In vitro Analysis of Drug Resistance

In vitro chemosensitivity (or chemoresistance) assays are routinely used in the laboratory for preclinical assessments of therapeutic efficacy. While many different assays have been used, there are 2 main types of assays: viability assays and proliferation assays. Viability assays analyze whether cells survive drug treatment; however, they do not typically demonstrate whether the cell retains the ability to proliferate. The simplest way to quantitate cell survival is to use live/dead cell assays such as cell counts using trypan blue. The trypan blue dye is actively pumped out of live cells but remains in dead cells giving them a blue color. This is a measure of membrane integrity and requires that each sample is individually counted. More sophisticated live/dead cell assays that use spectrophotometers or flow cytometers for quantitation are commercially available; however, they still only provide information as to how many cells survived treatment.

Viability assays that rely on the cell's metabolic activity are commonly used for high-throughput analysis of therapy resistance. These colorimetric assays utilize a dye that altered by the cell's metabolic activity. An example of this is the tetrazolium-type indicator dye 3-(4,5-dimethylthiazolyl-2)-2,5-diphe-nyltetrazolium bromide (MTT). Cells are plated in a 96-well plate, treated with a range of concentrations of the test drug, and MTT is added after an appropriate length of time. MTT is reduced to an insoluble formazan product by the mitochondria which is then solubilized with a detergent and the color is quantitated using a spectrophotometer [182]. The color is directly proportional to the number of viable (metabolizing) cells. Many assays have been developed that rely on similar mechanisms such as Alamar Blue [183] which has the advantage of leaving the cells alive so the same cells can be assayed over a number of days. The results are graphed with the drug concentration on the X-axis and the spectrophotometer result on the Y-axis. While relatively fast and easy to perform, the main disadvantage of these types of assays is that they only measure cell survival, they do not measure a cell's ability to divide. This may not provide an accurate indication of the drug's efficacy, as some drugs such as alkylating agents may damage a cell such that it survives and continues to metabolize until it has to divide. DNA damage prevents the cell from successfully dividing more than a few times.

Colony forming assays (CFAs) are more stringent assays for therapy resistance in that they require a cell to be able to divide in order to be counted as resistant to therapy. Cells are treated with a range of concentrations of the test drug, the cells are counted and an equal number of cells are plated into petri dishes, typically in triplicate. Cells are allowed to grow to form colonies of at least 50 cells and after the cells are fixed and stained, the colonies are counted. The results of a CFA done to analyze the BCNU resistance of a set of clones grown from a single tumor can be seen in Fig. 6.5. While some clones have essentially horizontal lines indicating resistance to BCNU, others are quite sensitive to the same doses of drug [184], demonstrating the heterogeneity present

FIGURE 6.5 Results of a colony forming assay demonstrating differential BCNU resistance in clones isolated from a single tumor. Clones with an essentially horizontal dose response curve are resistant. (Reprinted from [184]).

within a single tumor with regards to therapy resistance.

The ability to grow tumor cells as spheroids in the laboratory is allowing in vitro analyses of therapy resistance that takes into account the three-dimensional characteristics of tumors [111,185-188]. While these models have some obvious advantages, they also increase the number of variables inherent in the assay (reviewed in [189]), making it difficult to compare the results obtained in various laboratories.

Perhaps the most important question one must ask with regards to laboratory resistance assays is whether they should be used to identify patients that are likely to respond to a particular therapy. The American Society of Clinical Oncology (ASCO) organized a working group to compare various assays and determine if they should be used in a clinical setting [190]. They compared a subrenal capsule assay in which tumor cells are cultured and treated in the subrenal capsule of mice, CFAs, various viability assays and extreme drug resistance (EDR) assays in which cells are labeled with tritiated thymidine and exposed to concentrations of drugs equal to the maximum achievable concentrations in vivo. Resistance is indicated by continued uptake of tritiated thymidine. Their results demonstrated that none of these assays were currently suitable for routine incorporation into clinical trials. Similarly, Samson et al., [190] compared the results obtained from studies in which patients were treated based on the results of resistance assays versus those treated empirically. They concluded that randomized trials were needed to conclusively demonstrate the relative effectiveness of assay guided versus empiric therapy decisions. Haroun and coworkers [191] specifically examined extreme drug resistance in 64 brain tumors, most of which were obtained from patients who had previously been treated. They compared their results to response rates reported in the literature; however, it was not clear that the results of the EDR assays matched the in vivo results reported for these same drugs. Nevertheless, they conclude that this may still be a reasonable strategy to identify patients who will not respond to a particular therapy. This same assay was used by Parker et al., [31] in a prospective study of CPT-11 for the treatment of recurrent glioma. They correlated patient survival with the results of the EDR assay and found a significant correlation between the pre-treatment EDR results and both time to progression and overall survival, suggesting that this assay may have utility in clinical trials, although additional study is warranted.

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