvation (H/S); LC3; LCSCs; Liver cancer stem cells (LCSCs); PI; TEM; Tumor microenvironment; autophagy-related gene; chloroquine; fluorescein isothiocyannate; green fluorescent protein-linked LC3; hepatocellular carcinoma; hypoxia and nutrient starvation; liver cancer stem cells; microtubule-associated protein1 light chain 3; propidium iodide; transmission electron microscope