Le neoplasie neuroendocrine (NENs) sono un gruppo di neoplasie rare che originano dal sistema neuroendocrino diffuso. Le due principali NENs sono quelle di origine pancreatica e broncopolmonare (rispettivamente pNENs e BP-NENs) e, a tutt’oggi, non esiste ancora una terapia efficace per queste neoplasie. Il trattamento di prima scelta è la resezione chirurgica che però non è consentita nella maggior parte dei casi. Anche altre terapie vengono utilizzate per il trattamento delle NENs. Gli inibitori a bersaglio molecolare hanno richiamato un crescente interesse ed alcune molecole sono state approvate per il trattamento di questo tipo di neoplasie. In particolare, Everolimus, inibitore di mTOR, è stato approvato sia per il trattamento di NENs broncopolmonari che pancreatiche mentre Sunitinib, inibitore di recettori tirosin-chinasici, è stato approvato per il trattamento di NEN di origine pancreatica. Nonostante questi farmaci si siano rivelati efficaci nel prolungare il tempo libero da progressione di malattia, lo sviluppo di resistenza alla terapia può verificarsi anche dopo brevi periodi. La ricerca di trattamenti efficaci è quindi un punto cruciale e, in questo studio, abbiamo cercato di affrontare il problema sotto diversi punti di vista. Abbiamo iniziato il nostro studio cercando di identificare il sistema migliore per coltivare una linea di pNEN in 3D dato che questo metodo di coltura ha ampiamente dimostrato di essere efficace negli studi in vitro. Abbiamo preso in considerazione tre sistemi di coltura in 3D resi possibili dall’utilizzo di 3 piastre: piastre da 96 pozzetti con tecnologia “hanging drop”, piastre da 24 pozzetti con una superficie repellente e piastre da 96 pozzetti con un fondo “ultra low attachment” (ULA). I risultati ottenuti sono stati comparati in termini di facilità di coltura e di errore di riproducibilità indicando le piastre ULA come il metodo più efficace. Grazie a questi risultati, il metodo ULA è stato utilizzato anche nelle seguenti parti dello studio. Dopo aver individuato il miglior metodo di coltura in 3D, ci siamo domandati se il noto inibitore Sunitinib potesse essere efficace anche nel trattamento delle NENs di origine broncopolmonare. I risultati hanno indicato un effetto antiproliferativo del farmaco, contrastato dal co-trattamento con fattori di crescita. Grazie a questi risultati sono stati testati altri due inibitori, l’inibitore di IGF1R Linsitinib e l’inibitore di EGFR Erlotinib, in modo da poter comprendere se farmaci con un bersaglio molecolare più specifico possano diminuire la vitalità cellulare nonostante l’azione proliferativa fattori di crescita. I risultati hanno indicato che sia Linsitinib che Erlotinib hanno una forte azione inibitoria, nonostante il co-trattamento con fattori di crescita. Inoltre, l’azione combinata dei due farmaci è in grado di generare una forte riduzione della vitalità e una massiva attivazione caspasica. Nell’ultima parte del nostro lavoro è stata valutata l’influenza del micorambiente tumorale (TME) nel comportamento e nella resistenza ai farmaci delle NENs broncopolmonari. Due linee cellulari di BP-NEN sono state coltivate in 2D ed in 3D con o senza l’aggiunta di una linea cellulare di fibroblasti polmonari e, per poter perseguire al meglio il nostro scopo, sul sistema è stata valutata l’azione di due farmaci: Dinaciclib, inibitore di cicline e CDK, ed Everolimus. I risultati ottenuti hanno evidenziato un ruolo del TME nella sensibilità ai farmaci/sviluppo tumorale ed hanno inoltre indicato una forte azione inibitoria promossa da Dinaciclib su entrambe le linee di BP-NENs. In conclusione, in questo lavoro sono state ottenute diverse indicazioni per lo studio, la comprensione e la gestione delle NENs. Questi risultati preliminari possono pertanto porre le basi per future indagini mirate ad una maggiore conoscenza delle NENs e ad una terapia più efficace.

Neuroendocrine Neoplasms (NENs) are a group of malignancies arising from cells of the diffuse endocrine system. Pancreatic and broncho-pulmonary NENs (pNENs and BP-NENs, respectively) represent the two main NENs in terms of incidence and, since surgery is not possible in most of the cases, are orphan of an effective therapy. Beyond surgery, different therapeutic approaches are available for NENs management. Molecular targeted therapies gained during years growing interest and different agents have been approved for NENs treatment. In particular, Everolimus, an mTOR inhibitor, has been approved for the treatment of both pNENs and BP-NENs while Sunitinib, a multiple tyrosine kinase receptors (RTKs) inhibitor, has been approved for pNENs treatment. However, despite these agents have shown to improve progression free survival, mechanisms of acquired resistance may occur leading to the need of new approaches. Therefore, finding an effective therapy for NENs represents a crucial issue and, in this study, we tried to approach this problem in different ways. We started our work trying to identify, through the use of a pNEN cell line, the best method to culture NENs in 3D since this growth system has already demonstrated its validity with in vitro studies. The three methods taken into consideration involved different tools: a 96-well hanging drop plate, a 24-well plate with a cell repellent surface and an ultra low attachment 96-well plate with clear round bottom (ULA). Results were compared and analysed in terms of easiness/reproducibility error and have indicated ULA plates method as the best to culture NENs. Therefore, we used this method in the following parts of the study. After the assessment of the best 3D culture method, we started our investigation on BP-NENs resistance wondering whether the well-known RTKs inhibitor Sunitinib could have been effective in the treatment of BP-NENs and not only pNENs. Sunitinib showed to have an antiproliferative effect on both the BP-NENs cell lines taken into consideration. In addition, its action was counteracted by some growth factors co-tested with the drug. Thanks to these results we tested other agents, the IGF1R inhibitor Linsitinib and the EGFR inhibitor Erlotinib, in order to understand whether a drug with a more specific target could have overcome Sunitinib resistance. Results indicated that Linsitinib and Erlotinib were able to decrease cell viability despite the proliferative action promoted by growth factors and that their combination induced a strong antiproliferative effect and caspase activation. Thanks to these encouraging findings we started to investigate a complex topic: the influence of tumour microenvironment (TME) in BP-NENs behaviour and drugs resistance. Therefore, we cultured two BP-NENs cell lines in 2D and in 3D with or without the addition of a lung fibroblast cell line. Moreover, in order to pursue our aim, we evaluated the effects of a CDKs and cyclines inhibitor, Dinaciclib, on the described system comparing its action with the one displayed by Everolimus. Our preliminary results underlined the influence of fibroblasts on BP-NENs behaviour/drug sensitivity indicating Dinaciclib as an effective agent in the treatment of this malignancy. In conclusion our study provided several indications useful for NENs study, drugs resistance comprehension and malignancy management. These data are preliminary results that can be useful for further investigations in order to achieve a better NENs knowledge and more effective therapies.

Pancreatic and broncho-pulmonary neuroendocrine neoplasms in vitro studies: a route from culture systems evaluation to approved and innovative therapeutic approaches

BRESCIANI, Giulia
2020-02-14T00:00:00+01:00

Abstract

Neuroendocrine Neoplasms (NENs) are a group of malignancies arising from cells of the diffuse endocrine system. Pancreatic and broncho-pulmonary NENs (pNENs and BP-NENs, respectively) represent the two main NENs in terms of incidence and, since surgery is not possible in most of the cases, are orphan of an effective therapy. Beyond surgery, different therapeutic approaches are available for NENs management. Molecular targeted therapies gained during years growing interest and different agents have been approved for NENs treatment. In particular, Everolimus, an mTOR inhibitor, has been approved for the treatment of both pNENs and BP-NENs while Sunitinib, a multiple tyrosine kinase receptors (RTKs) inhibitor, has been approved for pNENs treatment. However, despite these agents have shown to improve progression free survival, mechanisms of acquired resistance may occur leading to the need of new approaches. Therefore, finding an effective therapy for NENs represents a crucial issue and, in this study, we tried to approach this problem in different ways. We started our work trying to identify, through the use of a pNEN cell line, the best method to culture NENs in 3D since this growth system has already demonstrated its validity with in vitro studies. The three methods taken into consideration involved different tools: a 96-well hanging drop plate, a 24-well plate with a cell repellent surface and an ultra low attachment 96-well plate with clear round bottom (ULA). Results were compared and analysed in terms of easiness/reproducibility error and have indicated ULA plates method as the best to culture NENs. Therefore, we used this method in the following parts of the study. After the assessment of the best 3D culture method, we started our investigation on BP-NENs resistance wondering whether the well-known RTKs inhibitor Sunitinib could have been effective in the treatment of BP-NENs and not only pNENs. Sunitinib showed to have an antiproliferative effect on both the BP-NENs cell lines taken into consideration. In addition, its action was counteracted by some growth factors co-tested with the drug. Thanks to these results we tested other agents, the IGF1R inhibitor Linsitinib and the EGFR inhibitor Erlotinib, in order to understand whether a drug with a more specific target could have overcome Sunitinib resistance. Results indicated that Linsitinib and Erlotinib were able to decrease cell viability despite the proliferative action promoted by growth factors and that their combination induced a strong antiproliferative effect and caspase activation. Thanks to these encouraging findings we started to investigate a complex topic: the influence of tumour microenvironment (TME) in BP-NENs behaviour and drugs resistance. Therefore, we cultured two BP-NENs cell lines in 2D and in 3D with or without the addition of a lung fibroblast cell line. Moreover, in order to pursue our aim, we evaluated the effects of a CDKs and cyclines inhibitor, Dinaciclib, on the described system comparing its action with the one displayed by Everolimus. Our preliminary results underlined the influence of fibroblasts on BP-NENs behaviour/drug sensitivity indicating Dinaciclib as an effective agent in the treatment of this malignancy. In conclusion our study provided several indications useful for NENs study, drugs resistance comprehension and malignancy management. These data are preliminary results that can be useful for further investigations in order to achieve a better NENs knowledge and more effective therapies.
ZATELLI, Maria Chiara
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11392/2488168
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