Astrocytes facilitate melanoma brain metastasis via secretion of IL-23.

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.

The metastatic microenvironment: Claudin-1 suppresses the malignant phenotype of melanoma brain metastasis.

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.

The metastatic microenvironment: Brain-derived soluble factors alter the malignant phenotype of cutaneous and brain-metastasizing melanoma cells.

The working hypothesis of this study is that the interactions between the brain microenvironment and melanoma cells determine metastasis formation at this organ site. The aim of the study was to evaluate the contribution of such interactions to the formation of brain metastasis in nude mice xenografted with human melanoma cells. An insight into these interactions is an essential prerequisite for the development of effective targeted therapy for melanoma brain metastasis. We assessed the effects of soluble factors present in supernatants of short-term cultures of normal mouse brain (referred here after as brain-derived soluble factors) on several characteristics linked to melanoma brain metastasis. It was found that brain-derived soluble factors affect differentially cutaneous and brain-metastasizing melanoma cells variants in vitro. Such factors enhanced the viability of cutaneous melanoma cells but caused an S phase arrest followed by apoptosis of brain-metastasizing cells. Brain-derived soluble factors enhanced migration of melanoma cells metastasizing to the brain, but did not affect the migration of the cutaneous variants. Such factors upregulated the expression of the chemokine receptor CCR4 in both cutaneous and brain-metastasizing melanoma cells. It is not unlikely that CCR4 ligands expressed in the brain interact with the CCR4-expressing melanoma cells thereby directing them to the brain. Brain-derived soluble factors enhanced the transmigration, across human brain endothelial cells of cutaneous but not of brain-metastasizing melanoma variants. This activity could promote the capacity of the cutaneous cells to metastasize to the brain.

The metastatic microenvironment: brain-residing melanoma metastasis and dormant micrometastasis.

Brain metastasis occurs frequently in melanoma patients with advanced disease whereby the prognosis is dismal. The underlying mechanisms of melanoma brain metastasis development are not well understood. We generated a reproducible melanoma brain metastasis model, consisting of brain-metastasizing variants and local, subdermal variants that originate from the same melanomas thus sharing a common genetic background. The brain-metastasizing variants were obtained by intracardiac inoculation. Brain metastasis variants when inoculated subdermally yielded spontaneous brain dormant micrometastasis. Cultured cells from the spontaneous brain micrometastasis grew very well in vitro and generated subdermal tumors after an orthotopic inoculation. Expression analysis assays indicated that the brain metastasis and micrometastasis cells expressed higher levels of angiopoietin-like 4, prostaglandin-synthesizing enzyme cyclooxygenase-2, matrix metalloproteinase-1 and preferentially expressed antigen in melanoma and lower levels of claudin-1 and cysteine-rich protein 61 than the corresponding cutaneous variants. The reproducible models of human melanoma metastasizing experimentally and spontaneously to the brain will facilitate the identification of novel biomarkers and targets for therapy and contribute to the deciphering of mechanisms underlying melanoma metastasis.

Ligand-binding Domain-activating Mutations of ESR1 Rewire Cellular Metabolism of Breast Cancer Cells.

PURPOSE: Mutations in the ligand-binding domain (LBD) of estrogen receptor α (ER) confer constitutive transcriptional activity and resistance to endocrine therapies in patients with breast cancer. Accumulating clinical data suggest adverse outcome for patients harboring tumors expressing these mutations. We aimed to elucidate mechanisms conferring this aggressive phenotype. EXPERIMENTAL DESIGN: Cells constitutively expressing physiologic levels of ER-harboring activating LBD mutations were generated and characterized for viability, invasiveness, and tumor formation in vivo. Gene expression profile was studied using microarray and RNAseq technologies. Metabolic properties of the cells were assessed using global metabolite screen and direct measurement of metabolic activity. RESULTS: Cells expressing mutated ER showed increased proliferation, migration, and in vivo tumorigenicity compared with cells expressing the wild-type ER (WT-ER), even in the presence of estrogen. Expression of the mutated ER was associated with upregulation of genes involved in invasion and metastases, as well as elevation of genes associated with tumor cell metabolism. Indeed, a metabolic examination revealed four distinct metabolic profiles: WT-ER-expressing cells either untreated or estrogen treated and mutated ER-expressing cells either untreated or estrogen treated. Pathway analyses indicated elevated tricarboxylic acid cycle activity of 537S-ER-expressing cells. Thus, while WT-ER cells were mostly glucose-dependent, 537S-ER were not addicted to glucose and were able to utilize glutamine as an alternative carbon source. CONCLUSIONS: Taken together, these data indicate estrogen-independent rewiring of breast cancer cell metabolism by LBD-activating mutations. These unique metabolic activities may serve as a potential vulnerability and aid in the development of novel treatment strategies to overcome endocrine resistance.

Attitudes toward integration of complementary and alternative medicine in primary care: perspectives of patients, physicians and complementary practitioners.

OBJECTIVE: Our study explored the attitudes of patients toward complementary and alternative medicine (CAM) use, their family physicians' role regarding CAM, and models for CAM referral and treatment. We compared patients' perspectives regarding integration of CAM into primary care with attitudes of primary care physicians (PCPs) and CAM practitioners. METHODS: We conducted a comprehensive literature review and focus group discussions to develop a questionnaire, which we gave to three groups: a random sample of patients receiving care at an academic family medicine clinic and PCPs and CAM practitioners employed in the largest health maintenance organization in Israel. RESULTS: A total of 1150 patients, 333 PCPs, and 241 CAM practitioners responded to our questionnaire. Compared with PCPs, patients expected their family physician to refer them to CAM, to have updated knowledge about CAM, and to offer CAM treatment in the clinic based on appropriate training. When asked about CAM integration into medical care, more patients expected to receive CAM in a primary care setting compared to PCPs' expectations of prescribing CAM (62% vs. 30%; p=0.0001). Patients, CAM practitioners, and PCPs expected family practitioners to generate CAM referrals in an integrative primary care setting (85.6% vs. 82.4% vs. 62.6%; p<0.0001). Patients supported CAM practitioners providing CAM treatments in the primary care setting, regardless of whether the practitioner held a medical degree (MD). Also, more patients than PCPs or CAM practitioners expected their family physician to provide CAM (28.2% vs. 14.5% vs. 3.8%; p<0.0001). CONCLUSION: Patients, PCPs, and CAM practitioners suggested that family physicians play a central role in CAM referral and, to a lesser extent, that they actually provide CAM treatment themselves. PRACTICE IMPLICATIONS: PCPs need to be aware of their present and future role in informed referral to CAM and, to a lesser degree, in providing CAM in integrative primary care clinics. With the increasing use of CAM, patients may expect their family physician to be more knowledgeable, skillful, and have a balanced approach regarding CAM use. In addition, practitioners should learn how to communicate effectively and better collaborate with CAM practitioners to the benefit of their patients.

Educating CAM practitioners about integrative medicine: an approach to overcoming the communication gap with conventional health care practitioners.

OBJECTIVE: To assess an educational initiative that teaches complementary and alternative medicine (CAM) students how to communicate more effectively with conventional physicians about CAM. DESIGN: We introduced an educational initiative in integrative medicine to CAM students in their final year of study, emphasizing evidence-based learning, patient-centered care, and communication skills with conventional health care providers. A precourse semistructured questionnaire and an anonymous open essay about the students' experiences at the end of the course were used as tools for assessment. The precourse questionnaires and the postcourse essays were evaluated, using content analysis for parallel responses to determine whether students' views changed during the course. RESULTS: We evaluated the experience in 62 students exposed to the initiative during 4 academic years, 2001-2005. We found that CAM students perceive that they need practical communication tools in order to communicate effectively with conventional practitioners. After the educational experience, the students confirmed that critical thinking training is important, and reported feeling more empowered and more confident in their work as well as in communicating with physicians. CONCLUSIONS: The results of this study suggest that CAM practitioners feel better equipped to communicate with conventional health care practitioners after exposure to a structured educational initiative that emphasizes critical thinking, patient-centered care, and communication skills with conventional practitioners.

[Attitudes of immigrants from the former Soviet Union towards complementary medicine].

BACKGROUND: The use of complementary and alternative medicine (CAM) in primary care in Israel is growing. Limited data is available on the influence of immigration and cross-cultural health-beliefs concerning CAM use. OBJECTIVES: This study explores the perspectives toward CAM of immigrants who came to Israel since 1990 from the former Soviet Union. RESEARCH DESIGN AND METHODS: Questionnaires were administered to a random sample of patients attending an urban academic primary care clinic located in Northern Israel. RESULTS: Out of 1146 participants in this study, 106 were immigrants from the former Soviet-Union. No statistical significance was found in the extent of CAM use in the last year among immigrants (63%) and non-immigrants (54%). Immigrants using CAM reported significantly less in reference to CAM practitioners and more use of herbal products. They also more significantly supported the idea of including an herbal therapist in the clinical staff at the medical center. Both groups expected their family physician to refer them to CAM, but immigrants had significantly higher expectations of their physician to provide CAM. Both groups anticipated an active role for their family physician in a future scenario of integrative care at the clinic. CONCLUSIONS: The use of CAM in primary care can be interpreted in a cross-cultural perspective. Patients who emigrated from the former USSR have unique perspectives toward CAM. The authors propose practical suggestions for primary care clinicians concerning anamnesis of immigrants regarding CAM. They also suggest health administrators take into account cross-cultural diversity when planning integration of CAM in primary care.

CCR4 is a determinant of melanoma brain metastasis.

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.

Prevalence and risk factors for carriage of extended-spectrum ß-lactamase-producing Enterobacteriaceae among patients prior to bowel surgery.

Prophylactic antibiotics are an important measure in preventing perioperative infection, Failure to cover multidrug-resistant pathogens may place carriers at increased risk of infection. We conducted a prospective, cross-sectional study in patients prior to bowel surgery to measure the carriage prevalence of extended-spectrum ß-lactamase-producing Enterobacteriaceae and identify risk factors for carriage in this population. During an 11-month period, 150 patients were eligible for inclusion. 27 patients (18%) were found to be carriers of extended-spectrum ß-lactamase-producing Enterobacteriaceae. Factors independently associated with carriage were immunosuppressive therapy (OR, 4.09; 95% CI 1.55-10.81; P = 0.005) and receipt of antibiotics in the prior 3 months (OR, 2.59; 95% CI 1.08-6.24; P = 0.033). Detection of a population at risk for carriage may help in devising and modifying appropriate antibiotic regimens for surgical prophylaxis in carriers of multidrug-resistant bacteria.

Incipient Melanoma Brain Metastases Instigate Astrogliosis and Neuroinflammation.

Malignant melanoma is the deadliest of skin cancers. Melanoma frequently metastasizes to the brain, resulting in dismal survival. Nevertheless, mechanisms that govern early metastatic growth and the interactions of disseminated metastatic cells with the brain microenvironment are largely unknown. To study the hallmarks of brain metastatic niche formation, we established a transplantable model of spontaneous melanoma brain metastasis in immunocompetent mice and developed molecular tools for quantitative detection of brain micrometastases. Here we demonstrate that micrometastases are associated with instigation of astrogliosis, neuroinflammation, and hyperpermeability of the blood-brain barrier. Furthermore, we show a functional role for astrocytes in facilitating initial growth of melanoma cells. Our findings suggest that astrogliosis, physiologically instigated as a brain tissue damage response, is hijacked by tumor cells to support metastatic growth. Studying spontaneous melanoma brain metastasis in a clinically relevant setting is the key to developing therapeutic approaches that may prevent brain metastatic relapse. Cancer Res; 76(15); 4359-71. ©2016 AACR.

Pilot study: fluvoxamine treatment for depression and anxiety disorders in children and adolescents with cancer.

OBJECTIVE: To evaluate the safety, tolerability, and benefit of fluvoxamine for the treatment of major depressive disorder or anxiety disorders in children and adolescents with cancer. METHOD: The study was conducted from 2001 to 2004 at a pediatric hematology-oncology center. Fifteen children and adolescents with cancer were treated with fluvoxamine 100 mg/day in an open prospective 8-week trial. Safety and tolerability were evaluated at baseline and at weeks 4 and 8 by blood tests and the Side Effects Checklist. Clinical benefit was assessed with the Clinical Global Impressions-Improvement, the Children's Depression Rating Scale-Revised, and the Pediatric Anxiety Rating Scale. RESULTS: Fluvoxamine was well tolerated by all subjects. Psychiatric symptoms improved significantly. CONCLUSIONS: In this open trial, fluvoxamine appeared to be well tolerated and was associated with a promising reduction in the depression and anxiety symptoms of pediatric patients with cancer.

Chemokine-chemokine receptor axes in melanoma brain metastasis.

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.