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Cancer Letters 397 (2017) 43e51

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Cancer Letters

journal homepage: www.elsevier .com/locate/canlet

Original Article

Galectin-1-driven upregulation of SDF-1 in pancreatic stellate cellspromotes pancreatic cancer metastasis

Dong Qian a, b, f, 1, Zipeng Lu a, b, 1, Qingcheng Xu a, b, g, 1, Pengfei Wu a, b, Lei Tian a, b,Liangtao Zhao a, b, Baobao Cai a, b, Jie Yin a, b, Yang Wu a, b, Kevin F. Staveley-O'Carroll c, d, e,Kuirong Jiang a, b, *, Yi Miao a, b, **, Guangfu Li c, d, ***

a Pancreas Center, The First Affiliated Hospital with Nanjing Medical University, Nanjing 210029, Chinab Pancreas Institute, Nanjing Medical University, Nanjing 210029, Chinac Department of Surgery, University of Missouri, Columbia, MO 65212, USAd Ellis Fischel Cancer Center, University of Missouri, Columbia, MO 65212, USAe Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65212, USAf Department of General Surgery, Affiliated Hospital of Nanjing University of TCM, Jiangsu Province Hospital of TCM, Nanjing 210029, Chinag Department of Gastroenterology, Subei People's Hospital, Clinical Medical School, Yangzhou University Affiliated Hospital, Yangzhou 225000, China

a r t i c l e i n f o

Article history:Received 24 July 2016Received in revised form11 March 2017Accepted 14 March 2017

Keywords:Galectin-1SDF-1Pancreatic stellate cellPancreatic cancerMetastasis

* Corresponding author. Pancreas Center, The FiNanjing Medical University, 300 Guangzhou Rd, GuProvince, 210029, China. Fax: þ86 025 68136989.** Corresponding author. Pancreas Center, The FiNanjing Medical University, 300 Guangzhou Rd, GuProvince, 210029, China. Fax: þ86 025 68136989.*** Corresponding author. Department of Surgery, ElHospital Drive, M272, University of Missouri-ColumbiFax: þ1 573 884 4585.

E-mail addresses: [email protected] (K.(Y. Miao), [email protected] (G. Li).

1 Dong Qian, Zipeng Lu and Qingchen Xu equally cofirst author.

http://dx.doi.org/10.1016/j.canlet.2017.03.0240304-3835/© 2017 Published by Elsevier Ireland Ltd.

a b s t r a c t

Galectin-1, mainly expressed in activated pancreatic stellate cells (PSCs), is involved in many importantcancer-related processes. However, very little is known how Galectin-1 modulates PSCs and subse-quently impacts pancreatic cancer cells (PCCs). Our chemokine antibody array and in vitro studiesdemonstrates that Galectin-1 induces secretion of stromal cell-derived factor-1(SDF-1) in PSCs by acti-vating NF-kB signaling. The secreted SDF-1 increases migration and invasion of PCCs. Knockdown ofGalectin-1 and inhibitor-mediated blockade of SDF-1 as well as its ligand CXCR4 and NF-kB verifies thefindings. In vivo experiment by knockdown of Galectin-1 in PSCs further demonstrates the conclusion.Collectively, the present studies demonstrate that Galectin-1-driven production of SDF-1 in PSCs throughactivation of NF-kB promotes metastasis in PDAC, offering a potential target in the treatment ofpancreatic cancer.

© 2017 Published by Elsevier Ireland Ltd.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) continues to be one ofthe most lethal human malignancies with a poor prognosis due tosystemic metastasis and a high recurrence rate. Interactions be-tween tumor and stromal cells play a critical role in tumor

rst Affiliated Hospital Withlou District, Nanjing, Jiangsu

rst Affiliated Hospital Withlou District, Nanjing, Jiangsu

lis Fischel Cancer Center, Onea, Columbia, MO 65212, USA.

Jiang), [email protected]

ntributed to this work as the

progression [1]. As a main component of the tumor stroma,pancreatic stellate cells (PSCs) may participate in the developmentof pancreatic cancer [2,3]. However, the interaction between PSCsand pancreatic cancer cells (PCCs) and the underlying mechanismsare poorly understood.

Chemokines and their receptors have been implicated in tumorgrowth and invasion. Stromal cell-derived factor-1 (SDF-1 orCXCL12) belongs to the CXC chemokine family and is the ligand ofCXCR4 [4]. In many recently published studies, it has becomeapparent that the SDF-1/CXCR4 axis is a critical mediator oftumorestromal interactions and it has been implicated in pro-moting themetastatic potential of breast, gastric, ovarian, prostate,lung and pancreatic cancer cells [5e10]. Elevated SDF-1 expressionin patients with pancreatic cancer is linked to a dismal prognosis[11]. SDF-1 induces PCC invasion and epithelialemesenchymaltransition through increased Smoothened (SMO) expression [12].Although the less production of SDF-1 is detected in PCC lines, therelatively higher expression is observed in pancreatic cancer tissue

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D. Qian et al. / Cancer Letters 397 (2017) 43e5144

D. Qian et al. / Cancer Letters 397 (2017) 43e51 45

samples [13,14], suggesting that upregulation of SDF-1 may be anintegral factor involved in tumorestromal interactions [15].

Galectin-1 is involved in multiple cancer-related processes,including immunosuppression, angiogenesis and metastasis[16,17]. Studies have proved the specific location of Galectin-1 instromal myofibroblasts in PDAC or other cancer tissue by immu-nohistochemistry and in situ hybridization [18,19]. Our previousstudy also reported that Galectin-1 is highly expressed in pancre-atic cancer tissues, especially in activated PSCs within the stroma ofcancer, which was associated with perineural invasion and poorprognosis in patients with pancreatic cancer [16,20]. Galectin-1also promotes the secretion of several kinds of chemokines [21]in pancreatic cancer. Our present studies aim to investigate howGalectin-1 modulates PSCs by chemokines and subsequently im-pacts tumor metastasis.

Materials and methods

Patients and pancreatic tissue samples

Pancreatic cancer tissue samples were obtained from patients undergoingpancreaticoduodenectomy for pancreatic cancer at the Pancreas Center, the FirstAffiliated Hospital of Nanjing Medical University, China. All patients providedwritten informed consent and the study was approved by the Ethical Committee ofNanjing Medical University.

Cell lines and culture

The PDAC cell lines (Panc-1, MiaPaCa-2, Bxpc-3 and Capan-1) were obtainedfrom the Pancreas Institute, Nanjing Medical University. The PDAC cell lines werepropagated and cultured in Dulbecco's Modified Eagle's Medium, supplementedwith 10% fetal bovine serum (FBS), 100 mg/ml penicillin and streptomycin in a hu-midified atmosphere of 5% CO2 at 37 �C. Activated human-derived cancer-associatedPSCs were isolated from primary pancreatic cancer tissue samples using the explantmethod [20]. The cell type was confirmed by immunohistochemical staining for a-SMA, vimentin, desmin and GFAP and by stellate cell morphology, as previouslydescribed [20].

Human chemokine antibody array assay

The levels of chemokines in the supernatants in the PSC cultures (derived fromthree patients) were determined using a RayBio Human Chemokine Antibody ArrayKit (RayBiotech; Norcross, GA, USA). Briefly, PSCs were cultured in a 6-well plate(3 � 105 cells/well) with 1 ml/well serum-free medium for 16 h and then treatedwith or without 10 mg/ml Recombinant Human Galectin-1 (rhGalectin-1; 450-39;PeproTech; Rocky Hill, NJ, USA) for 24 h. The treated and untreated supernatants(three treated vs. three control) were harvested and analyzed according to themanufacturer's instructions. The array was scanned by a laser scanner (InnoScan300 Microarray Scanner, INNOPSYS, France) using green channel (excitation fre-quency 532 nm).

Immunohistochemical staining

Immunohistochemical staining was performed as previously described [22,23].45 cases of pancreatic cancer samples were analyzed. The primary antibodies wereincubated as follows: Anti-Galectin 1 antibody (ab138513; 1:300; Abcam), Anti-SDF-1 (ab9797; 1:1000; Abcam). The results of immunohistochemical staining wereinterpreted by two experienced pathologists, The immunoreactivity level wasestimated by assessing the average staining intensity (on a scale of 0e3) and theproportion of positive cells (0,


NF-kB activity assay

About 1.5 � 104/well sh-Gal-PSCs were seeded into 24-well plates and cultued.Themediumwas replacedwith serum-freemedium containing rhGalectin-1 with orwithout the NF-kB inhibitor, BAY 11-7082 (Beyotime, China). After 24 h, NF-kBactivation was evaluated using an NF-kB activation nuclear translocation assay kit(Beyotime, China). Briefly, the cells were permeabilized and blocked to prevent non-specific staining and incubated with anti-NF-kB p65 at 4 �C overnight. They werethen incubated with secondary Cy3 goat anti-rabbit fluorescent antibody at roomtemperature for 1 h and stained with DAPI for 5 min. The cells were visualized undera fluorescent microscope (Nikon; Japan).

Total RNA was extracted from the PSCs for analysis by qRT-PCR; and the su-pernatants from cell culture were harvested for ELISA.

Statistical analysis

All experiments were performed in triplicate. Differences between groups weredetermined by t-test. The KaplaneMeier method and Log-rank test were used tocompare survival outcomes. c2 tests were used to analyze the correlations betweengalectin-1 and SDF-1 expression. SPSS v. 22.0 (SPSS Inc.; Chicago, IL, USA) was usedfor all of the statistical analyzes. Datawere presented as mean ± SD and P < 0.05 wasconsidered statistically significant.

Results

Galectin-1 stimulated production of SDF-1 & expression of SDF-1and Galectin-1

Activated human-derived cancer-associated PSCs were isolatedfromprimary pancreatic cancer tissue samples and the expression ofa-SMA, vimentin, desmin and GFAP were positive (Fig. 1A). Chemo-kine array analysiswas conductedwith serum-freemedia from three

Fig. 2. Expression of CXCR4 in PCCs & SDF-1 promoted migration and invasion of PC(Capan-1, BxPC-3, MiaPaCa-2, Panc-1). (B)Representative images and (C)quantification of aPCCs. (D)Representative images and (E)quantification of a matrigel invasion assay show200 � magnification; quantification was performed by counting cells from 10 random field

primary isolated PSC lines. As shown in Fig.1B, Galectin-1 treatmentresulted in 2.06-fold increase in signal intensity of SDF-1 level in thesupernatants. As shown in Fig. 1C, PSCs express Galectin-1 and SDF-1(PCR: Fig. 1Ca and Cb; WB: Fig. 1Cc; ELISA: Fig. 1Cd and Ce). Cor-relation analysis showed that SDF-1 expression was proportional togalectin-1 expression in pancreatic cancer tissues (Fig. 1D and E).

Expression of CXCR4 in PCCs & SDF-1 promoted migration andinvasion of PCCs

QRT-PCR and western blot analysis were used to compare themRNA and protein expression level between PCCs. The effect of theSDF-1/CXCR4 axis on cell migration and invasion in PCCs wasinvestigated. The results showed that treatment with recombinantSDF-1 protein significantly increased migration and invasion of thetumor cells in a dose-dependent manner. Blockade of CXCR4 withantagonist AMD3100 eliminated SDF-1-induced migration andinvasion. These findings were consistent with previous studies[12,15,24] and indicated that inhibition of CXCR4 could signifi-cantly reduce migration and invasion of PCCs (Fig. 2BeE).

Galectin-1 promoted PSC-augmented migration of pancreatic tumorcells via SDF-1

Knockdown of Galectin-1 through lentiviral transfection withGalectin-1 shRNA (sh-Gal-PSCs; Fig. 3A) suppressed SDF-1 mRNAexpression and secretion in the supernatants. These observations

Cs. (A)WB and qRT-PCR analysis show the expression levels of CXCR4 in four PCC linestranswell migration assay showing the effect of SDF-1 on the migration capability ofing the effect of SDF-1 on the invasive capability of PCCs. Images are shown ats; data represent mean ± SD from three independent experiments; *P < 0.05.


were verified by qRT-PCR and ELISA (Fig. 3B), demonstrating thatSDF-1 was a key target of Galectin-1. To determine whetherGalectin-1 mediate PSC-induced migration of PCCs by modulatingexpression of SDF-1, PSCs and PCCs were co-cultured in the pres-ence of an SDF-1 neutralizing antibody (SDF-1 ab). The resultsshowed that reduced migration of PCCs was detected when co-culturing with PSCs transfected with null control (sh-NC-PSCs)but not observed when co-culturing with PSCs transfected withsh-Gal-PSCs (Fig. 3C and D). To investigate the effect of CXCR4 inPCCs migration migration as SDF-1 can bind to CXCR4, antagonist-mediated blockade of CXCR4 with AMD3100 is conducted. Ourresults showed that sh-NC-PSC-induced migration of PCCs wasreduced by addition of AMD3100, but sh-Gal-PSC-inducedmigration was unaffected (Fig. 3E and F). These results demon-strated that expression of Galectin-1 in PSCs stimulated produc-tion of SDF-1 thereby promoting metastasis via the SDF-1-CXCR4interaction.

Knockdown of Galectin-1 in PSCs inhibited metastasis of PDAC cellsin vivo

To validate in vitro finding, in vivo experiments were carried outwith the nude mice. Mice inoculated with MiaPaCa-2 PDAC cellswere divided into three groups which received the followingdifferent treatments: no further treatment (Mia-2), the treatmentwith null control PSCs (Mia-2þsh-NC-PSCs) and Galectin-1-knockdown PSCs (Mia-2þsh-Gal-PSCs). In the survival model,inoculated mice in the group of Mia-2þsh-NC-PSCs died earlierthan the mice in other two groups (Fig. 4A). In the metastatic

Fig. 3. Galectin-1 promoted PSC-augmented migration of pancreatic tumor cells via SDFlevels of Galectin-1 in PSCs transfected with sh-Gal-PSCs compared to those transfected withELISA showing the expression levels of SDF-1 in PSCs transfected with sh-Gal-PSCs compquantification of transwell migration assays in a PSC-PCC co-culture system: (C, D)SDF-1 neffect on sh-Gal-PSCs induced cell migration. (E, F)Blockade of CXCR4 by AMD3100 s200 � magnification; quantification was performed by counting cells from 10 random field

model, whole body imaging and HE staining revealed that the nullcontrol group exhibited more metastatic lesions (Fig. 4BeD). Theseobservations indicated that production of Galectin-1 in PSCs maypromote metastasis in pancreatic cancer.

Galectin-1 stimulated production of SDF-1 via activation of NF-kB

Fluorescent microscopy shows increased levels of NF-kB/p65nuclei translocation and Western Blot shows IkB-a degradation insh-Gal-PSCs after stimulationwith Galectin-1. This effect is negatedby the addition of NF-kB inhibitor BAY 11-7082 (Fig. 5A and B),suggesting that Galetin-1-induced stimulation of PSCs involvedactivation of NF-kB. This observation was verified by qRT-PCR andELISA by showing that Galectin-1-stimulated secretion of SDF-1 inPSC following knockdown of Galectin-1 (sh-Gal-PSCs) could beinhibited by BAY 11-7082 (Fig. 5C).

In combination, these findings suggested that Galectin-1may bea critical regulator in PSC-mediated tumor metastasis in pancreaticcancer.

Discussion

Chemokine antibody array and genetic knockdown togetherwith blockade with antibody and antagonist are utilized in thepresent studies. The experiments demonstrate that Galectin-1 en-hances SDF-1 production in PSCs through activating NF-kB(Fig. 5D). The resultant PSCs interact with tumor cells to promotetheir migration, leading to the augmented tumor metastasis.

-1 (A) qRT-PCR assay, Western blot and ELISA assay showing the expression or secretionsh-NC-PSCs (null control); b-actin was used as a loading control. (B) qRT-PCR assay andared to those transfected with sh-NC-PSCs (null control). Representative images andeutralizing antibody suppresses sh-NC-PSC-induced cancer cell migration but has nouppresses sh-NC-PSC-induced cell migration. Representative images are shown ats; data represent mean ± SD from three independent experiments; *P < 0.05.


The importance of the tumor microenvironment in the devel-opment of cancer has been widely recognized [22,25]. Some com-ponents consisting of tumor microenvironment play an importantrole in influencing the outcome of the malignancy. The PDACenvironment contains many cells but predominantly PSCs [26].PSCs have been identified as a key source of abnormal levels ofcytokines, chemokines and growth factors, thereby providing amicroenvironment suitable for cancer invasion [21,27]. Persistentactivation of PSCs promotes malignant behavior in PDAC, such asrapid proliferation of tumor cells and local or distant metastasis[28,29]. Studies have also shown that PSC supernatants and PSC-conditioned medium promoted the invasion of PCCs [3,15,30],demonstrating that the paracrine is an important factor in PSC-PCCinteractions.

Galectin-1 is involved in a wide range of biological activitiesincluding apoptosis, migration, adhesion, malignant trans-formation, carcinogenesis, tumor angiogenesis, and immunosup-pression [23,31e37]. Pancreatic tumor cells have been found to

Fig. 4. Knockdown of Galectin-1 in PSCs inhibits metastasis of PCCs in vivo. (A) KaplaneM2þsh-NC-PSCs, and Mia-2þsh-Gal-PSCs. These show that mice in the Mia-2þsh-NC-PSCs gtumor in pancreas with no metastasis; (b) thoracic metastasis; (c) and liver metastasis. (C) HEthe Mia-2þsh-NC-PSC group exhibit more metastatic sites.

stimulate PSCs to express enhanced levels of Galectin-1 bysecreting cytokine transforming growth factor TGF-b1 [20,38]; thisin turn induced activation and proliferation of PSCs resulting in acycle that sustained the stromal reaction and promoted the ma-lignant behavior of PDAC [21,39,40]. We previously demonstratedthat Galectin-1 was overexpressed in PSCs leading to the forma-tion of fibroblasts surrounding the tumor cells and was associatedwith perineural invasion, advanced tumor stage, lower differen-tiation and poor prognosis in patients with pancreatic cancer[16,20,41]. Conversely, in our present study, silencing Galectin-1by lentiviral shRNA suppressed PSC-augmented migration andinvasion in vitro and inhibited tumor progression in vivo. Thesefindings further demonstrated the important role of Galectin-1 inPSC-PCC interaction, and suggested that targeting Galectin-1 inPSCs may offer a potential therapeutic strategy in the treatment ofpancreatic cancer.

Although chemokines play a crucial role in immune and in-flammatory reactions, they have an equally important role in the

eier curves compare survival time in mice inoculated with MiaPaCa-2 cells alone, Mia-roup had the shortest survival time. (B) Representative images showing (a) orthotopicstaining of (a) orthotopic tumor; (b) liver metastasis (c) thoracic metastasis (D)Mice in


development of a variety of cancers, such as melanomas, ovarian,breast, lung, and prostate cancers [26]. In addition to inducing theactivation of PSCs and promotes their proliferation, Galectin-1 alsopromotes the secretion of several kinds of chemokines [21].

Fig. 5. Galectin-1 stimulated production of SDF-1 via activation of NF-kB. (A)FluorescentBlot shows IkB-a degradation in sh-Gal-PSCs after stimulation with Galectin-1; this effect is n(nuclei; blue). (C)qRT-PCR assay and ELISA confirm that Galectin-1-stimulated secretion ofproposed pathway: Galectin-1 stimulates PSCs to produce enhanced levels of SDF-200 � magnification; data represent mean ± SD from three independent experiments; *P <referred to the web version of this article.)

However, there were no published studies fully investigating thechemokine inducer role of Galectin-1. By human chemokine anti-body array, we demonstrated that treatment of PSC supernatantswith Galectin-1 increased the secretion of chemokines in PSCs

microscopy shows increased levels of NF-kB/p65 nuclei translocation and (B) Westernegated by the addition of NF-kB inhibitor BAY 11-7082. NF-kB p65 antibody (red); DAPISDF-1 in PSCs can be inhibited by BAY 11-7082. (D) A schematic diagram showing the1, thereby promoting metastasis of PCCs. Representative images are shown at0.05. (For interpretation of the references to colour in this figure legend, the reader is


(Three cancer-activated PSC lines), especially SDF-1, suggesting thatSDF-1 could be a potential downstream mediator of Galectin-1.

Stromal cells are an important source of SDF-1 in pancreaticand other primary tumors [12,15,42e44]. Although Galectin-1 hasbeen shown to stimulate MCP-1 production in PSCs via activationof NF-kB [21], there are no published reports on its effect in pro-moting the secretion of SDF-1. Our exploration of the potentialmechanism indicated that this effect was induced via activation ofNF-kB. The role of SDF-1 and its receptor CXCR4 in the develop-ment of pancreatic or other kinds of cancers has been investigatedby various studies: the production of SDF-1 by stromal cellsinfluenced the growth andmalignant behavior of pancreatic tumorcells, whereas secretion of sonic hedgehog by pancreatic tumorcells promoted desmoplasia [6,45]; SDF-1 induced cell invasionand epithelialemesenchymal transition of PCCs [12]; SDF-1expressed in lymph node fibroblasts elevated lymph node me-tastases in oral squamous cell carcinoma [46]; paracrine SDF-1mediated the effect of PSCs on chemoresistance in pancreaticcancer [47]; SDF-1 played a pivotal role in perineural invasion ofPCCs and was associated with metastasis in pancreatic cancer [24];whereas, expression of CXCR4 mediated organ-specific metastasisof PCCs [48]. Our study supported these reports by demonstratingthat SDF-1 was predominantly expressed in PSCs and that CXCR4was predominately expressed in PCCs. We also validated theseprevious findings by demonstrating that SDF-1 promoted migra-tion and invasion of PCCs. It has been reported that SDF-1 can alsoexert its biological function by binding to another chemokine re-ceptor, CXCR7 [49]. To further confirmwhether the SDF-1-inducedmigration and invasion are dependent upon the activation ofCXCR4, we used a CXCR4 antagonist AMD3100 to block it, that inturn reduced the effect of SDF-1. Our study was the first study todemonstrate that SDF-1 was an important chemokine mediated byGalectin-1 in PSC. Although our results confirmed that SDF-1 wasimportant in the progression of pancreatic cancer, other chemo-kines mediated by Galectin-1 may also be involved. Studies onother potential chemokines have been scarce and further researchis required.

Our present studies demonstrate a pivotal role of Galectin-1 indriving SDF-1 production in PSCs through activation of NF-kBwhich promotes tumor metastasis. The findings provide innovativeinsight into the mechanism by which tumor microenvironmentmediates tumor metastasis and potential targets which can beapplied in the treatment of pancreatic cancer.

Funding

This work was supported by the National Natural Science Fundof China [grant number: No. 81272382, Kuirong Jiang, PI]; the Na-tional Natural Science Fund of China for Young Scholars [grantnumber: No. 81300351, Zipeng Lu, PI] and the “Six Talent Peaks”Project of Jiangsu Province [grant number: No. 2014-WSW-006,Kuirong Jiang, PI].

Conflict of interest

No conflicts of interest to declare.

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Galectin-1-driven upregulation of SDF-1 in pancreatic stellate cells promotes pancreatic cancer metastasisIntroductionMaterials and methodsPatients and pancreatic tissue samplesCell lines and cultureHuman chemokine antibody array assayImmunohistochemical stainingQuantitative reverse transcription polymerase chain reaction (qRT-PCR)Western blot analysisQuantification of Galectin-1 and SDF-1 by enzyme-linked immunosorbent assay (ELISA)Transfection of pancreatic satellite cells with lentiviral shRNAInvasion and migration assayIn vivo assaysNF-κB activity assayStatistical analysis

ResultsGalectin-1 stimulated production of SDF-1 & expression of SDF-1 and Galectin-1Expression of CXCR4 in PCCs & SDF-1 promoted migration and invasion of PCCsGalectin-1 promoted PSC-augmented migration of pancreatic tumor cells via SDF-1Knockdown of Galectin-1 in PSCs inhibited metastasis of PDAC cells in vivoGalectin-1 stimulated production of SDF-1 via activation of NF-κB

DiscussionFundingConflict of interestReferences

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