Malignant pleural mesothelioma (MPM) is a rare, aggressive malignancy affecting the pleura, the membrane lining the lungs, with a very poor prognosis. The current standard of care, besides surgery and/or radiotherapy in localized disease, is treatment with the chemotherapeutic agent cisplatin and pemetrexed, although their effectiveness remains limited, and they often leave space for the reoccurrence of chemotherapy-resistant mesothelioma. In this context, discovering treatments that can be substituted to chemotherapy or can effectively be combined with it is of utmost importance. In this project, 1520 FDA and EMA approved drugs were screened on the NCI-H28 mesothelioma cell line in vitro for their efficacy in killing tumor cells when used in combination with cisplatin. The best performing drugs (Riboflavin, Proglumide, Aminosalicylic acid, Gabapentin, Terfenadine, Propafenone, Oseltamivir) were further validated in vitro on two different mesothelioma cell lines to confirm their effectiveness and an attempt to understand their mechanism of action was made. In particular, the expression of CD24, OCT4 (cancer stem cell markers), ABCB1, ABCG2 (drug resistance markers), p21 (senescence marker), and BCL-XL (autophagy and apoptosis regulator) was analyzed upon treatment with the chosen drugs. No substantial difference in the expression of these markers between cells treated with cisplatin alone and cells treated with cisplatin plus each drug was observed, suggesting that other mechanisms are at play and further investigations are needed. Nonetheless, synergism was confirmed and scored by analysis with Lowe’s algorithm. The efficacy of the selected drugs was also tested on primary mesothelioma cells isolated from patients undergoing biopsies or surgery and treated with the different drugs combined with cisplatin. Interestingly, cells isolated from different patients showed sensitivity to different drugs. Furthermore, to validate the drug combinations’ effectiveness in a three-dimensional setting, mesothelioma spheroids were produced, and their size was evaluated upon treatment with the drugs combination. To conclude the project, evaluating the safety and effectiveness of the drugs combinations in an in vivo setting will be crucial. Therefore, an in vivo xenograft mouse model will be used to evaluate tumor size in untreated mice, cisplatin-treated mice, and mice treated with a combination of cisplatin and selected drugs.
Il mesotelioma pleurico maligno (MPM) è una rara e aggressiva forma di tumore che colpisce la pleura, la sierosa che riveste i polmoni, e ha una prognosi molto negativa. Lo standard di cura attuale, oltre all'intervento chirurgico e/o alla radioterapia nella malattia localizzata, è il trattamento con i chemioterapici cisplatino e pemetrexed, anche se la loro efficacia rimane limitata e spesso lascia spazio a recidive. In questo contesto, scoprire trattamenti che possano sostituire la chemioterapia o essere efficacemente combinati con essa è di massima importanza. In questo progetto, sono stati testati in vitro 1520 farmaci approvati dalla FDA e dall'EMA sulla linea cellulare di mesotelioma NCI-H28 per valutarne l'efficacia nell’eliminare le cellule tumorali quando utilizzati in combinazione con il cisplatino. I farmaci che hanno ottenuto i migliori risultati (Riboflavina, Proglumide, Acido Aminosalicilico, Gabapentin, Terfenadina, Propafenone, Oseltamivir) sono stati successivamente convalidati in vitro su due diverse linee cellulari di mesotelioma per confermare la loro efficacia e si è cercato di comprendere il loro meccanismo d'azione. In particolare, è stata analizzata l'espressione di CD24, OCT4 (marker di cellule staminali cancerose), ABCB1, ABCG2 (marker di chemioresistenza), p21 (marker di senescenza) e BCL-XL (regolatore dell'autofagia e dell'apoptosi) in risposta al trattamento con i farmaci scelti. Non è stata osservata una differenza sostanziale nell'espressione di questi marcatori tra le cellule trattate solo con il cisplatino e le cellule trattate con il cisplatino insieme a ciascun farmaco, suggerendo che altri meccanismi sono in gioco e ulteriori indagini sono necessarie. Tuttavia, è stata confermata la sinergia, che è stata valutata tramite analisi con algoritmo di Lowe. L'efficacia dei farmaci selezionati è stata testata anche su cellule primarie di mesotelioma isolate da pazienti sottoposti a biopsie o interventi chirurgici. È interessante notare che le cellule isolate da diversi pazienti hanno mostrato una diversa sensibilità ai vari farmaci. Inoltre, per convalidare l'efficacia delle combinazioni di farmaci in un ambiente tridimensionale, sono stati prodotti sferoidi di mesotelioma e ne è stata valutata la dimensione dopo trattamento con le varie combinazioni di farmaci. Per concludere il progetto, sarà cruciale valutare la sicurezza e l'efficacia delle combinazioni di farmaci in vivo. Pertanto, verrà utilizzato un modello murino per valutare le dimensioni del tumore in topi non trattati, topi trattati con cisplatino e topi trattati con una combinazione di cisplatino e farmaci selezionati.
High throughput screening per lo studio della patogenesi del mesotelioma maligno della pleura: un approccio di riposizionamento di farmaci per sviluppare nuove terapie di combinazione / Maiocchi, Serena. - (2024 Mar 07).
High throughput screening per lo studio della patogenesi del mesotelioma maligno della pleura: un approccio di riposizionamento di farmaci per sviluppare nuove terapie di combinazione.
MAIOCCHI, SERENA
2024-03-07
Abstract
Malignant pleural mesothelioma (MPM) is a rare, aggressive malignancy affecting the pleura, the membrane lining the lungs, with a very poor prognosis. The current standard of care, besides surgery and/or radiotherapy in localized disease, is treatment with the chemotherapeutic agent cisplatin and pemetrexed, although their effectiveness remains limited, and they often leave space for the reoccurrence of chemotherapy-resistant mesothelioma. In this context, discovering treatments that can be substituted to chemotherapy or can effectively be combined with it is of utmost importance. In this project, 1520 FDA and EMA approved drugs were screened on the NCI-H28 mesothelioma cell line in vitro for their efficacy in killing tumor cells when used in combination with cisplatin. The best performing drugs (Riboflavin, Proglumide, Aminosalicylic acid, Gabapentin, Terfenadine, Propafenone, Oseltamivir) were further validated in vitro on two different mesothelioma cell lines to confirm their effectiveness and an attempt to understand their mechanism of action was made. In particular, the expression of CD24, OCT4 (cancer stem cell markers), ABCB1, ABCG2 (drug resistance markers), p21 (senescence marker), and BCL-XL (autophagy and apoptosis regulator) was analyzed upon treatment with the chosen drugs. No substantial difference in the expression of these markers between cells treated with cisplatin alone and cells treated with cisplatin plus each drug was observed, suggesting that other mechanisms are at play and further investigations are needed. Nonetheless, synergism was confirmed and scored by analysis with Lowe’s algorithm. The efficacy of the selected drugs was also tested on primary mesothelioma cells isolated from patients undergoing biopsies or surgery and treated with the different drugs combined with cisplatin. Interestingly, cells isolated from different patients showed sensitivity to different drugs. Furthermore, to validate the drug combinations’ effectiveness in a three-dimensional setting, mesothelioma spheroids were produced, and their size was evaluated upon treatment with the drugs combination. To conclude the project, evaluating the safety and effectiveness of the drugs combinations in an in vivo setting will be crucial. Therefore, an in vivo xenograft mouse model will be used to evaluate tumor size in untreated mice, cisplatin-treated mice, and mice treated with a combination of cisplatin and selected drugs.File | Dimensione | Formato | |
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PhD_thesis_SM_FINAL_2023.pdf
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Descrizione: High throughput screening to model malignant pleural mesothelioma (MPM) pathogenesis: a drug repurposing approach to develop new combination therapies.
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