Isolation of Human Adipose-Derived Stem Cells.

Human adipose-derived stromal/stem cells (hASCs) are a promising source of adult stem cells used in numerous applications in regenerative medicine. We present the protocols from our laboratory for isolating and expanding hASCs. The isolation of hASCs involves the enzymatic digestion of adipose tissue and subsequent culturing of the isolated cells.

Spheroid Formation of Human Adipose-Derived Stem Cells Using a Liquid Overlay Technique.

Compared to two-dimensional monolayer culture, cells cultured in three-dimensional (3D) platforms provide a more biochemically and physiologically relevant environment to study cell-cell and cell-extracellular matrix interactions in vitro. Using the liquid overlay technique, a scaffold-free method to generate 3D spheroids from human adipose-derived stem cells is described.

Etiology and treatment of cancer-related secondary lymphedema.

Lymphedema and specifically cancer-related lymphedema is not the main focus for both patients and physicians dealing with cancer. Its etiology is an unfortunate complication of cancer treatment. Although lymphedema treatments have gained an appreciable consensus, many practitioners have developed and prefer their own specific protocols and this is especially true for conventional (manual) versus surgical treatments. This collection of presentations explores the incidence and genetics of cancer-related lymphedema, early detection and monitoring techniques, both conventional and operative treatment options, and the importance and role of exercise for patients with cancer-related lymphedema. These assembled presentations provide valuable insights into the challenges and opportunities presented by cancer-related lymphedema including the latest research, treatments, and exercises available to improve patient outcomes and quality of life.

Enhanced Angiogenesis in HUVECs Preconditioned with Media from Adipocytes Differentiated from Lipedema Adipose Stem Cells In Vitro.

Lipedema is a connective tissue disorder characterized by increased dilated blood vessels (angiogenesis), inflammation, and fibrosis of the subcutaneous adipose tissue. This project aims to gain insights into the angiogenic processes in lipedema using human umbilical vein endothelial cells (HUVECs) as an in vitro model. HUVECs were cultured in conditioned media (CM) collected from healthy (non-lipedema, AQH) and lipedema adipocytes (AQL). The impacts on the expression levels of multiple endothelial and angiogenic markers [CD31, von Willebrand Factor (vWF), angiopoietin 2 (ANG2), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMPs), NOTCH and its ligands] in HUVECs were investigated. The data demonstrate an increased expression of CD31 and ANG2 at both the gene and protein levels in HUVECs treated with AQL CM in 2D monolayer and 3D cultures compared to untreated cells. Furthermore, the expression of the vWF, NOTCH 4, and DELTA-4 genes decreased. In contrast, increased VEGF, MMP9, and HGF gene expression was detected in HUVECs treated with AQL CM cultured in a 2D monolayer. In addition, the results of a tube formation assay indicate that the number of formed tubes increased in lipedema-treated HUVECs cultured in a 2D monolayer. Together, the data indicate that lipedema adipocyte-CM promotes angiogenesis through paracrine-driven mechanisms.

Indications of Peripheral Pain, Dermal Hypersensitivity, and Neurogenic Inflammation in Patients with Lipedema.

Lipedema is a disease with abnormally increased adipose tissue deposition and distribution. Pain sensations have been described in the clinical evaluation of lipedema, but its etiology remains poorly understood. We hypothesized that pain sensitivity measurements and ex vivo quantitation of neuronal cell body distribution in the skin would be lipedema stage-dependent, and could, thus, serve to objectively characterize neuropathic pain in lipedema. The pain was assessed by questionnaire and peripheral cutaneous mechanical sensitization (von-Frey) in lipedema (n = 27) and control (n = 23) consenting female volunteers. Dermal biopsies from (n = 11) Stages 1-3 lipedema and control (n = 10) participants were characterized for neuronal cell body and nociceptive neuropeptide calcitonin gene-related peptide (CGRP) and nerve growth factor (NGF) distribution. Stage 2 or 3 lipedema participants responded positively to von Frey sensitization in the calf and thigh, and Stage 3 participants also responded in the arm. Lipedema abdominal skin displayed reduced Tuj-1+ neuronal cell body density, compared to healthy controls, while CGRP and NGF was significantly elevated in Stage 3 lipedema tissues. Together, dermal neuronal cell body loss is consistent with hyper-sensitization in patients with lipedema. Further study of neuropathic pain in lipedema may elucidate underlying disease mechanisms and inform lipedema clinical management and treatment impact.

Aberrant Expression of COX-2 and FOXG1 in Infrapatellar Fat Pad-Derived ASCs from Pre-Diabetic Donors.

Osteoarthritis (OA) is a degenerative joint disease resulting in limited mobility and severe disability. Type II diabetes mellitus (T2D) is a weight-independent risk factor for OA, but a link between the two diseases has not been elucidated. Adipose stem cells (ASCs) isolated from the infrapatellar fat pad (IPFP) may be a viable regenerative cell for OA treatment. This study analyzed the expression profiles of inflammatory and adipokine-related genes in IPFP-ASCs of non-diabetic (Non-T2D), pre-diabetic (Pre-T2D), and T2D donors. Pre-T2D ASCs exhibited a substantial decrease in levels of mesenchymal markers CD90 and CD105 with no change in adipogenic differentiation compared to Non-T2D and T2D IPFP-ASCs. In addition, Cyclooxygenase-2 (COX-2), Forkhead box G1 (FOXG1) expression and prostaglandin E2 (PGE2) secretion were significantly increased in Pre-T2D IPFP-ASCs upon stimulation by interleukin-1 beta (IL-1ß). Interestingly, M1 macrophages exhibited a significant reduction in expression of pro-inflammatory markers TNFα and IL-6 when co-cultured with Pre-T2D IPFP-ASCs. These data suggest that the heightened systemic inflammation associated with untreated T2D may prime the IPFP-ASCs to exhibit enhanced anti-inflammatory characteristics via suppressing the IL-6/COX-2 signaling pathway. In addition, the elevated production of PGE2 by the Pre-T2D IPFP-ASCs may also suggest the contribution of pre-diabetic conditions to the onset and progression of OA.

Short-Term Autophagy Preconditioning Upregulates the Expression of COX2 and PGE2 and Alters the Immune Phenotype of Human Adipose-Derived Stem Cells In Vitro.

Human adipose-derived stem cells (hASCs) are potent modulators of inflammation and promising candidates for the treatment of inflammatory and autoimmune diseases. Strategies to improve hASC survival and immunoregulation are active areas of investigation. Autophagy, a homeostatic and stress-induced degradative pathway, plays a crucial role in hASC paracrine signaling-a primary mechanism of therapeutic action. Therefore, induction of autophagy with rapamycin (Rapa), or inhibition with 3-methyladenine (3-MA), was examined as a preconditioning strategy to enhance therapeutic efficacy. Following preconditioning, both Rapa and 3-MA-treated hASCs demonstrated preservation of stemness, as well as upregulated transcription of cyclooxygenase-2 (COX2) and interleukin-6 (IL-6). Rapa-ASCs further upregulated TNFα-stimulated gene-6 (TSG-6) and interleukin-1 beta (IL-1ß), indicating additional enhancement of immunomodulatory potential. Preconditioned cells were then stimulated with the inflammatory cytokine interferon-gamma (IFNγ) and assessed for immunomodulatory factor production. Rapa-pretreated cells, but not 3-MA-pretreated cells, further amplified COX2 and IL-6 transcripts following IFNγ exposure, and both groups upregulated secretion of prostaglandin-E2 (PGE2), the enzymatic product of COX2. These findings suggest that a 4-h Rapa preconditioning strategy may bestow the greatest improvement to hASC expression of cytokines known to promote tissue repair and regeneration and may hold promise for augmenting the therapeutic potential of hASCs for inflammation-driven pathological conditions.

Adipose Stem Cells in Regenerative Medicine: Looking Forward.

Over the last decade, stem cell-based regenerative medicine has progressed to clinical testing and therapeutic applications. The applications range from infusions of autologous and allogeneic stem cells to stem cell-derived products. Adult stem cells from adipose tissue (ASCs) show significant promise in treating autoimmune and neurodegenerative diseases, vascular and metabolic diseases, bone and cartilage regeneration and wound defects. The regenerative capabilities of ASCs in vivo are primarily orchestrated by their secretome of paracrine factors and cell-matrix interactions. More recent developments are focused on creating more complex structures such as 3D organoids, tissue elements and eventually fully functional tissues and organs to replace or repair diseased or damaged tissues. The current and future applications for ASCs in regenerative medicine are discussed here.

Medicinal Plants Towards Modeling Skin Cancer.

Skin cancer remains a major cause of mortality worldwide. It can be divided into melanoma and non-melanoma cancer, which comprise mainly squamous cell carcinoma and basal cell carcinoma. Although conventional therapies have ameliorated the management of skin cancer, the search for chemopreventive compounds is still the most effective and safer strategy to treat cancer. Nowadays, chemoprevention is recognized as a novel approach to prevent or inhibit carcinogenesis steps with the use of natural products. Crude extracts of plants and isolated phytocompounds are considered chemopreventive agents since they harbor anti-inflammatory, antioxidant and anti-oncogenic properties against many types of diseases and cancers. In this review, we will discuss the therapeutic effect and preventive potential of selected medicinal plants used as crude extracts or as phytocompounds against melanoma and non-melanoma cutaneous cancers.

3D Spheroids Derived from Human Lipedema ASCs Demonstrated Similar Adipogenic Differentiation Potential and ECM Remodeling to Non-Lipedema ASCs In Vitro.

The growth and differentiation of adipose tissue-derived stem cells (ASCs) is stimulated and regulated by the adipose tissue (AT) microenvironment. In lipedema, both inflammation and hypoxia influence the expansion and differentiation of ASCs, resulting in hypertrophic adipocytes and deposition of collagen, a primary component of the extracellular matrix (ECM). The goal of this study was to characterize the adipogenic differentiation potential and assess the levels of expression of ECM-remodeling markers in 3D spheroids derived from ASCs isolated from both lipedema and healthy individuals. The data showed an increase in the expression of the adipogenic genes (ADIPOQ, LPL, PPAR-γ and Glut4), a decrease in matrix metalloproteinases (MMP2, 9 and 11), with no significant changes in the expression of ECM markers (collagen and fibronectin), or integrin A5 in 3D differentiated lipedema spheroids as compared to healthy spheroids. In addition, no statistically significant changes in the levels of expression of inflammatory genes were detected in any of the samples. However, immunofluorescence staining showed a decrease in fibronectin and increase in laminin and Collagen VI expression in the 3D differentiated spheroids in both groups. The use of 3D ASC spheroids provide a functional model to study the cellular and molecular characteristics of lipedema AT.

Short-Term Rapamycin Preconditioning Diminishes Therapeutic Efficacy of Human Adipose-Derived Stem Cells in a Murine Model of Multiple Sclerosis.

Human adipose-derived stem cells (ASCs) show immense promise for treating inflammatory diseases, attributed primarily to their potent paracrine signaling. Previous investigations demonstrated that short-term Rapamycin preconditioning of bone marrow-derived stem cells (BMSCs) elevated secretion of prostaglandin E2, a pleiotropic molecule with therapeutic effects in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), and enhanced immunosuppressive capacity in vitro. However, this has yet to be examined in ASCs. The present study examined the therapeutic potential of short-term Rapamycin-preconditioned ASCs in the EAE model. Animals were treated at peak disease with control ASCs (EAE-ASCs), Rapa-preconditioned ASCs (EAE-Rapa-ASCs), or vehicle control (EAE). Results show that EAE-ASCs improved clinical disease scores and elevated intact myelin compared to both EAE and EAE-Rapa-ASC animals. These results correlated with augmented CD4+ T helper (Th) and T regulatory (Treg) cell populations in the spinal cord, and increased gene expression of interleukin-10 (IL-10), an anti-inflammatory cytokine. Conversely, EAE-Rapa-ASC mice showed no improvement in clinical disease scores, reduced myelin levels, and significantly less Th and Treg cells in the spinal cord. These findings suggest that short-term Rapamycin preconditioning reduces the therapeutic efficacy of ASCs when applied to late-stage EAE.

Adipose Tissue-Derived Stem Cells Retain Their Adipocyte Differentiation Potential in Three-Dimensional Hydrogels and Bioreactors †.

Osteoarthritis (OA) is a common joint disorder with a significant economic and healthcare impact. The knee joint is composed of cartilage and the adjoining bone, a synovial capsule, the infrapatellar fat pad (IPFP), and other connective tissues such as tendons and ligaments. Adipose tissue has recently been highlighted as a major contributor to OA through strong inflammation mediating effects. In this study, methacrylated gelatin (GelMA) constructs seeded with adipose tissue-derived mesenchymal stem cells (ASCs) and cultured in a 3D printed bioreactor were investigated for use in microphysiological systems to model adipose tissue in the knee joint. Four patient-derived ASC populations were seeded at a density of 20 million cells/mL in GelMA. Live/Dead and boron-dipyrromethene/4',6-diamidino-2-phenylindole (BODIPY/DAPI) staining of cells within the constructs demonstrated robust cell viability after 28 days in a growth (control) medium, and robust cell viability and lipid accumulation in adipogenic differentiation medium. qPCR gene expression analysis and protein analysis demonstrated an upregulated expression of key adipogenesis-associated genes. Overall, these data indicate that ASCs retain their adipogenic potential when seeded within GelMA hydrogels and cultured within perfusion bioreactors, and thus can be used in a 3D organ-on-a-chip system to study the role of the IPFP in the pathobiology of the knee OA.

Increase in Leptin and PPAR-γ Gene Expression in Lipedema Adipocytes Differentiated in vitro from Adipose-Derived Stem Cells.

Lipedema is a painful loose connective tissue disorder characterized by a bilaterally symmetrical fat deposition in the lower extremities. The goal of this study was to characterize the adipose-derived stem cells (ASCs) of healthy and lipedema patients by the expression of stemness markers and the adipogenic and osteogenic differentiation potential. Forty patients, 20 healthy and 20 with lipedema, participated in this study. The stromal vascular fraction (SVF) was obtained from subcutaneous thigh (SVF-T) and abdomen (SVF-A) fat and plated for ASCs characterization. The data show a similar expression of mesenchymal markers, a significant increase in colonies (p < 0.05) and no change in the proliferation rate in ASCs isolated from the SVF-T or SVF-A of lipedema patients compared with healthy patients. The leptin gene expression was significantly increased in lipedema adipocytes differentiated from ASCs-T (p = 0.04) and the PPAR-γ expression was significantly increased in lipedema adipocytes differentiated from ASCs-A (p = 0.03) compared to the corresponding cells from healthy patients. No significant changes in the expression of genes associated with inflammation were detected in lipedema ASCs or differentiated adipocytes. These results suggest that lipedema ASCs isolated from SVF-T and SVF-A have a higher adipogenic differentiation potential compared to healthy ASCs.

Adipose Tissue-Derived Stem Cells: Immunomodulatory Effects and Therapeutic Potential.

Adipose-derived stem cells (ASCs) can self-renew and differentiate along multiple cell lineages. ASCs are also potently anti-inflammatory due to their inherent ability to regulate the immune system by secreting anti-inflammatory cytokines and growth factors that play a crucial role in the pathology of many diseases, including multiple sclerosis, diabetes mellitus, Crohn's, SLE, and graft-versus-host disease. The immunomodulatory effects and mechanisms of action of ASCs on pathological conditions are reviewed here.

Dilated Blood and Lymphatic Microvessels, Angiogenesis, Increased Macrophages, and Adipocyte Hypertrophy in Lipedema Thigh Skin and Fat Tissue.

Background and Aim: Lipedema is a common painful SAT disorder characterized by enlargement of fat primarily in the legs of women. Case reports of lipedema tissue samples demonstrate fluid and fibrosis in the interstitial matrix, increased macrophages, and adipocyte hypertrophy. The aims of this project are to investigate blood vasculature, immune cells, and structure of lipedema tissue in a cohort of women. Methods: Forty-nine participants, 19 controls and 30 with lipedema, were divided into groups based on body mass index (BMI): Non-Obese (BMI 20 to <30 kg/m2) and Obese (BMI 30 to <40 kg/m2). Histological sections from thigh skin and fat were stained with H&E. Adipocyte area and blood vessel size and number were quantified using ImageJ software. Markers for macrophages (CD68), mast cells (CD117), T cells (CD3), endothelial cells (CD31), blood (SMA), and lymphatic (D2-40 and Lyve-1) vessels were investigated by IHC and IF. Results: Non-Obese Lipedema adipocyte area was larger than Non-Obese Controls (p=0.005) and similar to Obese Lipedema and Obese Controls. Macrophage numbers were significantly increased in Non-Obese (p < 0.005) and Obese (p < 0.05) Lipedema skin and fat compared to Control groups. No differences in T lymphocytes or mast cells were observed when comparing Lipedema to Control in both groups. SMA staining revealed increased dermal vessels in Non-Obese Lipedema patients (p < 0.001) compared to Non-Obese Controls. Lyve-1 and D2-40 staining showed a significant increase in lymphatic vessel area but not in number or perimeter in Obese Lipedema participants (p < 0.05) compared to Controls (Obese and Non-Obese). Areas of angiogenesis were found in the fat in 30% of lipedema participants but not controls. Conclusion: Hypertrophic adipocytes, increased numbers of macrophages and blood vessels, and dilation of capillaries in thigh tissue of non-obese women with lipedema suggest inflammation, and angiogenesis occurs independent of obesity and demonstrates a role of altered vasculature in the manifestation of the disease.

Cx43 Expression Correlates with Breast Cancer Metastasis in MDA-MB-231 Cells In Vitro, In a Mouse Xenograft Model and in Human Breast Cancer Tissues.

Connexins regulate multiple cellular functions and are considered tumor suppressors. Connexin43 (Cx43) is frequently down-regulated in breast tumors. However, Cx43 regulation during cancer onset and metastasis is complex and context-dependent. We investigated the effect of Cx43 over-expression or knock-down on the metastatic potential of MDA-MB-231 breast cancer cells in vitro and in vivo and in human breast cancer tissues. MDA-MB-231 cells over-expressing (Cx43D) or down-regulating Cx43 (shCx43) were generated and used in proliferation, migration, and invasion assays. The regulation of genes/proteins implicated in progression, invasion and metastasis was assessed in vitro and in immune-compromized mice injected with MDA-MB-231, Cx43D or shCx43 cells. Primary tumor onset/growth, metastasis and overall survival of these animals was monitored and evaluated. In addition, Cx43 expression in human breast carcinoma samples was assessed by qPCR. Cx43 over-expression increased protein levels of epithelial markers E-cadherin and zonula occludens 1 expression and resulted in the sequestration of ß-catenin at the cell membrane, while Cx43 knock-down induced protein expression of the mesenchymal marker N-cadherin and an increased invasive potential of shCx43 cells. In vivo, in mice xenografted with breast cancer cells, Cx43 over-expression decreased tumor volume, attenuated cell metastasis to lungs and liver and increased overall mice survival. Importantly, the expression of Cx43 in triple negative human breast cancer tissues is also down-regulated. Collectively, Cx43 over-expression induced an epithelial-like phenotype in MDA-MB-231 cells and suppressed tumor growth and metastasis to secondary organs in vivo. In contrast, Cx43 knock-down in MDA-MB-231 cells induced a mesenchymal phenotype with increased cell invasion leading to an enhanced metastatic phenotype. These data provide evidence for a pivotal role of Cx43 in breast cancer metastasis and support the potential targeting of connexins in breast cancer therapy.

Anti-proliferative and anti-inflammatory activities of the sea cucumber Holothuria polii aqueous extract.

OBJECTIVE: Sea cucumbers are considered among the most important functional foods. Following bioassay guided fractionation, we assessed the anti-proliferative and anti-inflammatory activities of Holothuria polii (H. polii) extracts. METHODS: Sea cucumber ethanolic extract and the partially purified aqueous fractions were assessed for their anti-proliferative activities. These latter bioactivities were evaluated in the highly invasive MDA-MB-231 human breast cancer cells in two-dimensional and three-dimensional cultures using trypan blue exclusion assay. The tumor-suppressive effects of sea cucumber ethanolic extract and aqueous fractions were assayed by measuring the trans-well invasion of MDA-MB-231 cells and the expression of some epithelial mesenchymal transition markers using quantitative reverse-transcription polymerase chain reaction and western blot analysis. The anti-inflammatory activity of the aqueous fraction was tested by measuring the secreted levels of interleukin-6, nitric oxide, and matrix metalloproteinase 9 in endotoxin-induced mammary epithelial SCp2 cells and interleukin-1ß in phorbol-12-myristate-13-acetate-activated human monocytic THP-1 cells. RESULTS: Sea cucumber ethanolic extract and the aqueous fraction significantly decreased the proliferation of MDA-MB-231 cells by more than 50% at similar and noncytotoxic concentrations and caused an arrest in the S-phase of the cell cycle of treated cells. In contrast, petroleum ether, chloroform, ethyl acetate, and n-butanol organic fractions did not show any significant activity. Furthermore, sea cucumber ethanolic extract and aqueous fraction reduced the proliferation of MDA-MB-231 cells in three-dimensional cultures by more than 60% at noncytotoxic concentrations. In addition, treatment with these concentrations resulted in the loss of stellate outgrowths in favor of spherical aggregates and a 30% decrease in invasive properties. Both sea cucumber ethanolic extract and aqueous decreased the transcription of vimentin and the protein expression levels of vimentin and N-cadherin in three-dimensional cultures. The aqueous fraction decreased the levels of inflammatory markers interleukin-6, nitric oxide, and matrix metalloproteinase 9 in the mouse mammary SCp2 cells, and the level of interleukin-1ß produced by phorbol-12-myristate-13-acetate-activated THP-1 human monocytic cells. CONCLUSION: The data reveal for the first time promising anti-proliferative and anti-inflammatory activities in H. polii water extract in two-dimensional and three-dimensional culture models.

Intravitreal Adalimumab for the Control of Breakthrough Intraocular Inflammation.

PURPOSE: Investigate the efficacy of intravitreal adalimumab in breakthrough panuveitis in patients on systemic adalimumab for more than 3 months. METHODS: Retrospective study of patients on systemic adalimumab with breakthrough panuveitis requiring intravitreal adalimumab therapy. RESULTS: Seven eyes of four patients with Adamantiades-Behçet disease panuveitis were included and all were maintained on systemic adalimumab for 7.3 months (range 3-11) with inflammation controlled for 4.1 months (range 2-10) before breakthrough uveitis. The total number of attacks was 13 over 24.5 months (range 12-30). Resolution of attack was defined as return to baseline visual acuity with resolution of inflammatory markers. Three attacks resolved after only one injection and 10 attacks required an average of 2.4 injections (range 2-3). No systemic or ocular complications were noted. CONCLUSIONS: Intravitreal adalimumab warrants further investigation as a potentially effective, practical and safe adjunctive therapy for the control of breakthrough inflammation in select patients maintained on systemic adalimumab.

Cross-talk between intestinal epithelial cells and immune cells in inflammatory bowel disease.

Inflammatory bowel disease (IBD) involves functional impairment of intestinal epithelial cells (IECs), concomitant with the infiltration of the lamina propria by inflammatory cells. We explored the reciprocal paracrine and direct interaction between human IECs and macrophages (MΦ) in a co-culture system that mimics some aspects of IBD. We investigated the expression of intercellular junctional proteins in cultured IECs under inflammatory conditions and in tissues from IBD patients. IECs establish functional gap junctions with IECs and MΦ, respectively. Connexin (Cx26) and Cx43 expression in cultured IECs is augmented under inflammatory conditions; while, Cx43-associated junctional complexes partners, E-cadherin, ZO-1, and ß-catenin expression is decreased. The expression of Cx26 and Cx43 in IBD tissues is redistributed to the basal membrane of IEC, which is associated with decrease in junctional complex proteins' expression, collagen type IV expression and infiltration of MΦ. These data support the notion that the combination of paracrine and hetero-cellular communication between IECs and MΦs may regulate epithelial cell function through the establishment of junctional complexes between inflammatory cells and IECs, which ultimately contribute to the dys-regulation of intestinal epithelial barrier.

Anti-angiogenesis therapy and gap junction inhibition reduce MDA-MB-231 breast cancer cell invasion and metastasis in vitro and in vivo.

Cancer cells secrete VEGF, which plays a key role in their growth, invasion, extravasation and metastasis. Direct cancer cell-endothelial cell interaction, mediated by gap junctions, is of critical importance in the extravasation process. In this study, we evaluated avastin (Av), an anti-VEGF antibody; and oleamide (OL), a gap junction inhibitor, using MDA-MB-231 human breast cancer cells in vitro and a xenograft murine model in vivo. Results showed that Av/OL significantly decreased proliferation, induced cell cycle arrest and decreased migration and invasion of MDA-MB-231 cells in vitro. In addition, Av/OL significantly decreased homo and hetero-cellular communication interaction between MDA-MDA and MDA-endothelial cells, respectively. The expression levels of several factors including VEGF, HIF1α, CXCR4, Cx26, Cx43, and MMP9 were attenuated upon Av/OL treatment in vitro. On the other hand, avastin, but not oleamide, reduced tumor size of NSG mice injected subdermally (s.d.) with MDA-MB-231 cells, which was also associated with increased survival. Furthermore, Av but also OL, separately, significantly increased the survival rate, and reduced pulmonary and hepatic metastatic foci, of intravenously (i.v.) injected mice. Finally, OL reduced MMP9 protein expression levels, better than Av and in comparisons to control, in the lungs of MDA-MB-231 i.v. injected NSG mice. In conclusion, while avastin has anti-angiogenic, anti-tumor and anti-metastatic activities, oleamide has anti-metastatic activity, presumably at the extravasation level, providing further evidence for the role of gap junction intercellular communication (GJIC) in cancer cell extravasation.