© 2018. Published by The Company of Biologists Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License

(http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction

in any medium provided that the original work is properly attributed.

Synergistic anti-proliferative effect of mTOR inhibitor (rad001) plus gemcitabine on

cholangiocarcinoma by decreasing choline kinase activity

Gigin Lin, MD, PhD,1* Kun-Ju Lin, MD, PhD,2* Frank Wang, MClinEpid, FRACS4, Tse-Ching

Chen, MD, PhD,3 Tzu-Chen Yen, MD, PhD,2 and, Ta-Sen Yeh, MD, PhD4

Department of Medical Imaging and Intervention, Imaging Core Lab, Institute for Radiological

Research, Clinical Metabolomics Core Lab,1 Nuclear Medicine and Molecular Imaging Center,2

Pathology,3 and Surgery,4 Chang Gung Memorial Hospital at Linkou, Chang Gung University,

Taiwan

*Both had an equal contribution

Correspondence and reprint requests should be addressed to: Ta-Sen Yeh, MD, PhD

Department of Surgery, Chang Gung Memorial Hospital, 5 Fu-Hsing Street, Kwei-Shan Shiang,

Taoyuan, Taiwan. Tel, 886-3281200 Ext, 3219 Fax, 886-3-3285818

e-mail, tsy471027@adm.cgmh.org.tw

Keywords: Cholangiocarcinoma, choline kinase, Fas, gemcitabine, Rad001

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http://dmm.biologists.org/lookup/doi/10.1242/dmm.033050Access the most recent version at First posted online on 12 April 2018 as 10.1242/dmm.033050

Summary Statement

Rad001 plus gemcitabine exerts a synergistic antitumor effect on cholangiocarcinoma irrespective

of k-ras status, with underlying mechanisms involving activation of the death receptor,

mitochondrial pathways, and down-regulated choline kinase activity.

Abstract

Although gemcitabine plus cisplatin is the gold standard chemotherapy regimen for advanced

cholangiocarcinoma, the response rate has been disappointing. This study aims to investigate a

novel therapeutic regimen (gemcitabine plus rad001, an mTOR inhibitor) for cholangiocarcinoma.

Gemcitabine, oxaliplatin, cetuximab, and rad001 in various combinations were first evaluated in

vitro using six cholangiocarcinoma cell lines. In vivo therapeutic efficacies of gemcitabine, rad001

alone and combination were further evaluated using a xenograft mouse model and a chemically

induced orthotopic cholangiocarcinoma rat model. In the in vitro study, gemcitabine plus rad001

exhibited a synergistic therapeutic effect on the cholangiocarcinoma cells irrespective of the k-ras

status. In the xenograft study, gemcitabine plus rad001 showed the best therapeutic effect on tumor

volume change, which was associated with an increased caspase-3 expression, a decreased eIF4E

expression, as well as overexpression of both death receptor and mitochondrial apoptotic pathway-

related genes. In a chemically-induced cholangiocarcinoma-afflicted rat model, the gemcitabine

plus rad001 treatment suppressed tumor glycolysis as measured by 18F FDG micro-PET (positron

emission tomography). Also, an increased intra-tumoral free choline, a decreased

glycerophosphocholine and nearly undetectable phosphocholine levels were demonstrated by

proton NMR (nuclear magnetic resonance), supported by a decreased choline kinase expression on

Western blotting. We concluded that gemcitabine plus rad001 has a synergistic anti-proliferative

effect on the cholangiocarcinoma irrespective of the k-ras status. The antitumor effect is associated

with the flare-ups of both death receptor and mitochondrial pathways, as well as the down-

regulation of the choline kinase activity, resulting in a characteristic change of choline metabolism. D

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Introduction

Cholangiocarcinoma is a malignant biliary tract tumor that carries a very poor prognosis (de Groen

et al., 1999; Khan et al., 2005). Although uncommon worldwide, its incidence reaches 87 per

100,000 in the endemic areas including Israel, Japan, and South Eastern Asian countries such as

Taiwan (Rajagopalan et al., 2004; Yeh et al., 2005). Surgical resection is the only treatment

modality that offers a potential cure. Unfortunately, resection rates are low at the initial diagnosis

because of early intrahepatic spread, nodal involvement, vascular invasion, and distant metastasis.

Other therapeutic strategies including chemotherapy, radiation therapy, transplantation, and

photodynamic therapy have been developed, but none has shown significant survival benefit in

patients with advanced cholangiocarcinoma (de Groen et al., 1999; Khan et al., 2005). Gemcitabine,

with or without platinum compounds, is currently the most effective chemotherapy regimen for

advanced biliary tract cancer, although the response rate is only about 20%(Andre et al., 2004;

Eckel and Schmid, 2007; Valle et al., 2010; Valle et al., 2009). In addition, epidermal growth factor

receptor (EGFR) is activated by bile acids and has a role in the carcinogenesis of

cholangiocarcinoma through the induction of cyclooxygenase-2 expression via an MAPK cascade.

Accordingly, neutralizing antibodies that target the extracellular domain of EGFR (e.g., cetuximab)

have sporadically shown clinical efficacy in advanced cholangiocarcinoma (Paule et al., 2007).

Mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that regulates cell

growth and metabolism. mTOR pathway is dysregulated in various cancers including

cholangiocarcinoma (Chung et al., 2009; Ma and Blenis, 2009), making mTOR an important target

for the development of new anti-cancer drugs (Faivre et al., 2006). Everolimus (rad001), a 40-O-(2-

hydroxyethyl) derivative of rapamycin, conjugates with FK binding protein-12 (FKBP-12) to

inhibit the activation of mTOR. Although rad001 exhibits antitumor activity both as a single oral

agent and in combination with other anticancer agents (Mabuchi et al., 2007; Majumder et al., 2004;

Mondesire et al., 2004; Motzer et al., 2008; Piguet et al., 2008; Yan et al., 2006; Zhang et al., 2009),

its therapeutic effect on cholangiocarcinoma has yet been clarified.

Phosphocholine (PC) is both a precursor and a breakdown product of phosphatidylcholine, the most

abundant phospholipid in the biological membranes. Increased levels of PC and total choline, as

detected by 1H or 31P magnetic resonance spectroscopy (MRS), are characteristics of cancer cells

including breast, prostate, colon, lung, ovarian, and brain tumors (Ackerstaff et al., 2003; Iorio et al.,

2005; Venkatesh et al., 2012). The significance of these studies is that specific changes in the levels

of the phospholipid precursors and catabolites in the context of solid cancers might be a useful

biochemical indicator of tumor progression andor treatment response.

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This study aims to evaluate the therapeutic effects of rad001 and gemcitabine on

cholangiocarcinoma and elucidate the underlying molecular and metabolic mechanisms underlying

their antitumor activity.

Results

Selection of optimal drug regimens for cholangiocarcinoma cells

The corresponding proliferation indices of the six cholangiocarcinoma cell lines treated with

standard IC50 dosages of various therapeutic regimens are shown in Figure 1A. Gemcitabine

exhibited superior therapeutic efficacy against all cholangiocarcinoma cell lines as a single-agent

therapy compared with oxaliplatin, cetuximab, and rad001. Gemcitabine plus rad001 was the most

promising combination therapeutic regimen in comparison with gemcitabine plus oxaliplatin and

gemcitabine plus oxaliplatin plus cetuximab. The median effects analysis demonstrated that rad001

and gemcitabine had a synergistic effect on both k-ras mutation (HuCCT1, RBE) and wild-type

(TFK-1, YSCCC) cell lines with a CI < 1 (Figure 1B).

Rationale of mTOR inhibition for treatment of cholangiocarcinoma

To assess the pharmacological effect of rad001 and gemcitabine, four treatment groups were

assigned in the following in vitro and in vivo experiments: control, gemcitabine-treated (Gem),

rad001-treated (Rad), and gemcitabine plus rad001-treated (Gem+Rad) groups. In the in vitro study,

phosphorylation of p70S6K, but not p-p38MAPK, was significantly decreased in the Rad and

Gem+Rad groups in both HuCCT1and TFK-1 cells, indicating that Gem and Gem+Rad specifically

inhibited the downstream mTOR signaling irrespective of the k-ras status. Gem might not

synergistically enhance the effect of Rad to inhibit mTOR (Figure 1C). Regarding cell cycling, a

smaller percentage of HuCCT1 cells entered the G2/M phase in the Gem+Rad group compared with

the Gem group (10.2% vs. 13.6%, P < 0.05, Figure 1D, Table 1). Furthermore, the percentage of

apoptotic HuCCT1 cells in the Gem+Rad group was significantly increased compared with the

Gem group (30.1% vs. 4.4%, P < 0.01, Figure 1E).

Evaluation of therapeutic response using a xenograft model

Xenograft experiments were conducted to evaluate the measurable volumetric changes of the

cholangiocarcinoma treated with various drug regimens. The representative therapeutic response of

HuCCT1 xenograft model after various treatments for three weeks is shown in Figure 2A. The

therapeutic responses of HuCCT1 and TFK-1 xenograft models treated for three weeks were

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analyzed (Figure 2B). Gem + Rad regime conferred the most promising therapeutic response in

both HuCCT1 and TFK-1 xenograft models. Representative images of the caspase-3 and eIF4E

expression in the HuCCT1 xenograft are shown in Figure 2C. The expression of caspase-3 in the

control, Gem, Rad, and Gem + Rad groups was 2.1±0.7, 9.4±1.6, 5.1±1.1, and 14.5±2.3 /HPF,

respectively (P < 0.01). In contrast, the expression of eIF4E in the control, Gem, Rad, and Gem +

Rad groups was 14.2±1.3, 8.9±1.5, 11.3±2.3, and 4.6±0.7, respectively (P < 0.01).

Altered apoptotic signaling after gemcitabine and rad001 combination therapy

Based on the xenograft model, the percentage of Fas-positive HuCCT1 cells in the control, Gem,

Rad, and Gem+Rad groups as detected by flow cytometry were 13.7%, 30.6%, 23.6% and 44.5%,

respectively (P < 0.01), and the intensity of Fas-positive HuCCT1 cells was 7.2, 9.5, 8.4, and 10.5,

respectively (P < 0.01, Figure 3A). The expression of 6 apoptotic genes including Fas, caspase-8,

BID, APAF-1, XIAP, and caspase-3 using qPCR-derived from the treated HuCCT1 xenograft is

shown in Figure 3B. The expression of these six genes was modestly increased in the Gem and Rad

groups compared with the control group, whereas the expression of all six genes was significantly

increased in the Gem+Rad group.

Metabolic response of orthotopic cholangiocarcinoma rat model as detected by18F FDG-

microPET and 1H NMR

The representative in vivo metabolic response of cholangiocarcinoma-afflicted rats to various

treatments as detected by FDG-microPET is shown in Figure 4A. The Gem+Rad group had the

most significant metabolic response after two cycles of treatment (Figure 4B). The expression of

Glut-1, HK-II, HIF-1α, and VEGF mRNAs of the corresponding surgical specimens as detected by

qPCR is shown in Figure 4C. Of these, the expression of HK2 and Glu 1 mRNA in the Gem + Rad

group was reduced by half in comparison with the control group. The difference in the expression

of these genes correlated with the SUV changes on the FDG-PET. The tumor response in the

cholangiocarcinoma-afflicted rats was confirmed by histopathological examination. Results derived

from the histomorphological score and Ki-67 labeling index of the corresponding surgical specimen

also supported the findings in the FDG microPET study (Table 2).

The alteration of choline-associated metabolites in the treated cholangiocarcinoma-afflicted rats

was analyzed by 1H NMR, with a particular emphasis on the free choline, phosphocholine (PC) and

glycerophosphocholine (GPC) levels (Figure 5A). A significant increase in the free choline level of

the Gem+Rad group was associated with a dramatic disappearance of PC and a significantly

reduced level of GPC (Figure 5B). In contrast, the Gem group exhibited a significant increase in

the free choline level without significant changes of the PC and GPC levels. No significant change

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of the choline-associated metabolites was observed in the Rad group. Choline kinase (CK), an

enzyme that catalyzes the phosphorylation of free choline using ATP to produce PC and plays a

rate-limiting regulatory role in the phosphatidylcholine biosynthesis, was assayed to determine the

underlying mechanism that was responsible for the observed changes in the PC levels. Of the four

treatment groups, the CK level detected by Western blot was lowest in the Gem + Rad group

(Figure 5C-D).

Discussion

The presence of tyrosine kinase-Akt-mTOR axis in the carcinogenesis of cholangiocarcinoma

(Chung et al., 2009) has provided us with an opportunity to evaluate the therapeutic role of an

mTOR inhibitor, rad001, in the management of this disease. We have shown, in this study, that

rad001 resulted in a reduction of expression of the downstream molecules of mTOR such as

p70S6K-1, but not p38MAPK. Furthermore, it has been demonstrated for the first time that

gemcitabine plus rad001 exhibited a synergistic antitumor effect on cholangiocarcinoma

independent of the k-ras status. In contrast, the EGFR monoclonal antibody, cetuximab, only had a

modest therapeutic effect on the k-ras wild-type cholangiocarcinoma xenograft (TFK-1) but not the

k-ras mutated xenograft (HuCCT1). This finding is consistent with the previous clinical experience

on colorectal and non-small cell lung cancers (NSCLC).(Bardelli and Siena, 2010; Massarelli et al.,

2007) The in vivo therapeutic effect of gemcitabine plus rad001 was macroscopically evaluated and

confirmed by measuring changes in the tumor volume in the xenograft model, and assessing the

metabolic response in the orthotopic rat cholangiocarcinoma model using 18F FDG microPET.

Besides, the downstream molecules of mTOR (Ki 67, HIF-1, HK-II, Glut-1, and VEGF) were all

attenuated in the Rad + Gem group. This observation had been nicely reflected by the changes in

SUV on the18F-FDG microPET.

The available evidence in the literature suggests that gemcitabine primarily hits an intrinsic

(mitochondrial) apoptotic pathway in the NSCLC and pancreatic cancer to achieve its

pharmacological effect. Gemcitabine-mediated activation of caspase-8 is a late and mitochondrial-

dependent event which is independent of Fas-FasL signaling (Ferreira et al., 2000; Salvesen and

Duckett, 2002). Que et al. reported that cholangiocarcinoma cells synthesized FasL and Fas and that

FasL expressed by these cells induced host’s lymphocyte cell death (Que et al., 1999). Shimonishi

et al. further demonstrated up-regulation of FasL in the early stages and down-regulation of Fas in

the advanced stages of cholangiocarcinoma, the latter of which was responsible for evasion from

host immunity and disease progression (Shimonishi et al., 2000). To understand the mechanism by

which rad001 and gemcitabine acted synergistically to enhance apoptosis in the

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cholangiocarcinoma cells, we examined six key apoptotic genes that were involved in the extrinsic

and intrinsic cell death pathways. These included Fas, caspase-8, BID, APAF-1, XIAP, and caspase-

3. Among these 6 genes, the former two represent the death receptor (extrinsic) pathway, whereas

the latter three represent the mitochondrial (intrinsic) pathway, with both pathways intermediated by

BID (Salvesen and Duckett, 2002). Of particular importance, XIAP, an X-linked inhibitor of

apoptosis, is thought to directly inhibit certain caspases such as caspase-3, caspase-7 and caspase-9.

Shrikhande et al. showed that XIAP was overexpressed in pancreatic cancer and contributed to

chemoresistance (Shrikhande et al., 2006). Our data revealed that both death receptor-related (Fas

and caspase-8) and mitochondrial pathway-related genes (Apaf-1 and caspase-3) in the Gem + Rad

group were flared up by 10-folds in comparison with those of the Gem group, indicating that both

death receptor and mitochondrial pathways were recruited and co-activated by the combination

therapy. It is noteworthy that XIAP was simultaneously increased up to 10-fold in the Gem+Rad

group along with the pro-apoptotic genes in comparison with the Gem group. Our findings

suggested that XIAP plays a major role in arresting the apoptotic process and potentially

contributing to drug resistance.

Early detection of response to therapy is desirable but not easily achieved using the conventional

imaging techniques such as computed tomography or magnetic resonance imaging (MRI).

Biological cancer treatment might be associated with stagnation of cellular proliferation or cellular

clonal changes rather than obvious tumor shrinkage in the early stage of therapeutic response.

Cancer metabolism has emerged in recent studies as a powerful link between deregulated signaling

pathways and altered cellular metabolism. Specific endogenous metabolites and metabolic fluxes

within cells and tissues in vivo and in vitro can be readily identified using 31P, 1H, or 13C MRS. Of

them, derangement in the choline metabolism during carcinogenesis and treatment has been

increasingly recognized in various solid cancers (Ackerstaff et al., 2003). Choline kinase (CK)

catalyzes the phosphorylation of choline to yield PC in the biosynthesis of phosphatidylcholine, a

process known as the Kennedy pathway (Ackerstaff et al., 2003; Ramirez de Molina et al., 2008).

The enzyme is modulated by Ras and interacts with HIF 1- Ramirez de Molina et al., 2002a

It may also play a role in the cell cycle regulation (Gruber et al., 2012), MAPK and PI3K/Akt

signaling pathways (Yalcin et al., 2010), as well as acting as a potential biomarker for various solid

cancers (Ramirez de Molina et al., 2002b). Specific inhibitors against CK are to be anticipated soon

in the clinical trials as these agents have shown promising antitumor effects in the preclinical

studies (Ackerstaff et al., 2003; Al-Saffar et al., 2010; Ramirez de Molina et al., 2008; Ramirez de

Molina et al., 2002a). In the present study, we have implemented 1H NMR to detect alterations of

choline-associated metabolites in the treated cholangiocarcinoma-afflicted rats. The down-regulated

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CK activity, presenting as an accumulation of free choline and nearly undetectable level of PC in

the GEM + Rad group, was confirmed by a significantly decreased expression of CK on Western

blot. Such down-regulation of CK following mTOR inhibition was associated with HIF-1 and

PI3K/Akt /mTOR pathways, with the down-regulation of HIF-1 mRNA correlating to the

respective CK levels in the Gem+ Rad, Gem, and control groups. In line with our study, Glunde et

al. had demonstrated a similar process of CK regulation by the HIF-1 signaling in human prostate

cancer (Glunde et al., 2008). Furthermore, using a PI3K inhibitor, PI-103, Al-Saffar et al. were able

to demonstrate down-regulation of the CK levels in the prostate and colon cancers with a

corresponding decrease in the PC levels (Al-Saffar et al., 2010). Again, by silencing CK using

siRNA, Yalcin et al. showed that PC was reduced through the PI3K/ Akt/mTOR pathways, leading

to the inhibition of cervical cancer cell growth (Yalcin et al., 2010).

The change of GPC level after biological agent treatment is another interesting issue. Al-Saffar et al.

reported that blockade of PI3K with LY294002 (a relatively less potent, non-selective inhibitor of

PI3K) was associated with a decrease in the PC level as well as an elevation in the GPC content in

human breast cancer cells (Beloueche-Babari et al., 2006). In contrast, the authors noted that both

PC and GPC levels were below the detection limit of MRS when the HCT116 colon cancer cells

were treated instead with PI-103, a potent selective inhibitor of class 1 PI3K (Al-Saffar et al., 2010).

Accordingly, they concluded that the contradictory response of the GPC levels following LY294002

and PI-103 treatments was probably related to other off-target effects of the former inhibitor. In our

study, the characteristic profile of the choline metabolites in the cholangiocarcinoma cells treated by

gemcitabine plus rad001 was most likely a result of a potent and selective targeting against the

PI3K/Akt/mTOR signaling pathway. As there is an increase in choline and a decrease in its

downstream metabolite, PC, we expect to observe an accumulation of choline signal on 11C-choline

PET when the treatment is effective. A future in vivo imaging study using 11C-choline PET or

magnetic resonance spectroscopy imaging (MRSI) would better facilitate longitudinal monitoring

of the relevant biological response.

In conclusion, our preclinical study shows that gemcitabine plus rad001 exerts a synergistic

antitumor effect on cholangiocarcinoma irrespective of the k-ras status. The underlying mechanisms

of the antitumor activity are associated with the flare-ups of the death receptor and mitochondrial

pathways as well as the down-regulation of the choline kinase activity resulting in a specific change

of choline metabolism.

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Materials and Methods

Cholangiocarcinoma cell lines

Six cholangiocarcinoma cell lines used in this study (HuCCT1, SSP-25, RBE, YSCCC, TGBC-

24TKB, and TFK-1) were purchased from RIKEN Cell Bank (Tsukuba, Japan). The HuCCT1, SSP-

25, and RBE cell lines had a k-ras mutation, whereas the remaining three cell lines were k-ras wild-

type. Throughout our experiments, our in vitro studies were triplicated at least.

In vitro drug cytotoxicity assay

Oxaliplatin (TTY Biopharm, Taiwan), gemcitabine (Gemmis, TTY Biopharm), cetuximab (Erbitux;

Boehringer Ingelheim, Germany), and everolimus (rad001; Novartis, Switzerland), alone or in

combination, were first used to treat the cholangiocarcinoma cell lines in vitro. The half-maximal

inhibitory concentration (IC50) was determined using a cell proliferation reagent, WST-1 (Roche

Applied Science, Germany).

Analysis of interactions

A commercial software package (CalcuSyn; Biosoft, Cambridge, United Kingdom) was used to

perform median effect analysis as described by Chou and Talalay (Chou and Talalay, 1984). The

combination index (CI) was calculated using the formula: CI = (D)1/(Dx)1 + (D)2/(Dx)2, whereby

CI = 1 indicated an additive effect, CI >1 an antagonistic effect, and CI < 1 a synergistic effect.

Flow cytometry

Both adherent and floating cholangiocarcinoma cells were collected 72 hours after various drug

treatments for cell cycle analysis. Cells were passed through a fluorescence activated cell sorter

(FACS Caliber; BD Biosciences, San Jose, CA), and data were acquired using CellQuest (BD

Bioscience) software. The cell cycle was modeled using ModFit software (Venty Software,

Topsham, ME). The separation of cells into G0/G1, S, and G2/M phases was based on linear

fluorescence intensity after staining with propidium iodide. Cells were labeled with the Annexin V-

FITC Apoptosis Kit (BD Biosciences-Clontech) and analyzed on a FACS Caliber to detect

apoptosis. The cell surface expression of Fas after various drugs treatments for 16 hours was

assessed. Indirect immunofluorescence staining for Fas was performed with an IgG1 mouse anti-

Fas primary monoclonal antibody (DX2 clone, BD Biosciences) and an FITC-conjugated secondary

antibody (goat anti-mouse Ig) (BD Biosciences). The percentage and intensity of Fas-positive cells

were analyzed by the FACS Caliber.

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Bio-Plex phosphoprotein assay

Cholangiocarcinoma cells were treated with various drug regimens, and protein lysates were

prepared using the Cell lysis kit (Bio-Rad). Phospho-p38MAPK and phospho-p70S6 kinase were

detected by the Bio-Plex phosphoprotein and total protein assay kits (Bio-Rad) according to the

manufacturer’s protocols.

Xenograft mice model

To initiate tumor xenograft, cholangiocarcinoma cells, HuCCT1 and TFK-1 respectively, were

implanted subcutaneously (2x106 cells) in the flank of the Fox Chase SCID (severe combined

immunodeficiency) mice (BioLASCO Taiwan Co., Ltd). Six therapeutic regimens were given as

follows: gemcitabine 200mg/kg I.P. (intraperitoneal injection) + oxaliplatin 5mg/kg I.P. every week

(GEMOX); gemcitabine 200mg/kg I.P. + oxaliplatin 5mg/kg I.P. + cetuximab 2 mg/kg I.P. every

week (GEMOX + cetuximab); gemcitabine 200mg/kg I.P. every week (GEM); rad001 5mg/kg

orally twice every week (Rad); gemcitabine, 200mg/kg I.P. every week + rad001 5mg/kg orally

twice every week (GEM + Rad); and saline with the same volume I.P. every week (control group).

Tumor size did not exceed 1,000 mm3 (approximating 4% mouse body weight) in compliance with

the animal welfare regulations. At the end of the third week, the xenografts were measured and

retrieved. The surgical specimens were divided into two portions; half was kept in frozen and stored

at -80C and the remaining half was prepared as a paraffin block.

Immunohistochemical staining

Formalin-fixed, paraffin-embedded tissues were cut into 4-m sections and mounted on the glue-

coated slides. A modification of the avidin-biotin-peroxidase complex immunohistochemical

method was performed. Various primary antibodies (human caspase-3, and eukaryotic initiating

factor 4E [eIF4E]; and rat Ki-67) were applied. The expression of eIF4E was assessed using the

product of 2 scores (intensity % positive), whereas the expression of caspase-3 was represented as

positively stained cells per HPF. The Ki67 labeling index was calculated as positively stained cells

per 100 cells counted × 100%. Histopathological assessment of tumor response was performed by

both histomorphological regression analysis (Schneider et al., 2005) and Ki-67 labeling index. The

extent of histomorphological regression was divided into four categories: grade 1, 75–100% viable

residual tumor cells; grade 2, 50–75% viable residual tumor cells; grade 3, 25–50% viable residual

tumor cells; and grade 4, 0-25% viable residual tumor cells.

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Thioacetamide-induced cholangiocarcinoma-afflicted rat model

Male Sprague-Dawley rats weighed 180–200 g were housed in a temperature and humidity

controlled environment with free access to water and rat chow. Cholangiocarcinoma was induced

by oral administration of thioacetamide (0.03% w/w in water) for 24 weeks, as previously described

(Jan et al., 2004). The treatment regimens were as follows: (1) gemcitabine, 25 mg/kg I.P. every 2

weeks; (2) rad001, 5 mg/kg orally twice every 2 weeks; (3) gemcitabine, 25 mg/kg I.P. every 2

weeks + rad001, 5 mg/kg orally twice every 2 weeks; or (4) saline with the same volume I.P. every

2 weeks for the vehicle group. The animals underwent FDG micro-PET pre-treatment, and at 2 and

4 weeks after treatment, before being sacrificed for histological confirmation.

18F FDG-microPET (positron emission tomography) image to measure metabolic response

Rats had full access to drinking water at all times and were fasted overnight for 8 hours before

radiotracer injection. A dose of 18.5 MBq (0.5 mCi) 18F FDG was administered and the liver region

was scanned for 120 minutes (min) continuously after intravenous radiotracer injection. Image

analysis was carried out at the PMOD image analysis workstation (PMOD Technologies Ltd, Zurich,

Switzerland). Regions of interest were drawn over the liver tumor and nontumoral liver background.

The time-activity curve and tumor-to-liver background (T/L) ratio were calculated. The optimal

scanning time point was determined when the highest T/L ratio was reached. Standardized uptake

values (SUVs) were used to quantify tracer uptake according to the following formula:

SUV = Decay corrected tissue activity (Bq/mL)

Injected dose (Bq) / Body weight (g)

The SUVmax and SUVmean stood for maximal and mean SUV, respectively, within the VOIs.

Tumor counts and FDG uptake values were obtained in all animals (Yeh et al., 2008).

Quantification of intratumoral metabolites by using nuclear magnetic resonance (NMR)

Thioacetamide-induced rat cholangiocarcinoma tumor tissues (~100 mg) were extracted by dual

phase procedures as previously described (Ackerstaff et al., 2003; Iorio et al., 2005; Venkatesh et al.,

2012). Water-soluble extracts were freeze-dried and reconstituted in 700 l deuterated water (D2O,

Sigma-Aldrich), and the extracts (500 L) were then placed in 5mm NMR tubes. Fifty microliters

of 0.75% sodium 3-trimethylsilyl-2,2,3,3-tetradeuteropropionate (TSP) in D2O (Sigma-Aldrich)

was added to the samples for chemical shift calibration and quantification. 1H-NMR measurement

was performed on a 600MHz spectrometer (Bruker, Germany): 7500Hz spectral width, 16384 time

domain points, 128 scans, temperature 298K, acquisition time approximately 5 minutes. The water

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resonance was suppressed by a gated irradiation centered on the water frequency. Spectral

processing was carried out using the Bruker Topspin-2 software package to quantify the metabolites.

Quantitative real-time PCR (polymerase chain reaction) for human Fas, caspase-8, BID,

APAF-1, XIAP, and caspase-3, and rat Glut(glucose transporter)-1, HK(hexokinase)-II, HIF

(hypoxia- inducible factor) -1α, and VEGF

Total RNA (2 g) was treated with DNAse 1 (Invitrogen) in a 50 L reaction mixture. TaqMan

primers and probes were used for quantitative detection of human apoptotic genes (Fas, caspase-8,

BID, APAF-1, XIAP, and caspase-3). Rat Glut-1 (ABI assay ID, Rn01417099), HK-II

(Rn00562457), HIF-1 (Rn00577560), VEGF (Rn01511601), and GAPDH (Rn99999916) were

designed with Primer Express (ABI-Perkin-Elmer) using the GenBank accession number. cDNA

samples were mixed with 2× Universal TaqMan buffer containing the Taq enzyme, primers, and

probes to a total volume of 25 L. The thermal cycle conditions were 50C 2 min, 95C 10 min,

and 42 cycles of 95C 15 seconds and 60C 1 min. All PCRs and analyses were performed using an

ABI PRISM 7700 Sequence Detection System (Applied Biosystems). All samples were run in

triplicate.

Statistical analysis

All continuous variables are expressed as the mean (SD). Statistical analysis for continuous

variables was performed using Student's t-test or one-way ANOVA test where appropriate.

Intergroup comparisons of choline kinase were otherwise performed using a box plot, where the

data were expressed as the median value. A p value of <0.05 was considered a statistically

significant difference between tested data sets.

Competing interests

None to declare.

Funding

Supported by Chang Gung Medical Foundation grant CRRPG3F0021~2, CMRPG3G1091~2,

CMRPG3E1321~2, National Science Council (Taiwan) MOST 106-2314-B-182A-019-MY3, and

Department of Health, Taiwan (DOH99-TD-C-111-006).

Data availability

Detailed data are available at figshare: https://figshare.com/s/6e46c082a9a1f60d448b

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Tables

Table 1. Cell cycle distribution of HuCCT1 treated by different regimens

Phase Control (%) Gem (%) Rad (%) Gem+Rad (%)

G1 50.4 0.4 76.1± 2.5* 45.1± 2.4*† 71.9± 1.6*‡

S 28.0± 0.5 10.1± 2.2* 24.9± 2.5† 17.6± 1.9*†‡

G2/M 21.6± 0.4 13.6 0.9* 29.9± 0.4*† 10.2± 0.3*†‡

Gem, gemcitabine; Rad, rad001

*, †, and ‡ represented p< 0.05 when compared to control, Gem- and Rad-groups, respectively.

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Table 2. Assessment of tumor response of rat cholangiocarcinoma to chemo- and/or target

therapy

Groups Histomorphological

regression score

Ki67 labelling index

Control 3.60.5 6213%

Gem 2.70.7* 3611*

Rad 2.80.7* 3917*

Gem + Rad 1.70.7*†‡ 154*†‡

Gem, gemcitabine; Rad, rad001

*, †, and ‡ represented p< 0.05 when compared to control, Gem- and Rad-groups,

respectively.

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Figures

Figure 1 A. Proliferation index of 6 cholangiocarcinoma cell lines treated with different regimens

(n=4 in each group). We examined the corresponding proliferation indices based on the HuCCT1

cell line and applied the dosage to the other five cholangiocarcinoma cell lines treated with mono-

therapy and combined therapy, respectively. Ox, oxaliplatin; Gem, gemcitabine; Erb, cetuximab;

Rad, rad001. B. The interaction between rad001 and gemcitabine in cholangiocarcinoma cell lines

was determined by median effects analysis to derive a combination index (CI). C. Phosphorylation

of p70S6K and p38MAPK in HuCCT1 and TFK-1 cells detected by the Bio-Plex phosphoprotein

assay (n=4 in each group). *, p < 0.05 versus control group. D. Cell cycle changes of HuCCT1 cells

detected by flow cytometry (n=4 in each group). E. Percentage of apoptotic HuCCT1 cells detected

by Annexin V-FITC Apoptosis Kit (n=4 in each group).

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Figure 2 A. Representative features of tumor growth in HuCCT1 xenograft models after 3-week

treatment (n=8 in each group). B. Left, quantitative analysis of tumor growth in HuCCT1 xenograft

models after 3-week treatment. *, ** p < 0.05 and p < 0.01 versus control group, respectively; †, p

< 0.05 versus Rad group; ‡, p < 0.05 versus Gem group. Right, quantitative analysis of tumor

growth in TFK-1 xenograft models after 3-week treatment. (n=8 in each group) *, ** p < 0.05 and p

< 0.01 versus control group, respectively; †, p < 0.05 versus Gem group; ‡, p < 0.05 versus Gem +

cetuximab group. C. Representative expression of caspase-3 and eIF4E in HuCCT1 xenograft

model detected by immunohistochemistry (n=8 in each group). Original magnification, 400× for

caspase -3 and 40× for eIF4E.

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Figure 3 A. The percentages (red digits) and intensity (blue digits) of Fas-positive HuCCT1 in

control, gemcitabine-treated, rad001-treated, and gemcitabine plus rad001-treated groups detected

by flow cytometry (n=4 in each group). B. The expression of Fas, caspase-8, BID, APAF-1, XIAP,

and caspase-3 mRNA in the HuCCT1 xenograft model detected by quantitative real-time PCR (n=6

in each group). *, ** p < 0.05 and p < 0.01 versus control group, respectively. †, †† p < 0.05 and p

< 0.01 versus Gem group, respectively; ‡ p < 0.01 versus Rad group.

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Figure 4 A. Representative image of 18F FDG microPET on cholangiocarcinoma-afflicted rats (n=6

in each group). B. Metabolic response of cholangiocarcinoma-afflicted rats detected by18F FDG

microPET was quantitatively analyzed (n=6 in each group). G, Gemcitabine group; R, Rad001

group. *, ** p < 0.05 and p < 0.01 versus control group, respectively; †, p < 0.05 versus Rad group;

‡, p < 0.05 versus Gem group. C. Expression of Glut-1, HK-II, HIF-1α, and VEGF mRNAs in rat

cholangiocarcinoma detected by quantitative real-time PCR (n=6 in each group). * p < 0.05 versus

control group; †, p < 0.05 versus Rad group; ‡, p < 0.05 versus Gem group.

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Figure 5 A. Representative nuclear magnetic resonance (NMR) spectrum of choline-associated

metabolites in cholangiocarcinoma-afflicted rats. Cho, choline; PC, phosphocholine; GPC,

glycerophosphocholine. B. Relative fold-changes of intratumoral choline-associated metabolites

after various treatment for 4 weeks (n=6 in each group). G, Gemcitabine group; R, Rad001 group. *

p < 0.05 versus control group. C. Representative features of choline kinase of treated

cholangiocarcinoma-afflicted rats detected by Western blot. Gem, Gemcitabine group; Rad, Rad001

group. MCF-7, a breast cancer cell line, served as positive control; whereas actin served as internal

control. D. Quantitative analysis of choline kinase of treated cholangiocarcinoma-afflicted rats

detected by Western blot (n=6 in each group). G, Gemcitabine group; R, Rad001 group. * p= 0.021

vs Control; †p=0.021 vs G; ‡p=0.043 vs R.

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