Human Mesothelioma Tumors Research Articles

Development of mesothelioma-specific oncolytic immunotherapy enabled by immunopeptidomics of murine and human mesothelioma tumors

Malignant pleural mesothelioma (MPM) is an aggressive tumor with a poor prognosis. As the available therapeutic options show a lack of efficacy, novel therapeutic strategies are urgently needed. Given its T-cell infiltration, we hypothesized that MPM is a suitable target for therapeutic cancer vaccination. To date, research on mesothelioma has focused on the identification of molecular signatures to better classify and characterize the disease, and little is known about therapeutic targets that engage cytotoxic (CD8+) T cells. In this study we investigate the immunopeptidomic antigen-presented landscape of MPM in both murine (AB12 cell line) and human cell lines (H28, MSTO-211H, H2452, and JL1), as well as in patients’ primary tumors. Applying state-of-the-art immuno-affinity purification methodologies, we identify MHC I-restricted peptides presented on the surface of malignant cells. We characterize in vitro the immunogenicity profile of the eluted peptides using T cells from human healthy donors and cancer patients. Furthermore, we use the most promising peptides to formulate an oncolytic virus-based precision immunotherapy (PeptiCRAd) and test its efficacy in a mouse model of mesothelioma in female mice. Overall, we demonstrate that the use of immunopeptidomic analysis in combination with oncolytic immunotherapy represents a feasible and effective strategy to tackle untreatable tumors.

Transglutaminase is a mesothelioma cancer stem cell survival protein that is required for tumor formation.

Mesothelioma is a rare cancer of the mesothelial cell layer of the pleura, peritoneum, pericardium and tunica vaginalis. It is typically caused by asbestos, notoriously resistant to chemotherapy and generally considered incurable with a poor life expectancy. Transglutaminase 2 (TG2), a GTP binding regulatory protein, is an important cancer stem cell survival and therapy resistance factor. We show that TG2 is highly expressed in human mesothelioma tumors and in mesothelioma cancer stem cells (MCS cells). TG2 knockdown or TG2 inhibitor treatment reduces MCS cell spheroid formation, matrigel invasion, migration and tumor formation. Time to tumor first appearance is doubled in TG2 knockout cells as compared to wild-type. In addition, TG2 loss is associated with reduced expression of stemness, and epithelial mesenchymal transition markers, and enhanced apoptosis. These studies indicate that TG2 is an important MCS cell survival protein and suggest that TG2 may serve as a mesothelioma cancer stem cell therapy target.

DCLK1 is correlated with MET and ERK5 expression, and associated with prognosis in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive cancer for which more effective treatments are needed. In this study, strong to moderate staining of MET and ERK5 was detected in 67.1 and 48% of the analyzed 73 human mesothelioma tumors, and significant correlation of MET and ERK5 expression was identified (P<0.05). We evaluated the doublecortin-like kinase 1 (DCLK1) expression in human mesothelioma tumors. Our results showed that 50.7% of the immunohistochemistry analyzed human mesothelioma tumors have strong to moderate staining of DCLK1, and its expression is significantly correlated with MET or ERK5 expression (P<0.05). Also, the upregulation of DCLK1 is correlated with poor prognosis in MPM patients (P=0.0235). To investigate whether DCLK1 is downstream of MET/ERK5 signaling in human mesothelioma, the effect of DCLK1 expression was analyzed after treatments with either the MET inhibitor XL184 or the ERK5 inhibitor XMD8-92 in human mesothelioma cell lines. Our results showed that the MET inhibitor XL184 reduced the expression of phospho-ERK5 and DCLK1 expression in human mesothelioma cell lines. In addition, the ERK5 inhibitor XMD8-92 reduced the expression of phospho-ERK5 and DCLK1 expression in human mesothelioma cell lines. Furthermore, XML184 and XMD8-92 treatment impaired invasion and tumor sphere formation ability of H290 mesothelioma cells. These results suggest that DCLK1 is regulated by MET/ERK5 signaling in human mesothelioma, and the MET/ERK5/DCLK1 signaling cascade could be further developed into a promising therapeutic target against mesothelioma.

Low signal strength enhances the antitumor activity of human CAR Th17 cells

Abstract We reported the antitumor activity of human Th17 cells (engineered to express a chimeric antigen receptor (CAR) that recognizes mesothelin) regresses large human mesothelioma tumors when infused into mice, but it remains unknown what type and what level of co-signal strength impacts their functional fate and antitumor activity. To address this question, human Th17 cells were activated with a high (3 beads: 1 T cell), medium (1:10) or low (1:100) signal via CD3-beads coated with either CD28 or ICOS agonists. We discovered Th17 cells activated with low ICOS signal were highly multifunctional (IL-17A, IFN-γ, IL-2 and IL-22) and possessed a less differentiated phenotype (high CD62L and CD45RA expression) than cells activated with a high ICOS signal. In comparison to low ICOS signal strength, Th17 cells activated with low CD28 signal strength were poorly functional and terminally differentiated. Of clinical significance, in two distinct tumor models (human mesothelioma and murine melanoma), we found that Th17 cells regressed large tumors to a better extent when primed with a low or medium ICOS signal strength compared to if they were activated with a high ICOS signal strength, or with any level of CD28 signal strength. Our data is compelling given that most ACT clinical trials use high CD3/CD28 activation to expand patients’ cells. Specifically, our work suggests the use of a low bead to T cell ratio will result in a more simple and clinically feasible method to expand highly therapeutic T cells for the clinic.

Mesothelioma tumor cells modulate dendritic cell lipid content, phenotype and function.

Dendritic cells (DCs) play an important role in the generation of anti-cancer immune responses, however there is evidence that DCs in cancer patients are dysfunctional. Lipid accumulation driven by tumor-derived factors has recently been shown to contribute to DC dysfunction in several human cancers, but has not yet been examined in mesothelioma. This study investigated if mesothelioma tumor cells and/or their secreted factors promote increases in DC lipid content and modulate DC function. Human monocyte-derived DCs (MoDCs) were exposed to human mesothelioma tumor cells and tumor-derived factors in the presence or absence of lipoproteins. The data showed that immature MoDCs exposed to mesothelioma cells or factors contained increased lipid levels relative to control DCs. Lipid accumulation was associated with reduced antigen processing ability (measured using a DQ OVA assay), upregulation of the co-stimulatory molecule, CD86, and production of the tolerogenic cytokine, IL-10. Increases in DC lipid content were further enhanced by co-exposure to mesothelioma-derived factors and triglyceride-rich lipoproteins, but not low-density lipoproteins. In vivo studies using a murine mesothelioma model showed that the lipid content of tumor-infiltrating CD4+CD8α- DCs, CD4-CD8α- DCs DCs and plasmacytoid DCs increased with tumor progression. Moreover, increasing tumor burden was associated with reduced proliferation of tumor-antigen-specific CD8+ T cells in tumor-draining lymph nodes. This study shows that mesothelioma promotes DC lipid acquisition, which is associated with altered activation status and reduced capacity to process and present antigens, which may impair the ability of DCs to generate effective anti mesothelioma T cell responses.

Low PIAS3 expression in malignant mesothelioma is associated with increased STAT3 activation and poor patient survival.

Deregulation of STAT3 activation is a hallmark of many cancer cells, and the underlying mechanisms are subject to intense investigation. We examined the extent of PIAS3 expression in mesothelioma cells and human tumor samples and determined the functional effects of PIAS3 expression on STAT3 signaling. We evaluated the expression of PIAS3 in mesothelioma tumors from patients and correlated the expression levels with the course of the disease. We also measured the effects of enhanced PIAS3 activity on STAT3 signaling, cellular growth, and viability in cultured mesothelioma cells. Gene expression databases revealed that mesotheliomas have the lowest levels of PIAS3 transcripts among solid tumors. PIAS3 expression in human mesothelioma tumors is significantly correlated with overall survival intervals (P = 0.058). The high expression of PIAS3 is predictive of a favorable prognosis and decreases the probability of death within one year after diagnosis by 44%. PIAS3 expression is functionally linked to STAT3 activation in mesothelioma cell lines. STAT3 downregulation with siRNA or enhanced expression of PIAS3 both inhibited mesothelioma cell growth and induced apoptosis. Mesothelioma cells are sensitive to curcumin and respond by the induction of PIAS3. Corroborative evidence has been obtained from STAT3 inhibition experiments. Exposure of the cells to a peptide derived from the PIAS3 protein that interferes with STAT3 function resulted in apoptosis induction and the inhibition of cell growth. These results suggest that PIAS3 protein expression impacts survival in patients with mesothelioma and that PIAS3 activation could become a therapeutic strategy. Clin Cancer Res; 20(19); 5124-32. ©2014 AACR.

Sensitivity of malignant mesothelioma lacking Merlin to the FAK inhibitor VS-6063: Evaluation of merlin/NF2 status in clinical samples.

e18541 Background: Malignant pleural mesothelioma (MPM) is an aggressive tumor in the pleural lining of the lung often resulting from prior exposure to asbestos. MPM patients are usually diagnosed at an advanced stage of disease and the prognosis is poor. Median survival after diagnosis is 9 to 12 months and standard-of-care agents such as cisplatin and pemetrexed have only a modest impact on median survival time for MPM patients. New therapeutic modalities are urgently needed to improve the prognosis of MPM patients. 40-50% of MPM patients exhibit homozygous disruption of the NF2 tumor suppressor gene by mutation and/or deletion resulting in lack of expression of functional merlin protein. Methods: Proliferation of drug-treated mesothelioma cell lines in 3-dimensional (3D) Matrigel culture was assessed by MTS, and MPM xenograft growth was measured in vehicle- vs. FAK inhibitor-treated SCID mice. Since absence of merlin expression can theoretically result from several mechanisms including NF2 mutation and chromosome 22 abnormalities, we assessed NF2 gene deletion by FISH and merlin protein levels by IHC in the same human mesothelioma tumor samples. Results: Among a panel of mesothelioma cell lines in 3D culture, MPM lines lacking expression of merlin protein were found to be especially sensitive to the selective FAK inhibitor VS-6063. In contrast, MPM cell lines with wildtype merlin were less sensitive with EC50 values greater than 1 μM. Accordingly, oral dosing with a FAK inhibitor induced significant tumor growth inhibition in a merlin-negative mesothelioma model in mice. To enable the planned stratification of MPM patients by merlin status, an immunohistochemistry (IHC) assay has been optimized to quantify merlin protein levels. A merlin IHC H-score below the defined cutoff was associated with loss of at least one copy of chromosome 22, indicating that chromosomal deletion is an important mechanism of merlin loss in mesothelioma patients. Conclusions: These data support the clinical development of VS-6063 for treatment of malignant pleural mesothelioma patients stratified by merlin/NF2 status. A potentially pivotal mesothelioma trial is set to initiate in 2013.

Abstract 3446: FL118, a survivin/Mcl-1/XIAP/cIAP2-selective inhibitor, effectively inhibits human mesothelioma xenograft growth in animal models.

Abstract We have identified and characterized a novel small chemical molecule (designated FL118). Our in vitro studies showed that FL118 selectively inhibits the expression of survivin, Mcl-1, XIAP and cIAP2, while showing no inhibitory effects on control genes. In an intraperitoneal (i.p.) route, the maximum tolerated dose (MTD) of FL118 was found to be about 1.5 mg/kg at a weekly x 4 schedule in a Tween 80-comtaining formulation, FL118 showed superior antitumor activity in mouse models of human colon and head-&amp;-neck tumor xenografts (PLOS ONE. 2012, 7: e45571). However, we found that actually, the weekly x 4 schedule for FL118 in the Tween 80-containing formulation is the best schedule for showing the maximal antitumor activity for FL118. So further improvement of FL118 antitumor efficacy would have no rooms with this formulation. In this regard, we have developed a Tween 80-free formulation that fits the intravenously (i.v.) injection. Here we report our results with this formulation on human mesothelioma in animal models. Two mesothelioma cell lines (MSTO-211H, NCI-H226) were used to establish human xenograft in SCID mice. After the established human mesothelioma tumor in mice was sub-transplanted 2-3 times, the tumor masses were then used for testing FL118 efficacy and toxicity in a Tween 80-free formulation. We first determined FL118 MTD via i.v. routes in three commonly used schedules in clinical practice - every day for five times (daily x 5), every other day for five times (q2 x 5) and every week for 4 times (weekly x 4). Our result showed that the MTD for FL118 in the Tween 80-free formulation via i.v. increases 3-7 times in comparison with the FL118 MTD in the Tween 80 containing formulation. In the new formulation, FL118 reached 1.5 mg/kg, (daily x 5, q2 x 5) and 5 mg/kg (weekly x 4), respectively. Then we tested the efficacy of FL118 for inhibiting mesothelioma tumor growth and found that FL118 induced tumor regression in all three clinical compatible schedules, while the toxicity profile appears to be improved in comparison with the outcome from FL118 in the Tween 80-containing formulation. Specifically, our data showed that FL118 effectively inhibits MSTO-211H-derived tumor growth, induces tumor regression and even achieved a cure in a percentage of human mesothelioma xenograft, while the tumors in control mice without FL118 treatment reached the maximal size (∼2000 mg/mm3) allowed in less than five weeks. Additionally, FL118 also showed effective inhibition of NCI-H226-established tumor growth. Therefore, we conclude that we developed a clinical suitable formula for FL118 i.v. administration. We also, for the first time, demonstrated that FL118 possesses superior antitumor activity in animal models of human mesothelioma cancer cell line-established tumor xenograft, which provides a hope to use FL118 as a superior option to effectively treat mesothelioma in clinical practice. Citation Format: Fengzhi Li, Shousong Cao, Xiang Ling. FL118, a survivin/Mcl-1/XIAP/cIAP2-selective inhibitor, effectively inhibits human mesothelioma xenograft growth in animal models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3446. doi:10.1158/1538-7445.AM2013-3446

Efficacy of anti-insulin-like growth factor I receptor monoclonal antibody cixutumumab in mesothelioma is highly correlated with insulin growth factor-I receptor sites/cell

Insulin growth factor-I receptor (IGF-IR) is expressed in mesothelioma and therefore an attractive target for therapy. The antitumor activity of cixutumumab, a humanized monoclonal antibody to IGF-IR, in mesothelioma and relationship to IGF-IR expression was investigated using eight early passage tumor cells obtained from patients, nine established cell lines and an in vivo human mesothelioma tumor xenograft model. Although IGF-IR expression at the mRNA and protein level was present in all mesothelioma cells, using a quantitative ELISA immunoassay, there was considerable variability of IGF-IR expression ranging from 1 to 14 ng/mg of lysate. Using flow cytometry, the number of IGF-IR surface receptors varied from ≈ 2,000 to 50,000 sites/cell. Cells expressing >10,000 sites/cell had greater than 10% growth inhibition when treated with cixutumumab (100 μg/ml). Cixutumumab also induced antibody-dependent cell-mediated toxicity (>10% specific lysis) in cell lines, which had >20,000 IGF-IR sites/cell. Treatment with cixutumumab decreased phosphorylation of IGF-IR, Akt and Erk in cell lines, H226 and H28 having 24,000 and 51,000 IGF-IR sites/cell, respectively, but not in the cell line H2052 with 3,000 IGF-IR sites/cell. In vivo, cixutumumab treatment delayed growth of H226 mesothelioma tumor xenografts in mice and improved the overall survival of these mice compared to mice treated with saline (p < 0.004). Our results demonstrate that the antitumor efficacy of cixutumumab including inhibition of IGF-IR downstream signaling is highly correlated with IGF-IR sites/cell. A phase II clinical trial of cixutumumab is currently ongoing for the treatment of patients with mesothelioma.

Abstract 2514: CSPG4 as a target of antibody-based immunotherapy for malignant mesothelioma

Abstract Malignant mesothelioma (MM) is an aggressive tumor, resistant to conventional therapies, with median survival of 1 year from diagnosis. The lack of effective therapies available prompted us to implement antibody-based immunotherapy against novel targets and to investigate possible synergies with chemotherapy on MM. CSPG4 is involved in the onset and progression of melanoma and other tumors, as well as in neo-angiogenesis. CSPG4-specific monoclonal antibodies (mAb) have shown anti-tumor effects in both preclinical and clinical trials of melanoma. We hypothesized that targeting CSPG4 with a CSPG4-specific mAb would have therapeutic efficacy against MM. We found that chondroitin sulphate proteoglycan 4 (CSPG4) was aberrantly expressed in 6 out of 8 MM cell lines and in 24 out of 40 MM biopsies, with minimal expression in surrounding non-malignant cells. The expression of CSPG4 in MM was higher upon engagement of extracellular matrix components (ECM) and was associated with increased MM cell viability and motility. Silencing of CSPG4 expression by siRNAs supported this functional significance of CSPG4 in MM. Consistently, the CSPG4-specific mAb TP41.2 inhibited MM cell attachment to the ECM, resulting in decreased phosphorylation of FAK and AKT, decreased expression of cyclin D1 and apoptosis. Moreover, TP41.2 significantly reduced MM cell motility, migration and invasiveness, and inhibited MM growth in soft agar. TP41.2 prevented the outgrowth of human MM tumors in SCID mice and inhibited the growth of established MM xenografts, resulting in significantly extended survival of tumor-bearing mice. These results represent a novel approach for CSPG4 mAb-based immunotherapy of MM. Combinatorial strategies with chemotherapeutic agents and with mAbs targeting c-Met related receptor are ongoing, aimed at translating CSPG4-specific mAb into clinical setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2514. doi:1538-7445.AM2012-2514

IL-2/CD40-driven NK cells install and maintain potency in the anti-mesothelioma effector/memory phase

Murine and human mesothelioma tumors are susceptible to immunotherapy, particularly when immune adjuvants are delivered locally. We have shown that direct injection of IL-2 plus agonist anti-CD40 antibody induces regression of large mesothelioma tumors. These studies aimed to determine if NK cells contribute to IL-2/CD40 antibody-driven tumor eradication. We show that NK cells infiltrate developing mesothelioma tumors; however, their absence (in beige mice or in asialo GM(1) antibody-depleted C57BL/6J mice) does not alter tumor growth rates suggesting that they cannot function as effector cells in this microenvironment. Anti-CD40 antibody treatment did not alter the percent of NK cells in treated tumors or in draining lymph nodes (dLNs), and tumor resolution occurred in the absence of NK cells. However, a two-tumor model showed that NK cells contributed to CD40-driven systemic immunity leading to resolution of untreated distal tumors. IL-2 treatment led to increased proportions of NK cells in tumors and dLNs, and in the absence of NK cells, IL-2 lost its therapeutic effect. In contrast, the absence of NK cells did not reduce the anti-tumor activity of the IL-2/anti-CD40 antibody combination yet tumors recurred in NK-deficient mice and > 37% of tumor cell re-challenged mice were unable to provide protection, implying insufficient memory. Furthermore, untreated distal tumors in NK-depleted mice were less readily cured than in immunologically intact mice. These data show that NK cells infiltrate mesothelioma tumors, which, after local IL-2 and/or anti-CD40 antibody treatment, provide help for the acquisition and/or maintenance of systemic immunity and long-term effector/memory responses.

Abstract 646: Anti-tumor efficacy of IMC-A12, a fully human anti-insulin like growth factor 1 receptor (IGF-1R) antibody, against malignant mesothelioma (MM)

Abstract Introduction: MM is an aggressive cancer with limited treatment options and poor prognosis. Previous studies have shown that the interaction between IGF-1R and its ligands IGF-1 and IGF-2 play an important role in MM tumorogenesis. To exploit IGF-1R as a target for mesothelioma therapy we investigated the activity of IMC-A12 against early passage mesothelioma tumor cells, established MM cell lines and an in vivo human mesothelioma tumor xenograft model. Material and methods: Eight early passage mesothelioma tumor cells obtained from patients with mesothelioma and eight established MM cell lines were evaluated for IGF-1R expression by real time PCR, Western blotting, and a quantitative immunoassay. The cell surface IGF-IR expression (sites/cell) was analyzed by flow cytometry using QuantiBRITE-PE bead assay. The anti-proliferative effect of IMC-A12 and its ability to induce antibody mediated cell cytoxicity (ADCC) was evaluated in vitro using both early passage tumor cells as well as established cell lines. For our in vivo experiment, the mesothelioma cell line NCI-H226, stably transfected with the luciferase gene, was inoculated intraperitoneally (i.p) into athymic nude mice. Three days after tumor cell inoculation, mice received either IMC-A12 (50 mg/kg) or saline by i.p. injection twice weekly for 8 weeks and tumor growth was monitored using bioluminescence imaging. Results: All early passage as well as established MM cell lines showed IGF-1R expression at the mRNA and protein levels. Using a quantitative electrochemiluminescence immunoassay we determined that there is considerable variability of IGF-1R expression ranging from 1-12 ng/mg of cell lysate. Using flow cytometry we evaluated the number of IGF-1R cell surface receptors, which varied from ≈ 2,000-50,000 sites/cell. Cells expressing &amp;gt;10,000 sites/cell had greater than 10% (range, 10% -60%) growth inhibition when treated with IMC-A12 (100 μg/mL). Similarly, IMC-A12 treatment induced ADCC (&amp;gt; 10% specific lysis) in those cell lines with IGF-1R expression of &amp;gt; 10,000 sites/cell. In vivo IMC-A12 treatment delayed tumor growth in athymic nude mice bearing H226 tumors and improved the overall survival when compared to the control group (p&amp;lt; 0.05). Conclusion: Our results show that both early passage mesothelioma cells as well as established MM cell lines express IGF-1R but that there is a wide variability in the degree of IGF-1R expression. IMC-A12 exhibits anti-proliferative activity against these cell lines as well as induces ADCC, both of which are strongly co-related with IGF-1R cell surface expression. Single agent IMC-A12 also has significant in vivo anti-tumor activity. These results suggest IMC-A12 may be useful for treatment of MM. A phase II clinical trial of IMC-A12 has just opened for patient accrual. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 646. doi:10.1158/1538-7445.AM2011-646

A novel translational approach for human malignant pleural mesothelioma: heparanase-assisted dual virotherapy

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor that is related to asbestos exposure. MPM is characterized by rapid and diffuse local growth in the thoracic cavity, and it has a poor prognosis because it is often refractory to conventional therapy. Although MPM is an extraordinarily challenging disease to treat, locoregional virotherapy may be useful against this aggressive disease because of the accessibility by intrapleural virus delivery. In this study, we show that telomerase-specific, replication-selective adenovirus OBP-301 can efficiently infect and kill human mesothelioma cells by viral replication. Intrathoracic administration of virus significantly reduced the number and size of human mesothelioma tumors intrathoracically implanted into nu/nu mice. A high-definition, fluorescence optical imaging system with an ultra-thin, flexible fibered microprobe clearly detected intracellular replication of green fluorescent protein-expressing oncolytic virus in intrathoracically established mesothelioma tumors. As the extracellular matrix (ECM) may contribute to the physiological resistance of a solid tumor by preventing the penetration of therapeutic agents (including oncolytic viruses), we also examined whether the co-expression of heparanase, an endoglucuronidase capable of specifically degrading heparan sulfate, that influences the physiological barrier to macromolecule penetration, can modify the permeability of the ECM, resulting in profound therapeutic efficacy. Co-injection of OBP-301 and a replication-defective adenovirus (Ad-S/hep)-expressing heparanase resulted in more profound antitumor effects without apparent toxicity in an orthotopic pleural dissemination model. Our results suggest that intrathoracic dual virotherapy with telomerase-specific oncolytic adenovirus in combination with heparanase-expressing adenovirus may be efficacious in the prevention and treatment of pleural dissemination of human malignant mesothelioma.

Inhibition of the PI3K and mTOR survival pathways enhances apoptosis in human mesothelioma tumor fragment spheroids

Malignant mesothelioma exhibits significant resistance to apoptosis. We have characterized a novel in vitro model of human mesothelioma tissue grown in culture as tumor fragment spheroids to identify the mechanisms of resistance. Spheroids maintained many features of the original tumors, including the presence of viable proliferating mesothelioma cells, for up to 3 months. Mesothelioma cell monolayers or 2-week old human mesothelioma spheroids were exposed to the death receptor ligand TRAIL and cycloheximide (CHX) for 48 h and studied for apoptosis by expression of cleaved caspase 3 in mesothelioma (cytokeratin-positive) cells. Unlike the nearly 100% apoptosis in the monolayers, TRAIL plus CHX induced apoptosis in only 32 ± 4% (mean ± SEM) of mesothelioma cells within spheroids (versus 7 ± 1% in untreated spheroids). Apoptosis of mesothelioma cells in spheroids increased to 47 ± 6% by inhibition of the PI3K pathway with LY294002 and to 50 ± 17% by inhibition of the mTOR pathway with rapamycin. Inhibition of the pathways was confirmed with staining for p-Akt or p70-S6K, targets of the PI3K and mTOR pathways, respectively. Targeting these survival pathways therefore presents a promising approach for mesothelioma and perhaps other tumors resistant to apoptosis. This research was supported by an NIH R01 NCI-095671 to VCB.

A Novel In Vitro Model of Human Mesothelioma for Studying Tumor Biology and Apoptotic Resistance

Like many tumors, malignant mesothelioma exhibits significant chemoresistance and resistance to apoptosis in vivo that is not seen in current in vitro models. To study the mechanisms of this multicellular resistance, biologically relevant in vitro models are necessary. Therefore, we characterized and tested human mesothelioma tissue grown in vitro as tumor fragment spheroids. After 5-10 d in culture, fragments from each of 15 human mesothelioma tumors rounded into spheroids. The tumor fragment spheroids maintained multiple characteristics of the original tumors for up to 3 mo including the presence of viable mesothelioma cells, macrophages, and a collagen-rich stroma. In 14-d-old spheroids, mesothelioma cells showed the same proliferation rate and expression of a death receptor, DR5, as in the original tumor. To determine responses to treatment, we treated tumor fragment spheroids grown from three separate tumors with agents, TNF-related apoptosis-inducing ligand (TRAIL) plus cycloheximide, that induced near total apoptosis in three human mesothelioma cell lines (M28, REN, MS-1) grown as monolayers (94 +/- 6% apoptosis; mean +/- SEM). Compared with mesothelioma cells in monolayers, mesothelioma cells in the spheroids were resistant to TRAIL plus cycloheximide (32 +/- 4% apoptosis; mean +/- SEM). Apoptotic resistance of mesothelioma cells was significantly reduced by inhibiting either the PI3K/Akt pathway with LY294002 (47 +/- 6% apoptosis) or the mTOR pathway with rapamycin (50 +/- 17% apoptosis). We conclude that human mesothelioma can be maintained in vitro in a biologically relevant model that exhibits apoptotic resistance, thereby permitting study of its tumor biology and of novel approaches to therapy.

Gene therapy using adenovirus carrying the herpes simplex-thymidine kinase gene to treat in vivo models of human malignant mesothelioma and lung cancer.

Previous studies have shown adenoviral transfer of the herpes simplex virus thymidine kinase (HSVtk) gene followed by the anti-viral drug ganciclovir (GCV) can be used to successfully treat established human mesothelioma tumors growing within the peritoneal cavities of severe combined immune deficient (SCID) mice. These findings raised a number of questions important to the applicability, efficiency, and safety of this treatment strategy. In this report, we have further characterized the use of recombinant adenovirus carrying the HSVtk gene to treat mesothelioma and other localized malignancies. Our results indicate that the Ad.RSVtk/GCV system is effective in causing tumor regression in animals inoculated with another mesothelioma cell line and a lung cancer cell line and that animals with bulky disease can be successfully treated. Effects are seen at a wide range of virus doses and significant anti-tumor activity is present at doses of ganciclovir that are clinically achievable. Finally, this treatment approach appears safe, with limited dissemination of virus using a sensitive RT-PCR detection system. These studies further characterize the use of adenoviral transfer of the HSVtk gene to treat experimental mesothelioma and suggest that clinical trials using this approach may be feasible.

Treatment of experimental human mesothelioma using adenovirus transfer of the herpes simplex thymidine kinase gene.

The authors demonstrate the ability of an adenovirus vector expressing the herpes simplex thymidine kinase (HSVtk) gene to treat human malignant mesothelioma growing within the peritoneal cavity of severe combined immunodeficient (SCID) mice. Introduction of the HSVtk gene into tumor cells renders them sensitive to the antiviral drug ganciclovir (GCV). This approach has been used previously to treat experimental brain tumors. Although malignant mesothelioma is refractory to current therapies, its localized nature and the accessibility of the pleural space make it a potential target for a similar type of in vivo gene therapy using adenovirus. An adenovirus containing the HSVtk gene (Ad.RSVtk) was used to transduce mesothelioma cells in vitro. These cells were then injected into the flanks of SCID mice. Ad.RSVtk was also injected directly into the peritoneal cavity of SCID mice with established human mesothelioma tumors. Mice were subsequently treated for 7 days with GCV at a dose of 5 mg/kg. Mesothelioma cells transduced in vitro with Ad.RSVtk formed nodules when injected in the subcutaneous tissue. These tumors could be eliminated by the administration of GCV, even when as few as 10% of cells were transduced to express HSVtk (bystander effect). Administration of Ad.RSVtk into the peritoneal space of animals with established multifocal human mesothelioma followed by GCV therapy resulted in the eradication of macroscopic tumor in 90% of animals and microscopic tumor in 80% of animals when evaluated after 30 days. The median survival of animals treated with Ad.RSVtk/GCV was significantly longer than that of control animals treated with similar protocols. These results indicate that an adenoviral vector containing the HSVtk gene is effective in treating established malignant mesothelioma in an in vivo setting and raise the possibility of using adenovirus transfer of HSVtk for clinical trials in mesothelioma and other localized tumors.