Metformin Inhibits Cellular Proliferation and Bioenergetics in ... · Bioenergetics in Colorectal Cancer Patient– Derived Xenografts Nur-Aﬁdah Mohamed Suhaimi1,Wai Min Phyo1,

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Metformin Inhibits Cellular Proliferation andBioenergetics in Colorectal Cancer Patient–Derived XenograftsNur-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 againstvarious malignancies, including colorectal cancer. However, themajority of evidence, which was derived from cancer cell linesand 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 patientsto 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 PDXtumors by at least 50% (P < 0.05) when administered orally for24 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)

growth inhibition. Ex vivo culture of organoids generated fromPDX demonstrates that metformin inhibits growth by executingmetabolic changes to decrease oxygen consumption and acti-vating AMPK-mediated pathways. In addition, we also per-formed genetic characterizations of serial PDX samples withcorresponding parental tissues from patients using next-gener-ation sequencing (NGS). Our pilot NGS study demonstratesthat PDX represents a useful platform for analysis in cancerresearch because it demonstrates high fidelity with parentaltumor. Furthermore, NGS analysis of PDX may be useful todetermine genetic identifiers of drug response. This is the firstpreclinical study using PDX and PDX-derived organoidsto investigate the efficacy of metformin in colorectal cancer.Mol Cancer Ther; 16(9); 2035–44. �2017 AACR.

IntroductionAnticancer 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 andxenografts are useful in the laboratory setting, but may nottranslate well to the clinical setting (1). The failure of many cellline xenografts to accurately predict the clinical efficacy of anti-cancer agents is due to their inability to reflect the complexity andheterogeneity of human tumors (2). Substantial genetic diver-gence exists between primary tumor and the corresponding cellline derived from that tumor, as comparedwith a direct transplantof tumor into mice (3–5). Patient-derived xenografts (PDX), in

which patient-derived tumor fragments are directly implantedinto immunocompromised mice through serial passaging, are anincreasingly recognized tool for drug candidate evaluation. ThesePDXs are unlikely to have undergone extensive selection pressuretypically associated with cell line establishment as manifestedthrough genetic andmolecular changes (3), and are thus valuablefor drug studies.

We evaluated the performance of 5-fluorouracil (5-FU), thefirst-line treatment for colorectal cancer (6), along with theantidiabetic drug metformin. Epidemiologic studies have sug-gested the beneficial effects of metformin in cancer treatmentamong 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 metforminas an anticancer agent. Nonetheless, preclinical studies ofteninvolve supraphysiologic concentrations of metformin that arein excess of the therapeutic levels achieved in patients. Inhumans, the initial dose of metformin is 500 to 1,000 mg/daily, and subsequently, a maintenance dose of 2,000 mg/dailyis required. Studies performed on cell lines require elevateddoses of metformin (2–50 mmol/L) to elicit cellular response,while in vivo studies require up to 4 times the maximum clinicaldose in humans of 2,550 mg/day (15). Thus, it is anticipatedthat good relevant models are necessary for physiologic model-ing of metformin.

To the best of our knowledge, this is the first study evaluatingthe use of metformin on colorectal cancer PDX, a highly

1Institute 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. 6ConcordCancer Hospital Singapore, Singapore. 7National Cancer Centre Singapore,Singapore.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics 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: [email protected]

doi: 10.1158/1535-7163.MCT-16-0793

�2017 American Association for Cancer Research.

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relevant system for drug candidate testing. Using next-genera-tion sequencing (NGS), we interrogated the molecular profilesof these PDX to evaluate selection pressures initiated during thecourse of drug treatment. In addition, we aimed to identifymolecular signatures that influence response to metformin. Inparallel, we utilized colorectal cancer organoids to elucidate themechanistic effect of metformin.

Materials and MethodsCell culture maintenance, proliferation, and clonogenic assays

Cell lines were obtained in 2012 from ATCC directly whileisogenic 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-tionof cellswasnot performed.Cellswere cultured inDMEMwith10% fetal bovine serum and maintained in 37�C incubatorsupplemented with 5% CO2. The MycoAlert Mycoplasma Detec-tion Kit (Lonza) was used to test forMycoplasma contamination atdefined intervals, and the tests indicate that cell lines were free ofMycoplasma throughout the study.

The CellTiter96 AQueous One Solution Assay (Promega) wasused to assess proliferation of cells incubated with metformin(0–20 mmol/L) and/or 5-FU (10 mmol/L) in the presence ofhigh (4,500 mg/L) or low (1,000 mg/L) glucose. For clonogenicassay, cells harvested from exponential phase cultures wereplated in six-well plates. Seeding densities varied from 100 to300 cells per well. After 7 days, cells were washed with PBS,fixed with methanol and stained with Coomassie blue. Assayswere done in triplicate. Colonies were counted by two inde-pendent investigators.

Microsatellite instability (MSI) analysisMSI was determined using the MSI Analysis Kit version 1.2

(Promega). Briefly, DNA from matched normal and tumorspecimens was amplified using a panel of markers designedto 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 PDXFresh colorectal tumors were obtained from consenting

patients at Concord Cancer Hospital in accordance with pro-tocols approved by the Institutional Review Board. Tumorswere transplanted into severe combined immunodeficient(SCID) female mice aged 4 to 8 weeks (InVivos Singapore)under the approved protocol by the Institutional Animal Careand Use Committee. Tumor specimens were cut into 2 to 4mm3 pieces and implanted subcutaneously in the left flank ofthe mice. PDX was maintained by passaging into subsequentgenerations when tumor volumes reached approximately 1,000to 1,500 mm3. For treatment studies, tumors were expanded inthe left flanks of 5 to 12 mice (at least 5 evaluable tumors perarm). Mice were grouped into three arms: (i) control, (ii)metformin, or (iii) metformin and 5-FU when tumor volumesreached 100 to 200 mm3. Mice were treated daily with met-formin dissolved in drinking water (150 mg/kg, orally) and/orweekly with 5-FU (25 mg/kg, intraperitoneally) for 24 days.Mice were monitored daily for signs of toxicity and tumor sizewas evaluated twice weekly by caliper measurements using thefollowing formula: tumor volume inmm3¼ [length�width2]/2.

Tumor growth inhibition (TGI) was calculated as shown: TGI ¼[(tumor volume of control on day 24 � tumor volume of treatedon day 24)/(tumor volume of control on day 24� tumor volumeof control on day 0)] � 100.

At endpoint, tumors were harvested. A portion of each tumorwas fixed for histologic sections and hematoxylin and eosin(H&E) staining, while the remaining portions were snap-frozenor used for organoids derivation.

Organoids derivation and cellular respiration assayTumors from controlmicewere used for ex vivomeasurement of

cellular respiration. After removal of necrotic tissue, tumors werewashed in HBSS, minced and digested in digestion media (RPMImedia with 5X Penicillin-Streptomycin-Amphotericin B, 10mmol/L HEPES, 300 U/mL Collagenase, 100 U/mL Hyaluroni-dase, and 10 mmol/L Y-27632) for 2 hours at 37�C. Digestionmedia were removed and digested tissues were resuspended inculture 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/LY-27632), and filtered through 100- and 40-mm filter. Organoidswere collected from the 40- to 100-mm fraction, and the numberswere estimated.

Oxygen consumption rate (OCR) of organoids were measuredusing Seahorse Bioscience XF96 analyzer as described previously(16). Five hundred organoids were seeded overnight in a 96-wellcartridge and pretreated with 0.1 mmol/L or 1.0 mmol/L met-formin for 24 hours. Before measurement, the cells were washedandmedium was replaced. Reagents from the Cell Mito Stress Kit(Seahorse Bioscience) were injected into each well sequentiallyand serve as control.

Next-generation sequencingA customized Ion AmpliSeqColon Panel was designed to

amplify 513 regions covering 40 genes implicated in colorectalcancer (Supplementary Table S1). Briefly, the customized panelwas designed based on themultidimensional analysis of genomicprofiles in colorectal cancer published by The Cancer GenomeAtlas Network (17) and molecular subtype signatures (18–20).Hotspot mutations in genes of interest and other genes related toprobable actionable drug targets were identified and ranked. Tennanograms of DNA was used to generate libraries using the IonAmpliseq library preparation kit v2.0. The barcoded libraries werediluted to 7 pmol/L for template preparation using theOneTouch2 instrument and Ion PGMTemplate OT2 200 Kit. The resultingpooled librarieswere quality control checkedusing the Ion Spherequality control kit, and were then sequenced on the Ion TorrentPGM using a PGM 200 sequencing kit v2 and 318 Chip v2 (LifeTechnologies).

NGS variant analysisPointmutationswere identifiedwith the Torrent Suite Software

v3.0 and the Ion Torrent Variant Caller v4.0 Plugin using thesomatic high stringency parameters and the targeted and hotspotpipelines. A 5% allele frequency threshold and 500� minimumcoverage was set to report de novo mutations. All the variantsidentified were established by visualizing the data through IGV2.3 (Broad Institute). Concurrently, orthogonal verification ofvariant calls made was performed using the iCMDB (VishuoBiomedical) platform which provides variant annotation, as wellas Sanger sequencing for variant validation.

Mohamed Suhaimi et al.

Mol Cancer Ther; 16(9) September 2017 Molecular Cancer Therapeutics2036




Western blottingOne 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. Membraneswere blocked with 5% bovine serum albumin in Tris-bufferedsaline and incubated overnight at 4�C with 1:1,000 dilutions ofanti-phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-p70S6K (Thr389), anti-p70S6K, anti-phospho-AMPKa (Thr172),and anti-AMPKantibodies (Thermofisher).Membraneswere thenwashed and incubated with a 1:2,000 dilution of horseradishperoxidase-conjugated goat anti-rabbit secondary antibody.Immunoreactive bands were detected by chemiluminescenceusing 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 thecontrol mice after normalization to ß-actin.

Statistical analysisSample 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 ofat least 1,000 mm3. For all experiments, Mann–Whitney andKruskal–Wallis tests were used to determine the differencesbetween two groups, or three and more groups, respectively. Pvalues <0.05 were recognized as statistically significant.

ResultsGrowth inhibition of 5-FU and metformin on cancer cell lines

Metformin has been reported to preferentially inhibit thegrowth of p53-mutant cells by forcing a metabolic burden thatresult in selective toxicity (9, 21, 22). However, recent evidencesuggests that metformin requires p53 activity for metformin-induced growth inhibition (23, 24). To investigate whether met-formin affects tumorproliferation in a p53-dependentmanner,wescreened a panel of colorectal cancer cell lines with known p53statuses (Table 1), including a pair of isogenic cell lines HCT-116(p53-WT and p53-KO). Increasing concentration of 5-FU atmicro-molar levels results in a growth inhibition of all colorectal cancercells (Fig. 1A) while supraphysiologic concentrations of metfor-min were required to achieve 50% cell growth inhibition (relativeto 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 andp53-KO cells were similar uponmetformin treatment (P¼ 0.743);nonetheless, cell growth for both lines were severely impairedunder glucose-deprived conditions (Fig. 1C and D).

Approximately 15% of colorectal cancers displaymicrosatelliteinstability and are classified as microsatellite instable (MSI) ormicrosatellite stable (MSS). Characterization of colorectal cancercells for MSI/MSS status indicates that there was no difference inresponse to metformin between MSI and MSS cell lines. Essen-tially, our results indicate that there is non-differential growthinhibition tometformin, regardless ofmicrosatellite or p53 status.

PDX generationTwo 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 SCIDmice. Growth rates ranged from 8 to 15 weeks for the initialtransplantation to reach a tumor volume of 1,000 to 1,500mm3, and 3 to 8 weeks for subsequent passages. Histopathol-ogy of patient's primary tumors and various xenograft passagesshowed that the PDX models retained the original morphologyof the human primary tumor (Fig. 2A).

Non-differential inhibition of metformin on PDXTo evaluate tumor response in vivo, we utilized twoPDXmodels

withdiffering p53 andMSI/MSS status (Table 1).Mice treatedwithmetformin had a lower tumor burden as compared with controlgroup, regardless of p53 nor microsatellite status (Fig. 2B–D).Metformin was able to slow tumor growth initially, but at 12 daysposttreatment initiation, PDX tumors eventually progressed. After24 days of drug treatment, the average tumor volume of themetformin-treated group was reduced by 50% (P ¼ 0.056) and65% (P < 0.05) for p53-wild-type and p53-mutant PDX, respec-tively. A systemic effect ofmetformin on cell growthwas observedin the PDXs independent of tumor p53 status, with more pro-nounced growth inhibition being observed in the PDXs withmutant-p53.

When metformin was administered concurrently with a clin-ically acceptable dose of 5-FU, only an additional 4% (P¼ 0.767)tumor inhibition was observed in PDX-FIT-CRC-104; in contrast,a pronounced tumor growth inhibition of 85% (P ¼ 0.054) wasobserved in PDX-FIT-CRC-086.

Metformin activates the AMPK pathwayTo determine whether metformin influenced the AMPK and

mTOR signaling pathway in the PDX materials, we evaluated thephosphorylation of AMPK, mTOR, and p70S6K (Fig. 2E). Met-formin treatment activated AMPK as determined by phosphory-lation at Thr172, with a corresponding inhibition of mTORsignaling and downstream target phosphorylation of p70S6K. Intumors treated with metformin and 5-FU, AMPK was activated;interestingly, mTOR signaling appears to be activated and notsuppressed, 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). Drugsaffecting mitochondrial functions were used in parallel to dem-onstrate normal mitochondrial function. Metformin inhibitedOCR of both FIT-CRC-086 and FIT-CRC-104 (Fig. 3B and C,respectively) in a concentration-dependent manner. Consequentinjection of oligomycin did not suppress the OCR further in thesemetformin-treated organoids, while injection of FCCP, the

Table 1. p53 and microsatellite status of cell lines and PDX

Sample p53 status MSI/MSS

DLD-1 Mutant MSIHCT116 Wild-type MSSSK-CO-1 Wild-type MSSSW-480 Mutant MSSPDX FIT-CRC-086 Mutant MSSPDX FIT-CRC-104 Wild-type MSI

Metformin Inhibits Growth of Colorectal Cancer PDX

www.aacrjournals.org Mol Cancer Ther; 16(9) September 2017 2037




Figure 1.

In vitro effects of 5-fluorouracil (5-FU) andmetforminon colorectal cancer cells. 5-FU, the first-linetreatment for colorectal cancer, inhibits cellproliferation atmicromolar levels (A). Millimolar levelsof metformin are required to inhibit cell proliferationat 48 hours, with pronounced inhibition observed incombination with 10 mmol/L 5-FU (B). Glucosedeprivation accentuates growth inhibition bymetformin (C). Inhibition of colorectal cancer cellproliferation to 5-FU (D) and metformin isindependent of p53 status, in both HCT116 p53knockout (p53 KO) and HCT116 p53 wild-type (p53WT) cell lines, but with stronger degree of growthinhibition observed under glucose-deprivedconditions in the presence of metformin (E). Theabsolute number of colonies is presented as anaverage of triplicates (F).






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 respirationresulted in an increase in glycolysis, as measured by the extracel-lular acidification rate (ECAR; Fig. 3D and E). It was evident thatmetformin inhibits mitochondrial function as early as 4 hours(Fig. 3F and G).

Molecular profiling of circulating biomarkers in miceTo determine if tumor burden in vivo can be assessed using

alternative approaches that are viable in the clinical setting, weinvestigate the use of cfDNA in our models (25). CfDNA is aheterogeneous mixture of normal and tumor-associated cellpopulations (26, 27) which makes cfDNA analysis technicallychallenging. In xenografts, both human and mouse-derived cellpopulations are present, and the ability to distinguish these twocell populations is critical in order to assess the PDX response totreatment using cfDNA. Here, using a limited amount ofcfDNA, our assay can distinguish human and mice-derivedDNA efficiently (Supplementary Fig. S1), with limit of detec-tion of 10% human DNA in a background of mice DNA. NocfDNA was detected at week 0, at the point of tumor implan-tation. Subsequent evaluation of the cfDNA at different timepoints in the course of treatment indicates that human-derivedDNA could be detected in the mice blood, suggesting thatcfDNA could be a viable noninvasive biomarker for tumorburden.

Mutation profile of primary tumor and correspondingxenografts

Human samples and PDX were analyzed for variants/muta-tions by NGS using a panel of 40 genes commonly implicated incolorectal 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 parentaltumor and xenografts, but were not present in germline or normalcolon. Likewise, in FIT-CRC-104, mutations were observed inKRAS (A146T) andMLH1 (R100�) in parental tumor. Essentially,mutations remain conserved across PDX passages. An example ofa variant conserved in tumor and PDX as depicted by NGS arecorroborated by Sanger sequencing in Fig. 4B. Interestingly, wealso observed the presence of variants that are not reported inCOSMIC 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 sametreatment subset, individuals exhibit heterogeneity. Whetherthese new variants were a function of clonal selection of a subsetof tumor cells warrants further investigation.

One of the key objectives of performing NGS is to identifybiomarkers that may reflect response to metformin. Our analysis

indicates that there are no key colorectal cancer genes that areassociated with metformin's activity.

DiscussionMetformin 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, weobserved that the antiproliferative properties of metformin man-ifest at supraphysiologic conditions in cell lines (30). A recurrentfeature is that these cells are typically maintained in non-phys-iologic conditions that are optimized only for in vitro growth andproliferation. The excessive concentration of growth factors, insu-lin and glucose may account for the elevated doses of metforminrequired to elicit cellular responses and triggermetabolic stress. Assuch, it is virtually impossible to translate these supraphysiologicconditions in the clinical setting, especially when the pharmaco-kinetics of the drug is different in culture settings and in thehuman 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 tumormicroenvironment caused by the imbalance between nutrientsupply and oxygen consumption rate (31). In a study exploringthe role of the tumormicroenvironment in potentiating the effectof metformin, metformin is synthetically lethal with glucosewithdrawal in cancer cells (32). Essentially, this suggests that theclinically irrelevant, supraphysiologic levels needed to observemetformin's anticancer effects as reported bymany in vitro studiesmerely underlie the artefactual experimental conditions thatpoorly reflect actual glucose-starved in vivo conditions.

Most preclinical in vivomodels 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 andheterogeneity of human tumors. PDX, where surgically resectedtumor samples are engrafted directly into immunocompromisedmice, offer several advantages (33, 34). In our study, we haveshown that PDX tumors maintained the molecular, genetic, andhistologic heterogeneity typical of tumors of origin (35–39). PDXprovides an excellent platform to study cancer biology, analyzecancer therapeutics, and novel drug combinations. In our study,we demonstrated metformin efficacy in our PDX models. To thebest of our knowledge, ours represents the first study reported forcolorectal cancer PDX using metformin. Administering metfor-min at physiologic levels of 150mg/kg per day in ourmice, whichis equivalent to initial clinical dose of 500 to 1,000 mg/daily inhuman, is sufficient to inhibit tumor growth inPDX. Furthermore,we observed that the response to metforminmay be independentof p53 status and is inconsistent with the observations reportedearlier (9, 24). Conversely, studies conducted in breast cancerindicate that metformin's effects are independent of p53 status(40, 41). This contraindication suggests that p53 may not be areliable companion biomarker to predict response to metformin.

Table 2. Clinicopathologic information of patients with xenografts generated

Patient HistologyTumorsite

Tumorgrade

Tumortype

Tumor size(largest dimension in cm)

AJCCstage pT pN pM

Lymph nodeinvasion

FIT-CRC-086 Adenocarcinoma Rectum 1 Ulcerated 4 IVA 3 0 1a 0/7FIT-CRC-104 Mucinous adenocarcinoma Ascending colon 2 Localized 5 IIA 3 0 0 0/39






Studies from a recent phase II clinical trial evaluating metfor-min and 5-FU in patients with refractory colorectal cancer showedonly amildmedian progression-free survival (PFS) of 1.8monthsand median overall survival (OS) of 7.9 months, with a majorityof patients (78%) having disease progression before 8weeks (42).For 11 out of 50 patients (22%) having stable disease at the end of8 weeks, their median PFS and OS were 5.6 months and 16.2months, respectively. Likely, the modest benefits of metforminand 5-FU observed in this trial may be due to the nature of the

refractory colorectal cancer cases and that the benefits may besignificant in nonrefractory colorectal cancer cases.

Interestingly, in our study, we observed that combination of5-FUwithmetformin leads to a pronounced growth inhibition inthe p53-mutant PDX but not in p53-wild-type PDX. It may bepossible that p53 status plays a role in differential response tometformin 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

Figure 2.

Histomorphologic features of parental tumor are retained in patient-derived xenografts after serial passaging. Representative tumor cells are indicated by solidarrows; mucins are indicated by diamond arrowheads (A). Gross morphology of PDX-FIT-CRC-086 tumors treated with metformin and 5-FU (B). Tumor growthprofile 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 werenormalized to ß-actin and fold changes are quantified relative to vehicle control (F).






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OXYGEN CONSUMPTION RATEECAR vs TIME (Avg)






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 hoursinhibits oxygen consumption rate (OCR) relative to vehicle control for both FIT-CRC-086 (B) and FIT-CRC-104 (C), with corresponding increase in glycolysisfor 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 bothFIT-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/Lrotenone/antimycin A were used as mitochondrial stress controls.






deficient mismatch repair system or MSI do not benefit from5-FU–based adjuvant therapy in colorectal cancer (43, 44). Thus,our observations using PDX are in line with observationsobtained in the clinic, thus, emphasizing the crucial role PDXsplay as a clinically relevant model.

To demonstrate the integrity of PDX as suitable models, weperformed high-throughput NGS to characterize the genetic pro-files of parental tumor and PDX. We demonstrate that genes suchas TP53, APC, KRAS, and SMAD4, were mutated in the parentaltumor and conserved throughout serial passaging of the xeno-grafts. More importantly, in an effort to characterize biomarkersthat can serve as companion marker to predict metforminresponse, we compared the genetic differences between the twoPDX lines. Our NGS analysis suggests that there is lack of novelactionable genes that may reflect metformin's activity based ongenotype. Nonetheless, the key limitation in this particular anal-ysis is likely due to limited variation in genotype between the twoPDX lines.

To gain insight into possible mechanisms of metforminaction, we evaluated the frequently implicated AMPK signalingpathway (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 proteinsynthesis (46). Moreover, we observed that metformin inhibitsoxygen consumption in organoids derived from PDX, as earlyas 4 hours, with a concomitant increase in glycolysis. Takentogether, it is likely that treatment with metformin activates theAMPK pathway, which alters the mTOR signaling, and executesa metabolic change in the cells, which ultimately affects cellgrowth in the PDX.

We acknowledged that there are several limitations to ourcurrent study. Firstly, we restricted the use of PDX to subcuta-neous tumor models. Subcutaneous models are inferior toorthotopic models because the former do not represent appro-priate sites for human tumors, and may not be accuratelypredictive for drug evaluation (47). However, orthotopic tumormodels 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 thetumor. In anticipation of this limitation (should the need ariseto monitor tumor burden in orthotopic models), we assessednoninvasive circulating biomarkers (cfDNA) in our subcutane-ous PDX models. Using established techniques that we haveutilized previously for clinical cancer patients (48, 49), wedemonstrated that it is feasible to prove the presence ofhuman-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 pilotstudy involving metformin and PDX, banking on molecularprofiles derived from 2 colorectal cancer patients may be inade-quate for biomarker discovery process. Expanding the repertoireof PDX lines with different genetic make-up is likely to shedmoreinformation in future.While the creationof PDX is of clear interestas ameans to better define optimal therapy for colorectal cancer, itis undeniable that the high-throughput data are challenging tomanage and visualize. Thoughtful interpretation is necessary toconvert the knowledge derived from existing data in order tounderstand treatment outcomes.

In essence, our study demonstrated the utility of PDX inassessing drug efficacy viamolecular analysis,metabolic profiling,and bioinformatics approaches. This is the first study thatdescribes the use of metformin in colorectal cancer PDX andorganoids, providing direct evidence of metformin as an antican-cer agent in colorectal cancer.

Disclosure of Potential Conflicts of InterestM.-H. Tan has ownership interest in a patent filed for method for KRAS

mutation detection, owned by his employer, and licensed to industry. Nopotential conflicts of interest were disclosed.

Authors' ContributionsConception and design: N.-A. Mohamed Suhaimi, W.M. Phyo, H.Y. Yap,M.-H. TanDevelopment ofmethodology:N.-A.Mohamed Suhaimi, W.M. Phyo,W.J. TanAcquisition 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

Figure 4.

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






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. TanWriting, 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. TanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): W.J. Tan, L.A.P.Y. Tan, Z. Tiang, C.F. Wong,M.-H. TanStudy supervision: M.-H. Tan

AcknowledgmentsWe would like to thank Concord Cancer Hospital for the provision of the

tissue samples, and Vishuo Biomedical for the support in the bioinformaticsanalysis. We would like to thank Chua Teck Chiew Timothy, Ma Janise AnnKabiling 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

like to thank Chit Fang Cheok for the helpful discussion on the in vitrowork andShengyong Ng for the assistance in organoids derivation and culture.

Grant SupportThis work is supported by the Institute of Bioengineering and Nanotech-

nology and Agency for Science, Technology and Research, Singapore. M.H. Tanreceived 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 thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 22, 2016; revised March 29, 2017; accepted May 17,2017; published OnlineFirst May 22, 2017.

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2017;16:2035-2044. Published OnlineFirst May 22, 2017.Mol Cancer Ther Nur-Afidah Mohamed Suhaimi, Wai Min Phyo, Hao Yun Yap, et al.

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Metformin Inhibits Cellular Proliferation and Bioenergetics in ... · Bioenergetics in Colorectal Cancer Patient– Derived Xenografts Nur-Aﬁdah Mohamed Suhaimi1,Wai Min Phyo1,