Iodine-125 brachytherapy suppresses tumor growth and alters bone metabolism in a H1299 xenograft mouse model

The present study aimed to investigate the efficacy of Iodine-125 (I-125) brachytherapy in a mouse model of non-small cell lung cancer, to further explore the efficacy and appropriate method of implantation of the I-125 radioactive seed. This study also aimed to determine the impact of brachytherapy on bone metabolism. A total of 18 mice were used to establish H1299 xenograft models, and were randomly assigned to three groups. These included non-radioactive seed implantation (Sham IM), fractionated I-125 seed implantation (Fractionated IM) and single I-125 seed implantation (Single IM) groups. Mice were euthanized after 28 days of implantation. H&E staining, Ki67 immunohistochemistry, CD31 morphometric analysis and TUNEL immunofluorescence assays were respectively used to determine the histopathological changes, proliferation, micro-angiogenesis and apoptosis of tumors. In addition, bone volume and microstructure were evaluated using trabecular bone area (Tb.Ar), trabecular thickness (Tb.Th), trabecular number (Tb.N) and cortical thickness. Bone metabolic status was analyzed using histomorphometric staining of tartrate-resistant acid phosphate (TRAP) and alkaline phosphatase (ALP) expression in the femur, and using an ELISA assay to determine the expression of C-telopeptide of type 1 collagen (CTX-1) and procollagen type 1 n-terminal propeptide (P1NP) in the serum. Moreover, reverse transcription-quantitative PCR and western blotting were carried out for the analysis of bone remodeling-related gene expression in the bone tissue. Results of the present study demonstrated that compared with the Sham IM group, both the I-125 seed implantation groups, including Fractionated IM and Single IM, demonstrated significant therapeutic effects in both tumor volume and weight. More specifically, the most significant therapeutic effects on tumor inhibition were observed in the Fractionated IM group. Results of Ki67 and CD31 immunohistochemical staining suggested a notable reduction in tumor cell proliferation and micro-angiogenesis, and results of the TUNEL assay demonstrated an increase in tumor cell apoptosis. Although the cortical bone appeared thinner and more fragile in both I-125 seed implantation groups, no notable adverse changes in the morphology of the cancellous bone were observed, and the index of Tb.Ar, Tb.Th and Tb.n was not significantly different among Sham IM and I-125 implantation groups. However, alterations in bone metabolism were characterized by a decrease in CTX-1 and P1NP expression, accompanied by an increase in TRAP activity and a decrease in ALP activity. Results of the present study also demonstrated the notable suppression of osteocalcin and runt-related transcription factor 2. I-125 seed implantation may be an effective and safe antitumor strategy. Moreover, the use of fractionated implantation patterns based on tumor shape exhibited improved therapeutic effect on tumor suppression when the total number of I-125 seeds was equivalent along with reduced complications associated with bone loss.


Introduction
Lung cancer is a type of malignancy with high morbidity and mortality rates [1], and it is mainly divided into nonsmall cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Notably, NSCLC accounts for ~ 85% of all lung cancer cases. As the majority of patients with NSCLC are diagnosed at a locally advanced stage, the therapeutic outcomes are not optimal, and the overall five-year survival rate remains low [2][3][4]. For the treatment of early stage NSCLC, surgical resection remains the most common therapeutic approach, while radiotherapy and chemotherapy are the most common treatment options for locally advanced NSCLC [5]. The continuous development of novel cancer treatments has led to an increase in the use of brachytherapy (also known as internal radiation therapy), as a complementary technique to conventional surgery approaches, such as chemotherapy and radiotherapy. This approach has been used in the treatment of a variety of malignant tumors, such as bladder cancer, cervical carcinoma, oral cancer and lung cancer [6][7][8][9][10]. At present, I-125 seeds are used in numerous applications due to a high killing radius and relative biological effect. Previous clinical data demonstrated that local recurrence of stage I NSCLC was 2.0% in > 200 patients following sub-lobar resection combined with I-125 brachytherapy, which was 18.6% lower than in patients treated with sub-lobar resection alone [11]. For patients undergoing limited lung cancer resection, I-125 seed implantation along the margin of the resection resulted in a low local recurrence rate, which may prolong survival [12].
Although improvements have been made in the therapeutic efficacy of lung cancer treatment, prolonged anti tumor treatment is often associated with long-term negative effects on a patient's quality of life. The concept of cancer treatment-induced bone loss (CTIBL) has emerged in recent years and attracted a high level of attention from researchers. Results of previous studies demonstrated that patients with breast and prostate cancer may experience severe bone loss during hormone therapy, which can lead to osteoporosis and increase the risk of refractory fracture. Additionally, radiotherapy and cytotoxic chemotherapies may also lead to bone damage, whereas the risk of bone loss caused by brachytherapy remains to be fully elucidated [13][14][15][16]. The present study aimed to explore the therapeutic effects of I-125 brachytherapy with different implantation strategies in a lung cancer mouse model while focusing on the effects of I-125 brachytherapy on bone health.

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.

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% CO 2 .

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 1.0 × 10 7 /ml in medium without FBS. A total of 4 × 10 6 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 (mm 3 ) was calculated using the following formula: Tumor volume = (length×width 2 )/2, where length and width represent the longest and shortest tumor diameters, respectively.

Antitumor therapy using I-125 seed implantation
Following 28 days of tumor growth, the tumor volume reached an average of 700 mm 3 , and all xenograft mice were randomly divided into three groups, as follows: Nonradioactive seed implantation group (Sham IM group), I-125 radioactive seed fractionated implantation (Fractionated IM group; 2 × 2 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.

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.

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.

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, DNasefree 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).

Histomorphometric analysis of bone tissue
Femurs from bone tissues were fixed using 4% paraformaldehyde for 48 h 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 mm 2 of the total measurement area using ImageJ software (Bethesda, MD).

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.

Effects of I-125 brachytherapy on tumor growth
The H1299 xenograft tumor models of BALB/c mice were established within 4 weeks of injection with H1299 cells.
When the tumor volume reached ~ 500 mm 3 , all mice were randomly divided into three implantation groups, including the Sham IM, Fractionated IM and Single IM groups. A diagram presenting the experimental pattern is displayed in Fig. 1A. To assess the potential impact of antitumor therapy on overall physical condition, the weights of mice were measured regularly throughout the experiment. Results of the present study demonstrated that the body weights of mice in both antitumor therapy groups were lower than in the Sham IM group; however, there was no significant difference among the three groups (Fig. 1B). These results suggested that I-125 seed irradiation exerted a minor impact on the body weight of H1299 tumor-bearing mice (Fig. 1B). All mice survived for the duration of the experiment, and tumor growth was significantly inhibited in both antitumor groups compared with the Sham IM group (Fig. 1C, D, P < 0.001). The suppressive effects of H1299 lung cancer on growth was more pronounced in the Fractionated IM than the Single IM group, and tumor weight was notably decreased (Fig. 1D, P < 0.05).

Effect of I-125 brachytherapy on tumor proliferation, angiogenesis and cell apoptosis
To investigate the effects of I-125 brachytherapy on H1299 xenograft tumor models, xenograft tissues were obtained from all three groups. All tissues were stained to observe the tumor structure, markers of tumor cell proliferation, tumor angiogenesis and apoptosis. As shown in Fig. 2A, H & E staining was performed to detect pathological changes in the tumor architecture. Results of the present study demonstrated that in the Sham IM group, tumor cells displayed red-stained circular cytoplasm and blue-stained nuclei, an integrated structure and a highly ordered arrangement. In both antitumor therapy groups, the tumor structure exhibited a disordered arrangement, and this was most notable in the Fractionated IM group. Results of the present study demonstrated a loose and bubble-like dispersed structure in the Fractionated IM group. Therefore, these results suggested that I-125 brachytherapy may destroy tumor tissue integrity. To further investigate the proliferation of tumor cells, Ki67 staining was carried out. Results of the present study demonstrated that Ki-67 positive cells were significantly decreased in the antitumor therapy groups, and this decrease was highest in the Fractionated IM group (Fig. 2C, P < 0.001). Moreover, CD31 staining was carried out to visualize tumor  TTC TGC TCA CTC TGC TGA CCCT  CCT GCT TGG ACA TGA AGG CTT  Runx2  GAC TGT GGT TAC CGT CAT GGC  ACT TGG TTT TTC ATA ACA GCGGA  β-actin  GGC TGT ATT CCC CTC CAT CG  CCA GTT GGT AAC AAT GCC ATGT microvessels. Results of the present study demonstrated a significant decrease in the density of microvessels in both antitumor therapy groups, compared with the Sham IM group (Fig. 2D, P < 0.01). In addition, the morphology of the majority of microvessels in both antitumor therapy groups exhibited a non-luminal structure (lines or spots). TUNEL staining was also performed to determine the number of apoptotic cells in the tumor tissue (Fig. 2B). Results of the present study demonstrated an increased apoptotic rate in the antitumor therapy groups (Fig. 2E, P < 0.01). Collectively, results of the present study demonstrated that I-125 seed brachytherapy may inhibit tumor growth through inhibiting micro-angiogenesis and tumor cell proliferation, as well as inducing tumor cell apoptosis.

Impact of I-125 brachytherapy on bone structure
Results of the present study demonstrated that in the H1299 xenograft mouse models, I-125 brachytherapy promoted distal bone metabolism. Compared with the Sham IM group, the femur and tibia in both antitumor therapy groups appeared thinner and shortened. Moreover, H&E staining of the paraffin-embedded sections of the femur revealed that the trabecular bone formation near the growth plate was altered in both antitumor groups (Fig. 3A). Morphometric parameters, including trabecular number (Tb.N), trabecular bone area (Tb.Ar) and trabecular thickness (Tb.Th) were decreased to a certain extent; however, these results were not significant. Results of the present study demonstrated a significant decrease in cortical thickness (Ct.Th; Fig. 3B-E).

Impact of I-125 brachytherapy on bone remodeling markers
TRAP is a marker for bone resorption and osteoclast activity, and ALP is a marker of bone formation and osteogenic capacity. TRAP staining of paraffin-embedded sections of the femur demonstrated strong osteoclast activity in the Sham IM group, which suggested that transplanted tumors may alter the systemic inflammatory response. In addition, osteoclast activity in both I-125 implantation groups was elevated to some extent, and this was highest in the Fractionated IM group (Fig. 4A-B, P < 0.05). ALP staining of the femur sections demonstrated a significant decrease following antitumor treatment ( Fig. 4A and C, P < 0.05). In addition, results of the present study demonstrated that the In addition, the mRNA and protein expression levels of osteogenic marker genes, OCN and Runx2, were reduced in the Fractionated IM group and Single IM group compared to the control group. Compared with the Sham IM group, OCN mRNA and protein expression was markedly low in both antitumor groups, and the expression of Runx2 was notably reduced (Fig. 5C-D).

Discussion
Lung cancer remains the leading cause of cancer-related death worldwide and in China, of which NSCLC accounts for 85% and SCLC accounts for 15%. Further understanding the biology and molecular subtypes of NSCLC has led to the development of more specific treatment plans for lung Representative images of TUNEL-positive cells (green) and nuclei (blue; magnification, × 200). C Quantification of Ki67 immunostaining. D Quantification of the microvessel density. E Quantification of TUNEL-positive cells. Results are presented as the mean ± standard deviation. n = 6 in each group. **P < 0.01, ***P < 0.001 vs. Sham IM group. I-125, Iodine-125; IM implantation cancer at different pathological stages. Due to difficulties in the early diagnosis of lung cancer, the majority of patients with NSCLC are diagnosed at an advanced stage. As surgery is not optimal for patients with locally advanced cancer, the combined treatment of radiotherapy and chemotherapy are the main treatment options for locally advanced NSCLC [17,18]. However, the recurrence and sequelae of lung cancer following conventional radiotherapy and chemotherapy remain key issues [19]. In recent years, researchers have paid increasing attention to the consequences of anti tumor treatment, such as treatment-related adverse effects and the quality of life of the patient. CTIBL directly effects bone health, and is often associated with the treatment of multiple cancers, such as prostate and breast cancer [13,16,20,21]. Results of a previous study [22] demonstrated that adjuvant hormone therapy increases survival and induces bone loss in breast and prostate cancer, significantly increasing the susceptibility of bone fractures. However, the impact of certain emerging cancer treatments on bone health, including brachytherapy, are yet to be fully elucidated.
Early studies performed by Stephen W. Doggett demonstrated a novel treatment option; namely, CT-guided percutaneous palladium-103 ( 103 Pd) seed implantation for the treatment of lung cancer. Advantages of this treatment include low lung toxicity, no radiation shielding requirements and low costs [19]. More recently, seed implantation technology has been widely used in the treatment of various cancers. Furthermore, numerous studies have indicated that the γ-rays released continuously by the low-energy I-125 seeds led to DNA hypomethylation and radiation-induced apoptosis, which may be the key mechanisms underlying the therapeutic effects of seed brachytherapy [23,24]. The present study investigated the therapeutic effects of I-125 seed implantation on H1299 xenograft tumors. Results of the present study demonstrated that tumor growth of the I-125 brachytherapy group was significantly inhibited, while changes in tumor weight and volume were more pronounced in the Fractionated IM group. Subsequently, H&E staining of tumor sections demonstrated necrotic areas and the disordered morphology of the tumor tissue in both antitumor treatment groups. Further immunohistochemical staining of endothelial cell marker CD31 and proliferating cell marker Ki67 revealed that tumor cell proliferation and intratumor microvessel density were markedly decreased following I-125 seed implantation. Moreover, tumor cell apoptosis was also decreased, which is associated with the inhibition of tumor growth. Collectively, results of the present study indicated that I-125 brachytherapy may directly suppress tumor cell proliferation and angiogenesis.
Although I-125 seeds have demonstrated a promising efficacy in the treatment of tumors, the effects of I-125 brachytherapy on neighboring healthy tissues, including distal bone tissue, remain unclear. Results of previous studies have verified that radionuclides, such as Rhenium-186 and Strontium-89, can be used to treat bone metastasis in breast and prostate cancer. However, the majority of radionuclides are β-emitters and/or γ-emitters with medium to high strength penetrability, which may damage surrounding tissues [25]. Results of a previous study [22] demonstrated the short radiation track of I-125 seeds at 1.7 cm, and the limited regional high-dose irradiation exerted little to no damage on the surrounding healthy tissues. On the other hand, results of previous studies have revealed that patients with advanced pancreatic cancer often experience Fig. 4 Effects of I-125 brachytherapy on bone remodeling markers in H1299 xenograft mice models. A Histopathological staining of TRAP and ALP in paraffin-embedded bone tissue sections of the isolated femur. TRAP-positive osteoclasts are shown in dark purple/ red and ALP-positive osteoblasts are shown in brown (magnification, × 200). B Quantitative analysis of TRAP activity. C Quantitative analysis of ALP activity. Results are presented as the mean ± standard deviation. n = 6 in each group. *P < 0.05, **P < 0.01 vs. Sham IM group. I-125, Iodine-125; IM implantation; TRAP tartrate-resistant acid phosphatase; ALP alkaline phosphatase side effects and complications following the implantation of I-125 radioactive seeds, such as abdominal pain, nausea and vomiting [26]. Therefore, further investigations into the potential comorbidities associated with I-125 brachytherapy are required, including radiation-induced bone damage. In the present study, the femur and tibia of mice were separated, to observe the appearance at the end of the treatment. Results of the present study revealed significant bone damage following I-125 seed implantation. Notably, the femur and tibia of mice in both antitumor treatment groups became shorter, thinner, more brittle and easily broken, compared with the Sham IM group. In addition, the cortical thickness of the femur in both antitumor treatment groups was markedly reduced, and the reduction in the Single IM group was the most significant. In addition, morphometric parameters including Tb.N, Tb.Ar and Tb.Th were decreased to a certain extent; however, this difference was not significant. These results suggested that I-125 brachytherapy may exert reduced levels of deterioration on bone tissue compared with conventional external-beam radiotherapy, due to the shorter track length of β rays from I-125. However, further research is required.
Furthermore, bone-turnover indicators, including the bone resorption indicator CTX-1 and bone formation indicator P1NP, were decreased in the I-125 implantation groups, which reflected the suppression of bone metabolism. These results indicated that I-125 brachytherapy may affect the balance of bone remodeling in distal bone tissue, leading to bone damage. Moreover, TRAP, a marker for bone resorption and osteoclast activity, and ALP, a marker for bone formation and osteogenic capacity, were also observed to determine the effects of I-125 radioactive seed implantation on bone remodeling. These results revealed that TRAP-positive osteoclasts and ALP-positive osteoblasts were markedly decreased, accompanied by the reduced expression of osteogenic marker genes, OCN and Runx2. These results suggested that I-125 brachytherapy treatment may induce bone remodeling imbalances, leading to bone dysfunction. seed implantation. n = 3 in each group. **P < 0.01, ***P < 0.001 vs. Sham IM group. P1NP procollagen type 1 N-terminal propeptide; CTX-1 C-telopeptide of type 1 collagen; OCN osteocalcin; Runx2 runt-related transcription factor 2; I-125 Iodine-125; IM implantation However, reduced levels of deterioration of the bone tissue were observed, when compared with traditional focal external-beam radiotherapy.
Collectively, results of the present study indicated that I-125 brachytherapy may exhibit potential in the treatment of lung cancer, and the Fractionated IM protocol based on changes in tumor shape requires optimization. However, the risk of bone metabolic disorders following I-125 brachytherapy remain, and the prevention of CTIBL following brachytherapy is required.