Drug sensitivity profiling and molecular characteristics of cells from pleural effusions of patients with lung adenocarcinoma.
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ABSTRACT:
SUBMITTER: Otvos R
PROVIDER: S-EPMC4426949 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundPatients 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.MethodsPleural 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.ResultsWe 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.ConclusionsThe 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.
| S-EPMC4190467 | biostudies-literature
Project description:Briefly, we analyzed gene expression profiles in adherent cultures compared to sphere cultures from malignant pleural effusions (MPEs). MPEs could represent an opportunity to culture a wide variety of cancer cells from different donors. In this study, we set up culture conditions for cancer cells deriving from MPEs of several patients affected by the most frequent form of lung cancer, namely the subset of non small cell lung cancers (NSCLC) classified as Lung Adenocarcinomas, to show that MPEs are an excellent source of tumor initiating cells for the study of lung adenocarcinoma.
| S-ECPF-MEXP-3025 | biostudies-other
Project description:Malignant pleural effusions (MPEs) could represent an excellent source to culture a wide variety of cancer cells from different donors. In this study, we set up culture conditions for cancer cells deriving from MPEs of several patients affected by the most frequent form of lung cancer, namely the subset of non small cell lung cancers (NSCLC) classified as Lung Adenocarcinomas (AdenoCa) which account for approximately 40% of lung cancer cases. AdenoCa malignant pleural effusions gave rise to in vitro cultures both in adherent and/or in spheroid conditions in almost all cases analyzed. We characterized in greater detail two samples which showed the most efficient propagation in vitro. In these samples we also compared gene profiles of spheroid vs adherent cultures and identified a set of differentially expressed genes. Finally we achieved efficient tumor engraftment in recipient NOD/SCID mice, also upon inoculation of small number of cells, thus suggesting indirectly the presence of tumor initiating cells.
| S-EPMC3138755 | biostudies-literature
Project description:Adenocarcinoma (AdC) is the most common lung cancer subtype and is often associated with pleural effusion (PE). Its poor prognosis is attributable to diagnostic delay and lack of effective treatments and there is a pressing need in discovering new biomarkers for early diagnosis or targeted therapies. To date, little is known about lung AdC proteome. We investigated protein expression of lung AdC in PE using the isobaric Tags for Relative and Absolute Quantification (iTRAQ) approach to identify possible novel diagnostic/prognostic biomarkers. This provided the identification of 109 of lung AdC-related proteins. We further analyzed lumican, one of the overexpressed proteins, in 88 resected lung AdCs and in 23 malignant PE cell-blocks (13 lung AdCs and 10 non-lung cancers) using immunohistochemistry. In AdC surgical samples, lumican expression was low in cancer cells, whereas it was strong and diffuse in the stroma surrounding the tumor. However, lumican expression was not associated with tumor grade, stage, and vascular/pleural invasion. None of the lung cancer cell-blocks showed lumican immunoreaction, whereas those of all the other tumors were strongly positive. Finally, immunoblotting analysis showed lumican expression in both cell lysate and conditioned medium of a fibroblast culture but not in those of A549 lung cancer cell line. PE is a valid source of information for proteomic analysis without many of the restrictions of plasma. The high lumican levels characterizing AdC PEs are probably due to its release by the fibroblasts surrounding the tumor. Despite the role of lumican in lung AdC is still elusive, it could be of diagnostic value.
| S-EPMC4427354 | biostudies-literature
Project description:Briefly, we analyzed gene expression profiles in adherent cultures compared to sphere cultures from malignant pleural effusions (MPEs). MPEs could represent an opportunity to culture a wide variety of cancer cells from different donors. In this study, we set up culture conditions for cancer cells deriving from MPEs of several patients affected by the most frequent form of lung cancer, namely the subset of non small cell lung cancers (NSCLC) classified as Lung Adenocarcinomas, to show that MPEs are an excellent source of tumor initiating cells for the study of lung adenocarcinoma.
2011-12-02 | E-MEXP-3025 | biostudies-arrayexpress
Project description:A common symptom during late stage breast cancer disease is pleural effusion, which is related to poor prognosis. Malignant cells can be detected in pleural effusions indicating metastatic spread from the primary tumor site. Pleural effusions have been shown to be a useful source for studying metastasis and for isolating cells with putative cancer stem cell (CSC) properties. For the present study, pleural effusion aspirates from 17 metastatic breast cancer patients were processed to propagate CSCs in vitro. Patient-derived aspirates were cultured under sphere forming conditions and isolated primary cultures were further sorted for cancer stem cell subpopulations ALDH1+ and CD44+CD24-/low. Additionally, sphere forming efficiency of CSC and non-CSC subpopulations was determined. In order to genetically characterize the different tumor subpopulations, DNA was isolated from pleural effusions before and after cell sorting, and compared with corresponding DNA copy number profiles from primary tumors or bone metastasis using low-coverage whole genome sequencing (SCNA-seq). In general, unsorted cells had a higher potential to form spheres when compared to CSC subpopulations. In most cases, cell sorting did not yield sufficient cells for copy number analysis. A total of five from nine analyzed unsorted pleura samples (55%) showed aberrant copy number profiles similar to the respective primary tumor. However, most sorted subpopulations showed a balanced profile indicating an insufficient amount of tumor cells and low sensitivity of the sequencing method. Finally, we were able to establish a long term cell culture from one pleural effusion sample, which was characterized in detail. In conclusion, we confirm that pleural effusions are a suitable source for enrichment of putative CSC. However, sequencing based molecular characterization is impeded due to insufficient sensitivity along with a high number of normal contaminating cells, which are masking genetic alterations of rare cancer (stem) cells.
| S-EPMC5396869 | biostudies-literature
Project description:IntroductionThe use of patients' own cancer cells for in vitro selection of the most promising treatment is an attractive concept in personalized medicine. Human carcinoma cells from malignant pleural effusions (MPEs) are suited for this purpose since they have already adapted to the liquid environment in the patient and do not depend on a stromal cell compartment. Aim of this study was to develop a systematic approach for the in-vitro culture of MPEs to analyze the effect of chemotherapeutic as well as targeted drugs.MethodsMPEs from patients with solid tumors were selected for this study. After morphological and molecular characterization, they were cultured in medium supplemented with patient-derived sterile-filtered effusion supernatant. Growth characteristics were monitored in real-time using the xCELLigence system. MPEs were treated with a targeted therapeutic (erlotinib) according to the mutational status or chemotherapeutics based on the recommendation of the oncologists.ResultsWe have established a robust system for the ex-vivo culture of MPEs and the application of drug tests in-vitro. The use of an antibody based magnetic cell separation system for epithelial cells before culture allowed treatment of effusions with only moderate tumor cell proportion. Experiments using drugs and drug-combinations revealed dose-dependent and specific growth inhibitory effects of targeted drugs.ConclusionsWe developed a new approach for the ex-vivo culture of MPEs and the application of drug tests in-vitro using real-time measuring of cell growth, which precisely reproduced the effect of clinically established treatments by standard chemotherapy and targeted drugs. This sets the stage for future studies testing agents against specific targets from genomic profiling of metastatic tumor cells and multiple drug-combinations in a personalized manner.
| S-EPMC4993361 | biostudies-literature
Project description:Liquid biopsy analyzes the current status of primary tumors and their metastatic regions. We aimed to develop an optimized protocol for single-cell sequencing of floating tumor cells (FTCs) in pleural effusion as a laboratory test. FTCs were enriched using a negative selection of white blood cells by a magnetic-activated cell sorting system, and CD45-negative and cytokeratin-positive selection using a microfluidic cell separation system with a dielectrophoretic array. The enriched tumor cells were subjected to whole-genome amplification (WGA) followed by genome sequencing. The FTC analysis detected an EGFR exon 19 deletion in Case 1 (12/19 cells, 63.2%), and EML4-ALK fusion (17/20 cells, 85%) with an alectinib-resistant mutation of ALK (p.G1202R) in Case 2. To eliminate WGA-associated errors and increase the uniformity of the WGA product, the protocol was revised to sequence multiple single FTCs individually. An analytical pipeline, accurate single-cell mutation detector (ASMD), was developed to identify somatic mutations of FTCs. The large numbers of WGA-associated errors were cleaned up, and the somatic mutations detected in FTCs by ASMD were concordant with those found in tissue specimens. This protocol is applicable to circulating tumor cells analysis of peripheral blood and expands the possibility of utilizing molecular profiling of cancers.
| S-EPMC8088920 | biostudies-literature
Project description:Malignant pleural effusion (MPE) is a common complication of lung adenocarcinoma (LADC) which is associated with a dismal prognosis. We investigated the prognostic role of PD-L1 and other immunomodulators expression in the immune compartment of MPE immune composition. MPE cytologic cell blocks of 83 LADC patients were analysed for the mRNA expression of 770 cancer-immune genes by the NanoString nCounter platform. The expression of relevant immune cell lineage markers was validated by immunohistochemistry (IHC) using quantitative pathology. The mRNA immune profiling identified four MPE patient clusters (C). C1/2 (adaptive+, hot) showed better overall survival (OS) than C3/4 (adaptive-, cold). Additionally, cold immunity profiles (adaptive-), C4 (innate+) were associated with worse OS than C3 (innate-). High PD-L1 expression was linked to the regulation of T cell activation and interferon signalling pathways. Genes of pattern recognition receptor and type I interferon signalling pathways were specifically upregulated in the long-survival (≥90 days) patient group. Moreover, immunomodulators were co-activated and highly expressed in hot adaptive immunity patient clusters, whereas CD274 (PD-L1), TNFRSF9 (4-1BB), VEGFA (VEGF-A) and CD276 (B7-H3) were upregulated in the groups referred as cold. The patient cluster, age and PD-L1 expression were independent prognosticators for LADC MPE patients (p-value < 0.05). Our study sheds light on the variances of immune contexture regarding different PD-L1 expression and survival conditions. It revealed four distinct prognostic patient clusters with specific immune cell components and immunomodulator expression profiles, which, collectively, is supportive for future therapeutic and prognosis for cancer management.
| S-EPMC9577289 | biostudies-literature
Project description:Lung cancer is the leading cause of cancer-related mortality worldwide. Recent evidence indicates that tumors contain a subpopulation of cancer stem cells (CSCs) that are responsible for tumor maintenance and spread. CSCs have recently been linked to the occurrence of epithelial-to-mesenchymal transition (EMT). Neurotrophins (NTs) are growth factors that regulate the biology of embryonic stem cells and cancer cells, but still little is known about the role NTs in the progression of lung cancer. In this work, we investigated the role of the NTs and their receptors using as a study system primary cell cultures derived from malignant pleural effusions (MPEs) of patients with adenocarcinoma of the lung. We assessed the expression of NTs and their receptors in MPE-derived adherent cultures vs. spheroids enriched in CSC markers. We observed in spheroids a selectively enhanced expression of TrkB, both at the mRNA and protein levels. Both K252a, a known inhibitor of Trk activity, and a siRNA against TrkB strongly affected spheroid morphology, induced anoikis and decreased spheroid forming efficiency. Treatment with neurotrophins reversed the inhibitory effect of K252a. Importantly, TrkB inhibition caused loss of vimentin expression as well as that of a set of transcription factors known to be linked to EMT. These ex vivo results nicely correlated with an inverse relationship between TrkB and E-cadherin expression measured by immunohistochemistry in a panel of lung adenocarcinoma samples. We conclude that TrkB is involved in full acquisition of EMT in lung cancer, and that its inhibition results in a less aggressive phenotype.
| S-EPMC3713128 | biostudies-other