A total of 167 LARC patients were examined. All patients had newly diagnosed LARC and received neoCRT from January 2010 and December 2016 at The First Affiliated Hospital (Sun Yat-sen University) and Huaihe Hospital (Henan University). The inclusion criteria were presence of a single primary lesion, completion of standard neoCRT and receipt of radical surgical resection, and completion of adjuvant chemotherapy with a capecitabine, XELOX, or mFOLFOX6 regimen. Biopsy tissue samples were obtained from all patients before administration of neoCRT (5). The use of tissue blocks was approved by the Institutional Ethics Review Board of the Huaihe Hospital of Henan University and the First Affiliated Hospital of Sun Yat-sen University, and written consent was obtained from each patient.
The HCT116 and SW480 human CRC cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured according to ATCC guidelines. These cell lines were authenticated by short tandem repeat analysis at the China Center for Type Culture Collection (Wuhan, China). CRC cells with stable knockdown of XRCC2 (Sh-XRCC2) and with nonsilencing (vector) shRNAs were generated as previously described (5, 17). The knockdown efficiency of XRCC2 was determined using quantitative RT-PCR and western blotting.
Proteins were separated using 8–10% SDS-PAGE and then electrotransferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked for 1 h with 5% bovine serum albumin (BSA; Beyotime, Beijing, China) in TBS-T, and incubated with specific primary antibodies overnight at 4°C, followed by incubation with rabbit or mouse horseradish peroxidase-coupled secondary antibodies for 1 h. Antibody binding was detected using an enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA).
Quantitative real-time PCR
Total RNA was extracted and qRT-PCR was performed as described previously (5, 17). All experiments were performed at least 3 times.
Cells in confocal dishes were fixed in 4% paraformaldehyde, permeabilized using 0.5% Triton X-100, and blocked with 3% BSA. The cells were then incubated with anti-γH2AX (1:50, D17A3, Cell Signaling Technology [CST], Danvers, MA, USA) antibodies at 4°C overnight, followed by incubation with DyLight 488 AffiniPure Goat Anti-rabbit IgG (1:200, Abbkine, Redlands, CA, USA) for 1 h at room temperature in the dark. The samples were then costained with 4',6-diamidino-2-phenylindole (DAPI) and examined by confocal laser scanning microscopy. In each sample, the number of foci of γH2AX (a marker of DSBs) per nucleus was counted using a confocal microscope (Zeiss, Germany), and an average of 100 nuclei were analyzed for each sample.
Clonogenic cell survival assay
The clonogenic survival assay was performed as previously described (5). Briefly, cells were trypsinized to a single-cell suspension and then seeded into 6-well plates for 24 h before treatment. After addition of vehicle (Dimethylsulfoxide [DMSO]) or olaparib for 6 h, a varying dose of IR was applied (0–8 Gy), and the cells were then maintained in an incubator at 37°C with 5% CO2 for 10 to 14 days. The colonies were fixed with methanol and stained by crystal violet and colonies containing more than 50 cells were counted. All experiments were performed in triplicate and repeated three times.
For cell cycle analysis, cultured cells were harvested 72 h after treatment, fixed overnight with ice-cold 70% ethanol at −20°C, and then stained with 20 mg/mL propidium iodide (PI) staining buffer (1% Triton X-100 and 100 mg/mL RNase A) for 30 min. DNA content was determined using a FACSCalibur unit (Becton Dickinson, Franklin Lakes, NJ, USA) with ModFit LT version 2.0 software. All experiments were repeated at least three times.
Senescence-associated β-galactosidase assay
A kit for staining cell senescence using β-galactosidase (β-gal) was purchased from CST (#9860), and cells were fixed and stained according to the manufacturer's protocol. For the in vivo study, frozen sections of xenograft tumors (5-μm thick) were fixed in 2% glutaraldehyde and stained as in the in vitro experiments described above. Five images of stained cells from random fields were recorded using an inverted microscope, and blue-stained senescent cells and unstained cells in each image were counted using a computer. The positive percentages were calculated and presented using GraphPad Prism version 8.0.
IHC assays were performed as previously described (18). Briefly, 4-μm thick formalin-fixed, paraffin-embedded tissue sections were deparaffinized and rehydrated, followed by antigen retrieval and endogenous peroxidase inactivation. After blocking, the slides were incubated overnight at 4°C with anti-XRCC2 (1:200, #ab180752; Abcam), anti-PARP1 (1:200; #ab32138; Abcam), anti-Ki67(1:300; #9449; Cell Signaling Technology), or anti-γH2AX (1:100; D17A3; Cell Signaling Technology) antibodies and then with secondary antibodies (Vectastain ABC kit). Slides were stained with 3, 3-diaminobenzidine (DAB) and counterstained with hematoxylin.
Animals and in vivo studies
All animal experiments were approved by the Henan University Animal Care and Use Committee. Xenograft experiments were performed as previously described (19). Briefly, 6 × 106 SW480 cells were suspended in 100 μL of PBS and implanted subcutaneously into the right flanks of female BALB/c nude mice (4–5 weeks old, 15–18 g; SLRC Laboratory Animal Co). When the tumors had volumes of approximately 100 mm3, mice were randomized into four groups: DMSO, olaparib, RT+DMSO, or RT+olaparib. Mice received DMSO or olaparib via oral gavage once per day for 12 consecutive days. Mice in the RT+DMSO and RT+olaparib groups received fractionated radiotherapy (2 Gy every other day for 5 days) at 1 h after oral gavage; a lead plate was used for coverage to assure that IR was only applied to the xenograft region. Tumor growth was recorded using a digital caliper, and tumor volume was calculated as: 0.52 × width2 × length. Mice were euthanized and tumors were harvested 2 days after the last dose of olaparib or oralipib+RT. For long-term studies, mice received oralipib+RT therapy and were monitored for tumor growth until 30 days after RT.
One-way and two-way ANOVA tests were used for comparisons in the in vitro and in vivo experiments, and correction for multiple comparisons was performed using the Tukey or Sidak test, as appropriate. All values are expressed as means ± standard deviations (SDs). A P-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism version 8.0.