Despite the advances made in surgical and medical therapies, cancer is still a major cause of death in the world: patients often develop resistance to therapy and relapses due to the incomplete removal of cancer cells. In cancer therapy, the surgical tumour resection aims to remove the neoplastic tissue, along with a surrounding margin of normal non-tumoral tissue, in order to reduce the risk of relapses and improve patients’ survival. However, the procedure lacks an objective tool to intraoperative map the exact portion of tissue interested by the neoplastic growth. The aim of the present project was the preclinical development of an objective tool to label and circumscribe the tumour area, to guide surgeons in the intraoperative safe removal of cancer tissues. Here, we investigated novel drug delivery systems of natural origin named Extracellular Vesicles (EV) for the specific delivery of diagnostics at tumour sites, in order to allow the detection of tumours during the surgical procedure with increased sensitivity and accuracy. We encapsulated fluorescent dyes into EV from different biological sources and characterized the biodistribution of fluorescence in mouse models of cancer by in vivo and ex vivo imaging methodologies. In this experimental setting, we have identified EV isolated from cancer patients showing a selective tropism for neoplastic tissues; this tumour tropism is conserved among EV of tumoral origin and is independent from the tumour type and the species originating the vesicles. In PDX models we demonstrated that patient-derived EV recognize corresponding human tumours and deliver fluorescent agents inside cancer cells, providing a proof-of-principle demonstration of the possible translational use of patients’ EV in clinic. Taking advantage of omics technologies, we have identified a protein “signature” characterizing the PDEVs and candidate molecules for the mechanism of tumour targeting. From this signature we also discovered a novel promising biomarker for the prediction of CRC disease in liquid biopsy. In conclusion, this thesis presents the discovery and characterization of pathotropic nanoparticles derived from the blood of oncological patients as useful drug delivery tool to selectively target cancer cells. The biological properties of these autologous EV and their innate abilities to recognize tumours pave the way to the design of novel strategies of personalized diagnosis and therapies in cancer patients, where EV are loaded with diagnostics for the detection of neoplastic tissues, therapeutics (e.g. chemotherapeutic drugs) for targeted cancer therapies or theranostics for combined diagnostic and therapy.
Nonostante i progressi fatti nel campo delle terapie mediche e chirurgiche, il cancro rappresenta ancora una maggiore causa di morte nel mondo: i pazienti sviluppano spesso resistenza alle terapie antitumorali e recidive a causa della non completa deplezione di tutte le cellule tumorali. Tra le terapie antitumorali, la chirurgia mira alla rimozione completa del tessuto neoplastico, tipicamente asportato insieme ad un margine di tessuto sano non tumorale circostante allo scopo di ridurre il rischio di recidive ed aumentare la sopravvivenza dei pazienti. Tuttavia, ad oggi la procedura chirurgica di resezione tumorale manca ancora di uno strumento oggettivo in grado di mappare esattamente, nel contesto intraoperatorio, le porzioni di tessuto interessate dalla crescita neoplastica. Scopo di questo progetto era quindi lo sviluppo preclinico di uno strumento in grado di definire e circoscrivere in maniera oggettiva l’area tumorale, per guidare i chirurghi in una rimozione intraoperatoria più sicura del tessuto tumorale. Nello sviluppare questo progetto, ci siamo occupati dello studio di sistemi innovativi di origine naturale per la veicolazione di farmaci, chiamati Vescicole Extracellulari (EV), con i quali veicolare agenti diagnostici in maniera specifica ai siti tumorali, allo scopo di consentire la rilevazione di tumori durante l’operazione chirurgica con maggior sensibilità e precisione. Abbiamo incapsulato dei coloranti fluorescenti all’interno di vescicole extracellulari ottenute da diverse fonti biologiche e caratterizzato la biodistribuzione della fluorescenza veicolata in modelli tumorali murini tramite sistemi di imaging in vivo ed ex vivo. In questo contesto sperimentale abbiamo individuato vescicole extracellulari isolate da pazienti oncologici che mostrano un tropismo selettivo per i tessuti neoplastici; questo tropismo tumorale risulta conservato tra vescicole extracellulari di origine tumorale ed è indipendente sia dal tipo di tumore che dalla specie di origine delle vescicole. In modelli di xenotrapianto (PDX) abbiamo dimostrato che le vescicole derivate da pazienti sono in grado di riconoscere i corrispondenti tumori umani e veicolare i traccianti fluorescenti all’interno delle cellule tumorali stesse, fornendo così una prova di principio del possibile utilizzo delle vescicole di pazienti in clinica. Grazie all’utilizzo di tecniche omiche, abbiamo identificato una “firma” proteica caratterizzante le vescicole dei pazienti e delle molecole candidate per spiegare il meccanismo di targeting dei tumori. Da questa firma abbiamo inoltre scoperto un nuovo promettente biomarcatore per la predizione del tumore del colon-retto attraverso la biopsia liquida. In conclusione, questo lavoro di tesi presenta la scoperta e la caratterizzazione di nanoparticelle patotropiche derivate dal sangue di pazienti oncologici come utili strumenti per la veicolazione selettiva di sostanze attive alle cellule tumorali. Le proprietà biologiche di queste vescicole autologhe e le loro innate abilità di riconoscere i tumori aprono la strada allo sviluppo di nuove strategie per diagnosi e terapie personalizzate in pazienti oncologici, dove le vescicole extracellulari vengono caricate con agenti diagnostici per la rilevazione dei tessuti neoplastici, agenti terapeutici (es. farmaci chemioterapici) per terapie antitumorali mirate o agenti teranostici per diagnosi e terapia combinate.
TARGETED DELIVERY OF DIAGNOSTIC AGENTS TO NEOPLASTIC TISSUES: PRECLINICAL DEVELOPMENT OF NANOPARTICLES-BASED SYSTEMS FOR THE INTRAOPERATIVE LABELLING OF TUMOUR SITES / D. Crescenti ; tutor: P. Ciana ; coordinatore: G. D. Norata. Dipartimento di Scienze della Salute, 2022 Apr 04. 34. ciclo, Anno Accademico 2021.
TARGETED DELIVERY OF DIAGNOSTIC AGENTS TO NEOPLASTIC TISSUES: PRECLINICAL DEVELOPMENT OF NANOPARTICLES-BASED SYSTEMS FOR THE INTRAOPERATIVE LABELLING OF TUMOUR SITES
D. Crescenti
2022
Abstract
Despite the advances made in surgical and medical therapies, cancer is still a major cause of death in the world: patients often develop resistance to therapy and relapses due to the incomplete removal of cancer cells. In cancer therapy, the surgical tumour resection aims to remove the neoplastic tissue, along with a surrounding margin of normal non-tumoral tissue, in order to reduce the risk of relapses and improve patients’ survival. However, the procedure lacks an objective tool to intraoperative map the exact portion of tissue interested by the neoplastic growth. The aim of the present project was the preclinical development of an objective tool to label and circumscribe the tumour area, to guide surgeons in the intraoperative safe removal of cancer tissues. Here, we investigated novel drug delivery systems of natural origin named Extracellular Vesicles (EV) for the specific delivery of diagnostics at tumour sites, in order to allow the detection of tumours during the surgical procedure with increased sensitivity and accuracy. We encapsulated fluorescent dyes into EV from different biological sources and characterized the biodistribution of fluorescence in mouse models of cancer by in vivo and ex vivo imaging methodologies. In this experimental setting, we have identified EV isolated from cancer patients showing a selective tropism for neoplastic tissues; this tumour tropism is conserved among EV of tumoral origin and is independent from the tumour type and the species originating the vesicles. In PDX models we demonstrated that patient-derived EV recognize corresponding human tumours and deliver fluorescent agents inside cancer cells, providing a proof-of-principle demonstration of the possible translational use of patients’ EV in clinic. Taking advantage of omics technologies, we have identified a protein “signature” characterizing the PDEVs and candidate molecules for the mechanism of tumour targeting. From this signature we also discovered a novel promising biomarker for the prediction of CRC disease in liquid biopsy. In conclusion, this thesis presents the discovery and characterization of pathotropic nanoparticles derived from the blood of oncological patients as useful drug delivery tool to selectively target cancer cells. The biological properties of these autologous EV and their innate abilities to recognize tumours pave the way to the design of novel strategies of personalized diagnosis and therapies in cancer patients, where EV are loaded with diagnostics for the detection of neoplastic tissues, therapeutics (e.g. chemotherapeutic drugs) for targeted cancer therapies or theranostics for combined diagnostic and therapy.File | Dimensione | Formato | |
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phd_unimi_R12241.pdf
Open Access dal 10/09/2023
Descrizione: Tesi Ph.D.
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