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CytoMatrix for a reliable and simple characterization of lung cancer stem cells from malignant pleural effusions.

ABSTRACT: Cancer stem cells (CSCs) are a subpopulation with the properties of extensive self-renewal, capability to generate differentiated cancer cells and resistance to therapies. We have previously shown that malignant pleural effusions (MPEs) from patients with non-small-cell lung cancer (NSCLC) represent a valuable source of cancer cells that can be grown as three-dimensional (3D) spheroids enriched for stem-like features, which depend on the activation of the Yes-associated protein-transcriptional coactivator with PDZ-binding motif (YAP-TAZ)/Wnt-?catenin/stearoyl-CoA desaturase 1 (SCD1) axis. Here, we describe a novel support, called CytoMatrix, for the characterization of limited amounts of cancer cells isolated from MPEs of patients with NSCLC. Our results show that this synthetic matrix allows an easy and fast characterization of several epithelial cellular markers. The use of CytoMatrix to study CSCs subpopulation confirms that SCD1 protein expression is enhanced in 3D spheroids when compared with 2D adherent cell cultures. YAP/TAZ nuclear-cytoplasmic distribution analysed by CytoMatrix in 3D spheroids is highly heterogeneous and faithfully reproduces what is observed in tumour biopsies. Our results confirm and extend the robustness of our workflow for the isolation and phenotypic characterization of primary cancer cells derived from the lung MPEs and underscore the role of SCD1.

SUBMITTER: Bruschini S

PROVIDER: S-EPMC6916247 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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CytoMatrix for a reliable and simple characterization of lung cancer stem cells from malignant pleural effusions.

Bruschini Sara S di Martino Simona S Pisanu Maria Elena ME Fattore Luigi L De Vitis Claudia C Laquintana Valentina V Buglioni Simonetta S Tabbì Eugenio E Cerri Andrea A Visca Paolo P Alessandrini Gabriele G Facciolo Francesco F Napoli Christian C Trombetta Marcella M Santoro Antonio A Crescenzi Anna A Ciliberto Gennaro G Mancini Rita R

Journal of cellular physiology 20190809 3

Cancer stem cells (CSCs) are a subpopulation with the properties of extensive self-renewal, capability to generate differentiated cancer cells and resistance to therapies. We have previously shown that malignant pleural effusions (MPEs) from patients with non-small-cell lung cancer (NSCLC) represent a valuable source of cancer cells that can be grown as three-dimensional (3D) spheroids enriched for stem-like features, which depend on the activation of the Yes-associated protein-transcriptional c ...[more]

PMID: 31397494

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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.

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.

Project description:IntroductionPleural effusions frequently signal disseminated cancer. Diagnostic markers of pleural malignancy at presentation that would assess cancer risk and would streamline diagnostic decisions remain unidentified.MethodsA consecutive cohort of 323 patients with pleural effusion (PE) from different etiologies were recruited between 2013 and 2017 and was retrospectively analyzed. Data included history, chest X-ray, and blood/pleural fluid cell counts and biochemistry. Group comparison, receiver-operator characteristics, unsupervised hierarchical clustering, binary logistic regression, and random forests were used to develop the malignant pleural effusion detection (MAPED) score. MAPED was validated in an independent retrospective UK cohort (n = 238).ResultsFive variables showed significant diagnostic power and were incorporated into the 5-point MAPED score. Age > 55 years, effusion size > 50% of the most affected lung field, pleural neutrophil count 〈 2,500/mm3, effusion protein 〉 3.5 g/dL, and effusion lactate dehydrogenase > 250 U/L, each scoring one point, predicted underlying cancer with the area under curve(AUC) = 0.819 (P < 10-15) in the derivation cohort. The integrated discrimination improvement of MAPED scores showed an increase compared to cytology (p <0.001). Decision curve analysis indicated that the MAPED score generated net clinical benefit. In the validation dataset, the AUC of MAPED scores was 0.723 ( P = 3 × 10-9) for the MAPED score. Interestingly, MAPED correctly identified 33/42(79%) of cytology-negative patients that indeed had cancer.ConclusionsThe MAPED score identifies malignant pleural effusions with satisfactory accuracy and can be used complementary to cytology to streamline diagnostic procedures.Condensed abstractDiagnostic markers for malignant pleural effusions remain uncertain. The MAPED score identifies malignant pleural effusions and complements cytology and confers no additional risk to the patient or cost to the healthcare system.

Dataset Information

CytoMatrix for a reliable and simple characterization of lung cancer stem cells from malignant pleural effusions.

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CytoMatrix for a reliable and simple characterization of lung cancer stem cells from malignant pleural effusions.

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