The TNF-related apoptosis-inducing ligand (TRAIL) is a cell surface transmembrane protein primarily involved in immunosurveillance. It can also be released into the extracellular space, both as a transmembrane cargo of exosomes and as a less active soluble factor. Due to TRAIL's selective cytotoxic activity against cancer cells, many TRAIL receptor agonists have been developed as potential cancer therapies. However, these did not exhibit the expected outcomes in clinical trials, mainly due to poor pharmacokinetics and inability to induce receptor clustering. These issues could potentially be addressed by engineering cancer-associated fibroblasts (CAFs)—the most accessible cells within the tumor microenvironment (TME)—to produce full-length TRAIL via a gene therapy approach, as proposed in the AIRC-funded project “ANAKIN.” CAFs have recently emerged as promising targets for other anticancer therapies as well, but the development of such strategies is slowed down by the lack of well-characterized and standardized CAF preclinical models. This work aimed to develop and characterize an in vitro CAF model based on transforming growth factor beta (TGF-β) treatment of NIH/3T3 fibroblasts (NIH/3T3-CAFs) and to use this model to provide proof of concept that, regardless of the gene therapy vector used, the strategy proposed by the ANAKIN project could be effective. Cellular differentiation studies revealed that treated NIH/3T3 cells exhibit some CAF-like features within 24 hours of treatment with 5 ng/mL TGF-β, including specific marker expression and a highly glycolytic metabolic profile. These traits are maintained for at least five days after the removal of the stimulus. We therefore provided a protocol for the generation and sub-culturing of an easy-to-use, standardized, and readily available CAF model. However, since some phenotypes, such as enhanced proliferation and migration characteristics typical of CAFs, as well as an influence on cancer cell growth, were not observed, there are areas for improvement in CAF modeling. Regarding the validation of the feasibility of the “ANAKIN” strategy, we first optimized the production of a fluorescently tagged TRAIL variant in NIH/3T3-CAFs through transfection with commercial agents. Starting with plasmid DNA (pDNA) transfection, we subsequently selected mRNA transfection based on its higher efficiency and lack of toxicity. We then investigated the subcellular localization of both tagged and untagged TRAIL in transfected NIH/3T3-CAFs, ultimately abandoning the fusion protein due to its mislocalization. Finally, we explored the release of the native protein by TRAIL+ transfected NIH/3T3-CAFs and examined its cytotoxic effect on cancer cells. Our studies revealed that, following mRNA transfection, TRAIL does not reach the plasma membrane of NIH/3T3-CAFs. However, TRAIL can be released into the extracellular space, possibly as a membrane cargo of exosomes, but in insufficient quantities to exert an apoptotic effect on cancer cells. Therefore, to make the proposed strategy effective, it is essential to find a way to increase TRAIL release, other than its production, perhaps by promoting its trafficking to the plasma membrane.

Il ligando induttore di apoptosi correlato al TNF (TRAIL) è una proteina transmembrana espressa sulla superficie cellulare coinvolta principalmente in meccanismi di immunosorveglianza. Può anche essere rilasciato nello spazio extracellulare sia come proteina transmembrana associata agli esosomi sia come fattore solubile, meno attivo. Data l'attività citotossica selettiva del TRAIL nei confronti delle cellule tumorali, sono stati sviluppati numerosi agonisti dei recettori del TRAIL come potenziali terapie oncologiche. Tuttavia, questi si sono rivelati scarsamente efficaci negli studi clinici, principalmente a causa di problemi farmacocinetici e dell'incapacità di indurre l’oligomerizzazione dei recettori. Queste limitazioni potrebbero essere superate ingegnerizzando i fibroblasti associati al cancro (CAFs)—le cellule più accessibili all'interno del microambiente tumorale (TME)—per produrre il TRAIL di membrana tramite terapia genica, come proposto nel progetto “ANAKIN” finanziato dall’AIRC. I CAF sono emersi come promettenti cellule bersaglio anche per altre terapie antitumorali, il cui sviluppo è rallentato dalla mancanza di modelli preclinici di CAF ben caratterizzati e standardizzati. Questo lavoro si è proposto di sviluppare e caratterizzare un modello in vitro di CAF basato sul trattamento dei fibroblasti NIH/3T3 con il fattore di crescita trasformante beta (TGF-β) (NIH/3T3-CAFs) e di utilizzare questo modello per fornire una prova di concetto che, indipendentemente dal vettore di terapia genica utilizzato, la strategia proposta dal progetto “ANAKIN” possa essere efficace. Gli studi di differenziamento cellulare hanno dimostrato che entro 24 ore dal trattamento con 5 ng/mL di TGF-β le cellule NIH/3T3 trattate presentano alcuni fenotipi dei CAF, come l'espressione di marcatori specifici e un profilo metabolico altamente glicolitico. Questi fenotipi sono mantenuti per almeno cinque giorni dopo la rimozione dello stimolo. È stato quindi messo a punto un protocollo per la generazione e la coltivazione di un modello di CAF standardizzato, facile da usare e prontamente disponibile. Tuttavia, poiché alcune caratteristiche, come l'aumentata proliferazione e migrazione tipiche dei CAF, così come il loro effetto sulla crescita delle cellule tumorali, non sono state osservate, molto più essere fatto per ottenere un modello di CAF più rappresentativo. Per quanto riguarda la validazione del potenziale terapeutico della strategia del progetto “ANAKIN”, in primis abbiamo ottimizzato la produzione di una variante fluorescente del TRAIL – la proteina di fusione EGFP-TRAIL – nelle cellule NIH/3T3-CAF tramite trasfezione con agenti commerciali. Dalla trasfezione con DNA plasmidico (pDNA), abbiamo poi selezionato la trasfezione con mRNA per la sua maggiore efficienza e non tossicità. Abbiamo quindi investigato la localizzazione subcellulare di EGFP-TRAIL e TRAIL nelle cellule NIH/3T3-CAF trasfettate, abbandonando infine la proteina di fusione a causa della sua diversa localizzazione rispetto alla proteina non fusa. Infine, abbiamo esplorato il rilascio della proteina nativa dalle cellule NIH/3T3-CAF trasfettate ed esaminato il loro effetto citotossico sulle cellule tumorali. I nostri studi hanno rivelato che, a seguito della trasfezione con mRNA, il TRAIL non si localizza mai sulla membrana plasmatica delle cellule NIH/3T3-CAF. Può essere rilasciato nello spazio extracellulare, possibilmente come proteina transmembrana associata agli esosomi, ma in quantità insufficienti per esercitare un effetto apoptotico sulle cellule tumorali. Pertanto, per rendere efficace la strategia proposta, è essenziale trovare un modo per aumentare il rilascio di TRAIL, oltre che la sua stessa produzione, magari promuovendone la localizzazione sulla membrana plasmatica.

(2025). Development of a CAF model to investigate the potential of a TME-targeted TRAIL gene therapy approach for cancer eradication. (Tesi di dottorato, , 2025).

Development of a CAF model to investigate the potential of a TME-targeted TRAIL gene therapy approach for cancer eradication

BAIONI, CHIARA
2025

Abstract

The TNF-related apoptosis-inducing ligand (TRAIL) is a cell surface transmembrane protein primarily involved in immunosurveillance. It can also be released into the extracellular space, both as a transmembrane cargo of exosomes and as a less active soluble factor. Due to TRAIL's selective cytotoxic activity against cancer cells, many TRAIL receptor agonists have been developed as potential cancer therapies. However, these did not exhibit the expected outcomes in clinical trials, mainly due to poor pharmacokinetics and inability to induce receptor clustering. These issues could potentially be addressed by engineering cancer-associated fibroblasts (CAFs)—the most accessible cells within the tumor microenvironment (TME)—to produce full-length TRAIL via a gene therapy approach, as proposed in the AIRC-funded project “ANAKIN.” CAFs have recently emerged as promising targets for other anticancer therapies as well, but the development of such strategies is slowed down by the lack of well-characterized and standardized CAF preclinical models. This work aimed to develop and characterize an in vitro CAF model based on transforming growth factor beta (TGF-β) treatment of NIH/3T3 fibroblasts (NIH/3T3-CAFs) and to use this model to provide proof of concept that, regardless of the gene therapy vector used, the strategy proposed by the ANAKIN project could be effective. Cellular differentiation studies revealed that treated NIH/3T3 cells exhibit some CAF-like features within 24 hours of treatment with 5 ng/mL TGF-β, including specific marker expression and a highly glycolytic metabolic profile. These traits are maintained for at least five days after the removal of the stimulus. We therefore provided a protocol for the generation and sub-culturing of an easy-to-use, standardized, and readily available CAF model. However, since some phenotypes, such as enhanced proliferation and migration characteristics typical of CAFs, as well as an influence on cancer cell growth, were not observed, there are areas for improvement in CAF modeling. Regarding the validation of the feasibility of the “ANAKIN” strategy, we first optimized the production of a fluorescently tagged TRAIL variant in NIH/3T3-CAFs through transfection with commercial agents. Starting with plasmid DNA (pDNA) transfection, we subsequently selected mRNA transfection based on its higher efficiency and lack of toxicity. We then investigated the subcellular localization of both tagged and untagged TRAIL in transfected NIH/3T3-CAFs, ultimately abandoning the fusion protein due to its mislocalization. Finally, we explored the release of the native protein by TRAIL+ transfected NIH/3T3-CAFs and examined its cytotoxic effect on cancer cells. Our studies revealed that, following mRNA transfection, TRAIL does not reach the plasma membrane of NIH/3T3-CAFs. However, TRAIL can be released into the extracellular space, possibly as a membrane cargo of exosomes, but in insufficient quantities to exert an apoptotic effect on cancer cells. Therefore, to make the proposed strategy effective, it is essential to find a way to increase TRAIL release, other than its production, perhaps by promoting its trafficking to the plasma membrane.
PROSPERI, DAVIDE
CAFs; TGF-β; NIH/3T3; TRAIL; terapia genica
CAFs; TGF-β; NIH/3T3; TRAIL; mRNA therapy
BIO/10 - BIOCHIMICA
English
20-gen-2025
37
2023/2024
none
(2025). Development of a CAF model to investigate the potential of a TME-targeted TRAIL gene therapy approach for cancer eradication. (Tesi di dottorato, , 2025).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/535382
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