1.1 Materials
I-125 seeds were provided by Shanghai Xinke Pharmaceutical Co., Ltd. A single seed was 0.8 mm in diameter and 4.5 mm in length, with an average radioactivity of 0.8 mCi and a ~59.6 day half-life. Mouse procollagen type 1 n-terminal propeptide (P1NP) and C-telopeptide of type 1 collagen (CTX-1) ELISA kits were obtained from Novus Biologicals, Ltd. Hematoxylin-Eosin staining kit was from Solarbio. Antibodies against Ki67 and CD31 were purchased from Abcam.
1.2 Cell culture
Human NSCLC cell line H1299 was purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. H1299 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator with 5% CO2.
1.3 Establishment of H1299 xenograft tumor models of BALB/c mice
A total of 18 BALB/c mice (sex, female; age, 4-6 weeks; weight, 17-19 g) were purchased from The Department of Experimental Animals of Fudan University (Shanghai, China) and feeded them in a higher standard clean environment with warm temperature. All animal experiments were approved by The Committee for Ethical Use of Experimental Animals at Fudan University (Shanghai, China). H1299 cells were collected and adjusted to 1.0x107/ml in medium without FBS. A total of 4x106 cells were injected into the subcutaneous layer of the right side of a mouse. Body weight of mice and tumor size were regularly measured. Tumor volume (mm3) was calculated using the following formula: Tumor volume = (length x width 2)/2, where length and width represent the longest and shortest tumor diameters, respectively.
1.4 Antitumor therapy using I-125 seed implantation
Following 28 days of tumor growth, the tumor volume reached an average of 700 mm3, and all xenograft mice were randomly divided into three groups, as follows: Non-radioactive seed implantation group (Sham IM group), I-125 radioactive seed fractionated implantation (Fractionated IM group; 2x2 seed reimplantation) and I-125 radioactive seed single implantation (Single IM group; 4 seeds at one time). For the I-125 brachytherapy group, the tumor and surrounding skin were obtained from the mice and sterilized, followed by implantation of the I-125 seeds into the center of the tumor using a puncture needle. The Fractionated IM group was injected twice at an interval of 5 days. All mice were euthanized after 28 days of treatment. Subsequently, tumor xenografts were harvested. In addition, femurs and tibias were separated and collected for subsequent bone experiments.
1.5 H&E staining of tumor xenografts and bone tissue
To observe the effects of antitumor therapy, fixed tumor tissues were dehydrated, embedded in paraffin wax, and cut into 5-μm-thick sections, according to the standard immunohistochemical technique. Simultaneously, paraffin-embedded bone tissues were cut into 10-µm-thick sections. Prior to H&E staining, sections were deparaffinized with xylene, rehydrated using a descending ethanol series, washed with distilled water, and stained with H&E. Sections were observed by a light microscope following washing with 70% ethanol twice.
1.6 Immunohistochemical analysis of Ki67 and CD31 in tumor xenografts
For immunohistochemical staining, paraffin-embedded tumor sections were deparaffinized, followed by antigen repair and blocking of endogenous catalase. After washing with tris-buffered saline with Tween-20 (0.1%), sections were blocked with TBS solution containing 1% BSA at room temperature for 2 h. Subsequently, sections were incubated with the anti-Ki67 antibody (1:150; cat. no. ab15580; Abcam) and anti-CD31 antibody (1:200; cat. no. ab182981; Abcam) overnight at 4°C. Following washing with TBST, sections were incubated with horseradish peroxidase-labeled secondary antibody (1:50; cat. no. A0216; Beyotime Institute of Biotechnology) at 37°C for 1 h. Subsequently, diaminobenzidine substrate solution (cat. no. P0202; Beyotime Institute of Biotechnology) was used for coloration at room temperature for ~10 min. Hematoxylin (cat. no. C0107; Beyotime Institute of Biotechnology) counterstain was used for nuclei staining. Sections were dehydrated and sealed. A total of six randomly selected fields were photographed using a light microscope. Ki67-positive cells and CD31-positive microvessels were characterized as brown staining.
1.7 TUNEL staining of tumor xenografts
TUNEL staining of tumor sections was performed to detect the apoptosis of tumor cells following antitumor therapy. Following deparaffinization and alcohol rehydration, DNase-free proteinase K (20 μg/ml; cat. no. ST533;Beyotime Institute of Biotechnology) was added to the sections and incubated at 37°C for 15 min. After rinsing with PBS three times, sections were blocked using endoperoxidase at room temperature for 20 min (cat. no. P0100A;Beyotime Institute of Biotechnology), to inactivate the intrinsic peroxides. A total of 50 μl TUNEL solution was added and incubated at 37°C for 60 min. DAPI (cat. no. D523;Dojindo Laboratories, Inc.) of 0.5-10 μg/ml was used to counterstain nuclei. Six images of tunel-positive cells were randomly captured using a fluorescence microscope, and calculated using ImageJ software (Bethesda, MD).
1.8 Histomorphometric analysis of bone tissue
Femurs from bone tissues were fixed using 4% paraformaldehyde for 48h at room temperature, paraffin-embedded and cut into 5-µm-thick sections. H&E staining was used to observe the pathological changes of cortical bone and trabecular bone, as previously described. Histochemical staining of tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) were carried out using a commercially available kit (Sigma-Aldrich; Merck KGaA). The number of TRAP-positive osteoclasts were counted using Simple PCI software, and the densities of osteoclasts and ALP-positive cells were determined per mm2 of the total measurement area using ImageJ software (Bethesda, MD).
1.9 Measurement of bone-turnover markers in serum
Levels of serum P1NP (a sensitive biochemical marker of bone formation) were determined using Mouse Procollagen Type 1 N-Terminal Propeptide ELISA kit (cat. no. NBP2-76466; Novus Biologicals, Ltd.). Levels of serum CTX-1 (a marker of bone resorption) were detected using Mouse CTX-1 ELISA kit (cat. no. NBP2-69074; Novus Biologicals, Ltd.).
1.10 Reverse transcription-quantitative (RT-q) PCR
Total RNA was isolated from bone tissues using Simply P Total RNA Extraction Kit (cat. no. BSC52S1;Bioflux). A total of 1 μg total RNA was reverse-transcribed into cDNA using FastKing gDNA Dispelling RT SuperMix (cat. no. KR118-02; Tiangen Biotech Co., Ltd.). qPCR was performed using TB GreenTM Premix Ex Taq™ (cat. no. RR420A; Takara Bio, Inc.) and 20-μl PCR reactions on a Real-Time PCR System (Light Cycler 2.0, Roche Diagnostics GmbH). Primer sequences are displayed in Table 1.
1.11 Western blot analysis
Total protein was extracted from bone tissues using ice-cold cell lysis buffer (cat. no. P0013; Beyotime Institute of Biotechnology) containing protease inhibitors and quantified using a BCA Protein Assay kit. Electrophoresis using 12.5% SDS polyacrylamide gels (cat. no. PG113; Epizyme Biotech) was carried out to separate samples containing equivalent amounts of protein. Total proteins were transferred onto PVDF membranes (MilliporeSigma). Following washing with TBST (0.1% Tween 20), PVDF membranes were blocked using 5% non-fat milk for 30 min at room temperature, and subsequently incubated using a mouse monoclonal antibody against β-actin (1:2,000; cat. no. AF7018; Affinity Biosciences), a rabbit monoclonal antibody against osteocalcin (OCN; 1:1,000; cat. no. DF12303; Affinity Biosciences) and an antibody against runt-related transcription factor 2 (Runx2; 1:1,000; cat. no. AF5186; Affinity Biosciences) at 4°C overnight. Following washing with TBST, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies (1:2,000; Beyotime Institute of Biotechnology) at room temperature for 1 h. Chemiluminescent Biotin-labeled Nucleic Acid Detection kit (cat. no. D3308; Beyotime Institute of Biotechnology) was used to detect protein bands.
1.12 Statistical analysis
Statistical analysis of data was performed using SPSS Statistics 20 (IBM Corp.) and GraphPad Prism 5.01 (GraphPad Software, Inc.). Each experiment was repeated three times. Differences between groups were analyzed using one-way ANOVA. Values are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.