Article

Culture and Drug Profiling of Patient Derived Malignant Pleural Effusions for Personalized Cancer Medicine

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Introduction: The 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. Methods: MPEs 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. Results: We 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. Conclusions: We 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.
Determining tumor cell content in cultured malignant effusions. A-C. Second passage (P2) of a malignant effusion of a metastatic ovarian carcinoma. BerEP4 (B) and Calretinin (C) were used as epithelial and mesothelial markers, respectively. In this case, the number of mesothelial cells in passage 2 was very low (1–10%) (This case corresponds to the light blue line in the histogram of 2G, Ovarian Ca.). D-E. First passage (P1) of a malignant pleural effusion of an adenocarcinoma of the lung. TTF1 (nuclear) was used as specific marker for epithelial cells. F. In most of the effusions, relative tumor cell content decreased during the first passages in culture. Y-axis denotes relative tumor content as determined by the pathologist. G. Example of the tumor cell decrease in a malignant effusion from a patient with lung adenocarcinoma. In P0 and P1 most of the cells were from a pulmonary adenocarcinoma, as shown by TTF1 nuclear positivity (small box). In contrast, cells in P2 were mostly of non-epithelial origin (no specific nuclear TTF1 staining). Of note, mesothelial cells can change their morphology in culture and show pronounced atypia, and may be mistaken for tumor cells (green arrowhead). H. Substantial increase in tumor cells (EpCAM positive) and decrease in mesothelial cells (Calretinin positive) after enrichment with EpCAM antibody coated magnetic beads (MACS). Red arrowheads point towards epithelial tumor cells, green arrowheads towards mesothelial (non-neoplastic) effusion cells.
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... However, only modest gains have been made in long-term patient survival due to multidrug-resistant and highly aggressive characteristics of tumours growing in either pleural or peritoneal cavity. Screening of patients' own samples for in vitro chemo/immunotherapeutic agent selection enables the optimisation of individual therapeutic regimens [13]. To date, the utilisation of MSE 2D cell culture for drug screening and other experimental studies has already been demonstrated from literature evidences [8,13,14]. ...
... Screening of patients' own samples for in vitro chemo/immunotherapeutic agent selection enables the optimisation of individual therapeutic regimens [13]. To date, the utilisation of MSE 2D cell culture for drug screening and other experimental studies has already been demonstrated from literature evidences [8,13,14]. Moreover, in vitro MSE models generated either with scaffold-free techniques or with hydrogel matrix support were also used in few studies [15,16]. ...
... Therefore, for MSE cytologic diagnosis, Pan-CK is not be a proper marker for recognising tumour cells. Clearing of mesothelial cells using magnetic cell separation (MACS systems) might be crucial to establish pure cultures of MSE to be used for screening assays [13]. ...
Article
Full-text available
Background: Malignant serous effusion (MSE) denotes a manifestation of metastatic disease with typical high concentrations of both cancer and immune cells, making them an ideal resource for in vitro cytologic studies. Hence, the aim of the study was to investigate the features of 2D and 3D MSE culture systems as well as their feasibilities for in vitro drug screening. Methods: Pleural and peritoneal effusions from 8 patients were collected and processed for 2D monolayer and 3D hanging drop cell culture into GravityPLUS™ plates. Representative markers for cell components, proliferation rate and tumour classification were investigated by immunohistochemistry, followed by absolute quantification using a digitalised image analysis approach. Further, we implemented another 3D cell culture model based on a low attachment method for in vitro drug sensitivity testing of carboplatin, pemetrexed and pembrolizumab for 5 patients. Results: Monolayer cell culture was favourable for the growth of mesothelial cells, while hanging drop culture in GravityPLUS™ plates showed better ability for preserving cancer cells, inducing positive diagnostic markers expression and restraining the growth of mesothelial cells. For in vitro drug testing, MSE from five patients presented various drug sensitivities, and one case showed strong response to PD-1 checkpoint inhibition (pembrolizumab). For some patients, the application of combinatorial drugs had better therapeutic responses compared to monotherapy. Conclusions: Digitalised quantification of data offers a better understanding of different MSE culture models. More importantly, the proposed platforms are practical and amenable for performing in vitro chemo-/immunotherapeutic drug testing by using routine cytologic MSE in a personalised manner. Next to cell blocks, our work demonstrates the prognostic and predictive value of cytologic effusion samples.
... However, only modest gains have been made in long-term patient survival due to multidrug-resistant and highly aggressive characteristics of tumours growing in either pleural or peritoneal cavity. Screening of patients' own samples for in vitro chemo/immunotherapeutic agent selection enables the optimisation of individual therapeutic regimens [13]. To date, the utilisation of MSE 2D cell culture for drug screening and other experimental studies has already been demonstrated from literature evidences [8,13,14]. ...
... Screening of patients' own samples for in vitro chemo/immunotherapeutic agent selection enables the optimisation of individual therapeutic regimens [13]. To date, the utilisation of MSE 2D cell culture for drug screening and other experimental studies has already been demonstrated from literature evidences [8,13,14]. Moreover, in vitro MSE models generated either with scaffold-free techniques or with hydrogel matrix support were also used in few studies [15,16]. ...
... Therefore, for MSE cytologic diagnosis, Pan-CK is not be a proper marker for recognising tumour cells. Clearing of mesothelial cells using magnetic cell separation (MACS systems) might be crucial to establish pure cultures of MSE to be used for screening assays [13]. ...
Preprint
Full-text available
Background Malignant serous effusion (MSE) denotes a manifestation of metastatic disease with typical high concentrations of both cancer and immune cells, making them an ideal resource for in vitro cytologic studies. Hence, the aim of the study was to investigate the features of 2D and 3D MSE culture systems as well as their feasibilities for in vitro drug screening. Methods Pleural and peritoneal effusions from 8 patients were collected and processed for 2D monolayer and 3D hanging drop cell culture into GravityPLUS™ plates. Representative markers for cell components, proliferation rate and tumour classification were investigated by immunohistochemistry, followed by absolute quantification using a digitalised image analysis approach. Further, we implemented another 3D cell culture model based on a low attachment method for in vitro drug sensitivity testing of carboplatin, pemetrexed and pembrolizumab for 5 patients. Results Monolayer cell culture was favourable for the growth of mesothelial cells, while hanging drop culture in GravityPLUS™ plates showed better ability for preserving cancer cells, inducing positive diagnostic markers expression and restraining the growth of mesothelial cells. For in vitro drug testing, MSE from five patients presented various drug sensitivities, and one case showed strong response to PD-1 checkpoint inhibition (pembrolizumab). For some patients, the application of combinatorial drugs had better therapeutic responses compared to monotherapy. Conclusions Digitalised quantification of data offers a better understanding of different MSE culture models. More importantly, the proposed platforms are practical and amenable for performing in vitro chemo-/immunotherapeutic drug testing by using routine cytologic MSE in a personalised manner. Next to cell blocks, our work demonstrates the prognostic and predictive value of cytologic effusion samples.
... However, only modest gains have been made in long-term patient survival due to multidrug-resistant and highly aggressive characteristics of tumours growing in either pleural or peritoneal cavity. Screening of patients' own samples for in vitro chemo/immunotherapeutic agent selection enables the optimisation of individual therapeutic regimens [13]. To date, the utilisation of MSE 2D cell culture for drug screening and other experimental studies has already been demonstrated from literature evidences [8,13,14]. ...
... Screening of patients' own samples for in vitro chemo/immunotherapeutic agent selection enables the optimisation of individual therapeutic regimens [13]. To date, the utilisation of MSE 2D cell culture for drug screening and other experimental studies has already been demonstrated from literature evidences [8,13,14]. Moreover, in vitro MSE models generated either with scaffold-free techniques or with hydrogel matrix support were also used in few studies [15,16]. ...
... Therefore, for MSE cytologic diagnosis, Pan-CK is not be a proper marker for recognising tumour cells. Clearing of mesothelial cells using magnetic cell separation (MACS systems) might be crucial to establish pure cultures of MSE to be used for screening assays[13].The optimal growth conditions observed in 3D cell cultures supported the proliferation of tumour cells with a more reduced presence of mesothelial cells. The proportion of cells expressing diagnostic markers was also higher than corresponding 2D culture mirroring the histological analysis of the original MSE samples. ...
Preprint
Full-text available
Background Malignant serous effusion (MSE) denotes a manifestation of metastatic disease with typical high concentrations of both cancer and immune cells, making them an ideal resource for in vitro cytologic studies. Hence, the aim of the study was to investigate the features of 2D and 3D MSE culture systems as well as their feasibilities for in vitro drug screening. Methods Pleural and peritoneal effusions from 8 patients were collected and processed for 2D monolayer and 3D hanging drop cell culture into GravityPLUS™ plates. Representative markers for cell components, proliferation rate and tumour classification were investigated by immunohistochemistry, followed by absolute quantification using a digitalised image analysis approach. Further, we implemented another 3D cell culture model based on a low attachment method for in vitro drug sensitivity testing of carboplatin, pemetrexed and pembrolizumab for 5 patients. Results Monolayer cell culture was favourable for the growth of mesothelial cells, while hanging drop culture in GravityPLUS™ plates showed better ability for preserving cancer cells, inducing positive diagnostic markers expression and restraining the growth of mesothelial cells. For in vitro drug testing, MSE from five patients presented various drug sensitivities, and one case showed strong response to PD-1 checkpoint inhibition (pembrolizumab). For some patients, the application of combinatorial drugs had better therapeutic responses compared to monotherapy. Conclusions Digitalised quantification of data offers a better understanding of different MSE culture models. More importantly, the proposed platforms are practical and amenable for performing in vitro chemo-/immunotherapeutic drug testing by using routine cytologic MSE in a personalised manner. Next to cell blocks, our work demonstrates the prognostic and predictive value of cytologic effusion samples.
... In order to thoroughly evaluate and validate the utility of this real-time potency assay, herein the xCELLigence instrument is used for conducting diverse killing assays involving a variety of cancer target cells and immune effector cells. The resulting real-time impedance traces and their associated killing kinetics, as well as quantitative parameters such as EC 50 and KT 50 are used to derive mechanistic conclusions and potency ranks. Collectively, these results indicate that real-time potency assays using the xCELLigence system is an effective and efficient tool for both basic and applied immunotherapy research as well as monitoring CQA of various immunotherapies during manufacturing. ...
... After placing the E-Plate back inside the xCELLigence instrument, data acquisition was resumed to monitor NK92 cytotoxic activity based on the viability of the surface attached Raji cells, as reflected by Cell Index values. The xIMT software was used to plot the percentage of cytolysis and calculate parameters such as KT 50 . A parallel experiment was set up in which the Raji cells were incubated with NK92 cells in suspension only. ...
... Also, we have demonstrated the reproducibility and robustness of the assay for possible application as QC potency assay in manufacturing and for large screening campaigns (S1 Fig). Furthermore, the assay can also be potentially used with primary cancer cells [48][49][50] and adapted for in vitro clinical testing using the patient's own tumor primary cells along with immune cells harvested from the same patient or from donors. ...
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A growing understanding of the molecular interactions between immune effector cells and target tumor cells, coupled with refined gene therapy approaches, are giving rise to novel cancer immunotherapeutics with remarkable efficacy in the clinic against both solid and liquid tumors. While immunotherapy holds tremendous promise for treatment of certain cancers, significant challenges remain in the clinical translation to many other types of cancers and also in minimizing adverse effects. Therefore, there is an urgent need for functional potency assays, in vitro and in vivo, that could model the complex interaction of immune cells with tumor cells and can be used to rapidly test the efficacy of different immunotherapy approaches, whether it is small molecule, biologics, cell therapies or combinations thereof. Herein we report the development of an xCELLigence real-time cytolytic in vitro potency assay that uses cellular impedance to continuously monitor the viability of target tumor cells while they are being subjected to different types of treatments. Specialized microtiter plates containing integrated gold microelectrodes enable the number, size, and surface attachment strength of adherent target tumor cells to be selectively monitored within a heterogeneous mixture that includes effector cells, antibodies, small molecules, etc. Through surface-tethering approach, the killing of liquid cancers can also be monitored. Using NK92 effector cells as example, results from RTCA potency assay are very well correlated with end point data from image-based assays as well as flow cytometry. Several effector cells, i.e., PBMC, NK, CAR-T were tested and validated as well as biological molecules such as Bi-specific T cell Engagers (BiTEs) targeting the EpCAM protein expressed on tumor cells and blocking antibodies against the immune checkpoint inhibitor PD-1. Using the specifically designed xCELLigence immunotherapy software, quantitative parameters such as KT50 (the amount of time it takes to kill 50% of the target tumor cells) and % cytolysis are calculated and used for comparing the relative efficacy of different reagents. In summary, our results demonstrate the xCELLigence platform to be well suited for potency assays, providing quantitative assessment with high reproducibility and a greatly simplified work flow.
... In order to thoroughly evaluate and validate the utility of this real-time potency assay, herein the xCELLigence instrument is used for conducting diverse killing assays involving a variety of cancer target cells and immune effector cells. The resulting real-time impedance traces and their associated killing kinetics, as well as quantitative parameters such as EC 50 and KT 50 are used to derive mechanistic conclusions and potency ranks. Collectively, these results indicate that real-time potency assays using the xCELLigence system is an effective and effi- cient tool for both basic and applied immunotherapy research as well as monitoring CQA of various immunotherapies during manufacturing. ...
... Also, we have demonstrated the reproducibility and robustness of the assay for possible application as QC potency assay in manufacturing and for large screen- ing campaigns (S1 Fig). Furthermore, the assay can also be potentially used with primary cancer cells [48][49][50] and adapted for in vitro clinical testing using the patient's own tumor primary cells along with immune cells harvested from the same patient or from donors. ...
... Two other publications used xCELLigence for characterization of newly established cell lines from patient samples, offsetting them against parental tumor tissue or traditionally used carcinoma cell lines [57,58]. Finally, Ruiz et al. applied xCELLigence to patients' own cancer cells for the in vitro selection of the most promising treatment, in this case for human carcinoma cells from malignant pleural effusions [59]. This is an illustrative example of possible applications in precision medicine. ...
... Finally, the combination of patient cells and label-free technology can be used for clinical compound selection, for instance by measuring patient cell responses in vitro as means of selecting the most promising treatment. This has been demonstrated by profiling drug treatment responses of patient-derived malignant pleural effusions in a study by Ruiz et al. [59], with the aim to provide drug treatment of cancer in a personalized manner. ...
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Full-text available
Drug development requires physiologically more appropriate model systems and assays to increase understanding of drug action and pathological processes in individual humans. Specifically, patient-derived cells offer great opportunities as representative cellular model systems. Moreover, with novel label-free cellular assays, it is often possible to investigate complex biological processes in their native environment. Combining these two offers distinct opportunities for increasing physiological relevance. Here, we review impedance-based label-free technologies in the context of patient samples, focusing on commonly used cell types, including fibroblasts, blood components, and stem cells. Applications extend as far as tissue-on-a-chip models. Thus, applying label-free technologies to patient samples can produce highly biorelevant data and, with them, unique opportunities for drug development and precision medicine.
... Viability of the frozen cells allows broad molecular characterisation and functional drug sensitivity/resistance profiling for multiple predictive markers and therapy targets. [61][62][63] properly, first for the diagnosis and then for the therapy, and finally for future studies is the responsibility of cytopathology laboratories. ...
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Serous effusion fluid is one of the most commonly encountered specimens in routine cytopathology practice. It provides invaluable information about the patient and the clinical status; but to get the most of it, specimen handling and processing must be carried out properly. Cytomorphology is the basis of a successful analysis which should complemented by ancillary tests when needed. A wide spectrum of ancillary techniques ‐ ranging from immunocytochemistry and flow cytometry to different assays of molecular pathology ‐ can be applied to serous effusions. This article describes the acquisition and management of serous effusion fluids, methods for preservation and transportation, different techniques of cytopreparation, application of immunocytochemistry, flow cytometry, and fluorescence in‐situ hybridization (FISH), as well as DNA extraction for polymerase chain reaction (PCR) and next generation sequencing (NGS). Principles of bio‐banking of effusion samples are also discussed which is getting more important in correlation with the developments in personalized medicine. This article is protected by copyright. All rights reserved.
... To evaluate the utility of this system for analysis of targeted therapies, Ruiz et al. subjected two established non-small cell lung cancer cell lines H522 and H3122 to crizotinib treatment. The real-time monitoring system allowed them to determine the latency time of the drug effect on cell growth(14).Tumor cell lines with various proliferative rates were equally sensitive to orthovanadate cytotoxicity. Orthovanadate (Na3VO4) at concentrations greater than 5 µM has antineoplastic properties in vitro and in vivo(15).Within the concentration range of 1-20 µM, Na3VO4 demonstrated a time and dose-dependent inhibition of autocrine growth of the human carcinoma cell lines A549 (lung), HTB44 (kidney) and DU145 (prostate), as compared to appropriate controls (without Na3VO4). ...
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Objectives: Vanadium compounds have various pharmacologic effects and all available evidence reveals that the effects of vanadium compounds depend on many factors, mainly on the type of cells and dose. The proapoptotic or antiapoptotic effect of vanadium compounds depends strongly on the cell type. Materials and methods: In this study, the effects of vanadium pentoxide (V2O5) were investigated using several tumor cell lines: a colorectal cancer cell line (Colo-205), a human breast adenocarcinoma cell line (MCF-7), and a normal human fibroblast cell line. Five different concentrations of V2O5 between 25-200 µM were applied on the cells and xCELLigence real-time cell analysis was conducted to evaluate the impedance alterations. This study is the first to show V2O5's effects on Colo-205 and MCF-7 and human fibroblast cell lines in a real-time manner. Results: In the Colo-205 cell line, cell index (CI) alterations decreased slightly at 25 µM and 50 µM, and increased at 100 µM, 150 µM and 200 µM concentrations. In the MCF-7 cell line, CI alterations increased at all concentrations compared with the untreated control. However, in the healthy fibroblast cell line, the CI alterations decreased at all concentrations compared with the untreated control, which limits the use of V2O5 for its cytotoxic effect in vivo. Conclusion: The combination of conventional anticancer drugs can be used to increase the effectiveness and reduce the adverse effects of these drugs considering stages of cancer and cancer type. Our results suggest that V2O5 has disparate effects on several cancer cells at different concentrations.
... Malignant effusion cell culture of patients with different cancers have been used to assess the response to chemotherapeutic agents, to provide in vitro characterization of neoplastic cell lines, to investigate tumoral heterogeneity and to understand the role of epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) in metastasis and in high therapeutic failure rates (1)(2)(3)(4)(5)(6)(7)(8). ...
Article
The aim of the present study was to identify cell types in primary culture from malignant and non-malignant effusions. Effusion samples were subjected to cytology and culture. Immunocytochemistry was performed in cytological slides to evaluate malignancy (positivity for malignancy markers) and in culture slides for identification of cell types in growth. A total of 143 effusion samples (pleural n=76; peritoneal n=37; pericardial n=4; and peritoneal lavage n=26) were analyzed. Cell growth was observed in 34.9% of all samples and immunocytochemistry for identification of cell types in culture slides was conclusive in 90% of them. In non-malignant samples (n=28), growth of mesothelial cells, macrophages and of both cell types was identified in 82.14, 10.71 and 7.14%, respectively. In malignant samples (n=17, all carcinomas), growth of malignant epithelial cells and of both malignant epithelial and mesothelial cells was identified in 41.17 and 23.52%, respectively. In the remaining 35.29% of malignant samples, the only cells in growth were mesothelial and/or macrophages instead of malignant epithelial cells. In conclusion, in culture of malignant effusions, mesothelial cells may be simultaneously identified with malignant epithelial cells. Besides, mesothelial cells and macrophages may be the only cells identified in malignant effusion culture. Therefore, a broad panel of cell markers should be used for unmistakable identification of cells in studies of effusion primary culture. The ideal malignant effusion sample to obtain culture of neoplastic cells should be that without the presence of mesothelial cells and macrophages.
... Препараты добавляют через 1 сут к клеткам том 5 №3 / 2018 Онкология / Oncology аденокарциномы легкого человека, выделенных из злокачественной легочной эффузии. Это дает возможность построить динамические кривые и определить латентный период действия препарата на рост клеток [71]. ...
Article
The review considers methods for selecting chemo- and biopreparations, their combinations using 2D and 3D cell culture models to predict a personified response of patients with malignant neoplasms (ovarian, stomach, intestine, lung, lacteal, pancreas, head and neck cancer) to chemotherapy. Performance indicators (sensitivity, specificity, prolongation of total and disease-free survival) of chemosensitivity assessment methods in cultures of tumor cells are analyzed on the basis of their comparison with the effectiveness of chemotherapy of patients. The article highlights the advantages and disadvantages of the methods described, the prospects for their further application in clinical practice.
... Our results not only showed that MPE-derived NSCLC cell lines recapitulated representative genetic aberrations of the primary MPE-derived lung cancer cell lines retain the representative genetic mutations of human lung adenocarcinoma. Several studies have shown that MPE-derived tumor cell lines maintain the mutational profiles of the lung adenocarcinoma 6,9 . We performed both whole exome sequencing (WES) and targeted sequencing (CCP) on the 28 MPE-derived cell lines. ...
Article
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Malignant pleural effusion (MPE) is an independent determinant of poor prognostic factor of non-small cell lung cancer (NSCLC). The course of anchorage independent growth within the pleural cavity likely reforms the innate molecular characteristics of malignant cells, which largely accounts for resistance to chemotherapy and poor prognosis after the surgical resection. Nevertheless, the genetic and transcriptomic features with respect to various drug responses of MPE-complicated NSCLC remain poorly understood. To obtain a clearer overview of the MPE-complicated NSCLC, we established 28 MPE-derived lung cancer cell lines which were subjected to genomic, transcriptomic and pharmacological analysis. Our results demonstrated MPE-derived NSCLC cell lines recapitulated representative driver mutations generally found in the primary NSCLC. It also exhibited the presence of distinct translational subtypes in accordance with the mutational profiles. The drug responses of several targeted chemotherapies accords with both genomic and transcriptomic characteristics of MPE-derived NSCLC cell lines. Our data also suggest that the impending drawback of mutation-based clinical diagnosis in evaluating MPE-complicated NSCLS patient responses. As a potential solution, our work showed the importance of comprehending transcriptomic characteristics in order to defy potential drug resistance caused by MPE.
... Malignant pleural effusion (MPE) is observed in half of advanced NSCLC cases and is associated with a short survival [17]. MPE samples frequently contain numerous tumor cells, that allow for the determination of driver gene status and chemosensitivity [18][19][20]. In the present study, a panel of primary NSCLC lines from pleural effusions was employed to compare their chemosensitivity against fascaplysin with that for cisplatin. ...
Article
Full-text available
In the absence of suitable molecular markers, non-small cell lung cancer (NSCLC) patients have to be treated with chemotherapy with poor results at advanced stages. Therefore, the activity of the anticancer marine drug fascaplysin was tested against primary NSCLC cell lines established from pleural effusions. Cytotoxicity of the drug or combinations were determined using MTT assays and changes in intracellular phosphorylation by Western blot arrays. Fascaplysin revealed high cytotoxicity against NSCLC cells and exhibit an activity pattern different of the standard drug cisplatin. Furthermore, fascaplysin synergizes with the EGFR tyrosine kinase inhibitor (TKI) afatinib to yield a twofold increased antitumor effect. Interaction with the Chk1/2 inhibitor AZD7762 confirm the differential effects of fascplysin and cisplatin. Protein phosphorylation assays showed hypophosphorylation of Akt1/2/3 and ERK1/2 as well as hyperphosphorylation of stress response mediators of H1299 NSCLC cells. In conclusion, fascaplysin shows high cytotoxicity against pleural primary NSCLC lines that could be further boosted when combined with the EGFR TKI afatinib.
... Препараты добавляют через 1 сут к клеткам том 5 №3 / 2018 Онкология / Oncology аденокарциномы легкого человека, выделенных из злокачественной легочной эффузии. Это дает возможность построить динамические кривые и определить латентный период действия препарата на рост клеток [71]. ...
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Full-text available
The review considers methods for selecting chemo- and biopreparations, their combinations using 2D and 3D cell culture models to predict a personified response of patients with malignant neoplasms (ovarian, stomach, intestine, lung, lacteal, pancreas, head and neck cancer) to chemotherapy. Performance indicators (sensitivity, specificity, prolongation of total and disease-free survival) of chemosensitivity assessment methods in cultures of tumor cells are analyzed on the basis of their comparison with the effectiveness of chemotherapy of patients. The article highlights the advantages and disadvantages of the methods described, the prospects for their further application in clinical practice. Key words: in vitro methods of selection of chemotherapeutic drugs, malignant tumors, chemotherapeutic drugs in vitro prediction of an individual response to chemotherapy, advantages and disadvantages of the in vitro methods
... Some common antibodies that are conjugated to SPIONs include anti-EpCAM (epithelial-cell-adhesion molecule), anti-HER2 (human epidermal growth factor receptor 2) for HER2+ cancers (which may include breast, bladder, pancreatic, ovarian, gastric, and other cancers), and anti-CD63 (blocks phagocytosis and is commonly used to help identify extracellular vesicles). Antibodies may be conjugated for negative magnetophoresis as well, such as anti-CD45 to remove leukocytes from bulk patient samples [78][79][80][81][82][83][84][85]. Additional common SPION coatings include non-specific proteins or polysaccharides (e.g., serum albumin, dextran, chitosan) or hydrophilic inert polymers (e.g., polyethylene glycol, polyvinyl alcohol). ...
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Worldwide, there are currently around 18.1 million new cancer cases and 9.6 million cancer deaths yearly. Although cancer diagnosis and treatment has improved greatly in the past several decades, a complete understanding of the complex interactions between cancer cells and the tumor microenvironment during primary tumor growth and metastatic expansion is still lacking. Several aspects of the metastatic cascade require in vitro investigation. This is because in vitro work allows for a reduced number of variables and an ability to gather real-time data of cell responses to precise stimuli, decoupling the complex environment surrounding in vivo experimentation. Breakthroughs in our understanding of cancer biology and mechanics through in vitro assays can lead to better-designed ex vivo precision medicine platforms and clinical therapeutics. Multiple techniques have been developed to imitate cancer cells in their primary or metastatic environments, such as spheroids in suspension, microfluidic systems, 3D bioprinting, and hydrogel embedding. Recently, magnetic-based in vitro platforms have been developed to improve the reproducibility of the cell geometries created, precisely move magnetized cell aggregates or fabricated scaffolding, and incorporate static or dynamic loading into the cell or its culture environment. Here, we will review the latest magnetic techniques utilized in these in vitro environments to improve our understanding of cancer cell interactions throughout the various stages of the metastatic cascade.
... Препараты добавляют через 1 сут к клеткам том 5 №3 / 2018 Онкология / Oncology аденокарциномы легкого человека, выделенных из злокачественной легочной эффузии. Это дает возможность построить динамические кривые и определить латентный период действия препарата на рост клеток [71]. ...
Article
Full-text available
The review considers methods for selecting chemo- and biopreparations, their combinations using 2D and 3D cell culture models to predict a personified response of patients with malignant neoplasms (ovarian, stomach, intestine, lung, lacteal, pancreas, head and neck cancer) to chemotherapy. Performance indicators (sensitivity, specificity, prolongation of total and disease-free survival) of chemosensitivity assessment methods in cultures of tumor cells are analyzed on the basis of their comparison with the effectiveness of chemotherapy of patients. The article highlights the advantages and disadvantages of the methods described, the prospects for their further application in clinical practice.
Chapter
Ethical and practical reasons often restrict the use of human cells and tissues in pre-clinical studies; therefore, investigators employ research laboratory models. In vitro and in vivo models are essential and indispensable for the study of pleural diseases as they provide a simplified network of the human biology and can also replicate features of the human condition. Despite their simplicity, laboratory models provide major contributions towards our understanding of pleural diseases and have led to the discovery of perturbated molecular pathways and mechanisms. There are various available in vitro and in vivo models of pleural disease, each with their own strengths and limitations. Therefore, experimental assays have to be carefully designed and implemented to avoid data misinterpretation. Technological advances have improved the efficiency and potency of laboratory models and have introduced new research techniques. Thus, in vitro and in vivo models provide an effective pre-clinical research tool to elucidate pleural disease pathogenesis and progression.
Chapter
Spread of malignancies to serous cavities represents advanced stage disease that necessitates systemic treatment. Immunochemistry (IC) is an indispensable tool to ascertain malignancy in equivocal cases and to define the histological subtype or primary site of a metastatic tumor. This chapter focuses on the technical aspects of marker testing, molecular methods, and on predictive biomarkers that guide diagnosis and treatment of serous fluid diseases.
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Recent clinical trials have demonstrated the efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) in the treatment of patients with advanced metastatic non-small cell lung cancer. Most of these recent trials were conducted in patients with EGFR mutation-positive tumors. As our knowledge of the EGFR mutation and its resistant pathways develops, the complexity of the situation expands. This article briefly reviews the pivotal trials leading to approval of EGFR TKIs in the first-line setting for patients with EGFR mutation-positive non-small cell lung carcinomas. It discusses the historical use of EGFR TKIs after the first-line setting in unselected patients and briefly describes ongoing trials.
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Cancer cell lines are frequently used as in vitro tumour models. Recent molecular profiles of hundreds of cell lines from The Cancer Cell Line Encyclopedia and thousands of tumour samples from the Cancer Genome Atlas now allow a systematic genomic comparison of cell lines and tumours. Here we analyse a panel of 47 ovarian cancer cell lines and identify those that have the highest genetic similarity to ovarian tumours. Our comparison of copy-number changes, mutations and mRNA expression profiles reveals pronounced differences in molecular profiles between commonly used ovarian cancer cell lines and high-grade serous ovarian cancer tumour samples. We identify several rarely used cell lines that more closely resemble cognate tumour profiles than commonly used cell lines, and we propose these lines as the most suitable models of ovarian cancer. Our results indicate that the gap between cell lines and tumours can be bridged by genomically informed choices of cell line models for all tumour types.
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Daniel H., HovelsonAndrew S., McDanielAndi K., CaniBryan, JohnsonKate, RhodesPaul D., WilliamsSanthoshi, BandlaGeoffrey, BienPaul, ChoppaFiona, HylandRajesh, GottimukkalaGuoying, LiuManimozhi, ManivannanJeoffrey, SchagemanEfren, Ballesteros-VillagranaCatherine S., GrassoMichael J., QuistVenkata, YadatiAnmol, AminJaved, SiddiquiBryan L., BetzKaren E., KnudsenKathleen A., CooneyFelix Y., FengMichael H., RohPeter S., NelsonChia-Jen, LiuDavid G., BeerPeter, WyngaardArul M., ChinnaiyanSeth, SadisDaniel R., RhodesScott A., Tomlins. (2015) Development and Validation of a Scalable Next-Generation Sequencing System for Assessing Relevant Somatic Variants in Solid Tumors. Neoplasia 17, 385-399 CrossRef Richard, SullivanJeffrey, PeppercornKarol, SikoraJohn, ZalcbergNeal J, MeropolEitan, AmirDavid, KhayatPeter, BoylePhilippe, AutierIan F, TannockTito, FojoJim, SiderovSteve, WilliamsonSilvia, CamporesiJ Gordon, McVieArnie D, PurushothamPeter, NarediAlexander, EggermontMurray F, BrennanMichael L, SteinbergMark, De RidderSusan A, McCloskeyDirk, VerellenTerence, RobertsGuy, StormeRodney J, HicksPeter J, EllBradford R, HirschDavid P, CarboneKevin A, SchulmanPaul, CatchpoleDavid, TaylorJan, GeisslerNancy G, BrinkerDavid, MeltzerDavid, KerrMatti, Aapro. (2011) Delivering affordable cancer care in high-income countries. The Lancet Oncology 12, 933-980 CrossRef
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The use of advanced molecular profiling to direct the use of targeted therapy, such as tyrosine kinase inhibitors (TKIs) for patients with advanced-stage non-small-cell lung cancer (NSCLC), has revolutionized the treatment of this disease. However, acquired resistance, defined as progression after initial benefit, to targeted therapies inevitably occurs. This Review explores breakthroughs in the understanding and treatment of acquired resistance in NSCLC, focusing on EGFR mutant and ALK rearrangement-positive disease, which may be relevant across multiple different solid malignancies with oncogene-addicted subtypes. Mechanisms of acquired resistance may be pharmacological (that is, failure of delivery of the drug to its target) or biological, resulting from evolutionary selection on molecularly diverse tumours. A number of clinical approaches can maintain control of the disease in the acquired resistance setting, including the use of radiation to treat isolated areas of progression and adding or switching to cytotoxic chemotherapy. Furthermore, novel approaches that have already proven successful include the development of second-generation and third-generation inhibitors and the combination of some of these inhibitors with antibodies directed against the same target. With our increased understanding of the spectrum of acquired resistance, major changes in how we conduct clinical research in this setting are now underway.
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All patients with metastatic lung, colorectal, pancreatic or head and neck cancers who initially benefit from epidermal growth factor receptor (EGFR)-targeted therapies eventually develop resistance. An increasing understanding of the number and complexity of resistance mechanisms highlights the Herculean challenge of killing tumors that are resistant to EGFR inhibitors. Our growing knowledge of resistance pathways provides an opportunity to develop new mechanism-based inhibitors and combination therapies to prevent or overcome therapeutic resistance in tumors. We present a comprehensive review of resistance pathways to EGFR-targeted therapies in lung, colorectal and head and neck cancers and discuss therapeutic strategies that are designed to circumvent resistance.
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Receptor tyrosine kinases (RTKs) are activated by somatic genetic alterations in a subset of cancers, and such cancers are often sensitive to specific inhibitors of the activated kinase. Two well-established examples of this paradigm include lung cancers with either EGFR mutations or ALK translocations. In these cancers, inhibition of the corresponding RTK leads to suppression of key downstream signaling pathways, such as the PI3K (phosphatidylinositol 3-kinase)/AKT and MEK (mitogen-activated protein kinase kinase)/ERK (extracellular signal-regulated kinase) pathways, resulting in cell growth arrest and death. Despite the initial clinical efficacy of ALK (anaplastic lymphoma kinase) and EGFR (epidermal growth factor receptor) inhibitors in these cancers, resistance invariably develops, typically within 1 to 2 years. Over the past several years, multiple molecular mechanisms of resistance have been identified, and some common themes have emerged. One is the development of resistance mutations in the drug target that prevent the drug from effectively inhibiting the respective RTK. A second is activation of alternative RTKs that maintain the signaling of key downstream pathways despite sustained inhibition of the original drug target. Indeed, several different RTKs have been implicated in promoting resistance to EGFR and ALK inhibitors in both laboratory studies and patient samples. In this mini-review, we summarize the concepts underlying RTK-mediated resistance, the specific examples known to date, and the challenges of applying this knowledge to develop improved therapeutic strategies to prevent or overcome resistance.
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INTRODUCTION: Lung cancer is often diagnosed by cytology, necessitating predictive molecular marker analyses on cytological specimens. The gold standard for detection of predictive anaplastic lymphoma kinase (ALK)-rearrangements is fluorescence in situ hybridization (FISH), but FISH is both expensive and often challenging to interpret. The aim of our study was to investigate the accuracy of ALK immunocytochemistry (ICC) on cytological specimens of non-small-cell lung cancers (NSCLCs). METHODS: Forty-one cytological specimens with available ALK FISH results were retrospectively analyzed with the 5A4 monoclonal antibody (Novocastra; Leica Biosystems) on a fully automated slide stainer. The specimens were enriched for ALK FISH-positive NSCLCs (14 of 41; 34.1%). Evaluation of the ICC staining was performed blinded to the FISH results. The staining intensity and the percentage of stained cancer cells were recorded. Any ICC staining was regarded as a positive result. The ALK ICC results were compared with the FISH results. In case of a discrepancy the ICC-stained slide and the FISH signals were reviewed. RESULTS: ICC was evaluable on 40 of 41 specimens. Fifteen of 40 NSCLCs (37.5%) were ALK ICC-positive, with staining of the majority of cancer cells (median 100%; mean 82.3%). Twelve of the ICC-positive NSCLCs (80.0%) showed an intense staining (3+). Compared with the ALK FISH results, only one NSCLC was false-negative, and one false-positive by ICC, respectively. The sensitivity, specificity, and positive and negative predictive values for ALK ICC compared with ALK FISH were 93.3%, 96.0%, 93.3%, and 96%, respectively. CONCLUSION: ALK ICC is highly accurate for detecting ALK-rearranged NSCLCs.
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Recent discoveries of genomic alterations that underlie and promote the malignant phenotype, together with an expanded repertoire of targeted agents, have provided many opportunities to conduct hypothesis-driven clinical trials. The ability to profile each unique cancer for actionable aberrations by using high-throughput technologies in a cost-effective way provides unprecedented opportunities for using matched therapies in a selected patient population. The major challenges are to integrate and make biologic sense of the substantial genomic data derived from multiple platforms. We define two different approaches for the analysis, interpretation, and clinical applicability of genomic data: (1) the genomically stratified model originates from the "one test-one drug" paradigm and is currently being expanded with an upfront multicategorical approach following recent advances in multiplexed genotyping platforms; and (2) the comprehensive assessment model is based on whole-genome, -exome, and -transcriptome data and allows identification of novel drivers and subsequent therapies in the experimental setting. Tumor heterogeneity and evolution of the diverse populations of cancer cells during cancer progression, influenced by the effects of systemic treatments, will need to be addressed in the new scenario of early drug development. Logistical issues related to prescreening strategies and trial allocation, in addition to concerns in the economic and ethical domains, must be taken into consideration. Here we present a historical view of how increased understanding of cancer genomics has been translated to the clinic and discuss the prospects and challenges for further implementation of a personalized treatment strategy for human solid tumors.
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High-grade serous carcinoma (HGSC) is the most common and fatal form of ovarian cancer. While most tumors are highly sensitive to cytoreductive surgery and platinum- and taxane-based chemotherapy, the majority of patients experience recurrence of treatment-resistant tumors. The clonal origin and mutational adaptations associated with recurrent disease are poorly understood. We performed whole exome sequencing on tumor cells harvested from ascites at three time points (primary, first recurrence and second recurrence) for three HGSC patients receiving standard treatment. Somatic point mutations and small insertions and deletions were identified by comparison to constitutional DNA. The clonal structure and evolution of tumors were inferred from patterns of mutant allele frequencies. TP53 mutations were predominant in all patients at all time points, consistent with the known founder role of this gene. Tumors from all three patients also harbored mutations associated with cell cycle checkpoint function and Golgi vesicle trafficking. There was convergence of germline and somatic variants within the DNA repair, ECM, cell cycle control and Golgi vesicle pathways. The vast majority of somatic variants found in recurrent tumors were present in primary tumors. Our findings highlight both known and novel pathways that are commonly mutated in HGSC. Moreover, they provide the first evidence at single nucleotide resolution that recurrent HGSC arises from multiple clones present in the primary tumor with negligible accumulation of new mutations during standard treatment. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Cell-based assays are key tools in drug safety assessment. However, they usually provide only limited information about time-kinetics of a toxic effect and implementing multiple measurements is often complex. To overcome these issues we established an impedance-based approach which is able to differentiate cytostatic from cytotoxic drugs by recording time-kinetics of compound-effects on cells. NIH 3T3 fibroblasts were seeded on xCELLigence® E-plates and impedance was continuously measured over 5 days. The obtained results reflected cytotoxicity and cell proliferation, as confirmed by neutral red uptake in vitro. Based on known toxicants, we established an algorithm able to discriminate cytostatic, cytotoxic and non-toxic compounds based on the shape of the impedance curves. Analyzing impedance curve patterns of additional 37 compounds allowed the identification and differentiation of these distinct effects as results correlated well with previous in vivo findings. We show that impedance-based real-time cell analysis is a convenient tool to characterize and discriminate effects of compounds on cells in a time-dependent and label-free manner. The presented impedance assay could be used to further characterize toxicities observed in vivo or in vitro. Due to the ease of performance it may also be a suitable screening tool.
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ALK fusion genes occur in a subset of non-small-cell lung cancers (NSCLCs). We assessed the tolerability and activity of crizotinib in patients with NSCLC who were prospectively identified to have an ALK fusion within the first-in-man phase 1 crizotinib study. In this phase 1 study, patients with ALK-positive stage III or IV NSCLC received oral crizotinib 250 mg twice daily in 28-day cycles. Endpoints included tumour responses, duration of response, time to tumour response, progression-free survival (PFS), overall survival at 6 and 12 months, and determination of the safety and tolerability and characterisation of the plasma pharmacokinetic profile of crizotinib after oral administration. Responses were analysed in evaluable patients and PFS and safety were analysed in all patients. This study is registered with ClinicalTrials.gov, number NCT00585195. Between Aug 27, 2008, and June 1, 2011, 149 ALK-positive patients were enrolled, 143 of whom were included in the response-evaluable population. 87 of 143 patients had an objective response (60·8%, 95% CI 52·3-68·9), including three complete responses and 84 partial responses. Median time to first documented objective response was 7·9 weeks (range 2·1-39·6) and median duration of response was 49·1 weeks (95% CI 39·3-75·4). The response rate seemed to be largely independent of age, sex, performance status, or line of treatment. Median PFS was 9·7 months (95% CI 7·7-12·8). Median overall survival data are not yet mature, but estimated overall survival at 6 and 12 months was 87·9% (95% CI 81·3-92·3) and 74·8% (66·4-81·5), respectively. 39 patients continued to receive crizotinib for more than 2 weeks after progression because of perceived ongoing clinical benefit from the drug (12 for at least 6 months from the time of their initial investigator-defined disease progression). Overall, 144 (97%) of 149 patients experienced treatment-related adverse events, which were mostly grade 1 or 2. The most common adverse events were visual effects, nausea, diarrhoea, constipation, vomiting, and peripheral oedema. The most common treatment-related grade 3 or 4 adverse events were neutropenia (n=9), raised alanine aminotransferase (n=6), hypophosphataemia (n=6), and lymphopenia (n=6). Crizotinib is well tolerated with rapid, durable responses in patients with ALK-positive NSCLC. There seems to be potential for ongoing benefit after initial disease progression in this population, but a more formal definition of ongoing benefit in this context is needed. Pfizer.
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Personalised medicine is an emerging model that will revolutionise our current healthcare system. In the last decade, several genomic aberrations were discovered that are now used as predictive markers for treatment with targeted therapeutics. The technological advances in the last few years, such as the development of high resolution DNA microarrays or second generation sequencers, have led to a dramatic increase in the number of ongoing genomic profiling studies. These studies, in turn, are leading to an enormous number of detected genomic aberrations whose biological interpretation is still pending. This review will provide an overview on the current state of personalised medicine in cancer. Discussion of the use and development of the various technologies will help us to understand the opportunities and challenges that arise when novel technologies are implemented.
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Crizotinib is a dual MET and ALK inhibitor. Currently, clinical development of crizotinib is focused primarily on ALK rearranged non-small cell lung cancer (NSCLC). Here we report an NSCLC patient with de novo MET amplification but no ALK rearrangement who achieved a rapid and durable response to crizotinib indicating is also a bona fide MET inhibitor.
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Differentiating reactive mesothelial cells (RMs) from metastatic adenocarcinoma cells (MAC) in serous fluids based on cytomorphologic features alone can be very challenging. Various immunocytochemical (ICC) markers have been used to maximize the diagnostic accuracy, however, cytopathologists still encounter difficulties in effusion cytologic diagnosis. The aim of this study was to evaluate previous and recent ICC stains to identify the most sensitive and specific markers and the best panel for differentiating RM from MAC. Cell block sections from 41 MAC and 43 RM effusions cases were subjected to ICC staining for MOC-31, BerEp4, carcinoembryonic antigen (CEA), calretinin, HBME-1, CK5/6, and D2-40. For the MAC cases, the sensitivity of BerEp4, MOC-31, and CEA was 82.9, 92.6, and 17%, respectively, and the specificity was 95.3, 93, and 100%, respectively. For the RM cases, the sensitivity of calretinin, CK5/6, D2-40, and HBME-1 was 95.3, 27.9, 58.1, and 93%, respectively, and the specificity was 70.7, 73.1, 75.6, and 82.9%, respectively. The results show that BerEp4 and MOC-31 are highly sensitive and specific for detecting MAC, whereas calretinin and HBME1 are highly sensitive but only modestly specific for detecting RM cases (P < 0.05). Forced entry logistic regression revealed that using MOC-31, BerEp4, HBME-1, and calretinin, is an excellent panel for making correct diagnosis with 97.6% sensitivity in detecting MAC and 90.7% specificity in detecting RM. We conclude that adding a panel of MOC-31, BerEp4, calretinin, and HBME-1 immunostains to routine cytomorphologic features can greatly enhance the diagnostic accuracy of serous effusions.
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Sensitivity testing in ovarian cancer to individualize therapy remains an active area of interest and this has been renewed recently by results from several groups. The clinical results of assay-directed therapy are invariably better than would be expected from empirical treatment, but it has proved difficult to get these tests into practice. Several recent studies suggest that cellular individualized tumour response tests, particularly the ATP-based tumour chemosensitivity assay, can improve the chance of response and probably survival for individual patients. Most tumour response tests show excellent correlation with clinical resistance, but vary in their ability to predict sensitivity. Molecular assays of sensitivity and resistance are less developed in ovarian cancer than in breast cancer, but those using multiple gene signatures show considerable promise. Individualized therapy has the ability to guide the use of chemotherapy as well as newer agents. The development of companion diagnostics for drugs used in ovarian cancer has considerable potential for the future and such assays are already proving useful where there is clinical evidence of equivalence between possible treatments.
Article
Multicellular tumor spheroids (MCTS) are used as organotypic models of normal and solid tumor tissue. Traditional techniques for generating MCTS, such as growth on nonadherent surfaces, in suspension, or on scaffolds, have a number of drawbacks, including the need for manual selection to achieve a homogeneous population and the use of nonphysiological matrix compounds. In this study we describe a mild method for the generation of MCTS, in which individual spheroids form in hanging drops suspended from a microtiter plate. The method has been successfully applied to a broad range of cell lines and shows nearly 100% efficiency (i.e., one spheroid per drop). Using the hepatoma cell line, HepG2, the hanging drop method generated well-rounded MCTS with a narrow size distribution (coefficient of variation [CV] 10% to 15%, compared with 40% to 60% for growth on nonadherent surfaces). Structural analysis of HepG2 and a mammary gland adenocarcinoma cell line, MCF-7, composed spheroids, revealed highly organized, three-dimensional, tissue-like structures with an extensive extracellular matrix. The hanging drop method represents an attractive alternative for MCTS production, because it is mild, can be applied to a wide variety of cell lines, and can produce spheroids of a homogeneous size without the need for sieving or manual selection. The method has applications for basic studies of physiology and metabolism, tumor biology, toxicology, cellular organization, and the development of bioartificial tissue.
Article
This systematic review evaluates evidence comparing therapy guided by chemotherapy sensitivity and resistance assays with empiric chemotherapy, emphasizing survival outcomes. Prospective studies were sought comparing patients treated contemporaneously by assay-guided chemotherapy and empiric therapy. An initial MEDLINE search and a search performed by a Working Group of the American Society of Clinical Oncology were reviewed with attention to prespecified study selection criteria. This review identified 10 studies meeting selection criteria, plus one retrospective study, using seven different assays. Only two studies randomly assigned patients to assay-guided treatment or empiric treatment. Five of nine nonrandomized studies found significantly higher response rates for patients who received assay-guided therapy compared with those treated empirically. One of the two randomized trials found a significantly higher response rate in the assay-guided group. Four additional studies found response rates favoring assay-guided therapy, but comparisons did not achieve statistical significance. Two nonrandomized studies found overall survival to be significantly improved with assay-guided therapy. One randomized study used a cross-over design that made it difficult to determine whether survival differed between groups, while the other randomized trial found no difference in survival. Six studies provided no comparison of groups on baseline patient characteristics. Only one study reported adverse events data. While higher response rates for assay-guided therapy have been observed, differences may be attributable to bias or confounding. Little evidence on survival is available. These results do not establish the relative effectiveness of assay-guided treatment and empiric treatment. Randomized trials are needed.
Article
Serous effusions are a frequently encountered clinical manifestation of metastatic disease, with breast, ovarian, and lung carcinomas and malignant mesothelioma (MM) leading the list. Recently, extensive research has resulted in expansion of the antibody panel that is available for effusion diagnosis, thereby reducing the risk for error. Despite this progress, relatively little has been done in way of understanding the biology of cancer cells in effusions, especially those of nonovarian origin. The diagnosis of a malignant effusion signifies disease progression and is associated with a worse prognosis regardless of the tumor site of origin. However, survival is much more variable with ovarian cancer compared with other tumors. Furthermore, cancer cells of different origins differ considerably in their biology and have unique phenotypic and genotypic characteristics. This review summarizes the current knowledge in this field and presents a model for the study of tumor metastasis and disease progression, through large comparative studies of malignant cells in effusions, primary tumors, and solid metastases. The case also is made for potential applications of this rapidly evolving body of knowledge in the diagnosis, classification, and prediction of biological behavior of processes resulting in cryptic effusions at the clinical level.
Article
In this work, we assessed whether culture of uniformly seeded chondrocytes under direct perfusion, which supplies the cells with normoxic oxygen levels, can maintain a uniform distribution of viable cells throughout porous scaffolds several milimeters in thickness, and support the development of uniform tissue grafts. An integrated bioreactor system was first developed to streamline the steps of perfusion cell seeding of porous scaffolds and perfusion culture of the cell-seeded scaffolds. Oxygen tensions in perfused constructs were monitored by in-line oxygen sensors incorporated at the construct inlet and outlet. Adult human articular chondrocytes were perfusion-seeded into 4.5 mm thick foam scaffolds at a rate of 1 mm/s. Cell-seeded foams were then either cultured statically in dishes or further cultured under perfusion at a rate of 100 microm/s for 2 weeks. Following perfusion seeding, viable cells were uniformly distributed throughout the foams. Constructs subsequently cultured statically were highly heterogeneous, with cells and matrix concentrated at the construct periphery. In contrast, constructs cultured under perfusion were highly homogeneous, with uniform distributions of cells and matrix. Oxygen tensions of the perfused medium were maintained near normoxic levels (inlet congruent with 20%, outlet > 15%) at all times of culture. We have demonstrated that perfusion culture of cells seeded uniformly within porous scaffolds, at a flow rate maintaining a homogeneous oxygen supply, supports the development of uniform engineering tissue grafts of clinically relevant thicknesses.
Article
TGP (thermo-reversible gelation polymer) is a high molecular compound that has so-gel transmitting temperature of 221C Since solid cancer tissue grows in this polymer three-dimensionally, and fibroblasts scarcely grow in it, TGP is suitable for chemosensitivity assays for solid tumors. In this study, a chemosensitivity test using TGP was applied to recurrent gynecologic cancer patients in order to evaluate its utility and efficacy. In some ovarian cancer cases, expression of anticancer drug resistance-related proteins was also analyzed. Recurrent tumor tissues were surgically obtained with informed consent. After these tissues were minced and incubated for 4 days with CDDP, mitomycin C, 5-fluorouracil, paclitaxel, and CPT-11, the sensitivity against these drugs was estimated. Western blotting was performed in 8 recurrent ovarian cancer tissues in order to analyze the expression of Bcl-2, MRP2, BCRP, and GST-pi. The total evaluability rate of this assay was 90.6% (29/32). Sensitive drugs could be determined in 5 of 7 ascites samples (71.4%) and in 2 of 3 intra-tumoral fluid samples (66.7%). The overall clinical response rate of chemotherapy determined by these results was 50.0%. There were significant correlations between the IC50 of CDDP and Bcl-2, BCRP, GST-pi, and between that of 5-FU and MRP 2. Although this was a preliminary study, the chemosensitivity test using this new material appears to be useful for designing 'made-to order' salvage chemotherapy for pretreated recurrent gynecologic patients. In order to overcome multidrug resistance, the mechanisms of multidrug resistance should be further investigated.
The role of immunohistochemistry in the diagnosis of neoplastic pleural effusions. Romanian journal of morphology and embryology = Revue roumaine de morphologie et embryologie
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Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types
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Chemotherapy sensitivity and resistance assays: a systematic review
  • DJ Samson
Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types
  • JM Kelm