2, Ontario Cancer Institute, University Health Network
3, Samuel Lunenfeld Research Institute, Mount Sinai Hospital
4, Princess Margaret Hospital, University Health Network, Toronto, ON, Canada
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.
The colony formation assay (CFA) has been the gold standard for determining the effects of ionizing radiation therapy on in vitro cellular systems since first described by Puck and Marcus in 1956 (1). Despite being the gold standard, the CFA can be time consuming when counting the number of colonies manually under the microscope. For this reason, many different assays have been used in lieu of the CFA in order to assess the effects of cytotoxic agents on cancer cell growth in vitro. While many of these techniques are able to detect specific cellular processes such as apoptosis (2), proliferation (3), or senescence (4), the CFA is the only assay that monitors a cancer cell's ability to produce a viable colony after treatment. Unlike most other assays, the CFA is unbiased to the mode of cell death. It is able to detect the cytotoxic effect of an agent, regardless of mechanism, as long as the agent affects the cell's reproductive ability to form progenies.
Most of the advancements made in cancer therapy in recent years have resulted from the combination of previous individual modalities, such as radiation and chemotherapy. Chemotherapy with such agents as cisplatin, 5-fluorouracil, doxorubicin, temozolomide, or cetuximab have been combined with radiotherapy for the treatment of head and neck cancer (5), non-small cell lung carcinoma (6), glioblastoma (7), cervix (8), and bladder cancers (9), to name a few. Initially, discovery of such combinations was conducted in the laboratory using tissue culture as the primary testing platform. To assess novel potential combinations, the majority of experiments were performed in a traditional 6-well tissue culture plate CFA, with each plate representing a different combination of two potential treatments. Commonly, such experiments test up to six different doses of radiation (0–10 Gy) and up to seven different doses of drug (10). Using the traditional CFA, this would result in using 42 individual plates. Beyond the technical challenges of conducting an experiment with 42 individual plates, a significant amount of time would be required to manually count the colonies on all such plates. This underscores the need for a more modern approach to this assay. With recent improvements in fluorescent probes and high-content microscopy, it is now possible to adapt the traditional method to a more efficient approach using a 96-well plate and an automated colony counting algorithm. While this article describes this novel approach in evaluating combination therapies including radiation and chemical treatment, it can be extended to any combination of treatments including two different chemical treatments administered concurrently.
Another area of advancement in cancer research is the use of high-throughput screening (HTS) for the identification of novel anticancer compounds. There are two basic approaches to HTS. The forward chemical biology approach identifies a phenotype of interest, after treatment with chemical compounds, and the mechanism is subsequently elucidated (11). The reverse chemical biology approach identifies a target, and hits are selected as compounds that modulate that specified molecule (11). Both forward and reverse approaches have yielded clinically useful anticancer drugs. The forward chemical biology approach is illustrated by the use of paclitaxel, which was shown to be effective against tumors long before it was identified to target microtubules (12). The reverse chemical biology approach has been highly successful in the identification of the bcr-abl inhibitor, Imatinib (Novartis, Basel, Switzerland), used for treatment of chronic myelogenous leukemia (13), and the src-abl kinase inhibitor, Dasatinib (Bristol-Myers Squibb, New York, NY), used to treat imatinib-resistant chronic myelogenous leukemia (14).
In the current study, we have adapted the 6-well CFA to a more efficient 96-well CFA that will allow for rapid analysis of combination therapies and opens the potential for high-throughput drug screening using the CFA as the readout.Materials and Methods Cell Culture
Human head and neck squamous cell carcinoma FaDu cells were cultured in MEM-F15 medium containing 10% fetal bovine serum (FBS), 1 mM pyruvate, 1.5 g/L sodium bicarbonate, 100 mg/L penicillin, and 100 mg/L streptomycin. Human lung adenocarcinoma A549 cells were cultured in RPMI media containing 10% FBS, 100 mg/L penicillin, and 100 mg/L streptomycin.6-Well Colony Formation Assay
Cells were trypsinized and plated in 6-well dishes at different densities depending on the potency of the treatments (from 50 to 104 cells/well). Cells were allowed to attach overnight and then exposed to radiation therapy (0–16 Gy) or chemical treatment at the corresponding dilution. Forty-eight hours after chemical treatment, the media was replaced with fresh media, and the plates were incubated at 37°C. Seven to eleven days later, the cells were fixed and stained with 10% methylene blue in 70% ethanol. The number of colonies, defined as >50 cells/colony were counted, and the surviving fraction was calculated as the ratio of the number of colonies in the treated sample to the number of colonies in the untreated sample. Triplicate wells were set up for each condition.