Autophagy Contributes To The Survival Of CD133+Liver Cancer Stem Cells In The Hypoxic And Nutrient-deprived Tumor Microenvironment

Human hepatocellular carcinoma (HCC) which is common and aggressive in adultsis one of the leading causes about cancer-related death worldwide. Although better HCCdetection methods and treatments have improved disease prognosis at early stages, HCCremains largely incurable because of high recurrence rates and resistance to chemotherapy.The cancer stem cell (CSC) theory suggests that a subpopulation of self-renewingcells has the ability to differentiate into defined progenies, initiate and sustain tumorgrowth. A main principle of this theory is that there is a subpopulation of CSCs within thetumor that is resistant to therapy and is able to survive following treatment. Thus, theseclones of drug-resistant tumor cells grow and self-renew to promote recurrence of HCCtumors. CSCs play a role in tumorigenesis and may be responsible for patient prognosis.CD133has emerged as an important liver CSC marker. CD133+cells were shown topossess properties of cancer stem cells, generally characterized by their colony-formingefficiency, proliferative output, ability to form tumors in vivo, and the capacity forself-renew. Suetsugu and colleagues found that a sorted CD133+subpopulation of Huh7cells possessed higher proliferative and tumorigenic potential than their CD133-counterparts.To fully understand the behavior and characteristics of CSCs, the tumormicroenvironment must be considered. Such microenvironments tend to be ischemic andhypoxic. Since nutrients and oxygen are taken up by cells via distinct pathways, oxygendeficiency and nutrient deprivation do not necessarily have to co-occur. In fact, it isthought that low oxygen concentration is one of the earliest limitations that cancer cellshave to deal with. Components of the tumor microenvironment have been shown toinfluence CSC behavior, making them either more or less tumorigenic. Despite recentadvances, exactly how the microenvironment influences CSCs during tumor evolution andhow CSCs adapt to the microenvironment are still unclear.Ischemia and hypoxia commonly result in autophagy. Autophagy is an evolutionarilyconserved catabolic pathway and can protect cells during various types of stress. It isinvolved in the process that cells deliver their own protein and organelle to lysosomes fordegradation. A series of autophagy-related genes (ATGs) regulate the process of autophagy.Signaling involves the expression of Beclin1, Atg5, Atg7, P62and the conversion of LC3-I to LC3-II by binding to the membrane of autophagosomes after these vacuoles are formed.It is well known the role of autophagy in some cancer cells, but its role in liver CSCshas not been described. Here, we examine whether autophagy enables the survival ofhepatic tumorigenic cells despite exposed to the hypoxic and nutrient-deprived tumormicroenvironment. We performed in vitro and vivo experiments and examine levels ofautophagy in CD133+cells both under normal conditions and under hypoxic andnutrient-deprived (H/S) states.Part1CD133+cells exhibit lower sensitivity to H/S compared to CD133-cells.Hypoxia occurs before nutrient-deprived environment. To mimic this, we first culturedhepatocellular carcinoma cells under hypoxic condition for8h and then subjected them toH/S for16h to simulate the hypoxic and nutrient-deprived tumor microenvironment. Toexamine whether CD133+cells are more tolerant of these stressful conditions, weperformed immunofluorescent staining and flow cytometry analysis to show that CD133+cells are significantly enriched after hypoxia and nutrient starvation (H/S) in the humanHCC cell line Huh7. We sorted from Huh7cell line with flow cytometry to get CD133+and CD133-cells and examined the effect of H/S on cell death of CD133+and CD133-cells. Sorted CD133+cells showed higher survival, less apoptosis, and possess higherclonogenic ability under H/S compared to the CD133-population.Part2Autophagy involved in the tolerance of CD133+cells to H/S.To assess that the differential sensitivity of CD133+and CD133-subpopulations to H/Sdue to the different level of autophagy, we examined the various ATGs in both populations.Under H/S, electron microscopy revealed advanced autophagic vesicles in CD133+cells.Additionally, CD133+cells had higher autophagy levels as measured by both RT-qPCRand Western blotting. CD133+cells accumulated GFP-LC3puncta, which can be detectedby fluorescence microscopy. These data indicate that compared with CD133-cells,CD133+cells have higher basal and H/S-induced levels of autophagy. We next used theinhibitor chloroquine (CQ) to further find out whether autophagy contributes to the higherviability, lower rate of apoptosis, and increased clonogenic ability of CD133+cells underhypoxic and nutrient-deprived conditions, we examined the effect of CQ on cells viabilityand apoptosis following hypoxia for8h and H/S for16h. The autophagic inhibitor significantly increased apoptosis and decreased the clonogenic capacity of CD133+cellsunder H/S. Thus, autophagy is critically involved in mediating the survival of CD133+cells and promotes them to resist to H/S-induced apoptosis.Part3Autophagy is essential for liver CSCs maintenance and CD133+cells havegreater tumor-forming potential in mice, which is inhibited by CQ.We performed experiments to measure the effect of oxygen/nutrient-deprivedconditions on sphere-forming capability of Huh7cells and detect the role of autophagy inthis process. Pre-culturing in H/S enhanced the sphere-forming capacity of CD133+cells.Autophagy inhibition with CQ significantly decreased the sphere-forming capacity ofHuh7cells whether pre-cultured in normal medium or H/S for8h. Sorting of the Huh7cells to obtain CD133+and CD133-cells, and suspended them respectively, did not alterthe results. Furthermore and in contrast to CD133-cells, significantly larger hepatosphereswere observed in CD133+cells, which supports the greater sphere-forming capability ofliver CSCs in vitro. The data indicate that autophagy is essential for CD133+liver CSCmaintainance.We isolated CD133+and CD133-cells from Huh7cells by magnetic cellsorting and used them for the vivo tumorigenic experiments. We subcutaneously injectedeither CD133+or CD133-cells into nude mice to determine whether CD133+cells aremore tumorigenic in vivo. CD133+cells demonstrated an enhanced ability to initiate tumorformation when injected into immunodeficient mice compared to CD133-cells. To assesswhether autophagy influences tumorigenic potential in vivo, we compared thetumor-forming ability and growth rate of CD133-and CD133+cells with or without CQtreatment in mice. Administration of CQ caused significant tumor shrinkage. We observedthat there is a significant difference in tumor incidence between CD133+and CD133-cells.We subcutaneously injected four different doses of sorted cells and found that tumorincidence was increased with higher cell doses. Injection of CQ significantly inhibitedtumor formation. Thus, in vivo, autophagy may contribute to the maintenance oftumorigenic capacity of CD133+cells in the tumor microenvironment.To sum up, we conclude as follows:1、CD133+cells showed higher survival, less apoptosis, and possess higher clonogenicability under H/S compared to the CD133-population, and significantly enriched after hypoxia and nutrient starvation (H/S) in the human HCC cell line Huh7.2、Our results indicate that the involvement of autophagy in maintenance of CD133+liver cancer stem cells (LCSC) under the oxygen-and nutrient-deprived conditions that aretypical of the tumor microenvironment in HCC. Therefore, autophagy inhibitors may makeCSCs more sensitivity to the tumor microenvironment.3、 CD133+cells are able to adapt to an hypoxic and nutrient-deprivedmicroenvironment, which does not impair their self-renewal capacity, but instead confersenhanced sphere-forming ability via induction of autophagy.Therefore, autophagy isessential for CSC maintenance. CD133+cells were also found to have a higher ability oftumor-forming in vivo, which could be inhibited by CQ administration.