Published OnlineFirst May 22, 2017; DOI: 10.1158/1535-7163.
MCT-16-0793
Models and Technologies
Metformin Inhibits Cellular Proliferation and
Bioenergetics in Colorectal Cancer Patient–
Derived Xenografts
Nur-Afidah Mohamed Suhaimi1, Wai Min Phyo1, Hao Yun Yap1, Sharon Heng Yee Choy2,
Xiaona Wei1, Yukti Choudhury1, Wai Jin Tan1, Luke Anthony Peng Yee Tan1,
Roger Sik Yin Foo3,4, Suzanne Hui San Tan3, Zenia Tiang3, Chin Fong Wong5,
Poh Koon Koh6, and Min-Han Tan1,6,7
Abstract
There is increasing preclinical evidence suggesting that met-
formin, an antidiabetic drug, has anticancer properties against
various malignancies, including colorectal cancer. However, the
majority of evidence, which was derived from cancer cell lines
and xenografts, was likely to overestimate the benefit of met-
formin because these models are inadequate and require supra-
physiologic levels of metformin. Here, we generated patient-
derived xenograft (PDX) lines from 2 colorectal cancer patients
to assess the properties of metformin and 5-fluorouracil (5-FU),
the first-line drug treatment for colorectal cancer. Metformin
(150 mg/kg) as a single agent inhibits the growth of both PDX
tumors by at least 50% (P < 0.05) when administered orally for
24 days. In one of the PDX models, metformin given concur-
rently with 5-FU (25 mg/kg) leads to an 85% (P ¼ 0.054)
Introduction
Anticancer drug development is often impeded by a lack of
suitable preclinical models that are highly predictive of therapeu-
tic efficacy in humans. Most studies utilizing cell lines and
xenografts are useful in the laboratory setting, but may not
translate well to the clinical setting (1). The failure of many cell
line xenografts to accurately predict the clinical efficacy of anti-
cancer agents is due to their inability to reflect the complexity and
heterogeneity of human tumors (2). Substantial genetic diver-
gence exists between primary tumor and the corresponding cell
line derived from that tumor, as compared with a direct transplant
of tumor into mice (3–5). Patient-derived xenografts (PDX), in
1
Institute of Bioengineering and Nanotechnology Singapore, Singapore. 2Bio-
logical Resource Centre Singapore, Singapore. 3Genome Institute of Singapore,
Singapore. 4Cardiovascular Research Institute, National University Health Sys-
tems, Singapore. 5Tan Tock Seng Hospital Singapore, Singapore. 6Concord
Cancer Hospital Singapore, Singapore. 7National Cancer Centre Singapore,
Singapore.
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
Corresponding Author: Min-Han Tan, Institute of Bioengineering and Nano-
technology, 31 Biopolis Way, The Nanos #09-01, Singapore 138669. Phone: 65-
68247110; Fax: 65-64789080; E-mail: mhtan@ibn.a-star.edu.sg
doi: 10.1158/1535-7163.MCT-16-0793
2017 American Association for Cancer Research.
www.aacrjournals.org
Downloaded from mct.aacrjournals.org on May 27, 2021. © 2017 American Association for Cancer Research.
Molecular
Cancer
Therapeutics
growth inhibition. Ex vivo culture of organoids generated from
PDX demonstrates that metformin inhibits growth by executing
metabolic changes to decrease oxygen consumption and acti-
vating AMPK-mediated pathways. In addition, we also per-
formed genetic characterizations of serial PDX samples with
corresponding parental tissues from patients using next-gener-
ation sequencing (NGS). Our pilot NGS study demonstrates
that PDX represents a useful platform for analysis in cancer
research because it demonstrates high fidelity with parental
tumor. Furthermore, NGS analysis of PDX may be useful to
determine genetic identifiers of drug response. This is the first
preclinical study using PDX and PDX-derived organoids
to investigate the efficacy of metformin in colorectal cancer.
Mol Cancer Ther; 16(9); 2035–44. 2017 AACR.
which patient-derived tumor fragments are directly implanted
into immunocompromised mice through serial passaging, are an
increasingly recognized tool for drug candidate evaluation. These
PDXs are unlikely to have undergone extensive selection pressure
typically associated with cell line establishment as manifested
through genetic and molecular changes (3), and are thus valuable
for drug studies.
We evaluated the performance of 5-fluorouracil (5-FU), the
first-line treatment for colorectal cancer (6), along with the
antidiabetic drug metformin. Epidemiologic studies have sug-
gested the beneficial effects of metformin in cancer treatment
among diabetic patients (7, 8). This was supported by preclin-
ical studies, which demonstrate that metformin has antineo-
plastic activity in breast, colon, lung, and prostate cancer
(9–14), spurring significant interest to repurpose metformin
as an anticancer agent. Nonetheless, preclinical studies often
involve supraphysiologic concentrations of metformin that are
in excess of the therapeutic levels achieved in patients. In
humans, the initial dose of metformin is 500 to 1,000 mg/
daily, and subsequently, a maintenance dose of 2,000 mg/daily
is required. Studies performed on cell lines require elevated
doses of metformin (2–50 mmol/L) to elicit cellular response,
while in vivo studies require up to 4 times the maximum clinical
dose in humans of 2,550 mg/day (15). Thus, it is anticipated
that good relevant models are necessary for physiologic model-
ing of metformin.
To the best of our knowledge, this is the first study evaluating
the use of metformin on colorectal cancer PDX, a highly
2035
 Published OnlineFirst May 22, 2017; DOI: 10.1158/1535-7163.MCT-16-0793
Mohamed Suhaimi et al.
relevant system for drug candidate testing. Using next-genera-
tion sequencing (NGS), we interrogated the molecular profiles
of these PDX to evaluate selection pressures initiated during the
course of drug treatment. In addition, we aimed to identify
molecular signatures that influence response to metformin. In
parallel, we utilized colorectal cancer organoids to elucidate the
mechanistic effect of metformin.
Materials and Methods
Cell culture maintenance, proliferation, and clonogenic assays
Cell lines were obtained in 2012 from ATCC directly while
isogenic lines HCT116 p53þ/þ (wild-type, WT) and p53/
(knockout, KO) were kindly provided by Dr. Bert Vogelstein
(John Hopkins University, MD, USA). Independent authentica-
tion of cells was not performed. Cells were cultured in DMEM with
10% fetal bovine serum and maintained in 37 C incubator
supplemented with 5% CO2. The MycoAlert Mycoplasma Detec-
tion Kit (Lonza) was used to test for Mycoplasma contamination at
defined intervals, and the tests indicate that cell lines were free of
Mycoplasma throughout the study.
The CellTiter96 AQueous One Solution Assay (Promega) was
used to assess proliferation of cells incubated with metformin
(0–20 mmol/L) and/or 5-FU (10 mmol/L) in the presence of
high (4,500 mg/L) or low (1,000 mg/L) glucose. For clonogenic
assay, cells harvested from exponential phase cultures were
plated in six-well plates. Seeding densities varied from 100 to
300 cells per well. After 7 days, cells were washed with PBS,
fixed with methanol and stained with Coomassie blue. Assays
were done in triplicate. Colonies were counted by two inde-
pendent investigators.
Microsatellite instability (MSI) analysis
MSI was determined using the MSI Analysis Kit version 1.2
(Promega). Briefly, DNA from matched normal and tumor
specimens was amplified using a panel of markers designed
to amplify targeted microsatellite regions (BAT-25, BAT-26,
NR-21, NR-24, and MONO-27). Fragment analysis was per-
formed to assess the size of microsatellite repeats and deter-
mine MSI status.
Development of PDX
Fresh colorectal tumors were obtained from consenting
patients at Concord Cancer Hospital in accordance with pro-
tocols approved by the Institutional Review Board. Tumors
were transplanted into severe combined immunodeficient
(SCID) female mice aged 4 to 8 weeks (InVivos Singapore)
under the approved protocol by the Institutional Animal Care
and Use Committee. Tumor specimens were cut into 2 to 4
mm3 pieces and implanted subcutaneously in the left flank of
the mice. PDX was maintained by passaging into subsequent
generations when tumor volumes reached approximately 1,000
to 1,500 mm3. For treatment studies, tumors were expanded in
the left flanks of 5 to 12 mice (at least 5 evaluable tumors per
arm). Mice were grouped into three arms: (i) control, (ii)
metformin, or (iii) metformin and 5-FU when tumor volumes
reached 100 to 200 mm3. Mice were treated daily with met-
formin dissolved in drinking water (150 mg/kg, orally) and/or
weekly with 5-FU (25 mg/kg, intraperitoneally) for 24 days.
Mice were monitored daily for signs of toxicity and tumor size
was evaluated twice weekly by caliper measurements using the
following formula: tumor volume in mm3 ¼ [length  width2]/2.
2036 Mol Cancer Ther; 16(9) September 2017
Downloaded from mct.aacrjournals.org on May 27, 2021. © 2017 American Association for Cancer Research.
Tumor growth inhibition (TGI) was calculated as shown: TGI ¼
[(tumor volume of control on day 24  tumor volume of treated
on day 24)/(tumor volume of control on day 24  tumor volume
of control on day 0)]  100.
At endpoint, tumors were harvested. A portion of each tumor
was fixed for histologic sections and hematoxylin and eosin
(H&E) staining, while the remaining portions were snap-frozen
or used for organoids derivation.
Organoids derivation and cellular respiration assay
Tumors from control mice were used for ex vivo measurement of
cellular respiration. After removal of necrotic tissue, tumors were
washed in HBSS, minced and digested in digestion media (RPMI
media with 5X Penicillin-Streptomycin-Amphotericin B, 10
mmol/L HEPES, 300 U/mL Collagenase, 100 U/mL Hyaluroni-
dase, and 10 mmol/L Y-27632) for 2 hours at 37 C. Digestion
media were removed and digested tissues were resuspended in
culture media (Advanced DMEM/F12 with 5 Penicillin–Strep-
tomycin–Amphotericin B, 1 Glutamax, 1 B27, 1 N-2,
1 mmol/L N-Acetylcysteine, 50 ng/mL rhEGF, and 10 mmol/L
Y-27632), and filtered through 100- and 40-mm filter. Organoids
were collected from the 40- to 100-mm fraction, and the numbers
were estimated.
Oxygen consumption rate (OCR) of organoids were measured
using Seahorse Bioscience XF96 analyzer as described previously
(16). Five hundred organoids were seeded overnight in a 96-well
cartridge and pretreated with 0.1 mmol/L or 1.0 mmol/L met-
formin for 24 hours. Before measurement, the cells were washed
and medium was replaced. Reagents from the Cell Mito Stress Kit
(Seahorse Bioscience) were injected into each well sequentially
and serve as control.
Next-generation sequencing
A customized Ion AmpliSeqColon Panel was designed to
amplify 513 regions covering 40 genes implicated in colorectal
cancer (Supplementary Table S1). Briefly, the customized panel
was designed based on the multidimensional analysis of genomic
profiles in colorectal cancer published by The Cancer Genome
Atlas Network (17) and molecular subtype signatures (18–20).
Hotspot mutations in genes of interest and other genes related to
probable actionable drug targets were identified and ranked. Ten
nanograms of DNA was used to generate libraries using the Ion
Ampliseq library preparation kit v2.0. The barcoded libraries were
diluted to 7 pmol/L for template preparation using the OneTouch
2 instrument and Ion PGMTemplate OT2 200 Kit. The resulting
pooled libraries were quality control checked using the Ion Sphere
quality control kit, and were then sequenced on the Ion Torrent
PGM using a PGM 200 sequencing kit v2 and 318 Chip v2 (Life
Technologies).
NGS variant analysis
Point mutations were identified with the Torrent Suite Software
v3.0 and the Ion Torrent Variant Caller v4.0 Plugin using the
somatic high stringency parameters and the targeted and hotspot
pipelines. A 5% allele frequency threshold and 500 minimum
coverage was set to report de novo mutations. All the variants
identified were established by visualizing the data through IGV
2.3 (Broad Institute). Concurrently, orthogonal verification of
variant calls made was performed using the iCMDB (Vishuo
Biomedical) platform which provides variant annotation, as well
as Sanger sequencing for variant validation.
Molecular Cancer Therapeutics
 Published OnlineFirst May 22, 2017; DOI: 10.1158/1535-7163.MCT-16-0793
Western blotting
One hundred milligrams of snap-frozen tissues was lysed in
RIPA buffer containing protease and phosphatase inhibitors.
Protein extracts (50 mg) were electrophoresed on 4% to 12%
Bis-Tris gels and transferred to PVDF membranes. Membranes
were blocked with 5% bovine serum albumin in Tris-buffered
saline and incubated overnight at 4 C with 1:1,000 dilutions of
anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-
p70S6K (Thr389), anti-p70S6K, anti-phospho-AMPKa (Thr172),
and anti-AMPK antibodies (Thermofisher). Membranes were then
washed and incubated with a 1:2,000 dilution of horseradish
peroxidase-conjugated goat anti-rabbit secondary antibody.
Immunoreactive bands were detected by chemiluminescence
using the ECL Prime Western Blotting Kit (GE Healthcare).
Intensity of each immunoreactive band was quantified by den-
sitometry using Image J software, and expressed relative to the
control mice after normalization to ß-actin.
Statistical analysis
Sample size determination for in vivo studies was performed
using preliminary data generated from cell line xenografts a priori.
Briefly, the number of samples required per treatment group is 4,
at 95% confidence level and 80% power, for a desired effect size of
at least 1,000 mm3. For all experiments, Mann–Whitney and
Kruskal–Wallis tests were used to determine the differences
between two groups, or three and more groups, respectively. P
values <0.05 were recognized as statistically significant.
Results
Growth inhibition of 5-FU and metformin on cancer cell lines
Metformin has been reported to preferentially inhibit the
growth of p53-mutant cells by forcing a metabolic burden that
result in selective toxicity (9, 21, 22). However, recent evidence
suggests that metformin requires p53 activity for metformin-
induced growth inhibition (23, 24). To investigate whether met-
formin affects tumor proliferation in a p53-dependent manner, we
screened a panel of colorectal cancer cell lines with known p53
statuses (Table 1), including a pair of isogenic cell lines HCT-116
(p53-WT and p53-KO). Increasing concentration of 5-FU at micro-
molar levels results in a growth inhibition of all colorectal cancer
cells (Fig. 1A) while supraphysiologic concentrations of metfor-
min were required to achieve 50% cell growth inhibition (relative
to untreated cells) at 48 hours (Fig. 1B).
Under glucose-deprived conditions, the degree of growth inhi-
bition by metformin is higher in colorectal cancer lines (Fig. 1C).
There was no differential growth inhibition in response to 5-FU
(Fig. 1D) and metformin between the paired isogenic p53 lines
(P ¼ 0.206) as was previously reported, although cells with p53-
WT were inhibited to a greater extent under low glucose condi-
tions (P ¼ 0.06; Fig. 1E). The clonogenic ability of p53-WT and
p53-KO cells were similar upon metformin treatment (P ¼ 0.743);
nonetheless, cell growth for both lines were severely impaired
under glucose-deprived conditions (Fig. 1C and D).
Approximately 15% of colorectal cancers display microsatellite
instability and are classified as microsatellite instable (MSI) or
microsatellite stable (MSS). Characterization of colorectal cancer
cells for MSI/MSS status indicates that there was no difference in
response to metformin between MSI and MSS cell lines. Essen-
tially, our results indicate that there is non-differential growth
inhibition to metformin, regardless of microsatellite or p53 status.
www.aacrjournals.org
Downloaded from mct.aacrjournals.org on May 27, 2021. © 2017 American Association for Cancer Research.
Metformin Inhibits Growth of Colorectal Cancer PDX
Table 1. p53 and microsatellite status of cell lines and PDX
Sample p53 status MSI/MSS
DLD-1 Mutant MSI
HCT116 Wild-type MSS
SK-CO-1 Wild-type MSS
SW-480 Mutant MSS
PDX FIT-CRC-086 Mutant MSS
PDX FIT-CRC-104 Wild-type MSI
PDX generation
Two primary colorectal cancer tumors, FIT-CRC-086 and
FIT-CRC-104, with different clinicopathologic characteristics
(Table 2), were engrafted into 4 to 6 weeks old female SCID
mice. Growth rates ranged from 8 to 15 weeks for the initial
transplantation to reach a tumor volume of 1,000 to 1,500
mm3, and 3 to 8 weeks for subsequent passages. Histopathol-
ogy of patient's primary tumors and various xenograft passages
showed that the PDX models retained the original morphology
of the human primary tumor (Fig. 2A).
Non-differential inhibition of metformin on PDX
To evaluate tumor response in vivo, we utilized two PDX models
with differing p53 and MSI/MSS status (Table 1). Mice treated with
metformin had a lower tumor burden as compared with control
group, regardless of p53 nor microsatellite status (Fig. 2B–D).
Metformin was able to slow tumor growth initially, but at 12 days
posttreatment initiation, PDX tumors eventually progressed. After
24 days of drug treatment, the average tumor volume of the
metformin-treated group was reduced by 50% (P ¼ 0.056) and
65% (P < 0.05) for p53-wild-type and p53-mutant PDX, respec-
tively. A systemic effect of metformin on cell growth was observed
in the PDXs independent of tumor p53 status, with more pro-
nounced growth inhibition being observed in the PDXs with
mutant-p53.
When metformin was administered concurrently with a clin-
ically acceptable dose of 5-FU, only an additional 4% (P ¼ 0.767)
tumor inhibition was observed in PDX-FIT-CRC-104; in contrast,
a pronounced tumor growth inhibition of 85% (P ¼ 0.054) was
observed in PDX-FIT-CRC-086.
Metformin activates the AMPK pathway
To determine whether metformin influenced the AMPK and
mTOR signaling pathway in the PDX materials, we evaluated the
phosphorylation of AMPK, mTOR, and p70S6K (Fig. 2E). Met-
formin treatment activated AMPK as determined by phosphory-
lation at Thr172, with a corresponding inhibition of mTOR
signaling and downstream target phosphorylation of p70S6K. In
tumors treated with metformin and 5-FU, AMPK was activated;
interestingly, mTOR signaling appears to be activated and not
suppressed, although phosphorylation of p70S6K was inhibited
(Fig. 2F).
Metformin inhibits oxygen consumption rate (OCR)
For ex vivo measurement of oxygen consumption, organoids
derived from PDX were treated with metformin (Fig. 3A). Drugs
affecting mitochondrial functions were used in parallel to dem-
onstrate normal mitochondrial function. Metformin inhibited
OCR of both FIT-CRC-086 and FIT-CRC-104 (Fig. 3B and C,
respectively) in a concentration-dependent manner. Consequent
injection of oligomycin did not suppress the OCR further in these
metformin-treated organoids, while injection of FCCP, the
Mol Cancer Ther; 16(9) September 2017 2037
 Published OnlineFirst May 22, 2017; DOI: 10.1158/1535-7163.MCT-16-0793

Mohamed Suhaimi et al.

Figure 1.
In vitro effects of 5-fluorouracil (5-FU) and metformin
on colorectal cancer cells. 5-FU, the first-line
treatment for colorectal cancer, inhibits cell
proliferation at micromolar levels (A). Millimolar levels
of metformin are required to inhibit cell proliferation
at 48 hours, with pronounced inhibition observed in
combination with 10 mmol/L 5-FU (B). Glucose
deprivation accentuates growth inhibition by
metformin (C). Inhibition of colorectal cancer cell
proliferation to 5-FU (D) and metformin is
independent of p53 status, in both HCT116 p53
knockout (p53 KO) and HCT116 p53 wild-type (p53
WT) cell lines, but with stronger degree of growth
inhibition observed under glucose-deprived
conditions in the presence of metformin (E). The
absolute number of colonies is presented as an
average of triplicates (F).

2038 Mol Cancer Ther; 16(9) September 2017 Molecular Cancer Therapeutics

Downloaded from mct.aacrjournals.org on May 27, 2021. © 2017 American Association for Cancer Research.
 Published OnlineFirst May 22, 2017; DOI: 10.1158/1535-7163.MCT-16-0793
Table 2. Clinicopathologic information of patients with xenografts generated
Tumor Tumor Tumor
Patient Histology site grade
FIT-CRC-086 Adenocarcinoma Rectum 1 Ulcerated
FIT-CRC-104 Mucinous adenocarcinoma Ascending colon 2
mitochondrial uncoupler, rescues the respiration inhibition, albe-
it to a lesser extent in the metformin-treated organoids as com-
pared with control.
Correspondingly, metformin inhibition on cellular respiration
resulted in an increase in glycolysis, as measured by the extracel-
lular acidification rate (ECAR; Fig. 3D and E). It was evident that
metformin inhibits mitochondrial function as early as 4 hours
(Fig. 3F and G).
Molecular profiling of circulating biomarkers in mice
To determine if tumor burden in vivo can be assessed using
alternative approaches that are viable in the clinical setting, we
investigate the use of cfDNA in our models (25). CfDNA is a
heterogeneous mixture of normal and tumor-associated cell
populations (26, 27) which makes cfDNA analysis technically
challenging. In xenografts, both human and mouse-derived cell
populations are present, and the ability to distinguish these two
cell populations is critical in order to assess the PDX response to
treatment using cfDNA. Here, using a limited amount of
cfDNA, our assay can distinguish human and mice-derived
DNA efficiently (Supplementary Fig. S1), with limit of detec-
tion of 10% human DNA in a background of mice DNA. No
cfDNA was detected at week 0, at the point of tumor implan-
tation. Subsequent evaluation of the cfDNA at different time
points in the course of treatment indicates that human-derived
DNA could be detected in the mice blood, suggesting that
cfDNA could be a viable noninvasive biomarker for tumor
burden.
Mutation profile of primary tumor and corresponding
xenografts
Human samples and PDX were analyzed for variants/muta-
tions by NGS using a panel of 40 genes commonly implicated in
colorectal cancer. The mean sequencing depth across all experi-
ments was 1,385X for FIT-CRC-086 and 2,534X for FIT-CRC-104.
A summary of annotated variants is shown in Fig. 4A.
For FIT-CRC-086, mutations in APC (K1350 ), KRAS (G12V),
TP53 (R248Q), and SMAD4 (L495R) were observed for parental
tumor and xenografts, but were not present in germline or normal
colon. Likewise, in FIT-CRC-104, mutations were observed in
KRAS (A146T) and MLH1 (R100 ) in parental tumor. Essentially,
mutations remain conserved across PDX passages. An example of
a variant conserved in tumor and PDX as depicted by NGS are
corroborated by Sanger sequencing in Fig. 4B. Interestingly, we
also observed the presence of variants that are not reported in
COSMIC database. Using Sanger sequencing, we verified the non-
COSMIC variants that were detected (Fig. 4C). Nonetheless, non-
COSMIC variants were not present in every PDX. Within the same
treatment subset, individuals exhibit heterogeneity. Whether
these new variants were a function of clonal selection of a subset
of tumor cells warrants further investigation.
One of the key objectives of performing NGS is to identify
biomarkers that may reflect response to metformin. Our analysis
www.aacrjournals.org
Downloaded from mct.aacrjournals.org on May 27, 2021. © 2017 American Association for Cancer Research.
Metformin Inhibits Growth of Colorectal Cancer PDX
Tumor size AJCC Lymph node
type (largest dimension in cm) stage pT pN pM invasion
4 IVA 3 0 1a 0/7
Localized 5 IIA 3 0 0 0/39
indicates that there are no key colorectal cancer genes that are
associated with metformin's activity.
Discussion
Metformin has been extensively reported as a promising anti-
cancer agent in cancer prevention and therapy (9–12, 21, 22,
28, 29). In the present study, we evaluated the effects of metfor-
min using preclinical models. Similar to reported studies, we
observed that the antiproliferative properties of metformin man-
ifest at supraphysiologic conditions in cell lines (30). A recurrent
feature is that these cells are typically maintained in non-phys-
iologic conditions that are optimized only for in vitro growth and
proliferation. The excessive concentration of growth factors, insu-
lin and glucose may account for the elevated doses of metformin
required to elicit cellular responses and trigger metabolic stress. As
such, it is virtually impossible to translate these supraphysiologic
conditions in the clinical setting, especially when the pharmaco-
kinetics of the drug is different in culture settings and in the
human body.
Consequently, we observed that low glucose conditions accen-
tuated the growth inhibition by metformin. This is not surpris-
ing—glucose deprivation is a distinctive feature of the tumor
microenvironment caused by the imbalance between nutrient
supply and oxygen consumption rate (31). In a study exploring
the role of the tumor microenvironment in potentiating the effect
of metformin, metformin is synthetically lethal with glucose
withdrawal in cancer cells (32). Essentially, this suggests that the
clinically irrelevant, supraphysiologic levels needed to observe
metformin's anticancer effects as reported by many in vitro studies
merely underlie the artefactual experimental conditions that
poorly reflect actual glucose-starved in vivo conditions.
Most preclinical in vivo models have generally proven to be sub-
optimal for directing clinical application of new anticancer ther-
apies, largely due to their inability to reflect the complexity and
heterogeneity of human tumors. PDX, where surgically resected
tumor samples are engrafted directly into immunocompromised
mice, offer several advantages (33, 34). In our study, we have
shown that PDX tumors maintained the molecular, genetic, and
histologic heterogeneity typical of tumors of origin (35–39). PDX
provides an excellent platform to study cancer biology, analyze
cancer therapeutics, and novel drug combinations. In our study,
we demonstrated metformin efficacy in our PDX models. To the
best of our knowledge, ours represents the first study reported for
colorectal cancer PDX using metformin. Administering metfor-
min at physiologic levels of 150 mg/kg per day in our mice, which
is equivalent to initial clinical dose of 500 to 1,000 mg/daily in
human, is sufficient to inhibit tumor growth in PDX. Furthermore,
we observed that the response to metformin may be independent
of p53 status and is inconsistent with the observations reported
earlier (9, 24). Conversely, studies conducted in breast cancer
indicate that metformin's effects are independent of p53 status
(40, 41). This contraindication suggests that p53 may not be a
reliable companion biomarker to predict response to metformin.
Mol Cancer Ther; 16(9) September 2017 2039
 Published OnlineFirst May 22, 2017; DOI: 10.1158/1535-7163.MCT-16-0793

Mohamed Suhaimi et al.

Figure 2.
Histomorphologic features of parental tumor are retained in patient-derived xenografts after serial passaging. Representative tumor cells are indicated by solid
arrows; mucins are indicated by diamond arrowheads (A). Gross morphology of PDX-FIT-CRC-086 tumors treated with metformin and 5-FU (B). Tumor growth
profile of PDX-FIT-CRC-086 (C) and PDX-FIT-CRC-104 (D). Metformin triggers AMPK activation and inhibits mTOR signaling in PDX (E). Protein levels were
normalized to ß-actin and fold changes are quantified relative to vehicle control (F).

Studies from a recent phase II clinical trial evaluating metfor-
min and 5-FU in patients with refractory colorectal cancer showed
only a mild median progression-free survival (PFS) of 1.8 months
and median overall survival (OS) of 7.9 months, with a majority
of patients (78%) having disease progression before 8 weeks (42).
For 11 out of 50 patients (22%) having stable disease at the end of
8 weeks, their median PFS and OS were 5.6 months and 16.2
months, respectively. Likely, the modest benefits of metformin
and 5-FU observed in this trial may be due to the nature of the
refractory colorectal cancer cases and that the benefits may be
significant in nonrefractory colorectal cancer cases.
Interestingly, in our study, we observed that combination of
5-FU with metformin leads to a pronounced growth inhibition in
the p53-mutant PDX but not in p53-wild-type PDX. It may be
possible that p53 status plays a role in differential response to
metformin and 5-FU; however, it is also likely that 5-FU is non-
beneficial in the p53-wild-type PDX because this PDX has MSI.
Indeed, clinical observations have suggested that patients having a

2040 Mol Cancer Ther; 16(9) September 2017 Molecular Cancer Therapeutics

Downloaded from mct.aacrjournals.org on May 27, 2021. © 2017 American Association for Cancer Research.
 Published OnlineFirst May 22, 2017; DOI: 10.1158/1535-7163.MCT-16-0793

Metformin Inhibits Growth of Colorectal Cancer PDX

OXYGEN CONSUMPTION RATE OXYGEN CONSUMPTION RATE

B OCR
73
OCR vs TIME (Avg)

OLIGOMYCIN 1.0 µmol/L FCCP 1.0 µmol/L ROT\AA 0.5 µmol/L

C OCR
417 OLIGOMYCIN 1.0 µmol/L
OCR vs TIME (Avg)

FCCP 1.0 µmol/L ROT\AA 0.5 µmol/L

500 ORGANOIDS CONTROL (24 HRS) 500 ORGANOIDS CONTROL (24 HRS)
64 500 ORGANOIDS + 1.0 METFORMIN (24 HRS) 370 500 ORGANOIDS + 1.0 METFORMIN (24 HRS)
500 ORGANOIDS + 0.1 METFORMIN (24 HRS) 500 ORGANOIDS + 0.1 METFORMIN (24 HRS)

56 322

48 275
OCR pMoles/min

OCR pMoles/min
39 227

31 180

22 132
Control
14 85
0.1 mmol/L Met
1.0 mmol/L Met Control
5 37 0.1 mmol/L Met
1.0 mmol/L Met
–3 –10

–11 –58
0 15 30 45 60 75 91 106 121 136 151 0 15 30 45 60 75 90 105 120 135 151
TIME (min) TIME (min)

OXYGEN CONSUMPTION RATE OXYGEN CONSUMPTION RATE

D ECAR
ECAR vs TIME (Avg)
E ECAR
46
ECAR vs TIME (Avg)

30 OLIGOMYCIN 1.0 µmol/L FCCP 1.0 µmol/L ROT\AA 0.5 µmol/L OLIGOMYCIN 1.0 µmol/L FCCP 1.0 µmol/L ROT\AA 0.5 µmol/L
500 ORGANOIDS CONTROL (24 HRS)
500 ORGANOIDS CONTROL (24 HRS)
27 500 ORGANOIDS + 1.0 METFORMIN (24 HRS) 41 500 ORGANOIDS + 1.0 METFORMIN (24 HRS)
500 ORGANOIDS + 0.1 METFORMIN (24 HRS)
500 ORGANOIDS + 0.1 METFORMIN (24 HRS)

23 35

20 Control 30
1.0 mmol/L Met Control
ECAR mpH/min

24
ECAR mpH/min

16
0.1 mmol/L Met 0.1 mmol/L Met
13 19 1.0 mmol/L Met

9 13

6 8

2 2

–1 –3

–5 –9
0 15 30 45 60 75 91 106 121 136 151 0 15 30 45 60 75 90 105 120 135 151
TIME (min) TIME (min)

OXYGEN CONSUMPTION RATE OXYGEN CONSUMPTION RATE

F OCR
73
OCR vs TIME (Avg)
G OCR
196
OCR vs TIME (Avg)

OLIGOMYCIN 1.0 µmol/L FCCP 1.0 µmol/L ROT\AA 0.5 µmol/L OLIGOMYCIN 1.0 µmol/L FCCP 1.0 µmol/L ROT\AA 0.5 µmol/L
500 ORGANOIDS CONTROL (4 HRS)
500 ORGANOIDS CONTROL (4 HRS)
64 500 ORGANOIDS + 1 METFORMIN (4 HRS) 174 500 ORGANOIDS + 1 METFORMIN (4 HRS)
500 ORGANOIDS + 0.1 METFORMIN (4 HRS)
500 ORGANOIDS + 0.1 METFORMIN (4 HRS)

56 152

48 130
OCR pMoles/min

OCR pMoles/min

39 108

31 86

22 64

14 Control 41 Control
1.0 mmol/L Met 0.1 mmol/L Met
5 0.1 mmol/L Met 19
1.0 mmol/L Met

–3 –3

–11 –25
0 15 30 45 60 75 91 106 121 136 151 0 15 30 45 60 75 90 105 120 135 151
TIME (min) TIME (min)

Figure 3.
Organoids derived from PDX under 10 (left image) and 40 (right image) (A). Pretreatment with 0.1 mmol/L metformin or 1.0 mmol/L metformin for 24 hours
inhibits oxygen consumption rate (OCR) relative to vehicle control for both FIT-CRC-086 (B) and FIT-CRC-104 (C), with corresponding increase in glycolysis
for FIT-CRC-086 (D) and FIT-CRC-104 (E). Pretreatment with 0.1 mmol/L metformin and 1.0 mmol/L metformin inhibits OCR as early as 4 hours in both
FIT-CRC-086 (F) and FIT-CRC-104 (G). 1.0 mmol/L oligomycin, 1.0 mmol/L carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and 0.5 mmol/L
rotenone/antimycin A were used as mitochondrial stress controls.

www.aacrjournals.org Mol Cancer Ther; 16(9) September 2017 2041

Downloaded from mct.aacrjournals.org on May 27, 2021. © 2017 American Association for Cancer Research.
 Published OnlineFirst May 22, 2017; DOI: 10.1158/1535-7163.MCT-16-0793

Mohamed Suhaimi et al.

Figure 4.
Molecular characterization of germline DNA, parental tumor, serial passages of xenografts and xenografts treated with drugs. Variant allelic frequency is
reflected on the scale of 0 to 1 (A). An example of a COSMIC variant detected by next-generation sequencing (NGS) and independently verified by Sanger sequencing
(B), two variants that have not been reported in COSMIC are detected by NGS and Sanger sequencing (C).

deficient mismatch repair system or MSI do not benefit from
5-FU–based adjuvant therapy in colorectal cancer (43, 44). Thus,
our observations using PDX are in line with observations
obtained in the clinic, thus, emphasizing the crucial role PDXs
play as a clinically relevant model.
To demonstrate the integrity of PDX as suitable models, we
performed high-throughput NGS to characterize the genetic pro-
files of parental tumor and PDX. We demonstrate that genes such
as TP53, APC, KRAS, and SMAD4, were mutated in the parental
tumor and conserved throughout serial passaging of the xeno-
grafts. More importantly, in an effort to characterize biomarkers
that can serve as companion marker to predict metformin
response, we compared the genetic differences between the two
PDX lines. Our NGS analysis suggests that there is lack of novel
actionable genes that may reflect metformin's activity based on
genotype. Nonetheless, the key limitation in this particular anal-
ysis is likely due to limited variation in genotype between the two
PDX lines.
To gain insight into possible mechanisms of metformin
action, we evaluated the frequently implicated AMPK signaling
pathway (45). Indeed, our data showed that metformin trig-
gered phosphorylation of Thr172 on AMPKa, which conse-
quently led to downregulation of mTOR and its target p70S6K,
whose phosphorylation at Thr389 is necessary for protein
synthesis (46). Moreover, we observed that metformin inhibits
oxygen consumption in organoids derived from PDX, as early
as 4 hours, with a concomitant increase in glycolysis. Taken
together, it is likely that treatment with metformin activates the
AMPK pathway, which alters the mTOR signaling, and executes
a metabolic change in the cells, which ultimately affects cell
growth in the PDX.
We acknowledged that there are several limitations to our
current study. Firstly, we restricted the use of PDX to subcuta-
neous tumor models. Subcutaneous models are inferior to
orthotopic models because the former do not represent appro-
priate sites for human tumors, and may not be accurately
predictive for drug evaluation (47). However, orthotopic tumor
models are time-consuming and technically challenging. Fur-
thermore, it is difficult to monitor tumor growth, and sophis-
ticated micro-imaging techniques are required to visualize the
tumor. In anticipation of this limitation (should the need arise
to monitor tumor burden in orthotopic models), we assessed
noninvasive circulating biomarkers (cfDNA) in our subcutane-
ous PDX models. Using established techniques that we have
utilized previously for clinical cancer patients (48, 49), we
demonstrated that it is feasible to prove the presence of
human-derived DNA circulating in plasma.
Utilizing NGS approach could aid in predictive biomarker(s)
identification as a result of drug selection. Because this is a pilot
study involving metformin and PDX, banking on molecular
profiles derived from 2 colorectal cancer patients may be inade-
quate for biomarker discovery process. Expanding the repertoire
of PDX lines with different genetic make-up is likely to shed more
information in future. While the creation of PDX is of clear interest
as a means to better define optimal therapy for colorectal cancer, it
is undeniable that the high-throughput data are challenging to
manage and visualize. Thoughtful interpretation is necessary to
convert the knowledge derived from existing data in order to
understand treatment outcomes.
In essence, our study demonstrated the utility of PDX in
assessing drug efficacy via molecular analysis, metabolic profiling,
and bioinformatics approaches. This is the first study that
describes the use of metformin in colorectal cancer PDX and
organoids, providing direct evidence of metformin as an antican-
cer agent in colorectal cancer.
Disclosure of Potential Conflicts of Interest
M.-H. Tan has ownership interest in a patent filed for method for KRAS
mutation detection, owned by his employer, and licensed to industry. No
potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: N.-A. Mohamed Suhaimi, W.M. Phyo, H.Y. Yap,
M.-H. Tan
Development of methodology: N.-A. Mohamed Suhaimi, W.M. Phyo, W.J. Tan
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): N.-A. Mohamed Suhaimi, W.M. Phyo, H.Y. Yap,
S.H.Y. Choy, Y. Choudhury, W.J. Tan, L.A.P.Y. Tan, R.S.Y. Foo, S.H.S. Tan,
P.K. Koh, M.-H. Tan

2042 Mol Cancer Ther; 16(9) September 2017 Molecular Cancer Therapeutics

Downloaded from mct.aacrjournals.org on May 27, 2021. © 2017 American Association for Cancer Research.
 Published OnlineFirst May 22, 2017; DOI: 10.1158/1535-7163.MCT-16-0793
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): N.-A. Mohamed Suhaimi, W.M. Phyo, X. Wei,
L.A.P.Y. Tan, M.-H. Tan
Writing, review, and/or revision of the manuscript: N.-A. Mohamed Suhaimi,
W.M. Phyo, Y. Choudhury, W.J. Tan, L.A.P.Y. Tan, P.K. Koh, M.-H. Tan
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): W.J. Tan, L.A.P.Y. Tan, Z. Tiang, C.F. Wong,
M.-H. Tan
Study supervision: M.-H. Tan
Acknowledgments
We would like to thank Concord Cancer Hospital for the provision of the
tissue samples, and Vishuo Biomedical for the support in the bioinformatics
analysis. We would like to thank Chua Teck Chiew Timothy, Ma Janise Ann
Kabiling Lalic, Mukta Pathak, and Hannes Martin Hentze from A STAR Bio-
logical Resource Centre for their help and support in PDX work. We would also
References
1. Voskoglou-Nomikos T, Pater JL, Seymour L. Clinical predictive value of the
in vitro cell line, human xenograft, and mouse allograft preclinical cancer
models. Clin Cancer Res 2003;9:4227–39.
€
2. Ostman A, Augsten M. Cancer-associated fibroblasts and tumor growth –
bystanders turning into key players. Curr Opin Genet Dev 2009;19:67–73.
3. Ertel A, Verghese A, Byers S, Ochs M, Tozeren A. Pathway-specific differ-
ences between tumor cell lines and normal and tumor tissue cells. Mol
Cancer 2006;5:55.
4. Stein WD, Litman T, Fojo T, Bates SE. A Serial analysis of gene expression
(SAGE) database analysis of chemosensitivity: comparing solid tumors
with cell lines and comparing solid tumors from different tissue origins.
Cancer Res 2004;64:2805–16.
5. Leupin N, Kuhn A, H€ ugli B, Grob TJ, Jaggi R, Tobler A, et al. Gene
expression profiling reveals consistent differences between clinical
samples of human leukaemias and their model cell lines. Br J Haematol
2006;135:520–3.
6. de Gramont A, Figer A, Seymour M, Homerin M, Hmissi A, Cassidy J, et al.
Leucovorin and fluorouracil with or without oxaliplatin as first-line treat-
ment in advanced colorectal cancer. J Clin Oncol 2000;18:2938–47.
7. Evans JM, Donnelly LA, Emslie-Smith AM, Alessi DR, Morris AD. Metfor-
min and reduced risk of cancer in diabetic patients. BMJ 2005;330:
1304–5.
8. Morales DR, Morris AD. Metformin in cancer treatment and prevention.
Annu Rev Med 2014;1–13.
9. Buzzai M, Jones RG, Amaravadi RK, Lum JJ, DeBerardinis RJ, Zhao F, et al.
Systemic treatment with the antidiabetic drug metformin selectively
impairs p53-deficient tumor cell growth. Cancer Res 2007;67:6745–52.
10. Zi FM, He JS, Li Y, Wu C, Yang L, Yang Y, et al. Metformin displays anti-
myeloma activity and synergistic effect with dexamethasone in in vitro and
in vivo xenograft models. Cancer Lett 2015;356:443–53.
11. Memmott RM, Mercado JR, Maier CR, Kawabata S, Fox SD, Dennis PA.
Metformin prevents tobacco carcinogen–induced lung tumorigenesis.
Cancer Prev Res 2010;3:1066–76.
12. Lengyel E, Litchfield LM, Mitra AK, Nieman KM, Mukherjee A, Zhang Y,
et al. Metformin inhibits ovarian cancer growth and increases sensitivity to
paclitaxel in mouse models. Am J Obstet Gynecol 2015;212:479.e1-479.
e10.
13. Ben Sahra I, Laurent K, Loubat A, Giorgetti-Peraldi S, Colosetti P, Auberger
P, et al. The antidiabetic drug metformin exerts an antitumoral effect
in vitro and in vivo through a decrease of cyclin D1 level. Oncogene
2008;27:3576–86.
14. Zordoky BNM, Bark D, Soltys CL, Sung MM, Dyck JRB. The anti-prolifer-
ative effect of metformin in triple-negative MDA-MB-231 breast cancer
cells is highly dependent on glucose concentration: implications for cancer
therapy and prevention. Biochim Biophys Acta 2014;1840:1943–57.
15. Kinaan M, Ding H, Triggle CR. Metformin: an old drug for the treatment of
diabetes but a new drug for the protection of the endothelium. Med Princ
Pract 2015;24:401–15.
16. Krishnamurthy S, Ng VW, Gao S, Tan MH, Yang YY. Phenformin-loaded
polymeric micelles for targeting both cancer cells and cancer stem cells in
vitro and in vivo. Biomaterials 2014;35:9177–86.
www.aacrjournals.org
Downloaded from mct.aacrjournals.org on May 27, 2021. © 2017 American Association for Cancer Research.
Metformin Inhibits Growth of Colorectal Cancer PDX
like to thank Chit Fang Cheok for the helpful discussion on the in vitro work and
Shengyong Ng for the assistance in organoids derivation and culture.
Grant Support
This work is supported by the Institute of Bioengineering and Nanotech-
nology and Agency for Science, Technology and Research, Singapore. M.H. Tan
received grants from Biomedical Research Council (Diagnostics Grant & Stra-
tegic Positioning Fund SPF 2012/003 and SPF2015/002).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received November 22, 2016; revised March 29, 2017; accepted May 17,
2017; published OnlineFirst May 22, 2017.
17. Muzny DM, Bainbridge MN, Chang K, Dinh HH, Drummond JA., Fowler
G, et al. Comprehensive molecular characterization of human colon and
rectal cancer. Nature 2012;487:330–7.
18. De Sousa E Melo F, Wang X, Jansen M, Fessler E, Trinh A, de Rooij
LPMH. Poor-prognosis colon cancer is defined by a molecularly distinct
subtype and develops from serrated precursor lesions. Nat Med 2013;
19:614–8.
19. Sadanandam A, Lyssiotis CA, Homicsko K, Collisson EA, Gibb WJ,
Wullschleger S, et al. A colorectal cancer classification system that
associates cellular phenotype and responses to therapy. Nat Med
2013;19:619–25.
20. Budinska E, Popovici V, Tejpar S, D'Ario G, Lapique N, Sikora KO, et al.
Gene expression patterns unveil a new level of molecular heterogeneity in
colorectal cancer. J Pathol 2013;231:63–76.
21. Sarfstein R, Friedman Y, Attias-Geva Z, Fishman A, Bruchim I, Werner H.
Metformin downregulates the insulin/IGF-I signaling pathway and inhi-
bits different uterine serous carcinoma (USC) cells proliferation and
migration in p53-dependent or -independent manners. PLoS One
2013;8:e61537.
22. Ben Sahra I, Laurent K, Giuliano S, Larbret F, Ponzio G, Gounon P, et al.
Targeting cancer cell metabolism: the combination of metformin and 2-
deoxyglucose Induces p53-dependent apoptosis in prostate cancer cells.
Cancer Res 2010;70:2465–75.
23. Li P, Zhao M, Parris AB, Feng X, Yang X. p53 is required for metformin-
induced growth inhibition, senescence and apoptosis in breast cancer cells.
Biochem Biophys Res Commun 2015;464:1267–74.
24. Yi G, He Z, Zhou X, Xian L, Yuan T, Jia X, et al. Low concentration of
metformin induces a p53-dependent senescence in hepatoma cells via
activation of the AMPK pathway. Int J Oncol 2013;43:5.
25. Mohamed Suhaimi NA, Tan MH. Circulating tumor cells and circulating
tumor DNA in colorectal cancer. Expert Rev Precis Med Drug Dev
2016;1:181–94.
26. Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO, Hesch RD, et al.
DNA fragments in the blood plasma of cancer patients: quantitations and
evidence for their origin from apoptotic and necrotic cells. Cancer Res
2001;61:1659–65.
27. Tomochika S, Iizuka N, Watanabe Y, Tsutsui M, Takeda S, Yoshino S, et al.
Increased serum cell-free DNA levels in relation to inflammation are
predictive of distant metastasis of esophageal squamous cell carcinoma.
Exp Ther Med 2010;1:89–92.
28. Hirsch HA, Iliopoulos D, Tsichlis PN, Struhl K. Metformin selectively
targets cancer stem cells, and acts together with chemotherapy to block
tumor growth and prolong remission. Cancer Res 2009;69:7507–11.
29. Quinn BJ, Kitagawa H, Memmott RM, Gills JJ, Dennis PA. Repositioning
metformin for cancer prevention and treatment. Trends Endocrinol Metab
2013;24:469–80.
30. Martin-Castillo B, Vazquez-Martin A, Oliveras-Ferraros C, Menendez JA.
Metformin and cancer: doses, mechanisms and the dandelion and hor-
metic phenomena. Cell Cycle 2010;9:1057–64.
31. Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis? Nat
Rev Cancer 2004;4:891–9.
Mol Cancer Ther; 16(9) September 2017 2043
 Published OnlineFirst May 22, 2017; DOI: 10.1158/1535-7163.MCT-16-0793
Mohamed Suhaimi et al.
32. Menendez JA, Oliveras-Ferraros C, Cufí S, Corominas-Faja B, Joven J,
Martin-Castillo B, et al. Metformin is synthetically lethal with glucose
withdrawal in cancer cells. Cell Cycle 2012;11:2782–92.
33. Tentler JJ, Tan AC, Weekes CD, Jimeno A, Leong S, Pitts TM, et al. Patient-
derived tumour xenografts as models for oncology drug development.
Nat Rev Clin Oncol 2012;9:338–50.
34. Hidalgo M, Amant F, Biankin A V, Budinska E, Byrne AT, Caldas C, et al.
Patient-derived xenograft models: an emerging platform for translational
cancer research. Cancer Discov 2014;4:998–1013.
35. Garralda E, Paz K, L opez-Casas PP, Jones S, Katz A, Kann LM, et al.
Integrated next-generation sequencing and avatar mouse models for per-
sonalized cancer treatment. Clin Cancer Res 2014;20:2476–84.
36. Mattie M, Christensen A, Chang MS, Yeh W, Said S, Shostak Y, et al.
Molecular characterization of patient-derived human pancreatic tumor
xenograft models for preclinical and translational development of cancer
therapeutics. Neoplasia 2013;15:1138–50.
37. Klinghammer K, Raguse JD, Plath T, Albers AE, Joehrens K, Zakar-
neh A, et al. A comprehensively characterized large panel of head
and neck cancer patient-derived xenografts identifies the mTOR
inhibitor everolimus as potential new treatment option. Int J
Cancer 2014;2948.
38. Seol HS, Kang H, Lee SI, Kim NE, Kim TI, Chun SM, et al. Development and
characterization of a colon PDX model that reproduces drug responsive-
ness and the mutation profiles of its original tumor. Cancer Lett
2014;345:56–64.
39. Hao C, Wang L, Peng S, Cao M, Li H, Hu J, et al. Gene mutations in primary
tumors and corresponding patient-derived xenografts derived from non-
small cell lung cancer. Cancer Lett 2015;357:179–85.
40. Rattan R, Giri S, Hartmann LC, Shridhar V. Metformin attenuates ovarian
cancer cell growth in an AMP-kinase dispensable manner. J Cell Mol Med
2011;15:166–78.
2044 Mol Cancer Ther; 16(9) September 2017
Downloaded from mct.aacrjournals.org on May 27, 2021. © 2017 American Association for Cancer Research.
41. Pandiri I, Chen Y, Joe Y, Kim HJ, Park J, Chung HT, et al. Tristetraprolin
mediates the anti-proliferative effects of metformin in breast cancer cells.
Breast Cancer Res Treat 2016;156:57–64.
42. Miranda VC, Braghiroli MI, Faria LD, Bariani G, Alex A, Evangelista J, et al.
Phase 2 trial of metformin combined with 5-fluorouracil in patients with
refractory metastatic colorectal cancer. Clin Colorectal Cancer 2016;15:
321–328.e1.
43. Sargent DJ, Marsoni S, Monges G, Thibodeau SN, Labianca R, Hamilton SR,
et al. Defective mismatch repair as a predictive marker for lack of efficacy of
fluorouracil-based adjuvant therapy in colon cancer. J Clin Oncol
2010;28:3219–26.
44. Ribic CM, Sargent DJ, Moore MJ, Thibodeau SN, French AJ, Goldberg RM,
et al. Tumor microsatellite-instability status as a predictor of benefit from
fluorouracil-based adjuvant chemotherapy for colon cancer. N Engl J Med
2003;349:247–57.
45. Viollet B, Guigas B, Sanz Garcia N, Leclerc J, Foretz M, Andreelli F. Cellular
and molecular mechanisms of metformin: an overview. Clin Sci (Lond)
2012;122:253–70.
46. Zakikhani M, Blouin MJ, Piura E, Pollak MN. Metformin and rapamycin
have distinct effects on the AKT pathway and proliferation in breast cancer
cells. Breast Cancer Res Treat 2010;123:271–9.
47. Killion J, Radinsky R, Fidler I. Orthotopic models are necessary to predict
therapy of transplantable tumors in mice. Cancer Metastasis Rev
1998;17:279–84.
48. Lim LS, Hu M, Huang MC, Cheong WC, Gan ATL, Looi XL, et al. Microsieve
lab-chip device for rapid enumeration and fluorescence in situ hybridiza-
tion of circulating tumor cells. Lab Chip 2012;12:4388.
49. Mohamed Suhaimi NA, Foong YM, Lee DYS, Phyo WM, Cima I, Lee EX,
et al. Non-invasive sensitive detection of KRAS and BRAF mutation in
circulating tumor cells of colorectal cancer patients. Mol Oncol 2015;
9:850–60.
Molecular Cancer Therapeutics
 Published OnlineFirst May 22, 2017; DOI: 10.1158/1535-7163.MCT-16-0793

Metformin Inhibits Cellular Proliferation and Bioenergetics in

Colorectal Cancer Patient−Derived Xenografts
Nur-Afidah Mohamed Suhaimi, Wai Min Phyo, Hao Yun Yap, et al.

Mol Cancer Ther 2017;16:2035-2044. Published OnlineFirst May 22, 2017.

Updated version Access the most recent version of this article at:
doi:10.1158/1535-7163.MCT-16-0793

Supplementary Access the most recent supplemental material at:

Material http://mct.aacrjournals.org/content/suppl/2017/05/22/1535-7163.MCT-16-0793.DC1

Cited articles This article cites 47 articles, 12 of which you can access for free at:
http://mct.aacrjournals.org/content/16/9/2035.full#ref-list-1

Citing articles This article has been cited by 2 HighWire-hosted articles. Access the articles at:
http://mct.aacrjournals.org/content/16/9/2035.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at
Subscriptions pubs@aacr.org.

Permissions To request permission to re-use all or part of this article, use this link
http://mct.aacrjournals.org/content/16/9/2035.
Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
Rightslink site.

Downloaded from mct.aacrjournals.org on May 27, 2021. © 2017 American Association for Cancer Research.