Human Melanoma Lesions Research Articles

Abstract 4148: Extracellular matrix biophysical properties change during nevus to melanoma progression

Abstract Cutaneous malignant melanoma is one of the most aggressive forms of skin cancer and its major etiological risk factor is ultraviolet (UV) solar radiation. The extracellular matrix (ECM) has been shown to influence many of the hallmark characteristics of tumor development, survival, and progression across several types of cancer, including melanoma. Interactions between the tumor and the ECM often results in rearrangement, cross-linking, and deposit of specific ECM proteins, which can contribute to stiffening of the tissue and tumorigenesis. To simulate tumorigenesis, we have previously used a hepatocyte growth factor (HGF) transgenic mouse model, which develops melanoma nevii when exposed to UVB radiation. This model is highly representative of human melanoma lesions in terms of biological, genetic, and etiologic criteria. In general, microrheology involves injecting beads into a medium, tracking movement of the beads using two-photon microscopy, and using the movement to calculate the viscosity and elasticity of the medium. The physical properties of the ECM during tumorigenesis have not been well studied at the cellular level, and microrheology has rarely been used in vivo. In our in vivo microrheology study, we injected fluorescent beads into the skin of these transgenic mice. Then, we used intra-vital microscopy with particle tracking to image the beads and measure changes in ECM elasticity as nevii grow into melanoma tumors. These results, combined with transcriptomic analysis of ECM regulation and proteomic analysis of ECM signaling, could provide unique insights into components of the extracellular matrix that are involved in melanoma tumor development and offer opportunities for therapeutics to target oncogenesis. <!–EndFragment–> Citation Format: Aditya Kavuturu, Maira A. Constantino, Antonella Sassano, Glenn Merlino. Extracellular matrix biophysical properties change during nevus to melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4148.

Characterization of Mueller matrix elements for classifying human skin cancer utilizing random forest algorithm.

.Significance: The Mueller matrix decomposition method is widely used for the analysis of biological samples. However, its presumed sequential appearance of the basic optical effects (e.g., dichroism, retardance, and depolarization) limits its accuracy and application.Aim: An approach is proposed for detecting and classifying human melanoma and non-melanoma skin cancer lesions based on the characteristics of the Mueller matrix elements and a random forest (RF) algorithm.Approach: In the proposal technique, 669 data points corresponding to the 16 elements of the Mueller matrices obtained from 32 tissue samples with squamous cell carcinoma (SCC), basal cell carcinoma (BCC), melanoma, and normal features are input into an RF classifier as predictors.Results: The results show that the proposed model yields an average precision of 93%. Furthermore, the classification results show that for biological tissues, the circular polarization properties (i.e., elements , , , and of the Mueller matrix) dominate the linear polarization properties (i.e., elements , , , and of the Mueller matrix) in determining the classification outcome of the trained classifier.Conclusions: Overall, our study provides a simple, accurate, and cost-effective solution for developing a technique for classification and diagnosis of human skin cancer.

Abstract PR07: The role of keratinocyte desmoglein 1 in shaping the melanoma tumor niche

Abstract Melanoma is an aggressive cancer arising from transformation of melanocytes residing in the basal layer of the epidermis, where they are in direct contact with surrounding keratinocytes. Keratinocytes and melanocytes communicate through both contact and paracrine signaling, a niche called the pigmentary unit. Ultraviolet (UV) radiation-induced melanocyte mutations drive melanoma formation and progression, but the role of keratinocytes and an altered microenvironment in melanomagenesis is understudied. We showed that the keratinocyte-specific desmosomal cadherin, desmoglein 1 (Dsg1), is reduced in response to acute UV exposure. Here, we show that Dsg1 is progressively reduced in regions surrounding human dysplastic nevi, melanoma in situ, and stage I/II melanomas, preceding loss of E-cadherin that occurs in later stages. Since we previously showed that Dsg1 suppresses EGFR/Erk1/2 signaling and regulates keratinocyte cytokine profiles, we hypothesized that altered keratinocyte Dsg1 expression governs melanocyte behavior and transformation through paracrine signaling. Dsg1 knockdown via shRNA elevated phospho-EGFR, phospho-Stat3, phospho-ERK and nuclear NFκB, accompanied by increases in IL-1α, IL-1β, IL-6, and IL-8, and CXCL1 proinflammatory cytokine mRNAs and secretion of IL6, IL8, and CXCL1. Melanocytes grown in conditioned media from Dsg1-deficient keratinocytes increased MITF transcription, while AXL transcript levels remained unchanged, a signature associated with early-stage melanoma. Consistent with increased MITF, melanocytes cultured in conditioned media from Dsg1-depleted keratinocytes increased pigment secretion, reversible by inhibiting the melanocortin 1 receptor. Melanocytes incorporated into Dsg1-deficient 3D human skin equivalents mis-localized to suprabasal layers, similar to pagetoid behavior in early melanoma. This, combined with the loss of keratinocyte Dsg1 surrounding human melanoma lesions, led us to examine mechanisms of sustained Dsg1 loss in the tumor niche. Keratinocytes cultured in melanoma cell-conditioned media reduced Dsg1 expression, suggesting that Dsg1 downregulation is maintained by melanoma cells post-transformation. We postulate that keratinocyte Dsg1 downregulation may initially be part of the skin’s protective response against UV exposure through its role in increasing pigmentation. However, chronic Dsg1 loss may contribute to melanoma initiation and be important for the maintenance of the promelanoma tumor niche. This abstract is also being presented as Poster A02. Note: This abstract was not presented at the conference. Citation Format: Hope E. Burks, Christopher R. Arnette, Jennifer L. Koetsier, Joshua A. Broussard, Valarie Ogwo, Quinn R. Roth-Carter, Pedram Gerami, Jodi L. Johnson, Kathleen J. Green. The role of keratinocyte desmoglein 1 in shaping the melanoma tumor niche [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR07.

HERV-K activation is strictly required to sustain CD133+ melanoma cells with stemness features

BackgroundMelanoma is a heterogeneous tumor in which phenotype-switching and CD133 marker have been associated with metastasis promotion and chemotherapy resistance. CD133 positive (CD133+) subpopulation has also been suggested as putative cancer stem cell (CSC) of melanoma tumor. Human endogenous retrovirus type K (HERV-K) has been described to be aberrantly activated during melanoma progression and implicated in the etiopathogenesis of disease. Earlier, we reported that stress-induced HERV-K activation promotes cell malignant transformation and reduces the immunogenicity of melanoma cells. Herein, we investigated the correlation between HERV-K and the CD133+ melanoma cells during microenvironmental modifications.MethodsTVM-A12 cell line, isolated in our laboratory from a primary human melanoma lesion, and other commercial melanoma cell lines (G-361, WM-115, WM-266-4 and A375) were grown and maintained in the standard and stem cell media. RNA interference, Real-time PCR, flow cytometry analysis, self-renewal and migration/invasion assays were performed to characterize cell behavior and HERV-K expression.ResultsMelanoma cells, exposed to stem cell media, undergo phenotype-switching and expansion of CD133+ melanoma cells, concomitantly promoted by HERV-K activation. Notably, the sorted CD133+ subpopulation showed stemness features, characterized by higher self-renewal ability, embryonic genes expression, migration and invasion capacities compared to the parental cell line. RNA interference-mediated downregulation experiments showed that HERV-K has a decisive role to expand and maintain the CD133+ melanoma subpopulation during microenvironmental modifications. Similarly, non nucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine were effective to restrain the activation of HERV-K in melanoma cells, to antagonize CD133+ subpopulation expansion and to induce selective high level apoptosis in CD133+ cells.ConclusionsHERV-K activation promotes melanoma cells phenotype-switching and is strictly required to expand and maintain the CD133+ melanoma cells with stemness features in response to microenvironmental modifications.

CCN2 Expression by Tumor Stroma Is Required for Melanoma Metastasis

Metastatic melanoma has an extremely poor prognosis with few durable remissions. The secreted matricellular protein connective tissue growth factor (CCN2) is overexpressed in cancers including melanoma and may represent a viable therapeutic target. However, the mechanism underlying the contribution of CCN2 to melanoma progression is unclear. Herein, we use the highly metastatic murine melanoma cell line B16(F10) and syngeneic mice, in which CCN2 expression is knocked out in fibroblasts, to demonstrate that loss of CCN2, either in melanoma cells or in the niche, impedes the ability of melanoma cells to invade. Specifically, loss of CCN2 in melanoma cells diminished their ability to invade through collagen in vitro, and loss of fibroblast-derived CCN2 decreased spontaneous metastases of melanoma cells from the skin to the lungs in vivo. Proliferation and tumor growth were not affected by loss of CCN2. CCN2-deficient B16(F10) cells showed reduced expression of the matricellular protein periostin; addition of recombinant periostin rescued the in vitro invasion defect of these cells. Immunohistochemical analysis of CCN2-deficient mice confirmed loss of periostin expression in the absence of CCN2. CCN2 and periostin mRNA levels are positively correlated with each other and with the stromal composition of human melanoma lesions but not BRAF mutations. Thus, CCN2 promotes invasion and metastasis via periostin and should be further evaluated as a possible therapeutic target for BRAF inhibitor-resistant melanoma.

Tumour-derived SPARC drives vascular permeability and extravasation through endothelial VCAM1 signalling to promote metastasis.

Disruption of the endothelial barrier by tumour-derived secreted factors is a critical step in cancer cell extravasation and metastasis. Here, by comparative proteomic analysis of melanoma secretomes, we identify the matricellular protein SPARC as a novel tumour-derived vascular permeability factor. SPARC deficiency abrogates tumour-initiated permeability of lung capillaries and prevents extravasation, whereas SPARC overexpression enhances vascular leakiness, extravasation and lung metastasis. SPARC-induced paracellular permeability is dependent on the endothelial VCAM1 receptor and p38 MAPK signalling. Blocking VCAM1 impedes melanoma-induced endothelial permeability and extravasation. The clinical relevance of our findings is highlighted by high levels of SPARC detected in tumour from human pulmonary melanoma lesions. Our study establishes tumour-produced SPARC and VCAM1 as regulators of cancer extravasation, revealing a novel targetable interaction for prevention of metastasis.

Dual receptor-targeting 99mTc-labeled Arg-Gly-Asp-conjugated Alpha-Melanocyte stimulating hormone hybrid peptides for human melanoma imaging

IntroductionThe aim of this study was to examine whether the substitution of the Lys linker with the aminooctanoic acid (Aoc) and polyethylene glycol (PEG) linker could substantially decrease the non-specific renal uptake of 99mTc-labeled Arg-Gly-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) hybrid peptides. MethodsThe RGD motif {Arg-Gly-Asp-DTyr-Asp} was coupled to [Cys3,4,10, D-Phe7, Arg11]α-MSH3–13 via the Aoc or PEG2 linker to generate RGD-Aoc-(Arg11)CCMSH and RGD-PEG-(Arg11)CCMSH. The biodistribution results of 99mTc-RGD-Aoc-(Arg11)CCMSH and 99mTc-RGD-PEG2-(Arg11)CCMSH were examined in M21 human melanoma-xenografted nude mice. ResultsThe substitution of Lys linker with Aoc and PEG2 linker significantly reduced the renal uptake of 99mTc-RGD-Aoc-(Arg11)CCMSH and 99mTc-RGD-PEG2-(Arg11)CCMSH by 58% and 63% at 2h post-injection. The renal uptake of 99mTc-RGD-Aoc-(Arg11)CCMSH and 99mTc-RGD-PEG2-(Arg11)CCMSH was 27.93±3.98 and 22.01±9.89% ID/g at 2h post-injection. 99mTc-RGD-Aoc-(Arg11)CCMSH displayed higher tumor uptake than 99mTc-RGD-PEG2-(Arg11)CCMSH (2.35±0.12 vs. 1.71±0.25% ID/g at 2h post-injection). The M21 human melanoma lesions could be clearly visualized by SPECT/CT using 99mTc-RGD-Aoc-(Arg11)CCMSH as an imaging probe. ConclusionsThe favorable effect of Aoc and PEG2 linker in reducing the renal uptake provided a new insight into the design of novel dual receptor-targeting radiolabeled peptides.

439. Transcriptomic Analysis of Human Melanoma Lesions Resected for TIL Production Reveals the Presence of B Cells in Association With T Lymphocytes

439. Transcriptomic Analysis of Human Melanoma Lesions Resected for TIL Production Reveals the Presence of B Cells in Association With T Lymphocytes

Nicotinamide Inhibits Vasculogenic Mimicry, an Alternative Vascularization Pathway Observed in Highly Aggressive Melanoma

Vasculogenic mimicry (VM) describes functional vascular channels composed only of tumor cells and its presence predicts poor prognosis in melanoma patients. Inhibition of this alternative vascularization pathway might be of clinical importance, especially as several anti-angiogenic therapies targeting endothelial cells are largely ineffective in melanoma. We show the presence of VM structures histologically in a series of human melanoma lesions and demonstrate that cell cultures derived from these lesions form tubes in 3D cultures ex vivo. We tested the ability of nicotinamide, the amide form of vitamin B3 (niacin), which acts as an epigenetic gene regulator through unique cellular pathways, to modify VM. Nicotinamide effectively inhibited the formation of VM structures and destroyed already formed ones, in a dose-dependent manner. Remarkably, VM formation capacity remained suppressed even one month after the complete withdrawal of Nicotimamid. The inhibitory effect of nicotinamide on VM formation could be at least partially explained by a nicotinamide-driven downregulation of vascular endothelial cadherin (VE-Cadherin), which is known to have a central role in VM. Further major changes in the expression profile of hundreds of genes, most of them clustered in biologically-relevant clusters, were observed. In addition, nicotinamide significantly inhibited melanoma cell proliferation, but had an opposite effect on their invasion capacity. Cell cycle analysis indicated moderate changes in apoptotic indices. Therefore, nicotinamide could be further used to unravel new biological mechanisms that drive VM and tumor progression. Targeting VM, especially in combination with anti-angiogenic strategies, is expected to be synergistic and might yield substantial anti neoplastic effects in a variety of malignancies.

Combining Filter-Aided Sample Preparation and Pseudoshotgun Technology To Profile the Proteome of a Low Number of Early Passage Human Melanoma Cells

The performance of two proteomic sample preparation methods, "pseudoshotgun" (PSG) and filter-aided sample preparation (FASP) were compared in terms of the number of identified proteins, representation of cellular component GO (gene ontology) categories in the obtained list of proteins, and the efficiency of both methods in the proteomic analysis of a very low number of cells. Both methods were combined to obtain a proteomic profile of a short-term culture (passage 3) of melanoma cells, established in our laboratory from a human metastatic melanoma lesion. The data revealed that with FASP, usually more proteins are identified than with PSG when analyzing a higher number of cells (≥ 5000/injection), whereas PSG is favorable when analyzing only a very small amount of cells (250-500/injection). PSG and FASP, however, are complementary techniques, as combining both methods further increases the number of identified proteins. Moreover, we show that it is feasible to identify a substantial number of proteins from only 250 cells/injection that is equivalent to 60 ng of protein.

GLI2-Mediated Melanoma Invasion and Metastasis

The transforming growth factor-beta (TGF-beta) pathway, which has both tumor suppressor and pro-oncogenic activities, is often constitutively active in melanoma and is a marker of poor prognosis. Recently, we identified GLI2, a mediator of the hedgehog pathway, as a transcriptional target of TGF-beta signaling. We used real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine GLI2 expression in human melanoma cell lines and subsequently classified them as GLI2high or as GLI2low according to their relative GLI2 mRNA and protein expression levels. GLI2 expression was reduced in a GLI2high cell line with lentiviral expression of short hairpin RNA targeting GLI2. We assessed the role of GLI2 in melanoma cell invasiveness in Matrigel assays. We measured secretion of matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography and expression of E-cadherin by western blotting and RT-PCR. The role of GLI2 in development of bone metastases was determined following intracardiac injection of melanoma cells in immunocompromised mice (n = 5-13). Human melanoma samples (n = 79) at various stages of disease progression were analyzed for GLI2 and E-cadherin expression by immunohistochemistry, in situ hybridization, or RT-PCR. All statistical tests were two-sided. Among melanoma cell lines, increased GLI2 expression was associated with loss of E-cadherin expression and with increased capacity to invade Matrigel and to form bone metastases in mice (mean osteolytic tumor area: GLI2high vs GLI2low, 2.81 vs 0.93 mm(2), difference = 1.88 mm(2), 95% confidence interval [CI] = 1.16 to 2.60, P < .001). Reduction of GLI2 expression in melanoma cells that had expressed high levels of GLI2 substantially inhibited both basal and TGF-beta-induced cell migration, invasion (mean number of Matrigel invading cells: shGLI2 vs shCtrl (control), 52.6 vs 100, difference = 47.4, 95% CI = 37.0 to 57.8, P = .024; for shGLI2 + TGF-beta vs shCtrl + TGF-beta, 31.0 vs 161.9, difference = -130.9, 95% CI = -96.2 to -165.5, P = .002), and MMP secretion in vitro and the development of experimental bone metastases in mice. Within human melanoma lesions, GLI2 expression was heterogeneous, associated with tumor regions in which E-cadherin was lost and increased in the most aggressive tumors. GLI2 was directly involved in driving melanoma invasion and metastasis in this preclinical study.

Abstract 5621: MCSP/CD3-bispecific single-chain antibody construct engages CD4+ and CD8+ T cells for lysis of MCSP-expressing human uveal melanoma cells

Abstract Uveal melanoma is the most common cancer of the eye and metastasizes in approximately 50% of patients. None of the current therapies prevents the development of metastases or extends the survival time of these patients. Bi-specific T cell engager (BiTE) are based on recombinant single-chain antibody constructs. These molecules can transiently connect T cells to tumor cells, leading to concomitant T cell activation and serial lysis of tumor cells. Recently, a BiTE antibody bi-specific for CD19 and CD3 (blinatumomab) has shown very high response rates in patients with refractory non-Hodgkin's B cell lymphoma (100%) and B-precursor acute lymphocytic leukemia (80%). These findings suggest that the mode of BiTE antibody action may also be useful for treatment of solid tumors. Human melanoma-associated chondroitin sulfate proteoglycan (MCSP, also known as HMW-MAA, gp240, or CSPG4), is expressed on the surface of &amp;gt;90% of human cutaneous melanoma lesions and cell lines, with restricted distribution in normal tissues. MCSP expression has also been detected in uveal melanoma tumor-tissue sections and could serve as a target antigen for immunotherapeutic approaches. Here we report that primary and metastatic uveal melanoma cell lines express MCSP and that CD4+ and CD8+ T cells are activated to lyse MCSP-expressing uveal melanoma cells by a novel BiTE antibody, which is bispecific for MCSP and CD3 (MCSP-BiTE). The surface of uveal melanoma cells was stained by anti-MCSP monoclonal antibodies when analyzed by flow cytometry. Seven out of 9 primary and 1 out of 3 metastatic uveal melanoma cell lines expressed MCSP at various expression levels. For functional analysis, CD4+ and CD8+ T cells were isolated from peripheral blood mononuclear cells (PBMC) of healthy donors using magnetic bead sorting. Both PBMC and unstimulated CD8+ T cells specifically lysed MCSP+ uveal melanoma cells in the presence of MCSP-BiTE as determined by 51Cr-release assays. As detected by ELISA, unstimulated CD4+ T cells produced IFNγ in response to MCSP-expressing uveal melanoma cells in the presence of MCSP-BiTE, and redirected lysis was evident by microscopy. In contrast, co-culture of T cells with MCSP-expressing uveal melanoma cells alone, or in the presence of a control BiTE, did not result in cytolytic activity or IFNγ release. Furthermore, naïve CD4+ and CD8+ T cells upregulated expression of T cell activation markers CD25 and CD137 solely in response to MCSP-expressing uveal melanoma cells decorated with MCSP-BiTE. BiTE-induced T cell activation required the expression of MCSP, as co-culture of T cells with MCSP-negative uveal melanoma cells in combination with MCSP-BiTE did not result in T cell activation. In conclusion, MCSP-BiTE can induce CD4+ and CD8+ T cells against MCSP-expressing uveal melanoma cells and should therefore be tested as a novel immunotherapy for uveal melanoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5621.

Restoration of the Expression of Transports Associated with Antigen Processing in Human Malignant Melanoma Increases Tumor-Specific Immunity

The transporter associated with antigen processing (TAP) is essential for peptide delivery from the cytosol into the lumen of the endoplasmic reticulum (ER), where these peptides are loaded on HLA I molecules. Our previous study found that expressions of TAP were reduced in human malignant melanoma (MM) lesions and associated with histopathologic characteristics. In this study, we further investigate expressions of TAP and HLA class I antigen in three human MM cell lines. pEGFP-TAP1/TAP2/TAP1+TAP2 were used to restore the expressions of TAP in the antigen presentation pathway-deficient MM cell line A375. TAP1- and TAP1+TAP2-transfected A375 increased TAP1, TAP2, and HLA class I antigen expression and antigen presentation. TAP1- and TAP1+TAP2-transfected A375 exhibited a dramatic increase in Melan-A-specific cytotoxic T lymphocytes (CTLs) compared with TAP2-transfected A375 or empty vector. These CTLs were capable of killing TAP1- and TAP1+TAP2-transfected A375. TAP1+TAP2-transfected A375 generated the highest frequency of Melan-A-specific IL-12 and interferon (IFN)-gamma-producing CD8+ T cells compared with TAP1, TAP2, and empty vector. Therefore, TAP expression restores both antigen presentation and immunogenicity in A375 melanoma cells and concomitantly increases IL-12 and IFN-gamma production in tumor antigen-specific CTLs; TAP should be considered as a part of the immunotherapies for MM.

Tumor-Reactive CD4+ T Cell Responses to the Melanoma-Associated Chondroitin Sulphate Proteoglycan in Melanoma Patients and Healthy Individuals in the Absence of Autoimmunity

To avoid immune escape by down-regulation or loss of Ag by the tumor cells, target Ags are needed, which are important for the malignant phenotype and survival of the tumor. We could identify a CD4(+) T cell epitope derived from the human melanoma-associated chondroitin sulfate proteoglycan (MCSP) (also known as high m.w.-melanoma-associated Ag), which is strongly expressed on >90% of human melanoma lesions and is important for the motility and invasion of melanoma cells. However, MCSP is not strictly tumor specific, because it is also expressed in a variety of normal tissues. Therefore, self tolerance should prevent the induction of strong T cell responses against these Ags by vaccination strategies. In contrast, breaking self tolerance to this Ag by effectively manipulating the immune system might mediate antitumor responses, although it would bear the risk of autoimmunity. Surprisingly, we could readily isolate CD4(+) Th cells from the blood of a healthy donor-recognizing peptide MCSP(693-709) on HLA-DR11-expressing melanoma cells. Broad T cell reactivity against this Ag could be detected in the peripheral blood of both healthy donors and melanoma patients, without any apparent signs of autoimmune disease. In some patients, a decline of T cell reactivity was observed upon tumor progression. Our data indicate that CD4(+) T cells are capable of recognizing a membrane glycoprotein that is important in melanoma cell function, and it may be possible that the sizable reactivity to this Ag in most normal individuals contributes to immune surveillance against cancer.

Electrical impedance scanning: a new approach to skin cancer diagnosis.

Skin cancer diagnosis depends, to a great extent, on visual inspection and histopathological examination of excised tissues. The aim of this study is to evaluate the ability of electrical impedance scanning to differentiate between benign and malignant skin lesions. A preclinical study was conducted on 40 nude mice injected subcutaneously with a human melanoma strain. Impedance measurements were recorded every week to correlate electrical changes with tumor growth and histological findings. A clinical study was also performed on 178 human suspicious skin lesions before excision. The impedance measurements were correlated to the histopathological results. Normalized conductivity and capacitance, recorded on growing skin tumors in nude mice, were shown to change relative to lesion size. Necrosis, present in most of the larger lesions, was associated with a decrease in the electrical conductivity. Similar electrical parameters were used to classify human melanoma lesions with 92% sensitivity and 67% specificity. In addition, four out of five BCC lesions were correctly diagnosed. Moreover, dysplastic lesions were diagnosed with 91% sensitivity and 59% specificity. For comparison, physicians diagnosed melanoma lesions with 75% sensitivity and 87% specificity and dysplastic lesions with 46% sensitivity and 80% specificity. The animal study showed that electrical impedance measurements reflect morphological changes related to the growth of a cancerous skin lesion. These findings are in agreement with a preliminary clinical study. Electrical Impedance Scanning can therefore be considered as an objective and non-invasive tool for differentiation between benign and malignant skin lesions.

Downregulation of tapasin expression in progressive human malignant melanoma.

Tumor cells with alterations in the MHC class I peptide-loading complex are unable to load peptide antigens onto MHC class I molecules. These alterations result in destabilization of MHC class I expression on the tumor cell surface and thus play a critical role in escape from immunological recognition by the acquired cellular immune system. By forming physical links between class I heavy chains and TAP molecules, a component of the class I peptide-loading complex, tapasin, plays an important role in the assembly of MHC class I molecules with peptides in the endoplasmic reticulum. In the present study, we compared 104 human melanoma lesions representing different stages of tumor progression for their expression of tapasin in tumor cells by immunohistochemistry. Tapasin downregulation was significantly associated with tumor progression. Whereas 100% of melanomata in situ and 96.2% of primary melanomas with a Breslow index of <or=0.75 mm showed strong expression of tapasin, significant downregulation of tapasin was observed in 25% of primary melanomas with a Breslow index >0.75 mm as well as in 21.1% metastatic melanoma lesions. The downregulation of tapasin in advanced stages of human melanoma may reflect the accumulation of alterations in the antigen-presenting/processing machinery associated with neoplastic progression. These alterations may lead to failure of the acquired cellular immune system to control progression and metastatic spread of melanoma cells in vivo, and thus contribute to the immune escape phenotype of human melanoma cells.

A ras-mutated peptide targeted by CTL infiltrating a human melanoma lesion.

Ags derived from commonly mutated oncogenic proteins seem ideally suited as targets for tumor immunotherapy. Nonetheless, only a few mutated epitopes efficiently presented by human tumors have thus far been identified. We describe here an approach to identify such epitopes. This approach involves: 1) identifying tumors expressing a ras mutation and isolating the tumor-infiltrating lymphocytes (TIL); 2) transfecting COS cells to induce expression of unknown mutated peptides in the context of a patient's HLA class I molecules; and 3) screening epitope recognition by using TIL from the tumors expressing a ras mutation. By using this approach, there appeared to be a N-ras mutation (a glutamine-to-arginine exchange at residue 61 (Q61R)), detected in a melanoma lesion, which was recognized specifically by the autologous TIL in the HLA-A*0101 context. The ras peptide 55-64(Q61R) was the epitope of these TIL and was regularly presented by Q61R-mutated HLA-A*0101(+) melanoma cell lines. This peptide and its wild-type homolog (55-64(wt)) bound to HLA-A*0101 with similar affinities. However, only the mutated peptide could induce specific CTL expansion from PBL. All the CTL clones specific to the mutated peptide, failed to recognize the wild-type sequence on both COS and melanoma cells. These data thus show that oncogenic protein mutations can create shared tumor-specific CTL epitopes, efficiently presented by tumor cells, and that screening for oncogene-transfected COS cell recognition by TIL (from tumors containing mutations) is a powerful approach for the identification of these epitopes.

Dimerization Co-Factor of Hepatocyte Nuclear Factor 1/Pterin-4α-Carbinolamine Dehydratase Is Necessary for Pigmentation in Xenopus and Overexpressed in Primary Human Melanoma Lesions

Dimerization co-factor of hepatocyte nuclear factor 1 (HNF1)/pterin-4alpha-carbinolamine dehydratase (DCoH/PCD) is both a positive co-factor of the HNF1 homeobox transcription factors and thus involved in gene regulation as well as an enzyme catalyzing the regeneration of tetrahydrobiopterin. Dysfunction of DCoH/PCD is associated with the human disorders hyperphenylalaninemia and vitiligo. In Xenopus, overexpression of the protein during development induces ectopic pigmentation. In this study loss of function experiments using DCoH/PCD-specific antibodies demonstrated that the protein is also absolutely necessary for pigment cell formation in Xenopus. In normal human skin DCoH/PCD protein is weakly expressed in the basal layer of the epidermis that consists of keratinocytes and melanocytes. Whereas only 4 of 25 benign nevi reacted with DCoH/PCD-specific antibodies, high protein levels were detectable in melanoma cell lines and 13 of 15 primary malignant melanoma lesions. The comparison with the commonly used melanoma markers S100 and HMB45 demonstrated that DCoH/PCD has an overlapping but distinct expression pattern in melanoma lesions. In addition to human colon cancer, this is the second report about the overexpression of DCoH/PCD in human tumor cells indicating that the protein might be involved in cancerogenesis.

Coexpression of Integrin αvβ3 and Matrix Metalloproteinase-2 (MMP-2) Coincides with MMP-2 Activation: Correlation with Melanoma Progression

Tumor cell invasion and metastasis formation depend on both adhesive and proteolytic mechanisms. Previous studies have shown that expression of matrix metalloproteinase-2 and integrin alphavbeta3 correlate with melanoma progression. Recently, direct binding of matrix metalloproteinase-2 to alpha(v)beta3 was implicated in presenting activated matrix metalloproteinase-2 on the cell surface of invasive cells. In this study we investigated this, using the highly metastatic, alpha(v)beta3-negative melanoma cell lines MV3 and BLM, their beta3-transfected alpha(v)beta3 expressing counterparts, xenografts derived from these cell lines, and fresh human cutaneous melanoma lesions comprising all stages of melanoma progression. Expression and activation status of matrix metalloproteinase-2 were studied by reverse transcription-polymerase chain reaction, immunohistochemistry, western blotting, and zymographic analysis, respectively. Matrix metalloproteinase-2 protein expression in vitro was similar in both alpha(v)beta3-negative and alpha(v)beta3-positive cell lines Remarkable differences, however, exist in the localization of inactive and active matrix metalloproteinase-2. Soluble active matrix metalloproteinase-2 was detectable only in the conditioned medium of alpha(v)beta3-negative cell lines and undetectable in the alpha(v)beta3-positive cell lines. Conversely, active matrix metalloproteinase-2 was present exclusively on the cell surface of the alpha(v)beta3 expressing transfectants. Western blot analysis of other components that are involved in matrix metalloproteinase-2 activation showed that processing of proMT1-matrix metalloproteinase to the activated form was enhanced in beta3 transfectants, whereas secretion of tissue inhibitor of metalloproteinase-2 was decreased. In vivo, the presence of functionally active matrix metalloproteinase-2 was significantly higher in xenografts derived from the alpha(v)beta3 expressing MV3 and BLM cell lines. In human cutaneous melanoma lesions, neither matrix metalloproteinase-2 nor integrin alpha(v)beta3 is detectable in melanoma in situ as determined by immunohistochemistry. In contrast, the number of matrix metalloproteinase-2-positive and alphavbeta3-positive tumor cells was clearly increased in primary melanomas, and melanoma metastases. Double staining experiments and confocal laser microscopy demonstrated that the percentage of cells coexpressing matrix metalloproteinase-2 and alpha(v)beta3 increased in advanced primary melanomas and melanoma metastases. In addition, zymography showed that functionally active matrix metalloproteinase-2 was frequently present in melanoma metastases. In these lesions a high proportion of matrix metalloproteinase-2- and alphavbeta3-double-positive melanoma cells were detectable. Our study demonstrates that the presence of activated matrix metalloproteinase-2 correlates with expression of alpha(v)beta3 in human melanoma cells both in vitro and in vivo, and also in fresh human melanoma lesions. These findings strongly suggest that co-ordinated expression of both factors may be required for melanoma cell invasion and metastasis formation.

A role for heterologous gap junctions between melanoma and endothelial cells in metastasis.

F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. We found that connexin (Cx) 26 is upregulated in BL6 cells. To examine gap junction formation, we devised a coculture system, in which an opened vein segment was placed at the bottom of a culture dish and then dye-labeled melanoma cells were seeded onto it. Immunohistochemistry indicated that the vein segment preserved the integrity of the endothelial monolayer. In this system, BL6 cells could transfer dye into endothelial cells but F10 cells could not. Transfection with wild-type Cx26 rendered F10 cells competent for coupling with endothelial cells and as spontaneously metastatic as BL6 cells. Conversely, transfection with a dominant-negative form of Cx26 rendered BL6 cells deficient in coupling and less metastatic. In human melanoma lesions, the level of Cx26 expression was low in melanoma cells residing in the basal layer, but significantly upregulated in melanoma cells invading the dermis. The results suggested that Cx26 plays a role in intravasation and extravasation of tumor cells through heterologous gap junction formation with endothelial cells.