Mesothelioma Xenografts Research Articles

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.

A phase I trial using CD19 CAR-T expressing PD-1/CD28 chimeric switch-receptor for refractory or relapsed B-cell lymphoma.

7557 Background: The anti-CD19 chimeric antigen receptor (CAR)-T cell therapy has established efficacy in the treatment of refractory or relapsed (R/R) B-cell lymphoma. However, the existence of different immunosuppressive pathways such PD-1/PD-L1 pathway can restrict the full potential of CAR-T therapy. A recent study demonstrates that a novel PD-1/CD28 chimeric switch-receptor, comprising the extracellular domain of PD-1 and the transmembrane and cytoplasmic signaling domains of CD28, can be used a modality for augmenting antitumor activity of mesothelin- and prostate specific cancer antigen-specific CAR-T cells in either pleural mesothelioma or prostate cancer xenograft model. Here, we describe the first human trial of CD19-PD-1/CD28-CAR-T cells (a CD19-specific CAR-T expressing PD-1/CD28 chimeric switch-receptor) to evaluate safety and efficacy. Methods: This phase I, single-arm, open-label, multicenter trial enrolled patients (18-75 years) with R/R large B-cell lymphoma. PD-L1 expression was confirmed by immunohistochemistry using an anti-PD-L1 mAb (SP142), and confirmed by the review committee with a central evaluation. Seventeen patients received conditioning chemotherapy (cyclophosphamide [500 mg/m2] and fludarabine [30 mg/m2] on days -5, -4 and -3) and followed by CD19-PD-1/CD28-CART cells infusion at doses ranging 0.5 to 4 × 106 CAR+ T cells/kg on day 0. Response was assessed by F-FDG PET/CT at month 3 after CAR-T infusion, according to the International Working Group Response Criteria for Malignant Lymphoma, and duration of response was evaluated by ultrasonic and computed tomography every three months from month 3 to month 12. Results: At data cutoff, 17 patients had received CD19-PD-1/CD28-CART cells. Overall PD-L1 positivity was 15/17 (88.2%) and the mean percentage of PD-L1 positivity was 19.7% (range, 5-50%). Fourteen patients (82.35%) developed cytokine release syndrome (grade ≤ 2) and four (23.53%) developed neurotoxicity (grade = 1), which was reversible in all. The overall response rate was 58.8% (10/17) and complete remission rate was 41.2% (7/17). At a median follow-up 5 months, median overall survival for all patients was not reached. Conclusions: This trial showed the feasibility, controllable toxicities, and effective activity of CD19-PD-1/CD28-CART cells for treating patients with R/R B-cell lymphoma, providing the first proof-of-principle of the potential therapeutic value of targeting the PD-1/PD-L1 pathway in lymphoma in the clinical setting. Clinical trial information: NCT03258047.

Therapeutic efficacy evaluation of radioimmunotherapy with 90 Y-labeled anti-podoplanin antibody NZ-12 for mesothelioma.

Podoplanin is a type I transmembrane sialomucin‐like glycoprotein that is highly expressed in malignant mesothelioma. The rat‐human chimeric antibody NZ‐12 has high affinity for human podoplanin and antibody‐dependent cellular cytotoxicity and is applicable for radioimmunotherapy (RIT) to enhance the antitumor effect. In the present study, we evaluated the in vivo and in vitro properties of radiolabeled NZ‐12 and the antitumor effect of RIT with 90Y‐labeled NZ‐12 in an NCI‐H226 (H226) malignant mesothelioma xenograft mouse model. 111In‐labeled NZ‐12 bound specifically to H226 cells with high affinity, and accumulation was high in H226 tumors but low in major organs. RIT with 90Y‐labeled NZ‐12 significantly suppressed tumor growth and prolonged survival without body weight loss and obvious adverse effects. Higher podoplanin expression levels were observed in human mesothelioma specimens, suggesting higher tumor accumulation of 90Y‐labeled NZ‐12 in patients compared with the H226 tumor xenografts. Our findings suggest that 90Y‐labeled NZ‐12 is a promising RIT agent as a new therapeutic option for malignant mesothelioma that warrants further clinical studies to evaluate the dosimetry and efficacy in patients.

The effect of CELLFOODTM on radiotherapy or combined chemoradiotherapy: preclinical evidence.

Background:Based on previous observations that the nutraceutical CELLFOOD™ (CF), the ‘physiological modulator’ that aimed to make oxygen available ‘on demand’, inhibits the growth of cancer cells, this study was designed to investigate the role of CF in the regulation of hypoxia-inducible factor 1 alpha (HIF1α) and its correlated proteins, phosphoglycerate kinase 1 and vascular endothelial growth factor. Our idea was that CF, acting on HIF1α, in combination with current anticancer therapies could improve their effectiveness.Methods:To evaluate the effect of CF in association with radiotherapy and chemotherapy, different human cancer cell lines and mice with mesothelioma were analysed by tumour growth, clonogenic assay, western blot and immunohistochemical analysis.Results:CF in combination with radiation with or without cisplatin increases the death rate of cancer cells. In vivo, 70% of mice treated with CF before the mesothelioma graft did not show any tumour growth, indicating a possible preventive effect of CF. Moreover, in mouse mesothelioma xenografts, CF improves the effect of radiotherapy also in combination with chemotherapy treatment. Immunohistochemical analysis of tumour explants showed that HIF1α expression was reduced by the combination of CF and radiotherapy treatment and even more by the combination of CF and radiotherapy and chemotherapy treatment. Mechanistically, CF increases the fraction of oxygenated cells, making the radiotherapy more effective with a greater production of reactive oxygen species (ROS) that in turn, reduce the HIF1α expression. This effect is amplified by further increase in ROS from chemotherapy.Conclusions:Collectively, results from preclinical trials suggest that CF could be a useful intervention to improve the efficacy of radiotherapy or combined treatment strategies and could be a promising treatment modality to counteract cancer.

Biodistribution and Tumor Uptake of 67Ga-Nimotuzumab in a Malignant Pleural Mesothelioma Xenograft.

Malignant pleural mesothelioma (MPM) is the most common tumor of the pulmonary pleura. It is a rare and aggressive malignancy, generally associated with continuous occupational exposure to asbestos. Only a multimodal-approach to treatment, based on surgical resection, chemotherapy and/or radiation, has shown some benefits. However, the survival rate remains low. Nimotuzumab (h-R3), an anti-EGFR (epidermal growth factor receptor) humanized antibody, is proposed as a promising agent for the treatment of MPM. The aim of this research was to implement a procedure for nimotuzumab radiolabeling to evaluate its biodistribution and affinity for EGF (epidermal growth factor) receptors present in a mesothelioma xenograft. Nimotuzumab was radiolabeled with 67Ga; radiolabel efficiency, radiochemical purity, serum stability, and biodistribution were evaluated. Biodistribution and tumor uptake imaging studies by microSPECT/CT in mesothelioma xenografts revealed constant nimotuzumab uptake at the tumor site during the first 48 h after drug administration. In vivo studies using MPM xenografts showed a significant uptake of this radioimmunoconjugate, which illustrates its potential as a biomarker that could promote its theranostic use in patients with MPM.

P2.06-19 Targeting Polyamines as Potential Adjuvant Therapy in Malignant Pleural Mesothelioma Xenograft Models

Inhaling asbestos fibers is one of the commonest of malignant pleural mesothelioma (MPM). Although the import and use of asbestos have been restricted, the incidence of MPM is still rising due to a long lag time in malignant transformation. In 2004, the US Food and Drug Administration approved a combination of pemetrexed with cisplatin for treatment of unresectable MPM. At the same time, development of novel adjuvant therapeutic options for resected early-stage disease is also urgently needed. Ornithine decarboxylase (ODC) is highly expressed in 211H and H226 MPM xenografts and clinical tumor samples. Upregulation of ODC increases polyamine production and enhances tumor growth. a-difluoromethylornithine (DFMO) is a specific ODC inhibitor. Recent preclinical studies have demonstrated the adjuvant effect of DFMO in colon cancers using xenograft model. However, adjuvant effect of DFMO in MPM has not yet been studied. This study aims to disclose the adjuvant effect of DFMO in MPM xenograft models. The findings from this study will provide scientific foundation for future design of clinical trials of DFMO for adjuvant therapy in early disease for advanced MPM. Nude mice were fed with DFMO in drinking water 7 days before subcutaneous inoculation of 200,000 tumor cells (211H (biphasic) or H226 (epithelioid)). Mice with tumor size >600mm3 were considered reaching humane endpoint. Spermidine levels, protein expression, cytokines concentrations, NFkB translocation and apoptosis were investigated by Dot plot, Western blot, ELISA, immunofluorescence staining and TUNEL assay respectively. DFMO suppressed tumor growth in both xenografts. DFMO increased median survival from 49.5 days in control arm to 65 days in treatment arm in mice with 211H xenografts (p = 0.08), while from 44 days to 120 days in those with H226 xenografts (p = 0.0002). In H226 xenograft model, 43% of treated mice have not yet reached humane endpoint, mimicking long-term survival. Upon DFMO treatment, decrease in spermidine level, increase in nitrotyrosine content, nuclear translocation of NFkB, elevation of serum IL-6 and activation of apoptosis were observed in both xenografts. In addition, increase in nitrocysteine level, decrease in serum keratinocyte chemoattractant (KC), increase in serum TNF alpha, elevation of DNA lesion and inhibition of Akt/mTOR pathway were induced by DFMO in H226 xenografts, which may explain higher potency of DFMO in this xenograft. DFMO may have a potential role as adjuvant therapy in MPM especially epithelioid mesothelioma. Acknowledgment: This research was supported by Hong Kong Pneumoconiosis Compensation Fund Board, HKSAR.

Pirfenidone decreases mesothelioma cell proliferation and migration via inhibition of ERK and AKT and regulates mesothelioma tumor microenvironment in vivo

Malignant mesothelioma is an aggressive cancer with poor prognosis. It is characterized by prominent extracellular matrix, mesenchymal tumor cell phenotypes and chemoresistance. In this study, the ability of pirfenidone to alter mesothelioma cell proliferation and migration as well as mesothelioma tumor microenvironment was evaluated. Pirfenidone is an anti-fibrotic drug used in the treatment of idiopathic pulmonary fibrosis and has also anti-proliferative activities. Mesothelioma cell proliferation was decreased by pirfenidone alone or in combination with cisplatin. Pirfenidone also decreased significantly Transwell migration/invasion and 3D collagen invasion. This was associated with increased BMP pathway activity, decreased GREM1 expression and downregulation of MAPK/ERK and AKT/mTOR signaling. The canonical Smad-mediated TGF-β signaling was not affected by pirfenidone. However, pirfenidone blocked TGF-β induced upregulation of ERK and AKT pathways. Treatment of mice harboring mesothelioma xenografts with pirfenidone alone did not reduce tumor proliferation in vivo. However, pirfenidone modified the tumor microenvironment by reducing the expression of extracellular matrix associated genes. In addition, GREM1 expression was downregulated by pirfenidone in vivo. By reducing two major upregulated pathways in mesothelioma and by targeting tumor cells and the microenvironment pirfenidone may present a novel anti-fibrotic and anti-cancer adjuvant therapy for mesothelioma.

Gremlin-1 is a key regulator of the invasive cell phenotype in mesothelioma.

Malignant mesothelioma originates from mesothelial cells and is a cancer type that aggressively invades into the surrounding tissue, has poor prognosis and no effective treatment. Gremlin-1 is a cysteine knot protein that functions by inhibiting BMP-pathway activity during development. BMP-independent functions have also been described for gremlin-1. We have previously shown high gremlin-1 expression in mesothelioma tumor tissue. Here, we investigated the functions of gremlin-1 in mesothelioma cell migration and invasive growth. Gremlin-1 promoted mesothelioma cell sprouting and invasion into three dimensional collagen and Matrigel matrices. The expression level of gremlin-1 was linked to changes in the expression of SNAI2, integrins, matrix metalloproteinases (MMP) and TGF-β family signaling - all previously associated with a mesenchymal invasive phenotype. Small molecule inhibitors of MMPs completely blocked mesothelioma cell invasive growth. In addition, inhibitors of TGF-β receptors significantly reduced invasive growth. This was associated with reduced expression of MMP2 but not SNAI2, indicating that gremlin-1 has both TGF-β pathway dependent and independent mechanisms of action. Results of in vivo mesothelioma xenograft experiments indicated that gremlin-1 overexpressing tumors were more vascular and had a tendency to send metastases. This suggests that by inducing a mesenchymal invasive cell phenotype together with enhanced tumor vascularization, gremlin-1 drives mesothelioma invasion and metastasis. These data identify gremlin-1 as a potential therapeutic target in mesothelioma.

CAR T-cell immunotherapy of MET-expressing malignant mesothelioma

ABSTRACTMesothelioma is an incurable cancer for which effective therapies are required. Aberrant MET expression is prevalent in mesothelioma, although targeting using small molecule-based therapeutics has proven disappointing. Chimeric antigen receptors (CARs) couple the HLA-independent binding of a cell surface target to the delivery of a tailored T-cell activating signal. Here, we evaluated the anti-tumor activity of MET re-targeted CAR T-cells against mesothelioma. Using immunohistochemistry, MET was detected in 67% of malignant pleural mesotheliomas, most frequently of epithelioid or biphasic subtype. The presence of MET did not influence patient survival. Candidate MET-specific CARs were engineered in which a CD28+CD3ζ endodomain was fused to one of 3 peptides derived from the N and K1 domains of hepatocyte growth factor (HGF), which represents the minimum MET binding element present in this growth factor. Using an NIH3T3-based artificial antigen-presenting cell system, we found that all 3 candidate CARs demonstrated high specificity for MET. By contrast, these CARs did not mediate T-cell activation upon engagement of other HGF binding partners, namely CD44v6 or heparan sulfate proteoglycans, including Syndecan-1. NK1-targeted CARs demonstrated broadly similar in vitro potency, indicated by destruction of MET-expressing mesothelioma cell lines, accompanied by cytokine release. In vivo anti-tumor activity was demonstrated following intraperitoneal delivery to mice with an established mesothelioma xenograft. Progressive tumor regression occurred without weight loss or other clinical indicators of toxicity. These data confirm the frequent expression of MET in malignant pleural mesothelioma and demonstrate that this can be targeted effectively and safely using a CAR T-cell immunotherapeutic strategy.

Abstract 2085: Development of a novel targeted therapy for malignant mesothelioma carcinoma by a midkine inhibitor

Abstract Malignant pleural mesothelioma is an aggressive tumor of mesenchymal origin and is increasing worldwide as a result of widespread exposure to asbestos. The median survival of patients with mesothelioma from time of diagnosis ranges between 1 and 2 years. The mortality is expected to increase, at least until 2020, which is mainly due to the long latency (30-50 years) of the disease. Despite considerable advances in the understanding of its pathogenesis and etiology, malignant mesothelioma remains largely unresponsive to standard modalities of cancer therapy. Thus, there is an urgent need for new therapeutic options for mesothelioma.Midkine (MDK) is a heparin-binding growth factor that is highly expressed in many malignant tumors, including lung cancers. We have previously reported that a MDK inhibitor, iMDK, suppresses non-small cell lung cancer expressing MDK without harming normal cells. Importantly, iMDK inhibits the PI3 kinase / Akt pathway and induces apoptosis in MDK expressing non-small cell lung cancer cells. In the present study, we have investigated the antitumor effect of iMDK against malignant mesothelioma both in vitro and in vivo. 48 hours after treatment, iMDK dose-dependently inhibited cell growth of MDK expressing malignant mesothelioma cells. iMDK also suppressed colony formation of MSTO-211H mesothelioma cells. TUNEL positive cells were significantly increased in MSTO-211H cells 48 hours after iMDK treatment in a dose-dependent manner, confirming the induction of apoptosis in mesothelioma cells by iMDK. Combination treatment of iMDK and Bcl-2 inhibitor ABT-263 is more effective than each drug alone in MSTO-211H mesothelioma cells. Moreover, systemic administration of iMDK significantly inhibited tumor growth in a mesothelioma xenograft tumor in vivo. Inhibition of MDK with iMDK provides a potential therapeutic approach for the treatment of malignant mesothelioma that is driven by MDK. Citation Format: Takuya Fukazawa, Yuitaka Maeda, Tomoki Yamatsuji, Munenori Takaoka, Masakazu Yoshida, Naomasa Ishida, Miki Iwai, Etsuko Yokota, Takuro Yukawa, Minoru Haisa, Noriko Miyake, Tomoko Ikeda, Nagio Takigawa, Jeffery Whitsett, Yoshio Naomoto. Development of a novel targeted therapy for malignant mesothelioma carcinoma by a midkine inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2085. doi:10.1158/1538-7445.AM2017-2085

Growth suppressive effect of pegylated arginase in malignant pleural mesothelioma xenografts

BackgroundMalignant pleural mesothelioma (MPM) is a difficult-to-treat global disease. Pegylated arginase (BCT-100) has recently shown anti-tumor effects in hepatocellular carcinoma, acute myeloid leukemia and melanoma. This study aims to investigate the effects of PEG-BCT-100 in MPM.MethodsA panel of 5 mesothelioma cell lines (H28, 211H, H226, H2052 and H2452) was used to study the in vitro effects of BCT-100 by crystal violet staining. The in vivo effects of BCT-100 were studied using 211H and H226 nude mice xenografts. Protein expression (argininosuccinate synthetase, ornithine transcarbamylase, cleaved PARP, cleaved caspase 3, cyclins (A2, D3, E1 and H), CDK4 and Ki67) and arginine concentration were evaluated by Western blot and ELISA respectively. Cellular localization of BCT-100 was detected by immunohistochemistry and immunoflorescence. TUNEL assay was used to identify cellular apoptotic events.ResultsArgininosuccinate synthetase was expressed in H28, H226, and H2452 cells as well as 211H and H266 xenografts. Ornithine transcarbamylase was undetectable in all cell lines and xenograft models. BCT-100 reduced in vitro cell viability (IC50 values at 13–24 mU/ml, 72 h) across different cell lines and suppressed tumor growth in both 211H and H226 xenograft models. BCT-100 (60 mg/kg) significantly suppressed tumor growth (p < 0.01) with prolonged median survival (p < 0.01) in both xenograft models. Combining BCT-100 with pemetrexed or cisplatin conferred no additional benefits over single agents. Serum and intratumoral arginine levels were effectively decreased by BCT-100, associated with cytosolic accumulation of BCT-100 within tumor cells. Apoptosis (PARP cleavage in 211H xenografts; Bcl-2 downregulation, and cleavage of PARP and caspase 3 in H226 xenografts; positive TUNEL staining in both) and G1 arrest (downregulation of cyclin A2, D3, E1 and CDK4 in 211H xenografts; suppression of cyclin A2, E1, H and CDK4 in H226 xenografts) were evident with BCT-100 treatment. Furthermore, proliferative factor Ki67 was downregulated in BCT-100 treatments arms.ConclusionsBCT-100 suppressed tumor growth with prolonged median survival partially mediated by intratumoral arginine depletion resulting in apoptosis and G1 arrest in mesothelioma xenograft models. The findings provide scientific evidence to support further clinical development of BCT-100 in treatment of MPM.

Synthesis and Superpotent Anticancer Activity of Tubulysins Carrying Non-hydrolysable N-Substituents on Tubuvaline.

Synthetic tubulysins 24 a-m, containing non-hydrolysable N-substituents on tubuvaline (Tuv), were obtained in high purity and good overall yields using a multistep synthesis. A key step was the formation of differently N-substituted Ile-Tuv fragments 10 by using an aza-Michael reaction of azido-Ile derivatives 8 with the α,β-unsaturated oxo-thiazole 5. A structure-activity relationship study using a panel of human tumour cell lines showed strong anti-proliferative activity for all compounds 24 a-m, with IC50 values in the sub-nanomolar range, which were distinctly lower than those of tubulysin A, vinorelbine and paclitaxel. Furthermore, 24 a-m were able to overcome cross-resistance to paclitaxel and vinorelbine in two tumour cell lines with acquired resistance to doxorubicin. Compounds 24 e and 24 g were selected as leads to evaluate their mechanism of action. In vitro assays showed that both 24 e and 24 g interfere with tubulin polymerization in a vinca alkaloid-like manner and prevent paclitaxel-induced assembly of tubulin polymers. Both compounds exerted antimitotic activity and induced apoptosis in cancer cells at very low concentrations. Compound 24 e also exhibited potent antitumor activity at well tolerated doses on in vivo models of diffuse malignant peritoneal mesothelioma, such as MESOII peritoneal mesothelioma xenografts, the growth of which was not significantly affected by vinorelbine. These results indicate that synthetic tubulysins 24 could be used as standalone chemotherapeutic agents in difficult-to-treat cancers.

Pravastatin reduces radiation-induced damage in normal tissues.

Pravastatin is an inhibitor of 3-hydroxy-3-methyl- glutaryl-coenzyme A reductase that has been reported to have therapeutic applications in a range of inflammatory conditions. The aim of the present study was to assess the radioprotective effects of pravastatin in an experimental animal model. Mice were divided into two groups: The control group received ionizing radiation with no prior medication, while the pravastatin group received pravastatin prior to ionizing radiation. Pravastatin was administered orally at 30 mg/kg body weight in drinking water at 24 and 4 h before irradiation. Intestinal crypt epithelial cell survival and the incidence of apoptosis in the intestine and lung were measured post-irradiation. The effect of pravastatin on intestinal DNA damage was determined by immunohistochemistry. Finally, the effect of pravastatin on tumor response to radiotherapy was examined in a mouse mesothelioma xenograft model. Pravastatin increased the number of viable intestinal crypts and this effect was statistically significant in the ileum (P<0.0001). The pravastatin group showed significantly lower apoptotic indices in all examined parts of the intestine (P<0.0001) and tended to show reduced apoptosis in the lung. Pravastatin reduced the intestinal expression of ataxia-telangiectasia mutated and gamma-H2AX after irradiation. No apparent pravastatin-related differences were observed in the response of xenograft tumors to irradiation. In conclusion, pravastatin had radioprotective effects on the intestine and lung and reduced radiation-induced DNA double-strand breaks. Pravastatin may increase the therapeutic index of radiotherapy.

Intracavitary ‘T4 immunotherapy’ of malignant mesothelioma using pan-ErbB re-targeted CAR T-cells

Malignant mesothelioma remains an incurable cancer. We demonstrated that mesotheliomas expressed EGFR (79.2%), ErbB4 (49.0%) and HER2 (6.3%), but lacked ErbB3. At least one ErbB family member was expressed in 88% of tumors. To exploit ErbB dysregulation in this disease, patient T-cells were engineered by retroviral transduction to express a panErbB-targeted chimeric antigen receptor (CAR), co-expressed with a chimeric cytokine receptor that allows interleukin (IL)-4 mediated CAR T-cell proliferation. This combination is referred to as T4 immunotherapy. T-cells from mesothelioma patients were uniformly amenable to T4 genetic modification and expansion/enrichment thereafter using IL-4. Patient-derived T4+ T-cells were activated upon contact with a panel of four mesothelioma cell lines, leading to cytotoxicity and cytokine release in all cases. Adoptive transfer of T4 immunotherapy to SCID Beige mice with an established bioluminescent LO68 mesothelioma xenograft was followed by regression or eradication of disease in all animals. Despite the established ability of T4 immunotherapy to elicit cytokine release syndrome in SCID Beige mice, therapy was very well tolerated. These findings provide a strong rationale for the clinical evaluation of intracavitary T4 immunotherapy to treat mesothelioma.

OA02.05 Expression of miR-223 in Mesothelioma Xenografts Originates from Stromal Cells in the Tumor Microenvironment

Malignant pleural mesothelioma (MPM) is an aggressive cancer caused by asbestos exposure with limited therapeutic options. Dysregulated microRNAs play an important role in MPM biology and candidate microRNAs have been investigated as diagnostic and prognostic biomarkers or as a potential treatment targets. The role of miR-223 has previously been investigated in MPM tumor cells and was shown to act as a tumor suppressor by regulating cell mobility. Previous research indicated miR-223 to be primarily expressed by myeloid progenitor derived cells during differentiation of granulocytes and monocytes. This suggests miR-223 might have a more significant role in the inflammatory response during tumorigenesis. In this study we aimed to investigate the origin of miR-223 using mesothelioma xenograft and syngraft models. Human and mouse mesothelioma cell line-derived xenograft (MSTO-H211 and H226) and syngraft (AB1) models were established. MicroRNA profiles of xenografts were compared against profiles of their corresponding in vitro cultured cells to determine candidates. RT-qPCR using TaqMan MicroRNA assays was used to validate expression levels of miR-143-3p, miR-214-3p and miR-223-3p in tumor xenografts and syngrafts with those in corresponding cell lines in vitro. Species-specific ddPCR analysis was performed on RNA from xenograft tumors to determine the expression of human and mouse pri-miR-223. MicroRNA profiles of xenograft tumors showed significant upregulation (p < 0.05) of miR-143-3p, miR-214-3p and miR-223-3p compared to corresponding in vitro mesothelioma cell lines. Only miR-223 showed significant upregulation in both xenograft and syngraft tumors compared to corresponding in vitro mesothelioma cell lines (>10000-fold increase). Other microRNAs were not significantly different between cell lines and tumors. RNA isolated from xenograft tumors contained significantly more mouse pri-miR-223 than human pri-miR-223 (p < 0.001), with only minimal expression levels of human tumor pri-miR-223 within xenograft tumors. Mature miR-223 is significantly overexpressed in xenograft tumors compared to corresponding in vitro mesothelioma cell lines suggesting stromal contribution. Species-specific pri-miRNA confirmed miR-223 is almost exclusively expressed by the mouse stromal cells in xenograft tumors. Ultimately, localizing the expression of miR-223 to specific cell types (such as myeloid derived cells) through in situ hybridization should help identify a more biologically relevant role for miR-223 in the tumor microenvironment.

ISY-11-3 - Development of RNAi-based anticancer drug: What is problem for clinical application and how establish a useful RNAi drug

During about two decades, a number of therapeutic drugs focused to RNA interference (RNAi) have been developed, however these drugs have been still unsuccessful due to various problems such as targeted disease, drug delivery system, dosing route, stability in the body and so on. To resolve these problems, we focused to develop new drug delivery system using cationic liposome and short-hairpin RNAi molecule for thymidylate synthase (TS shRNA), an important rate-limiting enzyme of DNA biosynthesis in cancer. To avoid a rapid degradation in the blood and immunological incidence of TS shRNA conjugated with liposome (named as DFP-10825), intraperitoneal and/or intrathoracic administration of DFP-10825 were applied to evaluate the antitumor activity on disseminated ascetic ovarian, gastric and pancreatic cancer models and/or malignant mesothelioma xenografts implanted into intrathoracic cavity. Locally injected DFP-10825 resulted in potent antitumor activity and prolonged survival on these cancer models with relative long-time retention of DFP-10825 in the cavity. In addition, the combination of DFP-10825 (i.p.) and paclitaxel (i.p.), down-regulator of TS, or pemetrexed (i.p.), one of TS inhibitors, showed augmented antitumor efficacy and further prolonged survival in those cancer models. DFP-10825 is a stable freeze-dried preparation and capable of being prepared in a large amount at once. Our novel trial for RNAi-based anticancer therapy will have a clinical benefit to treat patients with disseminated peritoneal cancers. We cooperate with a biotechnology venture company, Delta-Fly Pharma and plan to enter a clinical trial within 1.5 years.

Abstract 3764: Trabectedin activity in patient-derived mesothelioma xenografts

Abstract Malignant pleural mesothelioma (MPM) incidence is increased over the past two decades and it is predicted to increase further in the next 20 years especially in developing countries where asbestos has not yet been banned. Surgery is not an option for the majority of patients due to the diffuse growth of MPM, making chemotherapy the treatment of choice, but prognosis remains very poor with a median survival time of 10-17 months. Trabectedin (ET-743, Yondelis) is a marine alkaloid used for the 2nd line therapy of ovarian cancer (combined with pegylated doxorubicin) and soft tissue sarcomas. It binds the DNA minor groove modulating transcription of cancer cells and has an anti-inflammatory effect modifying tumor microenvironment and reducing tumor associated macrophages. The aim was to test antitumor activity of trabectedin in newly established patient derived xenografts of MPM. MPM473, MPM484 and MPM487 xenografts were obtained in athymic nude mice by s.c. injection of 107 cells isolated from the pleural effusion of MPM patients and maintained by serial passages in mice. H&amp;E staining and immunohistochemistry were performed. Mice were randomized when tumor reached about 200 mg to receive trabectedin, cisplatin, pemetrexed, gemcitabine, and imatinib. Antitumor activity was expressed as T/C% were T and C are the mean tumor weight of treated and control mice, respectively. The three xenografts reproduced the morphology and the histochemical profile of the human tumors. As reported in the table trabectedin was able to induce a 50% reduction in tumor growth in MPM473 and MPM 487 xenografts and only a marginal effect in MPM484 xenograft. TreatmentDose (mg/kg)ScheduleBest T/C% (Day)MPM473MPM484MPM487Cisplatin5i.v. q7dx361.9 (69)66.1 (69)67.3 (39)Gemcitabine100i.p. q4dx583.2 (41)67.7 (48)49.7 (56)Imatinib200p.o. qdx1568.9 (76)89.4 (55)87.1 (39)Pemetrexed250i.p. q4dx477.4 (52)76.8 (49)84.3 (39)Trabectedin0.15i.v. q7dx351.0 (73)63.6 (69)51.3 (56) In conclusion we report three new MPM xenografts able to mimic the pathological features of human MPM. Although trabectedin showed only a moderate antitumor activity in our experimental models, its efficacy is comparable or better than that of the other drugs currently used for this disease. Studies are in progress in syngenic models that are more appropriate to test also the ability of trabectedin to modulate the immune system. Citation Format: Simonetta Andrea Licandro, Roberta Frapolli, Ezia Bello, Roberta Libener, Sara Orecchia, Federica Grosso, Maurizio D’Incalci. Trabectedin activity in patient-derived mesothelioma xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3764.

Synergistic anti-tumor efficacy of immunogenic adenovirus ONCOS-102 (Ad5/3-D24-GM-CSF) and standard of care chemotherapy in preclinical mesothelioma model.

Malignant mesothelioma (MM) is a rare cancer type caused mainly by asbestos exposure. The median overall survival time of a mesothelioma cancer patient is less than 1-year from diagnosis. Currently there are no curative treatment modalities for malignant mesothelioma, however treatments such as surgery, chemotherapy and radiotherapy can help to improve patient prognosis and increase life expectancy. Pemetrexed-Cisplatin is the only standard of care (SoC) chemotherapy for malignant mesothelioma, but the median PFS/OS (progression-free survival/overall survival) from the initiation of treatment is only up to 12 months. Therefore, new treatment strategies against malignant mesothelioma are in high demand. ONCOS-102 is a dual targeting, chimeric oncolytic adenovirus, coding for human GM-CSF. The safety and immune activating properties of ONCOS-102 have already been assessed in phase 1 study (NCT01598129). In this preclinical study, we evaluated the antineoplastic activity of combination treatment with SoC chemotherapy (Pemetrexed, Cisplatin, Carboplatin) and ONCOS-102 in xenograft BALB/c model of human malignant mesothelioma. We demonstrated that ONCOS-102 is able to induce immunogenic cell death of human mesothelioma cell lines in vitro and showed anti-tumor activity in the treatment of refractory H226 malignant pleural mesothelioma (MPM) xenograft model. While chemotherapy alone showed no anti-tumor activity in the mesothelioma mouse model, ONCOS-102 was able to slow down tumor growth. Interestingly, a synergistic anti-tumor effect was seen when ONCOS-102 was combined with chemotherapy regimens. These findings give a rationale for the clinical testing of ONCOS-102 in combination with first-line chemotherapy in patients suffering from malignant mesothelioma.

Reduction of estradiol in human malignant pleural mesothelioma tissues may prevent tumour growth, as implied by in in-vivo and in-vitro models.

This study aimed to investigate intratumoural estradiol and estrogen-receptors (ERα, ERβ and GPR30) in malignant pleural mesothelioma (MPM) to understand their function. Here, we report that immunohistochemistry of estradiol showed cytoplasmatic staining in 95% of fifty-seven human MPM samples with a trend toward a negative correlation between estradiol levels and the median post-diagnosis survival time. ERβ was only focally positive in 5.3% of cases, GPR30 and ERα were negative in our cases of MPM. GPR30 was detected mainly in glycosylated form in MPM cells. Moreover, G15, a GPR30 antagonist, induced MPM cell death. Altogether, these data suggest that MPM cells produce E2 interact with glycosylated forms of GPR30, and this facilitates tumour growth. Estradiol was found in MPM cells and plasma from mice mesothelioma xenografts. Concurrent reduction in tumour mass and plasmatic estradiol levels were observed in the mice treated with exemestane, suggesting that the reduction of E2 levels inhibit MPM growth. Thus, it appears that agents reducing estradiol levels could be useful to MPM therapy.

Pegylated arginase treatment in mesothelioma xenograft models.

e20083Background: Cancers with deficient argininosuccinate synthetase (ASS) and/or ornithine transcarbamylase (OTC) are biologically vulnerable to arginine depletion. Preliminary clinical data had ...