Cancer Therapy: Preclinical γH2AX Expression in Tumors Exposed to Cisplatin and Fractionated Irradiation

Purpose: Is retention of γH2AX foci useful as a biomarker for predicting the response of xenograft tumors to cisplatin with X-ray? Is a similar approach feasible using biopsies from patients with cervical cancer? Experimental Design: Mice bearing SiHa, WiDr, or HCT116 xenograft tumors were exposed to cisplatin and/or three daily doses of 2 Gy. Tumors were excised 24 h after treatment and single cells were analyzed for clonogenic fraction and retention of γH2AX foci. Tumor biopsies were examined using 47 paraffin-embedded sections from untreated tumors and 24 sections from 8 patients undergoing radiochemotherapy for advanced cancer of the cervix. Results: Residual γH2AX measured 24 h after cisplatin injection accurately predicted surviving fraction in SiHa andWiDr xenografts. When a clinically equivalent protocol using cisplatin and fractionated irradiation was employed, the fraction of xenograft cells lacking γH2AX ranked survival accurately but underestimated tumor cell kill. Residual γH2AX foci were detected in clinical samples; on average, only 25% of tumor nuclei exhibited one or more γH2AX foci before treatment and 74% after the start of treatment. Conclusion: γH2AX can provide useful information on the response of human tumors to the combination of cisplatin and radiation, but prediction becomes less accurate as more time elapses between treatment and tumor biopsy. Use of residual γH2AX as a biomarker for response is feasible when cell survival exceeds ∼20%, but heterogeneity in endogenous and treatment-induced γH2AX must be considered. Prediction of tumor response to radiation remains an important goal in radiotherapy (1, 2). However, prediction is complicated by the use of combined modality therapy and the lack of a simple but effective predictor of response in situ. Ionizing radiation and many cancer therapeutics cause phosphorylation of histone H2AX at sites of individual DNA double-strand breaks, providing an exceptionally sensitive method for detection of this critical lesion (3, 4). Production of γH2AX foci can be detected microscopically or by using flow cytometry, and the extent of phosphorylation has been used as a biomarker of both exposure and DNA repair capacity (5–9). The fraction of cells that retain of γH2AX foci, measured 24 h after exposure to either radiation (6) or cisplatin (10), is correlated with the fraction of cells that lose clonogenicity in SiHa and WiDr tumor cells. These in vitro results and the availability of biopsies from patients treated with cisplatin and radiation provided the motivation to examine the response of xenograft tumors to the combination of cisplatin and radiation and then to measure γH2AX expression in clinical biopsies. The presence of residual γH2AX was examined using three human tumor xenograft models growing subcutaneously in immunodeficient mice given a single dose of cisplatin, 2 Gy × three daily fractions of X-ray, or the combination. To determine the importance of tumor cell location relative to functional blood vessels, tumor cells were sorted based on the fluorescence intensity of Hoechst 33342, a perfusion marker (11). Sorted cells were analyzed for clonogenicity and for expression of γH2AX using flow or image cytometry. A preliminary clinical evaluation was done to determine the feasibility of staining formalin-fixed, paraffin-embedded slides for fluorescence detection of endogenous and treatment-induced γH2AX expression. Pretreatment excision biopsies were obtained from 47 patients, and on-treatment biopsies were obtained from 8 patients undergoing radiochemotherapy for advanced cancer of the cervix. Materials and Methods Cell lines and treatments. SiHa human cervical carcinoma cells and WiDr human colon carcinoma cells were purchased from the American Authors' Affiliation: Medical Biophysics Department, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada Received 12/4/08; revised 1/14/09; accepted 1/16/09; published OnlineFirst 4/28/09. Grant support: Canadian Cancer Society and Canadian Institutes for Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This articlemust therefore be herebymarked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Peggy L. Olive, Medical Biophysics Department, British Columbia Cancer Research Centre, 675 West 10th Avenue, Vancouver, British Columbia, Canada V5Z 1L3. Phone: 604-675-8031; Fax: 604-675-8049; E-mail: [email protected]. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-3114 3344 Clin Cancer Res 2009;15(10) May 15, 2009 www.aacrjournals.org Research. on July 31, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Type Culture Collection. HCT116 human colon carcinoma cells were kindly supplied by Dr. Bert Vogelstein. Cell lines were maintained with twice weekly subculture in MEM containing 10% fetal bovine serum. Multicell spheroids were initiated directly from SiHa or WiDr monolayers and were grown for ∼2 weeks in spinner culture flasks as described previously (12). Spheroids were exposed to 2 Gy X-ray daily for 2 weeks, and samples were taken immediately before the daily irradiation for analysis of clonogenic survival and residual γH2AX expression. Cisplatin (Mayne Pharma) was diluted from the 1 mg/mL stock solution just before use. Xenograft tumors. Approximately 5 × 10 SiHa, WiDr, or HCT116 cells were injected subcutaneously into the back of NOD/SCID immunodeficient mice. After 4 to 6 weeks when tumors had reached a size of 0.25 to 0.5 g, mice were injected intraperitoneally with 0 to 20 mg/kg cisplatin or mice were exposed to 300 kVp X-ray. For radiation fractionation experiments, exposure to 2 Gy was done daily for 3 days with mice restrained in a Plexiglass jig. Mice were housed in our approved animal holding facility and treated according to the guidelines of the Canadian Council on Animal Care. Fluorescence-activated cell sorting. To separate tumor cells close to or distant from functional blood vessels, mice were injected intravenously with 0.05 mL Hoechst 33342 (20 mg/mL stock; Sigma) 20 min before sacrifice. Tumors were then excised and a single-cell suspension was prepared by mincing followed by incubation for 30 min in a freshly prepared cocktail of trypsin, collagenase, and DNase (13). Cells were sorted based on Hoechst 33342 fluorescence using a Becton Dickinson FACS440 cell sorter with UV excitation. Gated populations representing the dimmest 1/6 of cells, brightest 1/6 of cells, or all of the tumor cells were obtained under sterile sorting conditions. Sorted cells were analyzed in duplicate for clonogenic cell survival using a conventional colony formation assay (14). A sample of 250,000 sorted cells was also fixed in 70% ethanol and examined for γH2AX antibody binding by flow and image analysis. Four to six tumors per sort group were analyzed in independent sets that included controls, radiation only, cisplatin only, and the combination. Flow cytometry for analysis of γH2AX. Antibody staining was done as described previously (15). Briefly, 5 × 10 cells were rehydrated for 10 min, centrifuged, and resuspended in mouse monoclonal anti-phospho-Ser histone H2AX antibody (1:500 dilution; Upstate). After 2 h, cells were rinsed and resuspended in secondary antibody [1:200 dilution of Alexa 488 goat anti-mouse IgG (H + L) F(ab′)2 fragment conjugate; Molecular Probes]. Cell pellets were finally resuspended in 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI; Sigma) in TBS. Samples were analyzed using a Coulter Elite flow cytometer with UV and 488 nm lasers for excitation. To account for differences in radiation-induced changes in cell cycle distribution that affect average γH2AX intensity measurements, γH2AX expression per cell was corrected for DNA content and results were expressed as a ratio of the signal intensity for irradiated versus unirradiated cells. Experiments were typically repeated