Melanoma Cell Spreading Research Articles

Direct anti-tumoral effects of sulfated fucans isolated from echinoderms: a possible role of neuropilin-1/β1 integrin endocytosis and focal adhesion kinase (FAK) degradation.

Hypercoagulability, a major complication of metastatic cancers, has usually been treated with heparins from natural sources, or with their synthetic derivatives, which are under intense investigation in clinical oncology. However, the use of heparin has been challenging for patients with risk of severe bleeding. While the systemic administration of heparins, in pre-clinical models, has shown primarily attenuating effects on metastasis, their direct effect on established solid tumors has generated contradictory outcomes. We investigated the direct antitumoral properties of two sulfated fucans isolated from marine echinoderms, FucSulf1 and FucSulf2, which exhibit anticoagulant activity with mild hemorrhagic potential. Unlike heparin, sulfated fucans significantly inhibited tumor cell proliferation (by ~30-50 percent), and inhibited tumor migration and invasion in vitro. We found that FucSulf1 and FucSulf2 interacted with fibronectin (FN) as efficiently as heparin, leading to loss of prostate cancer and melanoma cell spreading. The sulfated fucans increased the endocytosis of β1 integrin and neuropilin-1 (NRP-1) chains, two cell receptors implicated in FN-dependent adhesion. The treatment of cancer cells with both sulfated fucans, but not with heparin, also triggered intracellular focal adhesion kinase (FAK) degradation, with a consequent overall decrease in activated FAK levels. Finally, only sulfated fucans inhibited the growth of B16-F10 melanoma cells implanted in the dermis of syngeneic C57/BL6 mice. FucSulf1 and FucSulf2 arise from this study as candidates for the design of possible alternatives to long-term treatments of cancer patients with heparins, with the advantage of also controlling local growth and invasion of malignant cells.

Endosomal Localization of RacGAP Protein ARHGAP22 Regulates its GAP Activity in Human Melanoma Cells.

Rho small GTPases regulate cancer cell adhesion, migration and invasion through reorganization of the actin cytoskeleton. Rho GTPase Activating Protein 22 (ARHGAP22) is a Rac-specific GAP and suppresses Rac-dependent lamella formation and cell spreading. We have previously shown that ARHGAP22 localizes at endosomes in human melanoma A7 cells. The aim of the present study was to demonstrate the functional significance of its localization at the endosomes in melanoma cells. The lamella formation and cell spreading were monitored using human melanoma A7 cells. The effect of inhibition of endosome recycling pathway was examined. We found that dominant negative Rab11 S25N mutant inhibits RacGAP activity of ARHGAP22 and blocks ARHGAP22-dependent suppression of lamella formation and melanoma cell spreading. Furthermore, deletion of 19 amino acid residues at the C-terminal region of ARHGAP22 abolished the localization of ARHGAP22 at enlarged vesicles and stimulated RacGAP activity of ARHGAP22. The deletion mutant accumulated at enlarged vesicles when endosome recycling pathway was blocked either by co-transfection of the Rab11 S25N mutant or treatment of the cells with N-ethylmaleimide, which blocks endosomal vesicular fusion to the plasma membrane. On the other hand, deletion of the pleckstrin homology (PH) domain of ARHGAP22 abolished its RacGAP activity and localization at the plasma membrane. ARHGAP22 localizes at endosomes and is transported to the plasma membrane to inactivate Rac and suppresses lamella formation and spreading of melanoma cells.

Glycopolymers for Efficient Inhibition of Galectin-3: In Vitro Proof of Efficacy Using Suppression of T Lymphocyte Apoptosis and Tumor Cell Migration.

The development of efficient galectin-3 (Gal-3) inhibitors draws attention in the field of anti-cancer therapy, especially due to the prominent role of extra- and intracellular Gal-3 in vital processes of cancerogenesis, such as immunosuppression, stimulation of tumor cells proliferation, survival, invasion, apoptotic resistance, and metastasis formation and progression. Here, by combining poly-LacNAc (Galβ4GlcNAc)-derived oligosaccharides with N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers, we synthesized multivalent glycopolymer inhibitors with a high potential to target extracellular and intracellular Gal-3. The inhibitory capabilities of the best conjugate in the studied series were in the nanomolar range proving the excellent Gal-3 inhibitory potential. Moreover, thorough investigation of the inhibitory effect in the biological conditions showed that the glycopolymers strongly inhibited Gal-3-induced apoptosis of T lymphocytes and suppressed migration and spreading of colorectal, breast, melanoma, and prostate cancer cells. In sum, the strong inhibitory activity toward Gal-3, combined with favorable pharmacokinetics of HPMA copolymers ensuring enhanced tumor accumulation via the enhanced permeability and retention effect, nominate the glycopolymers containing LacdiNAc-LacNAc (GalNAcβ4GlcNAcβ3Galβ4GlcNAc) tetrasaccharide as promising tools for preclinical in anti-cancer therapy evaluation.

DNA Origami Nanoarrays for Multivalent Investigations of Cancer Cell Spreading with Nanoscale Spatial Resolution and Single-Molecule Control.

We present a strategy for the fabrication of biomimetic nanoarrays, based on the use of DNA origami, that permits the multivalent investigation of ligand-receptor molecule interactions in cancer cell spreading, with nanoscale spatial resolution and single-molecule control. We employed DNA origami to control the nanoscale spatial organization of integrin- and epidermal growth factor (EGF)-binding ligands that modulate epidermal cancer cell behavior. By organizing these multivalent DNA nanostructures in nanoarray configurations on nanopatterned surfaces, we demonstrated the cooperative behavior of integrin and EGF ligands in the spreading of human cutaneous melanoma cells: this cooperation was shown to depend on both the number and ratio of the selective ligands employed. Notably, the multivalent biochips we have developed allowed for this cooperative effect to be demonstrated with single-molecule control and nanoscale spatial resolution. By and large, the platform presented here is of general applicability for the study, with molecular control, of different multivalent interactions governing biological processes from the function of cell-surface receptors to protein-ligand binding and pathogen inhibition.

New small molecular weight antioxidants and pro-oxidants control melanoma cell proliferation and spreading in vitro

Melanoma, the most dangerous skin cancer, originates from the melanocytes and has a high tendency to invade neighboring tissues, and metastasize. Both antioxidant and pro-oxidant appear to be involved in modulating melanocyte transformation, melanoma progression and invasion. Consequently, potent antioxidants and pro-oxidant may prevent cell transformation and tumor progression. Skin melanoma the most common malignancy in United States there are 2 million cases diagnosed in US annually. Among the novel therapies there is the use of bioactive compounds, which have proven to show increase response (IRR) and overall survival (OS) rates. In fact, Dacarbazine is the only FDA approved chemotherapeutic bioactive compound for melanoma treatment. We have identified eight bioactive compounds with antioxidant and pro-oxidative activities as anti-melanoma/anti-invasion agents. They were previously found to posse’s in vitro antioxidant or pro-oxidant activity, Compounds 1, 2, 3, 4, 5, 6, 7 and 8 were found to be most potent anti-melanoma agent. These compounds are now tested for intracellular free radical quenching and ROS producing role in skin melanoma cells in vitro and their ability to reduce proliferation and spreading.

Abstract 93: Tissue-specific CRISPR in zebrafish identifies PVRL1 as a novel metastasis suppressor gene in melanoma

Abstract Defining factors that lead to the spread of cancer will have great impact on designing new treatment options. To search for such modifiers of melanoma, we developed a vector system based on the CRISPR/Cas9 technology of genome editing that targets genes specifically in the melanocytes of a zebrafish melanoma model. As a proof of principle for this system, we showed that pten inactivation in Tg(mitfa:BRAFV600E), p53-/- zebrafish significantly accelerated tumor onset. This system allows us to interrogate the functional relevance of recurrent genetic alterations found in cancer patients by next generation sequencing. Here, we identified a frequent focal deletion on chr11q23.3 by GISTIC analysis of the TCGA cohort of primary human melanomas. This region contains a gene called PVRL1 that is homozygously lost in 8% of tumors. PVRL1 encodes nectin-1, a transmembrane protein involved in adherens junctions. To assess the role of PVRL1 in melanoma in vivo, we injected Tg(mitfa:BRAFV600E), p53-/- zebrafish embryos with our melanocyte-specific CRISPR vector directed against pvrl1. We detected tumors earlier in fish expressing a vector targeting pvrl1 compared to fish expressing a control vector (median: 18 vs 20 weeks). Deep sequencing of the pvrl1 CRISPR sites in tumors confirmed gene inactivation through frameshift insertions or deletions. To further assess the in vivo phenotype of pvrl1-deficient cells, we designed a transplantation assay in which we injected 300,000 primary zebrafish melanoma cells subcutaneously in the back of irradiated, transparent recipients. In this assay, pvrl1 CRISPR melanoma cells consistently migrated away from the injection site, and eventually invaded internal tissues, whereas control cells remained localized in the dorsal region of transplanted fish. Thus pvrl1 loss favors the spreading of melanoma cells. To investigate the function of PVRL1 at the molecular level, we established pvrl1 knock-out zebrafish melanoma cell lines in culture and realized that pvrl1 was required for attachment and sustained growth in vitro in the absence of zebrafish serum. Nectin-1 was up-regulated upon serum stress in both zebrafish and human lines, presumably to support cell survival via cell-cell interactions. Additionally PVRL1 overexpression in human melanoma cell lines increased cell aggregation in vitro. Finally, we found that nectin-1 expression was higher in primary melanomas compared to benign nevi, but lower in metastases compared to primary melanoma, in human tissue-microarrays. Collectively, these data suggest a model whereby PVRL1 expression controls a balance between growth and invasion. Cancer cells deficient for PVRL1 are less fit but more prone to detach from the tumor under serum stress conditions. These results establish PVRL1 as a new metastasis suppressor gene in melanoma and cast light on the role of cancer cell adhesion in tumor spreading. Citation Format: Julien Ablain, Michelle Dang, Harriet Rothschild, Leonard I. Zon. Tissue-specific CRISPR in zebrafish identifies PVRL1 as a novel metastasis suppressor gene in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 93.

Nanoscale Tuning of VCAM-1 Determines VLA-4-Dependent Melanoma Cell Plasticity on RGD Motifs.

The biophysical fine-tuning of cancer cell plasticity is crucial for tumor progression but remains largely enigmatic. Although vascular cell adhesion molecule-1 (VCAM-1/CD106) has been implicated in melanoma progression, here its presentation on endothelial cells was associated with diminished melanoma cell spreading. Using a specific nanoscale modulation of VCAM-1 (tunable from 70 to 670 ligands/μm²) next to integrin ligands (RGD motifs) in a bifunctional system, reciprocal regulation of integrin α4 (ITGA4/VLA-4/CD49d)-dependent adhesion and spreading of melanoma cells was found. As the VCAM-1/VLA-4 receptor pair facilitated adhesion, while at the same time antagonizing RGD-mediated spreading, melanoma cell morphogenesis on these bifunctional matrices was directly regulated by VCAM-1 in a dichotomic and density-dependent fashion. This was accompanied by concordant regulation of F-actin cytoskeleton remodeling, Rac1-expression, and paxillin-related adhesion formation. The novel function of VCAM-1 was corroborated in vivo using two murine models of pulmonary metastasis. The regulation of melanoma cell plasticity by VCAM-1 highlights the complex regulation of tumor-matrix interactions.Implications: Nanotechnology has revealed a novel dichotomic function of the VCAM-1/VLA-4 interaction on melanoma cell plasticity, as nanoscale tuning of this interaction reciprocally determines adhesion and spreading in a ligand density-dependent manner. Mol Cancer Res; 16(3); 528-42. ©2017 AACR.

Intrinsic Response Towards Physiologic Stiffness is Cell-Type Dependent.

In the continuous search for better tissue engineering scaffolds it has become increasingly clear that the substrate properties dramatically affect cell responses. Here we compared cells from a physiologically stiff tissue, melanoma, to cells isolated from a physiologically soft tissue, brain. We measured the cell line responses to laminin immobilized onto glass or polyacrylamide hydrogels tuned to have a Young's modulus ranging from 1 to 390 kPa. Single cells were analyzed for spreading area, shape, total actin content, actin-based morphological features and modification of immobilized laminin. Both cell types exhibited stiffness- and laminin concentration-dependent responses on polyacrylamide and glass. Melanoma cells exhibited very little spreading and were rounded on soft (1, 5, and 15 kPa) hydrogels while cells on stiff (40, 100, and 390 kPa) hydrogels were spread and had a polarized cell shape with large lamellipodia. On rigid glass surfaces, spreading and actin-based morphological features were not observed until laminin concentration was much higher. Similarly, increased microglia cell spreading and presence of actin-based structures were observed on stiff hydrogels. However, responses on rigid glass surfaces were much different. Microglia cells had large spreading areas and elongated shapes on glass compared to hydrogels even when immobilized laminin density was consistent on all gels. While cell spreading and shape varied with Young's modulus of the hydrogel, the concentration of f-actin was constant. A decrease in laminin immunofluorescence was associated with melanoma and microglia cell spreading on glass with high coating concentration of laminin, indicating modification of immobilized laminin triggered by supraphysiologic stiffness and high ligand density. These results suggest that some cell lines are more sensitive to mechanical properties matching their native tissue environment while other cell lines may require stiffness and extracellular ligand density well above physiologic tissue before saturation in cell spreading, elongation and cytoskeletal re-organization are reached.

Quantitative comparison of the spreading and invasion of radial growth phase and metastatic melanoma cells in a three-dimensional human skin equivalent model.

BackgroundStandard two-dimensional (2D) cell migration assays do not provide information about vertical invasion processes, which are critical for melanoma progression. We provide information about three-dimensional (3D) melanoma cell migration, proliferation and invasion in a 3D melanoma skin equivalent (MSE) model. In particular, we pay careful attention to compare the structure of the tissues in the MSE with similarly-prepared 3D human skin equivalent (HSE) models. The HSE model is identically prepared to the MSE model except that melanoma cells are omitted. Using the MSE model, we examine melanoma migration, proliferation and invasion from two different human melanoma cell lines. One cell line, WM35, is associated with the early phase of the disease where spreading is thought to be confined to the epidermis. The other cell line, SK-MEL-28, is associated with the later phase of the disease where spreading into the dermis is expected.Methods3D MSE and HSE models are constructed using human de-epidermised dermis (DED) prepared from skin tissue. Primary fibroblasts and primary keratinocytes are used in the MSE and HSE models to ensure the formation of a stratified epidermis, with a well-defined basement membrane. Radial spreading of cells across the surface of the HSE and MSE models is observed. Vertical invasion of melanoma cells downward through the skin is observed and measured using immunohistochemistry. All measurements of invasion are made at day 0, 9, 15 and 20, providing detailed time course data.ResultsBoth HSE and MSE models are similar to native skin in vivo, with a well-defined stratification of the epidermis that is separated from the dermis by a basement membrane. In the HSE and MSE we find fibroblast cells confined to the dermis, and differentiated keratinocytes in the epidermis. In the MSE, melanoma cells form colonies in the epidermis during the early part of the experiment. In the later stage of the experiment, the melanoma cells in the MSE invade deeper into the tissues. Interestingly, both the WM35 and SK-MEL-28 melanoma cells lead to a breakdown of the basement membrane and eventually enter the dermis. However, these two cell lines invade at different rates, with the SK-MEL-28 melanoma cells invading faster than the WM35 cells.DiscussionThe MSE and HSE models are a reliable platform for studying melanoma invasion in a 3D tissue that is similar to native human skin. Interestingly, we find that the WM35 cell line, that is thought to be associated with radial spreading only, is able to invade into the dermis. The vertical invasion of melanoma cells into the dermal region appears to be associated with a localised disruption of the basement membrane. Presenting our results in terms of time course data, along with images and quantitative measurements of the depth of invasion extends previous 3D work that has often been reported without these details.

P 032 - Modulation of Melanoma Cell Proliferation and Spreading by Novel Small Molecular Weight Antioxidants

Melanoma, the most dangerous skin cancer, originates from the melanocytes and has a high tendency to invade neighboring tissues, and metastasize. Free radicals appear to be involved in the melanocyte transformation and melanoma progression as well as invasion. Consequently, potent antioxidants may prevent transformation and progression of the tumor. We have recently synthesized some small molecular weight antioxidant compounds of eleven different synthetic classes. These compounds were previously found to posses free radical quenching activity in an in vitro antioxidant assay. Compounds 1 and 2 were found to be most potent among them with IC50=42.9 ± 0.31, and 55.6 ± 2.1 µM, respectively, as compared to standard butylated hydroxy toluene with IC50=128.8 ± 2.1 µM. We are now testing these compounds for their intracellular free radical quenching role in skin melanoma cells in vitro and their ability to reduce proliferation and spreading. We are currently analyzing the influence of these compounds on the molecular pathways, involved in the proliferation-invasion inhibition.

Suspected vitreous seeding of uveal melanoma: relevance of diagnostic vitrectomy

AimTo review all cases of suspected vitreous seeding of treated or untreated uveal melanoma at our clinic and to compare clinical, cytological and histological findings with patients’ survival.MethodsRetrospective non-randomised study...

GPR4 decreases B16F10 melanoma cell spreading and regulates focal adhesion dynamics through the G13/Rho signaling pathway

The effect of acidosis, a biochemical hallmark of the tumor microenvironment, on cancer progression and metastasis is complex. Both pro- and anti-tumorigenic effects of acidosis have been reported and the acidic microenvironment has been exploited for specific delivery of drugs, imaging agents, and genetic constructs into tumors. In this study we investigate the spreading and focal adhesion of B16F10 melanoma cells that are genetically engineered to overexpress the pH-sensing G protein-coupled receptor GPR4. By using cell attachment assays we found that GPR4 overexpression delayed cell spreading and altered the spatial localization of dynamic focal adhesion complex, such as the localization of phosphorylated focal adhesion kinase (FAK) and paxillin, at acidic pH. The potential G-protein and downstream signaling pathways that are responsible for these effects were also investigated. By using the Rho inhibitor CT04 (C3 transferase), the Rho-associated kinase (ROCK) inhibitors Y27632 and thiazovivin, the myosin light chain kinase (MLCK) inhibitor staurosporine or a G12/13 inhibitory construct, cell spreading was restored whereas the inhibition and activation of the Gq and Gs pathways had little or no effect. Altogether our results indicate that through the G12/13/Rho signaling pathway GPR4 modulates focal adhesion dynamics and reduces cell spreading and membrane ruffling.

TNF-receptor-1 adaptor protein FAN mediates TNF-induced B16 melanoma motility and invasion

Background:Locomotion of cancer cells can be induced by TNF and other motogenic factors secreted by cells of the tumour microenvironment such as macrophages. Based on our recent findings that the TNF receptor adaptor protein FAN mediates TNF-induced actin reorganisation and regulates the directed migration of immune cells responding to chemotactic cues, we addressed the role of FAN in cancer cell motility and the formation of invadopodia, a crucial feature in tumour invasion.Methods:In B16 mouse melanoma cells, FAN was downregulated and the impact on FAN on cell motility and invasion was determined using in vitro assays and in vivo animal models.Results:Like FAN−/− murine embryonic fibroblasts, FAN-deficient B16 melanoma cells showed defective motility responses to TNF in vitro. In vivo FAN-deficient B16 melanoma cells produced significantly less disseminated tumours after i.v. injection into mice. Danio rerio used as a second in vivo model also revealed impaired spreading of FAN-deficient B16 melanoma cells. Furthermore, FAN mediated TNF-induced paxillin phosphorylation, metalloproteinase activation and increased extracellular matrix degradation, the hallmarks of functionally active invadopodia.Conclusion:The results of our study suggest that FAN through promoting melanoma cellular motility and tumour invasiveness is critical for the tumour-promoting action of TNF.

Melanoma Cell Invasiveness Is Promoted at Least in Part by the Epidermal Growth Factor–Like Repeats of Tenascin-C

Tenascin-C (TNC), overexpressed in invasive growths, has been implicated in progression of melanoma but the source and function of this molecule are not well defined. We found TNC expression at the front of invading melanoma cells, and that adding TNC to matrices enhances individual melanoma cell migration. As TNC is a multidomain protein, we examined the role of the TNC EGF-like repeats (EGFL) as these activate motogenic signaling cascades. We overexpressed a TNC fragment containing the assembly and EGFL domains of TNC (TNCEGFL). TNCEGFL-expressing melanoma cells had lower speed and persistence in 2D migration assays due to a shift in the adhesion-contractility balance, as expression of TNCEGFL delayed melanoma cell attachment and spreading. The less adhesive phenotype was due, in part, to increased ROCK signaling concomitant with MLC2 and MYPT phosphorylation. Inhibition of ROCK activity, which drives transcellular contractility, restored adhesion of TNCEGFL expressing melanoma cells and increased their migration in 2D. In contrast to the diminished migration in 2D, TNCEGFL-expressing melanoma cells had higher invasive potential in Matrigel invasion assays, with cells expressing TNCEGFL having amoeboid morphology. Our findings suggest that melanoma-derived TNC EGFL play a role in melanoma invasion by modulating ROCK signaling and cell migration.

Abstract 485: Epidermal growth factor-like repeats of Tenascin C promote melanoma cell invasion, possibly by enabling mesenchymal to ameboid migration transition

Abstract In malignant melanoma, the most dangerous skin cancer, Tenascin-C (TNC) is overexpressed, especially at the invasive fronts of invasive melanomas. We found that the migration and invasiveness of a set of melanoma cell lines correlates with levels of TNC expression suggesting, surprisingly, that the melanoma cells themselves provide much of this matrix component. Adding TNC to matrices enhanced individual cell migration in cells that did not express much TNC, whereas it did not enhance the rapid motility of the cells expressing high levels. As TNC is a multidomain protein, the signaling pathways that promote this motogenic invasiveness are not obvious. Herein, we examined the potential role of the EGF-like repeats of TNC that have been shown to preferentially activate motogenic-signaling cascades. To analyze the effects of EGFL signaling on melanoma migration and invasion, we overexpressed TNC fragment containing the assembly and EGFL domains of TNC (TNCEGFL). Interestingly, this led to impaired cell migration in in vitro wound healing assays and live cell tracking experiments. This was likely due to a shift in the adhesion-contractility balance during cell migration. TNCEGFL expressing cells presented a spindle-shaped morphology and formed cords in culture. Expression of TNCEGFL delayed melanoma cell attachment and spreading, as determined in cell adhesion and inverted centrifugation assays. This anti-adhesive phenotype was concomitant with increased MLC2 phosphorylation. Inhibition of ROCK activity, which drives transcellular contractility, restored adhesion of TNCEGFL expressing cells and improved migration in 2D. In distinction to the findings in 2D, TNCEGFL expressing cells had higher invasion potential in Matrigel invasion assays, with cells expressing high TNC having rounded “ameboid” morphology, underlying the importance of morphological shifts, and possibly ROCK signaling, in 3D movement through barrier matrices during invasion and dissemination. These results imply that TNCEGFL repeats are involved in melanoma invasion both by directly influencing cell migration and indirectly changing the tumor microenvironment composition, possibly allowing transition from mesenchymal to ameboid migrational mode. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 485. doi:1538-7445.AM2012-485

Keratinocyte-derived Laminin-332 Promotes Adhesion and Migration in Melanocytes and Melanoma

Melanocytes are highly motile cells that play an integral role in basic skin physiological processes such as wound healing and proper skin pigmentation. It has been postulated that surrounding keratinocytes contribute to melanocyte migration, but underlying mechanisms remain rather vague so far. In this study, we set out to analyze the specific potential contribution of keratinocyte components to melanocytes and melanoma cell migration-related processes. Our studies revealed that A375 human melanoma cell attachment, spreading, and migration are interestingly better supported by HaCaT keratinocyte extracellular matrix (ECM) than by self-derived A375 ECM. Moreover, HaCaT ECM caused increased integrin α6 expression, adhesion-mediated focal adhesion kinase phosphorylation, and focal adhesion formations. Similar effects were confirmed in human melanocytes. Furthermore, we found that keratinocyte-derived soluble factors did not appear to significantly contribute to these processes. Specific extrinsic factors that promoted melanoma migration were attributed to keratinocyte-derived laminin-332, whereas alternative ECM component such as laminin-111 and fibronectin functions appeared to have insignificant contributions. Taken together, these studies implicate extrinsic laminin-332 in promoting the high mobility property and perhaps invasiveness inherently characteristic of, and that are the menace of, melanocytes and melanomas, respectively.

Tailored excision of in-transit metastatic melanoma based on indocyanine green fluorescence lymphography

In-transit metastases of melanoma almost certainly results from lymphatic dissemination of melanoma cells to tissues between the primary melanoma site and the draining regional lymph nodes. Most in-transit metastases occur after lymphatic obstruction, due either to tumor involvement or to surgical removal. The treatment of in-transit metastatic melanoma has not been standardized, and should be tailored accordingly. We report a case of in-transit metastatic melanoma in head and neck after regional lymph node dissection. In-transit metastases in the cheek were excised with tailored surgical margins based on the evaluation of lymph flow using indocyanine green (ICG) fluorescence lymphography. ICG fluorescence lymphography has been reported recently in non-invasive sentinel lymph node mapping, and lymphaticovenous anastomosis. Using this system, not only sentinel lymph nodes and lymph vessels but also dermal lymphatic back flow caused by previous surgery is detectable as a white spot through fluorescence high-sensitivity near-infrared video camera system. This enables our detection of abnormal lymphatic flow, which indicates possible areas of melanoma cell spreading through the lymphatic system. Although a preliminary case report, we propose tailored excision of in-transit metastatic melanoma based on ICG fluorescence lymphography.

Moesin/ezrin: a specific role in cell metastasis?

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Why vitamin D for cancer patients?

Several epidemiological, pre-clinical and clinical studies support Vitamin D as a preventive and therapeutic cancer agent.BackgroundVitamin D and cancer: calcitriol, the biologically active form of vitamin D (1,25(OH)D), exerts its effects mainly through binding to nuclear vitamin D receptor (VDR). Calcitriol has been shown to be an anti-proliferative, pro-differentiation, pro-apoptotic agent and an inhibitor of cell migration. Animal and human in vitro studies strongly indicate that vitamin D may have benefits for many forms of cancer. Inadequate levels of circulating 25-hydroxy-vitamin D (25(OH)D) are associated with an increased risk and poor prognosis of several types of cancer.Vitamin D and melanoma: cutaneous malignant melanoma (CMM) represents a major public health issue: rates in Italy have almost doubled in the last decade and CMM is frequent among young adults. For resected stage II melanoma no standard adjuvant treatment exists and five-year overall survival is about 70%.Cultured melanoma cells can synthesize calcitriol from 25(OH)D and express the VDR. Moreover, 1,25(OH)D has anti-proliferative and pro-differentiation effects in human melanoma cells. 1,25(OH)D has been shown to induce apoptosis in human melanoma cell lines and has an inhibitory effect on the spreading of melanoma cells in vitro.Preliminary results on vitamin D: epidemiological data indicate that vitamin D deficiency is relatively common in Europe. In an Italian study, we found that 85% of the participants had insufficient levels of 25(OH)D. We have shown through a meta-analysis of randomized trials that vitamin D supplementation is associated with a significant reduction (7%) in total mortality in healthy subjects and an association between VDR and 25(OH)D and CMM progression has also been demonstrated. We have also reported significant associations between VDR polymorphisms and incidence of skin cancer. In early supplementation trials, the lack of effect on cancer incidence has been attributed to insufficient vitamin D supplementation, stressing the need to better study vitamin D bioavailability.In fact, a recent IARC report highlighted the need for new randomized trials, based on results from our meta-analyses on 25(OH)D serum levels and cancer risk.Clinical trial and biomarkers studies: in order to assess whether vitamin D supplementation could improve prognosis of CMM, an Italian multi-centre trial in stage II resected melanoma patients was planned to monitor changes in 25(OH)D. The study will address two important questions regarding the relationship between the biology of VDR and (1) vitamin D metabolism, and (2) CMM prognosis. This will involve investigating the association between VDR polymorphisms and Breslow thickness, the most important prognostic factor of CMM, and between 25(OH)D serum level, vitamin D supplementation and VDR.We will also evaluate at baseline whether VDR polymorphisms are associated with Breslow thickness and whether we obtain significant increase in 25(OH)D serum levels during the first year of supplementation. We will quantify the percentages of patients who have desirable levels of 25(OH)D and, if they don’t, the mean time to reach that level. The findings from this study will be of great interest because vitamin D could have anti-cancer benefits for a wide spectrum of cancers.

Canstatin gene electrotransfer combined with radiotherapy: preclinical trials for cancer treatment

Given as a prophylactic treatment, a single muscle electrogene transfer of plasmid coding canstatin fused to human serum albumin (CanHSA), slowed down the development of two xenografted human carcinomas from mammary (MDA-MB-231) and prostate origin (PC-3) in nude mice and delayed lung metastatic spreading of B16F10 melanoma cells in syngenic mice. No effect was observed with unfused canstatin. The long lasting circulating blood level of CanHSA (20 ng ml(-1)) resulted in a profound disorganization of the tumor blood vessel network. However, when used as a curative treatment, on well-established tumors, CanHSA electrogenetherapy was ineffective in reducing tumor growth. As radiation is known to increase the alpha v beta3 and alpha v beta5 integrins, which are canstatin receptors, to extend the use of CanHSA electrogenetherapy, as a curative treatment, we explored the combination of CanHSA and ionizing radiation. We demonstrated a better efficacy (P=0.01) of the bitherapy over irradiation alone, as a result of strong vessel disorganization and dramatic increase of tumor cells apoptosis. This extremely simple virus free curative protocol could open the door to potential clinical applications, especially for prostate cancer that often develops radioresistance.