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ICANCKR KlìSIiAKCM56.446(1-4463. Oclobcr I. I«W6| Use of the Comet Assay to Identify Cells Sensitive to Tirapazamine in Multiceli Spheroids and Tumors in Mice1 Peggy L. Olive,2 Charlene M. Vikse, and Judit P. Banáth Metileni /¿/fi/i/iv.v/'r.vDffitirniii'iii.Brìtì\liColumbiaCtintrr Ri'scturh Centre. 601 Wc\i 10th Avcniu', Vtinciniver. Rriiish Ctilitnihui V5Z IL3. Ctintulit ABSTRACT lution to detect subpopulations that differ in damage by as little as a factor of 2 (4). Because the tumor cells that survive treatment with a DNA- Tii apa/aminc. a bioreductive ilnii; preferentially toxic to hypoxic cells, damaging drug are likely to be those that do not harbor extensive DNA produces significant numbers of I)N \ single-strand breaks that can be de damage following treatment, a DNA damage-based assay is appealing as tected using; the alkaline comet assay. Our goal was to determine whether single-strand breaks measured using this assay could act as a surrogate end a surrogate for cell killing. point tor cell killing in multiceli spheroids and solid tumors in mice. In Tirapa/amine (SR4233) is a hioreductive drag that is preferentially spheroids composed of Chinese hamster V79 cells. \ViI)r human colon car toxic to hypoxic mammalian cells (5. 6). Phase I and II clinical trials are cinoma cells, or Sil la human cervical carcinoma cells, histograms of tail currently underway to evaluate the use of this drug in combination with moments (indicators of DNA damage in the comet assay) could he used to radiation and chemotherapy. We have previously used the alkaline comet identify the percentage of cells that sustained sufficient DNA damage to cause assay to measure DNA damage in multiceli spheroids, murine tumors, cell death after treatment with tirapazaminc. The proportion of comets with and normal tissues exposed to tirapa/amine (7. 8). This drug produces tail moments £20(i.e.. with damage comparable to that produced In about large numbers of DNA single-strand breaks in hypoxic cells even at 1U (.\ i correlated with cell survival irrespective of cell type, dose of lira pazamine, time of treatment, or position of cell in the spheroid. Single-cell nontoxic drug concentrations. Both strand breaks and cell killing increase suspensions from squamous cell carcinoma Nil tumors in C3H mice or Sil la with dose and time of exposure, suggesting that strand breaks might xenografts in severe combined ¡mmunodeficicnt mice were also analy/ed for correlate with (if not cause) cell killing. Within subpopulations ot sphe clonogcnicity and DNA damage. Again, the percentage of comets with tail roids and tumors, cell-sorting experiments indicated that cells that sus moments 220 was found to be a good predictor of cell killing for both tumor tained the most DNA damage were also the most likely to die following types, providing tumor samples were obtained no more than l h after i.p. treatment (7). However, if DNA damage is to be used as a surrogate for drug administration. Because tirapa/aminc is currently undergoing clinical cell killing, it is necessary to show that a specific amount of DNA damage trials, application of this procedure could provide an early indicator of is predictive for cell killing independent of the dose of the drug, hypoxic tumors likely to contain hypoxic. susceptible cells and also the extent of the status, time of exposure, or cell type. Therefore, we tested whether DNA response. damage by tirapa/amine. measured using the comet assay, could be an effective predictor of cell killing in multiceli spheroids and tumors in INTRODUCTION mice and might therefore have potential applicability in the clinic. Many genetic, pharmacological, and microenvironmental factors can influence the response of a tumor io a specific cytotoxic therapy. MATERIALS AND METHODS Although it is important to understand how all of these factors individually affect treatment outcome, a more immediate and practical Spheroid Culture. Chinese hamster V79-l71b lung fibrohlusts were main tained in exponential monolaver growth by subcultivation twice weekly in problem is to simply estimate the overall impact of all genetic and Eagle's MEM containing IO'* FBS. ' SiHa 1mm.m cervical carcinoma cells and epigenetic factors on tumor cell killing by a specific cytotoxic treat WiDr human colon carcinoma cells were purchased from the American Type ment. In an effort to develop a rapid assay to assess tumor suscepti Culture Collection and maintained in exponential growth by Iwice-weekly bility to treatment, we have examined the ability of the comet assay to Subcultivation in MEM plus l()r/r FBS. Spheroids were initiated by seeding act as a surrogate end point for cell killing. 2 X 104 cells/ml into Bélicospinner culture vessels containing MF.M plus \()ci In 1984, a single-cell gel electrophoresis method was developed that FBS. Spheroids were ted after 3 days and daily thereafter by replacing spent allowed measurement of DNA damage in individual cells (1). This medium with MEM plus 5*/i FBS supplemented with antibiotics. technique was later modified and adapted for image analysis, providing Spheroids were exposed to tirapa/amine in MEM plus 5'< F'BS in Bélico greater sensitivity and resolution of subpopulations (2. 3). In this method, glass spinner culture vessels. Vessels containing spheroids were equilibrated single cells are embedded in agarose on a microscope slide, then lysed to with l()r/i oxygen. 5r/< carbon dioxide, and 859Õnitrogen at 37°Cfor l h prior remove proteins, exposed to low-voltage electrophoresis. and stained to and during incubation with the drug. Following incubation, spheroids were washed and disaggregated by exposure in an orbital shaker at 37°Cfor 8-10 with a fluorescent DNA-binding dye. The amount of DNA that migrates is proportional to the number of DNA breaks present in the cell. This min in 0.25ÇÎtrypsindissolved in citrate saline. Single cells were centrifuged "comet" assay is versatile and able to detect DNA single-strand breaks, and resuspended in fresh cold medium. Cells were analy/ed lor clonogenicilv by plating cells in l(K)-mm tissue culture dishes containing 10 ml MEM plus double-strand breaks, or cross-links in virtually any cell type, provided 10% FCS. Colonies were counted X-14 days after treatment. Plating efficien that a single-cell suspension can be obtained (3). The method has also cies of cells from V79. SiHa. and WiDr spheroids were 0.64 ± 0.04, been applied to human tumor samples obtained by tine-needle aspiration 0.66 ±0.09. and 0.17 ±0.06 (mean ±SD). respectively. The same popula biopsy (4). a relatively safe and simple procedure, which is applicable to tions examined for clonogenicity were also examined for DNA damage using many human tumors. Not only is the comet method sensitive to low the alkali comet assay. levels of DNA damage, but more important, it provides sufficient reso- Tumor Cells. SCCVII squamous cell carcinoma cells were transplanted s.c. over the sacral region of inbred male C3H/HeN mice, approximately 30 g in weight. Tumors were used for experimentation approximately 2 weeks later Received 6/1X/96: accepted 7/30/96. The costs of publication of this article were defrayed in pan by the payment of page when they had reached a weight of 350-500 mg. SiHa human cervical charges. This article must therefore be hereby marked uJrenisemeni in accordance with carcinoma cells were transplanted s.c. in the back of CB-17 severe combined 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by a grant from the National Cancer Institute of Canada. 2 To whom requests for reprints should be addressed. Phone: (6()4) 877-6(XK). ext. 'The abbreviations used are: FBS. fetal bovine serum: SCCVII. squamous cell 3024: Fax: (604) 877-0743: E-mail: olive6»unixg.uhc.ca. carcinoma VII: CCD. charge coupled device. 4460 Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1996 American Association for Cancer Research. PREDICTINGTUMORRESPONSI;TOTIRAPAZAMINE immunodeficient mice. Tumors were used for experimentation approximately c 4-5 weeks later when they had reached a weight of 300-500 mg. O Tirapazumine (SR4233) was kindly provided by Dr. Martin Brown (Stan 10min ford University). Mice were injected i.p. with lirapazamine from a stock 30min solution of 1-2 mg/ml in PBS. One h after i.p. injection, mice were sacrificed, O) and tumors were rapidly excised and placed in ice-cold PBS. A single-cell C suspension was prepared from the entire tumor by finely mincing the tumor and incubating for 30 min with a mixture of trypsin. collagenase. and DNase 0.1 r as described previously (9). Cells were then filtered through 30-fim nylon 3 CO mesh, centrifuged. and resuspended in cold medium. Plating efficiency in MEM plus 10% PCS was 0.28 ±0.04 for cells from SCCVII tumors and -+—' 0.22 ±0.06 for SiHa cells. 0) 30 Hoechst 33342 Cell Sorting. During the final 20 min of drug exposure, 20 multiceli spheroids were incubated with 1 fig/ml Hoechst 33342 to selectively o recover cells from different depths within the spheroid (10). At the end of drug 10 exposure, spheroids were rinsed, disaggregated with trypsin. and sorted into 10 CO fractions according to Hoechst 33342 fluorescence concentration using a O Becton Dickinson FACS 440 cell sorter. O 20 40 60 Mice were injected i.v. with 0.1 ml of an 8-mg/ml stock solution of Hoechst 33342 in PBS; then 10 min later, animals were sacrificed, tumors were Tirapazamine removed, and a single-cell suspension was prepared for fluorescence-activated O cell sorting (II).

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