Growth patterns of NPC animal models
We found that tumours from cell line models: C17, C666-1 and NPC43 started to grow as early as week2 post implantation, while tumours from fragment implantation, Xeno76 and Xeno23, needed a relative longer time to initiate growth and to reach humane endpoint. To compare among models, a 4-week observation period was selected for each model when tumours reached a comparable size (~ 100 - 200mm3): week2 to week5 for C17/C666-1/NPC43/Xeno76; and week11 and week14 for slow growing model Xeno23. Within the observation period, all five models attained varying tumour volumes (Fig. 1a and b). Notably, tumours from NPC cell lines (C17 and C666-1) generally were found to have a faster growth rate and shorter doubling time compared with tumours grown from tumour fragment implantation (Xeno76 and Xeno23). C666-1 had the fastest tumour growth rate of 12.7 ± 0.4 (%/day) and tumour doubling time of 5.5 ± 0.2 days (Table 1), resulting in tumour oversize at a relative early time point. Xeno76 had a tumour growth rate of 7.3 ± 0.8 (%/day) and tumour doubling time of 10.1 ± 1.5 days. Xeno23 was the slowest in tumour growth, with a rate of 2.9 ± 0.6 (%/day) and tumour doubling time of 27.7 ± 4.8 days. After 3-4 weeks, the individual difference of tumour volume were relatively large within C17 and C666-1: ranging from 500-1100 mm3 for C17 and 600-900 mm3 for C666-1. However, this did not lead to a big standard deviation in tumour growth rate when fitting into the exponential growth curve, suggesting that the growth pattern is relative stable and distinct for each xenograft model.
Repeatability of [18F]FDG microPET/MR imaging system for SUV measurement
To investigate the metabolic activity of NPC xenograft models, we first tested the repeatability of the microPET/MR system. The glucose uptake of liver in mice was reported stable upon fasting condition in several studies [27, 28] and SUVmean_liver has been used as the background to normalize radiotracer accumulation . Thus, in our study SUVmean_liver was used to examine the repeatability of the microPET/MR imaging system. We analyzed the initial liver uptake and endpoint liver uptake for all five mice models and a total of 50 data points were included in the analysis. Bland-Altman plot (Fig. 2) showed mean difference between the two timepoints was 0.0196 (95% limit of agreement: -0.099 - 0.14). Coefficient of variation (CoV) for SUVmean_liver between the initial and endpoint was 6.95% ± 5.02%. All the data points were within the agreement interval. No significant differences were observed between the initial and endpoint liver uptake. Results show an excellent consistency of liver SUVmean, suggesting the microPET/MR imaging system is stable and able to produce repeatable results.
Metabolic patterns of NPC animal models
We monitored the longitudinal changes in tumour uptake and found highly variable tumour metabolic patterns across tumour types. SUVRmean and SUVRmax of C17 showed a continuous increasing pattern, while C666-1 showed a decreasing pattern (Fig. 3a) attributed to extensive necrosis confirmed by histology at the endpoint. For Xeno76, NPC43 and Xeno23, there were no obvious overall changes in tumour metabolism over time. To further investigate whether tumour metabolism may change in a later timepoint, we continued to monitor tumour uptake of Xeno76 till week8 when the mice reached humane endpoint, still we did not observe any significant changes in the tumour uptake. After 4-week observation, C17 has the highest SUVRmean (3.19 ± 0.92) and Xeno76 is the lowest (1.57 ± 0.28). There was a significant difference between C17 and Xeno76 in SUVRmean (p = 0.0476), suggesting the metabolic heterogeneity across NPC xenograft models. We also compared the change of SUVRmean over the 4-week period (Fig. 3b) and found good correlation between growth rate and absolute change in SUVRmean (Fig. 3c, r = 0.6340, p = 0.0009), regardless of tumour types and metabolic patterns. As expected, there was a larger standard deviation in SUVRmax than SUVRmean values due to the inherent characteristic of SUVmax compared to SUVmean measurements (Fig. 3b and d).
Relation between PET, H&E and Ki-67
To compare PET images and autoradiography with H&E staining, tumour was extracted at the endpoint and tumour slices went through histological analysis (Fig. 4a, b and c). The low uptake region shown on the PET images and autoradiography image was confirmed to be necrotic region by H&E staining (Fig. 4d). We then performed a correlation between tumour SUV and the area of the non-necrotic region represented by H&E staining, across all tumour types. Strong positive correlation was found between SUVRmean and the area of the non-necrotic region (Fig. 4e).Notably, this result revealed that the presence of necrotic regions is a cause of reduced overall [18F]FDG uptake in the tumour.
In addition, we analyzed the expression level of cell proliferation marker Ki-67 at the endpoint. Proliferating cells mainly accumulated in the outer layer of the tumours, which was consistent with the distribution of higher glycolytic regions on the PET images (Fig. 4a and c). Diverse expression levels of Ki-67 were found among xenograft models. The expression level of Ki-67 was the highest in C17 and the lowest in C666-1 (Fig. 4f).
In summary, we found that C17 has an optimal growth rate, high SUVRmean, and this result was consistent with histology, which is high Ki-67 level and little necrosis confirmed by H&E. C666-1 has a fast growth rate, relatively low Ki-67 level, low SUVRmean and much necrosis confirmed by H&E. NPC43 and Xeno76 have slow growth rates and also low Ki-67 level and SUVRmean, as well as extensive necrosis confirmed by H&E. Xeno23 has the slowest growth rate among these models, but it has a relative high Ki-67 level and high SUVRmean. Notably, C17 has a much higher endpoint SUVRmean compared to the other models which had similar SUVRmean (Fig. 4g).
As a proof of concept, we applied standard chemotherapy for NPC, cisplatin, and evaluated tumour growth rate, metabolism and cell proliferation marker Ki-67 before and after the treatment. Based on our findings of the NPC xenograft models, we selected C17 as the optimal tumour model for our purpose due to the satisfactory growth rate and high tumour metabolic activity which we observed over the 4-week period. 4 mg/kg cisplatin was administered for consecutive 4 weeks. The result of cisplatin treatment is shown in Fig. 5. During the treatment period, we observed a siginificant tumour inhibition effect by cisplatin. Statistical analysis showed significant differences in tumour volume (Fig. 5a and b) and SUVmean (Fig. 5c) between treatment group and vehicle group. Immunohistochemistry staining also confirmed the results. Our results showed a remarkably lower expression level of Ki-67 in cisplatin treated group, suggesting that tumour cells proliferation have been suppressed by cisplatin (Fig. 5d, e and f). Imaging using microPET/MR was able to demonstrate the metabolic and anatomical outcome of cisplatin treatment on the NPC model.