Hidden stressors in the clonogenic assay used in radiobiology experiments.

While clonogenic assays are extensively used in radiobiology, there is no widely accepted procedure for choosing the composition of the cell culture media. Cell line suppliers recommend a specific culture medium for each cell line, however a researcher will frequently customize this aspect of the protocol by supplementing the recommended support medium with additives. For example, many researchers add antibiotics, in order to avoid contamination of cells and the consequent loss of data, with little discussion of the influence of the antibiotics on the clonogenic survival of the cells. It is assumed that the effect of any variables in the growth medium on cell survival is taken into consideration by comparing the survival fraction relative to that of controls grown under the same conditions. In the search for better cancer treatment, the effect of various stressors on clonogenic cell survival is under investigation. This study seeks to identify and test potential stressors commonly introduced into the cell culture medium, which may confound the response to radiation.

Increased efficiency for performing colony formation assays in 96-well plates: novel applications to combination therapies and high-throughput screening.

The colony formation assay (CFA) is the gold standard for measuring the effects of cytotoxic agents on cancer cells in vitro; however, in its traditional 6-well format, it is a time-consuming assay, particularly when evaluating combination therapies. In the interest of increased efficiency, the 6-well CFA was converted to a 96-well format using an automated colony counting algorithm. The 96-well CFA was validated using ionizing radiation therapy on the FaDu (human hypopharyngeal squamous cell) and A549 (human lung) cancer cell lines. Its ability to evaluate combination therapies was investigated by the generation of dose-response curves for the combination of cisplatin and radiation therapy on FaDu and A549 cells. The 96-well CFA was then transferred to a robotic platform for evaluating its potential as a high-throughput screening (HTS) readout. The LOPAC1280 library was screened against FaDu cells, and eight putative hits were identified. Using the 96-well CFA to validate the eight putative chemicals, six of the eight were confirmed, resulting in a positive hit rate of 75%. These data indicate that the 96-well CFA can be adopted as an efficient alternative assay to the 6-well CFA in evaluating single and combination therapies in vitro, providing a possible readout that could be used on a HTS platform.

Resazurin assay of radiation response in cultured cells.

We describe use of resazurin reduction for measurement of cell response to irradiation as a simple and non-destructive assay that complements the conventional colony forming assay and can readily be applied to both adherent and non-adherent cell cultures. The resazurin method yields data comparable with the colony forming assay as well as to assay of DNA synthesis (BrdU incorporation), giving an OER (oxygen enhancement ratio) of 2.5 at 60% isoeffect level versus 3.1 for the colony forming assay. Intraday and interday precisions for the resazurin assay were 4.1% and 5.2%, respectively.

The use of L5178Y mouse lymphoma cells to assess the mutagenic, clastogenic and aneugenic properties of chemicals.

Guidelines have been proposed to assess the potential of chemicals to affect human health. Written into these guidelines is the requirement that information be submitted on mutagenic activity. Although regulatory agencies accept mutagenicity data from both the hprt and tk loci in mammalian cells, many studies suggest that the L5178Y mouse lymphoma assay at the thymidine kinase locus is likely to detect a greater spectrum of mutagenic lesions. Thus, there is increasing emphasis being placed on this assay in many proposed and published guidelines. The L5178Y mouse lymphoma suspension protocol produces both small and large colonies which are the products of mutants growing at different rates. There is a reduction in the proportion of slowly growing mutants with respect to the total population of cells when expression is carried out in suspension. This potentially leads to quantitatively inaccurate assessments of the mutagenic activity of chemicals. Therefore an in situ procedure was developed that more accurately assesses the mutagenic activity of chemicals by maximizing the detection of small colonies. Many guidelines recommend tests that assess the clastogenic activity of chemicals. Some regulatory agencies accept data from the mouse lymphoma mutation assay to detect clastogens if the protocol is optimized for the detection of small colonies or if colony sizing data are submitted. The conventional suspension assay protocol is not sufficiently validated for this purpose. The in situ protocol has greater potential to meet these requirements.(ABSTRACT TRUNCATED AT 250 WORDS)

Multi-center collaborative in vitro studies using a human cancer cell line: the EORTC Preclinical Therapeutic Models Group experience.

The EORTC Preclinical Therapeutic Models Group (PTMG), formerly known as the Clonogenic Assay Screening Study Group (CASSG), began experiments in 1982 to investigate the feasibility of doing collaborative studies using in vitro clonogenic assays. The human colon adenocarcinoma cell line WiDr was selected as the model with which all participating laboratories would work. During the course of these studies, representatives from 34 different institutions located in 10 European countries participated. The first two collaborative experiments attempted to standardize the assay techniques using a double layer agar clonogenic assay. The results indicated that it was not possible to truly standardize these techniques in an international setting. The overall results demonstrated that the WiDr cell line grew well under a variety of conditions and that cloning efficiencies were independent of cell concentration, provided that a concentration of about 3,000 cells/ml was not exceeded. For the next series of protocols, the overall objectives were modified so that each laboratory could use its preferred assay technique whereas the WiDr cells were standardized by being expanded in a single center and sent to participants as viable cells. Analysis of the pooled results indicated that there was steady improvement in reproducibility for the group as a whole with repetition of the protocol. Drug dose responses with doxorubicin and etoposide showed good reproducibility from experiment to experiment. The results with cisplatin suggested that the sensitivity of this cell line may be affected by the presence of reduced sulfhydryl in some tissue culture media. The overall experience of the group indicates that use of a cancer cell line can provide the framework within which collaborative in vitro studies can be performed, provided that the initial conditions such as cell and drug concentrations are carefully determined in advance through preliminary studies.

The density of clonogenic cells in human solid tumors.

Considering that tumors are maintained by clonogenic cells, and that the primary target in the therapy of cancer is the clonogenic cell, the density of clonogens in a tumor could become an important parameter in quantitating the response to therapy. Indirect methods for determining the density of clonogenic cells in human tumors based on the response of tumors to radiation suggest there are circa 1 X 10(5) clonogens per gram with a large range. Direct methods, based on the measurement of cloning efficiency of enzymatically disaggregated biopsies of human tumors in soft agar, suggest a clonogen density of approximately 1,500 clonogens per gram. As this value is inconsistent with the prior data, we chose to determine the density of clonogenic cells in human tumors by assaying the enzyme digest of biopsies of human tumors for clonogenic cells using an enriched monolayer clonogenic assay. We determined the average clonogen density to be 1.12 x 10(5) clonogens per gram with a large range. The agreement with the indirect method suggests that the enriched monolayer clonogenic assay supports the proliferation of the cell population responsible for maintaining the tumor.

Reduced variation in the clonogenic assay obtained by standardization of the cell culture conditions prior to drug testing on human small cell lung cancer cell lines.

An advantage of established tumor cell lines compared to fresh human tumor specimens used in sensitivity assessments is the possibility of repeated experiments. Ultimately a database of sensitivity profiles on a panel of cell lines can be made and the sensitivity to new drugs compared with historical data. A prerequisite of this strategy is a minimal interexperimental variation. The sensitivity of eight human small cell lung cancer cell lines to adriamycin, daunomycin, aclacinomycin A, and mitoxantrone was tested in the clonogenic assay. A covariation in the sensitivity to the drugs emphasized the importance of simultaneous drug testing on the same batch of cells. On one cell line (NCI-N592) the interexperimental variation was further evaluated and a significant correlation was found between preexposure culture conditions, size of S-phase, and sensitivity to adriamycin, daunomycin, and mitoxantrone. Rigorous standardization of the growth conditions prior to clonogenic assay reduced the variation in the sensitivity to adriamycin from a factor of five to only 10-15%. It is concluded that simultaneous experiments on the same batch of cells in drug comparisons should be used if possible. Specification and standardization of culture conditions are necessary in the comparison of drugs tested in different experiments. Inclusion of the same reference drug in all experiments may further increase the validity of comparisons in different experiments.

A colony assay for in vitro transformation by human T cell leukemia viruses type I and type II.

We report here the development of a rapid and quantitative method for measuring in vitro T cell transformation by human T cell leukemia viruses type I (HTLV-I) and type II (HTLV-II). This method is based on our finding that cocultivation of lethally irradiated HTLV-producing cells with peripheral blood lymphocytes (PBLs) preactivated for 24 hours with phytohemagglutinin and interleukin-2 (IL-2) induces colony formation in methylcellulose-containing medium. Colonies of about 200 cells can be clearly distinguished from background aggregates within four to six days after cocultivation. These colonies gradually increase in size and reach 300 to 1,000 cells within 14 days after cocultivation. Cells of these colonies were infected, as evidenced by expression of viral p19 antigen and the presence of HTLV proviral sequences. These cells proved to be transformed in terms of IL-2-independent continuous growth in liquid medium. Colony formation was found to depend in a linear fashion upon the percentage of the infected cells present in the irradiated cell population and is sufficiently sensitive to detect as few as 1% of virus-producing cells.

[Fundamental study and clinical testing of human tumor clonogenic assay (HTCA)].

Through our retrospective analysis of HTCA and clinical effects as well as fundamental studies, the following results were obtained: In this retrospective study, HTCA for lung cancer showed a predictive accuracy of 71%, a true positive rate of 50% and a true negative rate of 77%. To obtain good predictive accuracy, HTCA should be modified to provide conditions comparable to those in vivo with regard to drug concentration and drug exposure time. More precise analysis of the pharmacokinetics of anticancer agents might yield methodological improvement. A decrease in the chemosensitivity spectrum in vitro was observed after chemotherapy. This might be related to evidence that patients with prior chemotherapy exhibited a poor response rate to chemotherapy. There were no active anticancer agents against specimens with aplating efficiency of more than 0.04%. More extensive prospective trials will be necessary to determine the clinical value of HTCA. HTCA could be a superior assay for detecting the anti-tumor activity of new agents and a useful method for in vitro phase II study.