Intact Melanoma Cells Research Articles

Melanoma Cells Produce Large Vesicular-Bodies That Cause Rapid Disruption of Brain Endothelial Barrier-Integrity and Disassembly of Junctional Proteins.

It is known that many cells produce extracellular vesicles, and this includes a range of different cancer cell types. Here we demonstrate the profound effects of large vesicular-like bodies produced by melanoma cells on the barrier integrity of human brain endothelial cells. These vesicular-bodies have not been fully characterised but range in size from ~500 nm to >10 µm, are surrounded by membrane and are enzymatically active based on cell-tracker incorporation. Their size is consistent with previously reported large oncosomes and apoptotic bodies. We demonstrate that these melanoma-derived vesicular-bodies rapidly affect brain endothelial barrier integrity, measured using ECIS biosensor technology, where the disruption is evident within ~60 min. This disruption involves acquisition of the vesicles through transcellular uptake into the endothelial cells. We also observed extensive actin-rearrangement, actin removal from the paracellular boundary of the endothelial cells and envelopment of the vesicular-bodies by actin. This was concordant with widespread changes in CD144 localisation, which was consistent with the loss of junctional strength. High-resolution confocal imaging revealed proximity of the melanoma vesicular-bodies juxtaposed to the endothelial nucleus, often containing fragmented DNA themselves, raising speculation over this association and potential delivery of nuclear material into the brain endothelial cells. The disruption of the endothelial cells occurs in a manner that is faster and completely distinct to that of invasion by intact melanoma cells. Given the clinical observation of large vesicles in the circulation of melanoma patients by others, we hypothesize their involvement in weakening or priming the brain vasculature for melanoma invasion.

Pharmacophore Identification and Scaffold Exploration to Discover Novel, Potent, and Chemically Stable Inhibitors of Acid Ceramidase in Melanoma Cells.

Acid ceramidase (AC) hydrolyzes ceramides, which are central lipid messengers for metabolism and signaling of sphingolipids. A growing body of evidence links deregulation of sphingolipids to several diseases, including cancer. Indeed, AC expression is abnormally high in melanoma cells. AC inhibition may thus be key to treating malignant melanoma. Here, we have used a systematic scaffold exploration to design a general pharmacophore for AC inhibition. This pharmacophore comprises a 6 + 5 fused ring heterocycle linked to an aliphatic substituent via a urea moiety. We have thus identified the novel benzimidazole derivatives 10, 21, 27, and 30, which are highly potent AC inhibitors. Their chemical and metabolic stabilities are comparable or superior to those of previously reported AC inhibitors. Moreover, they are potent against endogenous AC in intact melanoma cells. These novel inhibitors merit further characterization and can serve as a promising starting point for the discovery of new antimelanoma therapeutics.

Identification of tumor-associated, MHC class II-restricted phosphopeptides as targets for immunotherapy

The activation and recruitment of CD4(+) T cells are critical for the development of efficient antitumor immunity and may allow for the optimization of current cancer immunotherapy strategies. Searching for more optimal and selective targets for CD4(+) T cells, we have investigated phosphopeptides, a new category of tumor-derived epitopes linked to proteins with vital cellular functions. Although MHC I-restricted phosphopeptides have been identified, it was previously unknown whether human MHC II molecules present phosphopeptides for specific CD4(+) T cell recognition. We first demonstrated the fine specificity of human CD4(+) T cells to discriminate a phosphoresidue by using cells raised against the candidate melanoma antigen mutant B-Raf or its phosphorylated counterpart. Then, we assessed the presence and complexity of human MHC II-associated phosphopeptides by analyzing 2 autologous pairs of melanoma and EBV-transformed B lymphoblastoid lines. By using sequential affinity isolation, biochemical enrichment, mass spectrometric sequencing, and comparative analysis, a total of 175 HLA-DR-associated phosphopeptides were characterized. Many were derived from source proteins that may have roles in cancer development, growth, and metastasis. Most were expressed exclusively by either melanomas or transformed B cells, suggesting the potential to define cell type-specific phosphatome "fingerprints." We then generated HLA-DRbeta1*0101-restricted CD4(+) T cells specific for a phospho-MART-1 peptide identified in both melanoma cell lines. These T cells showed specificity for phosphopeptide-pulsed antigen-presenting cells as well as for intact melanoma cells. This previously undescribed demonstration of MHC II-restricted phosphopeptides recognizable by human CD4(+) T cells provides potential new targets for cancer immunotherapy.

Visualization of Circulating Melanoma Cells in Peripheral Blood of Patients with Primary Uveal Melanoma

In patients with uveal melanoma, tumor cell dissemination and subsequent formation of metastases are confined mainly to the hematogenous route. Here, we sought to isolate circulating melanoma cells in peripheral blood of patients with primary uveal melanoma and clinically localized disease. Blood samples from 52 patients with clinically localized uveal melanoma and from 20 control individuals were prospectively collected before therapy of the primary tumor. Tumor cells expressing the melanoma-associated chondroitin sulfate proteoglycan were enriched by immunomagnetic cell sorting and visualized by immunocytologic staining. Results were compared with clinical data at presentation. In 10 of 52 patients [19%; 95% confidence interval (95% CI), 10-33%], between 1 and 5 circulating melanoma cells were detected in 50 mL peripheral blood. No melanoma-associated chondroitin sulfate proteoglycan-positive cells were detected in any of the 20 controls examined. The presence of tumor cells in peripheral blood was associated with ciliary body invasion [odds ratio (OR), 20.0; 95% CI, 3.0-131.7], advanced local tumor stage (OR, 6.7; 95% CI, 1.8-25.4), and anterior tumor localization (OR, 4.0; 95% CI, 1.2-12.7), all established factors for uveal melanoma progression. Immunomagnetic enrichment enables detection of intact melanoma cells in peripheral blood of patients with clinically localized ocular disease. Visualization and capturing of these cells provide a unique tool for characterizing potentially metastasizing tumor cells from a primary melanoma at an early stage of the disease.

Tumor cell and circulating markers in melanoma: Diagnosis, prognosis, and management

The search is on for biomarkers for use in the diagnosis, staging, prognosis, and management of patients with melanoma. As with many types of cancer, the hematogenous spread of melanoma is a bad prognostic sign, and many groups have attempted to detect circulating melanoma cells in patients with different stages of melanoma. Some studies have used direct extraction of intact tumor cells from the peripheral blood and others the detection of surrogate markers of circulating melanoma cells, such as tyrosinase or MART-1. However, a correlation between the detection of intact melanoma cells in the circulation and prognosis is controversial. Many other biomarkers have also been studied, including lactate dehydrogenase, S100, TA90, and C-reactive protein. Much progress has been made, and preliminary studies have shown promise with many of these markers. Finally, the detection of tumor-specific circulating DNA has shown promise as a prognostic and diagnostic marker of disease in melanoma as well. In this review we examine the most promising biomarkers for use in patients with cutaneous melanoma.

Morphologically intact melanoma cells may be detected in peripheral blood of melanoma patients.

The detection of circulating melanoma cells has been the subject of numerous investigations in recent years. We developed a cellular approach to identifying circulating melanoma cells in peripheral blood using immunomagnetic cell sorting. The examination covered 205 blood samples from 155 melanoma patients and 30 samples from healthy persons and nonmelanoma patients. After density gradient centrifugation, the interphase was incubated with the 9.2.27 antibody. Positive cells were labeled with magnetic microbeads and enriched by immunomagnetic cell sorting. Cells were stained using an alkaline phosphatase-anti-alkaline phosphatase assay and examined by light microscopy. In spiking experiments, melanoma cells seeded at a concentration of one melanoma cell per milliliter of whole blood could be detected reliably. Circulating melanoma cells were not found in 30 controls, nor were 9.2.27-positive cells found in 41 patients with primary malignant melanoma. In patients with regional lymph node metastases and disseminated disease, circulating 9.2.27-positive cells could be detected in 3 of 29 patients (10%) and 13 of 85 patients (15%) examined, respectively. We conclude that immunomagnetic cell sorting is a promising method with high sensitivity and specificity. The method is not suitable for early detection of metastases but is a valuable tool for further investigating the biological characteristics of circulating 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.

T cell-receptor V gene use by CD4+ melanoma-reactive clonal and oligoclonal T-cell lines.

Tumor-reactive CD4+ T cells can be isolated and expanded from the peripheral blood and tumor lesions of patients with melanoma. In contrast to CD8+ T cells, little is known about the antigens recognized by these CD4+ T cells. As a consequence, little is known about the diversity of the T-cell receptor (TcR) use by melanoma-reactive CD4+ T cells. To address these questions, a panel of clonal or highly oligoclonal CD4+ T-cell lines was established from a patient with metastatic melanoma. A CD4+ tumor-infiltrating lymphocyte (TIL) line was established that was highly oligoclonal and recognized only autologous melanoma cells but not allogeneic melanomas, suggesting the expression of a mutated or uniquely expressed antigen by this melanoma. The antigen recognized by the CD4+ TILs could be presented by intact melanoma cells or by autologous Epstein-Barr virus (EBV) B cells pulsed with melanoma cell lysates. A panel of CD4+ clonal and highly oligoclonal T-cell lines was isolated form peripheral blood mononuclear cells (PBMC) from this patient; these were also reactive with autologous melanoma cells or tumor extracts pulsed on autologous EBV B cells. Despite their reactivity with the autologous melanoma, we found no evidence of restricted TcR V gene use, because all six T-cell lines recognized antigen via different TcR alpha/beta rearrangements. Furthermore, there were no conserved amino acids in the CDR3 regions of these TcRs, indicating that multiple TcR clonotypes could mediate recognition of a single unique major histocompatibility (MHC) complex class II restricted melanoma antigen or that multiple MHC class II restricted melanoma antigens are expressed by the melanoma.

A new mouse model of experimental melanoma for vaccine and lymphokine therapy.

The annual incidence of malignant melanoma is estimated at 10-12 per 100,000 inhabitants in countries of central Europe and the United States, and alarmingly there has been a dramatic upward trend in that estimate. The B16 murine melanoma is a rapidly growing metastatic tumor of spontaneous origin, as are human malignant melanomas. Melanoma cells produce specific antigens which are uniquely different from normal cellular antigens, and the expression of such antigens is the cornerstone for preparation of anti-melanoma vaccines. One major problem in evaluating the effectiveness of vaccination and other biologic therapies is the variability of experimental tumor models. A new metastatic model of experimental melanoma which was developed in our laboratory imitates the major clinical stages of malignant metastatic melanoma: stage I, primary (local) tumor growth and bone marrow invasion; stage II, regional lymph node involvement; and stage III, metastasis to distant organs, such as the lungs. This model has been used successfully for screening vaccines constructed in our laboratory. Immunization with formalinized vaccines (of extracellular antigens, intact melanoma cells, or B700 antigen) or irradiated vaccines (of intact melanoma cells) partially inhibit primary melanoma tumor growth, reduce metastasis to regional lymph nodes and lungs, and significantly increase mean survival time. These anti-tumor effects were improved when polyvalent and monovalent vaccines were combined with IL-2 therapy. We also compared the immunogenic activity of vaccines made from B16 melanoma cells transfected with genes encoding murine IL-2 or GM-CSF, and effects on tumor bearing mice were compared with or without therapy using the corresponding lymphokines. In sum, comparison of antibody production, growth of primary melanoma tumors, number of surviving mice, mean survival time, and percent of mice with lung metastases, showed that the best course of immunotherapy involves vaccination of mice with irradiated B16 melanoma cells transfected to secrete GM-CSF, coupled with GM-CSF therapy.

ANTITUMOR EFFECTS OF POLYVALENT AND MONOVALENT VACCINES COUPLED WITH INTERLEUKIN-2 IN A METASTATIC MELANOMA MODEL

We have previously reported that preimmunization of mice with formalinized extracellular antigens (fECA) derived from melanoma cells, in combination with interleukin 2 (IL-2) treatment and surgical resection, decreased subsequent tumor growth and increased survival of mice in a new model for spontaneous metastasis of melanoma. In this study, we have modified the sequence of tumor growth and therapy to more closely mimic the clinical situation. Mice were challenged subcutaneously in the tail with 5 x 10(5) B16 F10 melanoma cells and, by day 21, all of them had developed localized melanoma tumors. The primary tumor-bearing tails of control and experimental animals were then resected distal to the base of the tail, and therapy of the mice was initiated the following day. Groups of mice received different polyvalent and monovalent murine melanoma vaccines (including native or formalin treated extracellular antigens, intact melanoma cells, or purified B700 antigen), with or without concomitant low doses of IL-2. The results demonstrate that the vaccine therapies elicited significant increases in survival of the mice, accompanied by reductions in the size of lymph nodes and in the number of pulmonary metastases. These effects, particularly with the intact melanoma cell vaccine, could be improved even further with concomitant IL-2 treatment.

Diversity of Melanoma Plasma Membrane Proteinases: Inhibition of Collagenolytic and Cytolytic Activities by Minocycline<xref ref-type="fn" rid="FN2">2</xref>

The tissue-destructive proteinases of B16-BL6 melanoma cells from C57BL/6 mice and subcellular fractions were examined. Cancer cell organelles were isolated following nitrogen cavitation with the use of sucrose density gradient centrifugation. Serine, cysteine, and metalloproteinases were assayed with the use of radiolabeled proteins and synthetic substrates. Tumor-induced red blood cell lysis was quantitated by measurement of the release of isotope from 59Fe-labeled red blood cells (RBC) cocultivated with melanoma cells; the RBC were from Wistar rats. Enzyme inhibitors with specificity toward different classes of proteinases were used in the above assays to categorize the enzymes responsible for substrate degradation. Results indicated that intact melanoma cells, cell organelles, and cytosol contain proteinases that can degrade collagen and gelatin and lyse normal RBC. Melanoma plasma membranes are highly enriched in collagenase, gelatinase, cysteine proteinase, plasminogen activator, and cytolytic activity. The inhibition of tumor collagenolytic, gelatinolytic, and cytolytic activities by EDTA and 1,10-phenanthroline but not by diisopropyl fluorophosphate and N alpha-p-tosyl-L-lysine chloromethyl ketone indicates that metalloproteinases are the active enzymes in these assays. Minocycline, a synthetic tetracycline with demonstrable inhibitory activity with other mammalian collagenases, also inhibited melanoma collagenolytic and cytolytic activities.

Differential effects of NAD, nicotinamide and related compounds upon growth and nucleoside incorporation in human cells

Two human melanoma cell lines, MM96 and MM127, were found to be highly sensitive to the toxicity of adenosine ( d 50 100–150 μg/ml) compared with other melanoma lines. HeLa cells and a lymphoblastoid line ( d 50 > 500 μg/ml). The MM127 line was also sensitive to NAD ( d 50 41 μg/ml) compared with the other lines ( d 50 > 400 μg/ml), and accumulated three-fold more NAD-derived isotopic label. Nicotinamide exhibited little toxicity in any cell type ( d 50 > 400 μg/ml); 25–100 μg/ml nicotinamide greatly increased the plating efficiency of melanoma cells and fibroblasts when low levels of foetal calf serum were used. The toxicity of DNA-damaging agents (alkylating agents and u.v.) in melanoma cells was not reduced in the presence of NAD, adenosine or nicotinamide. Studies of the effects of the latter compounds upon the incorporation of deoxynucleosides showed that: (a) melanoma cells have lower purine pools than fibroblasts; (b) [ 3H]deoxyguanosine incorporation was inhibited more than [ 3H]deoxyadenosine incorporation; (c) incorporation of [ 3H]deoxyadenosine and [ 3H]deoxyadenosine and [ 3H]deoxyguanosine into RNA was inhibited by adenosine, thus providing a method for determination of guanine-specific DNA repair; and (d) NAD enhanced thymidine incorporation in intact melanoma cells but not in fibroblasts, in a pattern similar to the release from template restriction previously reported for permeabilised tumour cells.

Production and characterization of monoclonal antibody to a melanoma specific glycoprotein.

An immunogen consisting of a 4M urea extract derived from human melanoma cells (M14), that was devoid of HLA-A,B,C, HLA-DR antigens and fibronectin was adsorbed to lens culinaris lectin-Sepharose 4B and used to immunize mice for production of monoclonal antibody to a melanoma-specific glycoprotein. Screening for hybridomas secreting antibodies to melanoma associated antigens was facilitated by use of a solid phase target antigen of chemically defined medium of melanoma cells (CDM). Use of these procedures allowed us to select 40 hybridomas secreting antibody which recognized determinants on melanoma cells not found on lymphoid cells. Further characterization of one of these hybridomas, 9.2.27, indicated that the antibody it secreted recognized a 240K dalton glycoprotein found on all melanoma cell lines tested but not on carcinoma, lymphoid, or fibroblastoid cultures. These results demonstrate the utility of soluble antigen preparations devoid of strongly immunogenic non tumor-specific molecules in the elicitation of tumor specific antibody. Preliminary results suggest that immunogens of this kind are superior to intact melanoma cells for production of tumor specific hybridomas.