Interaction between tumour and microenvironment - molecular mechanisms of cell migration in canine tumours

Different steps forward have been made in the recent years to identify the molecular determinants in carcinogenesis and the evidence of a multistep process where cancer cells accumulate multiple and consecutive genetic alterations has been formulated. Recently, tumour progression has been recognized as the product of a complex crosstalk between tumour cells and their surrounding and supporting tissue, named tumour stroma. This stroma is known to influence the growth of cancer and it is composed by several types of cells, including endothelial cells of blood and lymphatic circulation, stromal fibroblasts and a variety of bone marrow-derived cells, such as macrophages, mast cells, neutrophils, lymphocytes and mesenchymal stem cells. The supportive microenvironment is generate and modulated by cancer cells through the production and activation of stroma growth factors including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta). Concomitant with altered growth-factor expressions, induced by their autocrine and paracrine effect on the tumour and stromal cells, cancer cells are able to produce proteolytic enzymes, such as Matrix metalloproteinases (MMPs), which operate the remodelling of extracellular matrix (ECM) and basement membrane, thus activating cell-surface and ECM-bound growth factors. All these processes are described to contribute to the extensive crosstalk between the microenvironment and the cancer cells. Therefore, the microenvironment is implicated in the regulation of cell growth, determining angiogenesis, tumour invasion and metastasis, and impacting the outcome. Even if stromal cells are not malignant, their role in supporting cancer growth is vital to the survival of the tumour. For this purpose, cells of microenvironment have become an attractive target for therapeutic agents. The present project has been divided in different tasks to identify the molecular mechanisms implicated in cell migration, angiogenesis and tumour growth led by stroma cells and their crosstalk with cancer cells in different neoplasia in dog. Canine mammary tumour, cutaneous mast cell tumour, lymphoid leukaemia and lymphoma were selected for the study and gene expression profiling and proteomic analysis of different growth factors (VEGF-TGF-beta-PDGF) and MMPs were analyzed in association with their possible prognostic and predictive role and crosstalk. Several important results have obtained highlighting the background of the tumour progression and the role of microenvironment in veterinary oncology. Selected results are shown below: - MMP-2, MT1-MMP, MMP-9 were significantly involved in canine mammary tumour and a significant role of the stromal compartment was described; - MMP-9 and VEGF-A were associated with the histological tumour grade in cutaneous mast cell tumour; - MMP-9, MT1-MMP, TIMP-1 and VEGF were correlated in T-cell lymphoma and in dogs with higher stage; - A potential role of MT1-MMP and TIMP-2 in the pathogenesis of canine acute lymphoblastic leukaemia has been discovered; - In chronic lymphocytic leukaemia, residual normal leukocytes have shown a significative influence in the expression of MMP-9, MT1-MMP, VEGF and TIMPs; - Lymphoma and leukaemia in vitro model exhibited a significative discrepancy that enhanced the importance of microenvironment in vivo; - PDGF-B mRNA expression was identified in canine T-cell lymphoma and cutaneous lymphomas. A functional autocrine and/or paracrine loop of growth stimulation was proposed due to the co-expression of PDGFs and PDGFRs at different time point during disease. Therefore, the obtained results may significantly improve the understanding of cancerogenesis of the most frequent tumours in dogs. The summarized data here show a primary role for the microenvironment during carcinogenesis. Development of novel cancer therapies that target the process of metastasis formation, tumour growth and differentiation, by interfering with the ability of cancer cells to transmigrate into blood and lymph vessels and to invade the connective tissue, is widely expected in veterinary oncology. Further data are necessary to indicate that the use of chemopreventive agents to control the function and behaviour of cells in the microenvironment might be an important approach to the overall control of cancer

EEG-based mental workload neurometric to evaluate the impact of different traffic and road conditions in real driving settings

Car driving is considered a very complex activity, consisting of different concomitant tasks and subtasks, thus it is crucial to understand the impact of different factors, such as road complexity, traffic, dashboard devices, and external events on the driver’s behavior and performance. For this reason, in particular situations the cognitive demand experienced by the driver could be very high, inducing an excessive experienced mental workload and consequently an increasing of error commission probability. In this regard, it has been demonstrated that human error is the main cause of the 57% of road accidents and a contributing factor in most of them. In this study, 20 young subjects have been involved in a real driving experiment, performed under different traffic conditions (rush hour and not) and along different road types (main and secondary streets). Moreover, during the driving tasks different specific events, in particular a pedestrian crossing the road and a car entering the traffic flow just ahead of the experimental subject, have been acted. A Workload Index based on the Electroencephalographic (EEG), i.e., brain activity, of the drivers has been employed to investigate the impact of the different factors on the driver’s workload. Eye-Tracking (ET) technology and subjective measures have also been employed in order to have a comprehensive overview of the driver’s perceived workload and to investigate the different insights obtainable from the employed methodologies. The employment of such EEG-based Workload index confirmed the significant impact of both traffic and road types on the drivers’ behavior (increasing their workload), with the advantage of being under real settings. Also, it allowed to highlight the increased workload related to external events while driving, in particular with a significant effect during those situations when the traffic was low. Finally, the comparison between methodologies revealed the higher sensitivity of neurophysiological measures with respect to ET and subjective ones. In conclusion, such an EEG-based Workload index would allow to assess objectively the mental workload experienced by the driver, standing out as a powerful tool for research aimed to investigate drivers’ behavior and providing additional and complementary insights with respect to traditional methodologies employed within road safety research

The role of vascular endothelial growth factor and matrix metalloproteinases in canine lymphoma: in vivo and in vitro study

Background: Canine lymphoma represents the most frequent haematopoietic cancer and it shares some similarities with human non-Hodgkin lymphoma. Matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) play a coordinated role during invasion and proliferation of malignant cells; however, little is known about their role in canine haematologic malignancies. The aim of this study was to investigate the mRNA and protein expression of VEGF and the most relevant MMPs in canine lymphoma. Lymph node aspirates from 26 B-cell and 21 T-cell lymphomas were collected. The protein expression levels of MMP-9, MMP-2 and VEGF-A were evaluated by immunocytochemistry, and the mRNA levels of MMP-2, MMP-9, MT1-MMP, TIMP-1, TIMP-2, RECK, VEGF-A and VEGF-164 were measured using quantitative RT-PCR.Results: MT1-MMP, TIMP-1 and RECK mRNA levels were significantly higher in T-cell lymphomas than in B-cell lymphomas. Higher mRNA and protein levels of MMP-9 and VEGF-A were observed in T-cell lymphomas than in B-cell lymphomas and healthy control lymph nodes. A positive correlation was found between MMP-9 and VEGF-A in T-cell lymphomas. Moreover, MMP-9, MT1-MMP, TIMP-1 and VEGF-A were expressed at the highest levels in high-grade T-cell lymphomas.Conclusions: This study provides new information on the expression of different MMPs and VEGF in canine lymphoma, suggesting a possible correlation between different MMPs and VEGF, immunophenotype and prognosis

Matrix metalloproteinases and their inhibitors in canine mammary tumors

BACKGROUND: Malignant canine mammary tumors represent 50% of all neoplasms in female dogs. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be involved in tumor progression, and they are also associated with the reactive stroma, which provides structural and vascular support for tumor growth. RESULTS: MMP-2, MMP-9 and MT1-MMP were expressed at both the mRNA and protein levels in tumor samples. MMP-2 and MMP-9 immunohistochemical reactions were evident both in the epithelial tumor cells and in the stromal compartment to varying degrees; in particular, the intensity of the MMP-2 staining was stronger in the stromal fibroblasts close to epithelial tumor cells in simple carcinomas than in adenomas. These data were supported by gelatin-zymography; bands for the active form of MMP-2 were found in 94% of carcinoma samples, compared with 17% of benign tumor samples. The gene expression and immunohistochemical results for MT1-MMP were comparable to those for MMP-2. The immunoreactivity for MMP-13 and TIMP-2 was lower in carcinomas than in adenomas, confirming the mRNA data for MMP-13 and the other MMP inhibitors that were evaluated. The active form of MMP-9, but not the active form of MMP-2, was identified in the plasma of all of the tested dogs. CONCLUSIONS: Our findings suggest that MMP-9, MMP-2 and MT1-MMP, which are synthesized by epithelial cancer cells and cancer-associated fibroblasts, play an important role in malignant canine mammary tumors. The reduction of MMP-13 and TIMP-2 could also be a significant step in malignant transformation. MMP-2 and MT1-MMP could be further evaluated as future biomarkers for predicting the progression and prognosis of canine mammary tumors

EEG-Based Mental Workload Neurometric to Evaluate the Impact of Different Traffic and Road Conditions in Real Driving Settings

Car driving is considered a very complex activity, consisting of different concomitant tasks and subtasks, thus it is crucial to understand the impact of different factors, such as road complexity, traffic, dashboard devices, and external events on the driver’s behavior and performance. For this reason, in particular situations the cognitive demand experienced by the driver could be very high, inducing an excessive experienced mental workload and consequently an increasing of error commission probability. In this regard, it has been demonstrated that human error is the main cause of the 57% of road accidents and a contributing factor in most of them. In this study, 20 young subjects have been involved in a real driving experiment, performed under different traffic conditions (rush hour and not) and along different road types (main and secondary streets). Moreover, during the driving tasks different specific events, in particular a pedestrian crossing the road and a car entering the traffic flow just ahead of the experimental subject, have been acted. A Workload Index based on the Electroencephalographic (EEG), i.e., brain activity, of the drivers has been employed to investigate the impact of the different factors on the driver’s workload. Eye-Tracking (ET) technology and subjective measures have also been employed in order to have a comprehensive overview of the driver’s perceived workload and to investigate the different insights obtainable from the employed methodologies. The employment of such EEG-based Workload index confirmed the significant impact of both traffic and road types on the drivers’ behavior (increasing their workload), with the advantage of being under real settings. Also, it allowed to highlight the increased workload related to external events while driving, in particular with a significant effect during those situations when the traffic was low. Finally, the comparison between methodologies revealed the higher sensitivity of neurophysiological measures with respect to ET and subjective ones. In conclusion, such an EEG-based Workload index would allow to assess objectively the mental workload experienced by the driver, standing out as a powerful tool for research aimed to investigate drivers’ behavior and providing additional and complementary insights with respect to traditional methodologies employed within road safety research

Interaction between tumour and microenvironment - molecular mechanisms of cell migration in canine tumours

Different steps forward have been made in the recent years to identify the molecular determinants in carcinogenesis and the evidence of a multistep process where cancer cells accumulate multiple and consecutive genetic alterations has been formulated. Recently, tumour progression has been recognized as the product of a complex crosstalk between tumour cells and their surrounding and supporting tissue, named tumour stroma. This stroma is known to influence the growth of cancer and it is composed by several types of cells, including endothelial cells of blood and lymphatic circulation, stromal fibroblasts and a variety of bone marrow-derived cells, such as macrophages, mast cells, neutrophils, lymphocytes and mesenchymal stem cells. The supportive microenvironment is generate and modulated by cancer cells through the production and activation of stroma growth factors including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta). Concomitant with altered growth-factor expressions, induced by their autocrine and paracrine effect on the tumour and stromal cells, cancer cells are able to produce proteolytic enzymes, such as Matrix metalloproteinases (MMPs), which operate the remodelling of extracellular matrix (ECM) and basement membrane, thus activating cell-surface and ECM-bound growth factors. All these processes are described to contribute to the extensive crosstalk between the microenvironment and the cancer cells. Therefore, the microenvironment is implicated in the regulation of cell growth, determining angiogenesis, tumour invasion and metastasis, and impacting the outcome. Even if stromal cells are not malignant, their role in supporting cancer growth is vital to the survival of the tumour. For this purpose, cells of microenvironment have become an attractive target for therapeutic agents. The present project has been divided in different tasks to identify the molecular mechanisms implicated in cell migration, angiogenesis and tumour growth led by stroma cells and their crosstalk with cancer cells in different neoplasia in dog. Canine mammary tumour, cutaneous mast cell tumour, lymphoid leukaemia and lymphoma were selected for the study and gene expression profiling and proteomic analysis of different growth factors (VEGF-TGF-beta-PDGF) and MMPs were analyzed in association with their possible prognostic and predictive role and crosstalk. Several important results have obtained highlighting the background of the tumour progression and the role of microenvironment in veterinary oncology. Selected results are shown below: - MMP-2, MT1-MMP, MMP-9 were significantly involved in canine mammary tumour and a significant role of the stromal compartment was described; - MMP-9 and VEGF-A were associated with the histological tumour grade in cutaneous mast cell tumour; - MMP-9, MT1-MMP, TIMP-1 and VEGF were correlated in T-cell lymphoma and in dogs with higher stage; - A potential role of MT1-MMP and TIMP-2 in the pathogenesis of canine acute lymphoblastic leukaemia has been discovered; - In chronic lymphocytic leukaemia, residual normal leukocytes have shown a significative influence in the expression of MMP-9, MT1-MMP, VEGF and TIMPs; - Lymphoma and leukaemia in vitro model exhibited a significative discrepancy that enhanced the importance of microenvironment in vivo; - PDGF-B mRNA expression was identified in canine T-cell lymphoma and cutaneous lymphomas. A functional autocrine and/or paracrine loop of growth stimulation was proposed due to the co-expression of PDGFs and PDGFRs at different time point during disease. Therefore, the obtained results may significantly improve the understanding of cancerogenesis of the most frequent tumours in dogs. The summarized data here show a primary role for the microenvironment during carcinogenesis. Development of novel cancer therapies that target the process of metastasis formation, tumour growth and differentiation, by interfering with the ability of cancer cells to transmigrate into blood and lymph vessels and to invade the connective tissue, is widely expected in veterinary oncology. Further data are necessary to indicate that the use of chemopreventive agents to control the function and behaviour of cells in the microenvironment might be an important approach to the overall control of cancer.Negli ultimi anni nell’ambito dell’oncologia, diversi studi hanno identificato diverse molecole target implicate nella cancerogenesi e sono stati evidenziati numerosi processi attraverso cui le cellule tumorali sono in grado di accumulare alterazioni genetiche. Recentemente, la progressione del tumore è stata riconosciuta come il prodotto di un complesso crosstalk tra le cellule tumorali e il tessuto circostante, chiamato stroma tumorale. Questo stroma è noto per influenzare la crescita del tumore ed è composto da diverse tipologie cellulari, che comprendono cellule endoteliali della circolazione sanguigna e linfatica, fibroblasti stromali ed una varietà di cellule derivate dal midollo osseo, come macrofagi, mastociti, neutrofili, linfociti e cellule staminali mesenchimali. Ulteriormente, il microambiente di supporto è generato e modulato da cellule tumorali attraverso la produzione e attivazione di fattori di crescita prodotti dallo stroma stesso, come Vascular Endothelial Growth Factor (VEGF), Platelet-Derived Growth Factor (PDGF) e Transforming Growth Factor-beta (TGF). Concomitante all’alterata espressione di questi fattori e per il loro effetto autocrino e paracrino sulle cellule tumorali e su quelle stromali, le cellule neoplastiche iniziano a produrre enzimi proteolitici, come metalloproteasi di matrice (Matrix metalloproteinases - MMPs). Le MMPs operano il rimodellamento della matrice extra cellulare e della membrana basale, attivando così fattori di crescita legati alla superficie cellulare e alla matrice stessa. Tutti questi processi contribuiscono all’esteso crosstalk tra il microambiente e le cellule tumorali. Il microambiente quindi è implicato nella regolazione della crescita cellulare, determinando neoangiogenesi, invasione, metastasi tumorali e influenzando il risultato della terapia. Anche se le cellule stromali non sono considerabili fenotipicamente maligne, il loro ruolo nel sostenere la crescita della neoplasia è essenziale per la sopravvivenza del tumore. Con questo presupposto, le cellule del microambiente sono diventate un bersaglio attrattivo per diversi agenti terapeutici. Il progetto di ricerca è stato suddiviso in diverse fasi per identificare i meccanismi molecolari implicati nella migrazione cellulare, nell'angiogenesi e nella crescita neoplastica, da parte di cellule stromali e dal loro crosstalk con le cellule tumorali, in diverse neoplasie del cane. Per lo studio sono state selezionate le tipologie tumorali più frequenti nel cane: tumore mammario, mastocitoma cutaneo, leucemie linfoidi e linfoma, analizzando i profili di espressione genica e proteica di diversi fattori di crescita (VEGF-TGF-beta-PDGF) e delle MMPs, in associazione al loro crosstalk e ad un loro eventuale ruolo prognostico. Sono stati ottenuti importanti risultati evidenziando lo scenario della progressione tumorale e il ruolo del microambiente in oncologia veterinaria. E’ stato dimostrato che: - MMP-2, MT1-MMP, MMP-9 sono significativamente coinvolte nel tumore mammario ed è stato descritto un loro ruolo rilevante del compartimento stromale; - MMP-9 e VEGF-A sono associati al grado istologico nei mastocitomi cutanei; - MMP-9, MT1-MMP, TIMP-1 e VEGF sono correlate nel linfoma T e nei cani con linfoma con stadio clinico più alto; - MT1-MMP e TIMP-2 hanno un ruolo nella patogenesi nelle leucemie linfoblastiche acute; - Nella leucemia linfocitica cronica, i leucociti residui normali mostrano un'influenza significativa nell'espressione di MMP-9, MT1-MMP, VEGF e dei TIMPs; - Il linfoma e la leucemia nel modello in vitro mostrano una considerevole discrepanza per alcune MMPs e VEGF che avvalora l'importanza del microambiente in vivo; - L’espressione genica del PDGF-B è significativa nei linfomi T e nei linfomi cutanei. E’ stato inoltre proposto un loop funzionale autocrino e/o paracrino di stimolazione della crescita della neoplasia, dovuto alla co-espressione dei PDGFs e dei recettori in diversi tempi durante la malattia. I risultati ottenuti potrebbero migliorare significativamente la comprensione della cancerogenesi nei tumori più frequenti nel cane. I dati qui sintetizzati mostrano un ruolo primario del microambiente durante la carcinogenesi. Lo sviluppo di nuove terapie antitumorali che colpiscano il processo di formazione di metastasi, la crescita e la differenziazione della neoplasia, interferendo con la capacità delle cellule tumorali di trasmigrare nel sangue e nei vasi linfatici e di invadere il tessuto connettivo, sarà ampiamente perseguito in oncologia veterinaria nel futuro prossimo. Sono però necessari ulteriori studi per indicare se l'uso di agenti chemio-preventivi per controllare la funzione ed il comportamento delle cellule nel microambiente possa essere un importante approccio al controllo complessivo del cancro

The sample size matters: to what extent the participant reduction affects the outcomes of a neuroscientific research. A case-study in neuromarketing field

The sample size is a crucial concern in scientific research and even more in behavioural neurosciences, where besides the best practice it is not always possible to reach large experimental samples. In this study we investigated how the outcomes of research change in response to sample size reduction. Three indices computed during a task involving the observations of four videos were considered in the analysis, two related to the brain electroencephalographic (EEG) activity and one to autonomic physiological measures, i.e., heart rate and skin conductance. The modifications of these indices were investigated considering five subgroups of sample size (32, 28, 24, 20, 16), each subgroup consisting of 630 different combinations made by bootstrapping n (n = sample size) out of 36 subjects, with respect to the total population (i.e., 36 subjects). The correlation analysis, the mean squared error (MSE), and the standard deviation (STD) of the indexes were studied at the participant reduction and three factors of influence were considered in the analysis: the type of index, the task, and its duration (time length). The findings showed a significant decrease of the correlation associated to the participant reduction as well as a significant increase of MSE and STD (p < 0.05). A threshold of subjects for which the outcomes remained significant and comparable was pointed out. The effects were to some extents sensitive to all the investigated variables, but the main effect was due to the task length. Therefore, the minimum threshold of subjects for which the outcomes were comparable increased at the reduction of the spot duration

Esports and Visual Attention: Evaluating In-Game Advertising through Eye-Tracking during the Game Viewing Experience

In recent years, technological advances and the introduction of social streaming platforms (e.g., Twitch) have contributed to an increase in the popularity of esports, a highly profitable industry with millions of active users. In this context, there is little evidence, if any, on how users perceive in-game advertising (IGA) and other key elements of the game viewing experience (e.g., facecam and chat) in terms of visual attention. The present eye-tracking study aimed at investigating those aspects, and introducing an eye-tracking research protocol specifically designed to accurately measure the visual attention associated with key elements of the game viewing experience. Results showed that (1) the ads available in the game view (IGAs) are capable altogether to attract 3.49% of the users’ visual attention; (2) the chat section draws 10.68% of the users’ visual attention and more than the streamer’s face, known as a powerful attentional driver; (3) the animated ad format elicits higher visual attention (1.46%) than the static format (1.12%); and (4) in some circumstances, the visual attention elicited by the ads is higher in the “Goal” scenes (0.69%) in comparison to “No-Goal” scenes (0.51%). Relevant managerial implications and future directions for the esports industry are reported and discussed

List of recurrent MCRs in pre-treatment DLBCLs.

<p>*referred to Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111817#pone.0111817.s001" target="_blank">File S1</a>.</p><p>List of recurrent MCRs in pre-treatment DLBCLs.</p