Human Mesothelioma Cell Lines Research Articles

Abstract 1336: Verification of the effect of human allogeneic iPS cell-derived gene-engineered NK cells (eNK cells HLCN061) on mesothelioma

Abstract Malignant pleural mesothelioma is a malignant tumor that is caused by exposure to asbestos. Because it is a rare cancer, there are few drugs that can be used to treat it. Healios is developing iPSC-derived NK cells (eNK cells) (HLCN061) engineered with NKG2D, DAP10, IL-15, CD16, CCL19, and CCR2B genes for the treatment of refractory solid tumors. This study evaluated the antitumor effect of eNK cells against human malignant pleural mesothelioma cell lines.(Method) The antitumor effect of eNK cells on human malignant pleural mesothelioma cell lines in vitro was evaluated using the Viability/Cytotoxicity Multiplex Assay Kit (WST-8/LDH).In vivo, immunodeficient mice were subcutaneously inoculated with human malignant pleural mesothelioma cell lines. After subcutaneous tumor formation, eNK cells were inoculated into or near the tumor, the size of the subcutaneous tumor was measured, and the tumor volume was compared with that of an untreated group. After the study was completed, the tumors were removed and the tumors in the untreated group and the eNK cell-administered group were analyzed using RNA-seq.(Results) The antitumor effect of eNK cells against human malignant pleural mesothelioma cell lines was confirmed in vitro. In a mouse subcutaneous tumor model, the eNK cell treatment group suppressed tumor growth compared to the untreated group (p≦0.05). RNA-seq showed increased expression of granulysin, IL2RB, cathepsin W, etc. in subcutaneous tumors treated with eNK cells. Citation Format: Seiji Matsumoto, Yoshiko Fujita, Kumiko Goto, Yuka Sato, Fusako Nishigaki, Yasuyuki Higashi, Hironobu Kimura, Kouichi Tamura. Verification of the effect of human allogeneic iPS cell-derived gene-engineered NK cells (eNK cells HLCN061) on mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1336.

Abstract 384: SLC7A11 modulates sensitivity to the first-in-class mitochondrial peroxiredoxin 3 inhibitor thiostrepton (RSO-021) via a ferroptosis independent pathway

Abstract Mitochondrial peroxiredoxin 3 (PRX3) has been identified as an actionable cancer vulnerability that is currently being investigated in the first-in-human phase 1 clinical trial, MITOPE (NCT05278975). RSO-021 (thiostrepton or TS) is a redox-active drug that inhibits PRX3’s peroxidase activity via covalent adduction of its active site peroxidatic and resolving cysteine residues, forming an irreversible crosslink across the protein dimer. Covalent inhibition of PRX3 results in tumor cell death due to a diminished ability to remove high levels of mitochondrial reactive oxygen species (ROS) in cancer cells. To better understand the mechanism underpinning sensitivity and resistance, TS-tolerant human mesothelioma cell lines were generated under constant TS pressure and subjected to a drug-screen comprising chemotherapeutic agents, metabolic inhibitors, ROS modulating agents and ferroptosis-inducing compounds, to identify synergistic interactions compared with TS- sensitive cell lines. Erastin, an SLC7A11 inhibitor exhibited the greatest synergy when combined with TS, potentiated PRX3 covalent crosslinking and cellular ROS levels. Synergy with TS was enhanced by cystine depletion and could be phenocopied by siRNA knockdown of SLC7A11. SLC7A11 was up-regulated in TS-tolerant cells and resistance could be overcome with erastin. TS-tolerant cell lines have significantly reduced glutathione levels and slowed proliferation. Ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 did not inhibit TS or TS in combination with erastin. In summary, SLC7A11 up-regulation confers tolerance to TS through a mechanism involving a cysteine-dependent modulation of the redox state independent of canonical ferroptosis, that can be overcome by the addition of an SLC7A11 inhibitor. The correlation between SLC7A11 with response to TS in a 33-patient primary mesothelioma explant co-clinical trial, and patients enrolled into the MITOPE phase 1/2 trial will be presented. Citation Format: Terri Messier, Victoria Gibson, Aleksandra Bzura, Joanna Dzialo, Charlotte Poile, Stephanie Stead, Alexis Saaman, George N. Naumov, Dean A. Fennell, Brian Cunniff. SLC7A11 modulates sensitivity to the first-in-class mitochondrial peroxiredoxin 3 inhibitor thiostrepton (RSO-021) via a ferroptosis independent pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 384.

Long-Chain Acyl Coenzyme A Dehydrogenase, a Key Player in Metabolic Rewiring/Invasiveness in Experimental Tumors and Human Mesothelioma Cell Lines.

Cross-species investigations of cancer invasiveness are a new approach that has already identified new biomarkers which are potentially useful for improving tumor diagnosis and prognosis in clinical medicine and veterinary science. In this study, we combined proteomic analysis of four experimental rat malignant mesothelioma (MM) tumors with analysis of ten patient-derived cell lines to identify common features associated with mitochondrial proteome rewiring. A comparison of significant abundance changes between invasive and non-invasive rat tumors gave a list of 433 proteins, including 26 proteins reported to be exclusively located in mitochondria. Next, we analyzed the differential expression of genes encoding the mitochondrial proteins of interest in five primary epithelioid and five primary sarcomatoid human MM cell lines; the most impressive increase was observed in the expression of the long-chain acyl coenzyme A dehydrogenase (ACADL). To evaluate the role of this enzyme in migration/invasiveness, two epithelioid and two sarcomatoid human MM cell lines derived from patients with the highest and lowest overall survival were studied. Interestingly, sarcomatoid vs. epithelioid cell lines were characterized by higher migration and fatty oxidation rates, in agreement with ACADL findings. These results suggest that evaluating mitochondrial proteins in MM specimens might identify tumors with higher invasiveness.

Antitumoral effects of Bortezomib in malignant mesothelioma: evidence of mild endoplasmic reticulum stress in vitro and activation of T cell response in vivo

BackgroundMalignant mesothelioma (MM) is a rare tumor with a dismal prognosis. The low efficacy of current treatment options highlights the urge to identify more effective therapies aimed at improving MM patients’ survival. Bortezomib (Bor) is a specific and reversible inhibitor of the chymotrypsin-like activity of the 20S core of the proteasome, currently approved for the treatment of multiple myeloma and mantle cell lymphoma. On the other hand, Bor appears to have limited clinical effects on solid tumors, because of its low penetration and accumulation into tumor tissues following intravenous administration. These limitations could be overcome in MM through intracavitary delivery, with the advantage of increasing local drug concentration and decreasing systemic toxicity.MethodsIn this study, we investigated the effects of Bor on cell survival, cell cycle distribution and modulation of apoptotic and pro-survival pathways in human MM cell lines of different histotypes cultured in vitro. Further, using a mouse MM cell line that reproducibly forms ascites when intraperitoneally injected in syngeneic C57BL/6 mice, we investigated the effects of intraperitoneal Bor administration in vivo on both tumor growth and the modulation of the tumor immune microenvironment.ResultsWe demonstrate that Bor inhibited MM cell growth and induced apoptosis. Further, Bor activated the Unfolded Protein Response, which however appeared to participate in lowering cells’ sensitivity to the drug’s cytotoxic effects. Bor also affected the expression of EGFR and ErbB2 and the activation of downstream pro-survival signaling effectors, including ERK1/2 and AKT. In vivo, Bor was able to suppress MM growth and extend mice survival. The Bor-mediated delay of tumor progression was sustained by increased activation of T lymphocytes recruited to the tumor microenvironment.ConclusionsThe results presented herein support the use of Bor in MM and advocate future studies aimed at defining the therapeutic potential of Bor and Bor-based combination regimens for this treatment-resistant, aggressive tumor.

Small RNA-Seq Transcriptome Profiling of Mesothelial and Mesothelioma Cell Lines Revealed microRNA Dysregulation after Exposure to Asbestos-like Fibers

Environmental exposure to fibers of respirable size has been identified as a risk for public health. Experimental evidence has revealed that a variety of fibers, including fluoro-edenite, can develop chronic respiratory diseases and elicit carcinogenic effects in humans. Fluoro-edenite (FE) is a silicate mineral first found in Biancavilla (Sicily, Italy) in 1997. Environmental exposure to its fibers has been correlated with a cluster of malignant pleural mesotheliomas. This neoplasm represents a public health problem due to its long latency and to its aggression not alerted by specific symptoms. Having several biomarkers providing us with data on the health state of those exposed to FE fibers or allowing an early diagnosis on malignant pleural mesothelioma, still asymptomatic patients, would be a remarkable goal. To these purposes, we reported the miRNA transcriptome in human normal mesothelial cell line (MeT-5A) and in the human malignant mesothelioma cell line (JU77) exposed and not exposed to FE fibers. The results showed a difference in the number of deregulated miRNAs between tumor and nontumor samples both exposed and not exposed to FE fibers. As a matter of fact, the effect of exposure to FE fibers is more evident in the expression of miRNA in the tumor samples than in the nontumor samples. In the present paper, several pathways involved in the pathogenesis of malignant pleural mesothelioma have been analyzed. We especially noticed the involvement of pathways that have important functions in inflammatory processes, angiogenesis, apoptosis, and necrosis. Besides this amount of data, further studies will be designed for the selection of the most significant miRNAs to test and validate their diagnostic potential, alone or in combination with other protein biomarkers, in high-risk individuals' liquid biopsy to have a noninvasive tool of diagnosis for this neoplasm.

Water-Soluble Truncated Fatty Acid-Porphyrin Conjugates Provide Photo-Sensitizer Activity for Photodynamic Therapy in Malignant Mesothelioma.

Clinical trials evaluating intrapleural photodynamic therapy (PDT) are ongoing for mesothelioma. Several issues still hinder the development of PDT, such as those related to the inherent properties of photosensitizers. Herein, we report the synthesis, photophysical, and photobiological properties of three porphyrin-based photosensitizers conjugated to truncated fatty acids (C5SHU to C7SHU). Our photosensitizers exhibited excellent water solubility and high PDT efficiency in mesothelioma. As expected, absorption spectroscopy confirmed an increased aggregation as a consequence of extending the fatty acid chain length. In vitro PDT activity was studied using human mesothelioma cell lines (biphasic MSTO-211H cells and epithelioid NCI-H28 cells) alongside a non-malignant mesothelial cell line (MET-5A). The PDT effect of these photosensitizers was initially assessed using the colorimetric WST-8 cell viability assay and the mode of cell death was determined via flow cytometry of Annexin V-FITC/PI-stained cells. Photosensitizers appeared to selectively localize within the non-nuclear compartments of cells before exhibiting high phototoxicity. Both apoptosis and necrosis were induced at 24 and 48 h. As our pentanoic acid-derivatized porphyrin (C5SHU) induced the largest anti-tumor effect in this study, we put this forward as an anti-tumor drug candidate in PDT and photo-imaging diagnosis in mesothelioma.

Immune marker expression of irradiated mesothelioma cell lines.

Though immune checkpoint inhibition has recently shown encouraging clinical efficacy in mesothelioma, most patients do not respond. Combining immune checkpoint inhibition with radiotherapy presents an attractive option for improving treatment responses owing to the various immunomodulatory effects of radiation on tumors. However, the ideal dosing and scheduling of combined treatment remains elusive, as it is poorly studied in mesothelioma. The present study characterizes the dose- and time-dependent changes to expression of various immune markers and cytokines important to antitumor responses following irradiation of mesothelioma cell lines. Two murine (AB1, AE17) and two human (BYE, JU77) mesothelioma cell lines were treated with titrated gamma-radiation doses (1-8 Gy) and the expression of MHC class-I, MHC class-II and PD-L1 was measured over a series of post-irradiation timepoints (1-72 hours) by flow cytometry. Levels of cytokines IL-1α, IL-1β, IL-6, IL-10, IL-12p70, IL-17A, IL-23, IL-27, MCP-1, IFN-β, IFN-γ, TNF-α, and GM-CSF were measured by multiplex immunoassay in murine cell lines following 8 Gy radiation. Following irradiation, a dose-dependent upregulation of MHC-I and PD-L1 was observed on three of the four cell lines studied to varying extents. For all cell lines, the increase in marker expression was most pronounced 72 hours after radiation. At this timepoint, increases in levels of cytokines IFN-β, MCP-1 and IL-6 were observed following irradiation with 8 Gy in AB1 but not AE17, reflecting patterns in marker expression. Overall, this study establishes the dose- and time-dependent changes in immune marker expression of commonly studied mesothelioma cell lines following radiation and will inform future study into optimal dosing and scheduling of combined radiotherapy and immune checkpoint inhibition for mesothelioma.

Effects of Photon Radiation on DNA Damage, Cell Proliferation, Cell Survival, and Apoptosis of Murine and Human Mesothelioma Cell Lines

PurposeTo characterize the cellular responses of murine and human mesothelioma cell lines to different doses of photon radiation with a long-term aim of optimizing a clinically relevant in vivo model in which to study the interaction of radiation therapy and immunotherapy combinations. Methods and MaterialsTwo murine mesothelioma cell lines (AB1 and AE17) and 3 human cell lines (BYE, MC, and JU) were used in the study. Cells were treated with increasing doses of photon radiation. DNA damage, DNA repair, cell proliferation, and apoptosis at different time points after irradiation were quantified by flow cytometry, and cell survival probability was examined using clonogenic survival assay. ResultsDNA damage increased with escalating dose in all cell lines. Evident G2/M arrest and reduced cell proliferation were observed after irradiation with 8 Gy. DNA repair was uniformly less efficient at higher compared with lower radiation-fraction doses. The apoptosis dose response varied between cell lines, with greater apoptosis observed at 16 Gy with human BYE and murine AB1 cell lines but less for other studied cell lines, regardless of dose and time. The α/β ratio from the cell survival fraction of human mesothelioma cell lines was smaller than from murine ones, suggesting human cell lines in our study were more sensitive to a change of dose per fraction than were murine mesothelioma cell lines. However, in all studied cell lines, colony formation was completely inhibited at 8 Gy. ConclusionsA threshold dose of 8 Gy appeared to be appropriate for hypofractionated radiation therapy. However, the radiation therapy doses between 4 and 8 Gy remain to be systematically analyzed. These observations provide an accurate picture of the in vitro response of mesothelioma cell lines to photon irradiation and characterize the heterogeneity between human and murine cell lines. This information may guide in vivo experiments and the strengths and limitations of extrapolation from murine experimentation to potential human translation.

Organic extract of Geodia cydonium induces cell cycle block in human mesothelioma cells.

The serious side effects caused by chemotherapeutics and the development of cancer chemoresistance represent the most significant limitations in the treatment of cancer. Some alternative approaches have been developed in recent years, which are based on natural compounds, and have allowed important advances in cancer therapeutics. During the last 50 years, sponges have been considered a promising source of natural products from the marine environment, representing ~30% of all marine natural products. Among sponges, the Mediterranean species Geodia cydonium represents a potential source of these type of products with considerable biotechnological interest as pharmaceutical agents. The present study demonstrated the antiproliferative effect of an organic G. cydonium extract (GEOCYDO) against three human mesothelioma cell lines, MSTO-211H (MSTO), NCI-H2452 (NCI) and Ist-Mes2 (Mes2), which differ in their sensitivity (MSTO and NCI) and resistance (Mes2) to standard combined treatment with cisplatin and piroxicam. To this aim, the activity of the extract was evaluated by analyzing its effects on cell viability, cancer properties and cell cycle progression by means of colony formation assay, cell cycle analysis and protein expression analysis. The results revealed that in mesothelioma, this extract was able to reduce self-renewal, cell migration and it could induce cell cycle arrest in G0/G1 stage, thus blocking cell proliferation. In conclusion, to the best of our knowledge, the present results indicated for the first time that GEOCYDO can contain active compounds able to affect cell proliferation in mesothelioma, suggesting that it could be considered as a potential novel drug source for cancer treatment.

Dysregulation of microRNAs and tRNA-derived ncRNAs in mesothelial and mesothelioma cell lines after asbestiform fiber exposure

Experimental evidence demonstrated that fluoro-edenite (FE) can develop chronic respiratory diseases and elicit carcinogenic effects. Environmental exposure to FE fibers is correlated with malignant pleural mesothelioma (MPM). An early diagnosis of MPM, and a comprehensive health monitoring of the patients exposed to FE fibers are two clinical issues that may be solved by the identification of specific biomarkers. We reported the microRNA (miRNA) and transfer RNA-derived non coding RNA (tRNA-derived ncRNA) transcriptome in human normal mesothelial and malignant mesothelioma cell lines exposed or not exposed to several concentration FE fibers. Furthermore, an interactive mesothelioma-based network was derived by using NetME tool. In untreated condition, the expression of miRNAs and tRNA-derived ncRNAs in tumor cells was significantly different with respect to non-tumor samples. Moreover, interesting and significant changes were found after the exposure of both cells lines to FE fibers. The network-based pathway analysis showed several signaling and metabolic pathways potentially involved in the pathogenesis of MPM. From papers analyzed by NetME, it is clear that many miRNAs can positively or negatively influence various pathways involved in MPM. For the first time, the analysis of tRNA-derived ncRNAs molecules in the context of mesothelioma has been made by using in vitro systems. Further studies will be designed to test and validate their diagnostic potential in high-risk individuals' liquid biopsies.

3-Imino derivative-sulfahydantoins: Synthesis, in vitro antibacterial and cytotoxic activities and their DNA interactions

Sulfahydantoins are five-membered rings found in the structure of chemicals that exhibit antibacterial, anti-inflammatory, and anticonvulsant properties. They also activate serine protease enzymes that catalyze the hydrolysis of peptide bonds. Five 3-imino sulfahydantoin compounds were synthesized by using Strecker synthesis reaction with minor modifications. We used reflux of various aldehydes with excess sulfamide in 85% methanol in the presence of sodium cyanide. The spectroscopic properties of these compounds were studied in detail. Antibacterial activities of all synthesized new compounds against four Gram-positive (Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, Streptococcus mutans) and four Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella Enteritidis) bacteria were investigated by disc diffusion and microdilution method. pBR322 plasmid DNA binding abilities of compounds were investigated in vitro by agarose gel electrophoresis. In addition, the cytotoxic activities of the compounds against the human malignant pleural mesothelioma (SPC212) cell line were determined by the MTT method. The remarkable result in this study is that the synthesized compounds, especially 4b, 4d, and 4e, have significant biological activities. It has been demonstrated that these compounds, which cause DNA damage, also have an important antibacterial effect on both Gram-negative and Gram-positive bacteria when results compared with the control group antibiotics. Compound 4e exhibited the highest antibacterial potency against Streptococcus mutans (24.33 ± 0.57) from Gram-positive bacteria and Pseudomonas aeruginosa (24.66 ± 1.15) from Gram-negative bacteria. At the same time, MTT results determined that compounds 4b, 4d, and 4e showed cytotoxic activity against the SPC212 cells. In particular, compound 4b had a high cytotoxic effect, and the IC50 value was determined as 6.25 µM.

Abstract 1064: Antiproliferative effects of Tumor Treating Fields in human mesothelioma cell lines

Abstract Despite the increasing knowledge of the biology of malignant pleural mesothelioma (MPM), patients with this diagnosis still have a poor prognosis with a 5-year survival rate of lower than 10%. The effectiveness demonstrated by Tumor Treating Fields (TTFields) in combination with chemotherapy in preclinical experiments and in MPM patients (NCT02397928) affirms TTFields as a promising therapeutic tool. Knowledge of the mechanism of action of TTFields and its interaction with chemotherapeutic agents may contribute to the design of more effective clinical trials for MPM patients. A panel of primary MPM cells was isolated from pleural effusion and/or lavages of patients' thoracic cavity, before administration of therapeutic treatment, obtaining six cell lines from the 3 types of MPM cells: epithelioid CD473 and CD484, sarcomatoid CD60 and CD432, and biphasic CD487 and CD491. RNAseq analysis revealed that the transcriptional profiles of CD473 and CD60 cells were comparable with those derived from patient biopsies of the same histotype, representing clinically relevant MPM models. Antiproliferative effects induced by TTFields alone (1.12 V/cm; 150 KHz; for 96h duration) were studied in these MPM cell lines. Cell survival and cell cycle perturbations analyses during and after treatment were performed. Consistent with clinical observations, epithelioid MPM cells demonstrated greater sensitivity to TTFields than biphasic and sarcomatoid MPM cells. Cell cycle analysis revealed that the former cell type was blocked in G2M phase and was followed by formation of polyploid cells; while the latter cell types were only slightly affected by TTFields with a general delay in all cell cycle phases. Apoptotic cells were present in all treated samples, but while epithelioid and biphasic cell death was already observed during the first 24h of treatment, sarcomatoid cells needed to be treated for at least 24h before starting apoptotic pathways. The antiproliferative effect observed in epithelioid cells was persistent even after treatment cessation, whereas biphasic and sarcomatoid cells were able to reinitiate growth upon TTFields cessation. RNAseq experiments were performed in CD473 and CD60 cells at different times during TTFields application to explore transcriptional modifications that may explicate these varied MPM cell responses. Preliminary data suggested that TTFields induced a transcriptional response already detectable at earlier time points of treatment (8h; maximum effect at 24h). The number of differentially expressed genes was higher in CD473 relative to CD60 cells (1951 in CD473 and 336 in CD60 at 24h), involving several pathways, that need to be further investigated. These data demonstrate that TTFields induce specific effects on cell proliferation in varied subsets of mesothelioma cells and provide a mechanistic rationale for future therapies and combinations with other anticancer drugs for MPM treatment. Citation Format: Monica Lupi, Federica Mirimao, Nicolò Panini, Greta Piazza, Lara Paracchini, Laura Mannarino, Sergio Marchini, Federica Grosso, Serena Penpa, Antonio Maconi, Giovanni L. Ceresoli, Maurizio D'Incalci. Antiproliferative effects of Tumor Treating Fields in human mesothelioma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1064.

Abstract 1534: Mesothelin isoform 2 is a novel target for allogenic CARγδT cell therapy in solid tumors

Abstract Mesothelin (MSLN) is a tumor-associated antigen over-expressed on the cell surface of various malignant tumor cells, including majority of mesothelioma and ovarian cancer. MSLN-directed therapy has been intensively studied in preclinical and clinical settings. However, the clinical efficacy and safety of mesothelin-targeted therapy remains to be demonstrated. To identify a safe and effective target for CAR-T cell therapy in solid tumors, we used artificial intelligent platform developed with public and private RNA transcription database to identify cancer-associated antigens caused by alternative spicing. One of interesting targets is an alternative spicing isoform from MSLN. Human MSLN transcript has at least three isoforms. The isoform 1 is the predominant transcript detected in normal and tumor tissues and has been a promising target for cancer immunotherapy. The isoform 2 is the minor transcript using alternatively spliced exons producing 8 additional amino acids insertion compared to isoform 1. The isoform 3 produces a truncated and soluble MSLN. Our data demonstrated that MSLN isoform 2 is specifically expressed in ovarian cancers but not normal tissues, confirmed by Q-PCR studies. Furthermore, we generated mouse hybridoma antibodies specifically targeting MSLN isoform 2. The antibody specificity was screened and confirmed by ELISA-based reaction to MSLN isoform 2-specific peptide and flow cytometry-based binding to 293T cells overexpressing MSLN isoform 2 but not isoform 1 proteins. We further detected the endogenous MSLN isoform 2 expression in human mesothelioma cell line NCI H226 and further confirmed MSLN isoform 2 expression on human primary mesothelioma and ovarian cancer tissue but not normal tissues by immunohistochemistry staining. To study if targeting MSLN isoform 2 with chimeric antigen receptor (CAR) can control the tumor growth, we made CAR retrovirus construct and showed that genetically modified allogenic gamma-delta T cells expressing CAR for MSLN isoform 2 can kill human mesothelioma cells in vitro and in vivo. In summary, we have demonstrated that MSLN isoform 2 is a tumor-specific antigen, which can be targeted for CAR-T cell therapy. Citation Format: Xiaohong Wang, Yibin Chen, Anupama Gopisetty, Leonardo Mirandola, Lucia Piccotti, Quynh Nguyen, Maurizio Chiriva-Internati. Mesothelin isoform 2 is a novel target for allogenic CARγδT cell therapy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1534.

Functional inhibition of heat shock protein 70 by VER-155008 suppresses pleural mesothelioma cell proliferation via an autophagy mechanism.

Background: Pleural mesothelioma, a devastating asbestos‐associated malignancy, urgently requires a novel effective therapy. Heat shock protein 70 (HSP70), which is synthesized in the cell response to protein damage, is expected to be a new target for antitumor treatment. In addition to its well‐known protein refolding function, HSP70 regulates cell proliferation through different pathways, including PI3K/AKT/mTOR, and autophagy in malignant cells. In this study, we attempted to clarify the effects of VER‐155008, an HSP70 inhibitor, on pleural mesothelioma. Methods: Human pleural mesothelioma cell lines 211H, H2452 and H28 were cultured with VER‐155008, and protein expression, cell proliferation, colony formation, cell cycle, synergistic effect with cisplatin, and autophagy induction were analyzed. Results: In mesothelioma cell lines, VER‐155008 (5.0 μM or more) inhibited cell growth and colony formation, accompanied by G1 cell cycle arrest. According to western blot analysis, VER‐155008 reduced p‐AKT expression. However, VER‐155008 failed to show a synergistic effect with cisplatin on cell growth. Mesothelioma cells transfected with the novel plasmid pMRX‐IP‐GFP‐LC3‐RFP‐LC3ΔG, which was developed for the quantitative and statistical estimation of macroautophagy, showed enhanced macroautophagy upon treatment with VER‐155008 and gefitinib which is an EGFR‐tyrosine kinase inhibitor. In addition, fetal bovine serum deprivation induced macroautophagy was further enhanced by VER‐155008. Conclusions: On the basis of these results, functional HSP70 inhibition by VER‐155008 suppressed cell growth in pleural mesothelioma cells, accompanied by enhanced macroautophagy. HSP70 inhibition is thus expected to become a new strategy for treating mesothelioma.Key points Significant findings of the study In pleural mesothelioma cells, inhibition of HSP70 function by VER‐155008 suppressed cell proliferation accompanied by induction of autophagy which was synergistically enhanced under the starvation condition, whereas gefitinib, an EGFR‐TKI, did not show the same synergistic effect in autophagy. What this study adds The inhibition of HSP70 induced autophagy and suppressed cell proliferation in mesothelioma cells.

Mitochondrial ROS Induce Partial Dedifferentiation of Human Mesothelioma via Upregulation of NANOG.

The expression of pluripotency factors is a key regulator of tumor differentiation status and cancer stem cells. The purpose of this study was to examine the expression of pluripotency factors and differentiation status of human mesothelioma and the role of mitochondria in their regulation. We tested the expression of OCT4/POU5F1, NANOG, SOX2, PI3K-AKT pathway and BCL2 genes and proteins in 65 samples of human mesothelioma and 19 samples of normal mesothelium. Mitochondrial membrane potential, reactive oxygen species (ROS) generation and expression of pluripotency factors were also tested in human mesothelioma cell line. Human mesothelium and mesothelioma expressed SOX2, NANOG, PI3K and AKT genes and proteins and POU5F1 gene, whereby NANOG, SOX2 and phosphorylated (activated) AKT were upregulated in mesothelioma. NANOG protein expression was elevated in less differentiated samples of human mesothelioma. The expression of genes of PI3K-AKT pathway correlated with pluripotency factor genes. Mesothelioma cells had functional, but depolarized mitochondria with large capacity to generate ROS. Mitochondrial ROS upregulated NANOG and mitoTEMPO abrogated it. In conclusion, human mesothelioma displays enhanced expression of NANOG, SOX2 and phosphorylated AKT proteins, while elevated NANOG expression correlates with poor differentiation of human mesothelioma. Mitochondria of mesothelioma cells have a large capacity to form ROS and thereby upregulate NANOG, leading to dedifferentiation of mesothelioma.

Microwave-assisted ultra-fast synthesis of carbon quantum dots from linter: Fluorescence cancer imaging and human cell growth inhibition properties

The water dispersed-fluorescent carbon quantum dots (CDs) were synthesized using microwave-assisted hydrothermal process in the closed system from the waste cotton linter as a new carbon source. This method provided an ultra-fast, more effective, economical and easier synthesis for cancer-imaging applications compared to the other methods presented in literature. The morphological and optical properties of the hydrothermally produced carbon quantum dots were characterized by using FT-IR, transmission electron microscopy (TEM), SEM, EDX, UV–vis and fluorescent spectrophotometry techniques. An emission peak was observed at 420 nm when the CDs were excited at 376 nm. CDs were calculated to have an average particle diameter of 10.14 nm by TEM. In order to determine the usability of the CDs in cell imaging, two different concentrations (50 μL/mL and 100 μL/mL) of the CDs colloidal solution were applied to a human mesothelioma cell line, H2452, and human umbilical vein endothelial cells (HUVEC) for varying durations. While the fluorescent photos revealed that 2 h of exposure was sufficient for the cells to take the C-dots in and higher concentrations appeared brighter in the wavelengths studied (red channel (excitation/emission 586/646 nm), blue channel (390/446 nm) and the green channel (482/532 nm)), cell viability and proliferation assays indicated that the material was cytotoxic against the both cell lines and inhibited the cell growth in a time- and dose- dependent manner.

Cisplatin unleashes Toll-like receptor 3-mediated apoptosis through the downregulation of c-FLIP in malignant mesothelioma.

Toll-like receptor 3 (TLR3) is an immune receptor that behaves like a death receptor in tumor cells, thereby providing an original target for cancer therapy. The therapeutic potential of TLR3 targeting in malignant mesothelioma, an aggressive and incurable neoplasia of the pleura and peritoneum, has so far not been addressed. We investigated TLR3 expression and sensitivity of human mesothelioma cell lines to the synthetic dsRNA Poly(I:C), alone or in combination with cisplatin, the gold standard chemotherapy in mesothelioma. Activation of TLR3 by Poly(I:C) induced apoptosis of 4/8 TLR3-positive cell lines but not of TLR3-negative cell lines. The combined cisplatin/Poly(I:C) treatment enhanced apoptosis of 3/4 Poly(I:C)-sensitive cell lines and overcame resistance to Poly(I:C) or cisplatin alone in 2/4cell lines. Efficacy of the combined treatment relied on cisplatin-induced downregulation of c-FLIP, the main regulator of the extrinsic apoptotic pathway, leading to an enhanced caspase-8-mediated pathway. Of note, 6/6 primary cell samples isolated from patients with peritoneal mesothelioma expressed TLR3. Patient-derived cells were sensitive to Poly(I:C) alone while the combined cisplatin/Poly(I:C) treatment induced dramatic cell death. Our findings demonstrate that TLR3 targeting in combination with cisplatin presents an innovative therapeutic strategy in mesothelioma.

1852P - Effects of tumor treating fields (TTFields; 150 kHz) and cisplatin or pemetrexed combination therapy on mesothelioma cells in vitro and in vivo

BackgroundMalignant pleural mesothelioma (MPM) is an aggressive thoracic cancer mostly linked to asbestos exposure. The standard of care (SOC) therapy for unresectable MPM is cisplatin plus pemetrexed. Tumor Treating Fields (TTFields) therapy is an effective anti-neoplastic treatment modality delivered via noninvasive application of low intensity, intermediate frequency, alternating electric fields. We explored the potential use of TTFields alone and in combination with cisplatin or pemetrexed as an option for MPM. MethodsCells from human mesothelioma cell lines NCI-H2052 and MSTO-211H were treated at various TTFields frequencies for 72hrs using the InovitroTM System. The combined treatment of TTFields with cisplatin or pemetrexed was tested by applying TTFields at the optimal frequency together with various drug concentrations of cisplatin or pemetrexed. Cell count, clonogenic potential, and apoptosis induction were determined. TTFields (1.2 V/cm) were applied for 8 days to rats injected intrapleurally with IL-45 cells, and overall survival (OS) was determined. ResultsThe optimal TTFields frequency was 150kHz for both human cell lines. TTFields application (1.1 V/cm, 72hrs) at 150kHz led to a 45-51% reduction in cell counts and a 46-64% additional reduction in clonogenic potential. The combined treatment of TTFields and cisplatin or pemetrexed led to a significant reduction in cell count, apoptosis induction, and clonogenic potential as compared to each modality alone (P<0.0001(. TTFields significantly prolonged the survival of rats compared to control groups. Safety studies did not reveal any adverse events associated with 150kHz TTFields application to the rat torso. ConclusionsIn these in vitro and in vivo analyses, TTFields was demonstrated to be an effective treatment for mesothelioma. TTFields in combination with cisplatin or pemetrexed further enhanced treatment efficacy. These results are consistent with the recent STELLAR phase 2 study (EF-23 trial; NCT02397928) that showed improved OS for TTFields plus SOC compared to historical control, with no increase in systemic toxicity. Legal entity responsible for the studyNovocure. FundingNovocure. DisclosureM. Munster: Full / Part-time employment: Novocure; Shareholder / Stockholder / Stock options: Novocure. K. Gotlib: Full / Part-time employment: Novocure; Shareholder / Stockholder / Stock options: Novocure. R.S. Schneiderman: Full / Part-time employment: Novocure; Full / Part-time employment: Novocure. Y. Porat: Full / Part-time employment: Novocure; Shareholder / Stockholder / Stock options: Novocure. T. Voloshin: Full / Part-time employment: Novocure; Shareholder / Stockholder / Stock options: Novocure. S. Davidi: Full / Part-time employment: Novocure; Shareholder / Stockholder / Stock options: Novocure. A. Shteingauz: Full / Part-time employment: Novocure; Shareholder / Stockholder / Stock options: Novocure. N. Kaynan: Full / Part-time employment: Novocure; Shareholder / Stockholder / Stock options: Novocure. E. Zeevi: Full / Part-time employment: Novocure; Shareholder / Stockholder / Stock options: Novocure. M. Giladi: Full / Part-time employment: Novocure; Shareholder / Stockholder / Stock options: Novocure. E.D. Kirson: Full / Part-time employment: Novocure; Shareholder / Stockholder / Stock options: Novocure. U. Weinberg: Full / Part-time employment: Novocure; Shareholder / Stockholder / Stock options: Novocure. A. Kinzel: Full / Part-time employment: Novocure; Shareholder / Stockholder / Stock options: Novocure. Y. Palti: Shareholder / Stockholder / Stock options: Novocure.

Anti-cancer activity of grape seed semi-polar extracts in human mesothelioma cell lines

Malignant mesothelioma is a tumor that affects pleural surface and has very poor prognosis. The standard therapeutic modalities for this cancer have yielded unsatisfactory outcomes, therefore the development of alternative and effective therapies is currently an urgent requirement. Grapevine is a plant rich of bioactive compounds, known for its therapeutic effects. Here, we describe the anti-cancer activity of grape seeds semi-polar extracts of two Italian grape varieties (Aglianico and Falanghina) in mesothelioma in vitro. Seed extracts from both varieties induced intrinsic apoptosis in a dose and time-dependent manner in three different human mesothelioma cell lines. Global metabolic analysis of this fraction revealed a higher accumulation of phenylpropanoid precursors and proanthocyanidins and expression of genes involved in the aforementioned pathways. These findings suggest that new phenolic molecules from grape seeds could be viewed as new drugs to be used alone or in combinations with standard chemotherapeutics in mesothelioma treatment.

Curcumin C3 complex\xae/Bioperine\xae has antineoplastic activity in mesothelioma: an in vitro and in vivo analysis

BackgroundA major limitation in the treatment for malignant mesothelioma is related to serious side effects caused by chemotherapeutics and to the development of cancer-resistance. Advances in cancer therapies have been reached thanks to the introduction of alternative approaches, such as the use of phytochemicals. Curcumin-C3complex®/Bioperine® is a commercially standardized extract containing a ratio-defined mixture of three curcuminoids and piperine that greatly increase its bioavailability. Interestingly, the anticancer effect of this formulation has been described in different studies and several clinical trials have been started, but to our knowledge none refers to human mesothelioma.MethodsCurcumin-C3complex®/Bioperine® anticancer effect was evaluated in vitro in different human mesothelioma cell lines analysing cell proliferation, colony-forming assay, wound healing assays, invasion assay and FACS analysis. In vivo anticancer properties were analysed in a mesothelioma xenograft mouse model in CD1 Nude mice.ResultsCurcumin-C3complex®/Bioperine® in vitro induced growth inhibition in all mesothelioma cell lines analysed in a dose- and time-depended manner and reduced self-renewal cell migration and cell invasive ability. Cell death was due to apoptosis. The analysis of the molecular signalling pathway suggested that intrinsic apoptotic pathway is activated by this treatment. This treatment in vivo delayed the growth of the ectopic tumours in a mesothelioma xenograft mouse model.ConclusionsCurcumin-C3complex®/Bioperine® treatment strongly reduces in vitro tumorigenic properties of mesothelioma cells by impairing cellular self-renewal ability, proliferative cell rate and cell migration and delays tumor growth in xenograft mouse model by reducing angiogenesis and increasing apoptosis. Considering that curcumin in vivo synergizes drug effects, its administration to treatment regimen may help to enhance drug therapeutic efficacy in mesothelioma. Our results suggest that implementation of standard pharmacological therapies with novel compounds may pave the way to develop alternative approaches to mesothelioma.