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Syntenic genomic loci on human chromosome 8 and mouse chromosome 15 (mChr15) code for LY6/Ly6 (lymphocyte Ag 6) family proteins. The 23 murine Ly6 family genes include eight genes that are flanked by the murine Ly6e and Ly6l genes and form an Ly6 subgroup referred to in this article as the Ly6a subfamily gene cluster. Ly6a, also known as Stem Cell Ag-1 and T cell–activating protein, is a member of the Ly6a subfamily gene cluster. No LY6 genes have been annotated within the syntenic LY6E to LY6L human locus. We report in this article on LY6S, a solitary human LY6 gene that is syntenic with the murine Ly6a subfamily gene cluster, and with which it shares a common ancestry. LY6S codes for the IFN-inducible GPI-linked LY6S-iso1 protein that contains only 9 of the 10 consensus LY6 cysteine residues and is most highly expressed in a nonclassical spleen cell population. Its expression leads to distinct shifts in patterns of gene expression, particularly of genes coding for inflammatory and immune response proteins, and LY6S-iso1–expressing cells show increased resistance to viral infection. Our findings reveal the presence of a previously unannotated human IFN-stimulated gene, LY6S, which has a 1:8 ortholog relationship with the genes of the Ly6a subfamily gene cluster, is most highly expressed in spleen cells of a nonclassical cell lineage, and whose expression induces viral resistance and is associated with an inflammatory phenotype and with the activation of genes that regulate immune responses.

Soluble pulmonary factors have been reported to be capable of inhibiting the viability of cancer cells that metastasize to the lung, but the molecular identity was obscure. Here we report the isolation and characterization of the beta subunit of hemoglobin as a lung-derived antimetastatic factor. Peptide mapping in the beta subunit of human hemoglobin (HBB) defined a short C-terminal region (termed Metox) as responsible for activity. In tissue culture, both HBB and murine HBB2 mediated growth arrest and apoptosis of lung-metastasizing neuroblastoma cells, along with a variety of other human cancer cell lines. Metox acted similarly and its administration in human tumor xenograft models limited the development of adrenal neuroblastoma tumors as well as spontaneous lung and bone marrow metastases. Expression studies in mice indicated that HBB2 is produced by alveolar epithelial and endothelial cells and is upregulated in mice bearing undetectable metastasis. Our work suggested a novel function for HBB as a theranostic molecule: an innate antimetastasis factor with potential utility as an anticancer drug and a biomarker signaling the presence of clinically undetectable metastasis. Cancer Res; 77(1); 14–26. ©2016 AACR.

This file includes tables presenting quantitative Real-Time PCR oligonucleotide primers and chemically synthesized peptide segments of human HBB. It also contains figures presenting the influence of separated lung-derived factors on neuroblastoma cell viability, the beta subunit of hemoglobin is elevated in micrometastasis-bearing organs of inoculated mice, single channel confocal images of HBB2 expression in alveolar epithelial and endothelial cells, a high expression of HBB2 is seen in alveolar endothelial cells lining blood vessels of varied diameters in micrometastasis-bearing mice, the beta subunit isolated from native human hemoglobin inhibits neuroblastoma cell viability, human HBB and its derivative peptide Metox do not mediate hemolysis of red blood cells, an intravenous or intranasal treatment of Metox inhibits neuroblastoma local tumor growth and metastasis, and nitric oxide is not influenced by high HBB2 levels in the lungs and serum of micrometastasis-bearing mice.

This file includes supplemental experimental procedures. It includes a description of animals, antibodies, dialysis of lung-derived factors, in-vitro viability assays, blocking the inhibitory effect of lung-derived factors, quantitative Real-Time PCR, fixation and sectioning of frozen tissues, immunofluorescence of frozen tissue sections, protein extraction from mouse organs, western blotting, enzyme-Linked Immunosorbent assay, Isolation of primary endothelial and epithelial cells from mouse lungs, Incubation of HPF and HPEC with MicroNB cells in Transwell system, apoptosis assay, cell cycle analysis, ERK1/2, p38 and TAK1 phosphorylation, Solid-phase synthesis of human HBB segments, Conjugation of Metox peptide to FITC, Confocal microscopy for Metox cell entry, Flow cytometry for Metox cell entry, orthotopic inoculation of tumor cells, monitoring tumor growth and metastasis, RNA preparation and Reverse Transcription Polymerase Chain Reaction, quantitative real-time PCR, intravenous and intranasal treatment of Metox, hemolytic assay, and nitric oxide assay.

The tumor microenvironment (TME) is conceived as a dynamic interaction arena in which cancer cells interact with resident and recruited host cells, with their secreted factors and with the extracellular matrix (ECM). Moreover, non-cancerous cells in the TME interact with each other. The outcome of these multidirectional interactions is an ever-changing milieu in which the interaction partners acquire plasticity, altered phenotypes and signaling pathways. These TME events regulate, either positively or negatively, tumor survival, propagation and progression by a variety of mechanisms. It is up to the TME community to conceive and create novel means and technologies in order to target microenvironmental factors that impact, positively or negatively, tumor progression.

Cancer heterogeneity is a result of genetic mutations within the cancer cells. Their proliferation is not only driven by autocrine functions but also under the influence of cancer microenvironment, which consists of normal stromal cells such as infiltrating immune cells, cancer-associated fibroblasts, endothelial cells, pericytes, vascular and lymphatic channels. The relationship between cancer cells and cancer microenvironment is a critical one and we are just on the verge to understand it on a molecular level. Cancer microenvironment may serve as a selective force to modulate cancer cells to allow them to evolve into more aggressive clones with ability to invade the lymphatic or vascular channels to spread to regional lymph nodes and distant sites. It is important to understand these steps of cancer evolution within the cancer microenvironment towards invasion so that therapeutic strategies can be developed to control or stop these processes.

STAT1 is a critical effector and a target gene of interferon (IFN) signaling, and thus a central mediator of antiviral responses. As both a mediator and a target of IFN signals, STAT1 expression reports on, and determines IFN activity. Gene expression analyses of melanoma patient samples revealed varied levels of STAT1 expression, which highly correlated with expression of >700 genes. The ability of oncolytic viruses to exploit tumor-induced defects to antiviral responses suggests that oncolytic viruses may efficiently target a subset of melanomas, yet these should be defined. We modeled this scenario with murine B16F10 melanomas, immortalized skin fibroblasts as controls and a novel oncolytic virus, EHDV-TAU. In B16F10 cells, constitutive low expression of STAT1 and its target genes, which included intracellular pattern recognition receptors (PRRs), correlated with their inability to mount IFN-based antiviral responses upon EHDV-TAU challenge, and with potency of EHDV-TAU-induced oncolysis. This underexpression of interferon stimulated genes (ISGs) and PRRs, and the inability of EHDV-TAU to induce their expression, were reversed by epigenetic modifiers, suggesting epigenetic silencing as a basis for their underexpression. Despite their inability to mount IFN/STAT-based responses upon viral infection, EHDV-TAU infected B16F10 cells secreted immune-stimulatory chemokines. Accordingly, in vivo, EHDV-TAU enhanced intratumoral infiltration of cytotoxic T-cells and reduced growth of local and distant tumors. We propose that "STAT1 signatures" should guide melanoma virotherapy treatments, and that oncolytic viruses such as EHDV-TAU have the potential to exploit the cellular context of low-STAT1 tumors.

The conclusion derived from the information provided in this review is that disseminating tumor cells (DTC) collaborate with the microenvironment of a future metastatic organ site in the establishment of organ‐specific metastasis. We review the basic principles of site‐specific metastasis and the contribution of the cross talk between DTC and the microenvironment of metastatic sites (metastatic microenvironment [MME]) to the establishment of the organ‐specific premetastatic niche; the targeted migration of DTC to the endothelium of the future organ‐specific metastasis; the transmigration of DTC to this site and the seeding and colonization of DTC in their future MME. We also discuss the role played by DTC‐MME interactions on tumor dormancy and on the differential response of tumor cells residing in different MMEs to antitumor therapy. Finally, we summarize some studies dealing with the effects of the MME on a unique site‐specific metastasis—brain metastasis.

Melanoma metastasis to the brain is one of the most frequent extracranial brain tumors. Cell surface gangliosides are elevated in melanoma metastasis; however, the metabolic regulatory mechanisms that govern these specific changes are poorly understood in melanoma particularly brain metastases (MBM) development. We found ganglioside GD3 levels significantly upregulated in MBM compared to lymph node metastasis (LNM) but not for other melanoma gangliosides. Moreover, we demonstrated an upregulation of ST8SIA1 (GD3 synthase) as melanoma progresses from melanocytes to MBM cells. Using RNA‐ISH on FFPE specimens, we evaluated ST8SIA1 expression in primary melanomas (PRM) (n = 23), LNM and visceral metastasis (n = 45), and MBM (n = 39). ST8SIA1 was significantly enhanced in MBM compared to all other specimens. ST8SIA1 expression was assessed in clinically well‐annotated melanoma patients from multicenters with AJCC stage III B‐D LNM (n = 58) with 14‐year follow‐up. High ST8SIA1 expression was significantly associated with poor overall survival (HR = 3.24; 95% CI, 1.19–8.86, P = 0.02). In a nude mouse human xenograft melanoma brain metastasis model, MBM variants had higher ST8SIA1 expression than their respective cutaneous melanoma variants. Elevated ST8SIA1 expression enhances levels of cell surface GD3, a phenotype that favors MBM development, hence associated with very poor prognosis. Functional assays demonstrated that ST8SIA1 overexpression enhanced cell proliferation and colony formation, whereby ST8SIA1 knockdown had opposite effects. Icaritin a plant‐derived phytoestrogen treatment significantly inhibited cell growth in high GD3‐positive MBM cells through targeting the canonical NFκB pathway. The study demonstrates GD3 phenotype associates with melanoma progression and poor outcome., We demonstrated the upregulation of a ganglioside synthase, ST8SIA1 and GD3 expression in MBM, which can be suppressed by icaritin treatment. Icaritin inhibits NFκB‐mediated activation of ST8SIA1 by downregulating p50. These findings suggest that ST8SIA1 is a significant factor and potential target to MBM.

Syntenic genomic loci on human chromosome 8 (hChr8) and mouse chromosome 15 (mChr15) code for LY6/Ly6 (lymphocyte antigen 6) family proteins. The 23 murine Ly6 family genes include eight genes that are flanked by the murine Ly6e and Ly6l genes and form an Ly6 subgroup referred to here as the Ly6a subfamily gene cluster. Ly6a, also known as Sca1 (Stem Cell Antigen-1) and TAP (T-cell activating protein), is a member of the Ly6a subfamily gene cluster. No LY6 genes have been annotated within the syntenic LY6E to LY6L human locus. We report here on LY6S, a solitary human LY6 gene that is syntenic with the murine Ly6a subfamily gene cluster, and with which it shares a common ancestry. LY6S codes for the interferon-inducible GPI-linked LY6S-iso1 protein that contains only 9 of the 10 consensus LY6 cysteine residues and is most highly expressed in a non-classical cell population. Its expression leads to distinct shifts in patterns of gene expression, particularly of genes coding for inflammatory and immune response proteins, and LY6S-iso1 expressing cells show increased resistance to viral infection. Our findings reveal the presence of a previously un-annotated human interferon-stimulated gene, LY6S, which has a one to eight ortholog relationship with the genes of the Ly6a subfamily gene cluster, is most highly expressed in spleen cells of a non-classical cell-lineage and whose expression induces viral resistance and is associated with an inflammatory phenotype and with the activation of genes that regulate immune responses.One Sentence SummaryLY6S is a newly discovered human interferon-inducible gene associated with inflammation and with resistance to viral replication.

Cancer heterogeneity is a result of genetic mutations within the cancer cells. Their proliferation is not only driven by autocrine functions but also under the influence of cancer microenvironment, which consists of normal stromal cells such as infiltrating immune cells, cancer-associated fibroblasts, endothelial cells, pericytes, vascular and lymphatic channels. The relationship between cancer cells and cancer microenvironment is a critical one and we are just on the verge to understand it on a molecular level. Cancer microenvironment may serve as a selective force to modulate cancer cells to allow them to evolve into more aggressive clones with ability to invade the lymphatic or vascular channels to spread to regional lymph nodes and distant sites. It is important to understand these steps of cancer evolution within the cancer microenvironment towards invasion so that therapeutic strategies can be developed to control or stop these processes.

© The Author(s) 2021., Over the last decade, important clinical benefits have been achieved in cancer patients by using drug-targeting strategies. Nevertheless, drug resistance is still a major problem in most cancer therapies. Epithelial-mesenchymal plasticity (EMP) and tumour microenvironment have been described as limiting factors for effective treatment in many cancer types. Moreover, epithelial-to-mesenchymal transition (EMT) has also been associated with therapy resistance in many different preclinical models, although limited evidence has been obtained from clinical studies and clinical samples. In this review, we particularly deepen into the mechanisms of which intermediate epithelial/mesenchymal (E/M) states and its interconnection to microenvironment influence therapy resistance. We also describe how the use of bioinformatics and pharmacogenomics will help to figure out the biological impact of the EMT on drug resistance and to develop novel pharmacological approaches in the future., AF group has been supported by Plan Estatal I + D + i 2013 − 2016, co-funded by the Instituto de Salud Carlos III (ISCIII, Spain) under grant agreements PI13/00250 and PI18/00121 by Fondo Europeo de Desarrollo Regional (FEDER) “A way of Making Europe”; by “la Caixa” Foundation (ID 100010434) under the agreement (LCF/TR/CI19/52460016); by PRIS3 project from ACIS and Consolidation of Competitive Research (IN607B2020/14) from GAIN, both from Xunta de Galicia and by I.M.Q. San Rafael Foundation (A Coruña). The studies of IPW are supported by the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (Needham, MA, USA), the Sara and Natan Blutinger Foundation (West Orange, NJ, USA), the Fred August and Adele Wolpers Charitable Fund (Clifton, NJ, USA) and the James and Rita Leibman Endowment Fund for Cancer Research (New York, NY, USA). AB group is supported by the Croatian Science Foundation (CSF, Project No. IP-2016–06-1036). JDLR group is funded by the Instituto de Salud Carlos III (ISCIII, AES, Spain) with grants PI18/00591 and PT17/0009/0008, co-financed by the European Regional Development Fund (FEDER).

The melanoma‐microglia cross‐talk upregulates the expression of aldolase C (ALDOC) in melanoma cells from several patients. The response of melanoma cells from 2 different patients to ALDOC upregulation was diametrically divergent. Whereas several metastasis‐associated functions including brain metastasis formation were augmented in one melanoma cell line (Mel 1), these functions were attenuated in the other melanoma cell line (Mel 2)., Previous studies indicated that microglia cells upregulate the expression of aldolase C (ALDOC) in melanoma cells. The present study using brain‐metastasizing variants from three human melanomas explores the functional role of ALDOC in the formation and maintenance of melanoma brain metastasis (MBM). ALDOC overexpression impacted differentially the malignant phenotype of these three variants. In the first variant, ALDOC overexpression promoted cell viability, adhesion to and transmigration through a layer of brain endothelial cells, and amplified brain micrometastasis formation. The cross‐talk between this MBM variant and microglia cells promoted the proliferation and migration of the latter cells. In sharp contrast, ALDOC overexpression in the second brain‐metastasizing melanoma variant reduced or did not affect the same malignancy features. In the third melanoma variant, ALDOC overexpression augmented certain characteristics of malignancy and reduced others. The analysis of biological functions and disease pathways in the ALDOC overexpressing variants clearly indicated that ALDOC induced the expression of tumor progression promoting genes in the first variant and antitumor progression properties in the second variant. Overall, these results accentuate the complex microenvironment interactions between microglia cells and MBM, and the functional impact of intertumor heterogeneity. Since intertumor heterogeneity imposes a challenge in the planning of cancer treatment, we propose to employ the functional response of tumors with an identical histology, to a particular drug or the molecular signature of this response, as a predictive indicator of response/nonresponse to this drug.

Granulocyte-monocyte colony stimulating factor (GM-CSF) is used as an adjuvant in various clinical and preclinical studies with contradictory results. These were attributed to opposing effects of GM-CSF on the immune or myeloid systems of the treated patients or to lack of optimal dosing regimens. The results of the present study point to inter-tumor heterogeneity as a possible mechanism accounting for the contrasting responses to GM-CSF incorporating therapies. Employing xenograft models of human melanomas in nude mice developed in our lab, we detected differential functional responses of melanomas from different patients to GM-CSF both in vitro as well as in vivo. Whereas cells of one melanoma acquired pro metastatic features following exposure to GM-CSF, cells from another melanoma either did not respond or became less malignant. We propose that inter-melanoma heterogeneity as manifested by differential responses of melanoma cells (and perhaps also of other tumor) to GM-CSF may be developed into a predictive marker providing a tool to segregate melanoma patients who will benefit from GM-CSF therapy from those who will not.

Melanoma has the highest propensity to metastasize to the brain compared to other cancers, as brain metastases are found frequently high in patients who have prolonged survival with visceral metastasis. Once disseminated in the brain, melanoma cells communicate with brain resident cells that include astrocytes and microglia. Microglia cells are the resident macrophages of the brain and are the main immunological cells in the CNS involved in neuroinflammation. Data on the interactions between brain metastatic melanoma cells and microglia and on the role of microglia-mediated neuroinflammation in facilitating melanoma brain metastasis are lacking. To elucidate the role of microglia in melanoma brain metastasis progression, we examined the bidirectional interactions between microglia and melanoma cells in the tumor microenvironment. We identified the molecular and functional modifications occurring in brain-metastasizing melanoma cells and microglia cells after the treatment of each cell type with supernatants of the counter cell type. Both cells induced alteration in gene expression programs, cell signaling, and cytokine secretion in the counter cell type. Moreover, melanoma cells exerted significant morphological changes on microglia cells, enhanced proliferation, induced matrix metalloproteinase-2 (MMP-2) activation, and cell migration. Microglia cells induced phenotypic changes in melanoma cells increasing their malignant phenotype: increased melanoma proliferation, MMP-2 activity, cell migration, brain endothelial penetration, and tumor cells ability to grow as spheroids in 3D cultures. Our work provides a novel insight into the bidirectional interactions between melanoma and micoglia cells, suggesting the contribution of microglia to melanoma brain metastasis formation.

The concept that progression of cancer is regulated by interactions of cancer cells with their microenvironment was postulated by Stephen Paget over a century ago. Contemporary tumour microenvironment (TME) research focuses on the identification of tumour-interacting microenvironmental constituents, such as resident or infiltrating non-tumour cells, soluble factors and extracellular matrix components, and the large variety of mechanisms by which these constituents regulate and shape the malignant phenotype of tumour cells. In this Timeline article, we review the developmental phases of the TME paradigm since its initial description. While illuminating controversies, we discuss the importance of interactions between various microenvironmental components and tumour cells and provide an overview and assessment of therapeutic opportunities and modalities by which the TME can be targeted. In this Timeline article, Maman and Witz describe how much progress has been made in understanding how the tumour microenvironment influences tumour progression since its initial description, highlighting the controversies in the field and the potential of targeting components of the microenvironment for cancer therapy.

// Sivan Izraely 1 , Shlomit Ben-Menachem 1 , Orit Sagi-Assif 1 , Tsipi Meshel 1 , Diego M. Marzese 2 , Shuichi Ohe 2 , Inna Zubrilov 1 , Metsada Pasmanik-Chor 3 , Dave S.B. Hoon 2 and Isaac P. Witz 1 1 Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel 2 Department of Translational Molecular Medicine, John Wayne Cancer Institute at Providence Saint John’s Health Center, Santa Monica, CA, USA 3 Bioinformatics Unit, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Correspondence to: Isaac P. Witz, email: // Keywords : ANGPTL4, melanoma, brain, metastasis, TGFβ1 Received : March 15, 2017 Accepted : June 10, 2017 Published : July 05, 2017 Abstract In an ongoing effort to identify molecular determinants regulating melanoma brain metastasis, we previously identified Angiopoietin-like 4 (ANGPTL4) as a component of the molecular signature of such metastases. The aim of this study was to determine the functional significance of ANGPTL4 in the shaping of melanoma malignancy phenotype, especially in the establishment of brain metastasis. We confirmed that ANGPTL4 expression is significantly higher in cells metastasizing to the brain than in cells from the cutaneous (local) tumor from the same melanoma in a nude mouse xenograft model, and also in paired clinical specimens of melanoma metastases than in primary melanomas from the same patients. In vitro experiments indicated that brain-derived soluble factors and transforming growth factor β1 (TGFβ1) up-regulated ANGPTL4 expression by melanoma cells. Forced over-expression of ANGPTL4 in cutaneous melanoma cells promoted their ability to adhere and transmigrate brain endothelial cells. Over-expressing ANGPTL4 in cells derived from brain metastases resulted in the opposite effects. In vivo data indicated that forced overexpression of ANGPTL4 promoted the tumorigenicity of cutaneous melanoma cells but did not increase their ability to form brain metastasis. This finding can be explained by inhibitory activities of brain-derived soluble factors. Taken together these findings indicate that ANGPTL4 promotes the malignancy phenotype of primary melanomas of risk to metastasize to the brain.

Proteome profiling data of eight xenografted cutaneous and cerebellar metastasis of human melanoma cells were combined with eicosanoid and glutathione measurements as well as multiparametric morphologic analyses and immunofluorescence. The analyses revealed significant differences in the expression of molecules associated with metastatic properties, while lacking any commonalties when comparing the metastatic variants from four different donors. Apparently we lack appropriate meta-analysis strategies making use of the large number of presently identified metastasis-associated molecules for successful classification of cells., Graphical Abstract Highlights Comprehensive molecular profiling of cutaneous and cerebellar metastasis variants. Identification of differentially regulated metastasis-associated molecules. Evidence for individually distinct patterns of metastasis-associated molecules. Highlighting the evident need for establishing meta-analyses strategies., The prediction of metastatic properties from molecular analyses still poses a major challenge. Here we aimed at the classification of metastasis-related cell properties by proteome profiling making use of cutaneous and brain-metastasizing variants from single melanomas sharing the same genetic ancestry. Previous experiments demonstrated that cultured cells derived from these xenografted variants maintain a stable phenotype associated with a differential metastatic behavior: The brain metastasizing variants produce more spontaneous micro-metastases than the corresponding cutaneous variants. Four corresponding pairs of cutaneous and metastatic cells were obtained from four individual patients, resulting in eight cell-lines presently investigated. Label free proteome profiling revealed significant differences between corresponding pairs of cutaneous and cerebellar metastases from the same patient. Indeed, each brain metastasizing variant expressed several apparently metastasis-associated proteomic alterations as compared with the corresponding cutaneous variant. Among the differentially expressed proteins we identified cell adhesion molecules, immune regulators, epithelial to mesenchymal transition markers, stem cell markers, redox regulators and cytokines. Similar results were observed regarding eicosanoids, considered relevant for metastasis, such as PGE2 and 12-HETE. Multiparametric morphological analysis of cells also revealed no characteristic alterations associated with the cutaneous and brain metastasis variants. However, no correct classification regarding metastatic potential was yet possible with the present data. We thus concluded that molecular profiling is able to classify cells according to known functional categories but is not yet able to predict relevant cell properties emerging from networks consisting of many interconnected molecules. The presently observed broad diversity of molecular patterns, irrespective of restricting to one tumor type and two main classes of metastasis, highlights the important need to develop meta-analysis strategies to predict cell properties from molecular profiling data. Such base knowledge will greatly support future individualized precision medicine approaches.

Background: Intersecting a genome-wide expression profile of metastatic and nonmetastatic human neuroblastoma xenograft variants with expression profiles of tumours from stage 1 and 4 neuroblastoma patients, we previously characterised hexokinase 2 (HK2) as a gene whose expression was upregulated in both metastatic neuroblastoma variants and tumours from stage 4 neuroblastoma patients. Methods: Local and metastatic neuroblastoma cell variants as well as metastatic neuroblastoma cells genetically manipulated to downregulate the expression of HK2 were utilised for in vitro and in vivo examinations of the involvement of HK2 in neuroblastoma. Results: Hexokinase 2 expression and its activity levels were increased in neuroblastoma metastatic variants as compared with the local variants. The upregulation of HK2 confers upon the metastatic cells high resistance to the antiproliferative effect of the HK2 inhibitor 3-BrPa and to the chemotherapy agent Deferoxamine. The inhibition of HK2 transcript lowered the proliferation and motility of sh-HK2 cells as compared with sh-control cells. Mice that were inoculated with sh-HK2 cells had a lower incidence of local tumours, smaller tumour volumes and a diminished load of lung metastasis compared with mice inoculated with sh-control cells. Conclusions: Hexokinase 2 plays a significant role in shaping the malignant phenotype of neuroblastoma and influences the progression of this disease.

Paired like homeobox 2B (PHOX2B) is a minimal residual disease (MRD) marker of neuroblastoma. The presence of MRD, also referred to as micro-metastases, is a powerful marker of poor prognosis in neuroblastoma. Lung metastasis is considered a terminal event in neuroblastoma. Lung micro-metastatic neuroblastoma (MicroNB) cells show high expression levels of PHOX2B and possess a less malignant and metastatic phenotype than lung macro metastatic neuroblastoma (MacroNB) cells, which hardly express PHOX2B. In vitro assays showed that PHOX2B knockdown in MicroNB cells did not affect cell viability; however it decreased the migratory capacity of the MicroNB-shPHOX2B cells. An orthotopic inoculation of MicroNB-shPHOX2B cells into the adrenal gland of nude mice resulted in significantly larger primary tumors and a heavier micro-metastatic load in the lungs and bone-marrow, than when control cells were inoculated. PHOX2B expression was found to be regulated by methylation. The PHOX2B promoter in MacroNB cells is significantly more methylated than in MicroNB cells. Demethylation assays using 5-azacytidine demonstrated that methylation can indeed inhibit PHOX2B transcription in MacroNB cells. These pre-clinical data strongly suggest that PHOX2B functions as a suppressor of neuroblastoma progression.

Neural repair after stroke involves initiation of a cellular proliferative program in the form of angiogenesis, neurogenesis, and molecular growth signals in the surrounding tissue elements. This cellular environment constitutes a niche in which regeneration of new blood vessels and new neurons leads to partial tissue repair after stroke. Cancer metastasis has similar proliferative cellular events in the brain and other organs. Do cancer and CNS tissue repair share similar cellular processes? In this study, we identify a novel role of the regenerative neurovascular niche induced by stroke in promoting brain melanoma metastasis through enhancing cellular interactions with surrounding niche components. Repair-mediated neurovascular signaling induces metastatic cells to express genes crucial to metastasis. Mimicking stroke-like conditions in vitro displays an enhancement of metastatic migration potential and allows for the determination of cell-specific signals produced by the regenerative neurovascular niche. Comparative analysis of both in vitro and in vivo expression profiles reveals a major contribution of endothelial cells in mediating melanoma metastasis. These results point to a previously undiscovered role of the regenerative neurovascular niche in shaping the tumor microenvironment and brain metastatic landscape.

The development of melanoma brain metastasis is largely dependent on mutual interactions between the melanoma cells and cells in the brain microenvironment. Here, we report that the extracellular cysteine protease inhibitor cystatin C (CysC) is involved in these interactions. Microglia-derived factors upregulated CysC secretion by melanoma. Similarly, melanoma-derived factors upregulated CysC secretion by microglia. Whereas CysC enhanced melanoma cell migration through a layer of brain endothelial cells, it inhibited the migration of microglia cells toward melanoma cells. CysC was also found to promote the formation of melanoma three-dimensional structures in matrigel. IHC analysis revealed increased expression levels of CysC in the brain of immune-deficient mice bearing xenografted human melanoma brain metastasis compared to the brain of control mice. Based on these in vitro and in vivo experiments we hypothesize that CysC promotes melanoma brain metastasis. Increased expression levels of CysC were detected in the regenerating brain of mice after stroke. Post-stroke brain with melanoma brain metastasis showed an even stronger expression of CysC. The in vitro induction of stroke-like conditions in brain microenvironmental cells increased the levels of CysC in the secretome of microglia cells, but not in the secretome of brain endothelial cells. The similarities between melanoma brain metastasis and stroke with respect to CysC expression by and secretion from microglia cells suggest that CysC may be involved in shared pathways between brain metastasis and post-stroke regeneration. This manifests the tendency of tumor cells to highjack physiological molecular pathways in their progression.

Melanoma is the leading cause of skin cancer mortality. The major cause of melanoma mortality is metastasis to distant organs, frequently to the brain. The microenvironment plays a critical role in tumourigenesis and metastasis. In order to treat or prevent metastasis, the interactions of disseminated tumour cells with the microenvironment at the metastatic organ have to be elucidated. However, the role of brain stromal cells in facilitating metastatic growth is poorly understood. Astrocytes are glial cells that function in repair and scarring of the brain following injury, in part via mediating neuroinflammation, but the role of astrocytes in melanoma brain metastasis is largely unresolved. Here we show that astrocytes can be reprogrammed by human brain-metastasizing melanoma cells to express pro-inflammatory factors, including the cytokine IL-23, which was highly expressed by metastases-associated astrocytes in vivo. Moreover, we show that the interactions between astrocytes and melanoma cells are reciprocal: paracrine signalling from astrocytes up-regulates the secretion of the matrix metalloproteinase MMP2 and enhances the invasiveness of brain-metastasizing melanoma cells. IL-23 was sufficient to increase melanoma cell invasion, and neutralizing antibodies to IL-23 could block this enhanced migration, implying a functional role for astrocyte-derived IL-23 in facilitating the progression of melanoma brain metastasis. Knocking down the expression of MMP2 in melanoma cells resulted in inhibition of IL-23-induced invasiveness. Thus, our study demonstrates that bidirectional signalling between melanoma cells and astrocytes results in the formation of a pro-inflammatory milieu in the brain, and in functional enhancement of the metastatic potential of disseminated melanoma cells.

BRAF mutations are frequent in cutaneous melanomas, and BRAF inhibitors (BRAFi) have shown remarkable clinical efficacy in BRAF mutant melanoma patients. However, acquired drug resistance can occur rapidly and tumor(s) often progresses thereafter. Various mechanisms of BRAFi resistance have recently been described; however, the mechanism of resistance remains controversial. In this study, we developed BRAFi-resistant melanoma cell lines and found that metastasis-related epithelial to mesenchymal transition properties of BRAFi-resistant cells were enhanced significantly. Upregulation of EGFR was observed in BRAFi-resistant cell lines and patient tumors because of demethylation of EGFR regulatory DNA elements. EGFR induced PI3K/AKT pathway activation in BRAFi-resistant cells through epigenetic regulation. Treatment of EGFR inhibitor was effective in BRAFi-resistant melanoma cell lines. The study demonstrates that EGFR epigenetic activation has important implications in BRAFi resistance in melanoma.

International audience; We previously identified the chemokine receptor CCR4 as part of the molecular signature of melanoma brain metastasis. The aim of this study was to determine the functional significance of CCR4 in melanoma brain metastasis. We show that CCR4 is more highly expressed by brain metastasizing melanoma cells than by local cutaneous cells from the same melanoma. Moreover, we found that the expression of CCR4 is significantly higher in paired clinical specimens of melanoma metastases than in samples of primary tumors from the same patients. Notably, the expression of the CCR4 ligands, Ccl22 and Ccl17 is upregulated at the earliest stages of brain metastasis, and precedes the infiltration of melanoma cells to the brain. In-vitro, CCL17 induced migration and transendothelial migration of melanoma cells. Functionally, human melanoma cells over-expressing CCR4 were more tumorigenic and produced a higher load of spontaneous brain micrometastasis than control cells. Blocking CCR4 with a small molecule CCR4 antagonist in-vivo, reduced the tumorigenicity and micrometastasis formation of melanoma cells. Taken together, these findings implicate CCR4 as a driver of melanoma brain metastasis.

P-REX1 (PIP3-dependent Rac exchange factor-1) is a guanine nucleotide exchange factor that activates Rac by catalyzing exchange of GDP for GTP bound to Rac. Aberrant up-regulation of P-REX1 expression has a role in metastasis however, copy number (CN) and function of P-REX1 in cutaneous melanoma are unclear. To explore the role of P-REX1 in melanoma, SNP 6.0 and Exon 1.0 ST microarrays were assessed. There was a higher CN (2.82-fold change) of P-REX1 in melanoma cells than in melanocytes, and P-REX1 expression was significantly correlated with P-REX1 CN. When P-REX1 was knocked down in cells by P-REX1 shRNA, proliferation, colony formation, 3D matrigel growth, and migration/invasiveness were inhibited. Loss of P-REX1 inhibited cell proliferation by inhibiting cyclin D1, blocking cell cycle, and increased cell apoptosis by reducing expression of the protein survivin. Knockdown of P-REX1 expression inhibited cell migration/invasiveness by disrupting P-REX1/RAC1/PAK1/p38/MMP-2 pathway. Assessment of patient tumors and disease outcome demonstrated lower distant metastasis-free survival among AJCC stage I/II/III patients with high P-REX1 expression compared to patients with low P-REX1 expression. These results suggest P-REX1 plays an important role in tumor progression and a potential theranostic target.

Brain metastases occur frequently in melanoma patients with advanced disease whereby the prognosis is dismal. The underlying mechanisms of melanoma brain metastasis development are not well understood. Identification of molecular determinants regulating melanoma brain metastasis would advance the development of prevention and therapy strategies for this disease. Gene expression profiles of cutaneous and brain-metastasizing melanoma variants from three xenograft tumor models established in our laboratory revealed that expression of tight junction component CLDN1 was lower in the brain-metastasizing variants than in cutaneous variants from the same melanoma. The objective of our study was to determine the significance of CLDN1 downregulation/loss in metastatic melanoma and its role in melanoma brain metastasis. An immunohistochemical analysis of human cells of the melanocyte lineage indicated a significant CLDN1 downregulation in metastatic melanomas. Transduction of melanoma brain metastatic cells expressing low levels of CLDN1 with a CLDN1 retrovirus suppressed their metastatic phenotype. CLDN1-overexpressing melanoma cells expressed a lower ability to migrate and adhere to extracellular matrix, reduced tumor aggressiveness in nude mice and, most importantly, eliminated the formation of micrometastases in the brain. In sharp contrast, the ability of the CLDN1-overexpressing cells to form lung micrometastases was not impaired. CLDN1-mediated interactions between these cells and brain endothelial cells constitute the mechanism underlying these results. Taken together, we demonstrated that downregulation or loss of CLDN1 supports the formation of melanoma brain metastasis, and that CLDN1 expression could be a useful prognostic predictor for melanoma patients with a high risk of brain metastasis.

Recent data suggest that the mechanisms determining whether a tumor cell reaching a secondary organ will enter a dormant state, progress toward metastasis, or go through apoptosis are regulated by the microenvironment of the distant organ. In neuroblastoma, 60-70% of children with high-risk disease will ultimately experience relapse due to the presence of micrometastases. The main goal of this study is to evaluate the role of the lung microenvironment in determining the fate of neuroblastoma lung metastases and micrometastases. Utilizing an orthotopic mouse model for human neuroblastoma metastasis, we were able to generate two neuroblastoma cell populations-lung micrometastatic (MicroNB) cells and lung macrometastatic (MacroNB) cells. These two types of cells share the same genetic background, invade the same distant organ, but differ in their ability to create metastasis in the lungs. We hypothesize that factors present in the lung microenvironment inhibit the propagation of MicroNB cells preventing them from forming overt lung metastasis. This study indeed shows that lung-derived factors significantly reduce the viability of MicroNB cells by up regulating the expression of pro-apoptotic genes, inducing cell cycle arrest and decreasing ERK and FAK phosphorylation. Lung-derived factors affected various additional progression-linked cellular characteristics of neuroblastoma cells, such as the expression of stem-cell markers, morphology, and migratory capacity. An insight into the microenvironmental effects governing neuroblastoma recurrence and progression would be of pivotal importance as they could have a therapeutic potential for the treatment of neuroblastoma residual disease.

Brain metastasis confers an extremely unfavorable prognosis upon melanoma patients. The mechanisms underlying the homing of metastatic melanoma to the brain and survival of metastatic melanoma cells in the brain are unknown. Tumor cells, including melanoma, use chemokine receptor-ligand axes to home to specific organ sites. To identify chemokine receptors that might be involved in brain-targeted melanoma metastasis, we first established a chemokine receptor profile of cultured melanoma cells (3 cell lines of cutaneous melanoma and 5 cell lines of melanoma brain metastasis). The expression of the membrane-bound chemokine CX3CL1 by these lines was also determined. We show that out of 19 receptors tested, cultured melanoma cells express CCR3, CCR4, CXCR3, CXCR7, CX3CR1 and membrane CX3CL1. Utilizing cells from newly created variants of human melanoma xenografts, we found that the expression of CCR4 was significantly higher in one brain metastatic variant compared to its expression in the corresponding local variant. Local and metastatic variants stimulated with the CCR4 ligand, CCL22, showed a differential AKT phosphorylation pattern. These findings may suggest the involvement of CCR4 in the process of brain metastasis in human melanoma, and that CCR4 may be a novel molecular biomarker for the identification of melanoma cells likely to metastasize to the brain.

What has been will be again, what has been done will be done again; there is nothing new under the sun (Ecclesiastes 1:9) Stephen Paget was the conceptual father of the role played by the Tumor Microenvironment (TME) in tumor progression. The focus of this essay is the developmental phase of the post Paget TME research. Attempts will be made to highlight some of the pioneering work of scientists from the late sixties through the eighties of last century who laid the foundations for the contemporary scientific achievements of TME research but whose ground breaking studies are rarely cited. This review should serve as a small tribute to their great work.

This review will document that the selectin-selectin ligand axis is actively involved in tumor progression and drives this process. The involvement of selectins and their ligands in tumor progression takes place at three levels which will be reviewed: Interaction of tumor cells with platelets and leukocytes resulting in the formation of circulating emboli; interaction of tumor cells with endothelial cells leading to extravasation of the tumor cells; and utilization of reciprocal pro malignancy signals delivered by the selectins or by their ligands to interacting cells that express the corresponding co-receptor. We propose that the selectin-selectin ligand mediated interactions between cells in the tumor microenvironment constitute an axis of evil, that it be included in the list of pro malignancy factors, and that molecules associated with this axis serve as targets for cancer therapy.

A solid tumor is an ecosystem composed of tumor cells, resident and infiltrating nontumor cells, and molecules present in proximity to these cells. This ecosystem can be collectively described as the tumor microenvironment. Both the tumor cells as well as the neighboring nontumor cells take part in

CXCL10 was recently shown to exert antimalignancy functions by influencing the tumor microenvironment. Here, we have taken a different approach, investigating the effects of CXCL10 directly on tumor-promoting functions in colorectal carcinoma (CRC) cells. CXCL10 expression was detected in preferred metastatic sites of CRC (liver, lungs, and lymph nodes), and its CXCR3 receptor was expressed by eight CRC cell lines (detected: reverse transcription-PCR and/or flow cytometry). Detailed analysis was done on two cell lines derived from primary CRC tumors (SW480, KM12C) and their metastatic descendents (SW620 and KM12SM). The three known variants of CXCR3 (CXCR3-A, CXCR3-B, and CXCR3-alt) were detected in all four cell lines. CXCR3 expression was also observed on colorectal tumor cells in biopsies of CRC patients (immunohistochemistry). CXCL10 and CXCR3 expression were potently induced in CRC cells by Interferon γ and all four CRC cell lines responded to CXCL10 by extracellular signal-regulated kinase 1/2 dephosphorylation. The chemokine did not affect tumor cell growth or angiogenesis-related functions in the tumor cells, such as CXCL8 and vascular endothelial growth factor secretion. Importantly, CXCL10 significantly up-regulated invasion-related properties in CRC cells: It promoted matrix metalloproteinase 9 expression and induced CRC cell migration. Of note, CXCL10-induced migration was detected only in the two metastatic cells and not in their primary counterparts. Also, CXCL10 promoted the adhesion of metastatic cells to laminin. These results suggest that CXCL10 can be exploited by CRC cells toward their progression, thus possibly antagonizing the antimalignancy effects of the chemokine on the tumor microenvironment. Therefore, care should be taken when considering CXCL10 as a therapeutic antitumor modality for CRC treatment. [Cancer Res 2007;67(7):3396–405]

Melanoma brain metastasis is a complication with rising incidence. Despite the high rate of somatic mutations driving the initial stages of melanocyte transformation, the brain colonization requires a phenotypic reprogramming that is, in part, influenced by epigenomic modifications. This special report summarizes recent findings in the epigenomic landscape of melanoma progression to brain metastasis, with particular emphasis on the clinical utility of DNA methylation, chromatin modifications and ncRNA expression as theragnostic markers, as well as the significance of the metastatic microenvironment on melanoma brain metastasis epigenome.

Soluble pulmonary factors have been reported to be capable of inhibiting the viability of cancer cells that metastasize to the lung, but the molecular identity was obscure. Here we report the isolation and characterization of the beta subunit of hemoglobin as a lung-derived antimetastatic factor. Peptide mapping in the beta subunit of human hemoglobin (HBB) defined a short C-terminal region (termed Metox) as responsible for activity. In tissue culture, both HBB and murine HBB2 mediated growth arrest and apoptosis of lung-metastasizing neuroblastoma cells, along with a variety of other human cancer cell lines. Metox acted similarly and its administration in human tumor xenograft models limited the development of adrenal neuroblastoma tumors as well as spontaneous lung and bone marrow metastases. Expression studies in mice indicated that HBB2 is produced by alveolar epithelial and endothelial cells and is upregulated in mice bearing undetectable metastasis. Our work suggested a novel function for HBB as a theranostic molecule: an innate antimetastasis factor with potential utility as an anticancer drug and a biomarker signaling the presence of clinically undetectable metastasis. Cancer Res; 77(1); 14–26. ©2016 AACR.

V600E being the most common mutation in BRAF, leads to constitutive activation of the MAPK signaling pathway. The majority of V600E BRAF positive melanoma patients treated with the BRAF inhibitor vemurafenib showed initial good clinical responses but relapsed due to acquired resistance to the drug. The aim of the present study was to identify possible biomarkers associated with the emergence of drug resistant melanoma cells. To this end we analyzed the differential gene expression of vemurafenib-sensitive and vemurafenib resistant brain and lung metastasizing melanoma cells. The major finding of this study is that the in vitro induction of vemurafenib resistance in melanoma cells is associated with an increased malignancy phenotype of these cells. Resistant cells expressed higher levels of genes coding for cancer stem cell markers (JARID1B, CD271 and Fibronectin) as well as genes involved in drug resistance (ABCG2), cell invasion and promotion of metastasis (MMP-1 and MMP-2). We also showed that drug-resistant melanoma cells adhere better to and transmigrate more efficiently through lung endothelial cells than drug-sensitive cells. The former cells also alter their microenvironment in a different manner from that of drug-sensitive cells. Biomarkers and molecular mechanisms associated with drug resistance may serve as targets for therapy of drug-resistant cancer.

The research area of tumor microenvironment is considered, at present, to be an important factor in tumorigenesis and especially in tumor progression. The present mini review is focused on three principles characterizing the nature of the tumor microenvironment. We first discuss the regulatory functions of the tumor microenvironment and the complexity of the combinatorial signaling pathways operating in it. We then address the aspect that the tumor microenvironment incorporates both pro and anti malignancy factors and that a balance between these factors regulates tumor progression. Thirdly we provide evidence that the non-tumor cells in the tumor microenvironment and their products may be different from those of their counterparts residing in non-tumor microenvironments. The conclusion of this mini review is that the tumor microenvironment, by exerting regulatory functions and selective pressures drives cancer cells into one of several molecular evolution pathways thereby determining and shaping their malignancy phenotype.

The dual-specificity phosphatase Pyst2-L was found to be over expressed in leukocytes derived from AML and ALL patients as well as in certain other solid tumors and lymphoblastoid cell lines. Pyst2-L, binds and dephosphorylates both pERKs and pJNKs proteins, and thus, plays a role in regulating the MAP kinase signaling pathway. In the present study, a comparative genomic application was used and sequence analysis of multi-organisms databases were searched in order to identify genes homologous to Pyst2-L. The Xenopus laevis MAP kinase phosphatase X17c gene and the Yeast nitrogen starvation-induced protein phosphatase Yvh1p gene were revealed to be highly homologous with Pyst2-L. Both X17c and Yvh1p genes play a role in cell cycle regulation. A down regulated expression of the Yvh1p gene occurred in Saccharomyces cerevisiae that were synchronized to the G2-phase of the cell cycle by alpha-factor. In conformity with this result, a reduction in Pyst2-L expression levels was observed in G2-phase-synchronized Human K562 cells. Finally, we were able to show that cells in highly crowded cultures express high levels of the Pyst2-L phosphatase. These observations may indicate that low levels of the Pyst2-L phosphatase are essential for the G2-phase of the cell cycle and that this phosphatase might play a role in signaling cascades induced by cellular crowding.

About 70 years ago, Peyton Rous described the progression of cancer towards metastasis formation as "the process whereby tumors go from bad to worse". The interactions of tumor cells with endothelium are pivotal steps in this process. This review focuses on the role played by the selectins and their ligands in these interactions and especially in tumor cell extravasation. The working hypothesis of researchers studying tumor cell extravasation is that the tumor cells follow the extravasation strategy of leukocytes in their migration towards inflammatory sites. A significant portion of this review is, therefore, dedicated to the molecular mechanisms underlying leukocyte extravasation and to a comparison between the extravasation strategy employed by leukocytes and tumor cells. The review also summarizes some of the available data on signals generated by selectin-selectin ligand interactions.

Extravasation of tumor cells is a pivotal step in metastasis formation. This step is initiated by an interaction of extravasating tumor cells with endothelial cells. Among the molecules mediating tumor-endothelium interactions are selectins and their fucosylated ligands. In a previous study, we demonstrated that the fucose-generating FX enzyme regulates the expression of selectin ligands by B and T lymphocytes and by head and neck squamous cell carcinoma cells. It was also shown that the FX enzyme regulated important interaction parameters between these cancer cells and endothelial cells. The present study was aimed to determine whether the FX enzyme controls adhesive interactions between colorectal cancer cells and endothelial cells. The results clearly indicate that this is indeed the case. Overexpressing the FX enzyme by the transfer of FX cDNA to low FX-expressing colorectal cancer cells resulted in an increased adhesive capacity of the transfectants to activated endothelial cells and to recombinant E-selectin. Down-regulating FX levels in colorectal cancer cells expressing high levels of endogenous FX by transfection with small-interfering RNA resulted in a down-regulated expression of the selectin ligand sialyl Lewis-a and a decrease in the adhesive capacity of the transfectants to activated endothelial cells and to recombinant E-selectin. These transfection experiments also indicated that manipulating the levels of the FX enzyme affected global cellular fucosylation and altered the interaction of colorectal cancer cells with some extracellular matrix components such as fibronectin. We also found that highly metastatic colorectal cancer variants express higher levels of FX and of sialyl Lewis-a than low metastatic variants originating in the same tumors. These results lead us to hypothesize that the FX enzyme controls the capacity of colorectal cancer to extravasate and form metastasis. If this hypothesis will be confirmed the FX enzyme could become a target molecule for metastasis prevention.

The dual-specificity phosphatase Pyst2-L was found to be highly expressed in leukocytes derived from AML and ALL patients as well as in certain other solid tumors and lymphoblastoid cell lines. Recently, by use of the 5′-RNA ligation-mediated rapid amplification of cDNA ends (5′-RLM-RACE) technique, we sequenced and cloned the entire open reading frame (ORF) of Pyst2-L. In the present study we determined the effect of exogenous overexpression on Erk1/2 phosphorylation. It was demonstrated that overexpression of this phosphatase in HEK293 cells reduced the basal levels of phospho-Erk1/2 as compared to the same cells transfected with the wild-type vector. This reduction was concomitant with a growth retardation of the Pyst2-L-transfected cells. Treating Pyst2-L transfected cells with known activators of the MAPK signaling cascade such as TPA or stimulating them by serum, it was demonstrated that the up regulation of phospho-Erk1/2, caused by these activators, was only partially suppressed by the over expression of the Pyst2-L phosphatase in these cells. These results together with our previous ones showing that the TPA-induced up regulation of Pyst2-L mRNA was only partially inhibited by the use of a specific Mek1/2 inhibitor, lead us to ask whether the Pyst2-L phosphatase has a monogamous relationship with the Erk2 protein. To answer this question, we employed the pull-down method and showed that in addition to phospho-Erk1/2, recombinant Pyst2-L binds the phospho-JNK protein. These findings may raise new perspectives regarding the role played by this phosphatase in malignant cells and in activation processes.

In previous studies, we demonstrated that human neuroblastoma cells are equipped with the machinery to direct their homing to bone marrow. These tumor cells express the CXCR4 receptor for the bone marrow stroma-derived chemokine CXCL12 (SDF-1) and secrete the CXCL12 ligand. The present study was undertaken to explore possible differences in gene-expression patterns between neuroblastoma variants that over-express CXCR4 (designated STH cells) and those which express very little of this receptor (STL cells). The results of the study clearly indicate that these variants show a differential gene-expression profile. They differ in expression of some integrins such as VLA2, VLA3 and VLA6, of neuroendocrine-markers such as CD56 and synaptophysin, in the expression of c-kit and in the secretion of certain cytokines and growth factors such as TNFalpha, SDF-1, VEGF, IL-8, GM-CSF and IP-10. We hypothesize that these differences are due to an autocrine SDF-1alpha-CXCR4 axis.