| Breast cancer is a common female malignant tumor. In recent years, despite the rapid development of tumor diagnosis and treatment technologies, the mortality of breast cancer patients remains high. High metastatic malignant tumor is still incurable. The cancer stem cell hypothesis has opened a new chapter of the understanding of tumor. This theory presumes that cancer stem cells have the property of self-renewal, unlimited growth and differentiation potential.Though of small proportion, it may play a vital role during the process of tumor generation, development, recurrence, resistance and escape from the recognition and elimination by the immune system. So, study of the characters of stem cells will enable us to understand the mechanism of recrudescence and metastasis of malignant tumor.During the process of tumor generation and development, it shows a very complicated relationship between the immune system and tumor. Along with research going thoroughly, we become more aware of the interaction between them with immune system remolding the immunogenicity of tumor cell and tumor cell influencing the functioning of immune system in return which is named cancer immunoediting. It is divided into three processes (3 Es): elimination, equilibrium and escape. Cancer immunoediting theory indicates that the immune system has not only the protective functions of tumor resistance but also the tumor-sculpting functions which act during immunoselection and pressures low immunogenicity tumor variants to escape and to grow further. NK cells constitute the important component of the innate immune system and form the first 1ine of defence against pathogens or host cells that are stressed or cancerous.The NK cells can directly eliminate tumor cells without pre-sensitization and thus play a vital role in early stage organism tumor resistance and immune surveillance. The cytotoxicity of NK cells on tumor cells is mainly regulated by the combination of the signals produced by stimulative receptors and inhibitive receptors. Latest researches indicate that the relationship between NK cells and cancer cells is much more than killing and being killed while these two cells contact, with the existence of immunoediting resulting in the changes of receptor-ligand molecules and biologic behavior in both sides. In this study, we use the mixed cultivation of NK cells supernatant and BCSCs as the reaction system to investigate the antineoplastic effects of NK cells on BCSCs, the molecular mechanism of immunoediting between BCSCs and NK cells.This paper aims to discover the interaction between NK cells and BCSCs and to reveal the new mechanism of tumor cell escaping from the NK cells mediated immune surveillance thus to provide a new direction in tumor therapy.The contents and conclusions of this research mainly include the following aspects:1. Isolation and identification of BCSCs from MCF-7We successfully isolated BCSCs from MCF-7 cell line through FACS and these cells cultured in stem cell medium.were capable of forming cell spheres. The cell spheres,on one hand, expressed high levels of Oct-4 and Sox-2 , on the other hand they could become differentiated cells with expression of CK-18 andα-SMA when cultured in differentiation conditions (medium containing serum). Moreover, CD44+CD24- cells initiated xenografts in nude mice. The tumors size ,inoculating with non-CD44+CD24- cells, were significantly smaller than that of CD44+CD24- cells.2. Isolation of Human primary NK cells from PBMCPrimary NK cells were sorted from PBMC through FACS, the purity of the isolated NK cells(CD3-CD56+) assessed by flow cytofluorimetric analysis was over 90%.3. Detection of the antineoplastic effects of NK cells on BCSCsThe culture supernatant collected from NK cells which had been cultured for 2 days was co-cultured with CD44+CD24- subset cells or non-CD44+CD24- subset cells for 2 days. On the respective conditions of the E:T ratio as 5:1 and 20:1, the killing efficiency of NK cells on tumor cells was detected by 51Cr releasing assay. The results indicated that the immunoedited tumor cells were much less sensitive to NK cells than control. This suggested that the cancer immunoediting of BCSCs toward NK cells had influenced the killing efficiency of NK cells.4. Detection of the stimulating efficiency of immunoedited BCSCs toward NK cellsTheγ-IFN secretion level of NK cells which had been co-cultured with the immunoedited BCSCs for 24 hours was detected by ELISA.Likewise the CD107a expressing level was analysed by flow cytometry. These results showed a decreasing ability for the immunoedited tumor cells to stimulate NK cells to secretγ-IFN and express CD107a.5. Detection of the expression of NK cells stimulative / inhibitive ligands on the surface of the immunoedited BCSCsHaving co-cultured with the culture supernatant of NK cells for respectively 24 hours and 48 hours, the expressing levels of relative molecular on the surface of the immunoedited BCSCs were detected by flow cytometry. The results demonstrated that immunoedting by NK cell culture supernatant leads to a down regulation of the expression of MICA and MICB,the major cellular ligands for NKG2D,on the surface of immunoedited BCSCs. However, there was no difference in the expression of HLA-I and ULBPs.6. Effect of MMPi during the process of cancer immunoediting of NK cells and BCSCsBy adding into MMPi during the immunoediting process, we found that it can partially restore the depressed expression of MICA/B on the surface of the immunoedited BCSCs. Keeping on treating with MMPi, the expression of MICA/B were up-regulated,which suggested that the MMPs secreted by BCSCs can also restrain the expression of MICA/B by itself. Based on that result, we further detected the influence of MMPi on NK cell cytotoxicity. The processing of MMPi on NK cells can partially restore the suppression of the NK cells cytotoxicity on immunoedited BCSCs.Besides,MMPi can also significantly reinforce the sensitivity of BCSCs to NK cells. These two aspects comprehensively indicated that MMPs expressed by NK cells and BCSCs all participate in reducing the expressing level and escaping the NK cytotoxicity.In summary, the mutual immunoediting between NK cells and BCSCs may generate on the basis of the cross reaction of the NKG2DL-NKG2D signal system. Our research primarily revealed the molecular mechanism of this immunoedting,however,further study is still needed to explore the mechanism of cancer immunoedting between NK cells and other tumor cells. |
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