Immune Evasive Property Of And Differentiation Therapy Against Cancer Stem Cells

Malignant tumors are the leading causes of death around the world. The incidence of many types of malignancy keeps increasing in recent year, especially in developing countries, owing to the deteriorating environmental pollution. And this situation has been a great threat to the public health. Therapeutic failure in malignancies was attributed to our limited knowledge in the exact causes and mechanisms of tumorigenesis. Although genetic evidences supported monoclonal origin in tumors from most tissues, tumor cells were heterogeneous in aspects including microscopic morphology, gene expression profile, and responses to therapeutics.“Clonal evolution theory”of tumorigenesis holds that genetic and epigenetic changes accumulated randomly in progenies of a transformed progenitor cells and every single cell within tumor parenchyma was equally capable of tumor propagation. Therefore, every tumor cell should a targeted in tumor therapy and the main evaluation criteria for therapeutic effectiveness is shrinkage in tumor volume.However, the emerging“Cancer stem cell”model of tumorigenesis posted great challenge to the long-holed view in recent years. Cancer stem cells (CSCs) were defined as a subset of tumor cell within a tumor mass which was solely capable of regenerating the phenotype of parental tumors in serial transplantation in immune incompetent mice. Owing to its highly enriched tumorigenic potential, CSCs were also tentatively defined as tumor initiating cells (TICs). CSCs were differentiation blocked and most immature subpopulation of tumor cells. They shared many similar cellular characteristics with normal stem cell from the corresponding tissue, including indefinite self-renewal and multiple differentiations. At present, CSCs has been identified in a series of solid tumors using cell surface markers for normal stem cells. Among them, glioma stem cells and breast cancer stem cells are the most thoroughly defined CSCs.Identification of CSCs in solid tumors not only renewed the conventional theory of tumorigenesis but also challenged the current cancer therapeutic modalities. It was demonstrated that CSCs are resistant to chemotherapeutic agents and radiotherapy, therefore resulted therapeutic resistance after drug withdrawal in clinical settings. In addition, the existence of migrating/metastatic subgroup of CSCs in solid tumors rendered surgical resection unable to eliminate the culprit of tumor relapse. In light of this, CSCs are now regarded as a necessary and important cellular target in designing novel therapeutic maneuver against cancer. And much work is now being done to identify potential targets in CSCs and treatment approaches that have potential in eliminating CSCs. In this regard, successful treatment of acute myeloid leukemia (AML) by all trans retinoic acid provided a promising model in which AML CSCs can be eradicated by differentiation therapy. We presume that differentiation inducing agent against CSCs in solid tumors may also have potential for application in clinical settings. In addition, immune cells are capable of eliminating transformed cells based on molecular distinctions between malignant cells and corresponding normal cells. For example, Natural killer cells (NK), the main effector cell in innate immunity, can destroy tumor cells without preconditioning using tumor antigens, hence represent potential cytotoxic cell in adoptive infusion for cancer treatment. In the settings of breast cancer therapy, NK cells are important executor of antibody-dependent cellular cytotoxicity (ADCC) in treatment of Her positive breast cancer patients using trastuzumab (Herceptin). Clinical trials have shown that enhanced natural cytotoxicity in breast cancer patients were associated with good responses in trastuzumab therapy and improved tumor free survival. However, initially well-responding tumors often become resistant. It remains to be defined whether trastuzumab unresponsiveness is caused by resistance of breast CSCs to NK cell cytotoxicity.Therefore, this project focused on the exploration of therapeutic modalities targeting glioma stem cells and breast CSCs. The first section was to examine the differentiation inducing effect of Nordy on glioma stem cells and its underlying mechanisms of action. The second section was to test whether activated were capable of eliminating breast CSCs. We also explored the potential molecular target on which increased cell lysis of breast CSCs could be achieved by NK cells.A list of experiment data and main conclusions are as following.1. Isolation and characterization of CSCs from breast cancer and glioma(1) Flow cytometric sorting of CD44⁺CD24^- breast CSCs from MCF-7 cells and ALDH^high breast CSCs from primary breast tumor cell lines.?CD44⁺CD24^- and ALDH^high cells showed enhanced tumorosphere formation efficiency than their corresponding differentiated counterparts upon serial passage in serum free medium.?CD44⁺CD24^- cells were capable of forming more clones than non-CD44⁺CD24^- cells in vitro.?CD44⁺CD24^- cells showed preferential expression of stemness associated transcription factors Nanog?Oct4, and Sox2 and were absent in expression of luminal epithelial markers Muc1 and CK-18 and myoepithelial markers CK-14 and?-SMA.?CD44⁺CD24^- cells could be induced to express luminal and myoepithelial markers.?Subcutaneous injection of 5×103 CD44⁺CD24^- cells readily formed larger xenografts than 1×106 non-CD44⁺CD24^- cells.?Xenograft formed by CD44⁺CD24^- cell reproduced phenotype of parental luminal tumors.(2) Isolation and characterization of CSCs from primary glioma cells, glioma xenograft, and U87 cells.?Tumorospheres were obtained by culture in serum free medium, which showed high expression of glioma CSCs markers and stemness associated transcription factors and were capable of differentiation into multiple lineage of neuroepithelium.?Subcutaneous injection of 1×104 tumorospheres cells were capable of forming xenograft tumors, which resembled parental phenotype of gliomas and could be passaged serially in vivo.2. Lowered expression of MICA and MICB in breast CSC underlies its resistant properties to NK cell cytotoxicity(1) Resistance property of breast CSCs to NK cell cytotoxicity.?CD44⁺CD24^- cells were more capable of avoid clearance by NK cells in NOD/SCID mice or infused NK92 cells.?NK cell showed reduced cytotoxicity towards breast CSCs.?NK cell were readily attached with both breast CSCs and differentiated breast cancer cells.?NK cell cocultured with breast CSCs released less INF-?.?NK cell cocultured with breast CSCs showed reduced CD107a positive cells and expressed increased level of granzyme B.?After cycles of NK cell challenge, residual breast cancer cells showed increase percentages of CD44⁺CD24^- cells, ALDH^high and p-nanog/GFP+ cells respectively, which was concomitant with a higher tumorosphere formation efficiency.(2) Reduced MICA/B expression dominated differentially expressed NK cell activating ligands between breast CSCs and differentiated counterparts, resulting in resistant NK cell killing.?Inhibitory NK cell ligands HLA-ABC and HLA-E were expressed at a similar level between breast CSCs and differentiated counterparts.?Both breast CSCs and differentiated cells expressed various amounts of NKG2D ligands ULBP1-4 and MICA/B, NKp30/44/46 ligands, and DNAM-1 ligands Nectin-2 and PVR. However, Only MICA/B showed lowered expression in breast CSCs with other ligands expressed at a similar level between the two cellular populations.?Expression of MICA/B in breast CSCs increased profoundly after enhanced introduction using MICA/B expression vectors.?MICA/B overexpression breast CSCs stimulated more INF-?secretion in NK cells.?After cocultured with MICA/B overexpression breast CSCs, NK cell showed increased expression of CD107a and reduced intracellular granzyme B level.?MICA/B overexpression made breast CSCs sensitive to NK cell cytotoxicity.?MICA/B overexpression breast CSCs were readily destroyed by infused NK92 cells in NOD/SCID mice.3. Overexpression of miR-20a, miR-106a, and miR-320a repressed MICA/B expression in breast CSCs and contributed to its resistance to NK cell killing.(1) Overexpression of miR-20a, miR-106a, and miR-320a accounted for lowered MICA/B expression in breast CSCs.?Breast CSCs and differentiated cells expressed comparable level of MICA/B mRNAs.?miR-20a, miR-106a, and miR-320a were predicted to be regulating MICA/B expression by bioinformatics.?Breast CSCs expressed increase level of miR-20a, miR-106a, and miR-320a as compared with differentiated cells.?Dual luciferase assays confirmed that miR-20a and miR-106a can target MICA 3'UTR in a direct fashion, while miR-20a, miR-106a, and miR-320a can regulate MICB expression.?Inhibition of endogenous miRNA function in breast CSCs using anti miR-20a inhibitor and anti miR-106a inhibitor increased MICA protein expression, while miR-320a inhibitor transfection increased MICB expression.(2) MICA/B targeting miRNAs contributed to resistance of breast CSCs to NK cell killing.?Inhibition of either miR-20a, miR-106a, or miR-320a in breast CSCs increased INF-?secretion in NK cells, while MICA/B or NKG2D blocking antibodies abrogated the effect of anti-miRNA inhibitors.?After cocultured with anti-miRNA inhibitors transfected breast CSCs, NK cell showed increased expression of CD107a and reduced intracellular granzyme B level.?Anti-miRNA inhibitors transfected breast CSCs were sensitive to NK cell cytotoxicity.?Anti-miRNA inhibitors transfected breast CSCs showed increase clearance by infused NK92 cells in NOD/SCID mice.4. Differentiation inducing effect of Nordy on glioma CSCs(1) Nordy incubation induced astrocytic differentiation in glioma CSCs and reduced glioma CSCs frequency.?Nordy induced expression of an astrocytic marker GFAP, reduced mRNA expression level of stemness associated transcription factors, and resulted dynamic cell cycle changes characteristic of stem cell differentiation in glioma CSCs.?Nordy treated xenograft glioma cells in vitro showed reduced CD133⁺ fraction and tumorosphere formation efficiency.?Nordy was a potent inhibitor of arachidonate 5-lipoxygenase (ALOX-5) in vitro. ALOX-5 was preferentially expressed in glioma CSCs. Nordy incubation inhibited cell growth and clone formation in soft agar in glioma CSCs; however, these were rescued by treatment with an ALOX-5 metabolite LTB4. Shrinkage in CD133⁺ cells in xenograft glioma cells by Nordy in vitro was restored by incubation with LTB4.(2) Nordy inhibited tumorigeneicity of glioma CSCs and growth of glioma CSCs xenograft and induced a differentiation phenotype in xenograft glioma.?Glioma CSCs pretreated with Nordy in vitro showed prolonged tumor initiating latency, reduced tumor volume, a smaller pool of CD133⁺ cells, and a larger percentage of GFAP positive cells in NOD/SCID mouse.?Nordy injection in tumor bearing mice resulted in inhibited tumor growth, reduced CD133 positive fraction, and more GFPA positive cells.?BCNU treated xenografts showed tumor relapse on drug discontinuation, an increase in CD133⁺ cells, and less cells expressing GFAP.In conclusion, the above data suggested that(1) Breast CSCs were resistant to NK cell cytotoxicity both in vitro and in vivo. This was not due to inability of attach by NK cells, but reduced expression of NK cell activating ligands MICA/B in breast CSCs. Specific mi RNAs such as miR-20a, miR-106a, and miR-320a contributed reduced MICA/B expression in a 3'UTR dependent manner and resulted abrogated NK cell killing.(2) Nordy showed differentiation effect in glioma CSCs toward astrocytic lineage. ALOX-5 mediated the inhibitory effect of Nordy on cell growth and clone formation in glioma CSCs, and reduced CSCs frequency in xenograft cells. Nordy pretreatment reduced tumorigenic potential of glioma CSCs and inhibited growth of xenograft through direct elimination of glioma CSCs.