Neuroendocrine tumors (NETs) are a heterogeneous group of tumors generally characterized by a variable expression of five different somatostatin receptors (SSTR1 to SSTR5). SSTRs mediate anti-proliferative and anti-secretory effects of the natural agonist, the hormone somatostatin (SST). The therapeutic use of native SST in vivo is restricted due to its short half-life, therefore various SST analogs (SSAs) have been developed and are currently used for the treatment, both pharmacological and radiotherapeutical, of NETs, as well as their diagnostic imaging. First-generation SSAs (octreotide, lanreotide) had affinity almost exclusively for SSTR2. However, several NETs were found not to respond to treatment with these drugs, and this was associated to low expression of SSTR2. Indeed, it has been demonstrated that the response of NETs to treatment with SSAs depends on the expression pattern of the different SSTRs. Thus, to extend the efficacy of SSAs to more NETs, pasireotide was developed, an analog that binds with high affinity SSTR5, followed by SSTR3 and SSTR2. There is still intense research ongoing to identify novel SSAs with different affinities for the SSTRs to further broaden their application for treatment/imaging of additional NETs. The aim of this work was to assess the potential of novel SSAs as diagnostic (imaging) and therapeutic (medical therapy and radiotherapy) tools for NETs. In the first part of the thesis, we evaluated ITF2984, a SSA with higher affinity for SSTR3 when compared to octreotide, as a putative new therapy for pancreatic NETs. Specifically, we investigated the antiproliferative effects of ITF2984, in a neuroendocrine pancreatic well-differentiated tumor model (NT-3 cells), closely resembling typical primary pancreatic NETs, and showing a relatively high expression of SSTR3. We performed our studies using 3D spheroid cultures of NT-3 cells, a more physiological model when compared to 2D monolayer cultures. We observed a strong antitumor effect of ITF2984 upon treatment of NT-3 cells for a prolonged time (18 days), consisting in a reduction of both size and integrity of the spheroids. ITF2984 was more effective than the multi-receptor ligand pasireotide. These studies indicate that ITF2984 is highly effective against NET cells predominantly expressing SSTR3. In the second part of the thesis, we investigated two novel SSAs currently in early preclinical development, I.e. PTR-01, a SSTR3 selective agent, and STR-01, a pan-statin SSA showing the same affinity for SSTR2, SSTR3 and SSTR5. For these new drugs no information about the effect on hormone secretion, on cell viability and on receptor binding was available. We conducted our studies using two different NET models: GH3 pituitary NET cells and NT-3 pancreatic cells already introduced above. We first investigated the inhibitory effect of the two agents on growth hormone (GH) secretion using a well-established model: GH3 cells. Contrary to our expectations, we could not observe an effect of the two novel drugs on the secretion of GH, but we could also not reproduce the inhibitory effect of the control SSA octreotide on hormone secretion. Thus, our data on the ability of PTR-01 and STR-01 cannot be considered conclusive and additional studies need to be done to understand the reason for the lack of hormone reduction upon SSA administration. Then, we validated the specificity and selectivity of the binding of PTR-01 and STR-01 to SSTRs through radiolabeled-peptide binding assay using cells with known level of expression of the receptors. HEK293 cells stably transfected with SSTR2 or SSTR3, and NT-3 cells having a high expression of SSTRs (especially of SSTR3) showed consistent uptake of the radiolabelled derivative of the two novel SSAs, namely [68Ga]-DOTA-PTR-01 and [68Ga]-DOTA-STR-01. The binding of [68Ga]-DOTA-PTR-01 to NT-3 cells was particularly high, thereby confirming that this drug has a high affinity for SSTR3.
Nuovi analoghi della somatostatina come strumento teranostico nei tumori neuroendocrini. I tumori neuroendocrini (NET) sono un gruppo eterogeneo di tumori caratterizzati generalmente dalla presenza di cinque diversi recettori della somatostatina (da SSTR1 a SSTR5). Questi recettori mediano gli effetti antiproliferativi e antisecretori dell'agonista naturale, l'ormone somatostatina (SST). L'uso terapeutico della SST naturale in vivo è limitato dalla sua breve emivita e per questo motivo sono stati sviluppati vari analoghi della SST (SSA) utilizzati attualmente per il trattamento farmacologico e radioterapico dei NET, e per la loro diagnostica. I SSAs di prima generazione (octreotide, lanreotide) hanno una specificità quasi esclusiva per il recettore SSTR2. Tuttavia, alcuni NET non rispondono a questi composti a causa di una bassa espressione di SSTR2. Si è dimostrato infatti che la risposta dei NET al trattamento con SSAs dipende dal pattern di espressione dei diversi SSTRs. Pertanto, è stato sviluppato pasireotide, un analogo che si lega con alta affinità ai recettori SSTR5, seguito da SSTR3 e SSTR2. Attualmente, c’è un intenso lavoro di ricerca per identificare nuovi SSAs con affinità differenti, per ampliare l'efficacia dei SSAs su un maggior numero di NET. L'obiettivo di questo lavoro è stato valutare il potenziale di nuovi SSAs come strumenti diagnostici (imaging) e terapeutici (terapia medica e radioterapia) per i NET. Nella prima parte della tesi è stato valutato ITF2984, un SSA con maggiore affinità per il recettore SSTR3, come nuova terapia per i NET pancreatici. In particolare, sono stati studiati gli effetti antiproliferativi di ITF2984 su colture di sferoidi tridimensionali di cellule NT-3, un modello neuroendocrino di tumore pancreatico ben differenziato che mostra un'espressione relativamente elevata di SSTR3. È stato osservato un forte effetto antitumorale nel trattamento con ITF2984 per un tempo prolungato (18 giorni), con una riduzione sia delle dimensioni che dell'integrità degli sferoidi. ITF2984 è risultato essere più efficace di pasireotide, un SSA già utilizzato in clinica. Questi studi indicano che l'ITF2984 è altamente efficace nelle cellule NET che esprimono prevalentemente SSTR3. Nella seconda parte della tesi, abbiamo investigato due nuovi SSAs attualmente in fase precoce di sviluppo preclinico, ossia PTR-01, un agente selettivo per SSTR3, e STR-01, un SSA pan-statin che mostra la stessa affinità per SSTR2, SSTR3 e SSTR5. Per questi nuovi composti non erano disponibili informazioni sull'effetto sulla secrezione ormonale, sulla vitalità cellulare e sul legame del recettore. Abbiamo condotto i nostri studi utilizzando due diversi modelli di NET: le cellule pituitary NET GH3 e le NT-3 introdotte precedentemente. Inizialmente, abbiamo investigato il loro effetto inibitorio sulla secrezione dell'ormone della crescita (GH) utilizzando un modello ben stabilito: le cellule GH3. Contrariamente alle nostre aspettative, non abbiamo osservato un effetto dei due nuovi composti sulla secrezione di GH, ma non siamo neanche stati in grado di riprodurre l'effetto inibitorio del SSA di controllo octreotide. Pertanto, i nostri dati sull'effetto sulla secrezione di GH di PTR-01 e STR-01 non possono essere considerati conclusivi e sono necessari ulteriori studi per comprendere la ragione della mancanza di riduzione della secrezione dell’ormone, dopo il trattamento con SSAs. Successivamente, abbiamo validato la specificità e la selettività del legame di PTR-01 e STR-01 ai SSTRs, tramite un test di legame con peptide radiomarcato, utilizzando cellule con un livello noto di espressione dei recettori.
Novel somatostatin analogs as theranostic tool in neuroendocrine tumors
MARANGELO, ALESSANDRO
2021/2022
Abstract
Neuroendocrine tumors (NETs) are a heterogeneous group of tumors generally characterized by a variable expression of five different somatostatin receptors (SSTR1 to SSTR5). SSTRs mediate anti-proliferative and anti-secretory effects of the natural agonist, the hormone somatostatin (SST). The therapeutic use of native SST in vivo is restricted due to its short half-life, therefore various SST analogs (SSAs) have been developed and are currently used for the treatment, both pharmacological and radiotherapeutical, of NETs, as well as their diagnostic imaging. First-generation SSAs (octreotide, lanreotide) had affinity almost exclusively for SSTR2. However, several NETs were found not to respond to treatment with these drugs, and this was associated to low expression of SSTR2. Indeed, it has been demonstrated that the response of NETs to treatment with SSAs depends on the expression pattern of the different SSTRs. Thus, to extend the efficacy of SSAs to more NETs, pasireotide was developed, an analog that binds with high affinity SSTR5, followed by SSTR3 and SSTR2. There is still intense research ongoing to identify novel SSAs with different affinities for the SSTRs to further broaden their application for treatment/imaging of additional NETs. The aim of this work was to assess the potential of novel SSAs as diagnostic (imaging) and therapeutic (medical therapy and radiotherapy) tools for NETs. In the first part of the thesis, we evaluated ITF2984, a SSA with higher affinity for SSTR3 when compared to octreotide, as a putative new therapy for pancreatic NETs. Specifically, we investigated the antiproliferative effects of ITF2984, in a neuroendocrine pancreatic well-differentiated tumor model (NT-3 cells), closely resembling typical primary pancreatic NETs, and showing a relatively high expression of SSTR3. We performed our studies using 3D spheroid cultures of NT-3 cells, a more physiological model when compared to 2D monolayer cultures. We observed a strong antitumor effect of ITF2984 upon treatment of NT-3 cells for a prolonged time (18 days), consisting in a reduction of both size and integrity of the spheroids. ITF2984 was more effective than the multi-receptor ligand pasireotide. These studies indicate that ITF2984 is highly effective against NET cells predominantly expressing SSTR3. In the second part of the thesis, we investigated two novel SSAs currently in early preclinical development, I.e. PTR-01, a SSTR3 selective agent, and STR-01, a pan-statin SSA showing the same affinity for SSTR2, SSTR3 and SSTR5. For these new drugs no information about the effect on hormone secretion, on cell viability and on receptor binding was available. We conducted our studies using two different NET models: GH3 pituitary NET cells and NT-3 pancreatic cells already introduced above. We first investigated the inhibitory effect of the two agents on growth hormone (GH) secretion using a well-established model: GH3 cells. Contrary to our expectations, we could not observe an effect of the two novel drugs on the secretion of GH, but we could also not reproduce the inhibitory effect of the control SSA octreotide on hormone secretion. Thus, our data on the ability of PTR-01 and STR-01 cannot be considered conclusive and additional studies need to be done to understand the reason for the lack of hormone reduction upon SSA administration. Then, we validated the specificity and selectivity of the binding of PTR-01 and STR-01 to SSTRs through radiolabeled-peptide binding assay using cells with known level of expression of the receptors. HEK293 cells stably transfected with SSTR2 or SSTR3, and NT-3 cells having a high expression of SSTRs (especially of SSTR3) showed consistent uptake of the radiolabelled derivative of the two novel SSAs, namely [68Ga]-DOTA-PTR-01 and [68Ga]-DOTA-STR-01. The binding of [68Ga]-DOTA-PTR-01 to NT-3 cells was particularly high, thereby confirming that this drug has a high affinity for SSTR3.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/15436