Anti-tumor effect of endostatin in a sleep-apnea mouse model with tumor
Obstructive sleep apnea (OSA) is associated with cancer incidence and mortality. The underlying mechanism is unclear. This study aims to evaluate the influence of intermittent hypoxia (IH), a novel hallmark of OSA, on tumor and to access the anti-tumor effect of endostatin on a mouse model with OSA.
The C57BL/6 J mice were randomly classified into four groups: control (normoxia) (CTL), control plus endostatin (CTL + ED), IH, and IH plus endostatin (IH + ED). Mice in IH and IH + ED groups were subjected to IH 8 h per day in 5 weeks. Lewis lung cancer cells were injected into the flank of each mouse after 1 week of IH exposure. Endostatin was also intraperitoneally injected after tumor volume reached about 200 mm3. The maximum standard uptake values (SUVmax) were detected by micro-positron emission tomography–computed tomography (micro-PET–CT) imaging prior and post-endostatin administration. Microvessel density (MVD) and vascular endothelial growth factor (VEGF) were determined for evaluating the anti-tumor effect of endostatin among the normoxia and IH conditions.
Mice had higher SUVmax in the IH group than the CTL group (p < 0.01). When compared with mice in the CTL group, those in the IH group had significantly greater MVD values (p < 0.001). The SUVmax can be attenuated by endostatin both in the CTL (p < 0.01) and IH conditions (p < 0.001). When compared with CTL group, mice in the IH group had increased MVD values (p < 0.001) and VEGF expression both at mRNA (p < 0.05) and protein levels (p < 0.001 in western blotting results). Treatment with endostatin attenuated serum and tissue VEGF levels, lowering the MVD values. As compared to normoxia condition, the endostatin-therapeutic effects were more significant under the IH condition (p < 0.05 in western blotting results).
Micro-PET–CT imaging is a promising non-invasive technique to evaluate the tumor metabolic characteristics under IH condition in vivo. The anti-tumor effect of endostatin under IH condition is superior to that of the normoxia condition.
KeywordsIntermittent hypoxia Sleep apnea Tumor Endostatin Positron emission tomography
- CTL + ED
Control + endostatin
Epidermal growth factor receptor
- IH + ED
Intermittent hypoxia + endostatin
Integrated optical density
Micro-positron emission tomography–computer tomography
Obstructive sleep apnea
Quantitative real-time polymerase chain reaction
Maximum standard uptake values
Vascular endothelial growth factor
Obstructive sleep apnea (OSA) is a prevalent medical condition characterized by recurrent collapse of the upper airway during sleep. Its pathophysiological features are intermittent hypoxia (IH) and sleep fragmentation . A large number of data demonstrated that OSA is associated with cardiovascular, cognitive and metabolic morbidities [1, 2]. More recently, establishing the relationship between OSA and cancer is an active research topic in the medical community. On one side, high incidence and mortality of cancer in OSA population were detected [3, 4, 5, 6, 7]. On the other, researches also showed increased incidence of sleep disorder breathing among various types of cancer patients [8, 9]. OSA can accelerate cancer progress, aggressiveness, and mortality [3, 7, 10]. As a novel pathophysiological feature of OSA, IH is closely correlated with tumor [11, 12]. Both in vivo and in vitro studies confirmed that IH promotes various tumor growth, migration, invasiveness, and metastasis [13, 14, 15, 16, 17, 18].
Although preliminary studies implicated that IH leads to tumor progression, the potential molecular mechanisms are still far from conclusion. Furthermore, little work has been done to evaluate the efficacy of the anti-tumor drug on an animal model with OSA.
Nowadays, endostatin has been administrated as an effective therapy for cancer [19, 20]. Researchers found that endostatin enhances radioresponse by normalizing tumor vasculature and relieving hypoxia , and reduces vascular endothelial growth factor (VEGF) levels and microvessel density (MVD) on a cancerous mouse . Prior experimental studies delineated that IH enhances angiogenesis and the expression of VEGF [23, 24], which contributes to tumor growth, migration, invasiveness, and metastasis [13, 25, 26]. However, it remains unknown if the anti-tumor efficacy of endostatin under the IH condition is superior to that of the normoxia condition. Therefore, the goal of the present study was to evaluate the effect of endostatin on tumor progression in an IH mouse model mimicking OSA via micro-positron emission tomography (micro-PET) imaging.
Materials and methods
The protocol was approved by the Ethics Committee of Zhongshan Hospital, Xiamen University, and conducted in accordance with the Guide for the Care and Use of Laboratory Animals .
Animals and subgroups
Forty-eight 7-week-old male C57BL/6 mice were purchased from Chinese Academy of Science Laboratory Animals Center in Shanghai, China. Mice were randomly assigned to the following four groups (n = 12 in each group): control (CTL), control plus endostatin treatment (CTL + ED), intermittent hypoxia (IH), and intermittent hypoxia plus endostatin treatment (IH + ED). With the exception of the intermittent hypoxia period, all mice were housed in standard cages with 12:12-h light–dark cycle and fed and had tap water ad libitum. Body weight (g) of each mouse was measured every week via an electronic weighing scale.
Lewis lung carcinoma cell culture and tumor induction
Lewis lung carcinoma (LLC) cells were obtained from CoBioer Biosciences Co., Ltd. Shanghai, China, and were maintained in high-glucose DMEM and supplemented with 10% fetal bovine serum (GIBCO, USA). After 1 week of IH exposure, LLC cells were subcutaneously injected (1 × 106 LLC in 100 µl PBS of each mouse) into the right flank of each mouse. Tumor volume (V, mm3) was calculated by measuring the tumor length (L) and width (W) with an electronic caliper (V = W2 × L/2) every 5 days after 7 days of LLC injection.
Intermittent hypoxia exposure
Mice in the IH and IH + ED groups were subjected to IH environment. Exposure was conducted as previously described , with some modification. Briefly, 24 mice (IH and IH + ED groups) were caged in a self-made plexiglass chamber with one-way valves. The IH cycle included 50 s of nitrogen, resting for 10 s, oxygen for 10 s, and compressed air for 50 s. The oxygen concentration in the chamber fluctuated between normal (21 ± 1%) and nadir point (6 ± 1%) for every 120 s of one IH cycle.
When the tumor volume reached about 200 mm3, recombined human endostatin (Endostar, Simcere-Medgenn Bio-Pharmaceutical Co., Ltd, Shandong, China) was intraperitoneally injected (8 mg/kg) daily to those mice in the CTL + ED and IH + ED groups.
Micro-positron emission tomography–computed tomography (micro-PET–CT) and imaging analysis
Micro-PET–CT imaging of tumor-bearing mice (for each group n = 3) was implemented by a micro-PET scanner (Inveon, Siemens) when tumor volume increased about 200 mm3 (before drug injection). Before 18F-FDG injection, all mice were fasted for 12 h but had free access to water. Mice were intraperitoneally injected with 7.4 MBq (200 μCi) of 18F-FDG under 2% isoflurane anesthesia. Imaging commenced 60 min after injection. Mice were placed at the center of the field of view of the micro-PET scanner in the prone position. Data were reconstructed using a two-dimensional ordered subset expectation maximum algorithm. Imaging analysis was consistent with a previous report . ASIProVMTM software (CTI Concorde Microsystems) was used to collect the regions of interests (ROIs), while a computer automatically obtains an average PET unit as the uptake value. The maximal standardized uptake value (SUVmax) was calculated as the 18F-FDG uptake from the greatest intensity of the tumor image. The micro-PET–CT imaging was repeated after 15 days of endostatin injection.
After the IH exposure, mice were fasted overnight prior to terminal anesthesia. After being euthanized with 3% phenobarbital (30 mg/kg) intraperitoneal injection, mice were weighed. A total of 600 µl whole blood samples were collected by a direct cardiac puncture. Tumors were excised, weighed, frozen in liquid nitrogen, stored at −80 °C for further analysis, and fixed in buffered 10% formalin for histological examination.
Serum VEGF and endothelin-1 detection
Concentrations of VEGF (pg/ml) in serum were detected with ELISA, following the manufacturer’s protocol (R&D Systems, Minneapolis, USA).
Quantity real-time polymerase chain reaction
Total RNA was extracted from tumor tissues in each group with TRIzol reagent (Invitrogen, Carlsbad, USA). After synthesizing cDNA, quantity real-time polymerase chain reaction (qRT-PCR) was conducted in the 7500 PCR system (Applied Biosystems, Foster City, USA) in accordance with the protocol of manufacture (TaKaRa, Biotechnology, Dalian, China). The primers used in the RT-PCR were forward 5′-TGTACCTCCACCATGCCAAG-3′ and reverse 3′-TCTCAATCGGACGGCAGTAG-5′ for VEGF, forward 5′-GTGCTATGTTGCTCTAGACTTCG-3′ and reverse 3′-ATGCCACAGGATTCCATACC-5′ for β-actin which was used as internal control. The relative gene expression levels were calculated with 2−DDCt.
Total proteins were extracted from the tumor tissues with RIPA lysis buffer (Solarbio, Beijing, China) in a glass homogenizer on ice. The protein concentrations in the supernatant of homogenate were detected by the bicinchoninic acid method using a protein assay kit (Beyotime, Beijing, China). Equal amounts of protein in each group were subjected to 10% sodium dodecyl sulfate-PAGE, and then transferred to PVDF membranes. After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were then incubated with the following antibodies at 4 °C overnight: mouse anti-vascular endothelial growth factor (Santa Cruz Biotechnology, USA), and mouse anti-actin (Santa Cruz Biotechnology, USA). Membranes were washed and incubated with the appropriate secondary antibody for 1 h at room temperature. The bands were detected with an enhanced ECL kit (Clarity™ Western ECL Substrate, Bio-Rad).
Hematoxylin–eosin staining, immunohistochemistry, and microvessel density detection
The tumor tissue in the formalin was further embedded in paraffin and sectioned into 5-μm slices. Sections were stained with hematoxylin–eosin (HE). Immunohistochemistry (IHC) was performed using primary antibodies mentioned in western blotting. Integrated optical density (IOD) was measured using Image-Pro Plus (version 6.0; Medica Cybernetics, USA). Microvessel density (MVD) values were evaluated by immunohistochemical analysis of CD34 antibody (Santa Cruz Biotechnology, USA), and counted at a magnification of ×400 using the previous method . One isolated brown yellow vascular endothelial cell or cell cluster in the tumor tissue was considered as one microvessel. Vessels were counted in the three highest density areas. The mean of the MVD values represents the average number derived from high-power fields of each group.
The GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) and the SPSS statistical software version 21.0 (SPSS, Inc, Chicago, IL, USA) were performed for data analysis. All values are presented as mean ± standard deviation. Data were compared using one-way analysis of variance and post hoc test, or unpaired/paired T test was also conducted for further comparison between groups, appropriately. A p value less than 0.05 was indicated as a significant difference.
Body weight, tumor volume and tumor weight in each group
Effect of endostatin on VEGF
HE staining and MVD evaluation
The present study established two models of IH and tumor in mice. Micro-PET–CT was conducted to evaluate the influence of IH on tumor growth and the anti-tumor effect of endostatin under the normoxia and IH conditions. The results showed that IH accelerates tumor development and enhances the expression of VEGF. The micro-PET–CT results found high SUVmax values and necrotic area in the IH-exposed mice. The anti-tumor effect of endostatin under IH condition was superior to that of normoxia condition.
IH is a novel pathophysiological hallmark in OSA patients. Increasing attention has been paid to illustrating the exact relationship between IH and tumor [11, 12]. Almendros et al.  and Yoon et al.  demonstrated that IH facilitates tumor growth. Meanwhile, the previous studies both in vivo and in vitro also prove that IH induces migration and metastasis in different types of tumor [15, 16, 17, 18]. Our results were consistent with previous studies. The tumor weight and volume were greater in mice exposed to IH than those exposed to normoxia condition. VEGF levels in both serum and tumor tissue were increased after IH exposure, which was consistent with the previous study . The results of the present study indicated that OSA-liked IH can aggravate tumor growth, facilitating the angiogenesis in tumor tissue. The connection between IH and tumor progression was also confirmed by clinical investigation. Several studies exhibited high prevalence and mortality of cancer in OSA patients. Further analysis found that the polysomnographic parameters, such as apnea–hypopnea index and oxygen desaturation index, were closely associated with cancer incidence and mortality [3, 5, 6, 7].
Robust evidence elucidated that endostatin can inhibit endothelial proliferation and angiogenesis in tumor [33, 34]. Few studies addressed the therapeutic effect of endostatin under IH condition. The results of the present study confirmed that endostatin can slow down tumor development. More tumor necrotic area can be found after endostatin treatment. The MVD and VEGF levels were reduced by endostatin. Furthermore, the anti-tumor effect is more significant when tumor-bearing mice is subjected to the IH condition mimicking OSA. The findings of the present study were similar to several experimental and clinical studies. Zhu et al. , worked on the effect of endostatin on esophageal squamous cell cancer in mice and found that endostatin can suppress tumor growth, reducing MVD, and inhibiting the expression of VEGF in tumor tissue. These effects were more significant when endostatin combines with radiation therapy. The anti-cancer effect of endostatin possibly attributes to its normalized vasculature and reduced hypoxia . Another study  indicated that endostatin plus chemotherapy leads to a more potent inhibition of tumor growth. In human research, a randomized controlled trial from a research group in China found that the combination of vinorelbine–cisplatin with endostatin can increase therapeutic response in non-small cell lung cancer and bone metastasis patients . Two meta-analysis studies [19, 36] elicited that when compared to platinum-based chemotherapy, a combination of endostatin can improve chemotherapeutic response rate, disease control rate, and the time to progress, without increasing the incidence of adverse events. No data were found regarding the anti-tumor effect of endostatin among OSA population before; therefore, further investigation is required.
Compared with conservative CT scan, PET–CT is an imaging technique that allows for the non-invasive visualization and quantification of metabolic processes in vivo. PET using 18F-FDG shows the increased glycolytic rate of malignant cells in tumors . PET–CT can be used to evaluate tumor metabolic processes and treatment response . In the present study, we applied micro-PET–CT, in which 18F-FDG was used as a tracer, to assess the IH-exposed tumor features in live animals. High SUVmax values were observed in the tumor after IH exposure, while endostatin treatment can attenuate the SUVmax values and increase tumor necrotic area both under normoxia and IH conditions. The micro-PET–CT information from our study illustrated that increased tumor metabolic levels were observed in the IH condition. The reduced levels of tumor metabolism treated by endostatin were more obvious in the IH condition than in normoxia condition. Further clinical studies are warranted to figure out whether PET–CT can be conducted to evaluate the tumor metabolic levels in OSA patients.
The present study has its strength. Principally, it was the first study to determine the anti-tumor effect of endostatin in a mouse model mimicking OSA. Furthermore, the present study is the first to use micro-PET–CT to access the influence of IH on tumor and find out the anti-tumor effect of endostatin in live animals.
However, the present study has several limitations. First, when applying PET–CT to evaluate the anti-tumor effect of endostatin, we used 18F-FDG as PET–CT tracer, instead of those probes specific to epidermal growth factor receptor (EGFR), such as 18F-FBEM-Cys-ZEGFR:1907 and 64Cu-cetuximab-F(ab′)2. The specificity of 18F-FDG is possibly inferior to those EGFR probes. Additional study using specific EGFR probes as PET–CT tracers might be warranted to evaluate the anti-tumor effect of endostatin in an OSA mouse model. Second, only on-site tumor metabolic behavior was observed via micro-PET–CT, the metastatic lesion was not evaluated as the previous study [15, 16, 25]; therefore, further study is required. Third, we only judge the anti-tumor effect of endostatin alone under IH condition. Further study should be focused on the combined effects of endostatin plus chemotherapy or radiotherapy in OSA-mimicking animal or patients. Finally, as previous studies showed [14, 15, 16], the IH exposure time was relatively short in our current study (5 weeks); ongoing study is required to figure out the effect of long-term IH exposure on tumor mice.
The present study demonstrates that IH can aggravate tumor progress in a mouse model of sleep apnea, and the anti-tumor effect of endostatin under IH condition appears to be superior to that of normoxia condition.
X-B, Z, Y, Z, and H-Q, Z: conception and design. B-B, F, Y-Y, Y, Y-T, L, Y, W and Y-P, D: collection and assembly of data. X-B, Z, Y-Y, Y, L-D, C, X, L, C-Y, K, and H-Q, Z: data analysis and interpretation. Manuscript writing: all authors. Final approval of the manuscript: all authors.
This work was supported by Grant 3502Z20154019 for Fund from Xiamen Science and Technology Bureau, Grant 2018-2-65 for Youth Research Fund from Fujian Provincial Health Bureau, and Grant 2018J01393 for Fund from Natural Science Foundation of Fujian Province, China.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
The study was approved by the Ethics Commi ttee of Zhongshan Hospital, Xiamen University.
The research did not involve human participants.
Availability of data and material
All data generated or analyzed during this study are included in this published article.
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