Sviluppo di un sistema tridimensionale di coltura come modello preclinico di carcinoma del colon

Negli ultimi anni lo studio della biologia tumorale ha subito un progressivo sviluppo e molti aspetti fisiologici del microambiente tumorale sono stati chiariti, ma molto resta ancora da analizzare circa le modalità con cui il tumore interagisce con l’ambiente circostante e quali siano le caratteristiche molecolari di questo microambiente. Dal punto di vista tecnico, le colture cellulari bidimensionali (2D) riflettono solo parzialmente il pattern morfo-molecolare delle cellule tumorali umane ed inoltre non rispecchiano la complessità del microambiente in vivo. Nei sistemi in vivo, inoltre, lo sviluppo neoplastico differisce significativamente rispetto alle cellule coltivate in 2D, soprattutto per quanto riguarda la morfologia, la cinetica di crescita, l’espressione genica ed il grado di differenziazione. In questo scenario, le colture cellulari tridimensionali (3D) costituiscono un approccio alternativo e/o parallelo al 2D, esse sono dunque il punto di legame tra la coltura cellulare tradizionale e i modelli in vivo. L’utilizzo di sferoidi uni-cellulari e multi-cellulari si è rivelato un sistema efficiente per ottimizzare e superare le limitazioni legate ai sistemi convenzionali in vitro. Lo sferoide è costituito da cellule proliferanti nello strato periferico, che rispecchiano l’attivo turn-over delle cellule tumorali vicino ai capillari, e da cellule degli strati più interni che diventano, invece, quiescenti ed eventualmente muoiono, via apoptosi o necrosi, a causa della presenza di ipossia, man mano che il diametro dello sferoide aumenta. Al di sopra di 400-500µm di diametro si forma, infatti, un core necrotico, principalmente a causa della limitata diffusione di ossigeno e/o di nutrienti e all’accumulo di cataboliti e tossine. Tuttavia, nello studio di alcune tipologie neoplastiche, come ad esempio il carcinoma del colon, tale caratteristica potrebbe rappresentare un punto a favore più che un ostacolo, dal momento che la normale fisiologia del carcinoma del colon prevede la presenza di aree di tessuto altamente ipossiche e di tessuto necrotico. Al fine di creare un avanzato sistema 3D per la coltura di cellule del cancro del colon-retto in vitro, abbiamo comparato diversi metodi di coltura basati sulla tecnica “hanging drops”, cercando di ottimizzarli fino ad ottenere quello più efficiente per le nostre necessità e che mantenesse dimensioni e fenotipo costanti ed omogenee. Abbiamo inoltre valutato l’impatto di tali tecniche su linee cellulari di CRC inserite nel pannello di 60 linee cellulari del NCI, comparando fenotipo ed espressione genica delle colture in 3D rispetto alle tecniche di coltura tradizionali. Inoltre abbiamo analizzato il contributo della componente stromale sulla formazione e crescita degli sferoidi, utilizzando BM-MSC in co-coltura tridimensionale con le linee cellulari di CRC. I nostri risultati mettono in luce come l'istituzione di sistemi di coltura per una migliore integrazione tra le strutture in 3D, e maggiormente tra le cellule tumorali e la componente stromale, risulti fondamentale per uno studio più accurato della progressione tumorale, ma soprattutto per lo sviluppo e la validazione di nuovi farmaci antitumorali, che in questo modo possono esser testati anche su porzioni di tessuto che presentano ipossia e necrosi, andandone così a valutare la capacità di penetrazione in tutto il tessuto e la reale efficacia

Adult human cardiac stem cells become readily committed in pathological conditions: phenotypic and genetic evidence.

According to recent findings, pathological processes may deplete adult heart of stem cells. The scope of the present study was to assess proliferation, apoptosis and commitment of cardiac stem cells (CSCs) resident in normal (n=11) and pathological hearts with ischemic cardiomyopathy (n=20). Immunofluorescence and immunoblotting of CSCs isolated from normal and pathological hearts revealed higher expression of cardiac-specific markers in the latter. Proliferation of CSCs isolated from pathological hearts was 3.2±0.7-fold higher, while apoptosis induced by oxidative stress was 1.8±0.2-fold higher than in the normal cells. Asymmetric division, with unequal Notch distribution between daughter cells, occurred frequently in CSCs from ischemic hearts, while symmetric division was typical of normal cells. Gene expression was examined by stem cells specific PCR-based microarray. We confined our analysis to genes with at least 1.7-fold differential expression. Genes downregulated in pathological hearts belonged to the early stages of developmental process (snail1 homolog, jagged-1, ephrin b1), cell cycle control (mdm2, p15 and p16), neurogenesis, skeletal development, bone remodeling and cartilage development functional classes. Upregulated genes, among which OTF2, endothelin receptor, CD105 and MRG1, were involved in developmental maturation, mesenchymal cell differentiation, heart development and circulatory system processes. Major finding emerging from the analysis was the activation of the epithelial-mesenchymal transition and the upregulation of its inducer, TGFβ. The expression of genes related to TGFβ BMP-mediated signal transduction pathway was examined by PCR Array. The analysis confirmed the upregulation of genes as TGFβ-1 and -3, Nodal, ID1 and c-Myc, suggesting the activation of mesenchyme formation and CSCs differentiation towards cardiac cell lineages. In conclusion, CSCs in the normal and in the pathological heart differ in several respects and the differences reflect the activation of cardiac stem cells pool in the chronic pathological conditions. Given the apparent failure of intrinsic heart regeneration, further studies are warranted to optimize the strategies for CSCs application in regenerative medicine

Epicardium-derived cells and CD117-positive cells in the adult human heart: common origin through epithelial-mesenchymal transition.

Growing number of studies indicate epicardium-derived cells (EPDCs) as cardiac stem cells. While it is beyond doubt that these cells contribute to the normal developmentof the heart during organogenesis, it remains an open question whether mesothelial epicardial cells can undergo epithelial–mesenchymal transition, giving origin to cardiac primitive cells, in the adult human heart. We examined epicardium and subepicardium of the atria of human adult normal (n=11, mean age 41±12 years, 7 males, 4 females) and pathological hearts with chronic heart failure due to ischemic cardiomyopathy (n=22, mean age 55±5.5 years, 14 males, 8 females, mean ejection fraction 25±1%). Strikingly, only the normal adult human hearts were layered with epicardial cells. On the contrary, cell nuclei were absent from the surface lining of the diseased hearts. While normal epicardium resulted positive for cytokeratin 5/6, E-cadherin and Bves, in the pathological hearts the cells with epithelial markers were distributed among loose connective tissue of the subepicardium. Remarkably, these cells were CD117-positive and, when compared with subepicardium of normal heart, their number was 8.7-fold (p‹0.005) higher in the subepicardium of hearts with ischemic cardiomyopathy. In the PCR-based array of CD117-positive cells isolated from normal and pathological hearts, TGFβ and HGF receptor signaling pathways (both known inducers of EMT) resulted activated in the latter. Considering the hypothesis that EPDCs contribute to the cardiac CD117-positive cells population in the adult heart, fragments of epicardium of adult human cardiac atria were cultured on the extracellular matrix produced by cardiac fibroblasts, to obtain the outgrowth of the mesothelial cells forming epithelial sheets. When stimulated with TGFβ and HGF, intercellular contacts were lost and cells acquired mesenchymal characteristics and CD117 expression. Altogether, these results indicate that EPDCs enrich the pool of cardiac primitive cells that contribute to the regenerative properties of the adult human heart. It could be reasonably argued that CD117-positive and EPDCs represent the same primitive cell population

Induction of hypoxia and necrosis in multicellular tumor spheroids is associated with resistance to chemotherapy treatment

Culture of cancerous cells in standard monolayer conditions poorly mirrors growth in three-dimensional architectures typically observed in a wide majority of cancers of different histological origin. Multicellular tumor spheroid (MCTS) culture models were developed to mimic these features. However, in vivo tumor growth is also characterized by the presence of ischemic and necrotic areas generated by oxygenation gradients and differential access to nutrients. Hypoxia and necrosis play key roles in tumor progression and resistance to treatment. To provide in vitro models recapitulating these events in highly controlled and standardized conditions, we have generated colorectal cancer (CRC) cell spheroids of different sizes and analyzed their gene expression profiles and sensitivity to treatment with 5FU, currently used in therapeutic protocols. Here we identify three MCTS stages, corresponding to defined spheroid sizes, characterized by normoxia, hypoxia, and hypoxia plus necrosis, respectively. Importantly, we show that MCTS including both hypoxic and necrotic areas most closely mimic gene expression profiles of in vivo-developing tumors and display the highest resistance to 5FU. Taken together, our data indicate that MCTS may mimic in vitro generation of ischemic and necrotic areas in highly standardized and controlled conditions, thereby qualifying as relevant models for drug screening purposes

Musculoskeletal manifestations of childhood cancer and differential diagnosis with juvenile idiopathic arthritis (ONCOREUM): a multicentre, cross-sectional study

Summary Background Presenting symptoms of childhood cancers might mimic those of rheumatic diseases. However, the evidence available to guide differential diagnosis remains scarce. Preventing wrong or delayed diagnosis is therefore important to avoid incorrect administration of glucocorticoid or immunosuppressive therapy and worsening of prognosis. As such, we aimed to assess the prevalence and characteristics of presenting musculoskeletal manifestations in patients at cancer onset and to identify the factors that differentiate childhood malignancies with arthropathy from juvenile idiopathic arthritis. Methods We did a multicentre, cross-sectional study at 25 paediatric haemato-oncology centres and 22 paediatric rheumatology centres in Italy. We prospectively recruited patients who were younger than 16 years that were newly diagnosed with cancer or juvenile idiopathic arthritis. We excluded patients with glucocorticoid pre-treatment (>1 mg/kg per day of oral prednisone or equivalent for ≥2 consecutive weeks). We collected data for patients with a new diagnosis of cancer or juvenile idiopathic arthritis using an electronic case report form on a web-based platform powered by the Cineca Interuniversity Consortium. The primary outcome was to describe the frequency and characteristics of musculoskeletal manifestations at cancer onset; and the secondary outcome was to identify factors that could discriminate malignancies presenting with arthropathy, with or without other musculoskeletal symptoms, from juvenile idiopathic arthritis using multivariable logistic regression analysis. Findings Between May 1, 2015, and May 31, 2018, 1957 patients were eligible, of which 1277 (65%) had cancer and 680 (35%) had juvenile idiopathic arthritis. Musculoskeletal symptoms occurred in 324 (25% [95% CI 23·0–27·8]) of 1277 patients with cancer, of whom 207 had arthropathy. Patients with malignant bone tumours had the highest frequency of musculoskeletal symptoms (53 [80%] of 66), followed by patients with Langerhans histiocytosis (16 [47%] of 34), leukaemia (189 [32%] of 582), soft-tissue sarcomas (16 [24%] of 68), and neuroblastoma (21 [19%] of 109). In the 324 patients with cancer and musculoskeletal symptoms, the most common complaints were joint pain (199 [61%]), followed by limb bone pain (112 [35%]). Joint involvement had a prevalent monoarticular pattern (100 [48%] of 207) and oligoarticular pattern (86 [42%] had 2–4 joints involved and 20 [10%] had >4 joints involved), with the most frequently involved joints being the hip (88 [43%] of 207) and knee (81 [39%]). On multivariable analysis, limb bone pain was the independent variable most strongly associated with cancer (odds ratio [OR] 87·80 [95% CI 18·89–408·12]), followed by weight loss (59·88 [6·34–565·53]), thrombocytopenia (12·67 [2·40–66·92]), monoarticular involvement (11·30 [4·09–31·19]), hip involvement (3·30 [1·13–9·61]), and male sex (2·40 [1·03–5·58]). Factors independently associated with juvenile idiopathic arthritis were morning stiffness (OR 0·04 [95% CI 0·01–0·20]), joint swelling (0·03 [0·01–0·09]), and involvement of the small hand joints (0·02 [0–1·05]). Interpretation Our study provides detailed information about presenting musculoskeletal manifestations of childhood cancers and highlights the clinical and laboratory features that are most helpful in the differential diagnosis with juvenile idiopathic arthritis