Self-production Of CCL5 Promote Invasion And Migration Of Cancer Stem Cell In Ovarian Cancer

BackgroundOvarian cancer is the most lethal gynecological cancer and ranks as the fifth most common cause of cancer-related death in the word, about 70% of patients are diagnosis with stageâ…¢orâ…£ovarian cancer, which is characterize by distant metastases, so there is a need for a better understanding of the mechanisms involved in the spread of ovarian carcinoma. Growing evidence reveals that only a minority of cells within a tumor has the ability to propagate the disease. This rare fraction of self-renewing, and tumor initiating cells, termed as cancer stem cells (CSCs), solely CSCs are capable of generating tumor metastasis . However, how CSCs maintain the ability of invasion and migration, the most important properties of metastatic cells, still remains to be determined.Chemokines could specifically drive metastasis of CSCs. CSCs may indeed be involved in metastasis formation and, furthermore, be partially controlled by chemokine signaling.ObjectiveThe aim of this study is to determine the prognostic significance of chemokines and its receptors in migration and invasion ability of CSCs, and examine the correlation between self-produce of CCL5 and ovarian CSCs metastasis ability. Next we tested the possibility that the binding of self-production of CCL5 with its receptors on ovarian CSCs leading to NF-kappa B activation and MMP-9 upregulation is the main mechanism of metastatic property of ovarian cancer stem cells.Methods1. Ovarian CSCs were obtained from A2780 cell line by serum-free culture selection and from primary tumor tissues of ovarian cancer by CD133^-labeled maganetic activated cell sorting. CSCs were identified by stem cell surface marker (CD133), stem cell molecular marker (OCT-4, Nanog, etc), self-renewal, tumorgenesis in NOD/SCID mice. 2. The difference of chemokines and receptors expression between CSCs (A2780- derived CD133^-positive cells) and non-CSCs (A2780-derived CD133^-negative cells) were determined by human inflammation-related factots RT² Profiler^TM PCR Array, and then verified by real-time PCR and ELISA analysis in primary ovarian CSCs.3. Transwell experiments were used to determine the ability of invasion and migration of CSCs before and after blocking at different concentration of anti-CCL5, CCR1, CCR3, CCR5 antibody.4. Transwell experiments were used to determine the ability of invasion and migration of CSCs before and after blocking at different concentration of NF-kappa B inhibitor. ELISA array were used to determine the active of NF-kappa B before and after blocking of anti CCL5, CCR1, CCR3 antibody.5. MMP-9 expression in CSCs were tested with the use of real-time PCR before and after anti CCL5, CCR1, CCR3 antibody and inhibition of NF-kappa B.Results1. We obtained ovarian CSCs from A2780 cell line and cancer tissue defined by CD133 expression that displayed stem cell properties in vitro, such as self-renewing, anchorage-independent spheres; and highly tumorigenic in vivo. Ovarian CSCs had significant higher migration and invasive ability than that of common ovarian cancer cells2. The PCR-array data demonstrated that the expression of chemokines ligands (CCL2, CCL5, CLL27, CXCL16, etc. ) and chemokine receptors (CCR1, CCR3, CCR5, CXCR4, CCR7, etc ) are much stronger compared to non-CSCs. These upregultaed genes were successfully verified by real-time PCR, ELISA for chemokine ligands, flow cytometric analysis for chemokine receptors in primary ovarian cancer stem cells from five patients. Especially, the results of CCL5 and it's receptors (CCR1, CCR3 and CCR5) expression were consistent among five patients.3. The invasion and migration of CSCs could be blocked by anti-CCL5, CCR1 or CCR3, but not CCR5 blocking antibody, and indicated a dose-denpendent manner.4. Interestingly, we found the down-regulation of MMP-9 and NF-kappa B activity decrease in CSCs when blocking CCL5, CCR1 or CCR3, which is consistent to their effect on invasion and migration. Furthermore, NF-kappa B inhibitor not only significantly decreased the migration and invasion of CSCs, but also down-regulated MMP-9 expression in CSCs.Conclusions1. Here we demonstrated that CD133⁺ ovarian CSCs, isolated from primary cancer tissue or derived from A2780 cells, were highly tumorigenic and displayed the capacity for self-renewal. These results indicated that CD133 can be used as an independent CSC marker in ovarian cancer, consistent with previous reports. Most importantly, we have shown here that these CD133⁺ ovarian CSCs had significantly higher migratory and invasive abilities than non-CSCs2. We found that CCL5 and its receptors (CCR1, CCR3 and CCR5) showed significantly higher expression in ovarian CSCs than non-CSCs3. The effect of CCL5 on CSCs invasion and migration is predominantly mediated by CCR1 and CCR3.4. Our results suggest that the binding of self-production of CCL5 with CCR1 and CCR3 on ovarian cancer stem cells leading to NF-kappa B activation and MMP-9 upregulation is the main mechanism of metastatic property of ovarian cancer stem cells.5. Our results suggest that the binding of self-production of CCL5 with CCR1 and CCR3 on ovarian cancer stem cells leading to NF-kappa B activation and MMP-9 upregulation is the main mechanism of metastatic property of ovarian cancer stem cells.