Abstract
BackgroundAs an important stress-response mechanism, autophagy plays crucial role in the tumor formation and drug resistance of cancer cells including osteosarcoma (OS). OS cancer stem cells (CSCs) also are considered a key factor of tumorigenesis, drug resistance and tumor recurrence. However, the relationship between autophagy and OS CSCs still remains unclear.MethodsCD271+ OS CSCs and CD271- OS cells were isolated by magnetic activated cell sorting. The autophagy level was evaluated by the mRNA expression of autophagy genes, the protein level of LC3II and p62, and the mean number of GFP-LC3 dot per cell. Lentivirus-delivered specific shRNA was utilized to inhibit the corresponding gene expression. The cell viability was examined with CCK8 assay. The cell proliferation level was detected with BrdU staining assay. Cell death was determined by Annexin V/PI double staining of fluorescence activated cell sorting, lactate dehydrogenase release and caspase-3 activity. Tumorigenicity ability was evaluated by colony and sphere formation assay, the protein expression of stemness markers and tumor formation in nude mice.ResultsOur data indicated that CD271+ OS CSCs had a similar basic autophagy level with CD271- OS cells. Autophagy deficiency had no observable effects on the levels of cell proliferation and death both in CD271+ and CD271- OS cells under normal condition. However, CD271+ OS cells showed a higher autophagy activity than CD271- OS cells under hypoxia and low nutrient (LH) condition. Moreover, autophagy-deficient CD271+ OS cells lost the advantage of tolerance to LH condition compared to CD271- OS cells. Meanwhile, autophagy deficiency enhanced the sensitivity to chemotherapeutics in the CD271+ cells to the comparable level in the CD271- cells. More importantly, deficient-autophagy decreased the protein expression of stemness markers and caused the disappearance of the superiority in tumorigenicity in vitro and vivo in CD271+ OS cells.ConclusionThe results above demonstrated that autophagy contributes to the stem-like features of CD271+ OS CSCs. Inhibition of autophagy is a promising strategy in the CSCs-targeting OS therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-016-0297-5) contains supplementary material, which is available to authorized users.
Highlights
As an important stress-response mechanism, autophagy plays crucial role in the tumor formation and drug resistance of cancer cells including osteosarcoma (OS)
CD271+ OS cells have higher autophagy ability rather than basic autophagy level compared to CD271- OS cells To study the role of autophagy in OS cancer stem cells (CSCs), we isolated CD271+ OS cells and CD271- OS cells with Magnetic activated cell sorting (MACS) and examined their purities with Fluorescence Activated Cell Sorting (FACS) (Fig. 1a)
LC3 dots, an indicator of autophagosome formation, had a higher number in CD271+ OS cells than that in CD271- OS cells under low nutrients and hypoxia (LH) condition (Fig. 1d). These data demonstrated that there is no significant difference in the basic autophagy level between CD271+ OS CSCs and CD271- OS cells, CD271+ OS CSCs have higher autophagy activity than CD271- OS cells under LH condition
Summary
As an important stress-response mechanism, autophagy plays crucial role in the tumor formation and drug resistance of cancer cells including osteosarcoma (OS). OS cancer stem cells (CSCs) are considered a key factor of tumorigenesis, drug resistance and tumor recurrence. He current therapy strategy for OS consists of the addition of chemotherapy after surgical removal of tumor, and neoadjuvant chemotherapy followed by surgery. Novel therapeutic strategy for enhancing the chemotherapy sensitivity of the OS has yet to be explored. Cancer stem cells (CSCs) are seen as a subpopulation of self-renewing tumor cells, which can differentiate into the daughter tumor cells, have the lower sensitivity to chemotherapy and radiotherapy, and exhibit tumor reinitiating property. CD271+ cells showed many stem-like features including self-renew, the advantage of forming sarcospheres, drug resistance and tumorigenicity [10]
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