Literatura académica sobre el tema "Cellule di melanoma"

Melanoma is a kind of skin cancer caused by the irregular development of pigment-producing cells. Since melanoma detection efficiency is limited to different factors such as poor contrast among lesions and nearby skin regions, and visual resemblance among melanoma and non-melanoma lesions, intelligent computer-aided diagnosis (CAD) models are essential. Recently, computational intelligence (CI) and deep learning (DL) techniques are utilized for effective decision-making in the biomedical field. In addition, the fast-growing advancements in computer-aided surgeries and recent progress in molecular, cellular, and tissue engineering research have made CI an inevitable part of biomedical applications. In this view, the research work here develops a novel computational intelligence-based melanoma detection and classification technique using dermoscopic images (CIMDC-DIs). The proposed CIMDC-DI model encompasses different subprocesses. Primarily, bilateral filtering with fuzzy k-means (FKM) clustering-based image segmentation is applied as a preprocessing step. Besides, NasNet-based feature extractor with stochastic gradient descent is applied for feature extraction. Finally, the manta ray foraging optimization (MRFO) algorithm with a cascaded neural network (CNN) is exploited for the classification process. To ensure the potential efficiency of the CIMDC-DI technique, we conducted a wide-ranging simulation analysis, and the results reported its effectiveness over the existing recent algorithms with the maximum accuracy of 97.50%.

La neoplasia secondaria nella ghiandola parotide è un reperto sempre più frequente nella chirurgia parotidea. Vengono qui presentati i nostri risultati in quaranta anni di chirurgia parotidea, analizzando le modalità di metastasi in pazienti con lesioni metastatiche della ghiandola parotide, le procedure operatorie e la gestione dei pazienti. Sono stati esaminati retrospettivamente 772 casi consecutivi di chirurgia parotide in un ospedale universitario tra il 1975 e il 2015 e valutate le variazioni di incidenza e di gestione della patologia nel corso di quattro decenni (I: 1975-1985; II: 1986-1995; III: 1996-2005; IV: 2006-2015). Sono stati diagnosticati complessivamente 683 tumori della parotide, di cui il 15,8% (n = 108) sono rivelati essere di natura maligna; a loro volta, il 44% (n = 48) di tutte le lesioni maligne si sono rivelate essere metastasi. Si è inoltre potuto constatare come, con l’andare del tempo, i tumori maligni della ghiandola parotide abbiano incrementato la loro incidenza con un aumento dall’8% nel primo decennio, del 14% nel secondo, del 17% nel terzo fino al 21% nel quarto decennio. L’incidenza di metastasi alla ghiandola parotide è altresì ulteriormente aumentata dal 10% nella prima decade fino al 57% nell’ultimo decennio. Il 71% per cento dei pazienti era di sesso maschile e il 29% era di sesso femminile, con un’età compresa tra i 23 ei 93 anni (media di 68 anni). La diagnosi istopatologia più frequente era quella di metastasi di carcinoma a cellule squamose (79%). La grande maggioranza delle lesioni primarie era localizzata in lesioni sopra la clavicola (87%), delle quali 30 tumori primari erano localizzati nel cuoio capelluto e nella cute del collo. Nella maggior parte di questi casi, il tumore primario è stato rimosso tra 6 e 24 mesi prima della metastasi parotidea e i pazienti sono stato seguiti in modo subottimale. La gestione consisteva in intervento chirurgico di dissezione del collo. 48 pazienti (67%) sono stati sottoposti a terapia adiuvante, ma nonostante il trattamento multimodale aggressivo la malattia è progredita nella maggior parte dei casi, nel 57% dei casi di metastasi da carcinoma a cellule squamose cutaneo, nel 67% da metastasi di tumore primario della mucosa sopra la clavicola e l’83% dei casi di metastasi da primitivo infraclaveare. I tumori maligni parotidei registrano un progressivo aumento di incidenza, in gran parte dovuto ad un incremento delle lesioni metastatiche parotidee. I più frequenti tumori primitivi sono melanomi maligni precedentemente asportati, e i carcinomi a cellule squamose del cuoio capelluto e del collo precedentemente operati. Nonostante la terapia multimodale il tasso di recidiva e di progressione rimane alto. È auspicabile per i tumori della testa e del collo un programma di follow-up, come già in atto per i tumori della mucosa della testa e del collo.

ABSTRACT Mutations in CDK4 and its key kinase inhibitor p16INK4a have been implicated in the genesis and progression of familial human melanoma. The importance of the CDK4 locus in human cancer first became evident following the identification of a germ line CDK4-Arg24Cys (R24C) mutation, which abolishes the ability of CDK4 to bind to p16INK4a. To determine the role of the Cdk4 R24C germ line mutation in the genesis of other cancer types, we introduced the R24C mutation in the Cdk4 locus of mice by using Cre-loxP-mediated “knock-in” technology. Cdk4 R24C/R24C mouse embryo fibroblasts (MEFs) displayed increased Cdk4 kinase activity resulting in hyperphosphorylation of all three members of the Rb family, pRb, p107, and p130. MEFs derived from Cdk4 R24C/R24C mice displayed decreased doubling times, escape from replicative senescence, and escape sensitivity to contact-induced growth arrest. These MEFs also exhibited a high degree of susceptibility to oncogene-induced transformation, suggesting that the Cdk4 R24C mutation can serve as a primary event in the progression towards a fully transformed phenotype. In agreement with the in vitro data, homozygous Cdk4 R24C/R24C mice developed tumors of various etiology within 8 to 10 months of their life span. The majority of these tumors were found in the pancreas, pituitary, brain, mammary tissue, and skin. In addition, Cdk4 R24C/R24C mice showed extraordinary susceptibility to carcinogens and developed papillomas within the first 8 to 10 weeks following cutaneous application of the carcinogens 9,10-di-methyl-1,2-benz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). This report formally establishes that the activation of Cdk4 is sufficient to promote cancer in many tissues. The observation that a wide variety of tumors develop in mice harboring the Cdk4 R24C mutation offers a genetic proof that Cdk4 activation may constitute a central event in the genesis of many types of cancers in addition to melanoma.

Abstract The introduction of BRAF inhibitors (BRAFi) has improved response rate and overall survival of metastatic melanoma patients compared to standard chemotherapy. However, acquired drug resistance occurs in nearly all patients. The comprehension of cellular and molecular mechanisms underlying BRAFi resistance could help to identify novel actionable pathways in the treatment of BRAF-dependent tumors. VEGFA is an attractive target for combinatorial cancer therapy, and we have recently demonstrated in melanoma and CRC xenografts that targeting VEGFA enhanced the antitumor effect of BRAFi by normalizing the tumor vasculature, recruiting M1 macrophages, and inducing a remodeling of the extracellular matrix characterized by a reduction in collagen I and in cancer-associated fibroblasts (Comunanza et al., EMBO Mol Med 2017). Based on our previous proof of concept, obtained within an immunodeficient model, here we investigated the therapeutic effect of VEGFA targeting in association with PLX4720 (BRAFi) in a dedicated immune-competent model. D4M cells, a BRAFV600E-mutant melanoma murine cell line, were subcutaneously injected in syngeneic C57BL/6J mice. We demonstrated that the association of BRAFi with DC101 (antibody anti-VEGFR2) had a weak activity while we observed a synergistic antitumor effect when combined with B20 (murine anti-VEGFA neutralizing antibody). Although targeted inhibition of either BRAF or VEGFA delayed the tumor growth, only combined inhibition of both pathways resulted in the regression of initial tumor size, with an evident apoptotic effect, and delayed the onset of acquired resistance to the BRAF inhibition. Since the immune-suppressive role of VEGFA in tumors has been well characterized, we further investigated whether contrasting the VEGF effect along with simultaneous BRAF inhibition can turn into a promotion of both innate and adaptive immunity. Immune phenotype analysis revealed that the combinatorial regimen activated the host immune system, inducing the tumor infiltration of macrophages with tumor-suppressive features, NKs, CD4+ and CD8+ lymphocytes. Most of the infiltrating CD8+ lymphocytes in D4M tumors expressed high levels of PD-1 and none of the treatments significantly modulated PD-1 expression on T cells, suggesting a sustained T-cell exhaustion. We then postulated that the therapeutic effect obtained by the combinatorial VEGF blockade and BRAFi could be further enhanced by the association with an immune-checkpoint inhibitor targeting PD-1. Interestingly, we observed that the addition of anti-PD-1 blocking antibody boosted the antitumor effect and induced striking tumor volume regression in mice receiving the triplet therapy (BRAFi+anti-VEGFA+anti-PD1-1). Our findings provide biologic rationale to explore the association of immunotherapy in novel combinatorial approaches that could improve the clinical outcome exerted by oncogene-targeted therapy, and further investigation is warranted. Citation Format: Valentina Comunanza, Chiara Gigliotti, Gabriella Doronzo, Valentina Martin, Anna Gattuso, Dario Sangiolo, Federica Di Nicolantonio, Federico Bussolino. VEGF removal delays the onset of acquired resistance to target therapy and increases the efficacy of immune checkpoint inhibitors in BRAF-mutated melanoma [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 B17.

Two melanotic human melanoma cell lines, IRE 1 and IRE 2, and the lymphoma- and leukaemia-derived cell lines Raji and K 562, were exposed to different concentrations (from 5 × 10(-3) M to 10(-5) M) of phenols, both substrates (s) and non-substrates (ns) of tyrosinase, in the presence or absence of the oxygen-radical-scavenger enzymes superoxide dismutase, catalase and peroxidase. Monophenols were tyrosine (s), 4-hydroxyanisole (s) and butylated hydroxyanisole (ns); diphenols were L-3,4-dihydroxyphenylalanine (s), dopamine (3,4-dihydroxyphenethylamine) (s), terbutylcatechol (s), hydroquinone (s) and resorcinol (ns); triphenols were 6-hydroxydopa (3,4,6-trihydroxyphenylalanine) (s) and methyl gallate (s). Triphenols and o- and p-diphenols underwent complete oxidation in culture medium within 24 h of incubation and were significantly more toxic than monophenols and the m-diphenol resorcinol, which, under the same cultural conditions, were much more stable. No significant differences in percentage survival were found among the different cell lines for each drug tested. The major component of toxicity up to 24 h of di- and tri-phenols is due to toxic oxygen species acting outside the cells and not to cellular uptake of these phenols as such. In fact the addition of oxygen-radical-scavenger enzymes significantly (P less than 0.01) decreased the adverse effect of these drugs on all cell lines. The lower toxicity of monophenols and resorcinol as compared with that of di- and tri-phenols is due, in our opinion, to the fact that they are less oxidized under the conditions existing in the culture medium, and therefore do not produce sufficient levels of oxygen radicals. For these compounds, a primary intracellular action has to be taken into account to explain their cytotoxicity.

Abstract Abstract 1562 Our previous findings have made it clear that the significant clinical efficacy attained by dendritic cell-based vaccination in relapsed B-cell non-Hodgkin lymphoma (B-NHL) patients is firmly associated with multifaceted immunologic responses, including the development of anti-heat shock protein (HSP)105 humoral immunity (Di Nicola et al., Blood 2009 113:18–27; Zappasodi et al., Cancer Res. 2010 70:9062–9072; Zappasodi et al., Blood 2011 118:4421–4430). Human HSP105 is a high-molecular-weight chaperone constitutively expressed at low levels within the cytoplasm, and can also be induced in the nucleus by various forms of stress. It is overexpressed in several solid tumors, including melanoma, breast, thyroid and gastroenteric cancers. We have recently shown that this is also true for B-NHLs, in which HSP105 levels increase in function of their aggressiveness and proliferation index (Zappasodi et al., Blood 2011 118:4421–4430). Accordingly, in normal lymph nodes HSP105 expression is confined to the hyper-proliferating germinal center (GC) B cells, suggesting its involvement in the potentially oncogenic GC reactions. We have now set out to clarify the functional role of HSP105 in B-NHLs by stably silencing its expression in the Namalwa aggressive lymphoma cell line. Namalwa cells were infected by using a lentiviral vector carrying a HSP105-targeting pre-microRNA sequence and the Emerald Green Fluorescent Protein (EmGFP) gene, both under the human cytomegolovirus immediate early promoter, as well as the blasticidin resistance gene. Control cells were mock-infected with the empty vector. Infected cells were initially selected in the presence of blasticidin, and then single GFP+ cells were sorted on a flow cytometry device. In this way, we achieved 100% GFP+ subclones that displayed a specific constitutive down-regulation of HSP105, as there was no significant decrease in the expression of its cognate molecular homolog HSP70, or the other major cellular chaperone HSP90. Comparison of the in vitro proliferation rate of two silenced clones with that of the mock culture showed that the cell doubling time of both clones significantly increased and their in vitro growth was accordingly delayed (P= 0.01 and P= 0.04). Western blot analysis in 6 different silenced clones of the oncoproteins most frequently involved in B-NHLs revealed that BCL-6 and c-Myc were down-regulated in function of HSP105 knockdown levels, whereas in mock cells no modifications were detected with respect to their wild-type counterparts. Further strengthening the association between HSP105, BCL-6 and c-Myc expression, immunohistochemistry analysis of 50 primary human aggressive B-NHLs revealed that HSP105 expression, measured both as intensity and percentage of positive cells, was significantly higher in c-Myc- or BCL-6-dependent Burkitt (P= 0.0264) and diffuse large B-cell lymphomas (P= 0.0068) respectively than in other aggressive istotypes that do not overexpress these oncoproteins. These findings support the potential pro-tumorigenic cooperation of HSP105 with BCL-6 and c-Myc transcription factors. To find out whether counteracting HSP105 functions hampers in vivo lymphoma growth, we evaluated the tumor-forming capability of HSP105-silenced (siHSP105) or mock Namalwa cells subcutaneously injected into severe combined immunodeficient mice at serial 10-fold dilutions from 106 to 104 cells/injection (Figure 1). We found that HSP105 knockdown slightly delayed in vivo Namalwa tumor formation when 106 and 105 cells were injected. Noteworthy, no lesions appeared over 70-day observation in mice inoculated with 104 siHSP105 cells, whereas palpable tumors were present in 67% of the animals 24 days after injection of the mock cells (Figure 1). Overall, these results indicate that HSP105 may be a per se nononcogenic molecule that contributes to lymphomagenesis by facilitating the tumorigenic functions of key oncoproteins. They equally provide the rationale for developing HSP105 inhibitors as a novel strategy for improving the treatment of aggressive B-NHLs. Figure 1. In vivo tumor-forming capability of siHSP105 or mock Namalwa cells Figure 1. In vivo tumor-forming capability of siHSP105 or mock Namalwa cells Disclosures: Gianni: Hoffmann-La Roche: Consultancy, Honoraria.

Arsenic and Protein Expression: It might help to know the mechanism of As toxicity is described Introduction One of the largest public health problems at present is the drinking of water containing levels of Inorg-As that are known to be carcinogenic. The chronic ingestion of Inorg-As can results in skin cancer, urinary bladder cancer, lungs cancer, kidneys cancer, liver cancer, and cancer of other human organs 1-6. The molecular mechanisms of the carcinogenicity and toxicity of inorganic arsenic are not well understood 7–9. Many mechanisms of arsenic toxicity and carcinogenicity have been suggested 1, 7, 10 including chromosome abnormalities 11, oxidative stress 12, 13, altered growth factors 14, cell proliferation 15, altered DNA repair 16, altered DNA methylation patterns 17, inhibition of several key enzymes 18, gene amplification 19 etc. Some of these mechanisms result in alterations in protein expression. Proteomics is a powerful tool developed to enhance the study of complex biological system 20. This technique has been extensively employed to investigate the proteome response of cells to drugs and other diseases 21, 22. A proteome analysis of the Na-As (III) response in cultured lung cells found in vitro oxidative stress-induced apoptosis 23. In one of the study, hamsters were exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days and several protein spots were over expressed and several were under expressed in the livers and urinary bladders of hamsters (Fig.) 24, 25. Hamsters were exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days. The control hamsters were given tap water. The spot pairs of (A) equally expressed, (B) overexpressed, and (C) under expressed proteins in the liver tissues were shown. The amount of the protein is proportional to the volume of the protein peak. Transgelin was down-regulated, and GST-pi was up-regulated in the urinary bladder tissues of hamsters. In the liver tissues ornithine aminotransferase (OAT) was up-regulated, and senescence marker protein 30 (SMP 30), and fatty acid binding protein (FABP) were down-regulated. Down-regulation of transgelin has been noted in the urinary bladders of rats having bladder outlet obstruction 26. Ras-dependent and Ras-independent mechanisms can cause the down regulation of transgelin in human breast and colon carcinoma cell lines and patient-derived tumor samples 27. The loss of transgelin expression has been found in prostate cancer cells 28 and in human colonic neoplasms 29. It has been suggested that the loss of transgelin expression may be an important early event in tumor progression and a diagnostic marker for cancer development 26-29. Figure. Three-dimentional simulation of over-and under expressed protein spots in the livers of hamsters using Decyder software. Over-expression of GST-pi has been found in colon cancer tissues 30. Strong expression of GST-pi also has been found in gastric cancer 31, malignant melanoma 32, lung cancer 33, breast cancer 34 and a range of other human tumors 35. GST-pi has been up-regulated in transitional cell carcinoma of human urinary bladder 36. OAT has a role in regulating mitotic cell division and it is required for proper spindle assembly in human cancer cell 37. Ornithine amino transferase knockdown in human cervical carcinoma and osteosarcoma cells by RNA interference blocks cell division and causes cell death 37. It has been suggested that ornithine amino transferase has a role in regulating mitotic cell division and it is required for proper spindle assembly in human cancer cells 37. SMP 30 expressed mostly in the liver. By stimulating membrane calcium-pump activity it protects cells against various injuries 38. High levels of saturated, branched chain fatty acids are deleterious to cells and resulting in lipid accumulation and cytotoxicity. FABP expression has protected the cells against branched chain saturated fatty acid 39. Proteomics would be a powerful tool to know the unknown cellular mechanisms of arsenic toxicity in humans. References. NRC (National Research Council). (2001). Arsenic in Drinking Water. Update to the 1999 Arsenic in Drinking Water Report. National Academy Press, Washington, DC. Chen, C. J., Chen, C. , Wu, M. M., Kuo, T. L. (1992). Cancer potential in liver, lung, bladder, and kidney due to ingested inorganic arsenic in drinking water. Br. J. Cancer 66, 888-892. Hopenhayn-Rich, C., M.L. Biggs, A. Fuchs, et al. 1996. Bladder cancer mortality with arsenic in drinking water in Argentina. Epidemiology 7: 117–124. International Agency for Research on Cancer. (1987). In IARC Monograph on the Evaluation of Carcinogenicity Risk to Humans. 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BackgroundBecause conventional single-cell strategies rely on dissociating tissues into suspensions that lose spatial context,1 we developed Slide-TCR-seq to sequence both whole transcriptomes and TCRs with 10µm-spatial resolution, & applied it to renal cell carcinoma (ccRCC) treated with immune checkpoint inhibitors (ICI).MethodsSlide-TCR-seq combines Slide-seqV22 3—a 10µm-resolution spatial approach utilizing mRNA capture and DNA-barcoded beads—with sensitive targeted capture of TCR sequences (rhTCRseq,4 previously developed by our group), thereby enabling amplification of segments extending from upstream of CDR3 to the 3’-end of the TCR transcript (figure 1A). We tested Slide-TCR-seq first on OT-I murine spleen and then applied this methodology to 3 patients‘ pre-αPD-1 ccRCC samples5 and a post-αPD-1 metastasis to investigate the spatial, functional, and clonotypic organization of T cells in relationship to tumor using RCTD,6 spatial enrichment, and spatial expression analyses.ResultsUsing Slide-TCR-seq, we first recapitulated native spatial structure of OT-I mouse spleen (figure 1B-G). TCRα/β CDR3 sequences were detected on 37.1% of beads with Trac/Trbc2 constant sequences—comparable to other scTCRseq methods. Because the clonal and spatial context of TILs have been increasingly implicated in immunotherapy resistance, we used Slide-TCR-seq to analyze a lung ccRCC metastasis following αPD-1 therapy. We employed unsupervised clustering to delineate the tumor, intervening boundary, and lung compartments, and RCTD analyses to spatially map individual cell types; together recapitulating the architecture observed in corresponding histology (figure 2). We identified 1,132 unique clonotypes, with distinct spatial distributions spanning the tissue compartments. Eight clonotypes were significantly enriched in tumor, whereas 5 were depleted (all p<0.05) (figure 3). We then analyzed the relationships between the T cells’ clonotype, gene expression, and tumor infiltration depth among clonotypes. Using a T-cell geneset associated with poor response to ICI,7 we dichotomized T-clonotype beads by geneset expression, and found spatial segregation of this geneset’s expression both within and across clonotypes (figure 4). TCR-4—the most significantly tumor-enriched clonotype—and TCR-2 displayed high expression of the poor ICI response geneset near the tumor’s edge, but low expression deeper in the tumor compartment; indicating that there are transcriptionally distinct subpopulations of these clonotypes, which depended on the extent of their tumor infiltration.Abstract 76 Figure 1Slide-TCR-seq spatially localizes T cell receptors and transcriptome information. a. Schematic of Slide-TCR-seq, in which tissue is placed onto an in situ barcoded bead array. cDNA libraries prepared with Slide-seqV2 are split prior to fragmentation with one portion used for targeted amplification via rhTCRseq optimized for use with Slide-seq libraries. Slide-TCR-seq provides gene expression, cell type, and clonotype information in space. b. Serial sections of the OT-1 mouse spleen with hematoxylin and eosin stain show characteristic architecture of red pulp and white pulp separation. c. Spatial reconstruction of Slide-TCR-seq array for a corresponding section of OT-I mouse spleen, with RCTD immune cell type assignment. NK = natural killer. d. Gene expression gaussian-filtered heatmap for visualizing the spatial distribution of gene markers for marginal zone (Marco), red blood cells (RBCs; Gypa), and CD8 T cells (Cd8a). e and f. Comparing the spatial distribution of constant (left) and variable (right) sequences for TCRα (e) and TCRβ (f), with superimposed density plot. g. The fraction of beads that capture CDR3 variable sequences (y-axis) when constant UMIs are captured (x-axis) for TCRα (left, light blue) and TCRβ (right, dark blue), with the number of corresponding beads along the top axis. All scale bars: 500 µm.Abstract 76 Figure 2Slide-TCR-seq identifies spatial differences between T cell clonotypes in renal cell carcinoma. (a) H&E stain of a ccRCC metastasis to the lung following PD-1 blockade therapy. (b) The compartment assignment of lung (green), immune cell boundary (orange), and tumor (blue) by applying K-nearest neighbors to cell types determined by unsupervised clustering from Slide-TCR-seq of a sequential tissue section. (c) Spatial reconstruction of cell type identifies using RCTD anaysis of the Slide-TCR-seq data. (d) Spatial localization of T cell clonotypes (n=447 clonotypes, colored by clonotype) from the the Slide-TCR-seq data.Abstract 76 Figure 3Top: y-axis Significance of clonotype spatial distributions compared against all other clonotypes with at least ten beads per array from the ccRCC lung metastasis plotted against an x-axis of magnitude of tumor enrichment or depletion (data from n=3 replicate arrays, two one-tailed K-S tests). Bottom: Visualization of selected significant clonotypes, ordered by tumor enrichment, in tissue compartments for a single array (T cells within the tumor compartment are displayed as opaque, T cells within other compartments are displayed as translucent).Abstract 76 Figure 4Spatial and molecular heterogeneity in clonotype gene expression and tumor infiltration. a. The three axes — spatial localization, gene expression, and T cell clonotype — that Slide-TCR-seq can relate. b. Top: distribution of poor response to immune checkpoint inhibitor treatment (’PRI’) geneset7 expression across all clonotypes in the tumor region of the same post-PD1 inhibitor RCC lung metastasis from figures 2–3 (from a single replicate) with kernel density estimation. Yellow = clonotypes with lower than median PRI expression; purple = clonotypes with PRI expression greater than or equal to the median value. Bottom: localization of low (yellow) and high (purple) PRI geneset expression clonotypes within the tumor region (light blue) from the Slide-TCR-seq array shows their distinct spatial separation (light blue = tumor region, orange = boundary region, green = lung region). Scale bar: 500 µm. c. Smoothed histograms comparing the distance infiltrated into tumor by two-tailed K-S test comparing low (yellow) and high (purple) expression clonotypes, as dichotomized by median expression of PRI. d. Comparing distance infiltrated into tumor by two-tailed K-S test between low and high PRI expression T cells across those clonotypes with at least 20 beads (n=7 clonotypes).ConclusionsSlide-TCR-seq effectively integrates spatial transcriptomics with TCR detection at 10µm resolution, thereby relating T cells’ clonality and gene expression to their spatial organization in tumors. Our findings suggest that a clonotype’s T cells may exhibit mixed responses to ICI depending on their spatial localization. The heterogeneity among clonotypes, in both gene expression and organization, underscores the importance of studying the TCR repertoire with spatial resolution.AcknowledgementsWe are grateful to Irving A. Barrera-Lopez, Zoe N. Garcia, and Aziz Al’Khafaji for technical assistance.ReferencesGohil S, Iorgulescu JB, Braun D, Keskin D, Livak K. Applying high-dimensional single-cell technologies to the analysis of cancer immunotherapy. Nat Rev Clin Oncol 2021; 18:244–256.Stickels RR, Murray E, Kumar P, Li J, Marshall JL, Di Bella DJ, Arlotta P, Macosko EZ, Chen F. Highly sensitive spatial transcriptomics at near-cellular resolution with Slide-seqV2. Nat Biotechnol 2021 Mar;39(3):313–319.Rodriques SG, Stickels RR, Goeva A, Martin CA, Murray E, Vanderburg CR, Welch J, Chen LM, Chen F, Macosko EZ. Slide-seq: A scalable technology for measuring genome-wide expression at high spatial resolution. Science 2019 Mar 29;363(6434):1463–1467.Li S, Sun J, Allesøe R, Datta K, Bao Y, Oliveira G, Forman J, Jin R, Olsen LR, Keskin DB, Shukla SA, Wu CJ, Livak KJ. RNase H-dependent PCR-enabled T-cell receptor sequencing for highly specific and efficient targeted sequencing of T-cell receptor mRNA for single-cell and repertoire analysis. Nat Protoc 2019 Aug;14(8):2571–2594.Braun DA, Street K, Burke KP, Cookmeyer DL, Denize T, Pedersen CB, Gohil SH, Schindler N, Pomerance L, Hirsch L, Bakouny Z, Hou Y, Forman J, Huang T, Li S, Cui A, Keskin DB, Steinharter J, Bouchard G, Sun M, Pimenta EM, Xu W, Mahoney KM, McGregor BA, Hirsch MS, Chang SL, Livak KJ, McDermott DF, Shukla SA, Olsen LR, Signoretti S, Sharpe AH, Irizarry RA, Choueiri TK, Wu CJ. Progressive immune dysfunction with advancing disease stage in renal cell carcinoma. Cancer Cell 2021 May 10;39(5):632–648.Cable DM, Murray E, Zou LS, Goeva A, Macosko EZ, Chen F, Irizarry RA. Robust decomposition of cell type mixtures in spatial transcriptomics. Nat Biotechnol 2021 Feb 18. doi: 10.1038/s41587-021-00830-w. Epub ahead of print. PMID: 33603203.Sade-Feldman M, Yizhak K, Bjorgaard SL, Ray JP, de Boer CG, Jenkins RW, Lieb DJ, Chen JH, Frederick DT, Barzily-Rokni M, Freeman SS, Reuben A, Hoover PJ, Villani AC, Ivanova E, Portell A, Lizotte PH, Aref AR, Eliane JP, Hammond MR, Vitzthum H, Blackmon SM, Li B, Gopalakrishnan V, Reddy SM, Cooper ZA, Paweletz CP, Barbie DA, Stemmer-Rachamimov A, Flaherty KT, Wargo JA, Boland GM, Sullivan RJ, Getz G, Hacohen N. Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma. Cell 2018 Nov 1;175(4):998–1013Ethics ApprovalThis study was approved by MGB/DFCI/Broad institution’s Ethics Board; approval number 2019P000017.

Hamsters were exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days. Equal amounts of proteins from urinary bladder or liver extracts of control and arsenic-treated hamsters were labeled with Cy3 and Cy5 dyes, respectively. After differential in gel electrophoresis and analysis by the DeCyder software, several protein spots were found to be down-regulated and several were up regulated. Our experiments indicated that in the bladder tissues of hamsters exposed to arsenite, transgelin was down-regulated and GST-pi was up-regulated. The loss of transgelin expression has been reported to be an important early event in tumor progression and a diagnostic marker for cancer development [29-32]. Down-regulation of transgelin expression may be associated with the carcinogenicity of inorganic arsenic in the urinary bladder. In the liver of arsenite-treated hamsters, ornithine aminotransferase was up-regulated, and senescence marker protein 30 and fatty acid binding protein were down-regulated. The volume ratio changes of these proteins in the bladder and liver of hamsters exposed to arsenite were significantly different than that of control hamsters. Introduction Chronic exposure to inorganic arsenic can cause cancer of the skin, lungs, urinary bladder, kidneys, and liver [1-6]. The molecular mechanisms of the carcinogenicity and toxicity of inorganic arsenic are not well understood [7-9). Humans chronically exposed to inorganic arsenic excrete MMA(V), DMA(V) and the more toxic +3 oxidation state arsenic biotransformants MMA(III) and DMA (III) in their urine [10, 11], which are carcinogen [12]· After injection of mice with sodium arsenate, the highest concentrations of the very toxic MMA(III) and DMA(III) were in the kidneys and urinary bladder tissue, respectively, as shown by experiments of Chowdhury et al [13]. Many mechanisms of arsenic toxicity and carcinogenicity have been suggested [1, 7, 14] including chromosome abnormalities [15], oxidative stress [16, 17], altered growth factors [18], cell proliferation [19], altered DNA repair [20], altered DNA methylation patterns [21], inhibition of several key enzymes [22], gene amplification [23] etc. Some of these mechanisms result in alterations in protein expression. Methods for analyzing multiple proteins have advanced greatly in the last several years. In particularly, mass spectrometry (MS) and tandem MS (MS/MS) are used to analyze peptides following protein isolation using two-dimensional (2-D) gel electrophoresis and proteolytic digestion [24]. In the present study, Differential In Gel Electrophoresis (DIGE) coupled with Mass Spectrometry (MS) has been used to study some of the proteomic changes in the urinary bladder and liver of hamsters exposed to sodium arsenite in their drinking water. Our results indicated that transgelin was down-regulated and GST-pi was up-regulated in the bladder tissues. In the liver tissues ornithine aminotransferase was up-regulated, and senescence marker protein 30, and fatty acid binding protein were down-regulated. Materials and Methods Chemicals Tris, Urea, IPG strips, IPG buffer, CHAPS, Dry Strip Cover Fluid, Bind Silane, lodoacetamide, Cy3 and Cy5 were from GE Healthcare (formally known as Amersham Biosciences, Uppsala, Sweden). Thiourea, glycerol, SDS, DTT, and APS were from Sigma-Aldrich (St. Louis, MO, USA). Glycine was from USB (Cleveland, OH, USA). Acrylamide Bis 40% was from Bio-Rad (Hercules, CA, USA). All other chemicals and biochemicals used were of analytical grade. All solutions were made with Milli-Q water. Animals Male hamsters (Golden Syrian), 4 weeks of age, were purchased from Harlan Sprague Dawley, USA. Upon arrival, hamsters were acclimated in the University of Arizona animal care facility for at least 1 week and maintained in an environmentally controlled animal facility operating on a 12-h dark/12-h light cycle and at 22-24°C. They were provided with Teklad (Indianapolis, IN) 4% Mouse/Rat Diet # 7001 and water, ad libitum, throughout the acclimation and experimentation periods. Sample preparation and labelling Hamsters were exposed to sodium arsenite (173 mg) in drinking water for 6 days and the control hamsters were given tap water. On the 6th day hamsters were decapitated rapidly by guillotine. Urinary bladder tissues and liver were removed, blotted on tissue papers (Kimtech Science, Precision Wipes), and weighed. Hamster urinary bladder or liver tissues were homogenized in lysis buffer (30mMTris, 2M thiourea, 7M urea, and 4% w/w CHAPS adjusted to pH 8.5 with dilute HCI), at 4°C using a glass homogenizer and a Teflon coated steel pestle; transferred to a 5 ml acid-washed polypropylene tube, placed on ice and sonicated 3 times for 15 seconds. The sonicate was centrifuged at 12,000 rpm for 10 minutes at 4°C. Small aliquots of the supernatants were stored at -80°C until use (generally within one week). Protein concentration was determined by the method of Bradford [25] using bovine serum albumin as a standard. Fifty micrograms of lysate protein was labeled with 400 pmol of Cy3 Dye (for control homogenate sample) and Cy5 Dye (for arsenic-treated urinary bladder or liver homogenate sample). The samples containing proteins and dyes were incubated for 30 min on ice in the dark. To stop the labeling reaction, 1uL of 10 mM lysine was added followed by incubation for 10 min on ice in the dark. To each of the appropriate dye-labeled protein samples, an additional 200 ug of urinary bladderor liver unlabeled protein from control hamster sample or arsenic-treated hamster sample was added to the appropriate sample. Differentially labeled samples were combined into a single Microfuge tube (total protein 500 ug); protein was mixed with an equal volume of 2x sample buffer [2M thiourea, 7M urea, pH 3-10 pharmalyte for isoelectric focusing 2% (v/v), DTT 2% (w/v), CHAPS 4% (w/v)]; and was incubated on ice in the dark for 10 min. The combined samples containing 500 ug of total protein were mixed with rehydration buffer [CHAPS 4% (w/v), 8M urea, 13mM DTT, IPG buffer (3-10) 1% (v/v) and trace amount of bromophenol blue]. The 450 ul sample containing rehydration buffer was slowly pipetted into the slot of the ImmobilinedryStripReswelling Tray and any large bubbles were removed. The IPG strip (linear pH 3-10, 24 cm) was placed (gel side down) into the slot, covered with drystrip cover fluid (Fig. 1), and the lid of the Reswelling Tray was closed. The ImmobillineDryStrip was allowed to rehydrate at room temperature for 24 hours. First dimension Isoelectric focusing (IEF) The labeled sample was loaded using the cup loading method on universal strip holder. IEF was then carried out on EttanIPGphor II using multistep protocol (6 hr @ 500 V, 6 hr @ 1000 V, 8 hr @ 8000 V). The focused IPG strip was equilibrated in two steps (reduction and alkylation) by equilibrating the strip for 10 min first in 10 ml of 50mM Tris (pH 8.8), 6M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 0.5% (w/v) DTT, followed by another 10 min in 10 ml of 50mM Tris (pH 8.8), 6M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 4.5% (w/v) iodoacetamide to prepare it for the second dimension electrophoresis. Second dimension SDS-PAGE The equilibrated IPG strip was used for protein separation by 2D-gel electrophoresis (DIGE). The strip was sealed at the top of the acrylamide gel for the second dimension (vertical) (12.5% polyacrylamide gel, 20x25 cm x 1.5 mm) with 0.5% (w/v) agarose in SDS running buffer [25 mMTris, 192 mM Glycine, and 0.1% (w/v) SDS]. Electrophoresis was performed in an Ettan DALT six electrophoresis unit (Amersham Biosciences) at 1.5 watts per gel, until the tracking dye reached the anodic end of the gel. Image analysis and post-staining The gel then was imaged directly between glass plates on the Typhoon 9410 variable mode imager (Sunnyvale, CA, USA) using optimal excitation/emission wavelength for each DIGE fluor: Cy3 (532/580 nm) and Cy5 (633/670 nm). The DIGE images were previewed and checked with Image Quant software (GE Healthcare) where all the two separate gel images could be viewed as a single gel image. DeCyde v.5.02 was used to analyze the DIGE images as described in the Ettan DIGE User Manual (GE Healthcare). The appropriate up-/down regulated spots were filtered based on an average volume ratio of ± over 1.2 fold. After image acquisition, the gel was fixed overnight in a solution containing 40% ethanol and 10% acetic acid. The fixed gel was stained with SyproRuby (BioRad) according to the manufacturer protocol (Bio-Rad Labs., 2000 Alfred Nobel Drive, Hercules, CA 94547). Identification of proteins by MS Protein spot picking and digestion Sypro Ruby stained gels were imaged using an Investigator ProPic and HT Analyzer software, both from Genomic Solutions (Ann Arbor, MI). Protein spots of interest that matched those imaged using the DIGE Cy3/Cy5 labels were picked robotically, digested using trypsin as described previously [24] and saved for mass spectrometry identification. Liquid chromatography (LC)- MS/MS analysis LC-MS/MS analyses were carried out using a 3D quadrupole ion trap massspectrometer (ThermoFinnigan LCQ DECA XP PLUS; ThermoFinnigan, San Jose, CA) equipped with a Michrom Paradigm MS4 HPLC (MichromBiosources, Auburn, CA) and a nanospray source, or with a linear quadrupole ion trap mass spectrometer (ThermoFinnigan LTQ), also equipped with a Michrom MS4 HPLC and a nanospray source. Peptides were eluted from a 15 cm pulled tip capillary column (100 um I.D. x 360 um O.D.; 3-5 um tip opening) packed with 7 cm Vydac C18 (Vydac, Hesperia, CA) material (5 µm, 300 Å pore size), using a gradient of 0-65% solvent B (98% methanol/2% water/0.5% formic acid/0.01% triflouroacetic acid) over a 60 min period at a flow rate of 350 nL/min. The ESI positive mode spray voltage was set at 1.6 kV, and the capillary temperature was set at 200°C. Dependent data scanning was performed by the Xcalibur v 1.3 software on the LCQ DECA XP+ or v 1.4 on the LTQ [27], with a default charge of 2, an isolation width of 1.5 amu, an activation amplitude of 35%, activation time of 50 msec, and a minimal signal of 10,000 ion counts (100 ion counts on the LTQ). Global dependent data settings were as follows: reject mass width of 1.5 amu, dynamic exclusion enabled, exclusion mass width of 1.5 amu, repeat count of 1, repeat duration of a min, and exclusion duration of 5 min. Scan event series were included one full scan with mass range of 350-2000 Da, followed by 3 dependent MS/MS scans of the most intense ion. Database searching Tandem MS spectra of peptides were analyzed with Turbo SEQUEST, version 3.1 (ThermoFinnigan), a program that allows the correlation of experimental tandem MS data with theoretical spectra generated from known protein sequences. All spectra were searched against the latest version of the non redundant protein database from the National Center for Biotechnology Information (NCBI 2006; at that time, the database contained 3,783,042 entries). Statistical analysis The means and standard error were calculated. The Student's t-test was used to analyze the significance of the difference between the control and arsenite exposed hamsters. P values less than 0.05 were considered significant. The reproducibility was confirmed in separate experiments. Results Analysis of proteins expression After DIGE (Fig. 1), the gel was scanned by a Typhoon Scanner and the relative amount of protein from sample 1 (treated hamster) as compared to sample 2 (control hamster) was determined (Figs. 2, 3). A green spot indicates that the amount of protein from sodium arsenite-treated hamster sample was less than that of the control sample. A red spot indicates that the amount of protein from the sodium arsenite-treated hamster sample was greater than that of the control sample. A yellow spot indicates sodium arsenite-treated hamster and control hamster each had the same amount of that protein. Several protein spots were up-regulated (red) or down-regulated (green) in the urinary bladder samples of hamsters exposed to sodium arsenite (173 mg As/L) for 6 days as compared with the urinary bladder of controls (Fig. 2). In the case of liver, several protein spots were also over-expressed (red) or under-expressed (green) for hamsters exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days (Fig. 3). The urinary bladder samples were collected from the first and second experiments in which hamsters were exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days and the controls were given tap water. The urinary bladder samples from the 1st and 2nd experiments were run 5 times in DIGE gels on different days. The protein expression is shown in Figure 2 and Table 1. The liver samples from the 1st and 2nd experiments were also run 3 times in DIGE gels on different days. The proteins expression were shown in Figure 3 and Table 2. The volume ratio changed of the protein spots in the urinary bladder and liver of hamsters exposed to arsenite were significantly differences than that of the control hamsters (Table 1 and 2). Protein spots identified by LC-MS/MS Bladder The spots of interest were removed from the gel, digested, and their identities were determined by LC-MS/MS (Fig. 2 and Table 1). The spots 1, 2, & 3 from the gel were analyzed and were repeated for the confirmation of the results (experiments; 173 mg As/L). The proteins for the spots 1, 2, and 3 were identified as transgelin, transgelin, and glutathione S-transferase Pi, respectively (Fig. 2). Liver We also identified some of the proteins in the liver samples of hamsters exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days (Fig. 3). The spots 4, 5, & 6 from the gels were analyzed and were repeated for the confirmation of the results. The proteins for the spots 4, 5, and 6 were identified as ornithine aminotransferase, senescence marker protein 30, and fatty acid binding protein, respectively (Fig. 3) Discussion The identification and functional assignment of proteins is helpful for understanding the molecular events involved in disease. Weexposed hamsters to sodium arsenite in drinking water. Controls were given tap water. DIGE coupled with LC-MS/MS was then used to study the proteomic change in arsenite-exposed hamsters. After electrophoresis DeCyder software indicated that several protein spots were down-regulated (green) and several were up-regulated (red). Our overall results as to changes and functions of the proteins we have studied are summarized in Table 3. Bladder In the case of the urinary bladder tissue of hamsters exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days, transgelin was down-regulated and GST-pi was up-regulated. This is the first evidence that transgelin is down-regulated in the bladders of animals exposed to sodium arsenite. Transgelin, which is identical to SM22 or WS3-10, is an actin cross linking/gelling protein found in fibroblasts and smooth muscle [28, 29]. It has been suggested that the loss of transgelin expression may be an important early event in tumor progression and a diagnostic marker for cancer development [30-33]. It may function as a tumor suppressor via inhibition of ARA54 (co-regulator of androgen receptor)-enhanced AR (androgen receptor) function. Loss of transgelin and its suppressor function in prostate cancer might contribute to the progression of prostate cancer [30]. Down-regulation of transgelin occurs in the urinary bladders of rats having bladder outlet obstruction [32]. Ras-dependent and Ras-independent mechanisms can cause the down regulation of transgelin in human breast and colon carcinoma cell lines and patient-derived tumorsamples [33]. Transgelin plays a role in contractility, possibly by affecting the actin content of filaments [34]. In our experiments loss of transgelin expression may be associated or preliminary to bladder cancer due to arsenic exposure. Arsenite is a carcinogen [1]. In our experiments, LC-MS/MS analysis showed that two spots (1 and 2) represent transgelin (Fig. 2 and Table 1). In human colonic neoplasms there is a loss of transgelin expression and the appearance of transgelin isoforms (31). GST-pi protein was up-regulated in the bladders of the hamsters exposed to sodium arsenite. GSTs are a large family of multifunctional enzymes involved in the phase II detoxification of foreign compounds [35]. The most abundant GSTS are the classes alpha, mu, and pi classes [36]. They participate in protection against oxidative stress [37]. GST-omega has arsenic reductase activity [38]. Over-expression of GST-pi has been found in colon cancer tissues [39]. Strong expression of GST-pi also has been found in gastric cancer [40], malignant melanoma [41], lung cancer [42], breast cancer [43] and a range of other human tumors [44]. GST-pi has been up-regulated in transitional cell carcinoma of human urinary bladder [45]. Up-regulation of glutathione – related genes and enzyme activities has been found in cultured human cells by sub lethal concentration of inorganic arsenic [46]. There is evidence that arsenic induces DNA damage via the production of ROS (reactive oxygen species) [47]. GST-pi may be over-expressed in the urinary bladder to protect cells against arsenic-induced oxidative stress. Liver In the livers of hamsters exposed to sodium arsenite, ornithine amino transferase was over-expressed, senescence marker protein 30 was under-expressed, and fatty acid binding protein was under-expressed. Ornithine amino transferase has been found in the mitochondria of many different mammalian tissues, especially liver, kidney, and small intestine [48]. Ornithine amino transferase knockdown inhuman cervical carcinoma and osteosarcoma cells by RNA interference blocks cell division and causes cell death [49]. It has been suggested that ornithine amino transferase has a role in regulating mitotic cell division and it is required for proper spindle assembly in human cancer cells [49]. Senescence marker protein-30 (SMP30) is a unique enzyme that hydrolyzes diisopropylphosphorofluoridate. SMP30, which is expressed mostly in the liver, protects cells against various injuries by stimulating membrane calcium-pump activity [50]. SMP30 acts to protect cells from apoptosis [51]. In addition it protects the liver from toxic agents [52]. The livers of SMP30 knockout mice accumulate phosphatidylethanolamine, cardiolipin, phosphatidyl-choline, phosphatidylserine, and sphingomyelin [53]. Liver fatty acid binding protein (L-FABP) also was down- regulated. Decreased liver fatty acid-binding capacity and altered liver lipid distribution hasbeen reported in mice lacking the L-FABP gene [54]. High levels of saturated, branched-chain fatty acids are deleterious to cells and animals, resulting in lipid accumulation and cytotoxicity. The expression of fatty acid binding proteins (including L-FABP) protected cells against branched-chain saturated fatty acid toxicity [55]. Limitations: we preferred to study the pronounced spots seen in DIGE gels. Other spots were visible but not as pronounced. Because of limited funds, we did not identify these others protein spots. In conclusion, urinary bladders of hamsters exposed to sodium arsenite had a decrease in the expression of transgelin and an increase in the expression of GST-pi protein. Under-expression of transgelin has been found in various cancer systems and may be associated with arsenic carcinogenicity [30-33). Inorganic arsenic exposure has resulted in bladder cancer as has been reported in the past [1]. Over-expression of GST-pi may protect cells against oxidative stress caused by arsenite. In the liver OAT was up regulated and SMP-30 and FABP were down regulated. These proteomic results may be of help to investigators studying arsenic carcinogenicity. The Superfund Basic Research Program NIEHS Grant Number ES 04940 from the National Institute of Environmental Health Sciences supported this work. Additional support for the mass spectrometry analyses was provided by grants from NIWHS ES06694, NCI CA023074 and the BIOS Institute of the University of Arizona. Acknowledgement The Author wants to dedicate this paper to the memory of his former supervisor Dr. H. VaskenAposhian who passed away in September 6, 2019. He was an emeritus professor of the Department of Molecular and Cellular Biology at the University of Arizona. 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Le mutazioni oncogeniche di BRAF nel melanoma inducono l’inappropriata attivazione di ERK1/2 portando a proliferazione incontrollata. Il trattamento con gli inibitori di BRAF risulta in una rapida regressione delle metastasi. Comunque, le cellule di melanoma si adattano velocemente agli inibitori di BRAF portando a resistenza e a re-insorgenza di metastasi dopo 6-9 mesi. Lo scopo dello studio è stato indagare il ruolo di p53 nei meccanismi di resistenza indotti dal vemurafenib in linee cellulari di melanoma. I nostri dati indicano che il trattamento cronico con gli inibitori di BRAF favorisce la selezione di cellule con l’allele p53-72R portando eventualmente alla ri-attivvaszione di alcune proprietà trascrizionali e all’aumentata sintesi di citochine che controllano la resistenza secondaria ai BRAF, in maniera paracrina. Infatti i nostri dati dimostrano che un programma oncogenico trascrizionale p53-dipendente viene attivato nelle cellule resistenti a PLX4032 e modifica le proprietà delle cellule circostanti, promuovendo la loro resistenza agli inibitori di BRAF.

Le mutazioni oncogeniche di BRAF nel melanoma inducono l’inappropriata attivazione di ERK1/2 portando a proliferazione incontrollata. Il trattamento con gli inibitori di BRAF risulta in una rapida regressione delle metastasi. Comunque, le cellule di melanoma si adattano velocemente agli inibitori di BRAF portando a resistenza e a re-insorgenza di metastasi dopo 6-9 mesi. Lo scopo dello studio è stato indagare il ruolo di p53 nei meccanismi di resistenza indotti dal vemurafenib in linee cellulari di melanoma. I nostri dati indicano che il trattamento cronico con gli inibitori di BRAF favorisce la selezione di cellule con l’allele p53-72R portando eventualmente alla ri-attivvaszione di alcune proprietà trascrizionali e all’aumentata sintesi di citochine che controllano la resistenza secondaria ai BRAF, in maniera paracrina.

White adipose tissue (WAT) is considered as a complex organ with a central role in accumulation of lipids, and an important endocrine function with the secretion of several peptides as adipokines (leptin, adiponectin), cytokines and chemokines, tumor necrosis factor (TNF) and interleukin-6 (IL-6). Cancer is the leading cause of death in developed countries and the second leading cause of death in developing countries. The number of cancer cases is expected to rise due to the increasing aging population.
Epidemiological studies have shown that an increased risk of several cancers, including colon cancer, endometrial, breast, kidney, esophagus, pancreas, gallbladder, liver, and hematological malignancies, is associated with obesity. Moreover, this condition can lead to a reduction in the expected results from treatment, to a worse prognosis and to an increase of the cancer-associated mortality. Several studies have shown that in the white adipose tissue of obese subjects there is a decrease in the maturation of preadipocytes to adipocytes, as well as an imbalance between leptin and adiponectin; in addition, obesity is associated with hyperinsulinemia, hyperglycemia, insulin resistance, aberrant glucose metabolism, chronic inflammation and production of high levels of IGF-1, important risk factor for cancer.
Many studies have highlighted the complexity of the tumors and of their microenvironment. Tumor microenvironment is constituted by several different types of cells as immune system cells, cells of the vascular and lymphatic system (endothelial cells), fibroblasts, pericytes, adipocytes and stromal cells derived from adipose tissue. The role of adipose tissue, and more specifically of adipocytes, in cancer initiation, growth and metastatization is a relatively new area of investigation.
In tumors growing in a microenvironment dominated by adipocytes, it was observed that adipocytes disappear with an accumulation of fibroblast-like cells and subsequent formation of a desmoplastic stroma. Histological observations in some types of cancer, confirm that adipocytes localized at the tumor invasive front, become smaller and the number of fibroblast-like cells increases. It has been hypothesized that fibroblast-like cells could derived from dedifferentiation of adipocytes. In a previous in vitro study with 3T3-L1 cells differentiated to adipocytes, adipocytes promoted the growth, proliferation and survival of human breast cancer cells. However the role of adipocytes in the tumor microenvironment is only incompletely known; in particular in some types of tumors, as melanoma and pancreatic cancer, the role of adipocytes in cancer proliferation and invasiveness is not known. The main focus of this work was to study the interaction between adipocytes and cancer through a co-culture in vitro model. In particular, the study focused on the interaction between adipocytes and human pancreatic cancer cells and human melanoma cells, using a co-culture system between murine fibroblasts 3T3-L1 cell line differentiated to adipocytes and MiaPaca2 and A375 cell line respectively. Adipocytes co-cultured with both types of cells (human pancreatic cancer MiaPaca2 and human melanoma A375 cells) show a progressive loss of lipid content with more centralized nuclei and an elongated shape, similar to the fibroblasts morphology. Moreover, in both co-culture models, dedifferentiated adipocytes loste the adipocyte gene expression profile and acquire a gene profile of reprogramming cells. Finally, MiaPaca2 cells in co-culture showed an up-regulation of Wnt5a and greater activation of STAT3 compared to control; 3T3-L1 cells in co-culture had a greater ability to bind both c-Jun and AP-1, two proteins activated by the Wnt5a pathway; A375 cells in co-culture showed an increased migratory capacity compared to controls and a greater expression of β-catenin and LEF1. The plasticity of AT and the existence of dedifferentiation phenomena could bring new light into the complex relation between obesity, AT dysfunction and increased cancer risk.

INTRODUZIONE:Il melanoma è un tumore maligno di origine melanocitaria ad alta aggressività e rapida crescita cellulare. Sulla base del sistema di stadiazione dell’American Joint Committee on Cancer (AJCC) il parametro prognostico maggiormente considerato e validato statisticamente è lo spessore di Breslow. Il sistema dei fattori di crescita insulino-simile (IGFs, Insulin-like Growth Factors) riveste un ruolo particolarmente significativo nella crescita e nella funzionalità di molti tipi cellulari; questo sistema comprende gli insulin-like growth factors (IGFs), i recettori per IGF e le IGF binding proteins (IGFBPs). Queste proteine sono fondamentali nella patogenesi e nella progressione neoplastica; è noto, infatti, che elevati livelli sierici di IGF-1 sono associati ad un maggior rischio di sviluppare una neoplasia. Alterazioni a carico del sistema degli IGFs sono molto comuni nei processi tumorali; di conseguenza, sia gli IGFs che le IGFBPs potrebbero rappresentare dei nuovi marker tumorali utili sia per la diagnosi che per il follow up dei pazienti. Diversi studi epidemiologici hanno esaminato la relazione tra le concentrazioni sieriche di IGF e IGFBP-3 e l’incidenza dei tumori, sottolineando che sia gli IGFs che le IGFBPs potessero rappresentare degli specifici marker tumorali. MATERIALI E METODI:Da giugno 2007 a Novembre 2010, sono stati arruolati 48 pazienti affetti da melanoma cutaneo. Abbiamo effettuato anche delle colture cellulari, utilizzando 3 linee cellulari di melanoma umano: le Me-501, le LG e le Wm-793. Infine, abbiamo effettuato xenotrapianti di cellule di melanoma Me501 in topi SCID. RISULTATI:le concentrazioni di IGFBP-3 misurate mediante ELISA sono significativamente inferiori nei pazienti affetti da melanoma al IV stadio;la ridotta biodisponibilità di IGF-1 sierico è risultata correlata alla sopravvivenza dei pazienti;la progressione di malattia nei pazienti si accompagna a una riduzione dei livelli sierici di IGFBP-3; l’espressione di IGFBP-3 è maggiore nei melanomi primitivi rispetto alle metastasi dermiche;nel siero dei pazienti al IV stadio di malattia, la riduzione dei valori di IGFBP-3 è mediata da specifici processi proteolitici, assenti, invece, nel siero dei pazienti allo stadio 0-III; inoltre, nel siero dei pazienti al IV stadio sono presenti metalloproteasi 1, 2, 7 e 9;l’aggiunta di IGFBP-3 ricombinante al terreno riduce sia la motilità che la capacità migratoria delle cellule, mentre ne aumenta la capacità di melanogenesi;IGFBP-3 non ha, invece, effetti né sull’espressione né sulla stabilità della proteina Akt; questo suggerisce che l’iperattivazione di Akt rappresenti un importante evento nella transizione tra melanoma primitivo e metastatico, modulando la motilità e la capacità invasiva della cellula. Inoltre, il trattamento con IGFBP3 è in grado di provocare una riduzione della fosforilazione di Akt, quindi una sua ridotta attivazione; nel contempo, si osserva una riduzione della forma fosforilata, ossia inattiva, di GSK3-β, a cui si associa una riduzione dell’espressione della β-catenina. Infine, gli xenotrapianti di cellule di melanoma Me501 in topi SCID ci hanno consentito di dimostrare che il volume tumorale nei topi trattati è inferiore a quello del gruppo di controllo.

Dottorato di Ricerca in Farmacologia e Biochimica della Morte Cellulare, Ciclo XXII, a.a. 2009-2010
Cutaneous melanoma is an aggressive malignancy accounting for 4% of skin cancers but 80% of all skin-cancer related deaths. Its incidence is rapidly rising and advanced disease is notoriously treatment-resistant. The role of apoptosis in melanoma pathogenesis and chemoresistance is poorly characterized. Mutations in p53 occur infrequently and are not critical for tumour development. This may alternatively result from p53 upstream or downstream pathway defects or from alterations of p53 family co-activators, including the ASPP family members (Apoptosis Stimulating Proteins of p53). iASPP is the inhibitory member of the ASPP family. By binding p53, iASPP is believed to inhibit apoptosis in cancer, resulting in its oncogenic role. Recently it has been found highly expressed in several types of cancer, such as endometrial and hepatocellular carcinoma, acute leukemia and breast cancer. iASPP upregulation in some cases occurs with a concomitant downregulation of ASPP expression, the pro-apoptotic family member, thus providing a further option for targeting the p53 family in the treatment of cancers. So far, iASPP expression and its role in skin cancer is not yet been explored. Recently great attention has been given to DNA repair processes in melanoma, particularly to Mismatch Repair (MMR). This is a DNA damage repair mechanism, correcting bases mismatches due to replication errors or exogenous agents’ activity, whose defects have been demonstrated leading to genomic instability (microsatellite instability, MSI) frequently linked with cancer. MSI and altered expression of MMR factors such as MSH2 and MLH1 (both at mRNA and protein levels) has often been observed in primary and metastatic melanoma, compared to normal melanocytes and nevi. 8 Aim of this study is to investigate the role of iASPP in melanoma and particularly its involvement in DNA repair and apoptosis. In this work we used RT-PCR and western blot techniques to demonstrate that both MMR factors and iASPP were expressed at higher levels in several melanoma cell lines, mainly metastatic, compared to primary melanocytes extracted from human skin. We also observed a post-translational modification in the MSH2 protein (which is due at least to an ubiquitination) upon increased iASPP expression in three different melanoma cell lines, independently of p53 status. This results in an increase of DNA repair activity measured by MutS(MSH2/MSH6 complex) binding to a DNA bases mismatch. To confirm these results, we used a set of shRNAs targeting iASPP gene in metastatic melanoma cell line WM1158 and we found that the reduction of iASPP leads to a lower MSH2 protein expression, without affecting MLH1, and to a 50% reduction of MutS activity. Immunoprecipitation experiments showed that iASPP directly binds endogenous MSH2 and MLH1 in melanoma cells and this interaction was confirmed by immunostaining where iASPP partially co-localized with MMR factors in the nucleus of melanoma cells. Moreover, iASPP silencing and its consequent reduction in expression and activity of MMR factors, is able to sensitize melanoma cells to apoptosis induced by chemotherapeutic agent cisplatin. Taken together these results confirm the antiapoptotic role of iASPP and suggest a novel role of iASPP in melanoma, such as a modulator of MMR that may help in the future to explain further its oncogenic role in cancer. This study is also the first report available about iASPP expression in melanoma, highlighting the importance of investigating further this important target gene in such a chemoresistant disease. Future studies will be necessary to further elucidate the mechanism by which iASPP interferes with the MMR system and how it affects apoptosis and cell cycle progression in melanoma disease.
Università della Calabria

Exosomes (EXOs) are nanovesicles of diameter ranging between 50 to 140 nm, distinguished from other cell-derived vesicles by their origin, size, morphology and composition. Their stimulatory or inhibitory signaling activities are mediated by their content (mRNAs, microRNAs and proteins) that can be transferred from the cells of origin to recipient cells, influencing the surrounding microenvironment besides cell behavior. In this study we investigated EXO-mediated communications in two cancer models, melanoma and Ewing’s sarcoma. In view of our previous results demonstrating miR-221&222 as key factors for melanoma development and dissemination, we demonstrated that the EXO-mediated horizontal transfer of miR-222 was competent to deliver miR-222-associated properties increasing tumor malignancy. Melanoma-purified vesicles were characterized and investigated for the functionality of miR-222 in EXO-mediated tumorigenesis. Our data showed that EXOs secreted by miR-222-overexpressing cells induced a protumorigenic program in target cells, mainly through the upmodulation of the PI3K/AKT pathway. The reverse effects were obtained with EXOs recovered after inhibition of endogenous miR-221 and miR-222 by antagomir transfections. The possible differential significance of PI3K/AKT blockade in miR-222-transduced vs control cells was assessed by using BKM120, a pan inhibitor of PI3K. Results showed the capability of miR-222 overexpression to overcome BKM120-dependent effects. We then demonstrated the role of Ewing’s sarcoma-derived EXOs as mediators of signals involved in cancer growth, metastases and differentiation. Ewing’s sarcoma (EWS) is an aggressive childhood bone tumor characterized in the majority of cases by the presence of the fusion oncoprotein EWS-FLI1 and by high expression of the membrane glycoprotein CD99. These features, which are the necessary conditions for the pathogenesis of EWS, mediate tumor progression and maintain the cells in a dedifferentiated state. We evaluated the ability of EXOs, expressing or not CD99, to modulate the phenotype of EWS cells. We observed that the delivery of EXOs devoid of CD99 was sufficient to induce neural differentiation in EWS recipient cells through the inhibition of Notch-NF-kB signaling mediated by miR-34a overexpression. All together these observations would provide a significant step toward new biomarker discovery and innovative therapeutic options. These data on one side support miR-222 responsibility in the exosome-associated melanoma properties, on the other the role of CD99-shRNA/miR-34a-derived EXOs to induce differentiation in EWS, thus further indicating microRNAs as potential diagnostic, prognostic and eventually therapeutic biomarkers.