Abstract
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 40ā50% 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 (pā<ā0.01) 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.
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Introduction
Colorectal cancer is the third most common cause of cancer death worldwide according to the World Healthcare Organization1 and is the second most commonly diagnosed cancer in Canada according to the Canadian Cancer Society2. Colorectal cancer is responsible for approximately 14% of new cancer cases and 12% of cancer deaths in Canada3. According to Canadian Cancer Statistics (2016), 7% of men and 6% of women are expected to develop colorectal cancer during their lifetimes2. Moreover, an increasing incidence of colorectal cancer among young adults in Canada has been observed4.
5-Fluorouracil (5-FU) is one of the most widely used cytotoxic chemotherapies for treatment of a variety of solid tumours, including colorectal and breast cancer5,6,7,8,9. However, the treatment response rate (percentage of patients whose tumour shrinks or disappeared after treatment) for 5-FU based chemotherapy are relatively low5,10. Biochemical modulation of 5-FU has increased response rates up to 40ā50%5,11,12, while the influence on overall survival has been limited8. The improvement in overall survival was observed when 5-FU was modulated with irinotecan13,14. The current colorectal cancer mortality rates reflect ā at least in part ā resistance of individual tumours to 5-FU treatment. It has been clinically demonstrated that the ātrapping phenomenonā (the half-life of drug in the tumour is longer than 20āminutes) correlates with the clinical effectiveness of 5-FU chemotherapy15. Thus, a method to detect the responsiveness of a tumor to 5-FU in the earliest stages of treatment (i.e., sooner than six weeks) may enable effective targeting of 5-FU therapy to responsive tumours and also may prevent unnecessary exposure to cytotoxic chemotherapy in patients with 5-FU resistant tumours.
It is hoped that early identification of 5-FU resistant colorectal tumours will enable oncologists to choose treatment strategies more likely to improve patient survival and minimize unnecessary morbidity.
Several previous studies using MRI were conducted using Diffusion-Weighted Imaging (DWI) for early detection of the treatment response in patients with colorectal cancer16,17,18. 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-responders16. 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 methastasis17. 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 low19. Furthermore, DWI image are often susceptible to various artifacts like ghosting, blurring, ringing, distortions etc19,20.
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 Ī±-fluoro-Ī²-alanine (Fbal) that each display a different chemical shift (i.e., 5-FU and its metabolites have different resonant frequencies in the 19F magnetic resonance (MR) spectrum21,22,23,24,25,26,27,28. 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 model22,23,24,25 and in a mouse model26,27,28. The high natural abundance (approximately 100%) and large gyromagnetic ratio of Fluorine-19 (19F) lead to a strong observed signal of 5-FU. 19F MRI is a non-invasive and non-ionizing imaging technique. Another significant benefit of using 19F MRI is the absence of fluorinated compounds in the human body, thus there is no natural background signal. These characteristics together, make 19F 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-FU28. Also, CSI has been used for imaging the liver of patients with colorectal cancer and breast cancer29 and to study the metabolism of 5-FU in liver metastasis30,31. Although the metabolism of 5-FU was studied within tumours27,28,32 and liver29, the CSI imaging technique of 5-FU was not used for tumour resistivity detection. In addition, 19F 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 19F 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.
FigureĀ 1 represents the 19F CSI images acquired at 7.5(a & b) and 40āminutes (c & d) after bolus injection, superimposed on the 1H scans for two representative mice. HT-29 tumour cells were injected into mouse 1 (Fig.Ā 1a,c), whereas mouse 2 (Fig.Ā 1b,d) received H-508 tumour cells. 19F 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 40āminutes after bolus injection.
The SNR of the HT-29 tumor was 9 at 7.5āminutes after injection (Fig.Ā 1a). The SNR from the H-508 tumour voxel was 22.6 at the same time (Fig.Ā 1b). After 40āminutes, the SNR value from the H-29 tumour decreased slightly and was equal to 7.2 (Fig.Ā 1c), whereas the H-508 tumour SNR increased by 36% and was equal to 30.8 (Fig.Ā 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 7.5āminutes after bolus and 10 at 40āminutes after injection. Bladder SNR was equal to 24.7 and 25.7 for mouse 1 and mouse 2 respectively at 7.5āminutes 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.
FigureĀ 2 illustrates the 5-FU SNR time dependences in the tumour, liver, and bladder voxels from representative images of HTā29 (Fig.Ā 2a) and H-508 (Fig.Ā 2b) tumours shown in Fig.Ā 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 7.5āminutes after bolus. After 7.5āminutes, the SNR declined gradually.
FigureĀ 3 shows the 19F 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. 19F SNR values higher than 50 were thresholded to visually create necessary contrast in tumor voxels between the figures acquired at different times.
FigureĀ 3a,b were acquired at 5 and 70āminutes after bolus injection. The SNRs from the HT-29 and the H-508 voxels at 5āminutes after injection were 26.6 and 12.8 respectively (Fig.Ā 3a). After 70āminutes, 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 70āminutes after bolus. The SNR from the bladder voxels in Fig.Ā 3a was approximately 44, whereas after 70āminutes the SNR was 81.4, which is almost 2 times higher than the signal obtained at 5āminutes after the bolus (Fig.Ā 3b).
FigureĀ 4 shows the time curve evolution of SNR from both tumour types and the bladder. The HT-29 tumour signal increased during the first 10āminutes after 5-FU injection. Nevertheless, SNR decreased steadily throughout time from 10 to 70āminutes after bolus. On the contrary, the signal from the H-508 tumor increased gradually up to 22.5āminutes. The bladder signal grew steadily throughout the first 30āminutes 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 (pā<ā0.01). After 30āminutes, 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 ā amplitude āaā and time constant ābā (Eq.Ā 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. TableĀ 1 represents the values of the time constants ābā obtained for the HT-29 and H-508 singe-tumour mice. The values shown in TableĀ 1 has been used for statistical 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 valueā+ā/ā one standard deviation of the time constants for the HT-29 tumour in single-tumor mice was equal to -0.004āĀ±ā0.008āminā1. On the contrary, the mean time constant ābā for the H-508 tumour was equal to 0.018āĀ±ā0.015āminā1. The results are significantly different according to two-sample unpaired t-test (pā<ā0.01). Furthermore, according to one-sample t-test, the mean value for the H-508 tumour is significantly greater than 0 (pā<ā0.01). FigureĀ 5 shows a box chart analysis of time constants from both tumour types with a significant difference of pā<ā0.01 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 ābā for the H-508 and HT-29 tumors of dual-tumour cohort were equal to 0.006āĀ±ā0.009āminā1 and ā0.001āĀ±ā0.008āminā1 respectively. The difference in mean values was not statistically significant (pā>ā0.01). However, the Wilcoxon test showed that SNR curves of two different tumours from the dual-tumour animals were significantly different (pā<ā0.01).
Discussion
The results of this study illustrate the feasibility of detecting a 5-FU retention in different types of colorectal cancers using 19F 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 (pā<ā0.01) 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 (pā<ā0.01). A significant difference between time constant ābā for the dual-tumour animals was not observed (pā>ā0.01). It could be due to a small number of samples (nā=ā5). These results are consistent with the literature which shows that the HT-29 tumor demonstrates resistivity to 5-FU therapy while the H-508 tumor can be treated using 5-FU33,34,35.
We did not observe 5-FU ātrapping phenomenonā 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 70āminutes 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 dehydrogenase5,24. Indeed, Fig.Ā 2 illustrates the decay of the liver SNR with time. This time course corroborates the results, obtained in the rat model32. 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 tumour5,27,32, whereas Fbal can be found in the liver, tumor, and kidneys5,23,27,32. According to Otake (2011), Fnuc signal was observed 1āhour after bolus and Fbal signal was detected 10āminutes after injection32. In that study, the authors used a 7.0T animal MRI system. The absence of a 19F 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 40āminutes after bolus. Additionally, to improve the sensitivity of the RF coil, a phased array coil could be used for better coupling with 19F nuclei in the tumor and organs.
Overall, our studies demonstrate that 19F 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 19F 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 reagents were obtained from ATCC (Burlington, ON, Canada), Life Technologies (Burlington, Ontario, Canada), Becton Dickinson (St. Laurent, Quebec, Canada) or Sigma (St. Louis, MO). HT-29 cells were grown in McCoyās 5āA medium and NCI-H508 were grown in RPMI-1640 medium, with both cell lines supplemented with 10% FBS, 100U/ml Penicillin, 100āĀµg/ml streptomycin, and 2āmM glutamine. Cells were grown to 50ā75% confluency in T-75 flasks prior to injection into animals.
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 60ā80% confluency. Cells were trypsinized with 0.25% (w/v) Trypsin ā0.53āmM EDTA solution, resuspended in appropriate medium, and counted. 1āĆā106 human colon adenocarcinoma cells were mixed with a 1:1 ratio cold Matrigel (Corning Matrigel, Fisher Scientific) for each 100āĀµl bolus injection into 36ā40 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 1āĆā106 HT-29 or NCI-H508 cells using a BD Eclipse 27āG Ć 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 100āĀµl 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 ā 508 cells (9 mice) or both adenocarcinoma types (5 mice). All animal imaging was performed 12ā16 days post injection or when a caliper measurement of the volume of the tumours did not exceed either 450āĀ±ā75āmm3 for animals with single tumours or 600āĀ±ā75āmm3 in total for the dual-tumour mice. The mouse body weight was between 30ā37āg.
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 300āĀµl 5-FU (50āmg/ml) over the course of 2āminutes inside the magnet bore. During each MRI acquisition, animals were kept at 37āCĀ° with a temperature-controlled water-filled blanket (T/Pump, Gaymar).
MRI was performed using a clinical Philips 3.0āT Achieva whole-body scanner, equipped with a custom-built dual-tuned 1H/19F quadrature birdcage coil. Proton localization was performed using a multi-slice T1-weighted Turbo Spin Echo (TSE) pulse sequence with a Field of View (FOV) of 75āĆā75 mm2, TR/TEā=ā2000/55.19āms, slice thickness of 2āmm and Number of Signal Averages (NSA)ā=ā3. Acquisition matrix size was equal to 256āĆā256 which corresponds to the in-plane resolution of 0.29āmm. 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, 19F CSI images were acquired for up to 70āminutes with a time step of 2āminutes and 30āseconds. Mice that had one tumour type were studied using CSI with 8āĆā5 resolution, FOV of 20āĆā50 mm2 and NSAā=ā3, whereas mice with both tumour types were studied using 3āĆā5 matrix, FOV equal to 31āĆā18.6 mm2 and NSAā=ā9. All CSI images were acquired using spectral bandwidth of 32ākHz (266 ppm at 3.0āT) and TR/TEā=ā5000/4.27āms. 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).
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Acknowledgements
This work was supported by a grant from the Northern Ontario Academic Medicine Association (NOAMA). Lakehead University and Thunder Bay Regional Health Research Institute provided partial support and access to their facilities. We acknowledge Iain Ball and Peter Smylie for their contributions to the initial phases of this research. Yurii Shepelytskyi was supported by Ontario Graduate Scholarship.
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Y.S. and M.S.F. contributed equally to this work. Y.S. contributed to image reconstruction, data postprocessing and statistical analysis. M.S.F. contributed to data collection and experimental design. K.D. contributed to cell culturing and tumor growth as well as animal preparation, and experimental design. T.L. contributed to experimental design and data collection. M.S.A. and E.D. contributed to experimental design. All authors contributed to data analysis, manuscript writing and manuscript editing.
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Shepelytskyi, Y., Fox, M.S., Davenport, K. et al. In-Vivo Retention of 5-Fluorouracil Using 19F Magnetic Resonance Chemical Shift Imaging in Colorectal Cancer in a Murine Model. Sci Rep 9, 13244 (2019). https://doi.org/10.1038/s41598-019-49716-7
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DOI: https://doi.org/10.1038/s41598-019-49716-7
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