The Chemokine Receptor CX3CR1 Is Involved in the Neural Tropism and Malignant Behavior of Pancreatic Ductal Adenocarcinoma Federica Marchesi, 1 Lorenzo Piemonti, 4 Giuseppe Fedele, 5 Annarita Destro, 2 Massimo Roncalli, 2,9 Luca Albarello, 5 Claudio Doglioni, 5 Achille Anselmo, 1 Andrea Doni, 1 Paolo Bianchi, 3 Luigi Laghi, 3 Alberto Malesci, 3 Luigi Cervo, 8 MariaLuisa Malosio, 4 Michele Reni, 6 Alessandro Zerbi, 7 Valerio Di Carlo, 7 Alberto Mantovani, 1,10 and Paola Allavena 1 Departmentsof 1 ImmunologyandInflammation, 2 Pathology,and 3 Gastroenterology,IRCCSClinicalInstituteHumanitas,Rozzano,Milano,Italy; 4 Diabetes Research Institute and Departments of 5 Pathology, 6 Oncology, and 7 Surgery, IRCCS San Raffaele Scientific Institute; 8 Department of Neuroscience,MarioNegriInstitute;and 9 DepartmentofPathologyand 10 InstituteofPathology,UniversityofMilan,Milan,Italy Abstract Tumor perineural dissemination is a hallmark of human pancreatic ductal adenocarcinoma (PDAC) and represents a major source of local tumor recurrence after surgery. In this study, we provide in vitro and in vivo evidence that the chemokine receptor CX3CR1 may be involved in the neuro- tropism of PDAC cells to local peripheral nerves. Neoplastic cells from PDAC cell lines and surgical specimens express the chemokine receptor CX3CR1, absent in normal pancreatic ducts. Its unique ligand, the transmembrane chemokine CX3CL1, is expressed by neurons and nerve fibers. CX3CR1 + PDAC cell lines migrated in response to human recombinant CX3CL1 and specifically adhered to CX3CL1-expressing cells of neural origin via mechanisms involving activation of G proteins, B1 integrins, and focal adhesion kinase. In vivo experiments with transplanted PDAC showed that only CX3CR1-transfected tumor cells infiltrated the local periph- eral nerves. Immunohistochemistry of CX3CR1 in PDAC specimens revealed that 90% of the samples were positive with a heterogeneous pattern of expression. High receptor score was significantly associated with more prominent tumor perineural infiltration evaluated histologically (P = 0.026). Regression analyses (univariate and multivariate) showed that high CX3CR1 expression and perineural invasion were strongly associated with local and earlier tumor recurrence (P = 0.007). Collectively, this study shows that the CX3CR1 receptor may be involved in PDAC tumor neurotropism and is a relevant and independent risk factor to predict an early local tumor relapse in resected patients. Thus, the CX3CR1-CX3CL1 axis could represent a valuable therapeutic target to prevent tumor perineural dissemination in pancreatic cancer. [Cancer Res 2008;68(21):9060–9] Introduction Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer-related deaths in the Western world, is a highly aggressive and chemoresistant disease, with a 5-year survival rate of<5%.Thisisimprovedto20%inthesmallpercentageofpatients eligible to surgery (1, 2). PDAC extensively infiltrate surrounding structures;indeed,certainsitesthatpancreaticcancermayspread to eliminate surgery as treatment option. Depending on the localization of the primary tumor, preferential sites of metastasis arethelymphnodes,bigvesselsasportalvein,liver,andtheceliac plexus.Characteristicofthisneoplasiaisitspeculiarneurotropism: thegrowthoftumorfocialongintrapancreaticandextrapancreatic nervesisacommonpathologicfindingandamajorcauseoflocal tumor recurrence (3–6). Several studies have addressed the molecular mechanisms of pancreatictumorcelladhesiontonervefibers.Anumberofreports haveinvestigatedtheimportanceofneurotrophicfactorsexpressed by peripheral nerves in pancreatic tumor cell adhesion. The expressionofvariousneurotrophins(BDNF,NT-3,NT-4,NT-5)and their receptors (Trk A, B, C) in the perineural microenvironment suggestedtheirpossibleinvolvementintumorinvasion(7–9).More recently, members of the glial cell–derived neurotrophic factor family, including Artemin, were also shown to favor pancreatic cancer invasion of peripheral nerves (10) and promote the growth and survival of neoplastic cells (11). Chemokines and their receptors have been implicated in tumor growthandtumorcellinvasionofsurroundingorgans.Inparticular, chemokine receptors have been suggested to elicit cancer cell mobilizationandpromotedistantmetastasiswithacertaindegree of organ selectivity (12–15). Chemokine receptors expressed by PDAC include CCR6 (16), CXCR5 (17), and CXCR4 (18, 19). We reported that, in this neoplasia, CXCR4 not only has chemotactic function but also sustains tumor cell survival and proliferation (20). In the context of a transcriptional profiling aimed at exploring the chemokine system in pancreatic cancer, we found that the chemokinereceptorCX3CR1isup-regulatedinselectedPDACcell lines.CX3CR1isnormallyexpressedbyhematopoeticcells(21)and poorly studied in tumors, with few exceptions (22–24). The only ligand for CX3CR1 is the chemokine CX3CL1, also known as Fractalkine (25) or Neurotactin (26). CX3CL1 was originally describedasamembrane,aswellasasolublechemokineexpressed byneurons,nerveterminations,inadditiontoactivatedendothelial cells(25–28).Theinterestinthischemokinehasrecentlygaineda new impetus, resulting from the accumulation of data about its expression in several human diseases, including atherosclerosis (29, 30), allograft rejection (31), and age-related macular degener- ation(32).CX3CL1isoneofthemostexpressedchemokinesinthe nervoussystem;neuronsandastrocytesaremajorproducersofthe ligand, and microglia express the receptor (33, 34). A recent study highlighted the role of CX3CL1 in attenuating glial-induced Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). F.MarchesiandL.Piemonticontributedequallytothiswork.A.MantovaniandP. Allavena are senior and corresponding authors of this work. Requests for reprints: PaolaAllavena,IstitutoClinicoHumanitas,viaManzoni56, 20089 Rozzano (MI), Italy. Phone: 39-02-82245112; Fax: 39-02-82245101; E-mail: paola.allavena@humanitas.it. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-1810 Cancer Res 2008; 68: (21). November 1, 2008 9060 www.aacrjournals.org Research Article Research. on March 20, 2015. © 2008 American Association for Cancer cancerres.aacrjournals.org Downloaded from