Signal Transduction

Neurotransmitter Substance P Mediates PancreaticCancer Perineural Invasion via NK-1R in Cancer Cells

Xuqi Li1, Guodong Ma1, Qingyong Ma1, Wei Li1, Jiangbo Liu1, Liang Han1, Wanxing Duan1,Qinhong Xu1, Han Liu1, Zheng Wang1, Qing Sun1, Fengfei Wang2, and Erxi Wu2

AbstractPancreatic cancer significantly affects the quality of life due to the severe abdominal pain. However, the

underlying mechanism is not clear. This study aimed to determine the relationship between Substance P (SP) andpancreatic cancer perineural invasion (PNI) as well as themechanism of SPmediating pancreatic cancer PNI, whichcauses pain in patients with pancreatic cancer. Human pancreatic cancer cells and newborn dorsal root ganglions(DRG) were used to determine the expression of SP or NK-1R in pancreatic cancer cells and DRGs cells by QT-PCR and Western blotting. The effects of SP on pancreatic cancer cell proliferation and invasion were analyzedusingMTT assay and TranswellMatrigel invasion assay, respectively. Alterations in the neurotropism of pancreaticcancer cells were assessed by coculture system, whichmimics the interaction of tumor/neuron in vivo. SP is not onlywidely distributed in the neurite outgrowth from newborn DRGs but also expressed in MIA PaCa-2 and BxPC-3cells. NK-1R is found to be overexpressed in the pancreatic cancer cell lines examined. SP induces cancer cellproliferation and invasion as well as the expression of matrix metalloproteinase (MMP)-2 in pancreatic cancer cells,and NK-1R antagonists inhibit these effects. Furthermore, SP promotes neurite outgrowth and the migration ofpancreatic cancer cell cluster to the DRGs, which is blocked by NK-1R antagonists in the coculture model. Ourresults suggest that SP plays an important role in the development of pancreatic cancer metastasis and PNI, andblocking the SP/NK-1R signaling system is a novel strategy for the treatment of pancreatic cancer.Mol Cancer Res;11(3); 1–9. �2012 AACR.

IntroductionPancreatic cancer is a deadly disease with a mortality rate

very near to the incidence rate. A lack of early symptoms,explosive outcomes, short survival, and resistance to therapyare hallmarks of this type of cancer (1). The treatment ofpancreatic cancer has not improved substantially during thepast 30 years. Perineural invasion (PNI) in pancreatic canceris a common pathologic phenomenon. It has been reportedthat all of pancreatic tumors would reveal PNI if enoughsections are evaluated (2), of which up to 90% of patients

have intrapancreatic nerves infiltrated by tumor cells and69% have involvement of the extra-pancreatic nerve termi-nations. The presence of tumor cells in the perineuriumspace of local peripheral nerves in the pancreas may beassociated with a higher risk of retropancreatic tumor exten-sion, precluding curative resection, and promoting localrecurrence after tumor resection.Pancreatic cancer significantly affects the quality of life

due to the chronic symptoms and severe abdominal pain(3, 4). Substance P (SP) is an undecapeptide, released fromprimary sensory nerve fibers that belongs to the tachykininfamily, which has been implicated in a myriad of physiologicprocesses (5, 6). SP is widely expressed in the central andperipheral nervous system as well as in peripheral tissues suchas B and T cells in an autocrine or paracrine manner (7) andmacrophages (8). NK-1R, a receptor of SP, is overexpressedin several normal (9–11) and neoplastic cell types (12–18).SP regulates many biologic functions and has also beenimplicated in neurogenic inflammation, pain, and depres-sion (6, 19). Upon the binding of SP to its high affinityreceptor, NK-1R, SP initiates multiple biologic functionsincluding tumor cell proliferation, angiogenesis (9), andmigration, which are critical for tumor cells invasion andmetastasis (7, 10). These biologic functions can be reversedby the NK-1R antagonists. These reports suggest that SP/NK-1R signaling may play an important role in the cancerprogression and metastasis, as SP may be a mitogen in NK-

Authors' Affiliations: 1Department of Hepatobiliary Surgery, First AffiliatedHospital of Medical College, Xi'an Jiaotong University, Xi'an, Shaanxi,China; and 2Department of Pharmaceutical Sciences, North Dakota StateUniversity, Fargo, North Dakota

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

X. Li and G. Ma contributed equally to this work.

Corresponding Authors: Qingyong Ma, Department of HepatobiliarySurgery, First Affiliated Hospital of Medical College, Xi'an JiaotongUniversity, Xi'an, Shaanxi, China. Fax: 86-29-8532-3899; E-mail:qyma56@mail.xjtu.edu.cn; and Erxi Wu, Department of PharmaceuticalSciences, North Dakota State University, NDSU Dept 2665, Fargo, ND58105. Phone: 701-231-7250; Fax: 701-231-8333; E-mail:Erxi.Wu@ndsu.edu

doi: 10.1158/1541-7786.MCR-12-0609

�2012 American Association for Cancer Research.

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1R–expressing tumor cell types (10, 11). Therefore, target-ing the NK-1 receptor could be a promising approach fortreating patients with cancer, andNK-1 receptor antagonistscould improve cancer treatment.Friess and colleagues (12), in their pioneer work on the

expression of NK-1R, showed that the expression levels ofNK-1R mRNA and protein in human pancreatic cancersamples were increased 36.7- and 26.0-fold, respectively,compared with normal controls. Enhanced NK-1R levels inthe tumor tissues were associated with advanced tumor stageand poorer prognosis. SP analogs stimulate pancreatic cancercell growth, depending on the NK-1R expression level, andthis effect could be blocked by a selective NK-1R antagonist.These findings suggest that there may be a neuro-cancer cellinteraction in vivo (12).The pancreas is an organ with rich innervations that are

associated with PNI in pancreatic cancer (13). We reasonthat SP stimulatingNK-1R, which is overexpressed in tumorcells and in the tumor and peritumoral tissue (7), may be amolecular mechanism for tumor cells to develop PNI.To date, the relationship between SP and pancreatic

cancer metastasis and PNI has not been reported. Thepurpose of the present study was to test whether SP/NK-1R signaling could influence the progression of pancreaticcancer. Our data suggest that SP plays an important role inthe development of pancreatic cancer by inducingcell proliferation, metastasis, and PNI; and blocking theSP/NK-1R signaling may be a novel strategy for the treat-ment of pancreatic cancer.

Materials and MethodsCell lines, animals, and reagentsThe human pancreatic tumor cell lines MIA PaCa-2,

BxPC-3, CFPAC-1, HAPC, Panc-1, and SW1990 wereobtained from American Type Culture Collection (14).Newborn rats were purchased from the laboratory animalcenter of the Xi'an Jiaotong University (Xi'an, China).Dulbecco's modified Eagle's medium (DMEM) and FBSwere obtained from Invitrogen Life Technologies. Poly-clonal anti-NK-1R and polyclonal anti-SP antibodieswere bought from Sigma-Aldrich. A polyclonal anti-human matrix metalloproteinase (MMP)-2 antibodywas obtained from Santa Cruz Biotechnology. SP acetatesalt (Sigma-Aldrich) was dissolved in distilled waterand different concentrations of SP (5, 10, 50, 100 and120 nmol/L) were evaluated. (2S, 3S) 3-([3, 5-Bis(trifluoromethyl)phenyl] methoxy)-2-phenylpiperidinehydrochloride (L-733,060) was procured from TocrisCookson. N-acetyl-L-tryptophan-3,5-bis(trifluoromethyl)-benzyl ester (L-732,138) was purchased from Tocris Cook-son. Unless otherwise specified, all other reagents wereacquired from Invitrogen. All experiments were carried outin triplicate.

Cell lines and rat dorsal root ganglionsPancreatic cancer cells were maintained in DMEM

supplemented with 10% heat-inactivated FBS (Invitrogen),

50 U/mL penicillin G, 50 mg/mL streptomycin sulfate, and25 mmol/L glucose at 37�C in a humidified 5% CO2, 95%air atmosphere. Newborn rats were euthanized with carbondioxide and sterilized with 75% ethanol. Dorsal root gan-glions (DRG) from the lumbar areas were dissected, col-lected into medium (DMEM), stripped of meninges andnerve stumps, and plated into a drop of liquid Matrigel (BDBiosciences). After solidification, medium (DMEM con-taining 0, 5, 10, 50, or 100 nmol/L SP) was carefully addedand renewed every 2 days (15, 16).

Immunocytochemical stainingFor fluorescence immunocytochemistry, pancreatic

tumor cell lines and neurite outgrowths from newbornDRGs were fixed for 30 minutes in 4% paraformaldehydein PBS and the endogenous peroxidase activity wasquenched by 3% hydrogen peroxide. The specimens werepreblocked for 30 minutes with bovine serum albumin(BSA) at 37�C and incubated with primary antibody againstSP (1:100) or NK-1R (1:100) overnight at 4�C. Thenstaining was detected with fluorescein isothiocyanate(FITC)–conjugated goat anti-rabbit immunoglobulin G(IgG) antibody or CY3-conjugated goat anti-rabbit IgGantibody (Jackson Immuno Research). Slides were mountedand then examined by using a Nikon Instruments confocalmicroscope (Nikon Instruments Inc.).

Reverse transcription-PCR and real-time quantitativePCRTotal RNA from prostate cancer cells or DRGs were

extracted using a Fastgen200 Kit RNA isolation system(Fastgen) according to the manufacturer's protocol. TotalRNA was reverse-transcribed into cDNA using the Fermen-tas RevertAidTMKit (MBI Fermentas). The primer sequen-ces were as follows:

SP-F: 50-GACTCCTCTGACCGCTAC-30.SP-R: 50-AGACCTGCTGGATGAACT-30.NK-1R-F: 50-AGGTTCCGTCTGGGCTTCAA-30.NK-1R-R: 50-TCCAGGCGGCTGACTTTGTA-30.MMP-2-F: 50-GATGATGCCTTTGCTCGTGC-30.MMP-2-R: 50-CAAAGGGGTATCCATCGCCA-30.b-Actin-F: 50-GACTTAGTTGCGTTACACCCTTTCT-30.b-Actin-R: 50-GAACGGTGAAGGTGACAGCAGT-30.

Reverse transcription-PCR (RT-PCR) products wereresolved by using a 1.5% agarose gel. After each real-timequantitative PCR (QT-PCR), a dissociation curve analysiswas conducted. Relative gene expression was calculatedusing the 2�DDCt method reported previously (17). Eachmeasurement was carried out in triplicate.

Western blottingProteins were electrophoretically resolved on a denaturing

SDS polyacrylamide gel and electrotransferred onto nitro-cellulose membranes. Themembranes were initially blockedwith 5%nonfat drymilk in TBS for 2 hours and then probed

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with antibodies against SP, NK-1R, MMP-2, or b-actin.After coincubation with the primary antibodies at 4�Covernight, the membranes were hybridized with secondarygoat anti-mouse antibody or goat anti-rabbit antibody (Sig-ma-Aldrich) for 2 hours at room temperature. Immunopo-sitive bands were developed using an enhanced chemilumi-nescence (ECL) detection system (Amersham Bioscience).All analyses were done in duplicate.

MTT assayMIA PaCa-2 and BxPC-3 cells were seeded in 96-well

tissue culture plates at a density of 5,000 to 10,000 cells perwell 24 hours before serum starvation. After serum starvationfor 24 hours, cells were cultured in DMEM with differentconcentrations of SP (0, 5, 10, 50, 100, and 120 nmol/L), L-733,060 (0, 5, 10, 20, 30, and 40 mmol/L) or L-732,138 (0,20, 40, 60, 80, and100mmol/L) and incubated at 37�C.After24, 48, or 72 hours, the medium was removed, and MTTreagent was added to each well and incubated at 37�C for 4hours. The optical densities (OD) at 490 nm were measuredusing a microplate reader (BIO-TEC Inc.). The proliferationrate was defined as OD (cells plate)/OD (control plate).

Transwell Matrigel invasion assayAn invasion assay was conducted with a Millicell invasion

chamber (Millipore). The 8-mm pore inserts were coatedwith 25 mg of Matrigel (Becton Dickinson Labware). Afterserum starvation for 24 hours, MIA PaCa-2 and BxPC-3cells (5� 104) were seeded in the top chamber and mediumwith SP orNK-1R antagonists were also added to the bottomchamber to induce the cancer cell lines. The Matrigelinvasion chamber was incubated for 48 hours in a humid-ified tissue culture incubator. Noninvading cells wereremoved from the top of the Matrigel with a cotton-tippedswab. Invading cells on the bottom surface of filter were fixedin methanol and stained with Crystal Violet (Boster Bio-logical Technology Ltd.). The invasion ability was definedas the number of cells migrated to the bottom chamber.

Coculture assayCoculture experiments were carried out using a mod-

ified method based on previously described methods (15,16). The DRGs were kept on ice after collection inDMEM medium (Invitrogen) and subsequently seededon 24-well Petri dishes in 25 mL of Matrigel gel asdescribed earlier. BxPC-3 cells were suspended in 25 mLof solidified Matrigel and placed next to the DRG sus-pension. To exclude the possibility of a nonspecific guidedmigration of BxPC-3 cells, an additional 25 mL of Matrigelcontaining no neural cells was positioned on the oppositeside. The Petri dishes were then placed for 20 minutes inan incubator at 37�C saturated with 5% CO2 in a humidatmosphere to allow polymerization of the Matrigel. Aftersolidification, medium (DMEM containing SP or/andNK-1R antagonists) was added and renewed every 2 days.Photographic documentation of the 2 adjacent sides of thecell suspensions was conducted with an inverted lightmicroscope imaging system (Ti-E; Nikon Instruments

Inc.) and analytic system (NIS BR3.0; Nikon InstrumentsInc.).

Statistical analysisStatistical analysis was done with SPSS software (version

17.0, SPSS Inc.). Data were presented as themean� SEMof3 replicate assays. Differences between the groups wereanalyzed by ANOVA, followed by the Bonferroni correctionfor multiple comparisons. P < 0.05 was considered statis-tically significant. All experiments were repeated indepen-dently at least 3 times.

ResultsSP is mainly expressed in DRGs, whereas NK-1R isexpressed in pancreatic cancer cellsTo determine whether SP or NK-1R is expressed in

pancreatic cancer cells, we tested 6 pancreatic cancer celllines: MIA PaCa-2, BxPC-3, CFPAC-1, HAPC, Panc-1,and SW1990. As shown in Fig. 1A, the expression of SP inpancreatic cancer cells at mRNA level was low. Among the6 cell lines, the expression levels of NK-1R from high tolow are in the following order: Panc-1 > BxPC-3 >CFPAC-1 > SW1990 > HAPC > MIA PaCa-2 (Fig. 1B).The expression of NK-1R and SP was also tested by

Western blotting (Fig. 1C) and immunofluorescence (Fig.2) in BxPC-3, MIA PaCa-2, and DRGs. In the 2 pancreaticcancer cell lines MIA PaCa-2 and BxPC-3, the NK-1R wasvisualized as a single band 45 kDa. A higher expression levelof the NK-1R was present in BxPC-3. The expression ofSP was also detected in these cell lines, whereas the expres-sion level of SP was much higher in the neurite outgrowthfrom newborn DRG.

SP induces proliferation of pancreatic cancer cellsTo determine the effects of SP on pancreatic cancer cell

growth, we cultured BxPC-3 and MIA PaCa-2 cells in themedia containing increasing concentrations (5–120 nmol/L) of SP and the effects on cell proliferation were determinedusing MTT assay. The results showed that SP induced cellproliferation in BxPC-3 cells with statistical significance in adose-dependent manner after incubation for 24, 48, or 72hours with 5 to 100 nmol/L SP, whereas proliferation rate in120 nmol/L begins to drop (Fig. 3A). A similar effect wasobserved in MIA PaCa-2 cancer cells.

NK-1R antagonists inhibit pancreatic cancer growthWe next treated both BxPC-3 andMIA PaCa-2 cells with

2 different NK-1R antagonists: L-733,060 that showed highaffinity for the human NK-1R in vitro and L-732,138 thatshowed a competitive antagonism for the receptor (18).Treatment of pancreatic cancer cells with the antagonistsresulted in a concentration-dependent cytotoxicity in bothcell lines. The IC50 of L-733,060 in both pancreatic can-cer cell lines BxPC-3 and MIA PaCa-2 was approximately10 mmol/L. Furthermore, the IC50 of L-732,138 was approx-imately 60 mmol/L in BxPC-3, whereas it is 40 mmol/L inMIA PaCa-2 (Fig. 3B and C).

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SP/NK-1R signaling system promotes invasion ofpancreatic cancer cellsNext, we tested the effect of SP on pancreatic cancer cell

invasion in vitro. The results show that the number ofinvasive BxPC-3 and MIA PaCa-2 cells increased with theaddition of 100 nmol/L SP compared with the control andthis effect was significantly inhibited by NK-1R antagonists(Fig. 4A).We further determined the expression of metastatic-relat-

ed factor MMP-2 by QT-PCR (Fig. 4B) and Westernblotting (Fig. 4C). Interestingly, SP was able to promotethe expression of MMP-2, which was also counterbalancedby the NK-1R antagonists. Taken together, these resultssuggest that SP/NK-1R signaling system promotes theinvasion of pancreatic cancer cells, which can be mediatedby MMP-2.

SP promotes neurotropism in pancreatic cancer cellsTo determine if SP contributes to the neurotropism of

pancreatic cancer cell, BxPC-3 cancer cells, which express ahigher level of NK-1R were used to coculture with newbornrat DRGs in Matrigel in the addition of SP or/and NK-1Rantagonists for 8 days. We first measured the effect of SPlevels on neurite regeneration (Supplementary Fig. S1A–S1E). Newborn rat DRGs were cultured in the mediumcontaining various concentrations of SP (0, 5, 10, 50, or 100nmol/L) for 8 days. The neurite regeneration exhibited aslow but significant increase when the concentration of SPincreased from 5 to 100 nmol/L (Supplementary Fig. S1F).These results suggest that SP have a stimulating effect onneurites. We also observed that BxPC-3 cancer cells facingthe DRGs formed peak-like clusters (Supplementary Fig.S1G–S1I). The neurite outgrowth (Supplementary Fig. S1J

and S1K) extended to the clusters from the DRGs andprovided an invasive pathway for the clusters.In addition, our results showed that SP promotes pan-

creatic cancer cell clusters gradually migrating to the DRGand NK-1R antagonists could significantly reduce theseeffects (Fig. 5). These interesting phenomena in the newcoculture environment indicate that the nerve might play animportant role in the process of PNI.

DiscussionIn the current study, we showed that SP is highly

expressed in the neurite outgrowth from newborn DRGs.We found that NK-1R is overexpressed in the pancreaticcancer cell lines. Furthermore, we showed that SP inducespancreatic cancer cell proliferation and invasion withstatistically significant and these effects could be counter-balanced by NK-1R antagonists. We then showed that theneurite regeneration slowly was increased in response tovarious SP concentrations from 5 to 100 nmol/L in thecoculture system. Moreover, we showed that SP promotespancreatic cancer cell clusters gradually migrating to theDRG and SP-induced neurite regeneration extended tothe clusters from the DRGs, which provides an invasivepathway for the clusters. Taken together, to our knowl-edge, it is the first time to show that SP, which mediatesthe interaction between cancer cells and nerves, maypromote the proliferation, invasion, and neurotropism ofpancreatic cancer cells. SP-promoted PNI in pancreaticcancer may constitute a novel mechanism for how paindecreases the survival of patients with pancreatic cancer.Blocking the SP/NK-1R signaling system may provide anovel strategy for the pancreatic cancer therapeutics.

Figure 1. Expression of SP and NK-1R in pancreatic cancer cells andDRGs. A, themRNA level of SP andNK-1R in 6 pancreatic cancer celllines: MIA PaCa-2, BxPC-3,CFPAC-1, HAPC, Panc-1, andSW1990. B, analysis of expressionlevels of NK-1R in BxPC-3 andMIAPaCa-2 by QT-PCR. C, the proteinlevel of SP andNK-1R in DRGs andpancreatic cancer cell lines BxPC-3 and MIA PaCa.

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Traditionally, SP has been classified as a neurotransmitterthat exerts its effects on the periphery only by being releasedfromnerve endings.However, SP has been recently shown tobe expressed in non-neuronal cell types, including endothe-lial cells, macrophages and monocytes, eosinophils, lympho-cytes, and Leydig cells (7, 19). This suggests that it may notonly act as a neurotransmitter but also as a functionalregulator in an autocrine or paracrine manner.It has been shown that SP exerts promigratory effects on

many types of cancer cells such as colon carcinoma, small celllung cancer (20), and breast carcinoma (21). In the U-373MGastrocytoma cell line, SP stimulates mitogenesis by activatingthe mitogen-activated protein kinase (MAPK) pathway

through receptors of the NK-1 subtype, including extracellularsignal–regulated kinases 1 and 2 (ERK1/2), which translocateto the nucleus to induce cell proliferation and protect the cellfrom apoptosis (7, 22). SP also stimulates cell proliferation viatransactivation of the EGF receptor (EGFR; ref. 23) andactivation of Akt, resulting in the suppression of apoptosis (24).Patients with cancer usually do not succumb to the

primary tumor but to the development of metastases. Theactive migration of tumor cells is a crucial requirementfor metastasis and cancer progression. Entschladen andcolleagues (25) suggested that neurotransmitters are keyplayers in the regulation of tumor-cell migration. It has alsobeen shown that SP induces the migration of tumor cells in

Figure 2. Expression of SP and NK-1R protein in BxPC-3, MIA PaCa-2cells, and DRG. Cells were labeledwith fluorescence-conjugatedSP–specific antibody (green)and fluorescence-conjugated NK-1R–specific antibody (red) in bothpancreatic cancer cells (�200) andDRG (�100). Nucleus was stainedwith 40,6-diamidino-2-phenylindole(DAPI). Arrows indicate that both SPandNK-1Rwere expressed in cancercell plasma.

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breast and prostate cancer cells (26). Our results show thatSP/NK-1R signaling system also induces pancreatic cancercell line invasion. We showed that the proliferative andinvasive abilities of BxPC-3 and MIA PaCa-2 pancreaticcancer cells were increased with the addition of SP. NK-1Rantagonist could inhibit these effect of SP. SP may inducethe advancement of pancreatic cancer via a dual mechanisminvolving both proliferative and invasive properties.Friess and colleagues (12) showed new evidence on neuro-

cancer cell interaction. Moreover, it has been shown thattumor samples from patients with advanced stages exhibitsignificantly higherNK1-R levels and that such patients havepoorer prognosis. We also showed that the pancreatic cancer

cells BxPC-3 and MIA PaCa-2 express NK-1R. SP is higherexpressed in the surrounding cells (normal and/or stromaltumors, etc.) or nerve endings, and then interacts withpancreatic cancer cells. In our experiments, we found thatthe proliferative and invasive ability of pancreatic cancercells, especially in the SP nerve endings,may stimulate cancercell migration. Our observations suggest that SP promotesthe growth of neurite regeneration as well as pancreaticcancer cell invasion to the DRG and there is a SP–neuro-cancer cell interaction. Our coculture study shows that thehigh-SP tumor microenvironment enhances PNI. Takentogether, these data suggest that SP and NK-1R could playan important role in the development of metastasis.

Figure3. Theeffect of SPand/orNK-1Rantagonists on cancer cell proliferation. A, induction of cell proliferationof humanpancreatic tumor cell linesMIAPaCa-2 and BxPC-3 by SP at several nanomolar concentrations (0, 5, 10, 50, 100, and 120 nmol/L). In both cases, SP induced cell proliferation. Using the ANOVAtest, a significant difference between each group and the control group was found. �, P < 0.05 as compared with control group. The percentage ofgrowth inhibition of human pancreatic cancer BxPC-3 and MIA PaCa-2 cells treated by increasing concentrations (0–40 mmol/L) of L-733,060 (B) or(0–100 mmol/L) of L-732,138 (C) at 24, 48, and 72 hours. The concentrations of NK-1R antagonists less than IC50 were selected [L-733,060 (10 mmol/L) andL-732,138 (60 mmol/L in BxPC-3 and 40 mmol/L in MIA PaCa-2)].

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The degree of pain control is an important endpoint oftherapy, along with clinical outcome following surgical andmedical treatment of pancreatic cancer (27). Pain is a maincomplaint from patients with pancreatic cancer. SP isinvolved in peripheral pain generation and is associatedwith tumor formation. Specific SP antagonists (11, 28)have shown clinical efficacy in patients, acting as the fol-

lowing: analgesics (29), antidepressants (30), antiemetics(29), and neuroprotectors (31). Our results may in partexplain that pancreatic cancer patients suffer severe painand the expression of SP is associated pancreatic cancerpatients with advanced diseases and poor outcomes.In conclusion, our results show that SP, which mediates

the interaction between cancer cells and nerves, may

Figure 4. The effect of SP and/or NK-1R antagonists on cancer cell invasive ability. A, SP promotes the invasion of pancreatic cancer cells, which can beinhibited byNK-1Rantagonists L733, 060 andL-732,138. �,P< 0.05 as comparedwith control group; #,P <0.05 as comparedwith SPgroup. SP increases theexpression of MMP-2 at themRNA level (B) and protein level (C) that can be counterbalanced by the NK-1R antagonists. �, P < 0.05 as comparedwith controlgroup; #, P < 0.05 as compared with SP group.

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promote the proliferation, invasion, and neurotropism ofpancreatic cancer cells. These results suggest that SPpromotes PNI in pancreatic cancer, constituting a novelmechanism for how pain decreases the survival of patientswith pancreatic cancer. These findings suggest that block-ing the SP/NK-1R signaling system may provide a novelstrategy for the pancreatic cancer therapeutics.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: G. Ma, Q. Ma, W. Li, H. Liu, E. WuDevelopment of methodology: J. Liu, Q. Xu, Z. WangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. Li, G. Ma, Q. Ma, J. Liu, L. Han, W. Duan,Q. Xu, Z. WangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. Li, G. Ma, Q. Ma, J. Liu, W. Duan, Q. Sun,F. Wang, E. WuWriting, review, and/or revision of the manuscript: X. Li, G. Ma, Q. Ma, W. Li,J. Liu, L. Han, W. Duan, H. Liu, F. Wang, E. Wu

Administrative, technical, or material support (i.e., reporting or organizing data,constructing databases): G. Ma, Q. Ma, J. Liu, W. Duan, Q. Sun, E. WuStudy supervision: G. Ma, Q. Ma, E. Wu

AcknowledgmentsThe authors thank the staff of the Biology and Genetics Laboratory, Xi'an Jiaotong

University for their technical assistance in these studies. The contents of this report aresolely the responsibility of the authors and do not necessarily reflect the official views ofthe NIH, National Center for Research Resources (NCRR), or National Institute ofGeneral Medical Sciences (NIGMS).

Grant SupportThis study was supported by 13115 Major Project (2010ZDKG-49), National

Natural Science Foundation of China (81172360 and 81201824), and project grantsfrom the NCRR (P20 RR020151) and the NIGMS (P20 GM103505 and P30GM103332-01) from the NIH.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 20, 2012; revised November 30, 2012; accepted December 20,2012; published OnlineFirst January 23, 2013.

Figure 5. SP promotesneurotropism in BxPC-3 cells. Inthe coculture model, pancreaticcancer cell clusters graduallymigrated to the DRG and theneurite outgrowth extended to theclusters from the DRGs thatprovided an invasive pathway forthe clusters (A, originalmagnification �40). This trend wassignificantly enhanced by adding100 nmol/L SP (B). L-733,060 andL-732,138 could impair thepromoting effect of SP on cancercell cluster migration and neuriteoutgrowth(D and F). Adding NK-1Rantagonists alone in the coculturesystem could also reduce thenumber of migrated pancreaticcancer cell clusters to the DRG andthe length and density of neuriteoutgrowth (C and E). Arrowsindicate the outgrowth of neurites.�, P < 0.05 as compared withcontrol group; #, P < 0.05 ascompared with SP group.

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Substance P Mediates Pancreatic Cancer Perineural Invasion

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Published OnlineFirst January 23, 2013.Mol Cancer Res   Xuqi Li, Guodong Ma, Qingyong Ma, et al.   Perineural Invasion via NK-1R in Cancer CellsNeurotransmitter Substance P Mediates Pancreatic Cancer

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