Development Of Melanoma Metastases Research Articles

Abstract 2593: Spatial multi-omics profiling of tumor cell phenotypes in primary cutaneous melanoma at single-cell resolution

Abstract Primary cutaneous melanomas develop in stages from melanoma precursor cells that may progress to melanoma in situ (MIS) and eventually to invasive disease. Molecular changes, such as tumor cell state switches from melanocytic to undifferentiated and neural-crest-like phenotypes, have been associated with the development of melanoma metastases and therapeutic resistance. However, it is unknown whether cell states or gene expression changes enriched in advanced tumors exist at the premalignant stage and if they can elicit tumor-infiltrating lymphocytes (TILs). To address this gap, we analyzed a cohort of clinical samples from 43 patients with primary cutaneous melanoma using whole-slide cyclic multiplex immunofluorescence imaging (CyCIF), morphology-guided micro-regional RNA sequencing, and high-resolution 3D imaging. Each specimen contained multiple histologically-distinct regions representing a progression axis from normal to precursor to melanoma in situ to invasion. Subsequently, we examined the molecular profiles of both atypical melanocytes and tumor cells, and the gene expression and tumor microenvironmental (TME) changes that occurred during these progression stages. We identified major intra- and inter-specimen heterogeneity in the tumor intrinsic and extrinsic features across progression stages. Multiple tumor samples contained regions where publicly available gene signatures linked to both melanocytic and undifferentiated cell phenotypes were enriched. CyCIF analysis confirmed that various tumor cell phenotypes can coexist in close proximity to each other in early-stage untreated melanomas, and that the majority of undifferentiated tumor cells were located within regions of dermal invasion. Moreover, the cellular neighborhoods around undifferentiated tumor cells had a higher proportion of CD8+ T cells and cells of myeloid lineage compared to the neighborhoods around melanocytic tumor cell phenotypes. When assessing the localization and abundance of various tumor cell phenotypes, the levels of spatial and regional heterogeneity increased proportionally along normal to premalignant to malignant progression. Furthermore, the regional entropy of tumor cell phenotypes decreased along tumor depth in the majority (63%) of the samples, and even the melanoma in situ regions showed varying levels of spatial heterogeneity. Our in-depth multimodal molecular analyses highlight the presence of spatial heterogeneity among tumor cell phenotypes as well as the organization of early-stage primary melanomas, and are suggestive of tumor cell adaptation to the local environment. Citation Format: Tuulia Vallius, Edward Novikov, Ajit Nirmal Johnson, Shi Yingxiao, Roxanne Pelletier, Shishir Pant, Zoltan Maliga, Alyce Chen, Clarence Yapp, Sabrina Chan, Christine Lian, George F. Murphy, Sandro Santagata, Peter Sorger. Spatial multi-omics profiling of tumor cell phenotypes in primary cutaneous melanoma at single-cell resolution [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 2593.

Role of Annexin A1 Secreted by Neutrophils in Melanoma Metastasis.

Annexin A1 (AnxA1) is highly secreted by neutrophils and binds to formyl peptide receptors (FPRs) to trigger anti-inflammatory effects and efferocytosis. AnxA1 is also expressed in the tumor microenvironment, being mainly attributed to cancer cells. As recruited neutrophils are player cells at the tumor sites, the role of neutrophil-derived AnxA1 in lung melanoma metastasis was investigated here. Melanoma cells and neutrophils expressing AnxA1 were detected in biopsies from primary melanoma patients, which also presented higher levels of serum AnxA1 and augmented neutrophil-lymphocyte ratio (NLR) in the blood. Lung melanoma metastatic mice (C57BL/6; i.v. injected B16F10 cells) showed neutrophilia, elevated AnxA1 serum levels, and higher labeling for AnxA1 in neutrophils than in tumor cells at the lungs with metastasis. Peritoneal neutrophils collected from naïve mice were co-cultured with B16F10 cells or employed to obtain neutrophil-conditioned medium (NCM; 18 h incubation). B16F10 cells co-cultured with neutrophils or with NCM presented higher invasion, which was abolished if B16F10 cells were previously incubated with FPR antagonists or co-cultured with AnxA1 knockout (AnxA1-/-) neutrophils. The depletion of peripheral neutrophils during lung melanoma metastasis development (anti-Gr1; i.p. every 48 h for 21 days) reduced the number of metastases and AnxA1 serum levels in mice. Our findings show that AnxA1 secreted by neutrophils favors melanoma metastasis evolution via FPR pathways, addressing AnxA1 as a potential biomarker for the detection or progression of melanoma.

Ketogenic diets slow melanoma growth in vivo regardless of tumor genetics and metabolic plasticity

BackgroundGrowing evidence supports the use of low-carbohydrate/high-fat ketogenic diets as an adjunctive cancer therapy. However, it is unclear which genetic, metabolic, or immunological factors contribute to the beneficial effect of ketogenic diets. Therefore, we investigated the effect of ketogenic diets on the progression and metabolism of genetically and metabolically heterogeneous melanoma xenografts, as well as on the development of melanoma metastases in mice with a functional immune system.MethodsMice bearing BRAF mutant, NRAS mutant, and wild-type melanoma xenografts as well as mice bearing highly metastatic melanoma allografts were fed with a control diet or ketogenic diets, differing in their triglyceride composition, to evaluate the effect of ketogenic diets on tumor growth and metastasis. We performed an in-depth targeted metabolomics analysis in plasma and xenografts to elucidate potential antitumor mechanisms in vivo.ResultsWe show that ketogenic diets effectively reduced tumor growth in immunocompromised mice bearing genetically and metabolically heterogeneous human melanoma xenografts. Furthermore, the ketogenic diets exerted a metastasis-reducing effect in the immunocompetent syngeneic melanoma mouse model. Targeted analysis of plasma and tumor metabolomes revealed that ketogenic diets induced distinct changes in amino acid metabolism. Interestingly, ketogenic diets reduced the levels of alpha-amino adipic acid, a biomarker of cancer, in circulation to levels observed in tumor-free mice. Additionally, alpha-amino adipic acid was reduced in xenografts by ketogenic diets. Moreover, the ketogenic diets increased sphingomyelin levels in plasma and the hydroxylation of sphingomyelins and acylcarnitines in tumors.ConclusionsKetogenic diets induced antitumor effects toward melanoma regardless of the tumors´ genetic background, its metabolic signature, and the host immune status. Moreover, ketogenic diets simultaneously affected multiple metabolic pathways to create an unfavorable environment for melanoma cell proliferation, supporting their potential as a complementary nutritional approach to melanoma therapy.

Cytotoxic Activity and Lymphocyte Subtypes in Mice Selected for Maximal and Minimal Inflammatory Response after Transplantation of B16F10 and S91 Melanoma Cells.

AIRmax and AIRmin mice strains were selected according to the intensity of their acute inflammatory responsiveness. Previous studies have shown that AIR mice differ in their resistance to chemically induced skin tumors and in the development of melanoma metastases, in addition to differences in neutrophil and NK cells activity. In the present work, we aimed to evaluate whether the difference of susceptibility to murine melanoma is associated with NK cytotoxic activity against Yac.1 cells and lymphocyte subsets. Mice were subcutaneously inoculated with B16F10 or S91 melanoma cells. After 7, 14, or 30 days, the animals were euthanized to analyze the number of lymphocyte subsets, cytotoxic activity, and number of cytokine-producing spleen cells. AIRmax mice presented a higher number of CD4+/CD25+ cells than that of AIRmin mice following inoculation of B16F10 cells, whereas inoculation of S91 cells reduced CD4+/CD25+ and increased TCD8+ cell subsets in the AIRmax mice. AIRmax mice had a higher number of interleukin (IL)-10- and IL-12-producing cells and a lower number of interferon-γ–producing cells than those of AIRmin mice at 30 days. The cytotoxic activity of nonadherent spleen cells was similar in both the AIR strains. These results suggest that melanoma cells can induce different responses in AIR mice, possibly owing to alterations in regulatory mechanisms, such as the action of CD4+/CD25+ regulatory T cells and IL-10, in AIRmax mice.

The Antitumor Effect of Heparin is not Mediated by Direct NK Cell Activation.

Natural killer (NK) cells are innate lymphocytes responsible for the elimination of infected or transformed cells. The activation or inhibition of NK cells is determined by the balance of target cell ligand recognition by stimulatory and inhibitory receptors on their surface. Previous reports have suggested that the glycosaminoglycan heparin is a ligand for the natural cytotoxicity receptors NKp30, NKp44 (human), and NKp46 (both human and mouse). However, the effects of heparin on NK cell homeostasis and function remain unclear. Here, we show that heparin does not enhance NK cell proliferation or killing through NK cell activation. Alternatively, in mice models, heparin promoted NK cell survival in vitro and controlled B16-F10 melanoma metastasis development in vivo. In human NK cells, heparin promisingly increased interferon (IFN)-γ production in synergy with IL-12, although the mechanism remains elusive. Our data showed that heparin is not able to increase NK cell cytotoxicity.

Abstract 2736: AKT1E17K activates focal adhesion kinase and promotes melanoma brain metastasis

Abstract Hyper-activation of the PI3K/AKT signaling pathway occurs in most metastatic melanomas and increased PI3K/AKT pathway activity correlates with disease progression. The serine/threonine kinase, AKT, represents a major signaling hub within the pathway and consists of three highly conserved paralogs that have both distinct and overlapping functions. Activating mutations of AKT1 and AKT3 occur in human melanoma but their role in melanoma formation and metastasis remains unclear. Using an established melanoma mouse model, we evaluated the ability of constitutively active E17K, E40K, or Q79K mutants of each AKT paralog to promote tumor progression and metastasis in the context of BRAFV600E expression and loss of Cdkn2a and Pten. Expression of AKT1E17K promoted highly aggressive melanomas that metastasized to the lungs and brain. This metastatic phenotype was not significantly observed in the case of other mutant AKT-positive tumors, suggesting that the AKT paralogs have distinct, non-overlapping roles in the development of melanoma metastases. AKT1E17K-positive tumors showed AKT1E17K-dependent up-regulation of multiple focal adhesion (FA) factors, which are key components of focal adhesions and established stimulators of cell motility, as well as phosphorylation of focal adhesion kinase (FAK). Ectopic expression of AKT1E17K in non-metastatic melanoma cells increased cell invasion, a phenotype abrogated by pharmacological inhibition of AKT or FAK. These findings strongly suggest that one mechanism by which AKT1 promotes melanoma metastasis is through regulation and activation of proteins involved in focal adhesions. This has important implications for the development of therapeutic strategies aimed at preventing or treating disseminated disease. Citation Format: David A. Kircher, Kirby A. Trombetti, Mark R. Silvis, Gennie L. Parkman, Grant M. Fischer, Stephanie N. Angel, Christopher M. Stehn, Sean C. Strain, Allie H. Grossmann, Keith L. Duffy, Martin McMahon, Michael A. Davies, Michelle C. Mendoza, Matthew W. VanBrocklin, Sheri L. Holmen. AKT1E17K activates focal adhesion kinase and promotes melanoma brain metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2736.

Features associated with melanoma metastasis in Latvia.

Cutaneous melanoma (CM) is the most aggressive form of skin cancer, exhibits an increasing incidence worldwide and has a high potential to develop metastasis. The current study aimed to identify a set of parameters that may aid in predicting the probability and timing of the onset of CM metastasis. A retrospective analysis was performed using the archive data of 2,026 patients with CM that were treated at the Riga East University Hospital Latvian Oncology Centre, which is the largest oncological hospital in the country, between 1998 and 2015. A case-control study design was employed, where patients with metastasis (n=278) were used as the cases and patients without metastasis were used as the controls. The present study examined the associations between metastasis and potential risk factors and constructed multivariate models of features that predicted metastasis using stepwise regression. Time until metastasis was analyzed using negative binomial regression models. The results of the present study indicated an increase in the number of melanomas that developed metastases during 1998–2015. Tumor Breslow thickness was demonstrated to be significantly larger in patients with metastasis compared with those without (P=0.012). The presence of ulceration significantly increased the risk of metastases [odds ratio (OR), 1.66; 95% CI, 1.07-2.59; P=0.033]. The absence of pigment in melanoma tissues was indicated to lead to a greater likelihood of metastasis (OR, 2.14; 95% CI, 1.10-4.19; P=0.035). Shorter times from diagnosis until the onset of metastases were observed in older patients (incident rate ratio (IRR), 6.85; 95% CI, 2.43-19.30; P=2.78×10−4), and a borderline significant association was observed in those with ulcerated tumors (IRR, 1.33; 95% CI, 0.98-1.80; P=0.064). Therefore, the main features associated with the development of melanoma metastasis in Latvia were indicated to be tumor ulceration, absence of pigment and Breslow thickness.

Transcriptomic identification of miR-205 target genes potentially involved in metastasis and survival of cutaneous malignant melanoma

Cutaneous melanoma is an aggressive neoplasm and is responsible for the majority of skin cancer deaths. Several miRNAs are involved in melanoma tumor progression. One of them is miR-205, the loss of which contributes to the development of melanoma metastasis. We evaluated whole-genome mRNA expression profiling associated with different miR-205 expression levels in melanoma cells. Differential expression analysis identified 243 differentially expressed transcripts including inositol polyphosphate 5′-phosphatase-like protein-1 (INPPL1) and BTB/POZ Domain-Containing Protein 3 (BTBD3). INPPL1 and BTBD3 were downregulated when melanoma cells expressed miR-205, indicating that these genes are potential miR-205 targets. Additionally, the target prediction algorithm TargetScan revealed that INPPL1 and BTBD3 genes had predicted target sites of miR-205 in their 3′UTRs and functional analysis demonstrated that these genes were directly linked to miR-205. Interestingly, our clinical data showed that INPPL1 was significantly associated with lymph node metastasis-free survival (LNMFS), distant metastasis-free survival (DMFS) and melanoma specific survival (MSS). This study supports INPPL1 as a miR-205 target gene and, therefore, that the involvement of miR-205 in the metastatic dissemination of malignant melanoma is, at least in part, via INPPL1.

Prognostic factors for metastasis in cutaneous melanoma

BackgroundMelanoma is a malignant neoplasia that shows high mortality when diagnosed in advanced stages. Early identification of high-risk patients for the development of melanoma metastases is the main strategy to reduce mortality.ObjectiveTo assess the influence of eight epidemiological and histopathologic features on the development of metastases in patients diagnosed with primary cutaneous melanoma.MethodsOur historical cohort comprised patients with invasive primary cutaneous melanoma seen between 1995 and 2012 at a public university hospital and a private oncologic surgery institution in Southeastern Brazil. The following variables were analyzed: gender, age, family history of melanoma, site of the primary tumor, clinical and histologic subtype, Breslow thickness, histologic ulceration and the mitotic index. Kaplan-Meier univariate test and multivariate Cox proportional hazard analysis were used to assess factors associated with disease-free survival.ResultsFive hundred and fourteen patients were enrolled. The univariate analysis identified the following significant risk factors: gender, age, site of the tumor, clinical and histologic subtype, Breslow thickness, histologic ulceration and mitotic index. Multivariate analysis included 244 patients and detected four significant prognostic factors: male gender, nodular clinical and histologic subtype, Breslow thickness > 4mm, and histologic ulceration. The mitotic index was not included in this analysis.Study limitationsSmall number of patients in multivariate analysis.ConclusionsThe following prognostic factors to the development of melanoma metastasis were identified in the study: male gender, nodular histologic subtype, Breslow thickness > 4mm and ulceration.

MITF and BRN2 contribute to metastatic growth after dissemination of melanoma

Melanoma tumors are highly heterogeneous, comprising of different cell types that vary in their potential for growth and invasion. Heterogeneous expression of the Microphthalmia-associated Transcription Factor (MITF) and the POU domain transcription factor BRN2 (POU3F2) has been found in malignant melanoma. Changing expression of these transcription factors as the disease progresses has been linked to the metastatic mechanism of phenotype switching. We therefore investigated the effects of MITF and BRN2 expression in melanoma growth and metastasis. Depletion of MITF resulted in a cell population that had a slowed cell cycle progression, was less invasive in vitro and had hindered tumor and metastasis forming ability in mouse xenograft studies. BRN2 depletion left a cell population with intact proliferation and invasion in vitro; however metastatic growth was significantly reduced in the mouse xenograft model. These results suggest that the proliferative population within melanoma tumors express MITF, and both MITF and BRN2 are important for metastatic growth in vivo. This finding highlights the importance of BRN2 and MITF expression in development of melanoma metastasis.

Metformin Blocks Melanoma Invasion and Metastasis Development in AMPK/p53-Dependent Manner

Metformin was reported to inhibit the proliferation of many cancer cells, including melanoma cells. In this report, we investigated the effect of metformin on melanoma invasion and metastasis development. Using different in vitro approaches, we found that metformin inhibits cell invasion without affecting cell migration and independently of antiproliferation action. This inhibition is correlated with modulation of expression of proteins involved in epithelial-mesenchymal transition such as Slug, Snail, SPARC, fibronectin, and N-cadherin and with inhibition of MMP-2 and MMP-9 activation. Furthermore, our data indicate that this process is dependent on activation of AMPK and tumor suppressor protein p53. Finally, we showed that metformin inhibits melanoma metastasis development in mice using extravasation and metastasis models. The presented data reinforce the fact that metformin might be a good candidate for clinical trial in melanoma treatment.

Abstract IA29: Models of postsurgical early and late stage metastasis for improving preclinical adjuvant and metastatic therapy investigations

Abstract A long standing problem in anti-cancer drug development has been the limited value of preclinical mouse tumor models to reliably predict subsequent clinical activity. All too often highly encouraging preclinical results in mice are followed by complete failure in clinical trials, especially at the randomized phase III level. There are many possible reasons that have been postulated for this preclinical/clinical discrepancy. One which we have been studying for the past decade is the failure to use mouse models which duplicate the challenging circumstance of treating advanced (established) visceral metastatic disease after primary tumors have been surgically resected. Instead, preclinical treatment of established primary tumors or low volume (micro)metastatic disease, often confined to the lungs, have been the historical preclinical model norms. To address this problem we have developed several models of postsurgical advanced metastatic disease involving human tumor xenografts grown in SCID mice, including breast, colorectal or kidney cancer, and melanoma (1). This approach has been extended more recently for postsurgical adjuvant therapy of early stage microscopic metastatic disease (2,3). The established cell lines used for in vivo studies are variants previously selected in vivo for more aggressive spontaneous metastatic capability, which then are sometimes stably tagged with luciferase to permit whole body bioluminescent imaging. Using these models to evaluate the impact of several anti-cancer treatments, consisting mostly of antiangiogenic drugs and/or chemotherapy, either standard maximum tolerated dose or low-dose ‘metronomic’, has highlighted the critical contribution of the extent of metastatic disease to differential therapeutic outcomes, and the prospect of better correlation with clinical outcomes. For example, primary orthotopic tumors, e.g. breast cancer in the mammary fat pad, respond well to treatment with an antiangiogenic drug such as sunitinib, pazopanib or anti-VEGFR-2 antibodies whereas mice with advanced metastases in sites such as the liver or lungs do not, e.g. no prolongation of survival is observed (4). Adding chemotherapy, e.g. paclitaxel to sunitinib did not change the results, whereas adding DC101, an anti-VEGFR-2 antibody, to the same chemotherapy regimen did result in a modest survival improvement (thus mimicking the metastatic breast cancer E2100 phase III results of bevacizumab plus paclitaxel chemotherapy) (4). The observed lack of sunitinib efficacy alone or with chemotherapy when treating mice with advanced metastases mimics three failed phase III clinical trial results of this drug with or without chemotherapy in metastatic breast cancer patients (5). In addition, we have noted that successful treatment of mice with advanced systemic metastatic breast, melanoma or renal cell cancer, e.g. with low-dose metronomic chemotherapy plus an antiangiogenic drug such that overall survival is meaningfully prolonged, sometimes results in the emergence of overt spontaneous brain metastases(1,6,7). Thus, in mice, the brain appears to be a protective sanctuary for the survival and progressive growth of microscopic into macroscopic metastases, as already well known in the clinic. More recent studies have indicated how the brain microenvironment can contribute to the development of melanoma metastases in this organ environment e.g. the interaction of endothelins (ETs) with (elevated) endothelin receptor B expression by the brain melanoma metastatic variants (8). In summary, models of postsurgical advanced metastatic disease to undertake experimental therapeutic studies appear to have a greater degree of clinical relevance compared to most conventional primary tumor therapy models. We are now extending this approach to the development of postsurgical models of early stage microscopic metastatic disease to mimic adjuvant therapy in the clinic (2,9,10); some of our previous (2009) results indicated that adjuvant antiangiogenic therapy may actually worsen eventual survival outcomes of mice with early stage disease (2), a finding for which there is now some preliminary clinical support based on a recent phase III clinical trial assessing treatment of postsurgical early stage colorectal cancer patients with bevacizumab plus chemotherapy (11,12).

Human melanoma cells in the rhombencephalon of the chick embryo: a novel model for brain metastasis

Malignant melanoma has the highest propensity to metastasize to the brain of all primary neoplasms in adults. Here, we describe invasive growth and the development of melanoma metastases from suspensions of human melanoma cells in the brain of the chick embryo. Patient-derived melanoma cells and established melanoma cell lines were injected into the rhombencephalic brain vesicle of the two-day-chick embryo. After 48 and 96 h, tumor formation was studied in serial paraffin sections with melanoma-specific HMB45 and human-specific MIB1 proliferation markers. The majority of the cells injected into the embryonic liquor cavity perished. Only melanoma cells in newly formed aggregates or when attached to the dorsal roof plate escaped apoptosis. Local invasion occurred not in the ventral differentiating neural epithelium but only in the roof plate. Although after 48 h melanoma cells invaded the rhombencephalic roof plate profusely at both sides, after 96 h typically one large tumor developed in the midline between roof plate and the dorsal surface epithelium. From the tumor, single cells invaded the mesenchyme and blood vessels. Cell lines with different invasive properties retained their graded invasive behaviour. Maximally invasive cells formed continuous tracks via vessels and along nerve fibres. The central tumor in the roof plate of the chick embryo rhombencephalon resembles a metastatic nodule in the patients with melanoma. Penetration of the roof plate epithelium, tumor formation and invasion of surrounding tissues by single cells can reliably be reproduced. The chick embryo model can be used for molecular studies of early phases of melanoma brain metastasis.

Role of Tip60 in Human Melanoma Cell Migration, Metastasis, and Patient Survival

The tumor suppressor Tip60 regulates gene transcription, DNA damage response, apoptosis, and cancer development, but its role in melanoma is unknown. In this study, we investigated the expression pattern of Tip60 in melanoma and assessed its prognostic value. Using tissue microarrays consisting of 448 cases of melanomas (201 for the training set and 247 for the validation set) and 105 cases of nevi, we found that Tip60 expression was significantly reduced in metastatic melanoma compared to common nevi (P=0.045), dysplastic nevi (P=0.047), and primary melanoma (P=0.001). Kaplan-Meier survival curve and univariate Cox regression analyses showed that reduced Tip60 expression was associated with a poorer 5-year disease-specific survival in primary melanoma (P=0.016) and metastatic melanoma patients (P=0.027). Multivariate Cox regression analyses indicated that Tip60 expression was an independent prognostic marker for primary (P=0.024) and metastatic melanomas (P=0.035). In vitro wound healing assay showed that enforced Tip60 expression inhibited but Tip60 knockdown enhanced melanoma cell migration, suggesting that Tip60 might regulate melanoma metastasis. Finally, we showed that overexpression of Tip60 in melanoma cells resulted in significantly increased chemosensitivity. Our data indicate that Tip60 may serve as a potential biomarker for melanoma patient outcome as well as a potential therapeutic target.

Abstract 2423: B7-H3 contributes to increased metastatic potential of melanoma cells by interfering with the expression of metastasis-related genes

Abstract Metastatic malignant melanoma is a disease with a long time survival rate of only 5%. Since there is no effective treatment for advanced melanoma, detection of molecular markers involved in tumor progression and drug resistance can hopefully provide new insight in therapeutically aspects of this disease. We have been studying B7-H3 – a glycosylated transmembrane protein which has been shown to play a role in cancer progression through its immunoregulatory properties. B7-H3 is highly expressed in melanoma cell lines as well as in clinical melanoma specimens, and we have previously demonstrated a role of the protein in chemoresistance. Thus, in this study, we wanted to investigate the possible function of B7-H3 in melanoma metastasis development. We found that short hairpin RNA silencing of B7-H3 reduced the migratory potential as well as the matrigel-invasiveness of the malignant melanoma cells in vitro. In an in vivo experimental metastasis model, silencing of B7-H3 increased the symptom-free survival of nude mice and rats significantly, this in parallel with a lower fraction mice having microscopically detectable metastasis compared to control mice. The brain was the predominant organ with metastases and a clear difference between the nude mice injected with B7-H3 expressing cells and B7-H3 knock down cells was observed, with 10/14 and 6/17 mice having brain tumors, respectively. Furthermore, we found the expression of several metastasis-associated proteins, such as MMP-2, TIMP1 and TIMP2 to be regulated by B7-H3. In addition, the secretion of IL-8 was markedly reduced in the B7-H3 knock down cells as well as the phosphorylation of Stat3. Interestingly, activated Stat3 is known to play a role in brain metastasis. Taken together, our findings suggest that B7-H3 might be a potential therapeutic target for anti-metastasis therapy in human melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2423. doi:10.1158/1538-7445.AM2011-2423

Tonsil Metastasis from Cutaneous Melanoma: First Clinical Sign of Recurrence after Complete Lymph Node Dissection

The development of melanoma metastasis to the palatine tonsil is a very rare event, generally associated with advanced-stage disease and poor prognosis. A 29-year-old man presented with a melanoma metastasis to the right palatine tonsil 6 months after the surgical excision of an ulcerated nodular melanoma (tumor thickness 1.8 mm, Clark level IV) on the left shoulder. A metastatic sentinel lymph node had been removed from the left axilla, and a subsequent complete lymph node dissection had disclosed no further metastatic lymph nodes. Although staging tests had revealed multiple visceral metastases, a palliative tonsillectomy was performed. The patient died of metastases 5 months later. The case presentation suggests that careful examination of the head and neck should be part of the routine follow-up visit in all melanoma patients.

Antimetastatic activity of MONCPT in preclinical melanoma mice model

Previous study demonstrated that MONCPT, a topoisomerase I inhibitor, exhibited potent anti-proliferation and anti-angiogenesis activity in vitro and in vivo. In this study, we report the efficacy of MONCPT against the development of melanoma metastasis by an intravenous injection of green fluorescent protein-transfected mice melanoma carcinoma (B16F10-GFP) cells in C57BL/6 mice. MONCPT (2.0, 5.0 and 12.5mg/kg/2days) markedly decreased B16F10-GFP pulmonary metastases by 12.8%, 53.1% and 76.3%, respectively; whereas higher doses of MONCPT (31.0mg/kg/2days) significantly inhibited the tumor growth of B16F10 xenograft model. In the in vitro experiment, MONCPT suppressed the B16F10-GFP cell invasion and migration without affecting cell survival. Further studies demonstrated that MONCPT decreased the secretion of matrix metalloproteinase (MMP)-9 and VEGF, and reduced the protein expression of HIF-1α as well as the phosphorylation level of ERK in B16F10-GFP cells. These in vivo and in vitro results indicate that MONCPT possesses both the potent antimetastatic ability and the tumor growth-inhibition activity, and the dual function promises MONCPT as a potential therapeutic agent for tumor metastasis and tumor growth of melanoma carcinoma.

Behavior: A Relevant Tool for Brain‐immune System Interaction Studies

Neuroimmunomodulation describes the field focused on understanding the mechanisms by which the central nervous system interacts with the immune system, potentially leading to changes in animal behavior. Nonetheless, not many articles dealing with neuroimmunomodulation employ behavior as an analytical endpoint. Even fewer papers deal with social status as a possible modifier of neuroimmune phenomena. In the described sets of experiments, we tackle both, using a paradigm of social dominance and subordination. We first review data on the effects of different ranks within a stable hierarchical relationship. Submissive mice in this condition display more anxiety-like behaviors, have decreased innate immunity, and show a decreased resistance to implantation and development of melanoma metastases in their lungs. This suggests that even in a stable, social, hierarchical rank, submissive animals may be subjected to higher levels of stress, with putative biological relevance to host susceptibility to disease. Second, we review data on how dominant and submissive mice respond differentially to lipopolysaccharide (LPS), employing a motivational perspective to sickness behavior. Dominant animals display decreased number and frequency in several aspects of behavior, particularly agonistic social interaction, that is, directed toward the submissive cage mate. This was not observed in submissive mice that maintained the required behavior expected by its dominant mate. Expression of sickness behavior relies on motivational reorganization of priorities, which are different along different social ranks, leading to diverse outcomes. We suggest that in vitro assessment of neuroimmune phenomena can only be understood based on the behavioral context in which they occur.

Simultaneous blockade of VEGFR‐1 and VEGFR‐2 activation is necessary to efficiently inhibit experimental melanoma growth and metastasis formation

Metastasis continues to be the major cause of morbidity and mortality in malignant melanoma. In our study, we explored whether inhibition of VEGFR-1 or VEGFR-2 signaling conveys distinct suppressive effects on B16 melanoma subcutaneous growth and metastasis formation. The inhibition of VEGFR-1 or -2 alone had no significant influence on both melanoma growth and metastasis formation. In contrast, simultaneous blockade of VEGFR-1 and -2 signaling strongly suppressed progression in both B16 tumor models. There was no expression of VEGFR-1 or -2 detectable on the B16 cells used, excluding the melanoma cells as direct therapeutic targets. Analyzing the contribution of progenitor-like cells during melanoma metastasis formation, we observed an enhanced proliferation and mobilization of VEGFR-1+ myeloid and VEGFR-2+ endothelial cells with progenitor potential by the induction of melanoma lung metastasis, which was not influenced by interference with VEGFR signaling. These results indicate that the antimetastatic effects exerted by combined inhibition of VEGFR-1 and -2 signaling were mediated via targeting cell populations other than progenitors only. Sole inhibition of VEGFR-1 signaling led to a strong reduction of the CD45-positive inflammatory infiltrate in the tumor tissue. However, the formation of lung metastasis was not affected, indicating that inhibition of the inflammatory response was not sufficient to efficiently block B16 melanoma metastasis development. Taken together, our data suggest that in the utilized B16 tumor models the blockade of both the inflammatory and the VEGFR-2-dependent angiogenic response are necessary to effectively inhibit solid tumor growth and formation of lung metastasis by B16 melanoma cells.

β3 Integrin Expression in Melanoma Predicts Subsequent Metastasis

Previous studies have suggested that differential expression and/or activation of integrins facilitates metastatic progression in murine and human melanoma. While recent data show that the integrin αvβ3 is involved in tumor angiogenesis and that tumor growth may be abrogated by αvβ3 inhibitorsin vitro,the clinical significance of β3 integrin expression in human malignant melanoma is not known. To assess the prognostic value of β3 integrin expression, we examined primary cutaneous melanomas from 160 patients followed for a mean of 98 months or until death. We quantified the percentage of tumor area stained with β3 integrin Ab CD-61 using an image analyzer. β3 integrin expression was detected in 107/160 primary melanomas (69%). β3-integrin-positive (β3+) tumors were thicker (mean 2.98 ± 0.3 mm) than β3-integrin-negative (β3−) melanomas (mean 1.64 ± 0.2 mm) (P= 0.002). Patients with β3+ melanomas were more likely to relapse (57/107, 53%) and to die from disease (45/107, 42%) than those with β3− tumors (6/53, 11%; and 4/53, 8%, respectively) (P< 0.001). Overall survival was greater for β3− than for β3+ patients (mean 102 ± 9 vs 69 ± 6 months) (P= 0.001). These data show that β3 integrin expression in primary cutaneous melanoma predicts subsequent metastatic progression. Further study of β3 integrins in the development of melanoma metastases may yield new therapeutic strategies, as well as prognostic information, for the treatment of this cancer.