In-Vivo Retention of 5-Fluorouracil Using 19F Magnetic Resonance Chemical Shift Imaging in Colorectal Cancer in a Murine Model

Colorectal cancer is the third leading cause of cancer death worldwide. 5-Fluorouracil (5-FU) is one of the most commonly used chemotherapies for treatment of solid tumours, including colorectal cancer. The efficacy of treatment is dependent on tumour type and can only be determined six weeks after beginning chemotherapy, with only 40–50% of patients responding positively to the 5-FU therapy. In this paper, we demonstrate the potential of using Magnetic Resonance (MR) Chemical Shift Imaging (CSI) for in-vivo monitoring of 5-FU tumor-retention in two different colorectal tumour types (HT-29 & H-508). Time curves for 5-FU signals from the liver and bladder were also acquired. We observed significant differences (p < 0.01) in 5-FU signal time dependencies for the HT-29 and H-508 tumours. Retention of 5-FU occurred in the H-508 tumour, whereas the HT-29 tumour is not expected to retain 5FU due to the observation of the negative b time constant indicating a decline in 5FU within the tumour. This study successfully demonstrates that CSI may be a useful tool for early identification of 5-FU responsive tumours based on observed tumour retention of the 5-FU.

Several previous studies using MRI were conducted using Diffusion-Weighted Imaging (DWI) for early detection of the treatment response in patients with colorectal cancer [16][17][18] . It was found that the increase in Apparent Diffusion Coefficient (ADC) of tissue water within the tumour was significantly higher in responders compared to non-responders 16 . Marugami et al. was able to distinguish responders from non-responders 9 days after the initiation of chemotherapy based on the ADC changes in liver methastasis 17 . However, patients already received two infusions of 5-FU. Similar result was obtained by Lavdas et al. 18 . Although these results can be considered as an early detection of the tumour response, there are several limitations associated with DWI. Due to power requirements, hardware limitations and other external factors DWI accuracy is limited and the image quality is low 19 . Furthermore, DWI image are often susceptible to various artifacts like ghosting, blurring, ringing, distortions etc 19,20 .
It is hypothesized that the early monitoring of 5-FU retention at the site of colorectal tumours can indicate the tumour responsiveness to 5-FU chemotherapy. 5-FU metabolizes into fluorinated nucleotides (Fnuc) and α-fluoro-β-alanine (Fbal) that each display a different chemical shift (i.e., 5-FU and its metabolites have different resonant frequencies in the 19 F magnetic resonance (MR) spectrum [21][22][23][24][25][26][27][28] . All of them can be visualized using Magnetic Resonance Imaging (MRI). Chemical-Shift Selective images of 5-FU and its metabolites have been previously acquired in a rat model [22][23][24][25] and in a mouse model [26][27][28] . The high natural abundance (approximately 100%) and large gyromagnetic ratio of Fluorine-19 ( 19 F) lead to a strong observed signal of 5-FU. 19 F MRI is a non-invasive and non-ionizing imaging technique. Another significant benefit of using 19 F MRI is the absence of fluorinated compounds in the human body, thus there is no natural background signal. These characteristics together, make 19 F MRI a promising method for monitoring 5-FU retention immediately following a single chemotherapy treatment. This would be a significant improvement to the current method for evaluating the efficacy of chemotherapy based on the observation of reduced tumour size after prescribed chemotherapy treatment.
Chemical Shift Imaging (CSI) is an extension of MR spectroscopy (MRS), allowing metabolite information to be measured. This technique has already been used for imaging the small intestines of mice with orally administrated 5-FU 28 . Also, CSI has been used for imaging the liver of patients with colorectal cancer and breast cancer 29 and to study the metabolism of 5-FU in liver metastasis 30,31 . Although the metabolism of 5-FU was studied within tumours 27,28,32 and liver 29 , the CSI imaging technique of 5-FU was not used for tumour resistivity detection. In addition, 19 F CSI of 5-FU was not implemented as a clinical diagnostic modality.
The purpose of this study was to determine if there were observable differences between the signal to noise ratio (SNR) from colorectal tumours that are insensitive (HT-29) 33,34 and sensitive (H-508) 35 to 5-FU administration as a function of time. Significant differences between SNR values allow us to infer the potential utility of 19 F CSI to detect resistance of colorectal cancer to 5-FU treatment and potentially guide clinical personalized medicine.

Results
A total of 23 mice were imaged: 9 mice with the HT-29 tumour (non-responder), 9 mice with the H-508 tumour (responder), and 5 mice with both tumour types. For each animal, the SNR from the tumour, bladder and liver voxels were calculated.
The obtained values were used for plotting the SNR time-dependence curves to detect the presence or absence of 5-FU retention in given tumours. If any organ or tumor was bigger than a single voxel, a mean value of the SNR from all voxels containing the organ or tumor was calculated and used in the subsequent analysis. Figure 1 represents the 19 F CSI images acquired at 7.5(a & b) and 40 minutes (c & d) after bolus injection, superimposed on the 1 H scans for two representative mice. HT-29 tumour cells were injected into mouse 1 (Fig. 1a,c), whereas mouse 2 ( Fig. 1b,d) received H-508 tumour cells. 19 F SNR values higher than 50 (attributed to bladder uptake) were thresholded to create necessary contrast in tumour voxels between the images acquired at 7.5 and 40 minutes after bolus injection.
The SNR of the HT-29 tumor was 9 at 7.5 minutes after injection (Fig. 1a). The SNR from the H-508 tumour voxel was 22.6 at the same time (Fig. 1b). After 40 minutes, the SNR value from the H-29 tumour decreased slightly and was equal to 7.2 ( Fig. 1c), whereas the H-508 tumour SNR increased by 36% and was equal to 30.8 (Fig. 1d).
The liver SNR from the H-508 mouse decreased gradually from 17.8 to 8.3 throughout time. However, the SNR from the liver voxels in mouse 1 was equal to approximately 11 at 7.5 minutes after bolus and 10 at 40 minutes after injection. Bladder SNR was equal to 24.7 and 25.7 for mouse 1 and mouse 2 respectively at 7.5 minutes after injection. At the end of the time period, bladder SNR was equal to 86 for the HT-29 mouse and 58 for the H-508 mouse. Figure 2 illustrates the 5-FU SNR time dependences in the tumour, liver, and bladder voxels from representative images of HT-29 (Fig. 2a) and H-508 (Fig. 2b) tumours shown in Fig. 1. The HT-29 tumour SNR decreased steadily throughout time. On the contrary, the SNR from the H-508 tumour voxel grew gradually. The bladder signal steadily increased over time. The liver signal from the HT-29 mouse slightly oscillated around the mean value, which was equal to 10. However, the SNR of the liver voxels of mouse 2 significantly grew from 2.5 to 7.5 minutes after bolus. After 7.5 minutes, the SNR declined gradually. Figure 3 shows the 19 F CSIs of mouse 3 superimposed on proton scans. This mouse had both tumour types. The HT-29 tumour was injected into the left flank of the animal whereas the H-508 colorectal adenocarcinoma was injected into the right flank. There are no liver voxels on these CSI images. 19 F SNR values higher than 50 were thresholded to visually create necessary contrast in tumor voxels between the figures acquired at different times. Figure 3a,b were acquired at 5 and 70 minutes after bolus injection. The SNRs from the HT-29 and the H-508 voxels at 5 minutes after injection were 26.6 and 12.8 respectively (Fig. 3a). After 70 minutes, the SNR from the HT-29 tumor was 3.4-fold lower than the initial value and equal to 7.9. However, SNR from the H-508 tumour was equal to 31.1 at 70 minutes after bolus. The SNR from the bladder voxels in Fig. 3a was approximately 44,  www.nature.com/scientificreports www.nature.com/scientificreports/ whereas after 70 minutes the SNR was 81.4, which is almost 2 times higher than the signal obtained at 5 minutes after the bolus (Fig. 3b). Figure 4 shows the time curve evolution of SNR from both tumour types and the bladder. The HT-29 tumour signal increased during the first 10 minutes after 5-FU injection. Nevertheless, SNR decreased steadily throughout time from 10 to 70 minutes after bolus. On the contrary, the signal from the H-508 tumor increased gradually up to 22.5 minutes. The bladder signal grew steadily throughout the first 30 minutes and then leveled. The difference between the HT-29 and the H-508 SNR time curves was statistically significant according to Wilcoxon signed rank test (p < 0.01). After 30 minutes, bladder time dependency plateaued at a value of 83 with minor oscillations.
All tumour SNR time curves were fitted using an exponential function with two fitting parameters -amplitude "a" and time constant "b" (Eq. 1). One HT-29 tumour curve was excluded from analysis due to poor goodness of fit. In one of the animals which was injected with both tumour types, the H-508 tumour was not detected. Therefore, just the HT-29 tumour curve was measured. Table 1 represents the values of the time constants "b" obtained for the HT-29 and H-508 singe-tumour mice. The values shown in Table 1 has been used for statistical   (Fig. 3a), the H-508 (right tumour) (Fig. 3b) and the bladder of representative mouse which had both tumor types. The pink and green curves correspond to the exponential fit of the HT-29 and H-508 SNR values respectively. Notice that the HT-29 (non 5-FU responsive tumour) SNR dropped gradually during time period from 10 to 70 minutes after bolus. On the contrary, the H-508 (5-FU responsive tumour) SNR grew steadily throughout the first 22.5 minutes. (2019) 9:13244 | https://doi.org/10.1038/s41598-019-49716-7 www.nature.com/scientificreports www.nature.com/scientificreports/ evaluation of the obtained results. If the mean value of the observed time constants is positive and significantly different from a zero value, the tumour can successfully retain 5FU.
The mean value + /− one standard deviation of the time constants for the HT-29 tumour in single-tumor mice was equal to -0.004 ± 0.008 min −1 . On the contrary, the mean time constant "b" for the H-508 tumour was equal to 0.018 ± 0.015 min −1 . The results are significantly different according to two-sample unpaired t-test (p < 0.01). Furthermore, according to one-sample t-test, the mean value for the H-508 tumour is significantly greater than 0 (p < 0.01). Figure 5 shows a box chart analysis of time constants from both tumour types with a significant difference of p < 0.01 between the tumour types. Based on this statistical analysis, we can conclude that 5-FU uptake of studied tumors was significantly different.
The mean values of the time constant "b" for the H-508 and HT-29 tumors of dual-tumour cohort were equal to 0.006 ± 0.009 min −1 and −0.001 ± 0.008 min −1 respectively. The difference in mean values was not statistically significant (p > 0.01). However, the Wilcoxon test showed that SNR curves of two different tumours from the dual-tumour animals were significantly different (p < 0.01).

Discussion
The results of this study illustrate the feasibility of detecting a 5-FU retention in different types of colorectal cancers using 19 F CSI imaging. Time curves of the 5-FU signal acquired following bolus injection can reveal the difference in uptake of 5-FU in different tumour types. Time curves of the 5-FU signal acquired right after a bolus injection of the chemotherapy drug had different dynamics for two different types of human colon adenocarcinoma. Therefore, detection of tumour resistance to chemotherapy based on a 5-FU retention is possible approximately one hour after bolus injection. 5-FU SNR changes with time were found to be significantly different (p < 0.01) for the HT-29 and H-508 tumour types. Increasing 5-FU SNR throughout time is characteristic of the H-508 tumour, whereas the HT-29 tumour type SNR shows a tendency to decrease or to remain constant. This result was obtained for the single-tumor mice and it was statistically significant (p < 0.01). A significant difference between time constant "b" for the dual-tumour animals was not observed (p > 0.01). It could be due to a small number of samples (n = 5).
We did not observe 5-FU "trapping phenomenon" as described by Presant during the studies timeline from both tumour types. For the H-508 type, the signal did not reach steady-state, so we were not able to estimate the half-life time for the 5-FU in the tumor. However, the retention of 5-FU in H-508 tumour was observed over the studied time course. The HT-29 tumour is not expected to retain 5-FU due to the observation of the negative b time constant indicating a decline in 5-FU within the tumour. 5-FU SNR within the bladder increased steadily from injection time to 70 minutes after treatment. Nearly all of the bladder 5-FU SNR was observed to be stronger than the tumour and liver SNRs. This finding could be due to catabolism of 5-FU in the liver. The limiting factor of 5-FU metabolism in the liver could be attributable to the activity of the enzyme dihydrouracil dehydrogenase 5,24 . Indeed, Fig. 2 illustrates the decay of the liver SNR with time. This time course corroborates the results, obtained in the rat model 32 . The control animals which received no cell injection had the same SNR dynamics for the liver and bladder. Unfortunately, we were not able to observe the signals of 5-FU metabolites such as Fbal and Fnuc. 5-FU catabolizes into Fnuc in the tumour 5,27,32 , whereas Fbal can be found in the liver, tumor, and kidneys 5,23,27,32 . According to Otake (2011), Fnuc signal was observed 1 hour after bolus and Fbal signal was detected 10 minutes after injection 32 . In that study, the authors used a 7.0T animal MRI system. The absence of a 19 F signal from the fluorinated metabolites could be due to the small concentration of these metabolites in the organs and tumours. Our study time interval was likely too short to allow for a high concentration of catabolites to be produced. Future imaging of the metabolites Fbal and Fnuc requires further parameter optimization due to the low SNR of these metabolites even 40 minutes after bolus. Additionally, to improve the sensitivity of the RF coil, a phased array coil could be used for better coupling with 19 F nuclei in the tumor and organs.
Overall, our studies demonstrate that 19 F CSI imaging can be used to detect retention of 5-Fluorouracil in a murine model of colorectal cancer. This technique may extend prediction of patient tumour resistivity to 5-FU chemotherapy based on the pharmacokinetics of 5-FU at an early treatment stage, yielding an improvement in personalized cancer therapy. Implementing innovative imaging strategies that identify patients who respond to 5-FU would increase the efficacy of the treatment by allowing targeted matching of patients and chemotherapy agents in the neoadjuvant setting. This MRI-based technique could be readily implemented clinically as a means of non-invasively monitoring early response to 5-FU chemotherapy. Identifying resistant tumours early in cancer treatment will enable patients unlikely to respond to 5-FU therapy to rapidly alter treatment plans. The use of 19 F CSI to triage patients into different chemotherapy regimens could be a significant innovation and alter routine clinical practice.

Materials and Methods
Cell lines and culture conditions. All human colon adenocarcinoma cell lines, media, sera, and culture In Vivo cell implantation and tumour growth in immunodeficient mice. This study was approved by Lakehead University Animal Care Committee, and all procedures were done in compliance with the regulations of the Canadian Counsel on Animal Care (CCAC). HT-29 and NCI-H508 cells were grown in T-75 flasks between 60-80% confluency. Cells were trypsinized with 0.25% (w/v) Trypsin −0.53 mM EDTA solution, resuspended in appropriate medium, and counted. 1 × 10 6 human colon adenocarcinoma cells were mixed with a 1:1 ratio cold Matrigel (Corning Matrigel, Fisher Scientific) for each 100 µl bolus injection into 36-40 day-old male (Nu/Nu) nude mice.
Briefly, nude mice were aestheticized using 3% isoflurane and injected with 100ul of a 1:1 cold Matrigel solution of either 1 × 10 6 HT-29 or NCI-H508 cells using a BD Eclipse 27 G × 1/2 needle. Mice were injected under the skin in the left (HT-29) or right flank (NCI-H508), weighed, labelled by ear piercing, and immediately returned to cages. Mock mice were injected similarly with 100 µl of a 1:1 ratio of appropriate cold medium and Matrigel while control mice received no injection. The H-508 tumour grew more slowly than HT-29. Therefore, the dual-tumour mice were injected with HT-29 tumor 5 days post HT-508 cells injection. A total of 23 mice were injected with either HT-29 cells (9 mice), H -508 cells (9 mice) or both adenocarcinoma types (5 mice). All animal imaging was performed 12-16 days post injection or when a caliper measurement of the volume of the tumours did not exceed either 450 ± 75 mm 3 for animals with single tumours or 600 ± 75 mm 3 in total for the dual-tumour mice. The mouse body weight was between 30-37 g.

MRI acquisition.
Prior to the MRI scanning procedure, all animals were anesthetized using 5% isoflurane oxygen mixture and anesthesia was maintained at 2% with oxygen during all subsequent experiments. Mice were catheterized using a MTV-01 tail-vein catheter (SAI Instruments). All animals received a slow bolus injection of 300 µl 5-FU (50 mg/ml) over the course of 2 minutes inside the magnet bore. During each MRI acquisition, animals were kept at 37 C° with a temperature-controlled water-filled blanket (T/Pump, Gaymar).
MRI was performed using a clinical Philips 3.0 T Achieva whole-body scanner, equipped with a custom-built dual-tuned 1 H/ 19 F quadrature birdcage coil. Proton localization was performed using a multi-slice T 1  www.nature.com/scientificreports www.nature.com/scientificreports/ which corresponds to the in-plane resolution of 0.29 mm. The total number of slices was equal to 16 for each mouse. Proton scans were used to determine the location of the tumour, bladder and liver.
After the bolus injection, 19 F CSI images were acquired for up to 70 minutes with a time step of 2 minutes and 30 seconds. Mice that had one tumour type were studied using CSI with 8 × 5 resolution, FOV of 20 × 50 mm 2 and NSA = 3, whereas mice with both tumour types were studied using 3 × 5 matrix, FOV equal to 31 × 18.6 mm 2 and NSA = 9. All CSI images were acquired using spectral bandwidth of 32 kHz (266 ppm at 3.0 T) and TR/ TE = 5000/4.27 ms. The data sampling number was 1024, yielding a spectral resolution of 0.26 ppm.
Images were analyzed using a custom imaging processing program written in MATLAB R2016b (The Mathworks, Inc, Natick, MA). Statistical analysis. All SNR time curves were fitted by the exponential function where amplitude a and time constant b were fitting parameters. The time constant determines signal dynamics. If the time constant is positive, the signal will grow with time, conversely when it is negative, signal will decrease. Thus, time constant can be used as an indicator of 5-FU kinetics. A two-sample t-test was applied for b time constant to analyze the statistical significance of the fitted results for the single tumour animals. A one-sample t-test was applied to the b time constant of each group of single tumour animals to evaluate if the mean b value of each group was significantly different from 0. A Wilcoxon signed rank test has been used to evaluate the difference between SNR time curves obtained from different tumors in dual-tumor animals. OriginPro 2016 was used to conduct statistical analysis (OriginLab Corp., Northampton, MA).