Tumor specimens were obtained from patients at the Henan Provincial People's Hospital and the People's Hospital of Zhengzhou University (Zhengzhou, China). All patients gave informed consent for their tumor samples to be used. The present study was approved by the Internal Review and the Ethics Boards of Henan Provincial People's Hospital and the People's Hospital of Zhengzhou University. Tumor samples were isolated from a 47-year-old male patient with ccRCC during radical nephrectomy. Fresh tumors were minced, suspended in Dulbecco's Modified Eagle's Medium/nutrient mixture F-12 (DMEM/F12; Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA), and mixed with 300 U/ml collagenase I (Invitrogen, Thermo Fisher Scientific Inc.) and hyaluronidase (Calbiochem; EMD Millipore, Billerica, MA, USA), followed by overnight incubation at 37°C in 5% CO₂. Enzymatically disaggregated suspensions were filtered using a 40 µm cell strainer and washed twice with phosphate buffered saline (PBS), and red blood cells were lysed with ammonium chloride lysing buffer. The resulting single tumor cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in a humidified atmosphere containing 5% CO₂.

Cells were irradiated at room temperature using a ⁶⁰Co laboratory irradiator (Beijing Normal University, Beijing, China) at a dose rate of 1 Gy/min. The cultured cells were irradiated with a single dose of 3 Gy. For fractionated radiation, cells were either irradiated for 2–3 consecutive days, or sham-irradiated (controls). Irradiated and sham-irradiated cells were cultured for an additional 48 h and used in subsequent experiments.

Cells were plated at 1×10⁴/well in ultra-low-attachment 6-well plates and grown in serum-free DMEM/F12, supplemented with 20 ng/ml epidermal growth factor, 10 ng/ml human recombinant basic fibroblast growth factor-basic, and 1% B27 supplement (all from Invitrogen; Thermo Fisher Scientific, Inc.). The medium was changed every 2 days. Following 10 days in culture, colonies containing >20 cells were counted. To evaluate cell self-renewal ability, mammospheres were digested with 0.15% trypsin to be reseeded at 5×10³/well.

Side population (SP) analysis was performed as described by Goodell et al (16) with slight modifications. Briefly, the cells were suspended at a density of 1×10⁶ cells/ml in pre-warmed DMEM/F12, supplemented with 2% FBS (Invitrogen; Thermo Fisher Scientific, Inc.) and 10 mmol/l 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). This was followed by incubation with 5 mg/ml Hoechst 33342 (Invitrogen; Thermo Fisher Scientific, Inc.) with or without 50 µM verapamil (Sigma-Aldrich, St. Louis, MO, USA), an ABC transporter inhibitor, in the dark at 37°C for 90 min with interval mixing. Following staining, cells were washed twice with ice-cold PBS and resuspended in cold PBS. Flow cytometry analysis was subsequently performed using FACSAria II (Becton Dickinson; BD Biosciences, San Jose, CA, USA). Hoechst 33342 was stimulated using a 355 nm UV laser and detected using a 450/BP50 filter for blue fluorescence and 660/BP50 filter for red fluorescence.

Total RNA was obtained from cells using RNAiso Plus (Takara Biotechnology Co., Ltd. Dalian, China), and reverse transcription was performed according to Takara's protocol. qPCR was performed using a SYBR-Green I Master Mix kit (Takara Biotechnology Co., Ltd.) on the Bio-Rad IQ5 Real-Time-PCR reaction system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The relative amounts of mRNA were calculated from the comparative threshold cycle values using glyceraldehyde 3-phosphate dehydrogenase as a reference gene (all primers depicted in Table I). PCR was carried out with the following cycling conditions: 95°C for 2 min followed by 38 cycles of amplification (denaturation at 95°C for 15 sec, annealing at 58°C for 20 sec and extension at 72°C for 30 sec).

The study was approved by the Ethics Committee of Zhengzhou University (Zhengzhou, China). Briefly, 1×10⁵ cells suspended in 100 µl Matrigel (BD Biosciences) were injected subcutaneously into the left flank region of 4-week-old male NOD/SCID mice obtained from the Institute of Laboratory Animal Science, Peking University Health Science Center (Beijing, China). Tumor growth was monitored for 4 weeks following transplantation using a MOSAIC animal PET scanner (Philips Medical Systems, Inc., Bothell, WA, USA). For microPET imaging, the mice were subjected to fasting for 10 h prior to injection with fluorodeoxyglucose (¹⁸F-FDG), but were allowed free access to water. Mice were anesthetized intraperitoneally with 100 mg/kg pentobarbital (Sigma-Aldrich), and then injected intravenously with ~3.7 MBq ¹⁸F-FDG (Department of Nuclear Medicine, Peking University First Hospital, Beijing, China). To quantify the data, the area density of formed tumors was calculated using Gel-Pro Analyzer software ver. 3.0 (Media Cybernetics, Inc., Rockville, MD, USA).

For each experiment, a total of 600–1,000 cells were plated in 25 cm² flasks in triplicate and cultured in DMEM/F12 (supplemented with 10% FBS). To evaluate the DNA damage checkpoint response, primary ccRCC cells were cultured for 24 h before incubation with 100 nM AZD7762 (AstraZeneca R&D, Boston, MA, USA) for 1 h prior to irradiation and 24 h following radiation. The cells were cultured for an additional 9 days and subsequently fixed and stained with 0.5% crystal violet. Colonies containing >50 cells were counted. The clone formation efficiency was calculated as the ratio of the clone number to the seeded cell number.

Irradiated and sham-irradiated cells were pretreated with or without 100 nM AZD7762 (AstraZeneca R&D) for 1 h. The cells were harvested and washed with PBS 24 h following radiation, fixed in 70% ethanol for 30 min at 4°C, and stained with PBS containing 40 µg/ml RNaseA and 10 µg/ml propidium iodine, in the dark for 30 min. Cell cycle distribution was measured using the FACSCalibur™ flow cytometer (BD Biosciences), and data were analyzed using the BD CellQuest™ software ver. 3.1 (BD Biosciences).

Data are expressed as means ± SEM (n=3). The differences between SP cells were analyzed using Student's t-test. Statistical analyses were performed using SPSS software, ver. 17 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The intrinsic or acquired resistance of RCC cells to current therapeutic strategies severely limits their efficacy, and most patients with RCC succumb to the disease following the failure of current treatments. However, the molecular mechanisms contributing to the radioresistance of RCC tumors remain largely unknown. Therefore, the present study investigated whether TICs are enriched after receiving clinical fractions of radiation. Freshly isolated primary ccRCC cells were irradiated as follows: i) A single dose of 3 Gy on day 3 (R1); ii) 2 daily doses of 3 Gy on day 2 (R2); or iii) 3 daily doses of 3 Gy on day 1 (R3). Corresponding controls (CTR) were sham-irradiated on day 3. Following irradiation, the cells were incubated for an additional 48 h to simulate a typical weekend treatment gap.

Sphere formation is a well-described characteristic of TICs, reflecting their potential for self-renewal (17). Thus, the capacity for sphere formation by primary ccRCC cells following multiple rounds of fractionated radiation was evaluated in non-adherent and serum-starved medium. Mammospheres containing >20 cells were counted following 10 days in culture (Fig. 1A). The rate of sphere formation in cells from the CTR, R1, R2, and R3 groups were 5.59±0.90, 5.42±1.12, 7.86±1.07, and 10.98±2.47% respectively (Fig. 1B). The R3 group exhibited a ~2 fold higher frequency of mammosphere formation than that of the CTR group (Fig. 1B). The self-renewal capacity of primary formed mammospheres was analyzed among the four groups by dissociating the mammospheres into single cells, and reculturing them in tumor sphere medium. The rate of sphere formation in secondary mammospheres was 14.84±1.98% in the CTR, 15.18±2.10% in R1, 14.71±1.82% in R2, and 13.76±1.88% in R3 (Fig. 1B). The secondary frequencies of spherical colony formation were similar among all the groups, indicating that capacity for self-renewal was retained after irradiation.

TICs display conserved stem and progenitor cell phenotypes (7,18); therefore, the expression of embryonic stem cell (ES)-associated genes was evaluated to confirm the stemness phenotype of cells subjected to fractionated radiation. RT-qPCR results demonstrated that the R2 and R3 groups expressed ES marker genes, such as Bmi1, Nanog, Sox 2 and Oct4, more highly than CTR (Fig. 1C).

Tumorigenic capacity of TICs was determined by injecting immunocompromised mice with RCC cells, using a widely accepted assay (19,20). To determine whether irradiated cells exhibit higher tumorigenicity, the cells were injected subcutaneously into the left flank region of NOD/SCID mice. The Tumor growth of mice injected with cells from each group (CTR, R1, R2, and R3) was monitored at 4 weeks post-transplantation (Fig. 2A, indicated by arrow). The total densities of formed tumors were 3.29±1.16 in CTR, 4.28±0.79 in R1, 6.72±1.68 in R2, and 11.75±2.16 in R3 (×10,000; Fig. 2B). The total tumor densities formed in the R3 group were significantly higher than those formed in the CTR group (**P<0.01).

Previous studies have demonstrated that SPs within different cancer cells are less differentiated and have characteristics of stem/progenitor cells (7,16,21). In addition, it has been confirmed that SP cells in RCCs exhibit TIC characteristics (8,9,22). Fig. 2C presents the fraction of SP cells observed within each group. These were 3.84, 3.89, 4.97 and 6.82% in the CTR, R1, R2, and R3 groups, respectively. The frequency of SP cells increased from 3.84% in the CTR group to 6.82% in the R3 group (P<0.05; Fig. 2C), demonstrating enrichment of the SP fraction that occurred following fractionated radiation treatment.

The TIC subpopulation was enriched following fractionated radiation, suggesting that TICs within primary ccRCC mediate resistance to ionizing radiation. DNA damage leads to radiation-induced cell lethality; therefore the DNA damage checkpoint response serves an essential role in cellular radiosensitivity (23,24). To determine the role of the DNA damage checkpoint response in TIC radioresistance, the present study examined the expression of DNA damage checkpoint-associated genes including ataxia-telangiectasia-mutated serine/threonine kinase (ATM), ATM and Rad3-related serine/threonine kinase (ATR), checkpoint kinase 1 (Chk1), and Chk2. The expression levels of ATM, Chk1, and Chk2 were significantly higher in cells subjected to fractionated irradiation than in sham-irradiated cells (Fig. 3A), however, no significant difference in ATR gene expression was observed among the four groups. These data indicate that fractionated irradiated cells may activate checkpoint responses to a greater extent than sham-irradiated cells, suggesting that the radiation resistance of enriched TICs is due to increased checkpoint activation.

A novel checkpoint kinase inhibitor, AZD7762, was used to confirm the above hypothesis. Results from the clonogenic survival assay demonstrated that 100 nM AZD7762 exerted minimal cytotoxicity in the CTR group but yielded radiation enhancement effects that overcome the resistance of irradiated cells, significantly reducing the rate of clone formation in R2 and R3 cells (Fig. 3B and C; P<0.05). These data confirm that the preferential checkpoint response in radiation-enriched TICs is associated with cellular resistance to radiation.

Checkpoint activation induces cell-cycle arrest in order to repair damaged DNA. Therefore, the cell cycle distribution among the four groups was analyzed. Cell cycle analysis revealed induction of G2/M arrest following irradiation with 3×3 Gy; from 12.94±0.47 (CTR) to 32.57±1.58% (R3 group; Fig. 4A). This arrest was abolished by AZD7762 administration. AZD7762 specifically abrogated the G2 checkpoint as evidenced by an increased G1 cell population in groups R2 and 3 (Fig. 4B and C). These results indicate that cells exposed to fractionated radiation were arrested in the G2/M phase, due to the induction of the DNA damage checkpoint response, possibly via Chk1 and Chk2.