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Il tumore alla vescica è uno dei più diffusi a livello mondiale. Spesso non presenta sintomi e l’alta frequenza di tumori non muscolo invasivi ad alto grado è associata ad un elevato rischio di recidiva, con la necessità di controlli periodici, con impatti a livello economico. Le recidive portano alla progressione a tumori muscolo invasivi correlati ad una prognosi sfavorevole. I metodi di diagnosi più utilizzati sono la cistoscopia e la TURBT, quest’ultima utilizzata anche a scopo terapeutico. Tuttavia, spesso si verifica un’incompleta rimozione del tumore, dovuta ad una difficoltà nello stabilire l’entità della penetrazione, con aumento della probabilità di diagnosi errate e insorgenza di recidive. Diventa quindi cruciale identificare marcatori che permettano di assegnare, al momento della resezione, la corretta classificazione, migliorando la prognosi. La riprogrammazione del metabolismo è considerato un tratto distintivo della crescita tumorale, e lo studio dei processi metabolici alterati nella patologia può rappresentare un bersaglio clinico utilizzabile per nuove strategie terapeutiche. Questa tesi, nata dal progetto Horizon2020 AMPLITUDE (Advanced Multimodal Photonics Laser Imaging Tool for Urothelial Diagnosis in Endoscopy), riassume il percorso svoltosi nel corso di tre anni, volto a studiare le caratteristiche funzionali e metaboliche di un pannello di sei linee cellulari di tumore alla vescica, con lo scopo di trovare un marcatore indicativo della progressione tumorale. Una prima caratterizzazione delle linee cellulari cresciute in colture 2D ha rivelato un’eterogeneità intrinseca tra queste cellule. Le cellule muscolo invasive hanno mostrato un ampio spettro di proprietà morfo-funzionali che garantisce loro di manifestare un fenotipo più aggressivo rispetto alle linee non muscolo invasive. Allo stesso modo, anche il fenotipo metabolico si è rivelato altamente eterogeneo, senza correlazioni con la progressione tumorale. Tuttavia, è stato p, Bladder cancer (BC) is one of the most common malignancies worldwide. Symptoms are often absent and the frequent non muscle invasive (NMIBC) high grade tumors are associated to a high risk of relapses, resulting in high costs for patient follow-up. Frequent relapses often progress to muscle invasive tumors (MIBC), that result in a poor prognosis. Cystoscopy and transurethral resection of bladder tumor (TURBT) are the most used diagnostic methods. However, the frequent incomplete resections, related to the difficulty to establish the level of penetration of the tumor, cause most of the wrong diagnoses and relapse onsets. Therefore, identifying new markers is crucial to attribute the correct classification at the time of resection, improving patient’s prognosis. Energy metabolism reprogramming is an established cancer hallmark, and altered metabolic pathways can represent attractive clinical targets exploitable in new therapeutic strategies. This thesis, part of the Horizon2020 project AMPLITUDE (Advanced Multimodal Photonics Laser Imaging Tool for Urothelial Diagnosis in Endoscopy), represents the path followed these three years to study the functional and metabolic features of a panel of six bladder cancer cell lines, to find a marker for cancer progression. A first characterization of the cell lines grown in 2D models showed the intrinsic heterogeneity between these cells. MIBC cell lines showed a range of morpho-functional properties that allow them the manifestation of a more aggressive phenotype, compared to the two NMIBC cell lines. The metabolic phenotype as well showed high heterogeneity not relatable with the cancer progression, although some features correlated with the genetic background of the cell lines. From 2D models, we decided to grow cells forming spheroids, known to be more representative for the in vivo pathology, better simulating the architectural complexity of a tumor mass. The characterization of the 2D to 3D transition confirmed that the hete

Three-dimensional cancer models, such as spheroids, are increasingly being used to study cancer metabolism because they can better recapitulate the molecular and physiological aspects of the tumor architecture than conventional monolayer cultures. Although Agilent Seahorse XFe96 (Agilent Technologies, Santa Clara, CA, United States) is a valuable technology for studying metabolic alterations occurring in cancer cells, its application to three-dimensional cultures is still poorly optimized. We present a reliable and reproducible workflow for the Seahorse metabolic analysis of three-dimensional cultures. An optimized protocol enables the formation of spheroids highly regular in shape and homogenous in size, reducing variability in metabolic parameters among the experimental replicates, both under basal and drug treatment conditions. High-resolution imaging allows the calculation of the number of viable cells in each spheroid, the normalization of metabolic parameters on a per-cell basis, and grouping of the spheroids as a function of their size. Multivariate statistical tests on metabolic parameters determined by the Mito Stress test on two breast cancer cell lines show that metabolic differences among the studied spheroids are mostly related to the cell line rather than to the size of the spheroid. The optimized workflow allows high-resolution metabolic characterization of three-dimensional cultures, their comparison with monolayer cultures, and may aid in the design and interpretation of (multi)drug protocols.

Three-dimensional cancer models, such as spheroids, are increasingly being used to study cancer metabolism because they can better recapitulate the molecular and physiological aspects of the tumor architecture than conventional monolayer cultures. Although Agilent Seahorse XFe96 (Agilent Technologies, Santa Clara, CA, United States) is a valuable technology for studying metabolic alterations occurring in cancer cells, its application to three-dimensional cultures is still poorly optimized. We present a reliable and reproducible workflow for the Seahorse metabolic analysis of three-dimensional cultures. An optimized protocol enables the formation of spheroids highly regular in shape and homogenous in size, reducing variability in metabolic parameters among the experimental replicates, both under basal and drug treatment conditions. High-resolution imaging allows the calculation of the number of viable cells in each spheroid, the normalization of metabolic parameters on a per-cell basis, and grouping of the spheroids as a function of their size. Multivariate statistical tests on metabolic parameters determined by the Mito Stress test on two breast cancer cell lines show that metabolic differences among the studied spheroids are mostly related to the cell line rather than to the size of the spheroid. The optimized workflow allows high-resolution metabolic characterization of three-dimensional cultures, their comparison with monolayer cultures, and may aid in the design and interpretation of (multi)drug protocols.

Progranulin is a pleiotropic growth factor with important physiological roles in embryogenesis and maintenance of adult tissue homeostasis. While-progranulin deficiency is associated with a broad range of pathological conditions affecting the brain, such as frontotemporal dementia and neuronal ceroid lipofuscinosis, progranulin upregulation characterizes many tumors, including brain tumors, multiple myeloma, leiomyosarcoma, mesothelioma and epithelial cancers such as ovarian, liver, breast, bladder, adrenal, prostate and kidney carcinomas. The increase of progranulin levels in tumors might have diagnostic and prognostic significance. In cancer, progranulin has a pro-tumorigenic role by promoting cancer cell proliferation, migration, invasiveness, anchorage-independent growth and resistance to chemotherapy. In addition, progranulin regulates the tumor microenvironment, affects the function of cancer-associated fibroblasts, and modulates tumor immune surveillance. However, the molecular mechanisms of progranulin oncogenic function are not fully elucidated. In bladder cancer, progranulin action relies on the activation of its functional signaling receptor EphA2. Notably, more recent data suggest that progranulin can also modulate a functional crosstalk between multiple receptor-tyrosine kinases, demonstrating a more complex and context-dependent role of progranulin in cancer. Here, we will review what is currently known about the function of progranulin in tumors, with a focus on its molecular mechanisms of action and regulation.

Aims: The present investigation aimed at evaluating differences in psychiatric hospitalizations in Italy during and after the lockdown due to the novel coronavirus disease 2019 (COVID-19), compared to the same periods in 2018 and 2019. Methods: We obtained and analyzed anonymized data on psychiatric admissions (n = 4550) from 12 general hospital psychiatric wards (GHPWs) in different Italian regions (catchment area = 3.71 millions of inhabitants). Using a mixed-effects Poisson regression model, we compared admission characteristics across three periods: (a) March 1–June 30, 2018 and 2019; (b) March 1–April 30, 2020 (i.e., lockdown); and (c) May 1–June 30, 2020 (i.e., post-lockdown). Results: During the COVID-19 lockdown, there was a 41% reduction (IRR = 0.59; p < 0.001, CI: 0.45–0.79) in psychiatric admissions in the enrolled GHPWs with respect to the 2018 and 2019 control period. Conversely, admission rates in the post-lockdown period were similar to those observed in the control period. Notably, a consistent and significant reduction in psychiatric hospitalizations of older patients (aged >65 years) was observed in the lockdown (40%; IRR = 0.60; 95% CI: 0.44–0.82) and post-lockdown (28%; IRR = 0.72; 95% CI: 0.54–0.96) periods. Long-stay admissions (>14 days) increased (63%; IRR = 1.63; 95% CI: 1.32–2.02) during the lockdown and decreased by 39% thereafter (IRR = 0.61; 95% CI: 0.49–0.75). A significant 35% increase in patients reporting suicidal ideation was observed in the post-lockdown period, compared to the rate observed in the 2018 and 2019 control period (IRR = 1.35; 95% CI: 1.01–1.79). Conclusion: The COVID-19 lockdown was associated with changes in the number of psychiatric admissions, particularly for older patients and long-stay hospitalizations. Increased admission of patients reporting suicidal ideation in the post-lockdown period merits special attention. Further studies are required to gain insight into the observed phenomena.

No abstract available

Aims: The present investigation aimed at evaluating differences in psychiatric hospitalizations in Italy during and after the lockdown due to the novel coronavirus disease 2019 (COVID-19), compared to the same periods in 2018 and 2019. Methods: We obtained and analyzed anonymized data on psychiatric admissions (n = 4550) from 12 general hospital psychiatric wards (GHPWs) in different Italian regions (catchment area = 3.71 millions of inhabitants). Using a mixed-effects Poisson regression model, we compared admission characteristics across three periods: (a) March 1–June 30, 2018 and 2019; (b) March 1–April 30, 2020 (i.e., lockdown); and (c) May 1–June 30, 2020 (i.e., post-lockdown). Results: During the COVID-19 lockdown, there was a 41% reduction (IRR = 0.59; p < 0.001, CI: 0.45–0.79) in psychiatric admissions in the enrolled GHPWs with respect to the 2018 and 2019 control period. Conversely, admission rates in the post-lockdown period were similar to those observed in the control period. Notably, a consistent and significant reduction in psychiatric hospitalizations of older patients (aged >65 years) was observed in the lockdown (40%; IRR = 0.60; 95% CI: 0.44–0.82) and post-lockdown (28%; IRR = 0.72; 95% CI: 0.54–0.96) periods. Long-stay admissions (>14 days) increased (63%; IRR = 1.63; 95% CI: 1.32–2.02) during the lockdown and decreased by 39% thereafter (IRR = 0.61; 95% CI: 0.49–0.75). A significant 35% increase in patients reporting suicidal ideation was observed in the post-lockdown period, compared to the rate observed in the 2018 and 2019 control period (IRR = 1.35; 95% CI: 1.01–1.79). Conclusion: The COVID-19 lockdown was associated with changes in the number of psychiatric admissions, particularly for older patients and long-stay hospitalizations. Increased admission of patients reporting suicidal ideation in the post-lockdown period merits special attention. Further studies are required to gain insight into the observed phenomena.

Patients with severe psychotic disorders such as schizophrenia, schizoaffective, and bipolar disorders frequently suffer from concomitant substance use disorders (SUDs)–Dual Disorder (DD) patients. In order to better understand current practices for management of patients with psychotic episodes and concomitant SUD in Italy, we carried out a survey of psychiatrists on current routine practice among prescribers. These aspects can help to identify at-risk patients, improve current prescribing practices, and favor early intervention. An ad hoc survey of 17 questions was administered to psychiatrists via electronic polling and on-line distribution; 448 completed questionnaires were collected. Comorbid substance abuse was most frequently diagnosed within the context of anxiety disorder (46%), followed by bipolar disorder (25%), and schizophrenia/schizoaffective disorder (12%). The vast majority of respondents felt that patient management was becoming more complex due to substance abuse. The areas reported to be most affected in patients with SUD were functioning, interpersonal relations, and impulsivity, while sensory perception disorders, ideation, agitation, and impulsivity were the most frequently reported symptoms. In the acute setting, haloperidol was used as the first-line agent of choice followed by aripiprazole and olanzapine. In the maintenance phase, aripiprazole was the dominantly used first-line agent, followed by olanzapine. Almost half of respondents used long-acting agents, while about one-third did not. Among those prescribing long-acting agents, efficacy, control of impulsivity, and control of specific symptoms were cited as motivators, while in the maintenance phase, better adherence, and tolerability were mainly cited. From the responses to the present survey, it is clear that the respondents are aware of the problem of SUD in psychotic patients. While treatment be optimized in terms of the choice and formulation of antipsychotics, greater emphasis sho

Patients with severe psychotic disorders such as schizophrenia, schizoaffective, and bipolar disorders frequently suffer from concomitant substance use disorders (SUDs)–Dual Disorder (DD) patients. In order to better understand current practices for management of patients with psychotic episodes and concomitant SUD in Italy, we carried out a survey of psychiatrists on current routine practice among prescribers. These aspects can help to identify at-risk patients, improve current prescribing practices, and favor early intervention. An ad hoc survey of 17 questions was administered to psychiatrists via electronic polling and on-line distribution; 448 completed questionnaires were collected. Comorbid substance abuse was most frequently diagnosed within the context of anxiety disorder (46%), followed by bipolar disorder (25%), and schizophrenia/schizoaffective disorder (12%). The vast majority of respondents felt that patient management was becoming more complex due to substance abuse. The areas reported to be most affected in patients with SUD were functioning, interpersonal relations, and impulsivity, while sensory perception disorders, ideation, agitation, and impulsivity were the most frequently reported symptoms. In the acute setting, haloperidol was used as the first-line agent of choice followed by aripiprazole and olanzapine. In the maintenance phase, aripiprazole was the dominantly used first-line agent, followed by olanzapine. Almost half of respondents used long-acting agents, while about one-third did not. Among those prescribing long-acting agents, efficacy, control of impulsivity, and control of specific symptoms were cited as motivators, while in the maintenance phase, better adherence, and tolerability were mainly cited. From the responses to the present survey, it is clear that the respondents are aware of the problem of SUD in psychotic patients. While treatment be optimized in terms of the choice and formulation of antipsychotics, greater emphasis sho

Bladder cancer is one of the most prevalent deadly diseases worldwide. Grade 2 tumors represent a good window of therapeutic intervention, whose optimization requires high resolution biomarker identification. Here we characterize energy metabolism and cellular properties associated with spreading and tumor progression of RT112 and 5637, two Grade 2 cancer cell lines derived from human bladder, representative of luminal-like and basal-like tumors, respectively. The two cell lines have similar proliferation rates, but only 5637 cells show efficient lateral migration. In contrast, RT112 cells are more prone to form spheroids. RT112 cells produce more ATP by glycolysis and OXPHOS, present overall higher metabolic plasticity and are less sensitive than 5637 to nutritional perturbation of cell proliferation and migration induced by treatment with 2-deoxyglucose and metformin. On the contrary, spheroid formation is less sensitive to metabolic perturbations in 5637 than RT112 cells. The ability of metformin to reduce, although with different efficiency, cell proliferation, sphere formation and migration in both cell lines, suggests that OXPHOS targeting could be an effective strategy to reduce the invasiveness of Grade 2 bladder cancer cells.

Bladder cancer is one of the most prevalent deadly diseases worldwide. Grade 2 tumors represent a good window of therapeutic intervention, whose optimization requires high resolution biomarker identification. Here we characterize energy metabolism and cellular properties associated with spreading and tumor progression of RT112 and 5637, two Grade 2 cancer cell lines derived from human bladder, representative of luminal-like and basal-like tumors, respectively. The two cell lines have similar proliferation rates, but only 5637 cells show efficient lateral migration. In contrast, RT112 cells are more prone to form spheroids. RT112 cells produce more ATP by glycolysis and OXPHOS, present overall higher metabolic plasticity and are less sensitive than 5637 to nutritional perturbation of cell proliferation and migration induced by treatment with 2-deoxyglucose and metformin. On the contrary, spheroid formation is less sensitive to metabolic perturbations in 5637 than RT112 cells. The ability of metformin to reduce, although with different efficiency, cell proliferation, sphere formation and migration in both cell lines, suggests that OXPHOS targeting could be an effective strategy to reduce the invasiveness of Grade 2 bladder cancer cells.

Background: Recent evidence suggests that psychiatric symptoms share a common genetic liability across diagnostic categories. The present study investigated the effects of variants within previously identified relevant genes on specific symptom clusters, independently from the diagnosis. Methods: 1550 subjects affected by Schizophrenia (SCZ), Major Depressive Disorder or Bipolar Disorder were included. Symptoms were assessed using the Positive and Negative Syndrome Scale (PANSS) and the Hamilton Depression Rating Scale (HDRS). Principal component analysis and a further clinical refinement were used to define symptom clusters. Clusters scores were tested for association with 46 genetic variants within nine genes previously linked to one or more major psychiatric disorders by large genome wide association studies (ANK3, CACNA1C, CACNB2, FKBP5, FZD3, GRM7, ITIH3, SYNE1, TCF4). Exploratory analyses were performed in each disorder separately to further elucidate the SNPs effects. Results: five PANSS clusters (Negative; Impulsiveness; Cognitive; Psychotic; Depressive) and four HDRS clusters (Core Depressive; Somatic; Psychotic-like; Insomnia) were identified. CACNA1C rs11615998 was associated with HDRS Psychotic cluster in the whole sample. In the SCZ sample, CACNA1C rs11062296 was associated with PANSS Impulsiveness cluster and CACNA1C rs2238062 was associated with PANSS negative cluster. Discussion: CACNA1C rs11615998 was associated with psychotic symptoms (C-allele carriers have decreased psychotic-risk) independently from the diagnosis, in line with the evidence of a cross disorder effect of many risk variants. This gene was previously associated with SCZ and cross-disorder liability to psychiatric disorders. Our findings confirmed that deep phenotyping is pivotal to clarify the role of genetic variants on symptoms patterns.

Non-Communicable Diseases (NCDs) are among the most pressing global health problems of the twenty-first century. Their rising incidence and prevalence is linked to severe morbidity and mortality, and they are putting economic and managerial pressure on healthcare systems around the world. Moreover, NCDs are impeding healthy aging by negatively affecting the quality of life of a growing number of the global population. NCDs result from the interaction of various genetic, environmental and habitual factors, and cluster in complex ways, making the complex identification of resulting phenotypes not only difficult, but also a top research priority. The degree of complexity required to interpret large patient datasets generated by advanced high-throughput functional genomics assays has now increased to the point that novel computational biology approaches are essential to extract information that is relevant to the clinical decision-making process. Consequently, system-level models that interpret the interactions between extensive tissues, cellular and molecular measurements and clinical features are also being created to identify new disease phenotypes, so that disease definition and treatment are optimized, and novel therapeutic targets discovered. Likewise, Systems Medicine (SM) platforms applied to extensively-characterized patients provide a basis for more targeted clinical trials, and represent a promising tool to achieve better prevention and patient care, thereby promoting healthy aging globally. The present paper: (1) reviews the novel systems approaches to NCDs; (2) discusses how to move efficiently from Systems Biology to Systems Medicine; and (3) presents the scientific and clinical background of the San Raffaele Systems Medicine Platform.

Non-Communicable Diseases (NCDs) are among the most pressing global health problems of the twenty-first century. Their rising incidence and prevalence is linked to severe morbidity and mortality, and they are putting economic and managerial pressure on healthcare systems around the world. Moreover, NCDs are impeding healthy aging by negatively affecting the quality of life of a growing number of the global population. NCDs result from the interaction of various genetic, environmental and habitual factors, and cluster in complex ways, making the complex identification of resulting phenotypes not only difficult, but also a top research priority. The degree of complexity required to interpret large patient datasets generated by advanced high-throughput functional genomics assays has now increased to the point that novel computational biology approaches are essential to extract information that is relevant to the clinical decision-making process. Consequently, system-level models that interpret the interactions between extensive tissues, cellular and molecular measurements and clinical features are also being created to identify new disease phenotypes, so that disease definition and treatment are optimized, and novel therapeutic targets discovered. Likewise, Systems Medicine (SM) platforms applied to extensively-characterized patients provide a basis for more targeted clinical trials, and represent a promising tool to achieve better prevention and patient care, thereby promoting healthy aging globally. The present paper: (1) reviews the novel systems approaches to NCDs; (2) discusses how to move efficiently from Systems Biology to Systems Medicine; and (3) presents the scientific and clinical background of the San Raffaele Systems Medicine Platform.