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Anti-tumor efficacy of CKD-516 in combination with radiation in xenograft lung cancer mouse model

Background: To evaluate the anti-tumor efficacy of CKD-516 in combined with irradiation (IR) and examine tumor necrosis, delayed tumor growth, and expression of molecules involved in hypoxia and angiogenesis. Methods : A xenograft mouse model of lung cancer was established. The tumor was exposed to irradiation (IR) for 5 days per week. CKD-516 was administered with two treatment schedules (day 1 or days 1 and 5) at one hour after IR. After the administration, tumor tissues were stained with hematoxylin and eosin and pimonidazole. HIF1 , Glut-1, VEGF, CD31 and Ki-67 expression were evaluated by Immunohistochemical staining. Results: With short-term administration, IR and CKD-516+IR (d1) significantly reduced tumor size ( p = 0.0062 and p = 0.0051, respectively). CKD-516+IR groups were remarkably reduced blood vessels ( p < 0.005). In particular, CKD-516+IR (d1) resulted in the most extensive tumor necrosis, which was significantly increased with large hypoxic area ( p = 0.02) and decreased HIF1 , Glut-1, VEGF, and Ki-67 expressions. Long-term administration of CKD-516+IR reduced tumor size and delayed tumor growth. This combination also greatly reduced the number of blood vessels ( p = 0.0006) and significantly enhanced tumor necrosis ( p = 0.004). CKD-516+IR notably increased HIF1 expression ( p = 0.0047), but significantly diminished VEGF expression ( p = 0.0046). Conclusion: Taken together, our results demonstrate that CKD-516 in combination with IR can significantly enhance the anti-tumor efficacy compared to CKD-516 or IR alone in lung cancer xenograft mice.


Background
Lung cancer is one of the most common malignancies in both males and females. It is the major cause of cancer-related death worldwide [1]. Lung cancer is histologically classified into small-cell lung cancer (SCLS) and non-small-cell lung cancer (NSCLC). The latter accounts for approximately 85% of all lung cancers [2,3]. Squamous cell carcinoma (SCC) accounts for approximately 20-30% of NSCLC. It has poor prognosis due to limited treatment options [4].
Concomitant chemotherapy combined with radiation has been regarded as the standard treatment for locally advanced stage III NSCLC [5]. However, 5-year survival rate is lower than 30% due to severe toxicities caused by the multimodality treatment and frequent loco-regional recurrence and/or distant metastasis even after successful completion of treatment. Accordingly, it is urgent to develop a new treatment strategy that can not only enhance local effect, but also minimize side effects when anti-cancer drug is combined with radiation simultaneously or sequentially.
In contrast with normal cells that can recover rapidly in response to radiation, cancer cells are more sensitive to radiation so that they can become extinct. The anti-tumor activity of radiation is effective for local control. It has been applied for a variety of solid tumors, including lung cancer, head and neck cancer, and cervical cancer. However, hypoxic or acidic areas in cancer tissues are known to be highly resistant to radiation. In addition, tumor response to radiation therapy varies depending on tumor size [6,7].
Unlike normal tissue, blood vessels in tumor tissue are formed in complex structures with abnormal shapes. Such abnormal vascular structures in tumor tissue can become a hypoxic state which activates the expression of HIF1α, a hypoxia-4 inducible factor. Increased HIF1α induces more angiogenesis by increasing the expression of VEGF [8,9]. Ultimately, a series of these events give rise to local progression and distant metastasis through newly created blood vessels. In fact, about 50% of cancer patients receiving radiation become resistant over the time, with low oxygen tension in tumor tissue being the leading cause of local treatment failure.
So far, lots of studies on various forms of angiogenesis inhibitors have been done to overcome the resistance to radiation by effectively suppressing hypoxia-induced tumor angiogenesis. Vascular disrupting agent (VDA) is one of angiogenesis inhibitors with unique action mechanism by selectively targeting immature blood vessels in the center of tumor. Generally, they are classified into flavonoid vascular disrupting agents and tubulin polymerization inhibitors. Flavonoid vascular disrupting agents act on cytokines such as TNF and VEGF, leading to changed actin cytoskeleton, increased vascular permeability, and endothelial apoptosis.
Meanwhile, tubulin polymerization inhibitors can disrupt the tubulin network of cytoskeleton in endothelial cells, influence endothelial cell junction and actin cytoskeleton, and change vascular shape, resulting in increased vascular permeability [10].
In preclinical studies, efficacy and safety profile of VDAs have been reported [11][12][13][14]. Theoretically, radiation therapy is not so effective in locally controlling the hypoxic area of tumor tissue. Because these novel agents mainly affect blood vessels locating at central area of tumor, unlike cytotoxic anti-cancer drugs or other angiogenesis inhibitors, they may have a major advantage in compensating weak activity of radiation on the center of tumor when they are combined with radiation.
CKD-516, a novel tubulin polymerization inhibitor, can selectively bind to tubulin in endothelial cells of tumor vessels and block tubulin polymerization, thereby destroying the aberrant tumor vasculature. The intracellular process can lead to rapid decrease of tumor blood flow and nutrient supply for tumor cells, consequently inducing massive apoptotic tumor cell death.
In this study, we evaluated the anti-tumor efficacy of CKD-516 alone or in combination with radiation in short-term and long-term administration schedules in an in vivo mouse model. Balb/c nude mice were used in this study because they were known to be suitable for animal models for evaluating anticancer efficacy [16].
In addition, we investigated expression of signaling molecules involved in hypoxia and angiogenesis in tumor tissues.

Methods
Cell culture and reagents H520 (male, human squamous cell lung carcinoma) cell line was purchased from American Type Culture Collection (Manassas, USA). Cells were cultured in RPMI 1640 medium (Welgene, Korea) supplemented with 10% FBS, 200 U/mL penicillin, and 200 μg/mL streptomycin (Gibco, Korea). Cells were maintained at 37 °C in a 5% CO 2 incubator. CKD-516, a potent tubulin polymerization inhibitor, was obtained from Chong Kun Dang Research Institute [15]. Working concentrations were freshly prepared with 1xPBS.

Animals and Xenograft model
Male Balb/c nude mice (4 weeks of age, average weight 20 g) were purchased from Orient Bio (Seoul, Korea) and maintained under specific pathogen-free condition.

Drug treatment and irradiation
Mice were randomized with control and treatment groups. When tumor volume in mouse reached 500 ~ 700 mm 3 in diameter, mice were divided into four groups: 1) control group, injected with phosphate-buffered saline (PBS) weekly; 2) CKD-516, injected with CKD-516 at 3 mg/kg or 5 mg/kg; 3) irradiation (IR), treated with IR at 2 Gy or 4 Gy for 5 days per week; and 4) CKD-516 + IR, treated with both CKD-516 and IR. CKD-516 was administered with two treatment schedules by day 1 (d1) or days 1 and 5 (d1, 5) at one hour after IR. Shielding device was constructed for irradiating only the subcutaneous tumor, thereby avoiding whole body IR with consideration of the action mechanism of CKD-516. A 4-mm thick device in which a 50-mL tube could be inserted was placed into the mouse holder to be used for IR.
Toxicity during the period of IR was monitored based on body weight. Tumor volume was measured every other day using calipers throughout the experimental period.
Tumor volume was calculated based on the following formula: tumor volume = (length x width 2 ) x 2.

Analysis of tumor hypoxic area
Hypoxyprobe™ -1 plus kit (CHEMICON, Cat#: HP2-1000) was used to evaluate the hypoxic area in tumor tissue. Paraffin-embedded tissue sections (4 µm in thickness) were deparaffinized by xylene. Endogenous peroxidase activities were blocked by immersing these sections in methanol with 3% hydrogen peroxide for 5 min followed by washing with water and 1xTBST buffer (0.1% Tween 20 added in 1xTBS).

Statistical analysis
Results obtained from at least three independent experiments are presented as mean ± standard deviation. One-way analysis of variance (ANOVA) was used to determine differences between the control and treatment groups. P < 0.05 was considered statistically significant. All data were analyzed using Microsoft Excel 2010 for Windows 7 (Microsoft, Seoul, Korea).

Anti-tumor efficacy of CKD-516
We evaluated the anti-tumor efficacy of CKD-516 at two doses (3 mg/kg and 5 mg/kg) in H520 xenograft mice. In group 1 mice treated with 3 mg/kg of CKD-516, tumor growth was delayed until day 3. After that, tumor began to regrow. In group 2 mice treated with 5 mg/kg of CKD-516, tumor growth was inhibited from day 3.
Compared to the control, at the completion of drug administration, tumor sizes were reduced by 39.5% and 81.2% in groups 1 and 2, respectively (Fig. 1A). Additionally, we stained tumor tissues with Hoechst 33342 dye to examine morphological changes of blood vessels caused by CKD-516. Under a fluorescence microscope, we found that the morphology of blood vessels in mice treated with CKD-516 showed obvious changes (Fig. 1B). Based on these results, we selected the dose of CKD-516 at 3 mg/kg for the next study.
Anti-tumor efficacy of short-term treatment with CKD-516 alone or in combination with radiation We evaluated the anti-tumor efficacy of short-term administration with CKD-516 alone or in combination with radiation. At 24 hours after the completion of administration schedule, CKD-516 did not reduce tumor size. However, IR reduced it by 27.8% compared to the control ( Fig. 2A). When CKD-516 was combined with IR, tumor size was reduced by 28.6% in CKD-516 + IR (d1) and 27.9% in CKD-516 + IR (d1, 5). We also checked tumor size at 72 hours after the end of drug administration. CKD-516 reduced tumor size more at 72 hours than that at 24 hours.
Both IR alone and CKD-516 + IR (d1) delayed tumor growth. On the contrary, in CKD-516 + IR (d1, 5), tumor did grow again at 72 hours ( Fig. 2A). When tumor sizes were compared among four groups at the end of treatment, IR alone and CKD-516 + IR (d1) significantly reduced the tumor by 55.5% (p = 0.0062) and 58.5% (p = 0.0051), respectively. Notable body weight loss was observed in IR alone, CKD-516 + IR (d1), and CKD-516 + IR (d1, 5) groups by 15.4%, 13.7%, and 11.5%, respectively. In contrast, no changes in body weight were observed in the CKD-516 alone group (Fig. 2B). We counted the number of blood vessels and tumor necrosis area as well as tumor volume. As shown in Fig. 2C, the number of blood vessels stained with CD31 antibody was significantly diminished in CKD-516 group (52.1%, p = 0.0001) compared with the control. Both CKD-516 + IR (d1) and CKD-516 + IR (d1, 5) groups showed much more reduction in the number of blood vessels by 64.8% (p < 0.005) and 59.1% (p = 0.00016), respectively. We also analyzed tumor necrosis area in tumor tissue stained with H & E. IR significantly induced necrosis by 60.4% compared to the control (p = 0.004). CKD-516 + IR (d1) produced the most extensive tumor necrosis which was significantly increased by 66.0% (p = 0.02) compared to the control (Fig. 2D). However, tumor necrosis in CKD-516 alone or CKD-516 + IR (d1, 5) group did not significantly differ from that in the control.
Sustained tumor necrosis and hypoxia after short-term treatment 11 with CKD-516 in combination with radiation We investigated post-treatment effects of IR, CKD-516, and their combinations on tumor necrosis and hypoxic environment. The calculated tumor necrosis area (%) was the largest in IR alone (37.3%) at 24 hours from the beginning of treatment.

Expression of hypoxia-related molecules in mice after short-term treatment of CKD-516 in combination with radiation
We evaluated protein expression levels of hypoxia-related molecules (HIF1α, Glut-1, VEGF, and Ki-67) affecting the maintenance of hypoxic microenvironment in mice treated with CKD-516, IR, or their combinations (Fig. 4A). The expression of HIF1α, a classic marker for hypoxic condition, was the highest in CKD-516 group (57.6%) at 24 hours after drug administration (Fig. 4B). However, at 72 hours after treatment, it was the highest in IR group (68.1%). VEGF expression in IR alone was increased by 34.8% at 72 hours. In CKD-516 + IR (d1) group, it was significantly diminished from 22.0% to 7.0% (p = 0.019). The expression of Glut-1 was decreased as much as 20-30% for the analyzed area in all treatment groups at 24 hours after the end of drug administration. In IR alone, Glut-1 expression was decreased by 50.2% from 24 hours to 72 hours. It was greatly diminished in CKD-516 + IR (d1) group (81%, p = 0.0039). Ki-67 expression was the lowest (16.3%) in the CKD-516 alone group among four groups at 24 hours after the beginning of drug administration. However, at 72 hours, its expression was significantly declined in CKD-516 + IR (d1) and CKD-516 + IR (d1, 5) groups (86%, p = 0.0036 and 50.8%, p = 0.027, respectively).
Delayed tumor growth after long-term treatment of CKD-516 in combination with radiation We evaluated delayed tumor growth, tumor necrosis, and tumor hypoxia after longterm treatment with a combination of CKD-516 with IR compared to results from short-term treatment with a combination of CKD-516 and IR. Because weight loss and skin rash due to IR were frequently observed in short-term treatment, IR dose was decreased from 4 Gy to 2 Gy in the long-term combination schedule. Regarding tumor growth inhibition (TGI), both IR alone and CKD-516 + IR remarkably reduced tumor size (56.2%, p = 0.0091 and 71.2%, p = 0.007, respectively) at the end of administration ( Fig. 5A and Table 1). Additionally, we found sustained tumor growth delay even at 72 hours after the end of treatment, especially in the group of CKD-516 + IR (33.0% vs. 37.6%). No significant differences in body weight were found between CKD-516 and control groups (Fig. 5B). However, both IR alone and CKD-516 + IR groups displayed gradual decrease of body weight when the administration schedule progressed. We measured the number of blood vessels at 72 hours after the end of administration. Compared to the control, CKD-516, IR, and CKD-516 + IR significantly decreased the number of blood vessels by 38.4% (p = 0.003), 72.9% (p = 0.0002), and 84.2% (p = 0.0006), respectively (Fig. 5C). Conversely, tumor necrosis area was significantly expanded to 67% in IR group, 82% in CKD-516 group, and 84% in CKD-516 + IR group compared to the control (p = 0.02, p = 0.005, and p 13 = 0.004, respectively) (Fig. 5D).  (Fig. 6B). Glut-1 expression was increased in both IR and CKD-516 groups. However, it showed no significant change in CKD-516 + IR group (Fig. 6C). Ki-67 expression was greatly diminished by 4.3%, 4.4%, and 5.2% in IR, CKD-516, and CKD-516 + IR groups, respectively (data not shown).

Discussion
Chemotherapy combined with IR has been widely accepted as the standard treatment for locally advanced stage III NSCLC. However, hypoxic and acidic areas in the center of the tumor can lead to tolerance to radiation which is a major cause of treatment failure. In order to overcome IR-induced tolerance of hypoxic conditions, many studies have combined VDA or angiogenesis inhibitor with IR [17][18][19][20][21]. Although VDAs can cause rapid occlusion in the central tumor vessel, drug resistance to VDA can appear immediately. It might be attributed to remaining cancer cells acquiring nutrients and oxygen from marginal area of tumor [22]. Since 14 tumor growth becomes restored within a few hours after the administration of VAD [23,24], it is very important to combine VDA with other treatments to improve its anti-tumor efficacy. CKD-516 has been proven to have excellent activity in disrupting tumor vasculature in preclinical studies [25,26,27]. Its safety has also been confirmed in early clinical studies [28]. Results of the present study confirmed that high dose of CKD-516 (5 mg/kg) reduced tumor volume and increased tumor necrosis significantly more than a low dose (3 mg/kg). There was no noticeable change in body weight after low dose treatment.
However, gradual weight loss was observed after high dose treatment. Therefore, we used the administration dose of CDK-516 at 3 mg/kg for subsequent experiments. Based on preclinical data that VDA administration following IR was more effective for inhibiting tumor growth in breast cancer model [17], CKD-516 was also given one hour following IR in the present study.
After short-term treatment of CKD-516, IR, or their combinations for 1 week, both IR alone and CKD-516 + IR (d1) significantly reduced the tumor by more than 50%. In particular, CKD-516 + IR (d1) inhibited tumor growth up to 72 hours even after the end of administration. However, tumor did grow again in CKD-516 + IR (d1, 5). There was less tumor necrosis and hypoxia with higher expression of Glut-1 and Ki-67 in CKD-516 + IR (d1, 5) group compared to those in CKD-516 + IR (d1) group.
Interestingly, we found that expression of Ki-67 in rim area of tumor tissue in CKD-516 + IR (d1, 5) was increased (data not shown). Tumor is likely to regrow in CKD-516 + IR (d1, 5) at 72 hours after the end of drug administration.
In our study, CKD-516 + IR in combination significantly reduced blood vessels and Our results are contrary to prior literatures showing that expression levels of hypoxia-related proteins such as HIF1α, VEGF, and Glut-1 are increased in the presence of hypoxic condition [8,9,32,33]. Meanwhile, another study has shown that potent VEGF inhibitors including sunitinib and ziv-aflibercept can produce tumor necrosis and decrease expression levels of CD31 and Ki-67 in renal cell carcinoma PDX model [34]. The most likely explanation of our result was that CKD-516 + IR rapidly reduced tumor blood flow with excessive hypoxic condition, consequently leading to massive apoptotic tumor cell death with decreased expression of VEGF, Glut-1, and Ki-67.
In the present study, losses of body weight and skin rash were recorded in all IR- to 20%, their survival probability is lowered [35]. Furthermore, Balb/c nude mice responded more sensitively to IR than C57BL/6 mice [36]. One study has shown that when IR is given to colon cancer xenograft mice at 2 Gy for 5 days, tumor volume is reduced. However, body weight is not changed [37]. Therefore, we decreased IR dose to 2 Gy and applied it for the long-term treatment.
In the long-term treatment schedule, single IR and CKD-516 + IR significantly inhibited tumor growth with markedly reduced tumor. Moreover, CKD-516 + IR sustained tumor growth delay up to 72 hours even after the end of treatment. A previous report has shown that combination of CKD-516 and gemcitabine can greatly enhance its anti-cancer efficacy [29]. Another study has reported that tumor growth is restored from 3 days after a single administration of CKD-516. However, tumor growth is effectively inhibited until day 7 when CKD-516 is combined with doxorubicin [30]. These results strongly support our data that CKD-516 + IR Declarations

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Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
No potential conflict of interest was reported by the authors. After treatment with CKD-516 (day 1 or days 1 and 5) combined with daily IR for five consecu Figure 3 Short-term treatment of CKD-516 combined with IR induces persistent tumor necrosis and hy Sustained hypoxia by short-term treatment of CKD-516 combined with IR is associated with e Figure 5 Tumor growth is suppressed and delayed in H520 xenograft mice when CKD-516 is combined Figure 6 Changes in expression of hypoxia-related molecules after three cycles of CKD-516 combined 27 Supplementary Files This is a list of supplementary files associated with the primary manuscript. Click to download.