Isolation, Characterization Of Drug-resistant Lung Cancer Stem Cells And Identification The Role Of Bmi-1 In Regulating Its Self-renewal And Proliferation

IntroductionA small population of cells within malignant organ overrides others in hierarchy. This population was termed cancer-initiating cells or cancer stem cells (CSCs) for their high capacity for self-renewal, multi-lineage differentiation, and superior levels of malignancy. CSCs, first identified in acute leukemia, have now been isolated from several human malignancies, such as breast, brain, prostate, and ovarian, and also in lung cancer.The initial isolation of lung cancer stem cells (LCSCs) relied on a basic knowledge of normal stem cells in the bronchioalveolar duct junction, assays for their functionality, and expression of specific markers Sca+ and CD34+ on their cell membranes that provided robust mechanisms for identification of these elusive cells within induced mouse lung cancer. Later, cells co-expressing CD133+/ EpCAM+ have been isolated from lung cancer patients and are defined as human LCSCs. In the absence of such specific channels for their identification, LCSCs were also identified utilizing flow-cytometry-based side population cells sorting from lung cancer cell lines. However, those methods do not provide a direct model to assess how cancer stem cells recovered from chemotherapy hit. Yet another approach was the floating spheres culture of chemotherapy-resistant cells within tumors that were subsequently shown to be enriched in LCSCs.Lung cancer is the leading cause of malignancy worldwide. While advancements in early diagnosis and treatment have been made in the hope of improving survival, recurrence remains a major obstacle in achieving cure. Reported recurrence rates after complete surgical resection ranges from 30% to 75%. Accumulating evidence suggest that CSCs are thought to be responsible for tumor regeneration after chemotherapy for their high drug resistance and tumorigenicity. The potential of CSCs to proliferate and self-renewal is marvelous. Even 10 cells of CSCs can recapitulate a tumor in mouse model. Obviously, some signaling pathways regulating CSCs self-renewal and proliferation have been abnormal.In the haematopoietic system as well as in other normal tissues, normal stem cells perform asymmetric cell division, with one daughter cell retaining the stem cell identity while the other transient amplifying cells (TACs) is committed to differentiate. TACs undergo symmetric cell division to generate terminally differentiated cell populations that are destined to acquire the same fate. The ability of stem cells to undergo asymmetric cell division as a way to self-renew is a tightly regulated process, but may be disrupted in hyper-proliferative disease states such as cancer. It has been shown that mammary CSCs abandon asymmetric division and adopt the symmetric division which accelerates the self-renewal of CSCs and propagating tumorigenesis.The factors that determine the self-renewing capacity of CSCs, and how they regulate division pattern, are not yet understood. Knowledge of how mutations in molecules that influence asymmetric stem cell division will provide insight into tumorigenesis. A subset of proteins important for regulating asymmetric stem cell division are known tumor suppressors expressed in both invertebrate and vertebrate systems. The tumor suppressor gene p53, frequently mutated and silenced in lung cancer, has been reported a role in promoting mammary CSCs asymmetric division. Loss of p53 may lead to symmetric division in mammary CSCs. This suggested a role of p53 in cell fate determination and CSCs expansion. p53 is under the control of the p53-inducible gene MDM2 which functions as a feedback regulator of p53 by binding and promoting p53 ubiquitination and degradation. MDM2 gene is regulated by P14ARF, which is in turn suppressed by the Bmi1 gene. Bmi1, a member of the Polycomb group (PcG) gene family of chromatin modifiers and transcriptional repressors, is required for the proliferation of various differentiated cells and for the self-renewal of stem cells. The deficiency of Bmi1 suppresses hematopoietic malignancy, and leukemic stem cells lacking Bmi1 fail to propagate disease.In this study, we try to elucidate the mechanisms governing the outrageous growth pattern of drug-selected LCSCs (dsLCSCs) enriched by chemotherapy combined with serum free culture. We demonstrate that Bmi1 is consistently over-expressed in dsLCSCs. We further show silencing of Bmi1 suppress the growth of dsLCSCs and promote asymmetric division, and this effect will be reversed by targeted p53 inbihition. Our study reveals that Bmi1 is involved in controlling cell division of dsLCSCs in a p53 dependent manner, providing insight into the mechanism controlling the self-renewal pattern of chemo-therapy resistant LCSCs.Besides, we also report the method we developed to supply autologous feeder cells for LCSCs culture. These feeder cells are themselves derived from the lung cancer lesions, namely, autologous intratumoral fibroblasts (AIFs). To ensure that this feeder layer fully meets its in vitro requirements, we compared the growth rate, undifferentiated state, pluripotency of LCSCs grown over a prolonged period on AIFs feeders or in traditional feeder free medium. The undifferentiated LCSCs were assessed by limiting dilution assay (LDA) and immunofluorescence microscopy for undifferentiated markers, while pluripotency was assessed by the capacity of the LCSCs to form xenograft on NOD/SCID mouse. We found that AIFs feeder cells maintained LCSCs in a compact morphology and undifferentiated state after a prolonged culture of 16 weeks. These autologous-derived fibroblast feeders can be easily accessed by researchers and aid the development of LCSCs culture system, thus promoting the in vitro culture of LCSCs.Methods:1. Using autologuous fibroblasts as feeders for CD133+/EpCAM+ cells culture: Isolate CD133+/EpCAM+ cells from three lung cancer specimen by flow cytometry, and purify autologous fibroblasts from the same surgical sample depending on their differential adhesion during culture. CD133+/EpCAM+ cells were transfered onto autologous fibroblasts feeder after 3d culture in vitro when small spheres formed. Growth rate, undifferentiated state, pluripotency of CD133+/EpCAM+ cells grown over a prolonged period on fibroblasts feeders or in traditional feeder free medium were compared.2. Isolation and identification of LCSCs from cisplatin-resistant A549 cells: A549 lung cancer stem cell line was treated with cisplatin for 3d and majorities of cells were dead, a small proportion of cell gradually recovered after 4 week of culture. Serum free culture was used to selectively'starve'non-stem cells, however, stem cells can form spheres in this specific conditioned medium. After 3 passages, the third spheres were collected and the stem cell properties were investigated including stem cell marker, self-renewal ability, pluripotency and xenograft formation.3. Explore the role of Bmi-1 in regulating dsLCSCs self-renewal and proliferation: Real-time PCR and immuno-blot were carried out to detect the expression pattern of Bmi-1 in dsLCSCs. A lentivirus vector with a siRNA targeted at Bmi-1 was constructed to specificly degenerate Bmi-1 mRNA and to get a stable infected cell line, namely, LCSCsBmi1-. The effect of Bmi-1 on Self-renewal and proliferation of dsLCSCs was investigated using WST-1 assay, sphere forming assay, and xenograft formation. In addition, cell cycle assay was also detected using flow cytometry after Bmi-1 silence in dsLCSCs.4. Investigate cell division pattern in LCSCs and LCSCsBmi1-: PKH 26 staining and Numb localization was used to deterimine the cell division pattern of LCSCs and LCSCsBmi1-. Stem cell marker CD133 immunostaining was also used to demonstrate the results of PKH 26 staining and Numb localization. A primary study was also carried out to investigate the mechanism of Bmi-1 in regulating cell division pattern by detecting the effect of a downstream molecule p53 on cell division pattern.Results:1. LCSCs isolated from three clinical samples all showed vigorous growth on feeder cells for 16 weeks of continuous cultures with a doubling time of 41 - 47 h. The cells continued expressing stem cell marker CD133 and remained undifferentiated. Pluripotency was demonstrated by tumor formation in immunodeficient mice. In a feeder-free culture system, growth of LCSCs spheres was retarded and would cease when the diameter reached 100μm if immediate passage was not performed. Moreover, spontaneous differentiation was more frequently seen in serum-free culture system.2. Drug-selected LCSCs (dsLCSCs) were then defined phenotypically and functionally by their expression of cell surface marker, CD133, ABCG2 and their capacity for self-renewal, multi-potent differentiation, and induction of xenograft tumors in vivo. We detected approximately 0.5~1.3% of A549 cells were CD133+, ~20% of DSCs were CD133+, and > 85% of dsLCSCs were CD133+ in flow cytometry assays. ABCG2 immunostaining showed a strong fluorescence signal in dsLCSCs compared with a medium and weak signal in DSCs and A549 cells under the same background. In self-renewal assay, 85.34% of wells with single cell from tumor spheres formed a new set of spheres, while only 23.81% of wells with single cell from DSCs and 1.63% of parental A549 cells were able to form spheres. CK8 and CK18 were not detectable in dsLCSCs untilled dsLCSCs was induced to differentiate. Most importantly, we found dsLCSCs were more tumorigenic than DSCs and A549 cells.3. Bmi-1 expression is up-regulated in dsLCSCs cells compared with A549 cells both in mRNA and protein level. Following knockdown of Bmi-1, cell proliferation, tumor sphere self-renewal, and tumor formation in NOD/SCID mice was reduced.4. Bmi-1 promotes symmetric cell division of dsLCSCs. These results appear to confirm the existence of a Bmi-1/p14ARF/MDM2/p53 signaling axis. Furthermore, by specifically inhibiting p53 using PFTα, the effects of Bmi-1 on cell division were rescued.Conclusion:It is hypothesized that CSCs represent a valuable target for the prevention of tumor relapse, in the present study; we establish an in vitro model to measure the expansion of drug-resistant lung cancer stem cells after cisplain treatment. The results of this study demonstrates that using cisplatin treatment and combined with serum free condition medium culture is able to selectively collect lung cancer stem cells. Furthermore, the enhanced proliferation and self-renewal properties of LCSCs were demonstrated to be a result of over-expression of Bmi-1 which enhanced symmetric division to expand the stem cell pool. And we also found Bmi-1 down-stream protein p53 was identified as specific targets for affecting the symmetric self-renewal of dsLCSCs which might the mechanism Bmi-1 affecting cell division.