Generation of induced pluripotent stem cell (iPSC) lines from a Joubert syndrome patient with compound heterozygous mutations in C5orf42 gene.

We have generated new disease-specific induced pluripotent stem cell (iPSC) lines from skin fibroblasts obtained from a female patient with Joubert syndrome (JS) caused by compound heterozygous mutations in C5orf42 gene. The generated iPSCs offer an unprecedented opportunity to obtain iPSC-derived neurons to investigate the pathogenesis of JS in vitro and to develop therapeutic strategies

Establishment of three Joubert syndrome-derived induced pluripotent stem cell (iPSC) lines harbouring compound heterozygous mutations in CC2D2A gene.

We have developed Joubert syndrome (JS)-derived induced pluripotent stem cell (iPSC) lines from dermal fibroblasts biopsied from a female patient harbouring novel compound heterozygous mutations in CC2D2A gene. The newly established iPSC lines provide tremendous promises for development of JS-derived neuronal cell lines to uncover the molecular and cellular mechanisms underlying the pathogenesis of JS and to develop therapeutic interventions for treatment of JS

Epithelial ovarian cancer: a study on the autophagic and microenvironmental regulation of cancer stem cells and the pro-tumorigenic role of casein kinase 1 delta

Epithelial ovarian cancer (EOC), the most lethal gynecological malignancy, is normally treated with surgery followed by platinum and taxane-based chemotherapy. Despite an initial high response rate, the majority of patients eventually relapse and die. The cause could be ascribed to a subpopulation of cancer cells, named cancer stem cells (CSC), endowed with self-renewal, high tumorigenic and metastatic potential, and drug resistance. Therefore, CSC eradication could be envisioned as a successful treatment. Part of the work included in this thesis stemmed from the idea that ovarian CSC could enjoy some growth advantage, deriving from intrinsic properties or from autocrine/paracrine circuits operating in tumor microenvironment. Our group and others demonstrated that CD44+CD117+ cells are likely EOC CSC. Recent studies indicated autophagy as a mechanism to survive chemotherapy and that CSC in particular could rely on this metabolic process. For this reason, we decided to evaluate autophagy activation and the effects of its perturbation in CSC from EOC ascitic effusions and from patient-derived xenografts (PDX) and we found that they presented a higher basal autophagy level compared to non-CSC. Autophagy blockade with chloroquine impaired CSC viability, in vitro spheroid growth, and in vivo tumorigenic potential. In addition, autophagy inhibition and carboplatin treatment had a synergistic effect both in vitro and in vivo. Our results suggest a possible clinical application of the combined therapy in EOC as this approach would counteract tumor growth and impair the CSC compartment at the same time, thus reducing tumor relapse. As the microenvironment contribution to CSC maintenance is revealing its growing importance, we focused on SCF, the ligand of CD117 (or c-Kit). Indeed, the SCF/c-Kit axis regulates cell viability, proliferation and differentiation both in physiological conditions and in cancer. Notably, SCF exists both as a soluble and transmembrane protein. We found that the EOC ascites contained high amounts of the cytokine. Soluble SCF was detected only in tumor-associated fibroblasts and macrophages supernatants, whereas the membrane isoform was also expressed by cancer cells. Both SCF isoforms were effective in activating Akt pathway in CD117+ cells, while the tyrosine kinase inhibitor imatinib abolished this effect. Accordingly, results obtained in spheroid assays suggest that SCF stimulation could enhance the canonical CSC properties and imatinib pre-treatment inhibited them. Overall, SCF stimulation might help CSC to survive in selective culture conditions and, more extensively, could support CSC survival in patient ascitic fluid. Besides the role played by CSC in tumor outgrowth, the malignant cells forming the tumor bulk are sustained by pro-tumorigenic protein activation. Thus, we investigated the role of casein kinase 1 delta (CK1δ), member of a kinase family involved in many cellular processes, which is genetically amplified in ovarian cancer and overexpressed in other cancer types. Since CK1δ inhibition induced cell cycle arrest and apoptosis in a variety of tumor cell lines and delayed tumor growth in vivo, we evaluated the effects of CK1δ knockdown on two EOC cell lines (OVCAR3 and IGROV1) both in vitro and in vivo. Silenced cells grew more slowly and were less tumorigenic in vivo. Moreover, CK1δ knockdown sensitized ovarian cancer cells to carboplatin treatment. Interestingly, CK1δ-depleted OVCAR3 cells resulted more prone to migrate in vitro and more metastatic in vivo, but these results need further confirmation in IGROV1 cells. Since numerous small molecule inhibitors targeting CK1δ have been synthesized and tested, it would be important to unveil the molecular mechanism linking CK1δ to metastatic potential before these compounds enter the clinic for EOC management. This thesis encompasses three different topics, characterized by the common aim of investigating EOC biology from different perspectives, in order to highlight novel weak points of this deadly disease.Il carcinoma ovarico (EOC) è solitamente trattato tramite chirurgia e chemioterapia a base di platino e taxani. Nonostante un’iniziale risposta, nella maggior parte delle pazienti l’esito è la ricaduta e la morte. La causa può essere legata ad una popolazione di cellule cancerose, le cellule staminali tumorali (CSC), dotate di capacità di autorinnovamento, grande potenziale tumorigenico e metastatico e resistenza ai chemioterapici. Per questo, la loro eradicazione potrebbe essere una strategia terapeutica molto efficace. Parte di questo lavoro deriva dall’idea che le CSC godano di un vantaggio di crescita dovuto a caratteristiche intrinseche o a circuiti paracrini che si instaurano nel microambiente neoplastico. Il nostro gruppo ha dimostrato che le cellule CD44+CD117+ sono verosimilmente le CSC nell’EOC. Studi recenti hanno dimostrato che l’autofagia è un meccanismo di sopravvivenza alla chemioterapia e che le CSC potrebbero dipendere da questo processo. Abbiamo quindi valutato i livelli di autofagia e gli effetti causati da sue alterazioni su CSC isolate da asciti di pazienti con EOC e da xenotrapianti. Le CSC presentano un livello autofagico basale più alto rispetto alle non-CSC. Il blocco dell’autofagia compromette la vitalità delle CSC, la formazione di sferoidi in vitro e il loro potenziale tumorigenico in vivo. Inoltre, l’inibizione dell’autofagia e il trattamento con carboplatino (CPT) mostrano un effetto sinergico sia in vitro che in vivo. I nostri risultati suggeriscono una possibile applicazione clinica nell’EOC di tale terapia combinata, in quanto contrasterebbe la crescita tumorale colpendo al contempo il compartimento staminale, riducendo così il rischio di recidiva. Visto il contributo dato dal microambiente al mantenimento delle CSC, abbiamo posto la nostra attenzione su SCF, il ligando di CD117 (c-Kit). Infatti, l’asse SCF/c-Kit controlla la vitalità, la proliferazione e il differenziamento cellulare sia in contesti fisiologici che nel cancro. SCF esiste sia in forma solubile che associata alla membrana plasmatica. Abbiamo misurato alte concentrazioni della citochina in asciti delle pazienti affette da EOC. La forma solubile è rilevabile solo in surnatanti di macrofagi e fibroblasti associati al tumore, mentre quella di membrana è espressa anche da cellule tumorali. Entrambe le isoforme possono attivare la via di Akt in cellule CD117+, mentre l’inibitore tirosin-chinasico imatinib blocca questo effetto. Similmente, i saggi su sferoidi suggeriscono che la stimolazione mediata da SCF può potenziare le proprietà canoniche delle CSC, inibite dal pretrattamento con imatinib. La stimolazione da parte di SCF può quindi favorire la sopravvivenza delle CSC, e supportarle all’interno dell’ascite. A parte le CSC, il cancro è sostenuto dall’attivazione di proteine pro-tumorali. Per questo, abbiamo indagato il ruolo della casein chinasi 1 delta (CK1δ), coinvolta in molteplici processi cellulari, la cui amplificazione genica o iperespressione è stata descritta nell’EOC e in molte altre neoplasie. Poiché è noto che l’inibizione di CK1δ induce arresto del ciclo cellulare e apoptosi in linee cellulari tumorali e rallenta la crescita dei tumori in vivo, abbiamo valutato in vitro e in vivo gli effetti del silenziamento della CK1δ su due linee cellulari di carcinoma ovarico (OVCAR3 e IGROV1). Le cellule silenziate crescono più lentamente e sono meno tumorigeniche. Inoltre, il silenziamento di CK1δ sensibilizza le cellule al trattamento con CPT. Inoltre, le cellule OVCAR3 silenziate per CK1δ risultano più prone a migrare in vitro e più metastatiche in vivo. Poiché sono state sintetizzate numerose molecole che inibiscono la CK1δ, sarebbe importante delineare il meccanismo molecolare che lega la CK1δ al potenziale metastatico prima che questi composti possano entrare nella pratica clinica per il trattamento dell’EOC. Questa tesi è comprensiva di tre differenti macroaree, caratterizzate dal comune scopo di studiare la biologia dell’EOC da differenti punti di vista, al fine di portare alla luce nuovi punti deboli di questa malattia mortale

Eye model for floaters’ studies: production of 3D printed scaffolds

Floaters are aggregates of collagen that form inside the vitreous body. They originate by liquefaction of the vitreous and result in visual field distortions. In some cases, the patient discomfort is so severe that surgical removal is required, with the risk of consequent complications. However, floaters have yet to be fully studied and understood in the way and time they originate. The introduction of 3D printing boosted the development of new tools for scientific research such as bio-models. Bio-model applications range from surgical training to implants fabrication. Several models are also bioprinted as rigid scaffolds or bio inks containing living cells. Medical implants are often produced by Stereolithography (SLA) to build complex geometries and meet the desired mechanical properties with high-dimensional accuracies. This paper focuses on the optimization of the printing parameters of an SLA structure to obtain a scaffold with the required characteristic for a proper 3D cell culture and investigation. The model will be the starting point for a future study regarding the etiology and formation mechanism of eye floaters in cell culture. The studied 3D printing parameters are layer thickness, exposure time, and light blocker content added to a biocompatible resin. Due to the final application, the main required property of the scaffold is transparency that allows visual inspection under optical microscope. The selected samples showed a good biocompatibility and visibility under optical microscope, both promising results for long-term cell cultures

Autophagy inhibition reduces chemoresistance and tumorigenic potential of human ovarian cancer stem cells

Epithelial ovarian cancer (EOC) is one of the most malignant gynecological tumors with a high mortality rate owing to tumor relapse after anticancer therapies. It is widely accepted that a rare tumor cell population, known as cancer stem cells (CSC), is responsible for tumor progression and relapse; intriguingly, these cells are able to survive nutrient starvation (such as in vitro culture in the absence of glucose) and chemotherapy treatment. Recent data also indicated that chemotherapy resistance is associated with autophagy activation. We thus decided to investigate both in vitro and in vivo the autophagic activity and the effects of the perturbation of this pathway in CSC isolated from EOC ascitic effusions. Ovarian CSC, identified according to their CD44/CD117 co-expression, presented a higher basal autophagy compared with the non-stem counterpart. Inhibition of this pathway, by in vitro chloroquine treatment or CRISPR/Cas9 ATG5 knockout, impaired canonical CSC properties, such as viability, the ability to form spheroidal structures in vitro, and in vivo tumorigenic potential. In addition, autophagy inhibition showed a synergistic effect with carboplatin administration on both in vitro CSC properties and in vivo tumorigenic activity. On the whole, these results indicate that the autophagy process has a key role in CSC maintenance; inhibition of this pathway in combination with other chemotherapeutic approaches could represent a novel effective strategy to overcome drug resistance and tumor recurrence