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Oncotarget, 2017, Vol. 8, (No. 41), pp: 69756-69767
Research Paper
Application of in vivo imaging techniques to monitor therapeutic
efficiency of PLX4720 in an experimental model of microsatellite
instable colorectal cancer
Sarah Rohde1, Tobias Lindner2, Stefan Polei2, Jan Stenzel2, Luise Borufka1, Sophie
Achilles1, Eric Hartmann1, Falko Lange3, Claudia Maletzki4, Michael Linnebacher4,
Änne Glass5, Sarah Marie Schwarzenböck6, Jens Kurth6, Alexander Hohn6, Brigitte
Vollmar7, Bernd Joachim Krause6 and Robert Jaster1
1
Department of Medicine II, Division of Gastroenterology, Rostock University Medical Center, Rostock, Germany
2
Core Facility Multimodal Small Animal Imaging, Rostock University Medical Center, Rostock, Germany
3
Oscar-Langendorff-Institute of Physiology, Rostock University Medical Center, Rostock, Germany
4
Molecular Oncology and Immunotherapy, Department of General Surgery, Rostock University Medical Center, Rostock,
Germany
5
Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock University Medical Center, Rostock,
Germany
6
Department of Nuclear Medicine, Rostock University Medical Center, Rostock, Germany
7
Institute of Experimental Surgery, Rostock University Medical Center, Rostock, Germany
Correspondence to: Robert Jaster, email: robert.jaster@med.uni-rostock.de
Keywords: colorectal cancer, mouse model, PLX4720, 5’-fluorouracil, in vivo imaging
Received: February 01, 2017
Accepted: June 01, 2017
Published: July 15, 2017
Copyright: Rohde et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License
3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and
source are credited.
ABSTRACT
Objectives: Patient-derived tumor cell lines are a powerful tool to analyze the
sensitivity of individual tumors to specific therapies in mice. An essential prerequisite
for such an approach are reliable quantitative techniques to monitor tumor progression
in vivo.
Methods: We have employed HROC24 cells, grown heterotopically in NMRI
Foxn1nu mice, as a model of microsatellite instable colorectal cancer to investigate the
therapeutic efficiencies of 5’-fluorouracil (5’-FU) and the mutant BRAF inhibitor PLX4720,
a vemurafenib analogue, by three independent methods: external measurement
by caliper, magnetic resonance imaging (MRI) and positron emission tomography/
computed tomography (PET/CT) with 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG).
Results: Repeated measure ANOVA by a general linear model revealed that timedependent changes of anatomic tumor volumes measured by MRI differed significantly
from those of anatomic volumes assessed by caliper and metabolic volumes determined
by PET/CT. Over the investigation period of three weeks, neither 5’-FU, PLX4720 nor a
combination of both drugs affected the tumor volumes. Also, there was no drug effect on
the apparent diffusion constant (ADC) value as detected by MRI. Interestingly, however,
PET/CT imaging showed that PLX4720-containing therapies transiently reduced the
standardized uptake value (SUV), indicating a temporary response to treatment.
Conclusions: 5’-FU and PLX4720 were largely ineffective with respect to HROC24
tumor growth. Tumoral uptake of 18F-FDG, as expressed by the SUV, proved as a
sensitive indicator of small therapeutic effects. Metabolic imaging by 18F-FDG PET/CT
is a suitable approach to detect effects of tumor-directed therapies early and even in
the absence of morphological changes.
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sensitivity of the relevant methods with respect to an early
detection of drug effects. As experimental model, we
used HROC24 cells, a patient-derived CRC cell line of
low passage number that belongs to the CRC subgroup
of microsatellite instable (MSI) tumors [3]. Microsatellite
instability (observed in roughly 13 % of all CRCs) is
caused by defective DNA mismatch repair and represents
the fundamental molecular basis of sporadic hypermutated
CRCs and hereditary non-polyposis colorectal cancer
syndrome (Lynch syndrome; also known as HNPCC)
[11–13]. In a previous in vitro study, we could show
that HROC24 cells, which express oncogenic BRAF
(V600E), are highly sensitive to the growth-inhibitory
effects of the mutant BRAF inhibitor vemurafenib [4]. We
considered this finding of worth to be followed-up since
previous experimental studies, which did not specifically
focus on the subgroup of MSI CRCs, had suggested that
vemurafenib is apparently much less efficient in BRAF
mutant CRC than in malignant melanoma [14].
Recently, a phase II study revealed that single-agent
vemurafenib did not show meaningful clinical efficacy in
patients with BRAF V600E mutant colorectal cancer [15].
As part of combination therapies, however, the concept
of targeting the BRAF pathway remains viable [2, 15].
To challenge this strategy, we compared the efficiency of
monotherapies with the vemurafenib analogue PLX4720
and the routinely used cytostatic drug 5’-fluorouracil (5’FU) [16], respectively, to the efficiency of a combination
of both drugs.
INTRODUCTION
In 2016, colorectal carcinoma (CRC) represented
the third most common cancer in females and males
and the second leading cause of cancer-related deaths in
the United States [1]. While early detection of CRC is
associated with an excellent prognosis, there is a strong
need for improved therapies for locally advanced and
metastatic stages of the disease. A key aspect in this regard
is the development of personalized treatment strategies for
individual patients that take into account (1) the molecular
heterogeneity of the disease and (2) the rapidly growing
number of targeted therapeutics and options for tumordirected immune therapies [2].
Patient-derived tumor cell lines and xenografts
provide a versatile tool to study the individual tumor
biology and to analyze, understand and potentially
predict sensitivity and resistance of tumors to specific
therapies [3–6]. A common approach to mimic in vivo
conditions takes advantage of immunodeficient mice to
establish tumors in a heterotopic or orthotopic position,
followed by the evaluation of therapeutic approaches. A
critical factor for the success of such an approach is the
availability of reliable quantitative techniques to monitor
tumor progression or regression. While histological
investigations are largely restricted to end-point analyses,
external measurements with calipers (if applicable) are
quite inaccurate and do not provide information about
the internal structure of the tumor. Therefore, there is
a special need for molecular imaging techniques that
enable repeated investigations in living animals [7].
Two essential technologies in this field are small animal
magnetic resonance imaging (MRI) and positron emission
tomography/computed tomography (PET/CT). Major
advantages of MRI are the high resolution and an excellent
tissue contrast [7]. MRI not only ensures a precise
assessment of tumor size and localization, but, through
the measurement of apparent diffusion coefficient (ADC)
values, also provides insights into the biological structure
of tumor tissue during tumor progression [8]. PET, on the
other hand, is widely recognized as a key technology to
visualize, with high sensitivity, distinct molecular target
structures of a tumor. Integrated PET/CT provides the
additional advantage of co-registered molecular and
anatomic images, thereby compensating for the relatively
poor spatial resolution of PET alone [9]. One of the most
commonly used radiopharmaceuticals, 2-deoxy-2-(18F)
fluoro-D-glucose (18F-FDG), is a marker for the uptake of
glucose, which is an important parameter for tumor tissue
metabolism [10].
In this study, we have employed small animal
multiparametric MRI and 18F-FDG PET/CT along with
external caliper measurements and end-point analyses
by histopathology to evaluate an experimental therapy
of human CRC in mice. Specifically, we were interested
in the consistency of the different types of data and the
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RESULTS
Characterization of the heterotopic tumor model
Upon injection into the flanks of NMRI Foxn1nu
mice, HROC24 cells formed macroscopic tumors within
less than two weeks. Subsequently, the mice were
randomized into four experimental groups and treated for
three weeks with PLX4720, 5’-FU, both drugs, or vehicle
only. All mice of the control cohort and all individuals
except of one of each treatment group survived throughout
the course of the study. Further details are outlined in the
“materials and methods” section.
Over the entire period of investigation, the tumors
were accessible to external measurement by caliper and
clearly detectable both by MRI and PET/CT imaging
with 18F-FDG (Figure 1 and 2). Both imaging techniques
indicated a heterogeneous structure of the tumor with vital
tumor tissue in the peripheral zone and central necrotic
areas. Haematoxylin and eosin (H&E) staining of the
tumor tissue at the time of necropsy confirmed these
findings (Figure 3A). Cells expressing the proliferation
marker Ki-67 [17] (Figure 3B) were exclusively found in
the peripheral regions of the tumors. Apoptotic cells were
in general very scarce and almost exclusively found in the
transition zone between vital and necrotic areas (Figure 4).
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Assessment of anatomic and metabolic tumor
volumes
Apparent diffusion constant (ADC) and
standardized uptake value (SUV)
Anatomic tumor volumes assessed by caliper and
MRI as well as metabolic volumes determined by PET/
CT imaging (Figure 5A-5C) were subjected to analysis
of variance by means of general linear model-repeated
measures (GLM-RP). Here and in all related analyses,
there was no significant influence of tumor location (left
or right hind flank) so that for exploratory research all
tumors of mice with the same treatment regime could be
summarized in one group.
As expected, there was a highly significant effect
of the factor “time” in a way that the tumor volumes
increased over the period of investigation (p<0.001 for any
time points). In contrast, however, no significant difference
could be detected between the four experimental groups
(p=0.564), suggesting a lack of therapeutic effects.
Interestingly, further analyses revealed significant
differences between the (larger) changes of anatomic
volumes determined by MRI on one hand and the (smaller)
changes of anatomic volumes assessed by caliper as well
as metabolic volumes determined by PET/CT imaging
on the other hand (p<0.03 each). The differences are
illustrated in Figure 6 for all experimental groups over
time. In the case of the metabolic volumes, this finding is
likely to be explained by the fact that intratumoral regions
without uptake (central necrosis) were not considered for
volume calculation. Measurements by caliper might lead
to a misjudgement of tumor growth due to an inadequate
consideration of the third dimension (tumor depth).
ADC values are indicators of the magnitude of
diffusion of water molecules within the tissue. Decreases
of the cellular density are associated with increased ADC
values and may therefore suggest a therapeutic response
[18]. In this investigation, no significant changes of the
ADC over time in the context of treatment were observed
(Figure 7).
SUVs were expressed as mean values and used
as a parameter of normalized intratumoral radioactivity
concentrations [19]. In contrast to detections of anatomic/
metabolic volumes and ADC values, measurements of
mean SUV of tumors revealed differences between the
experimental groups (Figure 8): For all three cohorts with
specific therapies, tumoral mean SUVs were significantly
lower after completion of treatment (week 3) than before
its initiation (week 0). PLX4720-containing therapies
reduced the average SUV of tumors also at earlier
time points (weeks 1 and 2). For the control group, a
diminished SUV was observed in week 2 only. Moreover,
the comparison of treatments at individual time points
indicated that application of PLX4720 (alone or combined
with 5’-FU) was associated with a significantly lower
SUV in week 1 and week 2.
Together, these 18F-FDG PET/CT findings point to
a therapeutic effect of PLX4720-containing therapies. In
line with the transient nature of this effect, histological
evaluation at the time of necropsy revealed no differences
with respect to tumor morphology and the presence of
Figure 1: MR images of a mouse from the control group. top row: representative T2 weighted transversal images (red dotted line:
right tumor volume; orange dotted line: left tumor volume, * bladder, Δ spine, ~ gut); bottom row: ADC-maps derived from DWI for four
different time points (0, 1, 2 and 3 weeks) showing heterogeneous tumors with solid tumor mass (dark areas represent tissue with low ADC
values) and pervading necrotic/cystic areas (bright areas with high ADC values).
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apoptotic cells between the experimental groups (data
not shown). Furthermore, variations of the number of Ki67-positive cells among the groups were statistically not
significant (Figure 9).
prognosis of CRC patients [22] and has also been linked
to alterations of chemosensitivity. Specifically, the benefit
of an adjuvant treatment with 5’-FU, the most widely used
chemotherapeutic agent in advanced stage CRC patients,
is a matter of ongoing discussion [23]. How the MSI
status affects the response of CRC patients to targeted
therapeutics, such as mutant BRAF inhibitors, has not
been fully evaluated yet.
We have previously shown that low-passage
HROC24 cells, established from a microsatellite instable
CRC, are highly sensitive to PLX4720 in vitro [4].
The observation prompted us to ask if these findings
can be reproduced in a preclinical setting employing
NMRI Foxn1nu mice. We therefore took advantage of
traditional and advanced technologies to monitor tumor
DISCUSSION
Inhibition of mutant BRAF by specific kinase
inhibitors has proven a successful strategy in the treatment
of malignant melanoma [20, 21]. Although oncogenic
BRAF mutations are also present in a subgroup of CRC
patients, these patients do not benefit from mutant
BRAF-directed therapies to date [15]. MSI tumors
form a subclass of CRCs that is characterized by DNA
repair defects [11–13]. MSI is associated with a better
Figure 2: Representative PET/CT images (summed images; coronal and transaxial slices) at two experimental time
points (week 0, week 3). The images were derived from the same mouse as in Figure 1: A 15 min imaging scan was performed 60 min
after intravenous injection of 18F-FDG. The flank tumors are pointed out by arrows. HU; Hounsfield units; SUV; standardized uptake value.
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of PLX4720-containing therapies (with and without 5’FU). Since these effects were missed by the volumetric
measurements, we consider the detection of normalized
intratumoral radioactivity concentrations as the most
sensitive approach, and therefore PET/CT with 18F-FDG
as the method of choice for the detection of small/transient
therapeutic effects in the context of this study. Noteworthy,
metabolic imaging by 18F-FDG PET/CT proved suitable
to detect effects of tumor-directed therapies early in
the course of treatment and even in the absence of
morphological changes. The significance of 18F-FDG PET/
CT data, especially SUV measurements, for the preclinical
evaluation of antitumor drugs should therefore be further
elucidated in follow-up studies.
progression in vivo. Accordingly, we considered the
systematic comparison of the corresponding findings
as an equally important aim of this study. The results
of volumetric measurements by caliper, MRI and PET/
CT with 18F-FDG consistently showed that neither
PLX4720 and 5’-FU alone nor the combination of both
drugs displayed a significant effect on the growth of
HROC24 tumors over the investigation period of three
weeks. These negative findings are supported by the
results of ADC measurements and end-point analyses
(histological evaluation; Ki-67-staining), which did
also not indicate differences between the experimental
groups. Interestingly, however, measurements of mean
SUVs of the tumors revealed transient therapeutic effects
Figure 3: Representative H&E (A) and Ki-67 (B) stains of tumor tissue, exemplarily shown for a mouse of the control group. (A) Viable
colorectal cancer tissue in the peripheral region surrounding a central necrosis. (B) Ki-67-positive cells with brown-stained nuclei are
present in large numbers.
Figure 4: Typical M30 CytoDEATH stains of apoptotic cells, exemplarily shown for a mouse of the control cohort.
Apoptotic cells were very scarce in the periperal zone of the tumors (A), and somewhat more frequent (but still rare) in the transition zone
to the necrotic centre of the xenografts (B). Arrows point to positive-stained cells.
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There are limitations of our investigations that
need to be mentioned. Thus, heterotopic mouse models
provide only limited insights into the biology of human
CRC. Still, on the road towards a personalized treatment
of CRC they may represent an important bridge between
mere in vitro studies and clinical trials. Furthermore,
application of PLX4720 via chow, although representing
an established procedure, does not allow for an effective
Figure 5: Assessment of anatomic and metabolic tumor volumes. Mice carrying two flank tumors of HROC24 cells were treated
with PLX4720, 5’-FU, a combination of both drugs, or served as controls (n = 8-9 mice per experimental group). At the indicated time points,
anatomic tumor volumes were assessed employing a caliper (A) and measured by MRI (B), whereas metabolic tumor volumes were determined
by PET/CT (C). One hundred percent tumor volume corresponds to the tumor size prior to the start of treatment. Data are presented as mean ±
SEM (n = 12-18 samples per time point and method; variables: number of tumors per mouse that fulfilled the inclusion criteria, survival time of
the animals and availability of evaluable data). There were no statistically significant differences between the experimental groups.
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Figure 6: Comparison of volumetric data obtained by caliper, MRI and PET/CT. The data presented in Figure 5 were used
to calculate (by GLM-RP model) estimated marginal means ± SEM of anatomic and metabolic volumes over time for each of the four
experimental groups. A value of one hundred percent corresponds to tumor volumes prior to treatment (week 0).
Figure 7: ADC values of the tumors. At the indicated time points of treatment, mice of the four experimental groups (n = 8-9 per
treatment protocol) were subjected to MRI, and ADC values of the tumors were determined. Data are presented as mean ± SEM (n = 13-18
samples per time point; variables as described in Figure 5). There were no statistically significant differences between the experimental
groups.
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Figure 8: SUV analysis. Prior to the initiation of treatment and 1-3 weeks thereafter, mice of the four experimental groups (n = 8-9 per
treatment protocol) were subjected to PET/CT, and mean SUVs of the tumors ± SEM were determined (n=12-17 samples per time point;
variables as described in Figure 5). § p<0.05 vs. week 0 (Friedman test followed by Wilcoxon tests). * p<0.05 vs. controls and # p<0.05 vs.
combination of PLX4720 + 5’-FU (same time point; Kruskal-Wallis test followed by Mann-Whitney U tests).
Figure 9: Quantification of Ki-67-positive cells. Staining and cell counting were performed as described in the “materials and
methods” section. Data are presented as numbers of Ki-67-positive cells out of 300 tumor cells (mean ± SEM; n = 15-17 samples). There
were no statistically significant differences between the experimental groups.
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control of dosage and might result in an inefficient drug
concentration in the tumor tissue. Last but not least,
restrictions of the animal model (minimum and maximum
tumor sizes) limited the duration of therapy to three weeks,
leaving the possibility of enhanced or delayed drug effects
after longer periods of treatment.
As mentioned above, the effectiveness of 5’-FU in
microsatellite instable CRC is under debate. Specifically,
a systematic review revealed no statistically significant
effect of 5’-FU for microsatellite high (MSI-H) patients
for disease-free survival or overall survival. These findings
were in sharp contrast to the results for MSI-stable CRC,
where the drug significantly improved survival rates [23].
The complete failure of 5’-FU, at the established dose of
20 mg/kg [24], to inhibit growth of HROC24 tumors was
nevertheless unexpected and remains to be addressed in
future experiments.
In summary, we suggest SUV, determined by
PET/CT with 18F-FDG, as a sensitive parameter to
monitor drug responsiveness in a preclinical model of
CRC. Measurements of anatomic volumes by MRI and
metabolic volumes by PET/CT provide non-redundant
information and may complement each other in a
meaningful manner. The difference between changes of
anatomic volumes determined by MRI and by caliper
points to a systematic problem of the latter method, which
does not accurately consider the tumor depth. Although
5’-FU and PLX4720 displayed very limited effects in this
investigation, the concept of mutant BRAF inhibition in
CRC with MSI should be challenged in follow-up studies
with additional MSI cell lines and further inhibitors of the
BRAF pathway.
made to minimize suffering. Six to eight-weeks-old female
NMRI Foxn1nu mice were injected subcutaneously into
the left and right hind flank with 5×106 HROC24 cells.
Over the entire period of investigation, tumor growth
was monitored at least three times per week by external
measurement using a caliper, and volumes of outgrowing
tumors were evaluated according to the formula: width2 ×
length × 0.52 [25, 26].
On days 11-12 after cell injections, mice were
randomized into four experimental groups of 8-9
individuals and treatment was initiated: (1) Control
mice were fed a standard rodent chow diet (Broogarden,
Lynge, Denmark). The mice also received intraperitoneal
injections of phosphate-buffered saline twice a week. (2)
Animals of the 5’-FU group were injected twice a week at
a dose of 20 mg/kg and fed the standard rodent chow diet.
(3) A third group of mice obtained PLX4720 (Plexxikon
Inc., Berkeley, California, USA) provided through ad
libitum chow at a dose of 417 mg/kg (Broogarden). (4)
Mice of the fourth group received a combined treatment
with 5’-FU and PLX4720 as described above. After 21
days of treatment, mice were sacrificed by an overdose
of ketamine/xylazin hydrochloride and tumors were
harvested for further analysis.
One mouse of the PLX4720 group had to be
euthanized on day 15 of treatment before the tumor burden
became intolerable. Furthermore, one mouse of the 5’-FU
group and one mouse of the PLX4720 + 5’-FU group had
to be sacrificed on days 10 and 17, respectively, because of
progressive weight loss. In all these cases, the data of all
available time points were included into further analyses.
In contrast, tumors of less than 5 mm in length at all time
points (including day 0) were disregarded.
MATERIALS AND METHODS
Animal PET/CT imaging
Tumor model
Small animal PET/CT imaging with 18F-FDG was
performed 1-2 days prior to the initiation of treatment
and repeated on days 6-7, 13-14 and 20-21 of therapy
(four investigations per mouse in total). For all scans,
animals were anaesthetized by isoflurane (1.5–2.5 %)
supplemented with oxygen during preparations and
imaging sessions. Mice received a mean dose of 17.50
± 1.80 MBq 18F-FDG intravenously via a microcatheter
placed in a tail vein. After an uptake period of 60 min,
mice were imaged in prone position in a preclinical PET/
CT scanner (Inveon PET/CT, Siemens Medical Solutions,
Knoxville, TN, USA) for 15 min. The animals were kept at
constant temperature of 38°C by an electrical heating pad.
In addition, respiration was monitored during the whole
imaging period. For attenuation correction and anatomical
references whole body CT scans were acquired. Each PET
data set was corrected for random coincidences, dead time,
scatter and attenuation. CT images were reconstructed
with a Feldkamp algorithm. PET data were first Fourier
rebinned into a 2D dataset from which real-space images
The MSI cell line HROC24 was established from a
primary resection specimen of a 98 years old male CRC
patient and grown in culture as described before [3, 4].
HROC24 cells are mutant for BRAF (V600E) and APC but
wild-type for TP53 and KRAS [3].
NMRI Foxn1nu mice were bred in the animal
facility of the Rostock University Medical Center and
maintained in specified pathogen-free conditions. All
experiments were performed according to the guidelines
of the local animal use and care committee, which also
approved this study (Landesamt für Landwirtschaft,
Lebensmittelsicherheit und Fischerei MecklenburgVorpommern, permit number for the study: LALLF M-V/
TSD/7221.3-1-033/14). The mice had access to water
and standard laboratory chow (as specified below) ad
libitum. All animals received humane care according to
the German legislation on protection of animals and the
Guide for the Care and Use of Laboratory Animals (NIH
publication 86–23, revised 1985), and all efforts were
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were reconstructed with an ordered subset expectation
maximization (OSEM) algorithm with 16 subsets and
4 iterations. Data were decay-corrected to the time of
injection. Metabolic volumes and SUVs were determined
using PMOD v3.7 preclinical imaging software (PMOD
Technologies, Zurich, Switzerland).
Apoptotic cells were detected employing the M30
CytoDEATH assay (Roche Diagnostics, Mannheim,
Germany), which detects the caspase cleavage product
of cytokeratin 18. Therefore, deparaffinated tumor
sections were stained following the instructions of the
manufacturer. Subsequently, the slides were counterstained
with Mayer’s hemalaun solution, dehydrated by four short
incubations in ethanol and xylene (two times each) and
embedded in Pertex (MEDITE, Burgdorf, Germany). For
the detection of proliferating cells, expression of Ki-67 was
used as a surrogate marker [17]. Therefore, deparaffinated
sections of tumor tissue were stained employing antiKi-67 antibody (eBioscience, Frankfurt, Germany) and an
avidin-biotin-peroxidase (ABC) technique [29], followed
by counterstaining with Mayer’s hemalaun solution. Ki-67
positive-stained cells were quantified by evaluating 3 x
100 nuclei, located in three representative areas along the
invasive front of the tumor, per section.
Morphological and diffusion-weighted 7 Tesla
MRI
MRI was performed on anesthetized mice (1.5–2.5
% isoflurane in oxygen) at the same time points as PET/
CT (1-2 days before and 6-7, 13-14 and 20-21 days after
the initiation of therapy; four investigations per mouse
in total). Animal respiration rate and body temperature
were monitored continuously and respiration rate was
maintained between 35 and 50 breaths/min.
MR imaging of the mice was performed using a 7
Tesla small animal MRI scanner (BioSpec 70/30, 7.0 T,
440 mT/m gradient strength, Bruker, Ettlingen, Germany)
with a 1 H transmit resonator (86 mm Resonator, Bruker,
Ettlingen, Germany) and a 2-by-2 receive-only surface coil
array (Bruker, Ettlingen, Germany) positioned on the back
of the mice. The imaging protocol included morphological
T2-weighted turboRARE and diffusion-weighted (DWI)
imaging sequences. Tumor size was assessed in high
resolution T2-weighted images of transversal plane
(repetition time: due to respiratory gating approximately
4.200 ms; echo time: 26.0 ms; field of view: 42 mm × 24
mm; matrix: 351 × 200; voxel size: (0.12 × 0.12 × 0.75)
mm3; 35 to 50 slices depending on tumor size; acquisition
time: 10 min).
The ADC value was calculated by least square
monoexponential fit of the pixel-by-pixel signal intensity
for the different b-value images of a spin echo DWIsequence (four b values, b1- b4:(100, 350, 700, 1.000)
s/mm2, one b0 image and three orthogonal gradient
directions; repetition time: 2.000 ms; echo time: 25 ms;
field of view: 42 mm × 24 mm; matrix: 192 × 109; 35-50
slices of 0.75 mm per slice in transversal plane; acquisition
time: 30-35 min, depending on tumor dimensions and
number of slices).
Images and calculated ADC-maps were analyzed
employing ITK-Snap software (Penn Image Computing
and Science Laboratory “PICSL”, University of
Pennsylvania, USA) [27]. The tumor volume and ADC
evaluation is based on slice-wise region of interest
placement. ADC calculation was performed as described
by Jung et al. 2012 [28].
Statistical analyses
All data were processed using IBM SPSS Advanced
Statistics 22.0. Values were expressed as mean ± standard
error of the mean (SEM) for the indicated number of
samples. Anatomic and metabolic tumor volumes (related
to time point 0) were analyzed and compared with repeated
measure ANOVA for (factor) “treatment” and for (withinsubjects factors) “measuring method”, “time” and “tumor
location” employing GLM-RP. Here, estimated marginal
means and their standard errors are given.
For all other data, analysis of variance was performed
employing the Kruskal-Wallis test for unpaired samples and
the Friedman test for paired data, respectively. If appropriate,
subgroups were tested pairwise using the Mann-Whitney
U test (unpaired samples) or the Wilcoxon signed rank
sum test (paired data), two-sided. Normal distribution of
measurements was checked using the Kolmogorov-Smirnov
test. P < 0.05 (Bonferroni-adjusted for multiple testing) was
considered to be statistically significant.
ACKNOWLEDGMENTS
We gratefully acknowledge the excellent technical
assistance of Mrs. Katja Bergmann, Mrs. Romina Rauer
and Mrs. Anne Möller. The Core Facility Multimodal
Small Animal Imaging of the Rostock University Medical
Center is funded by the Deutsche Forschungsgemeinschaft
and EFRE (Europäischer Fonds für regionale
Entwicklung).
We thank Plexxikon Inc. for providing us with
PLX4720.
Histology and immunohistochemistry
For histology, tumors were fixed in 4%
formaldehyde phosphate buffer overnight and processed
for paraffin embedding. Routine H&E staining for
assessment of tumor histology was performed on 4 μm
sections using standard procedures.
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CONFLICTS OF INTEREST
The authors declare that there are no conflicts of
interest.
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GRANT SUPPORT
13. Guinney J, Dienstmann R, Wang X, de Reyniès A, Schlicker
A, Soneson C, Marisa L, Roepman P, Nyamundanda G,
Angelino P, Bot BM, Morris JS, Simon IM, et al. The
consensus molecular subtypes of colorectal cancer. Nat
Med. 2015; 21:1350-1356.
This work was supported by an intramural grant of
the Rostock University Medical Center.
14. Corcoran RB, Ebi H, Turke AB, Coffee EM, Nishino M,
Cogdill AP, Brown RD, Della Pelle P, Dias-Santagata D,
Hung KE, Flaherty KT, Piris A, Wargo JA, et al. EGFRmediated re-activation of MAPK signaling contributes
to insensitivity of BRAF mutant colorectal cancers to
RAF inhibition with vemurafenib. Cancer Discov. 2012;
2:227-235.
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