Given the fact that astrocytes are capable of modulating biologic properties and behavior of cancer cells,12 we hypothesized that they could induce formation of brain metastases through suppression of KISS1 expression among BrCa cells, entering into the brain. is further supported by the ability of KISS1, ectopically overexpressed from an adenoviral vector in MDA-MB-231Br cells with silenced expression of the endogenous gene, to revert invasive phenotype of those cells. Taken together, our results strongly suggest that human adult astrocytes can promote brain invasion of the brain-localized circulating breast cancer cells by upregulating autophagy signaling pathways via the CXCL12-(KiSS-1 metastasis-suppressor) gene deserves special attention. This gene encodes a 145-amino acid (aa) precursor peptide that becomes cleaved into several short peptides of 104-, 13- and 14 aa in length. KISS1 inhibits growth and invasion of osteosarcoma5 and prostate cancer cells.6 Whereas deficiency of KISS1 expression in tumor tissues is associated with cancer progression,7 overexpression of this protein can suppresses the formation of metastases8 via molecular mechanisms involving KISS1R9 and CXCR410 receptors. Although, our group11 and others12 have found a significant reduction in KISS1 expression in BrCa metastases to the brain relative to primary BrCa tumors, the precise role of in the development and progression of brain metastases remains unknown. The objective of this study was to investigate the role of in modulating brain metastases and to reveal the upstream and the downstream effectors of downregulation. Development of brain metastases is a result of complex interplay between the tumor cells and the tumor environment, 13 which is represented predominantly by normal astrocytes in the brain tissue. Astrocytes regulate the brain response to inflammation,14 maintain brain homeostasis15 and provide protection of neurons from hypoxia.16 Conversely, reactive astrocytes can play a mitogenic role by secreting interleukins and chemokines, such as CCL2 and CXCL12/SDF1, respectively. The latter can serve as a chemoattractant for highly metastatic CXCR4+ cells. 17 Elevated levels of Acetylcholine iodide CCL2 and CXCL12 expression have also been linked to tumor progression and development of metastases.18 Although normal astrocytes have been linked with tumor progression,14 the role of these cells in brain metastases is still unclear. Here we provide the first evidence that normal astrocytes can promote brain metastases through downregulation of KISS1 and activation of the autophagy survival pathway Acetylcholine iodide in circulating BrCa cells. Results Primary tumors release KISS1-expressing cancer stem cells into the bloodstream BrCa is represented by highly heterogeneous tumor types,19 each containing a distinctive population of cancer cells with stem cell properties,20 resistance to conventional BrCa therapies,21 and capability of migrating22 and initiating metastases in the brain. We hypothesized that blood-circulating cancer stem cells (CSC) with a self-renewal property and a KISS1-deficient phenotype could give rise to metastatic foci in the brain. To identify and isolate a population of circulating tumor cells (CTCs), we used a previously described23 MDA-MB-231 metastatic model of human BrCa in Acetylcholine iodide mice.24 We observed a strong association between primary Acetylcholine iodide tumor growth and number of CTCs in the blood (Fig.?S1A to D). Using an in vitro tumorigenicity analysis we showed that CD24?/LOW and CD44+ cells exhibit a 7.2- and 1.48-fold higher potential to form tumors as compared with CD24+ and CD44+ or parental cells, respectively (Fig.?S1E, 0.05), which highlights their potential for forming secondary tumors. In addition, flow cytometry together with the ALDEFLUOR assay25 (Fig.?S1F) demonstrate that the CD24?/LOW and CD44+ population of CSCs isolated from blood (CTC) is metabolically active. We also observed that the CD24? and CD44+ population of CTCs isolated from nude mice with established MDA-MB-231 mammary tumor xenografts exhibits a 4.7-fold higher expression of mRNA (Fig.?S1G), compared with parental CSCs ( 0.05). There was, however, no correlation between the mRNA levels and those of the gene,26 implicated in priming BrCa cells to the brain (Fig.?S1H). Cancer stem cells, identified in BrCa brain metastases by CD44, CD24 and FLOT2 markers, express low level of KISS1 Our next step was to characterize expression of KISS1 in a small subset of metastatic cells called cancer stem cells (CSCs), identified by expression of stem cell markers CD44 and CD2427 in the CX3CL1 primary brain metastatic tissue. Given the low expression levels of KISS1 in BrCa brain metastases,11 we hypothesized that high expression of KISS1 in CTCs entering the brain could be locally modulated by the brain tissue environment. As evident from Fig.?1A, RONC1 and RONC-BM35 tumor specimens are composed of either distinct.