Riassunto analitico
Background: Malignant Pleural Mesothelioma (MPM) is an aggressive and deadly tumor arising from the pleura’s mesothelial cells, usually following exposure to asbestos. In Italy, despite the abolishment of asbestos in 1992, evidence revealed an age-standardized incidence rate of 0.80, twofold the world rate of 0.39, and is expected to rise in the upcoming 15 years. MPM is usually diagnosed in advanced stages, and surgical resection is only possible for a minority of patients. Standard chemotherapy regimens with pemetrexed plus/minus platinum have resulted in a median overall survival of 12 months and only 7% of patients surviving 5 years after diagnosis. Sarcomatoid and biphasic histologic subtypes show a worse prognosis than epithelioid. Several therapeutic approaches have been tested, including angiogenesis inhibitors, synthetic lethal treatment, miRNA replacement, oncoviral therapies, and the rapidly advancing field of cell-based therapies. In recent years, the use of mesenchymal stromal/stem cells (MSC) has aroused great interest in oncology due to biological features including easy procedures for isolation and gene manipulation, as well as their typical tropism for tumor sites. Indeed, MSCs have proven to be an excellent delivery system for anticancer and therapeutic compounds. Since years our laboratory has developed an anticancer gene therapy approach based on MSCs genetically modified by lentiviral vectors to force the expression of soluble form of Tumor Necrosis Factor Related Apoptosis Inducing Ligand (sTRAIL).
In this study, we aim to investigate the sensitivity of different MPM histologic subtypes to MSC producing sTRAIL. More, we will focus on the TRAIL resistant MPM lines to investigate the molecular mechanisms of TRAL resistance and develop combinatorial strategies able to empower the therapeutic impact of MSC-sTRAIL.
Results: MPM cell lines (MSTO-211H; NCI-H28; NCI-H2052; NCI-H2452), were morphologically ad genetically characterized. MSTO-211H represent the biphasic subtypes, NCI-H28 and NCI-H2452 were epithelioid and NCI-H2052 can be classified as sarcomatoid. By cytofluorometric analysis we assessed the expression of Mesothelin and TRAIL receptors on all MPM cell lines. These analyses revealed that all cell lines express high levels of Mesothelin and DR5 receptor, while they are negative for DR4 and non-functional receptors (DcR1 and DcR2). The impact of sTRAIL-mediated cytotoxicity on all MPM lines was evaluated by in vitro dose-response assays. The apoptotic effect of sTRAIL makes it possible to discriminate between two TRAIL-sensitive (MSTO-211H; NCI-H28) and two TRAIL-resistant (NCI-H2052; NCI-H2452) cell lines, suggesting that resistance to sTRAIL in MPM is not dependent on receptor expression or related to a specific histologic subtype. In particular, RT-qPCR analyses revealed an up-regulated expression of anti-apoptotic genes cFLIP and Bcl-xL on NCI-H2052. Focusing on both TRAIL resistant MPM lines, we evaluated the apoptotic effect induced by the combination of sTRAIL with Cisplatin and Pemetrexed in 2D culture system. Notably, this approach was able to reverse resistance to sTRAIL in NCI-H205 only. The effectiveness of this combinatory strategy was further confirmed in 3D MPM models. Finally, the protein analysis indicated the key role of cFLIP as a driver of sTRAIL resistance in NCI-H2052.
Conclusion: Our findings shed light on the possibility of using a combinatory strategy based on chemo and MSC-sTRAIL approaches to enhance the antitumor effect in MPM. In particular, we demonstrated that treatment with Cisplatin and Pemetrexed can reverse resistance to sTRAIL by downregulating cFLIP protein expression in sarcomatoid NCI-H2052 cell line, which represent the most aggressive MPM subtype.
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