Malignant features of minipig melanomas prior to spontaneous regression.

In MeLiM minipigs, melanomas develop around birth, can metastasize, and have histopathologic characteristics similar to humans. Interestingly, MeLiM melanomas eventually regress. This favorable outcome raises the question of their malignancy, which we investigated. We clinically followed tens of tumors from onset to first signs of regression. Transcriptome analysis revealed an enrichment of all cancer hallmarks in melanomas, although no activating or suppressing somatic mutation were found in common driver genes. Analysis of tumor cell genomes revealed high mutation rates without UV signature. Canonical proliferative, survival and angiogenic pathways were detected in MeLiM tumor cells all along progression stages. Functionally, we show that MeLiM melanoma cells are capable to grow in immunocompromised mice, with serial passages and for a longer time than in MeLiM pigs. Pigs set in place an immune response during progression with dense infiltration by myeloid cells while melanoma cells are deficient in B2M expression. To conclude, our data on MeLiM melanomas reveal several malignancy characteristics. The combination of these features with the successful spontaneous regression of these tumors make it an outstanding model to study an efficient anti-tumor immune response.

Deciphering the immune reaction leading to spontaneous melanoma regression: initial role of MHCII+ CD163- macrophages.

The human cutaneous metastatic melanoma is the deadliest skin cancer. Partial, or less frequently complete spontaneous regressions could be observed, mainly mediated by T cells. Nevertheless, the underlying mechanisms are not fully unraveled. We investigated the first events of the immune response related to cancer regression in Melanoma-bearing Libechov Minipigs (MeLiM), a unique swine model of cutaneous melanoma that regresses spontaneously. Using a multiparameter flow cytometry strategy and integrating new clinical and histological criteria of the regression, we show that T cells and B cells are present only in the late stages, arguing against their role in the initial destruction of malignant cells. NK cells infiltrate the tumors before T cells and therefore might be involved in the induction of the regression process. Myeloid cells represent the main immune population within the tumor microenvironment regardless of the regression stage. Among those, MHCII+ CD163- macrophages that differ phenotypically and functionally compared to other tumor-associated macrophages, increase in number together with the first signs of regression suggesting their crucial contribution to initiating the regression process. Our study supports the importance of macrophage reprogramming in humans to improve current immunotherapy for metastatic melanoma.

River widening in mountain and foothill areas during floods: Insights from a meta-analysis of 51 European Rivers.

River widening, defined as a lateral expansion of the channel, is a critical process that maintains fluvial ecosystems and is part of the regular functioning of rivers. However, in areas with high population density, channel widening can cause damage during floods. Therefore, for effective flood risk management it is essential to identify river reaches where abrupt channel widening may occur. Despite numerous efforts to predict channel widening, most studies have been limited to single rivers and single flood events, which may not be representative of other conditions. Moreover, a multi-catchment scale approach that covers various settings and flood magnitudes has been lacking. In this study, we fill this gap by compiling a large database comprising 1564 river reaches in several mountain regions in Europe affected by floods of varying magnitudes in the last six decades. By applying a meta-analysis, we aimed to identify the types of floods responsible for more extensive widening, the river reach types where intense widening is more likely to occur, and the hydraulic and morphological variables that explain widening and can aid in predicting widening. Our analysis revealed seven groups of reaches with significantly different responses to floods regarding width ratios (i.e., the ratio between channel width after and before a flood). Among these groups, the river reaches located in the Mediterranean region and affected by extreme floods triggered by short and intense precipitation events showed significantly larger widening than other river reaches in other regions. Additionally, the meta-analysis confirmed valley confinement as a critical morphological variable that controls channel widening but showed that it is not the only controlling factor. We proposed new statistical models to identify river reaches prone to widening, estimate potential channel width after a flood, and compute upper bound width ratios. These findings can inform flood hazard evaluations and the design of mitigation measures.

A role for endothelial alpha-mannosidase MAN1C1 in radiation-induced immune cell recruitment.

Radiation therapy damages tumors and normal tissues, probably in part through the recruitment of immune cells. Endothelial high-mannose N-glycans are, in particular, involved in monocyte-endothelium interactions. Trimmed by the class I α-mannosidases, these structures are quite rare in normal conditions. Here, we show that the expression of the endothelial α-mannosidase MAN1C1 protein decreases after irradiation. We modeled two crucial steps in monocyte recruitment by developing in vitro real-time imaging models. Inhibition of MAN1C1 expression by siRNA gene silencing increases the abundance of high-mannose N-glycans, improves the adhesion of monocytes on endothelial cells in flow conditions and, in contrast, decreases radiation-induced transendothelial migration of monocytes. Consistently, overexpression of MAN1C1 in endothelial cells using lentiviral vectors decreases the abundance of high-mannose N-glycans and monocyte adhesion and enhances transendothelial migration of monocytes. Hence, we propose a role for endothelial MAN1C1 in the recruitment of monocytes, particularly in the adhesion step to the endothelium.

The Composition of Circulating Leukocytes Varies With Age and Melanoma Onset in the MeLiM Pig Biomedical Model.

Immunological research in pigs benefits from many improvements with a direct impact on the veterinary control of pig husbandry and on biomedical models. We compiled the available knowledge to develop gating strategies to monitor simultaneously all blood immune cell types by multicolor flow cytometry in Melanoblastoma-bearing Libechov Minipigs (MeLiM). The MeLiM pig spontaneously develops cutaneous melanomas that regress few months later. We monitored lymphoid and myeloid cell subsets in 3 to 21 weeks old pigs. Interestingly, neutrophils, type III monocytes (CD163+ CD14+ MHC II-) and CD4- CD8α- T cells are less abundant in oldest animals in contrast to eosinophils, type II monocytes (CD163- CD14low MHC II+), B cells, γδ T cells, CD4+ CD8α+ and CD4- CD8α+ T cells. Melanoma occurrence led to changes in the blood cell composition. Higher proportions of NK cells, CD4+ and CD4+ CD8α+ T cells, and CD21- B cells among B cells are found in young melanoma-bearing piglets, consistent with the immune-mediated spontaneous regression in the MeLiM model.

Impact of a CD4 gene haplotype on the immune response in minipigs.

The cluster of differentiation 4 (CD4) molecule functions as a co-receptor for MHC class II binding to TCR in T helper cells. A CD4 epitope deficiency was identified in the swine MeLiM (melanoblastoma-bearing Libechov minipig) strain, a model for spontaneous cutaneous melanoma development and regression. Extensive sequencing revealed a high genetic variability of CD4 and the existence of several haplotypes segregating in MeLiM. Forty polymorphisms were identified in the coding sequence, out of which 20 correspond to non-synonymous variants and 10 are located in the 3'UTR of CD4 transcripts. One of the haplotypes segregating in the MeLiM explained the epitope deficiency observed. An association analysis between CD4 genotype and several phenotypes related to tumor regression was performed in 267 animals. An association was evidenced between a MeLiM alternative CD4 haplotype and skin and eye depigmentation, as well as the extent of hair depigmentation. Also, seric IgG concentration was shown to be higher in pigs carrying the alternative haplotype at the homozygous state. In conclusion, the genetic variability of the CD4 gene is associated with immune response-related phenotypes in MeLiM minipigs.

Genetic and functional evaluation of MITF as a candidate gene for cutaneous melanoma predisposition in pigs.

Cutaneous melanoma arises from transformed melanocytes and is caused mainly by environmental effects such as ultraviolet radiation and to a lesser extent by predisposing genetic variants. Only a few susceptibility genes for cutaneous melanoma have been identified so far in human; therefore, animal models represent a valuable alternative for genetic studies of this disease. In a previous quantitative trait locus (QTL) study, several susceptibility regions were identified in a swine biomedical model, the MeLiM (Melanoblastoma-bearing Libechov minipig) pigs. This article details the fine-mapping of a QTL located on SSC13 (Sus scrofa chromosome 13) through an increase in marker density. New microsatellites were used to confirm the results of the first analysis, and MITF (microphthalmia-associated transcription factor) was selected as a candidate gene for melanoma development. A single-marker association analysis was performed with single-nucleotide polymorphisms (SNPs) spread over the locus, but it did not reveal a significant association with diverse melanoma-related traits. In parallel, MITF alternative transcripts were characterized and their expression was investigated in different porcine tissues. The obtained results showed a complex transcriptional regulation concordant with the one present in other mammals. Notably, the ratio between MITF+ and MITF- isoforms in melanoma samples followed the same pattern as in human tumors, which highlights the adequacy of the MeLiM pig as a model for human melanoma. In conclusion, although MITF does not seem to be the causal gene of the QTL initially observed, we do not exclude a prominent role of its transcription and function in the outbreak and evolution of the tumors observed in pigs.

Gene expression signature for spontaneous cancer regression in melanoma pigs.

Incomplete spontaneous regression of melanoma is common. However, complete melanoma regression is still a very rare phenomenon. Because melanoma is the most immunogenic human malignancy, the mechanisms leading to regression, based on accumulative evidence, are the host's immune responses. Unfortunately, therapies aiming to enhance the patient's natural immunity against melanoma have yet to meet their expectations. Reasons for failure include various immune escape mechanisms, induced by the tumor, that subsequently lead to tolerance. Here, we performed time-dependent gene expression profiling to unravel molecular changes involved in the transition of progressive melanoma to complete tumor regression using a porcine model. The melanoblastomabearing Libechov minipigs are highly suitable for this study because these animals exhibit naturally occurring and regressing melanomas. We were able to identify a molecular signature of the melanoma regression process. Genes regulated in this signature were associated with 1) cell cycle, 2) immune response, and 3) melanocyte differentiation. These genes may shed light on molecular mechanisms involved in complete melanoma regression and indicate what improvements are needed for successful antimelanoma therapy.

RelA repression of RelB activity induces selective gene activation downstream of TNF receptors.

TNF-alpha is a potent proinflammatory cytokine that regulates immune and inflammatory responses and programmed cell death. TNF-alpha stimulation causes nuclear translocation of several NF-kappaB dimers, including RelA/p50 and RelB/p50. However, contrary to RelA, RelB entering the nucleus in response to TNF-alpha cannot bind to DNA in mouse embryonic fibroblasts, strongly suggesting that RelB DNA-binding activity is modulated by additional nuclear mechanisms. Here, we demonstrate that TNF-alpha promotes the association of RelA with RelB in the nucleus and that TNF-alpha-induced RelA/RelB heterodimers do not bind to kappaB sites. Remarkably, we show that RelA serine-276, the phosphorylation of which is induced by TNF receptor ligation, is crucial for RelA/RelB complex formation and subsequent inhibition of RelB DNA binding. In the absence of RelA phosphorylation on serine-276, TNF-alpha stimulation leads to a strong increase in the expression of endogenous NF-kappaB-responsive genes, such as Bcl-xL, whose transcriptional up-regulation is mainly controlled by RelB. Our findings demonstrate that RelA has a major regulatory role serving to dampen RelB activity in response to TNF-alpha and define a previously unrecognized mechanism that represents an essential step leading to selective NF-kappaB target gene expression.