Dissemination Of Melanoma Cells Research Articles

Abstract 4997: MMP14 as PTP4A3 downstream target is involved in uveal melanoma cell dissemination

Abstract Abstract: Uveal melanoma is the most common intraocular malignancy in adults, representing 5% of all melanomas. Although at diagnosis over 95% of patients have disease limited to the eye, about 50% will develop metastases after a median period of 36 months. High expression levels of Protein Tyrosine Phosphatase 4A3 (PTP4A3), a dual phosphatase, is highly predictive of metastasis development and PTP4A3 overexpression in uveal melanoma cells increases their in vitro migration and in vivo invasiveness. The PTP4A3 mechanism of action in the tumoral process is poorly understood. We identified the metalloprotease MMP14 as an interacting partner of PTP4A3. MMP14 is more abundant at the cell membrane in PTP4A3 invasive cells compared to control cells (inactive catalytic site mutant GFP-PTP4A3(C104S) or EGFP) and inhibition of MMP14 expression decreases PTP4A3-induced cell invasiveness. The MMP14-differential cell membrane localization in presence of PTP4A3 is currently investigated by studying the trafficking of MMP14 in these cells. Citation Format: Selma Maacha. MMP14 as PTP4A3 downstream target is involved in uveal melanoma cell dissemination. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4997. doi:10.1158/1538-7445.AM2014-4997

Regulatory and Functional Connection of Microphthalmia-Associated Transcription Factor and Anti-Metastatic Pigment Epithelium Derived Factor in Melanoma

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor superfamily, has potent anti-metastatic effects in cutaneous melanoma through its direct actions on endothelial and melanoma cells. Here we show that PEDF expression positively correlates with microphthalmia-associated transcription factor (MITF) in melanoma cell lines and human samples. High PEDF and MITF expression is characteristic of low aggressive melanomas classified according to molecular and pathological criteria, whereas both factors are decreased in senescent melanocytes and naevi. Importantly, MITF silencing down-regulates PEDF expression in melanoma cell lines and primary melanocytes, suggesting that the correlation in the expression reflects a causal relationship. In agreement, analysis of Chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq) data sets revealed three MITF binding regions within the first intron of SERPINF1, and reporter assays demonstrated that the binding of MITF to these regions is sufficient to drive transcription. Finally, we demonstrate that exogenous PEDF expression efficiently halts in vitro migration and invasion, as well as in vivo dissemination of melanoma cells induced by MITF silencing. In summary, these results identify PEDF as a novel transcriptional target of MITF and support a relevant functional role for the MITF-PEDF axis in the biology of melanoma.

MT1‐MMP modulates melanoma cell dissemination and metastasis through activation of MMP2 and RAC1

Metastatic melanoma remains the deadliest of all skin cancers with a survival rate at five years of less than 15%. MT1-MMP is a membrane-associated matrix metalloproteinase that controls pericellular proteolysis and is an important, invasion-promoting, pro-tumorigenic MMP in cancer. We show that deregulation of MT1-MMP expression happens as early as the transition from nevus to primary melanoma and continues to increase during melanoma progression. Furthermore, MT1-MMP expression is associated with poor melanoma patient outcome, underscoring a pivotal role of MT1-MMP in melanoma pathogenesis. We demonstrate that MT1-MMP is directly required for melanoma cells to metastasize, as cells deprived of MT1-MMP fail to form distant metastasis in an orthotopic mouse melanoma model. We show that MT1-MMP affects cell invasion by activating its target MMP2. Importantly, we demonstrate, for the first time, that activation of MMP2 by MT1-MMP is required to sustain RAC1 activity and promote MT1-MMP-dependent cell motility. These data highlight a novel MT1-MMP/MMP2/RAC1 signaling axis in melanoma that may represent an intriguing molecular target for the treatment of invasive melanoma.

Dual role of sphingosine kinase-1 in promoting the differentiation of dermal fibroblasts and the dissemination of melanoma cells

Despite progress in the understanding of the biology and genetics of melanoma, no effective treatment against this cancer is available. The adjacent microenvironment has an important role in melanoma progression. Defining the molecular signals that control the bidirectional dialog between malignant cells and the surrounding stroma is crucial for efficient targeted therapy. Our study aimed at defining the role of sphingosine-1-phosphate (S1P) in melanoma-stroma interactions. Transcriptomic analysis of human melanoma cell lines showed increased expression of sphingosine kinase-1 (SPHK1), the enzyme that produces S1P, as compared with normal melanocytes. Such an increase was also observed by immunohistochemistry in melanoma specimens as compared with nevi, and occurred downstream of ERK activation because of BRAF or NRAS mutations. Importantly, migration of melanoma cells was not affected by changes in SPHK1 activity in tumor cells, but was stimulated by comparable modifications of S1P-metabolizing enzymes in cocultured dermal fibroblasts. Reciprocally, incubation of fibroblasts with the conditioned medium from SPHK1-expressing melanoma cells resulted in their differentiation to myofibroblasts, increased production of matrix metalloproteinases and enhanced SPHK1 expression and activity. In vivo tumorigenesis experiments showed that the lack of S1P in the microenvironment prevented the development of orthotopically injected melanoma cells. Finally, local tumor growth and dissemination were enhanced more efficiently by coinjection of wild-type skin fibroblasts than by fibroblasts from Sphk1(-/-) mice. This report is the first to document that SPHK1/S1P modulates the communication between melanoma cells and dermal fibroblasts. Altogether, our findings highlight SPHK1 as a potential therapeutic target in melanoma progression.

Development and characterization of a clinically relevant mouse model of melanoma brain metastasis

Development and characterization of a clinically relevant mouse model of melanoma brain metastasis

Cyr61 is a target for heparin in reducing MV3 melanoma cell adhesion and migration via the integrin VLA-4

The integrin VLA-4 is important for the metastatic dissemination of melanoma cells. We could recently show that heparin can block VLA-4 binding, which contributes, next to blocking P- and L-selectin, to the understanding of antimetastatic activities of heparin. The matricellular ligand Cyr61, secreted by numerous tumours, is responsible for increased tumourigenicity and metastasis. This has been attributed to Cyr61 binding to, and thus activating integrins. However, a VLA-4/Cyr61 axis has not yet been reported. Since Cyr61 possesses heparin binding capabilities, Cyr61 can be supposed as potential target for heparin to indirectly interfere with integrin functions. The present in vitro studies address (i) the existence of a Cyr61/VLA-4 axis and (ii) the functional relevance of heparin interference via Cyr61. The C-terminal module III of Cyr61 could be exposed as nanomolar affine binding site for VLA-4. A shRNA-based knockdown of Cyr61 in MV3 human melanoma cells reduced VLA-4-mediated cell binding to VCAM-1, migration on fibronectin, and integrin signalling functions significantly. Using a biosensor approach we provide insight into heparin interference with this process. The low-molecular-weight heparin tinzaparin, but not the pentasaccharide fondaparinux, binds module IV of Cyr61 with micromolar affinity. But tinzaparin cannot interfere with Cyr61 accumulation onto syndecan-4, indicating different Cyr61 binding sites for heparin and other GAGs. Nonetheless, tinzaparin affects the VLA-4 binding and signalling functions selectively via Cyr61 already at very low concentration most likely by blocking the cellular secreted free Cyr61. This study emphasises Cyr61 as promising, and hitherto not considered target for heparin to selectively influence integrin functions.

LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

BackgroundPatients with metastatic melanoma have a poor median rate of survival. It is therefore necessary to increase our knowledge about melanoma cell dissemination which includes extravasation, where cancer cells cross the endothelial barrier. Extravasation is well understood during travelling of white blood cells, and involves integrins such as LFA-1 (composed of two chains, CD11a and CD18) expressed by T cells, while ICAM-1 is induced during inflammation by endothelial cells. Although melanoma cell lines cross endothelial cell barriers, they do not express LFA-1. We therefore hypothesized that melanoma-endothelial cell co-culture might induce the LFA-1/ICAM ligand/receptor couple during melanoma transmigration.MethodsA transwell approach has been used as well as blocking antibodies against CD11a, CD18 and ICAM-1. Data were analyzed with an epifluorescence microscope. Fluorescence intensity was quantified with the ImageJ software.ResultsWe show here that HUVEC-conditioned medium induce cell-surface expression of LFA-1 on melanoma cell lines. Similarly melanoma-conditioned medium activates ICAM-1 expression in endothelial cells. Accordingly blocking antibodies of ICAM-1, CD11a or CD18 strongly decrease melanoma transmigration. We therefore demonstrate that melanoma cells can cross endothelial monolayers in vitro due to the induction of ICAM-1 and LFA-1 occurring during the co-culture of melanoma and endothelial cells. Our data further suggest a role of LFA-1 and ICAM-1 in the formation of melanoma cell clumps enhancing tumor cell transmigration.ConclusionMelanoma-endothelial cell co-culture induces LFA-1 and ICAM-1 expression, thereby favoring in vitro melanoma trans-migration.

The inhibition of the integrin VLA-4 in MV3 melanoma cell binding by non-anticoagulant heparin derivatives

IntroductionThe integrin VLA-4-mediated binding is important for the metastatic dissemination of melanoma cells. Recently we found that heparin possesses a binding capacity to VLA-4. This could contribute to the heparin function to attenuate metastasis in a selectin-dependent manner. Aiming to a purposive, anti-adhesive heparin application, structural requirements of heparin for VLA-4 recognition have to be elucidated. Materials and methodsA series of non-anticoagulant heparin derivatives were investigated concerning their inhibitory capacities for VLA-4 mediated binding of human melanoma MV3 cells to VCAM-1 under physiological flow conditions in vitro. A surface acoustic wave biosensor was applied to detect kinetic constants of selected derivatives binding to both, VLA-4 or P- and L-selectin. ResultsExperimental metastasis of MV3 cells in mice confirmed the relevance of VLA-4 for metastatic dissemination. LMWHs (enoxaparin, tinzaparin) efficiently blocked VLA-4 cell binding, dominantly via the integrin`s α-chain. Desulfation at 2-O-position, N-acetylation or a size smaller than tetradecasaccharide disfavoured VLA-4 inhibition. Glycol-splitting of heparin and thus higher chain flexibility is a tolerable parameter. A derivative with 50% 6-O-desulfation appeared promising and exceeded tinzaparin in VLA-4 inhibition, both compounds displayed binding affinities to VLA-4 in the low micromolar range. ConclusionsThese findings provide structure-activity relationships for heparin VLA-4 binding, which partly differ from P- and L-selectin requirements. The data confirm that anti-coagulative and anti-adhesive function of heparin can be distinguished favouring applications of non-anticoagulant heparins in antimetastatic approaches without the risk of bleeding complications. The 50% 6-O-desulfated heparin-derivative appears promising to further evaluate the interference with selectin and VLA-4 binding functions in vivo.

Rap1-GTP-interacting Adaptor Molecule (RIAM) Protein Controls Invasion and Growth of Melanoma Cells

The Mig-10/RIAM/lamellipodin (MRL) family member Rap1-GTP-interacting adaptor molecule (RIAM) interacts with active Rap1, a small GTPase that is frequently activated in tumors such as melanoma and prostate cancer. We show here that RIAM is expressed in metastatic human melanoma cells and that both RIAM and Rap1 are required for BLM melanoma cell invasion. RIAM silencing in melanoma cells led to inhibition of tumor growth and to delayed metastasis in a severe combined immunodeficiency xenograft model. Defective invasion of RIAM-silenced melanoma cells arose from impairment in persistent cell migration directionality, which was associated with deficient activation of a Vav2-RhoA-ROCK-myosin light chain pathway. Expression of constitutively active Vav2 and RhoA in cells depleted for RIAM partially rescued their invasion, indicating that Vav2 and RhoA mediate RIAM function. These results suggest that inhibition of cell invasion in RIAM-silenced melanoma cells is likely based on altered cell contractility and cell polarization. Furthermore, we show that RIAM depletion reduces β1 integrin-dependent melanoma cell adhesion, which correlates with decreased activation of both Erk1/2 MAPK and phosphatidylinositol 3-kinase, two central molecules controlling cell growth and cell survival. In addition to causing inhibition of cell proliferation, RIAM silencing led to higher susceptibility to cell apoptosis. Together, these data suggest that defective activation of these kinases in RIAM-silenced cells could account for inhibition of melanoma cell growth and that RIAM might contribute to the dissemination of melanoma cells.

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.

Metastasis: Alone or Together?

Summary Recent studies of carcinoma progression reveal the distinct routes of dissemination of discrete carcinoma cell populations and suggest that melanoma cell dissemination is linked to differentiation rather than stemness status.

Dormancy of metastatic melanoma

Metastatic dormancy of melanoma has not received sufficient attention, most likely because once detectable, metastasis is almost invariably fatal and, understandably, the focus has been on finding ways to prolong life of patients with overt recurrences. Nevertheless, analysis of the published clinical and experimental data on melanoma indicates that some aspect of melanoma biology imitate traits recently associated with dormancy in other solid cancers. Among them the ability of some melanomas to disseminate early during primary tumor progression and once disseminated, to remain undetected (dormant) for years. Comparison of cutaneous and uveal melanoma indicates that, in spite of being of the same origin, they differ profoundly in their clinical progression. Importantly for this discussion, between 40 and 50% of uveal melanoma remain undetected for longer than a decade, while less than 5% of cutaneous melanoma show this behavior. Both types of melanoma have activating oncogene mutations that provide autonomous pro-proliferative signals, yet the consensus is that those are not sufficient for tumor progression. If that is the case, it is possible to envision that signals from outside the tumor cell, (microenvironment) shape the fate of an individual disseminated cell, regardless of an oncogene mutation, to progress or to pause in a state of dormancy. To stimulate further debate and inquiry we describe here a few examples of potential signals that might modify the fate of disseminated cell and provide brief description of the current knowledge on dormancy in other cancers. Our hope is to convince the reader that disseminated melanoma cells do enter periods of prolonged dormancy and that finding ways to induce it, or to prolong it, might mean an extension of symptoms-free life for melanoma patients. Ultimately, understanding the biology of dormancy and the mechanisms of dormant cell survival, might allow for their specific targeting and elimination.

Melanoma-Specific Memory T Cells Are Functionally Active inRetTransgenic Mice without Macroscopic Tumors

We previously reported that bone marrows of breast cancer patients contained tumor antigen-specific CD8(+) T cells with central or effector memory phenotype. Using a recently developed ret transgenic mouse melanoma model, we now show that bone marrows and tumors of transgenic mice contain high frequencies of CD8(+) T cells specific for the melanoma antigen tyrosinase-related protein 2 and showing mostly effector memory phenotype. Moreover, increased numbers of bone marrow tyrosinase-related protein-2-specific effector memory CD8(+) T cells are also detected in transgenic animals older than 20 weeks with disseminated melanoma cells in the bone marrow and lymph nodes but showing no visible skin tumors and no further melanoma progression. After a short-term coincubation with dendritic cells generated from the bone marrow and pulsed with melanoma lysates, bone marrow memory T cells from mice without macroscopic melanomas produced IFN-gamma in vitro and exerted antitumor activity in vivo after adoptive transfer into melanoma-bearing mice. Our data indicate that functionally active bone marrow-derived melanoma-specific memory T cells are detectable at the phase of microscopic tumor load, suggesting that thereby they could control disseminated melanoma cells.

Choroidal melanoma endoresection, dandelions and allegory-based medicine

In this issue, Garcia-Arumi et al report their experience with endoresection of 38 choroidal melanomas (see page 1040) .1 Readers of this article might be wondering who in their right...

Favorable outcome in clinically stage II melanoma patients is associated with the presence of activated tumor infiltrating T‐lymphocytes and preserved MHC class I antigen expression

In this study we investigated whether the presence of specific populations of tumor infiltrating lymphocytes (TILs) in diagnostic primary melanoma biopsies are related to outcome in clinically stage II melanoma patients. Moreover, we investigated whether the presence of TILs correlates with expression of MHC class I antigen and MHC class II antigen on tumor cells and/or tumor infiltrating antigen presenting cells. Diagnostic primary melanoma samples of 15 patients with an unfavorable outcome were compared with 20 patients with favorable outcome. Patients were matched for age, gender and Breslow thickness. Biopsies were examined for the presence of granzyme B+, CD8+, CD4+ and CD56+ TILs and for expression of MHC class I antigen and MHC class II antigen on tumor and/or tumor infiltrating cells. A favorable clinical outcome was strongly associated with the presence of GrB+ and CD4+ TILs, with expression of MHC class I antigen on tumor cells and with expression of MHC class II antigen on intratumoral antigen presenting cells. These data strongly support the notion that in melanoma patients the cellular immune response is a major factor in preventing melanoma cell dissemination.

Stromal cell-derived factor-1alpha promotes melanoma cell invasion across basement membranes involving stimulation of membrane-type 1 matrix metalloproteinase and Rho GTPase activities.

Tissue invasion by tumor cells involves their migration across basement membranes through activation of extracellular matrix degradation and cell motility mechanisms. Chemokines binding to their receptors provide chemotactic cues guiding cells to specific tissues and organs; they therefore could potentially participate in tumor cell dissemination. Melanoma cells express CXCR4, the receptor for the chemokine stromal cell-derived factor-1alpha (SDF-1alpha). Using Matrigel as a model, we show that SDF-1alpha promotes invasion of melanoma cells across basement membranes. Stimulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) activity by SDF-1alpha was necessary for invasion, involving at least up-regulation in the expression of this metalloproteinase, as detected in the highly metastatic BLM melanoma cell line. Moreover, SDF-1alpha triggered the activation of the GTPases RhoA, Rac1, and Cdc42 on BLM cells, and expression of dominant-negative forms of RhoA and Rac1, but not Cdc42, substantially impaired the invasion of transfectants in response to SDF-1alpha, as well as the increase in MT1-MMP expression. Furthermore, CXCR4 expression on melanoma cells was notably augmented by transforming growth factor-beta1, a Matrigel component, whereas anti-transforming growth factor-beta antibodies inhibited increases in CXCR4 expression and melanoma cell invasion toward SDF-1alpha. The identification of SDF-1alpha as a potential stimulatory molecule for MT1-MMP as well as for RhoA and Rac1 activities during melanoma cell invasion, associated with an up-regulation in CXCR4 expression by interaction with basement membrane factors, could contribute to better knowledge of mechanisms stimulating melanoma cell dissemination.

Disseminated melanoma cells in blood and bone marrow. Significance and detection by potential tumor markers

As the majority of primary malignant melanomas can be cured by surgical excision, the prognosis of melanomas is dependent on whether tumor cells have disseminated orare capable of doing so at the time of surgery. A prospective and valid detection of this minimal residual disease is not currently possible. The most important known so-called markers of melanoma disease, tyrosinase, S100 and MIA, all are more likely to be present in patients with more advanced disease. A valid prognostic effect has only been shown for S100 in patients with already identified metastatic disease. Further prospective studies are required to determine the potential gain of information by routine determination of these markers in melanoma patients.

A novel strategy in the elimination of disseminated melanoma cells: chimeric receptors endow T cells with tumor specificity.

The application of immunotherapy to the treatment of micrometastases of melanoma has attracted growing interest in recent years. This trend reflects, at least in part, the disappointing results of conventional chemotherapy, the identification of melanoma-associated antigens suitable to be used as targets for immunotherapy, and the significant progress in our understanding of molecular processes involved in the development of an immune response. Because of the general belief that T cell immunity plays a major part in the control of tumor growth, we have recently applied a novel strategy to target cytolytic T cells to melanoma cells. The strategy bypasses the requirement of presentation of melanoma-associated-antigen-derived peptides by the major histocompatibility complex to the T cell receptor complex by permanent grafting of T cells with a recombinant, chimeric T cell receptor. The extracellular moiety of the grafted receptor contains the antigen-binding domain, consisting of a single-chain antibody fragment (scFv) derived from a monoclonal antibody specific for the high-molecular-weight melanoma-associated antigen (HMW-MAA). The intracellular receptor moiety contains the cellular activation domain, consisting of the gamma-signaling chain derived from the Fc epsilon RI receptor. Cytotoxic T cells grafted with the chimeric anti-HMW-MAA scFv-gamma signaling receptor specifically lyse HMW-MAA-positive melanoma cells in a human leukocyte antigen class I-independent fashion. The chimeric T cell receptor strategy is designed to eliminate disseminated tumor cells by the power of cytolytic T cells that physiologically penetrate tissues and that are specifically activated by the grafted receptor after binding to antigen-positive melanoma cells.

Analysis of melanoma cells in peripheral blood by reverse transcription-polymerase chain reaction for tyrosinase and MART-1 after mononuclear cell collection with cell preparation tubes: a comparison with the whole blood guanidinium isothiocyanate RNA isolation method

Melanoma cell detection in peripheral blood by tyrosinase reverse transcription-polymerase chain reaction (RT-PCR) is usually performed on RNA isolated from whole blood using a guanidinium isothiocyanate (GITC)/phenol extraction method or from Ficoll Hypaque isolated mononuclear cells. The first method contains environmentally harmful reagents, and the second is laborious in the preanalytical steps. Cell preparation tubes (CPTs) are ready-to-use Ficoll Hypaque-based tubes that avoid the time-consuming and critical loading on Ficoll Hypaque. We examined whether CPTs can be used to determine melanoma cell dissemination in peripheral blood. We first investigated whether melanoma cells were retained in the mononuclear cell layer. All six morphologically different melanoma cell lines studied in the spiking experiments were retained in the upper layer. In further experiments, we were able to detect low dilutions of added SK-MEL-28 cells more consistently after nested RT-PCR for tyrosinase or MART-1 in the RNA isolated from mononuclear cells from CPTs than from RNA isolated with the GITC method. In addition, RNA was extracted from paired blood samples from 24 analysable stage III and stage IV melanoma patients and analysed for the presence of tyrosinase and MART-1 RNA using both the CPT/RNeasy and the whole blood/GITC method. The quality of the CPT/RNeasy RNA was better than the RNA isolated from whole blood with GITC/phenol. However, the RT-PCR results were less unequivocal: MART-1 mRNA was more often detected with CPTIRNeasy compared with whole blood/GITC (six versus three), whereas tyrosinase mRNA was found less often in CPT/RNeasy RNA (two versus eight). Taken together these results suggest that the CPT isolation method is suitable for the isolation of mononuclear cells, including melanoma cells.

Dissemination of melanoma cells within electrocautery plume

Background: The observed occurrence of port site recurrence in laparoscopic surgery for malignant disease has stimulated interest in the dissemination of tumor cells during surgery. Study of electrocautery smoke has revealed the presence of large particles and viable viruses. The purpose of this study was to determine if viable malignant cells are present in suspension within the electrocautery plume. Methods: Pellets of B16-F0 mouse melanoma cells were cauterized and the plume collected into culture medium. In part 1 of this study, the trypan blue assay was used to assess cell viability immediately after collection and 7 days later. A cautery current of 30 W was applied for 5 minutes. In part 2, the tetrazolium (MTT) viability assay was used to assess cell viability after cauterization of tumor pellets at 10, 20, and 30 W for 5 seconds. Results: Although intact melanoma cells were identified with the trypan blue assay immediately after plume collection, no viable cells were seen at 7 days using this assay. In part 2, viable melanoma cells were present in the culture wells at 7 days. Lower fulguration currents appeared to yield higher cell counts: 2,250 cells/well at 10 W, 2,100 cells/well at 20 W, and 1,800 cells/well at 30 W. Conclusions: Results of this study confirm that application of electrocautery to a pellet of melanoma cells releases these cells into the plume. These cells are viable and may be grown in culture. This release of malignant cells may explain the appearance of port metastases at sites that are remote from the surgical dissection or that were never in direct contact with the tumor.