Putative cancer stem cells in malignant pleural mesothelioma show resistance to cisplatin and pemetrexed

Malignant pleural mesothelioma (MPM) is a lethal cancer of the mesothelium with high chemotherapeutic resistance via unknown mechanisms. A prevailing hypothesis states that cancer stem cells (CSCs) persist in tumors causing relapse after chemotherapy, thus, rendering these cells as critical targets responsible for tumor resistance and recurrence. We selected candidate CSC markers based on expansion under hypoxic conditions, a hallmark for the selection of chemoresistant cells; and investigated the expression of CSC markers: CD133, Bmi-1, uPAR and ABCG2 in three MPM cell lines and normal mesothelial cells by quantitative RT-PCR. Furthermore, we evaluated the chemotherapeutic resistance associated with each CSC marker by determining the change in CSC marker-mRNA levels as an index of drug-resistance following treatment with either cisplatin or pemetrexed. We demonstrate the expression of CSC markers: CD133, Bmi-1, uPAR and ABCG2 in both normal and MPM cell lines. Bmi-1+, uPAR+ and ABCG2+ cells show a distinct role in conferring chemoresistance to cisplatin and pemetrexed in the malignant setting. By contrast, these markers have no apparent participation in chemoresistance to drug treatments in normal mesothelial cells. Intriguingly, CD133 revealed chemoresistant properties in both normal mesothelial and malignant pleural mesothelioma cells. This study provides evidence of putative CSCs conferring drug-resistance to cisplatin and pemetrexed in MPM cell lines. Specific targeting of these drug-resistant cells, while considering the functional heterogeneity of the MPM subtypes, may contribute to more focused and effective chemotherapeutic regimens for malignant pleural mesothelioma.

Photodynamic drug delivery enhancement in tumours does not depend on leukocyte-endothelial interaction in a human mesothelioma xenograft model

The pre-treatment of tumour neovessels by low-level photodynamic therapy (PDT) improves the distribution of concomitantly administered systemic chemotherapy. The mechanism by which PDT permeabilizes the tumour vessel wall is only partially known. We have recently shown that leukocyte-endothelial cell interaction is essential for photodynamic drug delivery to normal tissue. The present study investigates whether PDT enhances drug delivery in malignant mesothelioma and whether it involves comparable mechanisms of actions.

Long term survival after trimodal therapy in malignant pleural mesothelioma

Trimodal therapy results in long term survival in a small fraction of patients with malignant pleural mesothelioma, particularly in patients having epithelial histology, R0-resection and no nodal involvement. This study analyses the outcome after trimodal therapy including extrapleural pneumonectomy.

p53-induced apoptosis occurs in the absence of p14(ARF) in malignant pleural mesothelioma

Malignant pleural mesotheliomas (MPMs) are usually wild type for the p53 gene but contain homozygous deletions in the INK4A locus that encodes p14(ARF), an inhibitor of p53-MDM2 interaction. Previous findings suggest that lack of p14(ARF) expression and the presence of SV40 large T antigen (L-Tag) result in p53 inactivation in MPM. We did not detect SV40 L-Tag mRNA in either MPM cell lines or primary cultures, and treatment of p14(ARF)-deficient cells with cisplatin (CDDP) increased both total and phosphorylated p53 and enhanced p53 DNA-binding activity. On incubation with CDDP, levels of positively regulated p53 transcriptional targets p21(WAF), PIG3, MDM2, Bax, and PUMA increased in p14(ARF)-deficient cells, whereas negatively regulated survivin decreased. Significantly, p53-induced apoptosis was activated by CDDP in p14(ARF)-deficient cells, and treatment with p53-specific siRNA rendered them more CDDP-resistant. p53 was also activated by: 1) inhibition of MDM2 (using nutlin-3); 2) transient overexpression of p14(ARF); and 3) targeting of survivin using antisense oligonucleotides. However, it is noteworthy that only survivin downregulation sensitized cells to CDDP-induced apoptosis. These results suggest that p53 is functional in the absence of p14(ARF) in MPM and that targeting of the downstream apoptosis inhibitor survivin can sensitize to CDDP-induced apoptosis.

Tocopherol-associated protein-1 accelerates apoptosis induced by alpha-tocopheryl succinate in mesothelioma cells

Alpha-tocopheryl succinate (alpha-TOS), a redox-silent analogue of vitamin E, induces apoptosis in multiple cell lines in a selective manner, by activating the intrinsic pathway. Since it is a highly hydrophobic compound, it may require a carrier protein for its trafficking to intracellular targets like mitochondria. We studied the role of the ubiquitous tocopherol-associated protein-1 (TAP1 or sec14-like 2) in apoptosis induction by alpha-TOS in malignant mesothelioma (MM) cells. Over-expression of TAP1 in MM cells sensitised them to apoptosis by low doses of alpha-TOS which were sub-apoptotic for the parental cells. Apoptosis induced in TAP1-over-expressing cells was mitochondria- and caspase-dependent, as suggested by dissipation of mitochondrial trans-membrane potential and inhibition by zVAD-fmk, respectively. Binding assays showed affinity of alpha-TOS for TAP1. Finally, TAP1 over-expressing cells accumulated alpha-TOS at higher levels compared to their normal counterparts. We suggest that TAP1 may act as an intracellular shuttle for alpha-TOS, promoting apoptosis initiated by this vitamin E analogue, as shown here for MM cells.

Multicenter trial of neo-adjuvant chemotherapy followed by extrapleural pneumonectomy in malignant pleural mesothelioma

BACKGROUND: The aim of this multicenter trial was to prospectively evaluate neo-adjuvant chemotherapy followed by extrapleural pneumonectomy (EPP) and radiotherapy, including quality of life as outcome. PATIENTS AND METHODS: Eligible patients had malignant pleural mesothelioma of all histological types, World Health Organization performance status of zero to two and clinical stage T1-T3, N0-2, M0 disease considered completely resectable. Neo-adjuvant chemotherapy consisted of three cycles of cisplatin and gemcitabine followed by EPP. Postoperative radiotherapy was considered for all patients. RESULTS: In all, 58 of 61 patients completed three cycles of neo-adjuvant chemotherapy. Forty-five patients (74%) underwent EPP and in 37 patients (61%) the resection was complete. Postoperative radiotherapy was initiated in 36 patients. The median survival of all patients was 19.8 months [95% confidence interval (CI) 14.6-24.5]. For the 45 patients undergoing EPP, the median survival was 23 months (95% CI 16.6-32.9). Psychological distress showed minor variations over time with distress above the cut-off score indicating no morbidity with 82% (N = 36) at baseline and 76% (N = 26) at 3 months after surgery (P = 0.5). CONCLUSIONS: The observed rate of operability is promising. A median survival of 23 months for patients undergoing EPP compares favourably with the survival reported from single center studies of upfront surgery. This approach was not associated with an increase in psychological distress.

Low prevalence of SV40 in Swiss mesothelioma patients after elimination of false-positive PCR results

The association of simian virus 40 (SV40) with malignant pleural mesothelioma is currently under debate. In some malignancies of viral aetiology, viral DNA can be detected in the patients' serum or plasma. To characterize the prevalence of SV40 in Swiss mesothelioma patients, we optimized a real-time PCR for quantitative detection of SV40 DNA in plasma, and used a monoclonal antibody for immunohistochemical detection of SV40 in mesothelioma tissue microarrays. Real-time PCR was linear over five orders of magnitude, and sensitive to a single gene copy. Repeat PCR determinations showed excellent reproducibility. However, SV40 status varied for independent DNA isolates of single samples. We noted that SV40 detection rates by PCR were drastically reduced by the implementation of strict room compartmentalization and decontamination procedures. Therefore, we systematically addressed common sources of contamination and found no cross-reactivity with DNA of other polyomaviruses. Contamination during PCR was rare and plasmid contamination was infrequent. SV40 DNA was reproducibly detected in only 4 of 78 (5.1%) plasma samples. SV40 DNA levels were low and not consistently observed in paired plasma and tumour samples from the same patient. Immunohistochemical analysis revealed a weak but reproducible SV40 staining in 16 of 341 (4.7%) mesotheliomas. Our data support the occurrence of non-reproducible SV40 PCR amplifications and underscore the importance of proper sample handling and analysis. SV40 DNA and protein were found at low prevalence (5%) in plasma and tumour tissue, respectively. This suggests that SV40 does not appear to play a major role in the development of mesothelioma.

Individual versus standard quality of life assessment in a phase II clinical trial in mesothelioma patients: feasibility and responsiveness to clinical changes

In patients with malignant pleural mesothelioma undergoing a multimodality therapy, treatment toxicity may outweigh the benefit of progression-free survival. The subjective experience across different treatment phases is an important clinical outcome. This study compares a standard with an individual quality of life (QoL) measure used in a multi-center phase II trial.

The role of muscle flap in preventing bronchus stump insufficiency after pneumonectomy for malignant pleural mesothelioma in high-risk patients

Bronchus stump insufficiency (BSI) is one of the major complications after pneumonectomy; we analyzed all patients who underwent extra pleural pneumonectomy (EPP) for malignant pleural mesothelioma (MPM) in order to detect the role of muscle flap (MF) on preventing early and late stump insufficiency. From January 2000 until December 2005, there were 42 patients admitted with MPM for further intervention at our institution. Thirty patients were suitable for surgery and thus received a multimodal treatment with neo-adjuvant chemotherapy using Cisplatin and Gemcitabin (Gemzar), EPP followed by 54 Gray (Gy) adjuvant radiotherapy. Data were collected from the surgical and oncological records. There were 37 male patients (88%), the median age was 65 years (range 40-83 years). Seven (17%) patients had concomitant diseases. Forty patients (95%) had asbestos exposition. The operative procedures were EPP with muscle flap through an anterolateral thoracotomy. Univariate and multivariate analyses were done. One patient (3%) died on the 2nd postoperative day due to lung embolism. Mild complications were noticed in the early postoperative phase in 8 (25%) patients. There was no early or late stump insufficiency during the 15-month follow-up. Surgical techniques using muscle flap seems to play a major role in the prevention of bronchus stump insufficiency especially after neo-adjuvant chemotherapy.

Cisplatin-resistant cells in malignant pleural mesothelioma cell lines show ALDH(high)CD44(+) phenotype and sphere-forming capacity

BACKGROUND Conventional chemotherapy in malignant pleural mesothelioma (MPM) has minimal impact on patient survival due to the supposed chemoresistance of cancer stem cells (CSCs). We sought to identify a sub-population of chemoresistant cells by using putative CSC markers, aldehyde dehydrogenase (ALDH) and CD44 in three MPM cell lines; H28, H2052 and Meso4. METHODS The Aldefluor assay was used to measure ALDH activity and sort ALDH(high) and ALDH(low) cells. Drug-resistance was evaluated by cell viability, anchorage-independent sphere formation, flow-cytometry and qRT-PCR analyses. RESULTS The ALDH(high) - and ALDH(low) -sorted fractions were able to demonstrate phenotypic heterogeneity and generate spheres, the latter being less efficient, and both showed an association with CD44. Cis- diamminedichloroplatinum (II) (cisplatin) treatment failed to reduce ALDH activity and conferred only a short-term inhibition of sphere generation in both ALDH(high) and ALDH(low) fractions of the three MPM cell lines. Induction of drug sensitivity by an ALDH inhibitor, diethylaminobenzaldehyde (DEAB) resulted in significant reductions in cell viability but not a complete elimination of the sphere-forming cells, suggestive of the presence of a drug-resistant subpopulation. At the transcript level, the cisplatin + DEAB-resistant cells showed upregulated mRNA expression levels for ALDH1A2, ALDH1A3 isozymes and CD44 indicating the involvement of these markers in conferring chemoresistance in both ALDH(high) and ALDH(low) fractions of the three MPM cell lines. CONCLUSIONS Our study shows that ALDH(high) CD44(+) cells are implicated in conveying tolerance to cisplatin in the three MPM cell lines. The combined use of CD44 and ALDH widens the window for identification and targeting of a drug-resistant population which may improve the current treatment modalities in mesothelioma.

Neoadjuvant chemotherapy and extrapleural pneumonectomy of malignant pleural mesothelioma with or without hemithoracic radiotherapy (SAKK 17/04): a randomised, international, multicentre phase 2 trial

BACKGROUND Postoperative hemithoracic radiotherapy has been used to treat malignant pleural mesothelioma, but it has not been assessed in a randomised trial. We assessed high-dose hemithoracic radiotherapy after neoadjuvant chemotherapy and extrapleural pneumonectomy in patients with malignant pleural mesothelioma. METHODS We did this phase 2 trial in two parts at 14 hospitals in Switzerland, Belgium, and Germany. We enrolled patients with pathologically confirmed malignant pleural mesothelioma; resectable TNM stages T1-3 N0-2, M0; WHO performance status 0-1; age 18-70 years. In part 1, patients were given three cycles of neoadjuvant chemotherapy (cisplatin 75 mg/m(2) and pemetrexed 500 mg/m(2) on day 1 given every 3 weeks) and extrapleural pneumonectomy; the primary endpoint was complete macroscopic resection (R0-1). In part 2, participants with complete macroscopic resection were randomly assigned (1:1) to receive high-dose radiotherapy or not. The target volume for radiotherapy encompassed the entire hemithorax, the thoracotomy channel, and mediastinal nodal stations if affected by the disease or violated surgically. A boost was given to areas at high risk for locoregional relapse. The allocation was stratified by centre, histology (sarcomatoid vs epithelioid or mixed), mediastinal lymph node involvement (N0-1 vs N2), and T stage (T1-2 vs T3). The primary endpoint of part 1 was the proportion of patients achieving complete macroscopic resection (R0 and R1). The primary endpoint in part 2 was locoregional relapse-free survival, analysed by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00334594. FINDINGS We enrolled patients between Dec 7, 2005, and Oct 17, 2012. Overall, we analysed 151 patients receiving neoadjuvant chemotherapy, of whom 113 (75%) had extrapleural pneumonectomy. Median follow-up was 54·2 months (IQR 32-66). 52 (34%) of 151 patients achieved an objective response. The most common grade 3 or 4 toxic effects were neutropenia (21 [14%] of 151 patients), anaemia (11 [7%]), and nausea or vomiting (eight [5%]). 113 patients had extrapleural pneumonectomy, with complete macroscopic resection achieved in 96 (64%) of 151 patients. We enrolled 54 patients in part 2; 27 in each group. The main reasons for exclusion were patient refusal (n=20) and ineligibility (n=10). 25 of 27 patients completed radiotherapy. Median total radiotherapy dose was 55·9 Gy (IQR 46·8-56·0). Median locoregional relapse-free survival from surgery, was 7·6 months (95% CI 4·5-10·7) in the no radiotherapy group and 9·4 months (6·5-11·9) in the radiotherapy group. The most common grade 3 or higher toxic effects related to radiotherapy were nausea or vomiting (three [11%] of 27 patients), oesophagitis (two [7%]), and pneumonitis (two [7%]). One patient died of pneumonitis. We recorded no toxic effects data for the control group. INTERPRETATION Our findings do not support the routine use of hemithoracic radiotherapy for malignant pleural mesothelioma after neoadjuvant chemotherapy and extrapleural pneumonectomy. FUNDING Swiss Group for Clinical Cancer Research, Swiss State Secretariat for Education, Research and Innovation, Eli Lilly.

Human lung-derived mesenchymal stem cell-conditioned medium exerts in vitro antitumor effects in malignant pleural mesothelioma cell lines.

BACKGROUND The soluble factors secreted by mesenchymal stem cells are thought to either support or inhibit tumor growth. Herein, we investigated whether the human lung-derived mesenchymal stem cell-conditioned medium (hlMSC-CM) exerts antitumor activity in malignant pleural mesothelioma cell lines H28, H2052 and Meso4. METHODS hlMSC-CM was collected from the human lung-derived mesenchymal stem cells. Inhibition of tumor cell growth was based on the reduction of cell viability and inhibition of cell proliferation using the XTT and BrdU assays, respectively. Elimination of tumor spheroids was assessed by the anchorage-independent sphere formation assay. The cytokine profile of hlMSC-CM was determined by a chemiluminescence-based cytokine array. RESULTS Our data showed that hlMSC-CM contains a broad range of soluble factors which include: cytokines, chemokines, hormones, growth and angiogenic factors, matrix metalloproteinases, metalloproteinase inhibitors and cell-cell mediator proteins. The 48- and 72-hour hlMSC-CM treatments of H28, H2052 and Meso4 cell lines elicited significant decreases in cell viability and inhibited cell proliferation. The 72-hour hlMSC-CM incubation of H28 cells completely eliminated the drug-resistant sphere-forming cells, which is more potent than twice the half maximal inhibitory concentration of cisplatin. CONCLUSIONS Our findings indicate that the cell-free hlMSC-CM confers in vitro antitumor activities via soluble factors in the tested mesothelioma cells and, hence, may serve as a therapeutic tool to augment the current treatment strategies in malignant pleural mesothelioma.

A microfluidic platform for chemoresistive testing of multicellular pleural cancer spheroids

This study reports on a microfluidic platform on which single multicellular spheroids from malignant pleural mesothelioma (MPM), an aggressive tumor with poor prognosis, can be loaded, trapped and tested for chemotherapeutic drug response. A new method to detect the spheroid viability cultured on the microfluidic chip as a function of the drug concentration is presented. This approach is based on the evaluation of the caspase activity in the supernatant sampled from the chip and tested using a microplate reader. This simple and time-saving method does only require a minimum amount of manipulations and was established for very low numbers of cells. This feature is particularly important in view of personalised medicine applications for which the number of cells obtained from the patients is low. MPM spheroids were continuously perfused for 48 hours with cisplatin, one of the standard chemotherapeutic drugs used to treat MPM. The 50% growth inhibitory concentration of cisplatin in perfused MPM spheroids was found to be twice as high as in spheroids cultured under static conditions. This chemoresistance increase might be due to the continuous support of nutrients and oxygen to the perfused spheroids.