Individual human tumors in short-term micro-organ cultures: chemosensitivity testing by fluorescent cytoprinting (original) (raw)
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Chemosensitivity testing of human gliomas using a fluorescent microcarrrier technique
Journal of Neuro Oncology, 1990
This report describes a new fluorescent microcarrier cytostasis assay. Human glioma cell lines and primary cultures were attached to microcarrier tissue culture beads and treated with various chemotherapeutic drugs. After treatment, the cells were labelled with two vital fluorescent dyes in order to measure cellular viability. The uptake of hydroethidine and Hoechst 33342 was evaluated alone and in combination as probes for determining metabolic activity and cellular proliferation. Hydroethidine was found to be superior when compared to trypan blue and tritiated thymidine. The use of the microcarrier technique allows for the direct cellular measurement of fluorescence without the need of extensive extraction procedures. The fluorescent assay is a sensitive, rapid and an effective way to screen for potential antiproliferative compounds.
Chemosensitivity testing of human gliomas using a fluorescent microcarrier technique
Journal of Neuro-Oncology, 1990
This report describes a new fluorescent microcarrier cytostasis assay. Human glioma cell lines and primary cultures were attached to microcarrier tissue culture beads and treated with various chemotherapeutic drugs. After treatment, the cells were labelled with two vital fluorescent dyes in order to measure cellular viability. The uptake of hydroethidine and Hoechst 33342 was evaluated alone and in combination as probes for determining metabolic activity and cellular proliferation. Hydroethidine was found to be superior when compared to trypan blue and tritiated thymidine. The use of the microcarrier technique allows for the direct cellular measurement of fluorescence without the need of extensive extraction procedures. The fluorescent assay is a sensitive, rapid and an effective way to screen for potential antiproliferative compounds.
British Journal of Haematology, 2012
The response of the tumour microenvironment to anti-cancer drugs can influence treatment efficacy. Current drug-screening methodologies fail to distinguish and quantify simultaneously the concomitant effect of drugs on the tumour stroma and cancer cells. To overcome this limitation we have developed a fluorescence-based experimental model that employs mCherrylabelled stromal cells (e.g. bone marrow fibroblastic stromal cells) cocultured in direct contact with enhanced green fluorescent protein-labelled tumour cell lines for accurate assessment of proliferation and viability in both cell compartments and adhesion of tumour cells. Additionally, we used fluorescence-based image analysis to determine morphological changes that correlate with cell function (e.g. morphology of the actin cytoskeleton and nuclearity of osteoclasts to predict their bone resorption activity). Using this platform we have revealed that dexamethasone induces HS5 fibroblast proliferation and contact with multiple myeloma cells via a process involving Src/ c-Abl kinases. Osteoclasts also inhibited dexamethasone-induced apoptosis in myeloma cells while retaining their normal morphology and functionality in bone resorption. Myeloma resistance to dexamethasone mediated by HS5 cells and osteoclasts was reversed by treatment with the Src/c-Abl inhibitor dasatinib but not with bortezomib. This new experimental platform provides a more precise screening of new therapeutics for improved efficacy of tumour cell killing within the bone marrow microenvironment.
A rapid fluorescent screening method for cellular sensitivity to anti-cancer compound
Cytotechnology, 2012
The tetrazolium salts (MTT, XTT, MTS, WST) based colorimetric assay or resazurin based fluorimetric assay are currently typical methods for cell sensitivity determination to anticancer compounds. We presented here a new rapid method for this purpose. This method uses a fluorescent dye named DCFH-DA which is previously taken as a intracellular probe for measurement of H 2 O 2 levels within a cell. The application basis for this method lies in two facts: the membrane permeable feature of the final metabolite of DCFH-DA inside a cell, and the linearity relationship between cell number and H 2 O 2 level. The results showed that there was a perfect association between cell number and fluorescent intensity determined by the DCFH-DA method, no matter whether using resuspended or adherent cells, and further 50% concentration of inhibition (IC 50) comparison between data obtained by DCFH-DA method or MTT method using a positive known anticancer compound Baicalin showed that there were no significant differences in cellular sensitivity determination to compound Baicalin though there existed a relatively higher coefficient of variation of IC 50 by the DCFH-DA method than that by the MTT method. Thus our data indicate that DCFH-DA might not only be a fine reagent for determination of H 2 O 2 levels in cells but also an ideal fluorescent dye for cellular sensitivity test of anti-cancer compounds, and may be suitable for primary high-throughput drugs screening. Keywords DCFH-DA Á MTT Á Anti-cancer compound Á Cell sensitivity Á Baicalin Abbreviations MTT 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5di-phenytetrazoliumromide XTT 3 0-[1-[(phenylamino)-carbonyl]-3,4tetrazolium]-bis(4-methoxy-6nitro)benzenesulfonic acid hydrate MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium WST-1 4-(3-4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3benzenedisulfonate DCFH-DA 2 0 ,7 0-dichlorfluorescein-diacetate H2DCF 2 0 ,7 0-dichlorodihydrofluorescin DCF 2 0 ,7 0-dichlorofluorescein DMSO Dimethyl sulfoxide PBS Phosphate-buffered saline Electronic supplementary material The online version of this article (
In vivo Models for Experimental Therapeutics Relevant to Human Cancer
Cancer Research, 2004
About 50 scientists attended, with 8 invited speakers coming from Germany, the Netherlands, and various institutions in the United States. The meeting focused primarily on novel imaging approaches to cancer experimental therapeutics using animal models and pharmacodynamic (PD) end points. At the end of the presentations, a roundtable discussion was held in an attempt to reach consensus on a recommendation for optimal use of these modern imaging modalities. This report summarizes the meeting and provides a forum through which the cancer research community can provide input on the issues addressed in the roundtable discussion.
In Vivo/Ex Vivo and In Situ Assays Used in Cancer Research: A Brief Review
Toxicologic Pathology, 2009
Predicting whether a potential new anticancer agent will have a positive therapeutic index in patients remains a challenge. This brief review provides examples of preclinical in vivo/ex vivo and in situ assays used to assess the therapeutic potential of experimental anticancer therapeutics. Excision assays involving removal of tumor, bone marrow, and other tissues from the host after treatment to determine the effects of therapy in ex vivo assays are important preclinical tools. The survival of malignant cells from tumors treated in vivo and then excised is often determined by colony formation (CFU) in culture. When mice bearing in vivo alkylating agent—resistant tumors were treated with anticancer drugs such as cyclophosphamide, the survival pattern of bone marrow granulocyte-macrophage-colony forming units (CFU-GM) paralleled tumor cell survival. When TNP-470 and minocycline, an antiangiogenic combination, were added to treatment with cytotoxic anticancer therapies, tumor response...
Three tumor sensitivity tests evaluated with mouse tumors
Stem Cells, 1987
The predictive value of three types of tumor sensitivity tests was evaluated using mouse tumors. Sensitivities of osteosarcoma C22LR, Lewis lung and M2661 carcinoma were determined for the following drugs: DNA interacting or alkylating agent (doxorubicin, cisplatin, 1,3-bis(2-chloroethyl)-1-nitrosourea, melphalan), antimetabolite (5-fluorouracil, methotrexate) and microtubule inhibitor (vinblastine, vincristine). Volume measurements of the subcutaneously growing tumors after treatment with the same drugs were considered to be the traditional reference system with which the results of the in vitro clonogenic assay, the labeled precursor incorporation assay and the subrenal capsule assay were compared.Results obtained with the in vitro clonogenic assay were highly reproducible. With the 1-h drug exposure technique the predictive accuracy was 71%. This result is in the same range as those found by others for human tumors. Predictive accuracy after continuous drug exposure was only 25%.Vinblastine, vincristine and cisplatin caused no inhibition of labeled precursor incorporation. However, the assay is too unreliable to use, due to the extreme variability when used with the other drugs.From 31 consecutively performed duplicate tests in the subrenal capsule assay, nine showed opposite results. This degree of disagreement between duplicate test results was considered too high to make reliable predictions of tumor sensitivity with this assay.
Radiation Oncology Investigations, 1993
We have compared the effectiveness of three short‐term proliferative assays [3(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction, methylene blue staining, and [3H]‐thymidine incorporation] in predicting the results of clonogenic assay of radiosensitivity in five mammalian cell lines (SCCVII, AA8, V3, FME, and MM‐96). Cells were cultured in 96‐well microculture plates and a lead wedge was employed to provide a range of radiation doses (60Co source) up to 10 Gy. Radiation dosimetry was determined by a novel modification of the Fricke/thiocyanate dosimetry technique to allow direct determination in 96‐well plates. Cells were cultured following irradiation to determine clonogenic survival curves and to calculate the survival at 2 Gy (SF2), the initial slope of the fitted linear quadratic function (α), and the mean inactivation dose (D). A broad range of radiosensitivity was obtained (SF2 = 0.14–0.92) with the clonogenic assay. All three proliferation assays provi...
A comparison of three assays used for the in vitro chemosensitivity testing of human tumours
British journal of cancer, 1984
In this study cell lines have been used to determine the level of correlation between three assays which are in use for in vitro prediction of human tumour chemosensitivity. The methods which were compared included a clonogenic assay, a monolayer assay and a short-term biochemical assay. The results indicated that the monolayer and clonogenic assays were either directly comparable or could be made comparable by reducing the drug exposure time in the monolayer assay. The biochemical assay also gave comparable results for 3 of the 5 drugs tested. It was concluded that although the 3 assays did not produce identical dose-response curves, the assays were equally valid when used for predictive testing because selection of cut-off points which were based on retrospective correlations between in vitro sensitivity data and response data, as established by other authors, compensated for differences in sensitivity between the assays.