The multi-well assay we describe here produces survival measurements on a multi-log scale comparable to the clonogenic survival assay. As described in the results section, BCNU sensitivities of the suspension cell lines TK6 and MT1 are remarkably similar to that measured by the clonogenic survival assay. Moreover, the TK6+MGMT cell line is identified as being resistant to BCNU treatment as expected [15
]. These results highlight three important aspects of our assay: i) The assay can measure sensitivity and resistance equally well, thus yielding accurate results that reflect those obtained from the clonogenic survival assay; ii) the multi-well assay is capable of measuring cell sensitivity on a multi-log scale, thus showing the large dynamic range the assay has in contrast to other available quick assays; iii) the assay can be used to measure cell survival of both suspension and adherent cell lines to cytotoxic agents, thus increasing its applicability.
The multi-well format of the assay has a reduced setup time allowing one to simultaneously assay either a large number of cell lines, doses or agents. This greatly improves cell survival measurement efficiency to yield results in a fraction of the time and in a less labor-intensive manner as compared to the clonogenic survival assay. As demonstrated, the assay enabled us to measure the sensitivity of a panel of 24 genetically diverse cell lines to multiple doses of BCNU in biological triplicates, and thus generate survival profiles for the panel of cell lines. The results showed a wide range of sensitivities across genetically diverse cell lines, enabling us to identify cell lines with extreme BCNU sensitivity or resistance for further study (Valiathan et. al.
in preparation). In our previous study monitoring MNNG-sensitivity across the panel of 24 cell lines, growth measurements at one time point following exposure to one dose was used to assess sensitivity, and we observed only a 9-fold change in sensitivity from most sensitive to most resistant [22
]. The approach described here, gave us the relative sensitivity of the cell lines to within three orders of magnitude, analogous to clonogenic survival assays, within a fraction of the time. Furthermore, comparing the most sensitive and most resistant cells lines from our previous studies with MNNG and the current study with BCNU, allows us to assess whether the sensitivity of cells to cytotoxic agents is specific to the class of cytotoxic agents used, or simply represents the response to cytotoxic damage in general. As an example, while cell line 6 was shown to be the most sensitive to MNNG in our previous report, this cell line clusters with the least sensitive cell lines after BCNU treatment. In contrast, cell line 4 appears to be among the most sensitive in both cases. The difference observed in cell line 6 may reflect differences in the ability to repair DNA interstrand crosslinks that are induced by BCNU but not by MNNG. Currently, we are investigating the mechanisms of BCNU resistance and sensitivity in the two most resistant and two most sensitive cell lines (Valiathan et. al.
The ability to gather proliferation data from a small number of cells and with higher throughput, as compared to traditional clonogenic survival assays, has the potential to be used to measure the sensitivity of cell sub-populations that have previously proven difficult to analyze. Glioblastoma is both the most common and most malignant form of brain cancer. Despite aggressive therapy, this tumor frequently displays a resistant phenotype. A sub-population of cells, dubbed glioblastoma stem cells, have been hypothesized to contribute to this resistant phenotype [23
]. Glioblastoma stem cells are identified and enriched from primary tumor samples due to their ability to form suspended cell aggregates, termed neurospheres, in certain culture conditions [24
]. The protocol described here would enable screening for compounds to target this resistant sub-population regardless of whether or not they can form colonies.
In addition to inducing cell death, many cytotoxic agents affect regular cell cycle progression, with cells undergoing arrest either in the G1, S or G2/M phases of the cell cycle. The nature of the arrest changes with the drug or cell line used, and provides insight into mechanisms of drug action and possible ways to modify cell sensitivity to a particular drug. The data obtained using our assay showed that the BCNU-induced decrease in surviving U87MG glioblastoma cells is accompanied by a concomitant arrest in late S/G2 phase of the cell cycle (). Previous studies have shown that U87MG cells show an accumulation of cells in late S or G2/M after BCNU treatment [25
] (assayed by PI staining and cell cycle profile analysis by flow-cytometry). Traditional clonogenic survival assays, as well as more recent high-throughput survival assays, yield no information on possible cell cycle arrest from the drug treatment. Any such cell cycle effects have been determined by separate experiments usually using flow cytometry. In comparison, results from the multi-well assay we describe here are rich in cell-cycle information, and provide added insight into the long-term cell cycle effects of the drug treatment.
In conclusion, we have presented a rapid and efficient method that takes advantage of current flow cytometry technology and properties of proliferating cells to measure the sensitivity of both suspension and adherent cells to cytotoxic agents. The flexibility of the method, its large dynamic range and its broad applicability makes it a powerful tool with great potential in many different applications including both small and large scale screening of sensitivity of numerous cell lines to known or suspected toxic agents.