Ex vivo evaluation of tumor cell specific drug responses in malignant pleural effusions.

The effect of chemotherapy may be improved by combining the most effective drugs based on testing the sensitivity of the individual tumor ex vivo. Such estimations of tumor cells from effusions have so far not been implemented in the clinical routine as a basis for individualized choice of therapy. One obstacle for such analyses is the admixture of benign cells that might obscure the results. In this paper we test and compare two ways of performing the analysis specifically on tumor cells. First we enrich the tumor cells, using antibody labeled magnetic separation, and measure the effects of subsequent drug exposure with the metabolic activity assays WST-1 and alamar blue. The second way of estimating drug effects specifically on tumor cells employs multi parameter flow cytometry, measuring apoptosis with the propidium iodide / AnnexinV technique and, particularly for pemetrexed, possible effects on cell cycle progression in immunologically identified tumor cells. The two techniques produce similar results, indicating a possible use in personalized medicine. The possible predictive role of the analysis remains to be shown.

Drug sensitivity profiling and molecular characteristics of cells from pleural effusions of patients with lung adenocarcinoma.

We propose to assess the therapeutic value of biomarker-guided individualized chemotherapy in patients with metastasizing lung adenocarcinoma. In this study, we used primary cells from pleural effusions from sixteen patients diagnosed with adenocarcinomas originating in the lung and from four patients with no malignant diagnosis. The ex vivo drug sensitivity of primary cells was assessed for 32 chemotherapeutical drugs. Linear regression analyses were performed to examine possible correlations between the drug sensitivity, overall survival and expression of ERCC1 and RRM1. The ex vivo drug sensitivity profiles of the patients revealed considerable heterogeneity in drug response. Vinblastine, vinorelbine, paclitaxel and actinomycin D showed high efficiency against 50% of the tested primary cells. Significant correlation was detected between the ex vivo sensitivity to platinum based drugs and gemcitabine and the level of ERCC1 and RRM1. No significant correlation was however seen between overall survival and drug sensitivity. The heterogeneity of the drug response suggests that optimal care of the adenocarcinoma patients should include the determination of drug sensitivity of the primary cells and would benefit to use personalized therapy.

Characterization and drug sensitivity profiling of primary malignant mesothelioma cells from pleural effusions.

BACKGROUND: Patients with malignant mesothelioma have a poor prognosis and only 40% respond to first line treatment; a combination of pemetrexed and cisplatin or carboplatin. We used primary malignant mesothelioma cells and an ex vivo chemosensitivity assay with future purpose to predict best choice of treatment. The clinical outcome of these patients might be predicted by measuring drug sensitivity. METHODS: Pleural effusions containing primary malignant mesothelioma cells were received from the diagnostic routine. We characterized and tested the chemosensitivity of 18 malignant samples and four benign samples from 16 different patients with pleural effusions. Cells were seeded in a 384-well plate for a robotized ex vivo testing of drug sensitivity to 32 different drugs. The primary cells were further characterized by immunocytochemistry to evaluate the proportion of malignant cells and to study the RRM1 and ERCC1 reactivity, two proteins associated with drug resistance. RESULTS: We observed great individual variability in the drug sensitivity. Primary cell isolates were affected by between one and ten drugs, and resistant to the remaining tested drugs. Actinomycin D and daunorubicin were the two drugs effective in most cases. Adjusting efficiency of individual drugs for varying proportion of tumor cells and to the average effect on benign cells correlated with effect of pemetrexed, cisplatin and survival time. General drug sensitivity, proportion of malignant cells and reactivity to RRM1 correlated to each other and to survival time of the patients. CONCLUSIONS: The proportion of malignant cells and RRM1 reactivity in the pleural effusions correlate to drug sensitivity and survival time. The variability in response to the commonly used chemotherapies emphasizes the need for tests that indicate best individual choice of cytotoxic drugs. The efficiency of the obtained results should preferably be corrected for admixture of benign cells and effects of given drugs on benign cells.

Affixing N-terminal α-helix to the wall of the voltage-dependent anion channel does not prevent its voltage gating.

The voltage-dependent anion channel (VDAC) governs the free exchange of ions and metabolites between the mitochondria and the rest of the cell. The three-dimensional structure of VDAC1 reveals a channel formed by 19 ß-strands and an N-terminal α-helix located near the midpoint of the pore. The position of this α-helix causes a narrowing of the cavity, but ample space for metabolite passage remains. The participation of the N-terminus of VDAC1 in the voltage-gating process has been well established, but the molecular mechanism continues to be debated; however, the majority of models entail large conformational changes of this N-terminal segment. Here we report that the pore-lining N-terminal α-helix does not undergo independent structural rearrangements during channel gating. We engineered a double Cys mutant in murine VDAC1 that cross-links the α-helix to the wall of the ß-barrel pore and reconstituted the modified protein into planar lipid bilayers. The modified murine VDAC1 exhibited typical voltage gating. These results suggest that the N-terminal α-helix is located inside the pore of VDAC in the open state and remains associated with ß-strand 11 of the pore wall during voltage gating.