Correlation of predictive markers and phenotype interactions to drug sensitivity.

<p>Interaction coefficients and p-values from the multivariate regression and ANOVA analysis for the suggested predictive markers. Gemcitabine and ERCC1 present the largest coefficients.</p

Variation in Drug Sensitivity of Malignant Mesothelioma Cell Lines with Substantial Effects of Selenite and Bortezomib, Highlights Need for Individualized Therapy

<div><p>Background</p><p>Malignant mesothelioma cells have an epithelioid or sarcomatoid morphology, both of which may be present in the same tumor. The sarcomatoid phenotype is associated with worse prognosis and heterogeneity of mesothelioma cells may contribute to therapy resistance, which is often seen in mesothelioma. This study aimed to investigate differences in sensitivity between mesothelioma cell lines to anti-cancer drugs. We studied two novel drugs, selenite and bortezomib and compared their effect to four conventional drugs. We also investigated the immunoreactivity of potential predictive markers for drug sensitivity; Pgp, MRP-1, ERCC1, RRM1, TS, xCT and proteasome 20S subunit.</p><p>Materials and methods</p><p>We treated six mesothelioma cell lines with selenite, bortezomib, carboplatin, pemetrexed, doxorubicin or gemcitabine as single agents and in combinations. Viability was measured after 24 and 48 hours. Immunocytochemistry was used to detect predictive markers.</p><p>Results</p><p>As a single agent, selenite was effective on four out of six cell lines, and in combination with bortezomib yielded the greatest response in the studied mesothelioma cell lines. Cells with an epithelioid phenotype were generally more sensitive to the different drugs than the sarcomatoid cells. Extensive S-phase arrest was seen in pemetrexed-sensitive cell lines. MRP-1 predicted sensitivity of cell lines to treatment with carboplatin and xCT predicted pemetrexed effect.</p><p>Conclusions</p><p>The observed heterogeneity in sensitivity of mesothelioma cell lines with different morphology highlights the need for more individualized therapy, requiring development of methods to predict drug sensitivity of individual tumors. Selenite and bortezomib showed a superior effect compared to conventional drugs, motivating clinical testing of these agents as future treatment regime components for patients with malignant mesothelioma.</p></div

Sensitivity of mesothelioma cell lines to different cytotoxic drugs.

<p>Treated cells were normalized to untreated cells and cell viability was measured using the WST-1 assay after 24 (red) and 48 hours (blue). Results are mean values of at least three independent experiments with four replicates in each. Error bars denote the 95% confidence intervals. Cell lines are presented with increasing length/width ratios from left to right and divided into three sub-groups according to their phenotype, epithelioid cell line on the left and sarcomatoid to the right on grey background, biphasic in the middle.</p

Morphological characteristics of the malignant mesothelioma cell lines.

<p>Characteristic micrographs presenting the different cell lines with increasing length/width ratios from A to F. A: Epitheliod STAV-AB cells. B: Biphasic M-14-K cells. C: Biphasic STAV-FCS cells. D: Biphasic ZL-34 cells. E: Biphasic JL-1 cells. F: Sarcomatoid DM-3 cells. Scale bar = 100 µm.</p

Antibodies used in these experiments.

<p>Suppliers: 1 = Leica Microsystems GmbH, Wetzlar, Germany, 2 = Thermo Fisher Scientific Inc, Waltham, MA, USA. 3 = Abcam, Cambridge, UK.</p

Effects of conventional drug combinations on mesothelioma cells.

<p>Cells were treated with combinations of the conventional drugs, normalized to untreated cells and viability was measured with the WST-1 assay after 24 (red) and 48 hours (blue). Mean values of at least three independent experiments with four replicates in each are presented. Error bars denote the 95% confidence intervals. Cell lines are divided into three sub-groups with increasing length/width ratios from left to right and according to their phenotype; biphasic in the middle, epithelioid cell line on the left and sarcomatoid to the right on grey background.</p

Correlation between immunoreactivity of predictive markers and drug sensitivity.

<p>The sensitivity of mesothelioma cell lines to different drugs plotted against the immunoreactivity of different predictive markers, presented together with results from the linear regression analyses. Statistical significance was accepted at p<0.05 and was seen for MRP-1 immunoreactivity and sensitivity of cell lines to carboplatin treatment (A) and for xCT and pemetrexed effect (B). Abbreviations: Pgp = P-glycoprotein, MRP-1 = Multidrug resistance-associated protein 1, ERCC1 = Excision repair cross-complementing rodent repair deficiency, complementation group 1, RRM1 = Ribonucleotide reductase M1, TS = Thymidylate synthase, xCT = x_c^- cystine transporter and 20S P = 20S proteasome.</p

Characteristics of mesothelioma cell lines.

<p>Length/width ratios are average with standard deviation. Epithelioid and sarcomatoid cell lines in bold. Abbreviations: RPMI 1640 = Gibco RPMI 1640 medium, AB = Human AB-serum, FBS = Fetal Bovine Serum, BS = Bovine Serum and NCTC-109 = NCTC-109 medium.</p

Cell cycle distribution after 48 hours of pemetrexed treatment.

<p>All cells were stained with PI. Cells were normalized according to FlowJo algorithms (% of max), considering the different amount of live cells in controls and treated cells. Mesothelioma cell lines arranged with increasing length/width ratios from A to F. One representative experiment is shown for each cell line with control cells marked in grey and 48 hours pemetrexed treated cells in black. STAV-AB (A), M-14-K (B), STAV-FCS (C) and Jurkat cells (G, used as positive control) show an S-phase arrest.</p

Effects of drug combinations with bortezomib on mesothelioma.

<p>Viability of cells measured using the WST-1 assay after 24 (red) and 48 hours (blue) of treatment with bortezomib in combination with the four conventional drugs and normalized to untreated cells. Results are shown as mean values of at least three independent experiments with four replicates in each experiment. Error bars denote the 95% confidence intervals. Cell lines are presented with increasing length/width ratios from left to right and divided into three sub-groups according to their phenotype, epithelioid cell line on the left and sarcomatoid to the right on grey background, biphasic in the middle.</p