K Kolluri

Systemic but not topical TRAIL-expressing mesenchymal stem cells reduce tumour growth in malignant mesothelioma

Malignant pleural mesothelioma is a rare but devastating cancer of the pleural lining with no effective treatment. The tumour is often diffusely spread throughout the chest cavity, making surgical resection difficult, while systemic chemotherapy offers limited benefit. Bone marrow-derived mesenchymal stem cells (MSCs) home to and incorporate into tumour stroma, making them good candidates to deliver anticancer therapies. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic molecule that selectively induces apoptosis in cancer cells, leaving healthy cells unaffected. We hypothesised that human MSCs expressing TRAIL (MSCTRAIL) would home to an in vivo model of malignant pleural mesothelioma and reduce tumour growth. Human MSCs transduced with a lentiviral vector encoding TRAIL were shown in vitro to kill multiple malignant mesothelioma cell lines as predicted by sensitivity to recombinant TRAIL (rTRAIL). In vivo MSC homing was delineated using dual fluorescence and bioluminescent imaging, and we observed that higher levels of MSC engraftment occur after intravenous delivery compared with intrapleural delivery of MSCs. Finally, we show that intravenous delivery of MSCTRAIL results in a reduction in malignant pleural mesothelioma tumour growth in vivo via an increase in tumour cell apoptosis.

LRIG1 regulates cadherin‐dependent contact inhibition directing epithelial homeostasis and pre‐invasive squamous cell carcinoma development

Epidermal growth factor receptor (EGFR) pathway activation is a frequent event in human carcinomas. Mutations in EGFR itself are, however, rare, and the mechanisms regulating EGFR activation remain elusive. Leucine‐rich immunoglobulin repeats‐1 (LRIG1), an inhibitor of EGFR activity, is one of four genes identified that predict patient survival across solid tumour types including breast, lung, melanoma, glioma, and bladder. We show that deletion of Lrig1 is sufficient to promote murine airway hyperplasia through loss of contact inhibition and that re‐expression of LRIG1 in human lung cancer cells inhibits tumourigenesis. LRIG1 regulation of contact inhibition occurs via ternary complex formation with EGFR and E‐cadherin with downstream modulation of EGFR activity. We find that LRIG1 LOH is frequent across cancers and its loss is an early event in the development of human squamous carcinomas. Our findings imply that the early stages of squamous carcinoma development are driven by a change in amplitude of EGFR signalling governed by the loss of contact inhibition.

Mesenchymal stromal cell delivery of full-length tumor necrosis factor-related apoptosis-inducing ligand is superior to soluble type for cancer therapy.

Background aims Mesenchymal stromal cell (MSC) delivery of pro-apoptotic tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an attractive strategy for anticancer therapy. MSCs expressing full-length human TRAIL (flT) or its soluble form (sT) have previously been shown to be effective for cancer killing. However, a comparison between the two forms has never been performed, leaving it unclear which approach is most effective. This study addresses the issue for the possible clinical application of TRAIL-expressing MSCs in the future. Methods MSCs were transduced with lentiviruses expressing flT or an isoleucine zipper-fused sT. TRAIL expression was examined and cancer cell apoptosis was measured after treatment with transduced MSCs or with MSC-derived soluble TRAIL. Results The transduction does not adversely affect cell phenotype. The sT-transduced MSCs (MSC-sT) secrete abundant levels of soluble TRAIL but do not present the protein on the cell surface. Interestingly, the flT-transduced MSCs (MSC-flT) not only express cell-surface TRAIL but also release flT into medium. These cells were examined for inducing apoptosis in 20 cancer cell lines. MSC-sT cells showed very limited effects. By contrast, MSC-flT cells demonstrated high cancer cell-killing efficiency. More importantly, MSC-flT cells can overcome some cancer cell resistance to recombinant TRAIL. In addition, both cell surface flT and secreted flT are functional for inducing apoptosis. The secreted flT was found to have higher cancer cell-killing capacity than either recombinant TRAIL or MSC-secreted sT. Conclusions These observations demonstrate that MSC delivery of flT is superior to MSC delivery of sT for cancer therapy.

TRAIL delivery by MSC-derived extracellular vesicles is an effective anticancer therapy

ABSTRACT Extracellular vesicles (EVs) are lipid membrane-enclosed nanoparticles released by cells. They mediate intercellular communication by transferring biological molecules and therefore have potential as innovative drug delivery vehicles. TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis of cancer cells. Unfortunately, the clinical application of recombinant rTRAIL has been hampered by its low bioavailability and resistance of cancer cells. EV-mediated TRAIL delivery may circumvent these problems. Mesenchymal stromal cells (MSCs) produce EVs and could be a good source for therapeutic EV production. We investigated if TRAIL could be expressed in MSC-derived EVs and examined their cancer cell-killing efficacy. EVs were isolated by ultracentrifugation and were membranous particles of 50–70 nm in diameter. Both MSC- and TRAIL-expressing MSC (MSCT)-derived EVs express CD63, CD9 and CD81, but only MSCT-EVs express surface TRAIL. MSCT-EVs induced apoptosis in 11 cancer cell lines in a dose-dependent manner but showed no cytotoxicity in primary human bronchial epithelial cells. Caspase activity inhibition or TRAIL neutralisation blocked the cytotoxicity of TRAIL-positive EVs. MSCT-EVs induced pronounced apoptosis in TRAIL-resistant cancer cells and this effect could be further enhanced using a CDK9 inhibitor. These data indicate that TRAIL delivery by MSC-derived EVs is an effective anticancer therapy.

Mesenchymal Stem Cells as Vectors for Lung Cancer Therapy

Despite recent advances in treatment, lung cancer accounts for one third of all cancer-related deaths, underlining the need of development of new therapies. Mesenchymal stem cells (MSCs) possess the ability to specifically home into tumours and their metastases. This property of MSCs could be exploited for the delivery of various anti-tumour agents directly into tumours. However, MSCs are not simple delivery vehicles but cells with active physiological process. This review outlines various agents which can be delivered by MSCs with substantial emphasis on TRAIL (tumour necrosis factor-related apoptosis-inducing ligand).

CADM1 inhibits squamous cell carcinoma progression by reducing STAT3 activity

Although squamous cell carcinomas (SqCCs) of the lungs, head and neck, oesophagus, and cervix account for up to 30% of cancer deaths, the mechanisms that regulate disease progression remain incompletely understood. Here, we use gene transduction and human tumor xenograft assays to establish that the tumour suppressor Cell adhesion molecule 1 (CADM1) inhibits SqCC proliferation and invasion, processes fundamental to disease progression. We determine that the extracellular domain of CADM1 mediates these effects by forming a complex with HER2 and integrin α6β4 at the cell surface that disrupts downstream STAT3 activity. We subsequently show that treating CADM1 null tumours with the JAK/STAT inhibitor ruxolitinib mimics CADM1 gene restoration in preventing SqCC growth and metastases. Overall, this study identifies a novel mechanism by which CADM1 prevents SqCC progression and suggests that screening tumours for loss of CADM1 expression will help identify those patients most likely to benefit from JAK/STAT targeted chemotherapies.

Cryopreservation of human mesenchymal stromal cells expressing TRAIL for human anti-cancer therapy.

Background aims Mesenchymal stromal cells (MSCs) are being extensively researched for cell therapy and tissue engineering. We have engineered MSCs to express the pro-apoptotic protein tumor necrosis factor–related apoptosis inducing ligand (TRAIL) and are currently preparing this genetically modified cell therapy for a phase 1/2a clinical trial in patients with metastatic lung cancer. To do this, we need to prepare a cryopreserved allogeneic MSCTRAIL cell bank for further expansion before patient delivery. The effects of cryopreservation on a genetically modified cell therapy product have not been clearly determined. Methods We tested different concentrations of dimethyl sulfoxide (DMSO) added to the human serum albumin ZENALB 4.5 and measured post-thaw cell viability, proliferation ability and differentiation characteristics. In addition, we examined the homing ability, TRAIL expression and cancer cell–killing capacities of cryopreserved genetically modified MSCs compared with fresh, continually cultured cells. Results We demonstrated that the post-thaw viability of MSCs in 5% DMSO (v/v) with 95% ZENALB 4.5 (v/v) is 85.7 ± 0.4%, which is comparable to that in conventional freezing media. We show that cryopreservation does not affect the long-term expression of TRAIL and that cryopreserved TRAIL-expressing MSCs exhibit similar levels of homing and, importantly, retain their potency in triggering cancer cell death. Conclusions This study shows that cryopreservation is unlikely to affect the therapeutic properties of MSCTRAIL and supports the generation of a cryopreserved master cell bank.

Comprehensive pharmacogenomic profiling of malignant pleural mesothelioma identifies a subgroup sensitive to FGFR inhibition.

Purpose: Despite intense research, treatment options for patients with mesothelioma are limited and offer only modest survival advantage. We screened a large panel of compounds in multiple mesothelioma models and correlated sensitivity with a range of molecular features to detect biomarkers of drug response. Experimental design: We utilized a high-throughput chemical inhibitor screen in a panel of 889 cancer cell lines, including both immortalized and primary early-passage mesothelioma lines, alongside comprehensive molecular characterization using Illumina whole-exome sequencing, copy-number analysis and Affymetrix array whole transcriptome profiling. Subsequent validation was done using functional assays such as siRNA silencing and mesothelioma mouse xenograft models. Results: A subgroup of immortalized and primary MPM lines appeared highly sensitive to FGFR inhibition. None of these lines harbored genomic alterations of FGFR family members, but rather BAP1 protein loss was associated with enhanced sensitivity to FGFR inhibition. This was confirmed in an MPM mouse xenograft model and by BAP1 knockdown and overexpression in cell line models. Gene expression analyses revealed an association between BAP1 loss and increased expression of the receptors FGFR1/3 and ligands FGF9/18. BAP1 loss was associated with activation of MAPK signaling. These associations were confirmed in a cohort of MPM patient samples. Conclusions: A subgroup of mesotheliomas cell lines harbor sensitivity to FGFR inhibition. BAP1 protein loss enriches for this subgroup and could serve as a potential biomarker to select patients for FGFR inhibitor treatment. These data identify a clinically relevant MPM subgroup for consideration of FGFR therapeutics in future clinical studies. Clin Cancer Res; 24(1); 84–94. ©2017 AACR.

S60 Mesenchymal stem cells expressing TRAIL induce apoptosis in malignant pleural mesothelioma

Background Malignant pleural mesothelioma (MPM) is an aggressive fatal cancer caused by asbestos exposure. Current treatments are ineffective with an average survival of 4–18 months. Mesenchymal stem cells (MSCs) migrate to tumours and incorporate into tumour stroma making them good vehicles for the delivery of anti-cancer therapies. TNF-related apoptosis inducing ligand (TRAIL) is a transmembrane protein that selectively induces apoptosis in malignant cells without affecting healthy tissues. In this study I tested the hypothesis that MSCs modified to express TRAIL (MSCTRAIL) would cause MPM cell death. Methods Human MSCs were transduced with a lentiviral vector containing TRAIL IRES-GFP under the control of a tetracycline dependent promoter. Successful transduction was measured using flow cytometry and immunoblotting. The biological activity of MSCTRAIL was determined using co-culture experiments. 5×105 MPM cells were stained withDiI and plated with 5×105 MSCTRAIL cells. After 24 h doxycycline (10 μg/ml) was added to induce TRAIL production and left for 48 h. Both cells and supernatant were collected and stained with Annexin V and DAPI to detect apoptosis and death respectively onflow cytometry. Results MSCs were successfully transduced with TRAIL with 96% showing GFP positivity on flow cytometry. Seven human MPM cell lines were tested with 6/7 (86%) being sensitive to MSCTRAIL. JU77 was highly sensitive with an increase in apoptosis from 10.32±2.34 to 48.73±4.3 (percentage ± SEM, p≤0.0001), while MSTO-211H and ONE 58 showed increases from 3.46±0.81to 27.68±1.1 and 8.92±0.05 to 32.93±1.08 respectively (p≤0.001). Conclusions MSCs can be successfully transduced to produce TRAIL and can induce significant levels of apoptosis in the majority of MPM cell lines tested.

S127 Chemotherapy sensitises Malignant pleural mesothelioma cells to undergo MSC-TRAIL induced apoptosis

Introduction Malignant pleural mesothelioma (MPM) is a highly aggressive, incurable, chemoresistant tumour. Recent studies have shown that Mesenchymal stem cells (MSC) can home to and incorporate into the tumour stroma. Their tumour tropism can be used to deliver Tumour necrosis factor related apoptosis inducing ligand (TRAIL), a transmembrane protein that selectively induces apoptosis in transformed cells. However, not all tumours are sensitive to TRAIL. TRAIL works through triggering the extrinsic apoptotic pathway while conventional chemotherapeutic agents act by triggering the intrinsic apoptotic pathway. We hypothesised the crosstalk between these two pathways could be exploited by combining chemotherapy and MSC-TRAIL in MPM tumour cell lines. Methods MSC were engineered to express TRAIL using a lentiviral plasmid vector. A Tetracycline (Tet)-inducible system was used as a backbone to control the expression of TRAIL. Apoptosis induced by recombinant TRAIL, MSC-TRAIL in MPM cell lines on combination with Vorinostat, a chemotherapeutic agent, was measured by Annexin-V/DAPI based flow cytometry. Results The combination of recombinant TRAIL and Vorinostat act synergistically to induce apoptosis in MPM cell lines. Recombinant TRAIL and Vorinostat, as monotherapies induce 7.17% and 51.35% apoptosis in an MPM cell line JU77 respectively. In CRL2081 and ONE58 cell lines, recombinant TRAIL induces 56.75% and 13.41% apoptosis while Vorinostat leads to 78.95% and 43.97% apoptosis respectively. The combination of recombinant TRAIL and Vorinostat shows an increased amount of apoptosis in JU77, CRL2081 and ONE58 cell lines at 80.77%, 96.6% and 77.27% respectively (Table 1). Abstract S127 Table 1. Apoptosis induced by recombinant TRAIL and MSC-TRAIL on combination with Vorinostat rTRAIL Vorinostat rTRAIL and Vorinostat MSCTRAIL not activated MSCTRAIL Activated MSCTRAIL not Activated + Vorinostat MSCTRAIL Activated + Vorinostat Ju77 7.71% 51.35% 80.77% 10.32% 48.73% 47.44%% 77.7% CRL2081 56.75% 78.95% 96.6% 37.3% 57.63% 81.45% 90.93% One58 13.41% 43.97% 79.27% 10.88% 53.8% 49.25% 77.8% MPM cells are treated with recombinant Trail (100ng/ml) and Vorinostat (2.5µM). MSC are plated in 1:1 ratio with tumour cells. MSC are activated with doxycycline to induce TRAIL expression. Similar synergistic affect was observed when TRAIL expressing MSCs were co-cultured with Vorinostat treated MPM cell lines. MSC-TRAIL induced apoptosis in JU77 (48.73%), CRL2081 (57.63%) and ONE58(53.8%). Combined treatment of Vorinostat and MSC-TRAIL significantly increased apoptosis to 77.7% in JU77, 90.93% in CRL2081 and 77.8% in ONE58 cells (Table 1). Conclusion The combination of Vorinostat and recombinant TRAIL acts synergistically to induce apoptosis in malignant plural mesothelioma cells. Similar affect is observed with the combination of MSC-TRAIL and Vorinostat. This study indicates that Mesenchymal stem cells can be used as vectors for delivery of TRAIL and upon combination with Vorinostat, could be a potential therapy for malignant pleural mesothelioma.