17-AAG loaded PMMA bone cement inhibits tumor growth
All 18 NCG mice survived without postoperative bleeding, wound infection, or other complications. Initial tumor volumes in the control, PMMA, and 17-AAG-PMMA groups were 444.60±217.51mm3, 480.23±161.40mm3, and 442.68±139.63mm3, respectively, and did not differ significantly. After surgical intervention (Figure 1), the tumor volume was measured twice weekly for 4 weeks (Supplement Table 1). This analysis revealed gradual growth in tumor volume over time (Figure 2A). Tumor growth was slow in all groups for first 3-6 days after operation, but accelerated thereafter. Beginning from the 1st week of the operation, tumor growth rate was highest in the control group, followed by the PMMA group, while the 17-AAG-PMMA group grew slowest. From the 10th day after operation, tumor growth rate in the control group was significantly higher than in the other 2 groups, while growth rate in the PMMA and 17-AAG-PMMA groups remained almost the same 1 week after the operation. There was difference between the control group and the PMMA group, and has significant difference between the control group and the 17-AAG-PMMA group.
The Kaplan-Meier survival curves of three groups of mice were generated (Figure 2B). This analysis found that mice in the 17-AAG-PMMA group prolonged than those in the PMMA group. Euthanasia performed when the tumor exceeds 2500mm3. Log-rank test revealed statistically significant differences in survival rate among the control groups and 17-AAG-PMMA group, and there were not significant between other two groups.
17-AAG loaded PMMA bone cement affects tumor growth rate
From the 1st to 3rd day after operation, tumor growth rate in the control group was significantly higher than in the 17-AAG-PMMA group, but there was no significant difference between the control group and PMMA group. There was no significant difference in the tumor growth rate between 4-6 and 7-10 days after operation. From the 11th to 13th and 14th to 17th day after operation, tumor growth rate in control group was higher than in the PMMA and 17-AAG-PMMA group. There were no significant differences between the groups at 18-20 days after operation but at 21-24 days after operation, the tumor growth rate in control group was significantly higher than in the simple bone cement and drug-loaded bone cement group. At 25-27 days after operation, there was no significant difference in tumor growth rate among the 3 groups (p>0.05, Supplement Table 2). Overall mean tumor growth rate analysis in the 3 groups found that tumor growth rate in the 17-AAG-PMMA and PMMA group was significantly slower relative to control group (p=<0.05, Figure 2C).
Comparison of tumor necrosis area
The intact tumor was cut perpendicularly along the long axis coronal plane (Figure 2D). The ratio of tumor section necrotic area to tumor section area was calculated on Image J and found to be 16.6±12.9%, 28.9±9.8% and 34.7±11.0%, in the control, PMMA, and 17-AAG-PMMA group respectively (Figure 2E). The ratio in the control group was significantly lower than in 17-AAG-PMMA group (p=<0.05). There was no significant difference between the control and PMMA group or PMMA and 17-AAG-PMMA group. HE staining revealed uniform mononuclear or multinucleated monoclonal plasma cells (dark purple), and more necrotic tissue around the bone cement (light pink), with the necrotic area largest in the 17-AAG-PMMA group, followed by PMMA group and smallest in the control group (Figure 2F).
17-AAG loaded PMMA bone cement reduced tumor angiogenesis.
Upon tannic acid staining and perfusion, the tumor was removed and the blood vessels observed. In the 3 groups (Figure 3A-C), vascular endothelial cells in the tumor stained grayish-black, and their branches, course, and distribution were clearly visible. Growth of tumor blood vessels is vigorous, and the distribution of blood vessels at all levels was irregular. Ten visual fields were randomly taken in each group on an optical microscope and counted small arteries and veins (>100μm), microarteries and veins (10-100μm), and capillaries (<10μm) (Supplement Table 3). The total number of blood vessels in control, PMMA, and 17-AAG-PMMA group was 96.80±7.22, 82.10±6.47, 42.40±8.15 respectively. The number of capillaries was 82.10±5.09, 73.10±7.33, and 38.00±7.29, respectively. The number of microarteries and veins was 12.70±4.00, 8±1.63, and 3.30±1.49, respectively. The number of small arteries and veins was 2±1.56, 1±1.05, and 1.10±1.19, respectively (Figure 3D-E). There were differences in the total number of blood vessels between control group and 17-AAG-PMMA group. There was no difference between control group and PMMA group. There were difference in the number of capillaries and microarteries between control group and 17-AAG-PMMA group.
17-AAG loaded PMMA bone cement promotes tumor apoptosis
Immunohistochemical staining revealed that Bax, Bcl-2, and active Caspase-3 are localized in the cytoplasm of myeloma cells (Figure 4A). In each group, 12 visual fields were randomly selected under a 40× objective lens (Figure 4B). Average optical density (AOD) of the positive areas in the visual field was calculated on image J (Supplement Table 4). Bax and active Caspase-3 expression in the 17-AAG-PMMA group was significantly higher than in controls, while Bcl-2 expression was significantly lower than in the control group. Active Caspase-3 expression in the PMMA group was significantly higher than in control group. While Bcl-2 expression was significantly lower relative to the control group. Bax expression did not differ significantly between the 2 groups. Active Caspase-3 expression in 17-AAG-PMMA group was significantly higher than in the PMMA group but Bcl-2 expression in 17-AAG-PMMA group was significantly lower than in the PMMA group.
17-AAG loaded PMMA bone cement does not affect body weight and anal temperature in mice
Mouse body weight (Supplement Table 5) and anal temperature (Supplement Table 6) were measured twice weekly (Tuesday and Friday). Body weight of mice in the control, PMMA and 17-AAG-PMMA groups did not vary significantly (Figure 4C,). The mouse anal temperature was monitored (Figure 4D). There was no significant increase in anal temperature among the 3 groups, and there was no redness and swelling, no exudation, no suppuration.
17-AAG loaded PMMA bone cement does not affect organ weight
After the mice were sacrificed, we stripped mice heart, liver, spleen, lungs, bilateral kidneys, and brain. Use an electronic balance to weigh the internal organs. The weights of the heart, spleen, lung, kidney, and brain had no significantly difference between the PMMA and 17-AAG-PMMA group (Figure 4E, and Supplement Table 7)
Latex vascular perfusion
During tumor-bearing mice dissection, we observed that mouse axillary vessels sent out large branches from both sides of the head and tail of the tumor, which then enter the tumor from the periphery. These two vessels are the main blood suppliers to subcutaneous MM tumor in this model. In the branch vessels on the head side of the tumor, it was observed that a blood vessel enters the skin of the mouse’s back. In the branch vessels on the caudal side of the tumor, no branch vessels were observed entering the skin, but several tertiary vessels originating from the trunk of the branch vessels were observed to enter the tumor (Figure 5A-D).