The Study Of Quiescent Colon Cancer Stem Cells Identification And IFN-γ Targeted Therapy

BackgroundColorectal cancer (CRC) is one of the most prevalent cancers globally and the leading causes of cancer-related deaths due to therapy resistance and metastasis. Along with the progression in cancer research, people found that tumor was comprised with distinct subpopulations with different functions and biological features. Cancer stem cells (CSCs) were considered as a small slow-cycling subpopulation of cells within tumor that possessed the capacity to self-renew and produce cancer cells of heterogeneous lineages, which could be modulated by Hedghog, Wnt and BMP pathway. CSCs are resistant to many current anticancer treatments, such as chemotherapy and radiotherapy, which are the main cause of therapy resistance and tumor relapse.Unlike germinal stem cells, most adult stem cells and CSCs were in quiescent or slow-cycling states which were termed as quiescent cancer stem cells (QCSCs). Because of the distinct metabolic and cell cycle traits, QCSCs usually revealed dull to traditional therapies and resulted in tumor recurrence after being awaken. Intestine is a unique organ that has two different stem cells:active division stem cells (ASC) which are responsible for the epithelial cells’ renewal every three days, and dormant stem cells (DSC) which are just gradually recognized in recent years. Researchers found both ASCs and DSCs gained the ability of differentiation to various intestinal cells. However, only DSCs could rebuild the intestine structure after radioactive damage, and partial DSCs differentiate to ASCs in this repair process. Therefore, DSCs could be considered as the original source of intestinal cells. Currently, the studies on quiescent colon cancer stem cells (QCCSCs) were very limited. Based on the CSC theory and compelling evidences, we supposed QCCSCs were the dominant cause of tumor initiation, chemoresistance and tumor relapse.From the perspective of time, dormanance or quiescence is a transient states, a cell exiting from active proliferation has four non-proliferating options:senescence, apoptosis, differentiation and quiescence. In other words, it is not universal that all quiescent cells can be referred as QCSC, but only the quiescent cells with stem cell-like traits can transit between quiescent and active division states reversibly. Besides, although active division cells may be the neighbor of quiescent cells, there are enormous differences existed, such as metabolic states and gene expression, mainly due to the epigenetic modulation. Among various epigenetic mechanisms, microRNA can take the core position.MicroRNA (miRNA) are18-25nucleotides non-coding endogenous RNA that negatively regulates gene expression at the post transcriptional level by inhibiting mRNA translation or promoting mRNA degradation. miRNAs are deemed to have widespread regulatory activity in a broad range of developmental processes and are implicated in diverse diseases, including cancer. miRNA can act as either an oncogene or a tumor suppressor in cancer:miR-155activate while let-7repress cancer related cell signaling pathway; miR-200c could inhibit the self-renewal of breast cancer stem cell through targeting BMI1; miR-20c enhanced the chemoresistance and EMT of cancer stem cells by upregulating AKT pathway and repress the expression of PTEN. However, how miRNA affect the biological feature of QCCSCs is still unknown.CSCs are both structurally and functionally distinct from the other cells within a tumor mass, and quiescent cancer stem cells are regarded as the core part of the CSCs, which play central role in tumor progression. Herein, we focused on the targeted therapy and miRNA regulatory mechanisms toward QCCSCs, which could supplement the epigenetic modulatory mechanisms in CCSCs, but also provide novel strategy and potential therapeutic target in colon cancer treatment.MethodsIn order to isolate QCSCs, several colon cancer cell lines which derived from ATCC and original colon cancer specimens were membrane-stained with PKH dye and cultured in serum free media, and PKHhi cells were then isolated through FACS and thereafter, their stemness were validated by series functional experiments (chemoresistance, colosphere formation, xenographt formation and stemness gene detection). Based on the above results, the sensitivity to IFN-y’s apoptotic effect and expression level of IFN-y receptors were evaluated among PKHhi, PKHlow and PKHneg cells through FCM, western-blot and immunofluorescence assay. Thereafter, the IFN-y related apoptotic pathway were also assessed by western-blot and immunofluorescence. For illuminating the underlying epigenetic mechanism in QCCSCs, we assessed miRNA expression profiles in PKHhi and rest cells using miRNA microarrays. With the combination of bioinformatic analysis and microarray result, the target gene of miR-4666-3p was predicted and validated with qRT-PCR, western-blot and dual luciferase reporter assay. Additionally, the effect of miR-4666-3p overexpression for the self-renewal and differentiation of QCCSCs were also investigated. Result1. PKHhi cells reveal slow-cycling and stem cell-like traits.Through membrane staining of PKH and serum-free sphere culture system, we termed cells with high PKH fluorescence intensity (>103) as quiescent colon cancer cells (PKHhi cells), and the rest part was divided into PKHlow and PKHneg subpopulations based on their PKH fluorescence intensity (PKHlow102-103; PKHneg<102). The FCM analysis of PY-Hoeschest double staining revealed most PKHhi cells stopped at GO phase, while PKHlow and PKHneg cells were at division or pre-division phase. Thereafter, PKHhi, PKHlow and PKHneg cells were acquired through FACS respectively with PKH and7-AAD double staining, then the stemness of PKHhi cells were validated by serious sphere formation assay, chemoresistance assay, stem cells related genes detection and xenographt assay.2. PKHhi cells are distinct subpopulation of cancer stem cell.The expression of CSCs related cell surface markers was evaluated on PKHhi, PKHlow and PKHneg cells. It was remarkable that all markers were preferentially expressed in PKHhi cells (CD133, CD44+CD24+, ALDH1+), and these expressions were greatly decreased with the label intensity shedding. However, the cells with positive traditional markers expression just take a small fraction of PKHhi cells. Besides, Lgr5+cells were deemed to be CSCs’ subset with active dividing features. Then we compared the relationship between Lgr5+cells and PKHhi cells, the results revealed that PKHhi cells barely expressed Lgr5and vice-verse. Furthermore, continuous observation with confocal found the expression of Lgr5gradually increased in PKHhi cells which entered into the cell cycle. All the above evidence indicated quiescent cancer stem cell was a distinct subset of CSCs.3. IFN-y exerts specific cytotoxicity against QCCSCs.FCM analysis with Annexin-V and7-AAD staining showed the sphere formation of colon cancer cells were greatly inhibited with IFN-y treatment, which implied a specific therapeutic effect of IFN-y against colon CSCs. Therefore, we evaluated the sensitivity of PKHhi, PKHlow and PKHneg cells with chemotherapy (Oxaliplatin, Oxa) and IFN-y treatment respectively. Although PKHhi subset revealed dull to Oxa treatment, but it was found vulnerable to IFN-γ induced cell death, and PKHlow and PKHneg cells were found totally opposite result. Besides, both in vitro and in vivo experiments showed a synergistic effect of IFN-γ and Oxa, which could eradicate both QCCSCs and non-QCCSCs simultaneously.4. QCCSCs express higher levels of IFN-γ receptors.Upon the above results, we sought to determined the detailed mechanisms behind these responses. we evaluated IFN-yR expression in the PKHhi, PKHlow and PKHneg cells. The FCM analyses highlighted the different IFN-yRa and IFN-yRP expression in the three cell populations; expression of two IFN-yR subunits was considerably higher in the PKHhi subset but lower in the PKHIow and PKHneg subsets. Thereafter western-blot and confocal analysis found the IFN-y related apoptotic pathway was highly activated in PKHhi cells, and neutralization of IFN-y receptor markedly attenuated the different response among PKHhi, PKHlow and PKHneg cells. In conclusion, all these evidences strongly suggested that increased IFN-yR expression in the PKHhi subset played a key role in PKHhi cells’ sensitivity to the apoptotic function induced by IFN-y.5. miR-4666-3p is less expressed in QCCSCs.Compelling evidence showed epigenetic regulation was essential for cancer stem cells’ self-renewal, proliferation and differentiation, therefore, we tried to illuminate the underlying cause of the different expression of IFN-yR between QCCSCs and the rest parts with microRNA detection. Enormous differences were found within the microarray results of PKHhi cells and the rest parts. A microRNA that predictive targeted IFN-γRα/β, miR-4666-3p was found to be less expressed in PKHhi cells but much higher in the relative differentiated cells, which indicated its anti-CSC function. With western-blot analysis and dual luciferase reporter assay, we identified IFN-γRα/β to be a functional downstream target of miR-4666-3p in CRC.6miR-4666-3p suppresses the self-renewal and promotes the differentiation of QCCSCs.We established both miR-4666-3p and anti-miR-4666-3p stable colon cancer cell lines (SW620, P1) to study the biological functions of miR-4666-3p. The in vitro experiment showed miR-4666-3p over-expression could suppress the serious sphere formation of QCSCs and sternness gene expression, meanwhile potentiated its chemotherapy sensitivity and differentiation; besides, xenographt assay also found the anti-tumor effect of miR-4666-3p. All of these findings support the suppressive role of miR-4666-3p in CRC.ConclusionQuiescent cancer stem cells were deemed to be a distinct subset of cancer stem cell, and responsible for the cancer chemo-resistance and tumor relapse. Herein, we isolated QCCSCs via PKH cell membrane staining and serum free culture, thereafter a series of experiments verified its cancer stem cell-like characterisitics, which referred as QCCSCs. Meanwhile, we found QCCSCs expressed higher level of IFN-y receptors which induced its great sensitivity to the apoptotic effect of IFN-y. This intriguing phenomenon drove us to investigate the upstream mechanism, and we found miR-4666-3p which targeted both IFN-yRl and IFN-yR2, that specifically less expressed in PKHh1cells. Furthermore, miR-4666-3p showed the ability to suppress the self-renewal of QCCSCs and induced their differentiation. Our work not only provided a better understanding of the epigenetic regulatory mechanism in CCSCs, but also facilitate the gradual development of novel cancer therapeutic strategy.