Anti-tumor efficacy of CKD-516
We evaluated the anti-tumor efficacy of CKD-516 at 3 mg/kg and 5 mg/kg in H520 xenograft mice. The molecular structure of CKD-516 is shown in Figure 1A. Following the completion of drug treatment, we found that tumor volumes were reduced by 39.5 % and 81.2 % (p = 0.002) in the 3 mg/kg and 5 mg/kg CKD-516 groups, respectively, compared to the vehicle (1.26 ± 0.58 cm3 and 0.35 ± 0.14 cm3 vs. 2.1 ± 0.32 cm3) (Fig. 1B).
Additionally, we stained tumor tissues with Hoechst33342 to study the morphological changes caused by CKD-516 to the tumor vasculature. Using fluorescence microscopy, we observed obvious morphological changes in the blood vessel shapes of mice treated with CKD-516 (Fig. 1D). Although a high dose of CKD-516 (5 mg/kg) markedly reduced tumor volume compared to the low dose (3 mg/kg), continuous body weight loss was evident following high-dose treatment (Fig. 1C). Based on these data, we chose to use 3 mg/kg CKD-516 for subsequent experiments.
Anti-tumor efficacy of short-term CKD-516 monotherapy or combination therapy with IR
We evaluated the anti-tumor efficacy of the short-term administration of CKD-516 alone or in combination with IR. To determine whether anti-tumor efficacy persisted even after treatment stopped, we compared tumor volumes 24 h and 72 h after the end of treatment.
Twenty-four hours after treatment was stopped, CKD-516 alone did not induce any additional reduction in tumor volume (1.49 ± 0.70 cm3). However, IR treatment alone showed a further 27.8 % reduction in tumor volume compared to the vehicle (1.11 ± 0.07 cm3 vs. 1.52 ± 0.66 cm3, p = 0.001; Fig. 2A). In contrast, 72 h after the end of treatment, monotherapy with IR and CKD-516 markedly decreased tumor volumes by 55.5 % (1.11 ± 0.1 cm3, p = 0.006) and 49.2 % (1.26 ± 0.65 cm3, p = 0.021), respectively, compared to vehicle treatment (2.49 ± 0.78 cm3). However, between 24 h and 72 h, IR alone did not change tumor volume, but CKD-516 alone reduced tumor volume 0.84-folds compared to the vehicle.
We combined IR and CKD-516 treatment in two different treatment schedules; IR (5 times per week) and CKD-516 once on day 1 (CKD-516+IR (d1)) or twice on day 1 and 5 [CKD-516+IR (d1, 5)] (Additional file 1). Compared to vehicle treatment, CKD-516+IR (d1) reduced tumor volume by 28.6 % at 24 h and by 58.5 % at 72 h (p = 0.049), but there were no notable changes in tumor volume between 24 h and 72 h (1.08 ± 0.03 cm3 vs. 1.03 ± 0.04 cm3). CKD-516+IR (d1, 5) also significantly reduced tumor volume by 27.9 % at 24 h (1.11 ± 0.04 cm3, p = 0.024) and by 32.2 % at 72 h (1.68 ± 1.0 cm3, p = 0.032). The tumor volumes of mice treated with CKD-516+IR (d1) and CKD-516+IR (d1, 5) 24 h after the end of treatment were similar to IR alone. Interestingly, after 72 h the tumor volume was increased by 1.51-folds in CKD-516+IR (d1, 5) treated mice. Tumor growth inhibition (TGI) and tumor growth delay (TGD) values of CKD-516 alone or CKD-516+IR combinations were similar to those of IR alone at 24 h. However, of the two combinations, only CKD-516+IR (d1) enhanced both TGI (58.5 %) and TGD (30.6 %) at 72 h (p = 0.049).
Notable body weight loss of 15.4 %, 13.7 %, and 11.5 %, was observed following IR, CKD-516+IR (d1), and CKD-516+IR (d1, 5) treatment, respectively. In contrast, no changes in body weight were observed in the mice treated with CKD-516 alone (Fig. 2B). After counting the number of blood vessels, we found that the number of positively CD31 stained blood vessels were significantly reduced in mice treated with CKD-516 alone (52.1 %, p < 0.001) compared to the vehicle (Fig. 2C). Mice treated with CKD-516+IR (d1) and CKD-516+IR (d1, 5) showed a 64.8 % (p < 0.005) and 59.1 % (p < 0.001) reduction in the number of blood vessels, respectively. We also analyzed tumor necrosis areas in tumor tissues stained with H&E and found that IR significantly increased necrosis by 60.4 % compared to the vehicle (p = 0.004). Interestingly, CKD-516+IR (d1) treatment induced the most extensive tumor necrosis (66.0 %, p = 0.02) compared to the vehicle (Fig. 2D). However, tumor necrosis in the CKD-516 alone or CKD-516+IR (d1, 5) groups did not differ significantly from the vehicle.
Sustained tumor necrosis and hypoxia following short-term combination treatment with CKD-516 and IR
We investigated the post-treatment effects of monotherapy and combination therapy with IR and CKD-516 on tumor necrosis and on the hypoxic tumor microenvironment. We observed the largest tumor necrosis area (%) in mice treated with IR alone (37.3 %) 24 h after treatment. However, no further changes were detected at the 72 h timepoint (37.1 %; Fig. 3A). Treatment with both CKD-516 alone and CKD-516+IR (d1) produced larger areas of tumor necrosis (41.3 %, p = 0.049 and 47.1 %, p = 0.004, respectively) after 72 h. Additionally, 24 h after the end of treatment we measured areas of hypoxia and found the following: 56.6 % in CKD-516+IR (d1), 42.0 % in CKD-516+IR (d1, 5), and 34.4 % in IR alone. After 72 h, the hypoxic areas rapidly decreased from 34.3 % to 6.9 % in mice treated with IR alone (p < 0.001). However, this increased from 56.6 % to 64.9 % in CKD-516+IR (d1) treated mice.
Expression of hypoxia-related proteins in mice following short-term combination treatment with CKD-516 and IR
We evaluated the expression of hypoxia-related proteins (HIF-1α, Glut-1, VEGF, and Ki-67), which are involved in the maintenance of the hypoxic tumor microenvironment, in mice treated with CKD-516 and IR alone, and in combination (Fig. 4A). The expression of HIF-1α, a classic marker for hypoxia, was the highest in mice treated with CKD-516 alone (57.6 %) 24 h after treatment (Fig. 4B). However, 72 h after treatment, HIF-1α expression was highest in mice treated with IR alone (68.1 %). VEGF expression increased by 34.8 % 72 h after treatment with IR alone. In CKD-516+IR (d1) treated mice, VEGF expression decreased significantly from 22.0 % to 7.0 % (p = 0.019). Glut-1 expression decreased as much as 20 – 30 % for the analyzed areas in all treatment groups 24 h after drug treatment. In mice treated with IR alone, Glut-1 expression decreased by 50.2 % from 24 h to 72 h. Additionally, Glut-1 expression was greatly reduced in the CKD-516+IR (d1) group (81 %, p = 0.004). Out of the four treatment groups, Ki-67 expression was the lowest (16.3 %) in mice treated with CKD-516 alone 24 h after drug administration. However, Ki-67 expression decreased significantly in the CKD-516+IR (d1) and CKD-516+IR (d1, 5) groups (86 %, p = 0.004 and 50.8 %, p = 0.027, respectively) 72 h post-treatment.
Delayed tumor growth after long-term combination treatment with CKD-516 and IR
We evaluated the effect on delayed tumor growth, tumor necrosis, and tumor hypoxia following short-term and long-term CKD-516 and IR combination treatment. Since weight loss and skin rash due to IR were frequently observed in short-term treatment (Additional file 2 B and C), the IR dose was lowered from 4 Gy to 2 Gy in the long-term combination treatment schedule.
We found that 24 h and 72 h after the end of treatment, IR alone decreased tumor volumes by 52 % (1.45 ± 0.36 cm3 vs. 3.02 ± 1.08 cm3) and by 56.2 % (1.58 ± 0.44 cm3 vs. 3.61 ± 1.30 cm3), respectively, compared to the vehicle. However, following treatment with CKD-516 alone tumor volumes did not differ significantly from vehicle treatment (2.73 ± 0.9 cm3 vs. 3.23 ± 1.02 cm3, respectively).
Even though CKD-516+IR did not make any significant change of tumor volume between 24 h (1.18 ± 0.04 cm3) and 72 h (1.04 ± 0.39 cm3) following the end of treatment, the tumor tended to decrease, unlike other groups. Compared to IR alone, CKD-516+IR reduced tumor volumes by 1.23-folds after 24 h and by 1.52-folds reduction after 72 h (p <0.001). Furthermore, when compared to CKD-516 alone, the combination treatment significantly reduced tumor volumes by 2.31-folds after 24 h (vs. 2.73 ± 0.9 cm3, p = 0.001) and by 3.11-folds after 72 h (vs. 3.23 ± 1.02 cm3, p < 0.001).
We found that compared to mice treated with IR or CKD-516 alone, the TGI and TGD values (%) 72 h after CKD-516+IR treatment were 1.27- and 1.93-folds higher than those of IR alone (71.2 % vs. 56.2 %, p = 0.001 and 28.2 % vs. 14.9 %, p = 0.004, respectively). Compared to mice treated with CKD-516 alone, the TGI values of CKD-516+IR at 24 h and 72 h were 6.34- and 6.78-folds higher (p = 0.002), and TGD values were 14.47- and 20.57-folds higher (p = 0.001), respectively (Table 1).
No significant differences in body weight were found between mice treated with CKD-516 alone and the vehicle group (Fig. 5B). However, both IR alone and CKD-516+IR groups showed a gradual decrease in body weight as the administration schedule progressed. After measuring the number of blood vessels 72 h after the end of treatment, we found that compared to the vehicle, CKD-516 alone, IR alone, and CKD-516+IR groups had a significantly reduced number of blood vessels (38.4 %, p = 0.003; 72.9 %, p < 0.001; and 84.2 %, p < 0.001, respectively; Fig. 5C). Conversely, the tumor necrosis area increased significantly to 67 % in the IR alone group, 82 % in the CKD-516 alone group, and 84 % in the CKD-516+IR group compared to the vehicle group (p = 0.02, p = 0.005, and p = 0.004, respectively; Fig. 5D).
Hypoxia-related protein expression in mice following long-term combination treatment with CKD-516 and IR
HIF-1α expression increased significantly by 64 % in the CKD-516 alone group (p = 0.002) and by 65 % in the CKD-516+IR group (p = 0.005) compared to the vehicle (Fig. 6A). VEGF expression in both IR alone and CKD-516 alone groups was similar to that in the vehicle group. However, VEGF expression was significantly lower (41 %, p = 0.046) in the CKD-516+IR group (Fig. 6B). Glut-1 expression was upregulated in mice treated with IR alone and CKD-516 alone. However, there were no significant changes in the CKD-516+IR group (Fig. 6C). Ki-67 expression was greatly diminished in the IR alone, CKD-516 alone, and CKD-516+IR groups (4.3 %, 4.4 %, and 5.2 %, respectively. Data not shown).