Caratterizzazione strutturale e funzionale dei fenotipi vascolari nel tumore polmonare

La crescita e lo sviluppo dei tumori solidi è dipendente dalla presenza di un adeguato supporto vascolare senza il quale il tumore rimane inattivo e incapace di metastatizzare. Il ruolo dell'angiogenesi nella progressione dei tumori polmonari è stato largamente descritto e molte strategie terapeutiche hanno come obiettivo l’inibizione del processo angiogenico. Scopo di questo studio è stata la valutazione, a livello tissutale, della presenza di riarrangiamenti strutturali dei vasi sanguigni e linfatici nel carcinoma polmonare umano. E’ stata inoltre studiata l'incidenza di cellule progenitrici vascolari riconosciute attraverso l’espressione del recettore di PDGF. Inoltre, abbiamo espanso in vitro popolazioni di cellule endoteliali ematiche e linfatiche ottenute dalle porzioni distale e neoplastica di polmoni, allo scopo di caratterizzare fenotipi vascolari anche dal punto di vista funzionale. Lo studio è stato condotto su 13 pazienti affetti da carcinoma polmonare e su dodici polmoni di pazienti in cui non era stata documentata alcuna patologia a carico del polmone, utilizzati come controllo (CTRL). Sul tessuto polmonare, utilizzato sia per isolamento cellulare che per la valutazione istologica, è stata effettuata un'analisi morfometrica ed immunoistochimica. Le porzioni tumorale (T) e distale (Dist) sono state considerate separatamente. Abbiamo potuto documentare una maggiore incidenza delle strutture vascolari (capillari e arteriole) nella porzione tumorale, sia rispetto alla porzione distale che rispetto ai campioni di controllo. Inoltre, la distribuzione di cellule progenitrici vascolari è risultata essere significativamente modificata nei campioni patologici. In particolare, i dati hanno mostrato una significativa deplezione di cellule PDGFRpos nelle porzioni distali mentre la loro incidenza era notevolmente aumentata nell'area neoplastica. Cellule endoteliali ematiche (BEC) e linfatiche (LEC) sono state caratterizzate mediante immunocitochimica. Abbiamo valutato sia l'espressione dei marcatori endoteliali che la presenza di recettori tirosin-chinasici (VEGFR2, VEGFR3, PDGFR). Su queste popolazioni cellulari sono state valutate anche la capacità angiogenica e migratoria mediante saggi funzionali in vitro. Il nostro studio può avere diverse implicazioni. Primo, l'alterazione strutturale e i cambiamenti nella distribuzione delle strutture vascolari possono rappresentare un potenziale indice prognostico del carcinoma polmonare. Inoltre, la nostra capacità di isolare le cellule endoteliali umane polmonari neoplastiche e normali può rappresentare un punto di partenza per studi finalizzati allo sviluppo di specifiche strategie terapeutiche mirate ad interferire con il microambiente tumorale.The growth and development of solid tumors is critically dependent on a functional vascular supply in the absence of which tumors remain dormant and unable to metastasize. As with all solid tumors, the role of angiogenesis is well established in the progression of lung cancers and the development of new therapeutic strategies designed to inhibit tumor-induced angiogenesis should prevent the tumor growth and metastasis holding great promise for the advancement of lung cancer management. The aim of the present study was to characterize at tissue level the structural rearrangements of blood and lymphatic vessels in human lung cancer. The incidence of vascular progenitor cells through the evaluation of PDGF- receptor expression was also investigated. In addition, we expanded in vitro Hematic and Lymphatic endothelial cell populations from normal and neoplastic human lungs in order to characterize vascular phenotypes and to evaluate their functional properties. The study was performed on 13 patients affected by lung cancer and on twelve lungs from patients lacking pathologic states used as control (CTRL). On lung tissue, utilized both for cell isolation and histologic assessment, was performed morphometric and immunohistochemical analysis, sharing samples in tumoral portion (T) and distal area (Dist). We could document an increased incidence of vascular structures (capillaries and arterioles) in tumor area, when compared to the distal portion and to control samples. Moreover, vascular progenitor cell distribution was significantly altered in pathologic samples. In particular, our data showed that in distal portions there was a significative depletion of PDGFRpos cells, on the other hand their incidence was markedly increased within the neoplastic area. Hematic (BEC) and Lymphatic Endothelial Cells (LEC) were characterized by immunocytochemistry. We evaluated the expression of endothelial marker and we also assessed the presence of Tyrosine Kinase Receptors (i.e. VEGFR2, VEGFR3, PDGFR). The angiogenic and migration abilities of endothelial cell population were also investigated. Our results may open new prospectives in the development of new therapeutic strategies designed to interfere with tumor microenvironment which plays an important role in cancer growth, since the vascular niche supports the proliferation of neoplastic cells

IGF-1 loaded injectable microspheres for potential repair of the infarcted myocardium

The use of injectable scaffolds to repair the infarcted heart is receiving great interest. Thermosensitive polymers, in situ polymerization, in situ cross-linking, and self-assembling peptides are the most investigated approaches to obtain injectability. Aim of the present work was the preparation and characterization of a novel bioactive scaffold, in form of injectable microspheres, for cardiac repair. Gellan/gelatin microspheres were prepared by a water-in-oil emulsion and loaded by adsorption with Insulin-like growth factor 1 to promote tissue regeneration. Obtained microspheres underwent morphological, physicochemical and biological characterization, including cell culture tests in static and dynamic conditions and in vivo tests. Morphological analysis of the microspheres showed a spherical shape, a microporous surface and an average diameter of 66 ± 17mm (under dry conditions) and 123 ± 24 mm (under wet conditions). Chemical Imaging analysis pointed out a homogeneous distribution of gellan, gelatin and Insulin-like growth factor-1 within the microsphere matrix. In vitro cell culture tests showed that the microspheres promoted rat cardiac progenitor cells adhesion, and cluster formation. After dynamic suspension culture within an impeller-free bioreactor, cells still adhered to microspheres, spreading their cytoplasm over microsphere surface. Intramyocardial administration of microspheres in a cryoinjury rat model attenuated chamber dilatation, myocardial damage and fibrosis and improved cell homing. Overall, the findings of this study confirm that the produced microspheres display morphological, physicochemical, functional and biological properties potentially adequate for future applications as injectable scaffold for cardiac tissue engineering

Functionalised peptide hydrogel for the delivery of cardiac progenitor cells.

Heart failure (HF) remains one of the leading causes of death worldwide; most commonly developing after myocardial infarction (MI). Since adult cardiomyocytes characteristically do not proliferate, cells lost during MI are not replaced. As a result, the heart has a limited regenerative capacity. There is, therefore, a need to develop novel cell-based therapies to promote the regeneration of the heart after MI. The delivery and retention of cells at the injury site remains a significant challenge. In this context, we explored the potential of using an injectable, RGDSP-functionalised self-assembling peptide - FEFEFKFK - hydrogel as scaffold for the delivery and retention of rat cardiac progenitor cells (CPCs) into the heart. Our results show that culturing CPCs in vitro within the hydrogel for one-week promoted their spontaneous differentiation towards adult cardiac phenotypes. Injection of the hydrogel on its own, or loaded with CPCs, into the rat after injury resulted in a significant reduction in myocardial damage and left ventricular dilation

Integrated MRI–Immune–Genomic Features Enclose a Risk Stratification Model in Patients Affected by Glioblastoma

Simple Summary: Despite crucial scientific advances, Glioblastoma (GB) remains a fatal disease with limited therapeutic options and a lack of suitable biomarkers. The unveiled competence of the brain immune system together with the breakthrough advent of immunotherapy has shifted the present translational research on GB towards an immune-focused perspective. Several clinical trials targeting the immunosuppressive GB background are ongoing. So far, results are inconclusive, underpinning our partial understanding of the complex cancer-immune interplay in brain tumors. High throughput Magnetic Resonance (MR) imaging has shown the potential to decipher GB heterogeneity, including pathologic and genomic clues. However, whether distinct GB immune contextures can be deciphered at an imaging scale is still elusive, leaving unattained the non-invasive achievement of prognostic and predictive biomarkers. Along these lines, we integrated genetic, immunopathologic and imaging features in a series of GB patients. Our results suggest that multiparametric approaches might offer new efficient risk stratification models, opening the possibility to intercept the critical events implicated in the dismal prognosis of GB. Abstract: Background: The aim of the present study was to dissect the clinical outcome of GB patients through the integration of molecular, immunophenotypic and MR imaging features. Methods: We enrolled 57 histologically proven and molecularly tested GB patients (5.3% IDH-1 mutant). Two- Dimensional Free ROI on the Biggest Enhancing Tumoral Diameter (TDFRBETD) acquired by MRI sequences were used to perform a manual evaluation of multiple quantitative variables, among which we selected: SD Fluid Attenuated Inversion Recovery (FLAIR), SD and mean Apparent Diffusion Coefficient (ADC). Characterization of the Tumor Immune Microenvironment (TIME) involved the immunohistochemical analysis of PD-L1, and number and distribution of CD3+, CD4+, CD8+ Tumor Infiltrating Lymphocytes (TILs) and CD163+ Tumor Associated Macrophages (TAMs), focusing on immune-vascular localization. Genetic, MR imaging and TIME descriptors were correlated with overall survival (OS). Results: MGMT methylation was associated with a significantly prolonged OS (median OS = 20 months), while no impact of p53 and EGFR status was apparent. GB cases with high mean ADC at MRI, indicative of low cellularity and soft consistency, exhibited increased OS (median OS = 24 months). PD-L1 and the overall number of TILs and CD163+TAMs had a marginal impact on patient outcome. Conversely, the density of vascular-associated (V) CD4+ lymphocytes emerged as the most significant prognostic factor (median OS = 23 months in V-CD4high vs. 13 months in V-CD4low, p = 0.015). High V-CD4+TILs also characterized TIME of MGMTmeth GB, while p53mut appeared to condition a desert immune background. When individual genetic (MGMTunmeth), MR imaging (mean ADClow) and TIME (V-CD4+TILslow) negative predictors were combined, median OS was 21 months (95% CI, 0–47.37) in patients displaying 0–1 risk factor and 13 months (95% CI 7.22–19.22) in the presence of 2–3 risk factors (p = 0.010, HR = 3.39, 95% CI 1.26–9.09). Conclusion: Interlacing MRI–immune–genetic features may provide highly significant risk-stratification models in GB patients

Advanced CT imaging features reflect distinct tissue immune profiles and exhibit high prognostic impact on NSCLC

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Combined Inhibition of CDK4/6 and PI3K/AKT/mTOR Pathways Induces a Synergistic Anti-Tumor Effect in Malignant Pleural Mesothelioma Cells.

Malignant pleural mesothelioma (MPM) is a progressive malignancy associated to the exposure of asbestos fibers. The most frequently inactivated tumor suppressor gene in MPM is CDKN2A/ARF, encoding for the cell cycle inhibitors p16INK4a and p14ARF, deleted in about 70% of MPM cases. Considering the high frequency of alterations of this gene, we tested in MPM cells the efficacy of palbociclib (PD-0332991), a highly selective inhibitor of cyclin-dependent kinase (CDK) 4/6. The analyses were performed on a panel of MPM cell lines and on two primary culture cells from pleural effusion of patients with MPM. All the MPM cell lines, as well as the primary cultures, were sensitive to palbociclib with a significant blockade in G0/G1 phase of the cell cycle and with the acquisition of a senescent phenotype. Palbociclib reduced the phosphorylation levels of CDK6 and Rb, the expression of myc with a concomitant increased phosphorylation of AKT. Based on these results, we tested the efficacy of the combination of palbociclib with the PI3K inhibitors NVP-BEZ235 or NVP-BYL719. After palbociclib treatment, the sequential association with PI3K inhibitors synergistically hampered cell proliferation and strongly increased the percentage of senescent cells. In addition, AKT activation was repressed while p53 and p21 were up-regulated. Interestingly, two cycles of sequential drug administration produced irreversible growth arrest and senescent phenotype that were maintained even after drug withdrawal. These findings suggest that the sequential association of palbociclib with PI3K inhibitors may represent a valuable therapeutic option for the treatment of MPM

Injectable gellan/gelatin microspheres for the treatment of heart failure

Injectable scaffolds are receiving a great interest as strategies to repair the infarcted myocardium. The aim of this work was the preparation and characterization of injectable microspheres for myocardial tissue engineering. Gellan/gelatin microspheres were prepared by a single water-in oil emulsion, using phosphatidylcholine as surfactant. The obtained particles underwent morphological, physicochemical and functional investigation. Loading with IGF-1 was then performed by adsorption. Loaded and unloaded particles, in combination with rat cardiac progenitor cells, were injected after cryoinjury on the rat heart. The morphological analysis showed a spherical shape and microporous surface. The infrared analysis confirmed the presence of interactions among the functional groups of the two biopolymers. Injectability through a narrow needle was verified. Preliminary in vivo tests, performed by three intramyocardial injections of cell loaded microspheres in a rat model of myocardial injury, documented that progenitor cells homed to the damaged myocardium and IGF-1 functionalization increased their engraftment. The results obtained showed adequate morphological, physicochemical, functional and biological properties for application as injectable scaffold for myocardial repair. This work was supported by the European Commission FP7 Programme, grant 214539

Development of Biomimetic Alginate/Gelatin/Elastin Sponges with Recognition Properties toward Bioactive Peptides for Cardiac Tissue Engineering

In recent years, there has been an increasing interest toward the covalent binding of bioactive peptides from extracellular matrix proteins on scaffolds as a promising functionalization strategy in the development of biomimetic matrices for tissue engineering. A totally new approach for scaffold functionalization with peptides is based on Molecular Imprinting technology. In this work, imprinted particles with recognition properties toward laminin and fibronectin bioactive moieties were synthetized and used for the functionalization of biomimetic sponges, which were based on a blend of alginate, gelatin, and elastin. Functionalized sponges underwent a complete morphological, physicochemical, mechanical, functional, and biological characterization. Micrographs of functionalized sponges showed a highly porous structure and a quite homogeneous distribution of imprinted particles on their surface. Infrared and thermal analyses pointed out the presence of interactions between blend components. Biodegradation and mechanical properties appeared adequate for the aimed application. The results of recognition tests showed that the deposition on sponges did not alter the specific recognition and binding behavior of imprinted particles. In vitro biological characterization with cardiac progenitor cells showed that early cell adherence was promoted. In vivo analysis showed that developed scaffolds improved cardiac progenitor cell adhesion and differentiation toward myocardial phenotypes

Low vagally-mediated heart rate variability and increased susceptibility to ventricular arrhythmias in rats bred for high increased susceptibility anxiety

In humans, there is a documented association between anxiety disorders and cardiovascular disease. Putative underlying mechanisms may include an impairment of the autonomic nervous system control of cardiac function. The primary objective of the present study was to characterize cardiac autonomic modulation and susceptibility to arrhythmias in genetic lines of rats that differ largely in their anxiety level. To reach this goal, electrocardiographic recordings were performed in high-anxiety behavior (HAB, n = 10) and low-anxiety behavior (LAB, n = 10) rats at rest, during stressful stimuli and under autonomic pharmacological manipulations, and analyzed by means of time- and frequency-domain indexes of heart rate variability. During resting conditions, HAB rats displayed a reduced heart rate variability, mostly in terms of lower parasympathetic (vagal) modulation compared to LAB rats. In HAB rats, this relatively low cardiac vagal control was associated with smaller heart rate responsiveness to acute stressors compared to LAB counterparts. In addition, beta-adrenergic pharmacological stimulation induced a larger incidence of ventricular tachyarrhythmias in HABs compared to LABs. At sacrifice, a moderate increase in heart-body weight ratio was observed in HAB rats. We conclude that high levels of anxiety-related behavior in rats are associated with signs of i) impaired autonomic modulation of heart rate (low vagally-mediated heart rate variability), ii) poor adaptive heart rate responsiveness to stressful stimuli, iii) increased arrhythmia susceptibility, and iv) cardiac hypertrophy. These results highlight the utility of the HAB/LAB model for investigating the mechanistic basis of the comorbidity between anxiety disorders and cardiovascular disease