Melanoma-Bearing Libechov Minipig (MeLiM): The Unique Swine Model of Hereditary Metastatic Melanoma

Weiss S.A., Wolchok J.D., Sznol M.

Abstract Melanoma is among the most sensitive of malignancies to immune modulation. Although multiple trials conducted over decades with vaccines, cytokines, and cell therapies demonstrated meaningful responses in a small subset of patients with metastatic disease, a true increase in overall survival (OS) within a randomized phase III trial was not observed until the development of anti–CTLA-4 (ipilimumab). Further improvements in OS for metastatic disease were observed with the anti–PD-1–based therapies (nivolumab, pembrolizumab) as single agents or combined with ipilimumab. A lower bound for expected 5-year survival for metastatic melanoma is currently approximately 35% and could be as high as 50% for the nivolumab/ipilimumab combination among patients who would meet criteria for clinical trials. Moreover, a substantial fraction of long-term survivors will likely remain progression-free without continued treatment. The hope and major challenge for the future is to understand the immunobiology of tumors with primary or acquired resistance to anti–PD-1 or anti–PD-1/anti–CTLA-4 and to develop effective immune therapies tailored to individual patient subsets not achieving long-term clinical benefit. Additional goals include optimal integration of immune therapy with nonimmune therapies, the development and validation of predictive biomarkers in the metastatic setting, improved prognostic and predictive biomarkers for the adjuvant setting, understanding mechanisms of and decreasing toxicity, and optimizing the duration of therapy.

Roberts M.R., Asgari M.M., Toland A.E.

Background Genome-wide association studies (GWAS) have identified thousands of susceptibility variants, although most have been associated with small individual risk estimates that offer little predictive value. However, combining multiple variants into polygenic risk scores (PRS) may be more informative. Multiple studies have developed PRS composed of GWAS-identified variants for cutaneous cancers. This review highlights data from these studies. Objectives To review published GWAS and PRS studies for melanoma, cutaneous squamous cell carcinoma (cSCC) and basal cell carcinoma (BCC), and discuss their potential clinical utility. Methods We searched PubMed and the National Human Genome Research Institute-European Bioinformatics Institute GWAS catalogue to identify relevant studies. Results Results from 21 GWAS (11 melanoma, 3 cSCC, 7 BCC) and 11 PRS studies are summarized. Six loci in pigmentation genes overlap between these three cancers (ASIP/RALY, IRF4, MC1R, OCA2, SLC45A2 and TYR). Additional loci overlap for cSCC/BCC and BCC/melanoma, but no other loci are shared between cSCC and melanoma. PRS for melanoma show roughly two-to-threefold increases in risk and modest improvements in risk prediction (2-7% increases). PRS are associated with twofold and threefold increases in risk of cSCC and BCC, respectively, with small improvements (2% increase) in predictive ability. Conclusions Existing data indicate that PRS may offer small, but potentially meaningful, improvements to risk prediction. Additional research is needed to clarify the potential utility of PRS in cutaneous carcinomas. Clinical translation will require well-powered validation studies incorporating known risk factors to evaluate PRS as tools for screening. What's already known about this topic? Over 50 susceptibility loci for melanoma, basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC) have been identified in genome-wide association studies (GWAS). Polygenic risk scores (PRS) using variants identified from GWAS have also been developed for melanoma, BCC and cSCC, and investigated with respect to clinical risk prediction. What does this study add? This review provides an overview of GWAS findings and the potential clinical utility of PRS for melanoma, BCC and cSCC.

Warner A.B., McQuade J.L.

We discuss how potentially modifiable factors including obesity, the microbiome, diet, and exercise may impact melanoma development, progression, and therapeutic response. Obesity is unexpectedly associated with improved outcomes with immune and targeted therapy in melanoma, with early mechanistic data suggesting leptin as one mediator. The gut microbiome is both a biomarker of response to immunotherapy and a potential target. As diet is a major determinant of the gut microbiome, ongoing studies are examining the interaction between diet, the gut microbiome, and immunity. Data are emerging for a potential role of exercise in reducing hypoxia and enhancing anti-tumor immunity, though this has not yet been well-studied in the context of contemporary therapies. Recent data suggests energy balance may play a role in the outcomes of metastatic melanoma. Further studies are needed to demonstrate mechanism and causality as well as the feasibility of targeting these factors.

Prouteau, André

Despite recent genetic advances and numerous ongoing therapeutic trials, malignant melanoma remains fatal, and prognostic factors as well as more efficient treatments are needed. The development of such research strongly depends on the availability of appropriate models recapitulating all the features of human melanoma. The concept of comparative oncology, with the use of spontaneous canine models has recently acquired a unique value as a translational model. Canine malignant melanomas are naturally occurring cancers presenting striking homologies with human melanomas. As for many other cancers, dogs present surprising breed predispositions and higher frequency of certain subtypes per breed. Oral melanomas, which are much more frequent and highly severe in dogs and cutaneous melanomas with severe digital forms or uveal subtypes are subtypes presenting relevant homologies with their human counterparts, thus constituting close models for these human melanoma subtypes. This review addresses how canine and human melanoma subtypes compare based on their epidemiological, clinical, histological, and genetic characteristics, and how comparative oncology approaches can provide insights into rare and poorly characterized melanoma subtypes in humans that are frequent and breed-specific in dogs. We propose canine malignant melanomas as models for rare non-UV-induced human melanomas, especially mucosal melanomas. Naturally affected dogs offer the opportunity to decipher the genetics at both germline and somatic levels and to explore therapeutic options, with the dog entering preclinical trials as human patients, benefiting both dogs and humans.

Lorentzen H.F.

Treatment of advanced melanoma has undergone a paradigm shift over the last 10-15 years. The frustrating results of studies on medical treatment ten years ago have been replaced by studies constantly improving survival in patients with advanced melanoma. Immune checkpoint inhibitors belong to one group of treatments and targeted therapy to another. Fifty percent of melanomas are BRAF mutation positive. Normally, the mitogen activated protein kinase or MAP kinase (Ras-BRAF-MEK-Erk chain) pathways translate external signals to intracellular growth and proliferation. In BRAF mutated melanoma cells, the mutated BRAF kinase is excessively active leading to autonomous proliferation and cancerous growth. This kinase can be blocked by BRAF-inhibitors. If given to BRAF negative melanoma patients, it may lead to disease progression because Ras is not inhibited in these cells. Development of Squamous cell carcinomas as a serious adverse event to BRAF inhibition may be caused by similar mechanisms in non BRAF mutated keratinocytes. A spontaneous and paradoxical loss of effect is seen with BRAF inhibitors due to various ways melanoma cells bypass BRAF. This is somewhat counteracted by the addition of a MEK1/2 inhibitor. Overall progression-free survival has increased from a median of two months for chemotherapy, via 7-8 months for BRAF inhibitor to 10-14 months for newer BRAF and MEK inhibitor combination therapy.

Cagan R.L., Zon L.I., White R.M.

Cancer has joined heart disease as the leading source of mortality in the US. In an era of organoids, patient-derived xenografts, and organs on a chip, model organisms continue to thrive with a combination of powerful genetic tools, rapid pace of discovery, and affordability. Model organisms enable the analysis of both the tumor and its associated microenvironment, aspects that are particularly relevant to our understanding of metastasis and drug resistance. In this Perspective, we explore some of the strengths of fruit flies and zebrafish for addressing fundamental cancer questions and how these two organisms can contribute to identifying promising therapeutic candidates.

Yu C., Liu X., Yang J., Zhang M., Jin H., Ma X., Shi H.

Metastatic melanoma is the most aggressive and obstinate skin cancer with poor prognosis. Variant novel applicable regimens emerged during the past decades intensively, while the most profound approaches are oncogene-targeted therapy and T-lymphocyte mediated immunotherapy. Although targeted therapies generated remarkable and rapid clinical responses in the majority of patients, acquired resistance was developed promptly within months leading to tumor relapse. By contrast, immunotherapies elicited long-term tumor regression. However, the overall response rate was limited. In view of the above, either targeted therapy or immunotherapy cannot elicit durable clinical responses in large range of patients. Interestingly, the advantages and limitations of these regimens happened to be complementary. An increasing number of preclinical studies and clinical trials proved a synergistic antitumor effect with the combination of targeted therapy and immunotherapy, implying a promising prospect for the treatment of metastatic melanoma. In order to achieve a better therapeutic effectiveness and reduce toxicity in patients, great efforts need to be made to illuminate multifaceted interplay between targeted therapy and immunotherapy.

Kozar I., Margue C., Rothengatter S., Haan C., Kreis S.

Melanoma is an aggressive malignancy originating from pigment-producing melanocytes. The development of targeted therapies (MAPK pathway inhibitors) and immunotherapies (immune checkpoint inhibitors) led to a substantial improvement in overall survival of patients. However, the long-term efficacy of such treatments is limited by side effects, lack of clinical effects and the rapidly emerging resistance to treatment. A number of molecular mechanisms underlying this resistant phenotype have already been elucidated. In this review, we summarise currently available treatment options for metastatic melanoma and the known resistance mechanisms to targeted therapies. A focus will be placed on "phenotype switching" as a mechanism and driver of drug resistance, together with an overview of novel approaches to circumvent resistance. A large body of recent data and literature suggests that tumour progression and phenotype switching could be better controlled and development of resistance prevented or at least delayed, by combining drugs targeting fast- and slow-proliferating cells.

Cizkova J., Sinkorova Z., Strnadova K., Cervinkova M., Horak V., Sinkora J., Stepanova K., Sinkora M.

Using a porcine model, we describe Melanoma-Associated CD4+CD8hi T-lymphocytes (MATL) in peripheral blood that increase during melanoma regression. These MATL possess the CD4+CD8hi phenotype and they have their direct counterparts in Tumor Infiltrating Lymphocytes (TIL) isolated from melanoma loci. Both MATL and CD4+CD8hi TIL have a similar expression of selected markers indicating that they represent effector/memory αβ T-cell subset. Moreover, although TIL also contain CD4-CD8+ T-cells, only CD4+CD8hi TIL expand during melanoma regression. Importantly, TIL isolated from different pigs and different melanoma loci among the same pig have similar composition of CD4/CD8 subsets, indicating that the composition of the MATL and TIL compartment is identical. Analysis of sorted cells from regressing pigs revealed a unique MATL subpopulation with mono-specific T-cell receptor that was further analyzed by sequencing. These results indicate that pigs regressing melanomas possess a characteristic population of recirculating T-cells playing a role in tumor control and regression.

Potrony M., Puig‐Butille J.A., Ribera‐Sola M., Iyer V., Robles‐Espinoza C.D., Aguilera P., Carrera C., Malvehy J., Badenas C., Landi M.T., Adams D.J., Puig S.

Background: Germline mutations in telomere-related genes such as POT1 and TERT predispose individuals to familial melanoma. Objectives: To evaluate the prevalence of germline mutations in POT1 and TERT in a large cohort of Spanish melanoma-prone families (at least two affected first- or second-degree relatives). Methods: Overall, 228 CDKN2A wild-type melanoma-prone families were included in the study. Screening of POT1 was performed in one affected person from each family and TERT was sequenced in one affected patient from 202 families (26 families were excluded owing to DNA exhaustion/degradation). TERT promoter sequencing was extended to an additional 30 families with CDKN2A mutation and 70 patients with sporadic multiple primary melanoma (MPM) with a family history of other cancers. Results: We identified four families with potentially pathogenic POT1 germline mutations: a missense variant c.233T>C (p.Ile78Thr); a nonsense variant c.1030G>T (p.Glu344*); and two other variants, c.255G>A (r.125_255del) and c.1792G>A (r.1791_1792insAGTA, p.Asp598Serfs*22), which we confirmed disrupted POT1 mRNA splicing. A TERT promoter variant of unknown significance (c.-125C>A) was detected in a patient with MPM, but no germline mutations were detected in TERT promoter in cases of familial melanoma. Conclusions: Overall, 1·7% of our CDKN2A/CDK4-wild type Spanish melanoma-prone families carry probably damaging mutations in POT1. The frequency of TERT promoter germline mutations in families with melanoma in our population is extremely rare.

Rabbie R., Ferguson P., Molina‐Aguilar C., Adams D.J., Robles‐Espinoza C.D.

Melanoma is characterised by its ability to metastasise at early stages of tumour development. Current clinico-pathologic staging based on the American Joint Committee on Cancer criteria is used to guide surveillance and management in early-stage disease, but its ability to predict clinical outcome has limitations. Herein we review the genomics of melanoma subtypes including cutaneous, acral, uveal and mucosal, with a focus on the prognostic and predictive significance of key molecular aberrations. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Rachidi S., Li H., Wallace K., Li Z., Balch C., Lautenschlaeger T.

Platelets play roles in malignancy, wound healing, and immunity. Nevertheless, their significance in postoperative outcomes is not established. This is a retrospective cohort study of 100,795 patients undergoing cancer surgery in 2010 and 2014 in >500 hospitals. Patients were stratified into five groups based on preoperative platelet counts. Multivariable logistic regression was used to determine the risk of 30-day mortality, morbidities, readmission, and prolonged hospitalization using the mid-normal group as a reference. We adjusted for demographic variables, comorbidities, and operation complexity. In the 2014 cohort, multivariable analysis showed that mortality was higher in patients with thrombocytopenia (OR 1.49, 95% CI [1.23-1.81]), high-normal platelets (OR 1.29, [1.06-1.55]), and thrombocytosis (OR 1.78, [1.45-2.19]). Composite postoperative morbidity followed a similar trend with thrombocytopenia (OR 1.34, [1.25-1.43]), high-normal counts (OR 1.41, [1.33-1.49]), and thrombocytosis (OR 2.20, [2.05-2.36]). Concordantly, the risks of prolonged hospitalization and 30-day readmission followed the same pattern. These results were validated in a large colon cancer cohort from the 2010 database. In conclusion, platelet count is a prognostic indicator in cancer surgeries. This could be related to the role of platelets in wound healing and immunity on one hand, and propagating malignancy on the other.

Hartman R.I., Lin J.Y.

Melanoma is an increasingly common cancer in the United States, although mortality has likely stabilized. Diagnosis relies on a skilled practitioner with the aid of dermoscopy and initial local surgical management is a mainstay of treatment. Recent changes in staging emphasize continued use of sentinel lymph node biopsy to aid in prognostication although routine complete lymph node dissection has fallen out of favor. Advances in systemic treatment options, including targeted and immunotherapy, have dramatically changed the treatment paradigm for advanced melanoma and improved outcome. Prevention via sun protection remains a critical tool in efforts to limit the burden of this disease.

McQuade J.L., Daniel C.R., Helmink B.A., Wargo J.A.

Although novel therapies, including immunotherapy, have dramatically improved outcomes for many patients with cancer, overall outcomes are heterogeneous and existing biomarkers do not reliably predict response. To date, predictors of response to cancer therapy have largely focused on tumour-intrinsic features; however, there is growing evidence that other host factors (eg, host genomics and the microbiome) can substantially affect therapeutic response. The microbiome, which refers to microbiota within a host and their collective genomes, is becoming increasingly recognised for its influence on host immunity, as well as therapeutic responses to cancer treatment. Importantly, microbiota can be modified via several different strategies, affording new angles in cancer treatment to improve outcomes. In this Review, we examine the evidence on the role of the microbiome in cancer and therapeutic response, factors that influence and shape host microbiota, strategies to modulate the microbiome, and present key unanswered questions to be addressed in ongoing and future research.

Margolis N., Markovits E., Markel G.

This decade has introduced drastic changes in melanoma therapy, predominantly due to the materialization of the long promise of immunotherapy. Cytotoxic T cells are the chief component of the immune system, which are targeted by different strategies aimed to increase their capacity against melanoma cells. To this end, reprogramming of T cells occurs by T cell centered manipulation, targeting the immunosuppressive tumor microenvironment or altering the whole patient. These are enabled by delivery of small molecules, functional monoclonal antibodies, different subunit vaccines, as well as living lymphocytes, native or genetically engineered. Current FDA-approved therapies are focused on direct T cell manipulation, such as immune checkpoint inhibitors blocking CTLA-4 and/or PD-1, which paves the way for an effective immunotherapy backbone available for combination with other modalities. Here we review the biology and clinical developments that enable melanoma immunotherapy today and in the future.

Bilger A., Lambert P.F.

Oh D., Hong N., Eun K., Lee J., Cai L., Kim M., Choi H., Jawad A., Ham J., Park M.G., Kim B., Lee S.C., Moon C., Kim H., Hyun S.

AbstractMelanoma is a serious type of skin cancer that originates from melanocytes. Rodent melanoma models have provided valuable insights into melanoma pathology; however, they often lack applicability to humans owing to genetic, anatomical, physiological, and metabolic differences. Herein, we developed a transgenic porcine melanoma model that closely resembles humans via somatic cell nuclear transfer (SCNT). Our model features the conditional oncogenes cassettes, TP53R167H and human BRAFV600E, controlled by melanocyte-specific CreER recombinase. After SCNT, transgenic embryos developed normally, with the capacity to develop porcine embryonic stem cells. Seven transgenic piglets with oncogene cassettes were born through embryo transfer. We demonstrated that Cre recombination-mediated oncogene activation remarkably triggered the mitogen-activated protein kinase pathway in vitro. Notably, intradermal injection of 4-hydroxytamoxifen activated oncogene cassettes in vivo, resulting in melanocytic lesions resembling hyperpigmented nevi with increased proliferative properties similar to early human melanomas. This melanoma-inducing system, heritably transmitted to offspring, supports large-scale studies. The novel porcine model provides a valuable tool for elucidating melanoma development and metastasis mechanism, advancing translational medicine, and facilitating preclinical evaluation of new anticancer drugs.

Bilger A., Ward-Shaw E.T., Lee D.L., King R.E., Newton M.A., Buehler D., Matkowskyj K.A., Sundberg J.P., Rong H., Lambert P.F.

AbstractDevelopment of invasive cancer in mammals is thought to require months or years after initial events such as mutation or viral infection. Rarely, invasive cancers regress spontaneously. We show that cancers can develop and regress on a timescale of weeks, not months or years. Invasive squamous cell carcinomas developed in normal adult, immune-competent mice as soon as 2 weeks after infection with mouse papillomavirus MmuPV1. Tumor development, regression or persistence was tissue- and strain-dependent. Cancers in infected mice developed rapidly at sites also prone to papillomavirus-induced tumors and cancers in humans – the throat, anus, and skin – and their frequency was increased in mice constitutively expressing the papillomavirus E5 oncogene, which MmuPV1 lacks. Cancers and dysplasia in the throat and anus regressed completely within 4-8 weeks of infection; however, skin lesions in the ear persisted. T-cell depletion in the mouse showed that regression of throat and anal tumors requires T cells. We conclude that papillomavirus infection suffices for rapid onset of invasive cancer, and persistence of lesions depends on factors including tissue type and host immunity. The speed of these events should promote rapid progress in the study of viral cancer development, persistence, and regression.Summary Graphic

Veverkova K., Pavelicova K., Vlcnovska M., Vejvodova M., Horák V., Kanicky V., Adam V., Vaculovic T., Vaculovicova M.

Effective detection and sensitive quantification of disease markers enable a better understanding of processes leading to disease development. Therefore, we present an approach that combines the excellent selective recognition capabilities...

Pinto C., Aluai-Cunha C., Santos A.

Currently, the most progressively occurring incident cancer is melanoma. The mouse is the most popular model in human melanoma research given its various benefits as a laboratory animal. Nevertheless, unlike humans, mice do not develop melanoma spontaneously, so they need to be genetically manipulated. In opposition, there are several reports of other animals, ranging from wild to domesticated animals, that spontaneously develop melanoma and that have cancer pathways that are similar to those of humans. The influence of the gut microbiome on health and disease is being the aim of many recent studies. It has been proven that the microbiome is a determinant of the host's immune status and disease prevention. In human medicine, there is increasing evidence that changes in the microbiome influences malignant melanoma progression and response to therapy. There are several similarities between some animals and human melanoma, especially between canine and human oral malignant melanoma as well as between the gut microbiome of both species. However, microbiome studies are scarce in veterinary medicine, especially in the oncology field. Future studies need to address the relevance of gut and tissue microbiome for canine malignant melanoma development, which results will certainly benefit both species in the context of translational medicine.

Fu Z., Ni D., Cai S., Li H., Xiong Y., Yang R., Chen C.

Multimodal nanocatalytic therapy has shown promising potential in cancer treatments. However, to design an effective multimodal nanocatalytic modality, with a deep understanding of synergistic therapy, remains a grand challenge. Herein, we propose an innovative “three-in-one” multimodal strategy with integrated photothermal/photodynamic/chemodynamic modalities for tumor-specific treatment, by rationally constructing a multifunctional two-dimensional (2D) nanoplatform based on the black phosphorous (BP)/Pd nanosheets (NSs) via in-situ growth. Interestingly, such a hybridization of different types of 2D NSs mediates formation of the Pd-P coordination bonds. With strong interfacial Pd-P interaction, the heterostructured NSs display enhanced cascade enzymatic activities in tumor microenvironment, leading to sufficient generation of reactive oxygen species (ROS) as therapeutic species including cytotoxic superoxide (O 2 •− ) radicals by successive catalase-/oxidase-catalysis, and deleterious singlet oxygen ( 1 O 2 ) radicals under light irradiation. The incorporation of Pd also improved the photothermal activity of the material. Furthermore, by facile functionalization with fluoro-polyethyleneimine (FPEI) and loading of cytosine-phosphate-guanine (CpG) as an immune adjuvant, the versatilities of this unique 2D hetero-nanoplatform are further extended, strikingly improving therapeutic efficacy for tumor cells, and simultaneously avoiding significant damage to normal tissues. The work provides useful insights into the design of new 2D nanoplatforms for multimodal tumor treatment. A novel “three-in-one” multimodal strategy is developed for effective tumor-specific treatment, by integrating photothermal/photodynamic/chemodynamic modalities into a versatile two-dimensional nanotherapeutic platform, based on heterostructured black phosphorous/Pd nanosheets with strong Pd-P interaction. It provides new perspectives to design and construct multifunctional nanomedicines for tumor therapy. • Facile preparation of BP/Pd nanosheets with eco-friendliness under mild conditions. • Superior cascade catalysis and improved photodynamic/photothermal effect by Pd-P interaction. • Enhanced stability, biocompatibility and cell uptake by fluoro-polyethyleneimine. • Increased TNF-α release of cells by loading of cytosine-phosphate-guanine. • Excellent antitumor efficacy with good biosafety.

Boettcher A.N., Tuggle C.K.

Ananbeh H., Novak J., Juhas S., Juhasova J., Klempir J., Doleckova K., Rysankova I., Turnovcova K., Hanus J., Hansikova H., Vodicka P., Kupcova Skalnikova H.

(1) Background: Huntington’s disease (HD) is rare incurable hereditary neurodegenerative disorder caused by CAG repeat expansion in the gene coding for the protein huntingtin (HTT). Mutated huntingtin (mHTT) undergoes fragmentation and accumulation, affecting cellular functions and leading to neuronal cell death. Porcine models of HD are used in preclinical testing of currently emerging disease modifying therapies. Such therapies are aimed at reducing mHTT expression, postpone the disease onset, slow down the progression, and point out the need of biomarkers to monitor disease development and therapy efficacy. Recently, extracellular vesicles (EVs), particularly exosomes, gained attention as possible carriers of disease biomarkers. We aimed to characterize HTT and mHTT forms/fragments in blood plasma derived EVs in transgenic (TgHD) and knock-in (KI-HD) porcine models, as well as in HD patients’ plasma. (2) Methods: Small EVs were isolated by ultracentrifugation and HTT forms were visualized by western blotting. (3) Results: The full length 360 kDa HTT co-isolated with EVs from both the pig model and HD patient plasma. In addition, a ~70 kDa mutant HTT fragment was specific for TgHD pigs. Elevated total huntingtin levels in EVs from plasma of HD groups compared to controls were observed in both pig models and HD patients, however only in TgHD were they significant (p = 0.02). (4) Conclusions: Our study represents a valuable initial step towards the characterization of EV content in the search for HD biomarkers.

Vaníčková L., Do T., Vejvodová M., Horák V., Hubálek M., Emri G., Zemánková K., Pavelicová K., Křížková S., Faltusová V., Pompeiano A., Vaculovičová M., Zítka O., Vaculovič T., Adam V.

Here we developed a powerful tool for comprehensive data collection and mapping of molecular and elemental signatures in the Melanoma-bearing Libechov Minipig (MeLiM) model. The combination of different mass spectrometric methods allowed for detail investigation of specific melanoma markers and elements and their spatial distribution in tissue sections. MALDI-MSI combined with HPLC-MS/MS analyses resulted in identification of seven specific proteins, S100A12, CD163, MMP-2, galectin-1, tenascin, resistin and PCNA that were presented in the melanoma signatures. Furthermore, the ICP-MS method allowed for spatial detection of zinc, calcium, copper, and iron elements linked with the allocation of the specific binding proteins.

Mekadim C., Skalnikova H.K., Cizkova J., Cizkova V., Palanova A., Horak V., Mrazek J.

The microbiome alterations are associated with cancer growth and may influence the immune system and response to therapy. Particularly, the gut microbiome has been recently shown to modulate response to melanoma immunotherapy. However, the role of the skin microbiome has not been well explored in the skin tumour microenvironment and the link between the gut microbiome and skin microbiome has not been investigated in melanoma progression. Therefore, the aim of the present study was to examine associations between dysbiosis in the skin and gut microbiome and the melanoma growth using MeLiM porcine model of melanoma progression and spontaneous regression. Parallel analysis of cutaneous microbiota and faecal microbiota of the same individuals was performed in 8 to 12 weeks old MeLiM piglets. The bacterial composition of samples was analysed by high throughput sequencing of the V4-V5 region of the 16S rRNA gene. A significant difference in microbiome diversity and richness between melanoma tissue and healthy skin and between the faecal microbiome of MeLiM piglets and control piglets were observed. Both Principal Coordinate Analysis and Non-metric multidimensional scaling revealed dissimilarities between different bacterial communities. Linear discriminant analysis effect size at the genus level determined different potential biomarkers in multiple bacterial communities. Lactobacillus, Clostridium sensu stricto 1 and Corynebacterium 1 were the most discriminately higher genera in the healthy skin microbiome, while Fusobacterium, Trueperella, Staphylococcus, Streptococcus and Bacteroides were discriminately abundant in melanoma tissue microbiome. Bacteroides, Fusobacterium and Escherichia-Shigella were associated with the faecal microbiota of MeLiM piglets. Potential functional pathways analysis based on the KEGG database indicated significant differences in the predicted profile metabolisms between the healthy skin microbiome and melanoma tissue microbiome. The faecal microbiome of MeLiM piglets was enriched by genes related to membrane transports pathways allowing for the increase of intestinal permeability and alteration of the intestinal mucosal barrier. The associations between melanoma progression and dysbiosis in the skin microbiome as well as dysbiosis in the gut microbiome were identified. Results provide promising information for further studies on the local skin and gut microbiome involvement in melanoma progression and may support the development of new therapeutic approaches.

Su Y., Wu F., Song Q., Wu M., Mohammadniaei M., Zhang T., Liu B., Wu S., Zhang M., Li A., Shen J.

Nanozyme-based catalytic therapy, an emerging therapeutic pattern, has significantly incorporated in the advancement of tumor therapy by generating lethal reactive oxygen species. Nevertheless, most of the nanozymes have mono catalytic performances with H2O2 in the tumor microenvironment (TME), which lowers their therapeutic efficiency. Herein, we design a newly-developed single-atom Fe dispersed N-doped mesoporous carbon nanospheres (SAFe-NMCNs) nanozyme with high H2O2 affinity for photothermal-augmented nanocatalytic therapy. The SAFe-NMCNs nanozyme possesses dual enzyme-mimic catalytic activity which not only acts as a catalase-mimic role to achieve ultrasonic imaging in tumor site by O2 generation, but also exhibits the superior peroxidase-mimic catalytic performance to generate •OH for nanocatalytic therapy. Besides, the SAFe-NMCNs nanozyme with strong optical absorption in the second near-infrared (NIR-II) region shows excellent photothermal conversion performance. The peroxidase-mimic catalytic process of SAFe-NMCNs nanozyme is realized using density functional theory (DFT). Both in vitro and in vivo results indicate that the SAFe-NMCNs nanozyme can efficiently suppress tumor cells growth by a synergistic therapy effect with photothermal-augmented nanocatalytic therapy. The work developed a single-atom-coordinated nanozyme with dual-enzyme catalytic performance and achieve hyperthermia-augmented nanocatalytic therapy effect, can open a window for potential biological applications.

Helke K., Nelson K., Sargeant A.

The choice of animal species in nonclinical research should always be carefully considered and justified. Pigs and minipigs are considered good models for humans because they share many important characteristics with man. Swine exhibit particularly close resemblance to humans with respect to the anatomy of the skin, cardiovascular system, the majority of the gastrointestinal tract, urogenital system, and the brain. In addition, there are similarities between these two species in drug metabolism and in physiological parameters. In drug development, the intended clinical route of dosing should be applied in nonclinical studies, wherever possible. All general routes of product administration [i.e., dermal (topical and intradermal), intramuscular and intravenous, oral, and subcutaneous] are feasible in pigs and minipigs. Furthermore, all generally required durations of dosing can be completed, with up to 12 months of daily dosing, depending on the route of administration. The minipig should be considered a relevant test species and is fully accepted for toxicity studies by regulatory authorities worldwide. This chapter provides an overview of the use of pigs in toxicologic research, describes spontaneous pathology findings and key models of disease, and discusses animal welfare issues relevant to use of the pig in research.

Matias M., Pinho J.O., Penetra M.J., Campos G., Reis C.P., Gaspar M.M.

Melanoma is recognized as the most dangerous type of skin cancer, with high mortality and resistance to currently used treatments. To overcome the limitations of the available therapeutic options, the discovery and development of new, more effective, and safer therapies is required. In this review, the different research steps involved in the process of antimelanoma drug evaluation and selection are explored, including information regarding in silico, in vitro, and in vivo experiments, as well as clinical trial phases. Details are given about the most used cell lines and assays to perform both two- and three-dimensional in vitro screening of drug candidates towards melanoma. For in vivo studies, murine models are, undoubtedly, the most widely used for assessing the therapeutic potential of new compounds and to study the underlying mechanisms of action. Here, the main melanoma murine models are described as well as other animal species. A section is dedicated to ongoing clinical studies, demonstrating the wide interest and successful efforts devoted to melanoma therapy, in particular at advanced stages of the disease, and a final section includes some considerations regarding approval for marketing by regulatory agencies. Overall, considerable commitment is being directed to the continuous development of optimized experimental models, important for the understanding of melanoma biology and for the evaluation and validation of novel therapeutic strategies.

Burroughs D., Diaz S., Lorch G., Yang C., Lakritz J.

A 7-month-old, male castrated mixed-breed pig presented for evaluation of a 3-month history of cutaneous pigmented nodular lesions, multifocal leukoderma and leukotrichia. Skin biopsies were performed from pigmented nodules and depigmented skin for histopathology. Additional skin biopsies were collected aseptically for bacterial and fungal tissue cultures. Histopathologic diagnosis was a benign melanocytic neoplasm (melanocytoma) and marked vitiligo. Spontaneous regression of melanocytomas did not occur after 1 year of age as previously reported to occur in several swine breeds. Topical imidazoquinoline amine cream was applied to one melanocytoma three times a week for a month. No clinical improvement in either the melanocytoma or vitiligo occurred. In humans, vitiligo is associated with both neoplastic and benign melanocytic neoplasms; this case illustrates the presence of melanoma-associated leukoderma in a domestic mixed-breed pig and emphasises the importance of meticulous skin examination in swine patients with the development of vitiligo.

Du F., Liu L., Wu Z., Zhao Z., Geng W., Zhu B., Ma T., Xiang X., Ma L., Cheng C., Qiu L.

The diversity, complexity, and heterogeneity of malignant tumor seriously undermine the efficiency of mono-modal treatment. Recently, multi-modal therapeutics with enhanced antitumor efficiencies have attracted increasing attention. However, designing a nanotherapeutic platform with uniform morphology in nanoscale that integrates with efficient chem-/sono-/photo-trimodal tumor therapies is still a great challenge. Here, new and facile Pd-single-atom coordinated porphyrin-based polymeric networks as biocatalysts, namely, Pd-Pta/Por, for chem-/sono-/photo-trimodal tumor therapies are designed. The atomic morphology and chemical structure analysis prove that the biocatalyst consists of atomic Pd-N coordination networks with a Pd-N2 -Cl2 catalytic center. The characterization of peroxidase-like catalytic activities displays that the Pd-Pta/Por can generate abundant •OH radicals for chemodynamic therapies. The ultrasound irradiation or laser excitation can significantly boost the catalytic production of 1 O2 by the porphyrin-based sono-/photosensitizers to achieve combined sono-/photodynamic therapies. The superior catalytic production of •OH is further verified by density functional theory calculation. Finally, the corresponding in vitro and in vivo experiments have demonstrated their synergistic chem-/sono-/photo-trimodal antitumor efficacies. It is believed that this study provides new promising single-atom-coordinated polymeric networks with highly efficient biocatalytic sites and synergistic trimodal therapeutic effects, which may inspire many new findings in reactive oxygen species-related biological applications across broad therapeutics and biomedical fields.