Role of CXCL13-CXCR5 Crosstalk Between Malignant Neuroblastoma Cells and Schwannian Stromal Cells in Neuroblastic Tumors

Published OnlineFirst June 3, 2011; DOI: 10.1158/1541-7786.MCR-10-0367

Molecular Cancer Angiogenesis, Metastasis, and the Cellular Microenvironment Research

Federica Del Grosso1, Simona Coco1, Paola Scaruffi1, Sara Stigliani1, Francesca Valdora6, Roberto Benelli2, Sandra Salvi3, Simona Boccardo3, Mauro Truini3, Michela Croce4, Silvano Ferrini4, Luca Longo1,5, and Gian Paolo Tonini1

Abstract Neuroblastoma is a stroma-poor (SP) aggressive pediatric cancer belonging to neuroblastic tumors, also including ganglioneuroblastoma and ganglioneuroma, two stroma-rich (SR) less aggressive tumors. Our previous gene-expression profiling analysis showed a different CXCL13 mRNA expression between SP and SR tumors. Therefore, we studied 13 SP and 13 SR tumors by reverse transcription quantitative real-time PCR (RT-qPCR) and we found that CXCR5b was more expressed in SP than in SR and CXCL13 was predominantly expressed in SR tumors. Then, we isolated neuroblastic and Schwannian stromal cells by laser capture microdissection and we found that malignant neuroblasts express CXCR5b mRNA, whereas Schwannian stromal cells express CXCL13. Immunohistochemistry confirmed that stroma expresses CXCL13 but not CXCR5. To better understand the role of CXCL13 and CXCR5 in neuroblastic tumors we studied 11 neuroblastoma cell lines and we detected a heterogeneous expression of CXCL13 and CXCR5b. Interestingly, we found that only CXCR5b splice variant was expressed in both tumors and neuroblastoma lines, whereas CXCR5a was never detected. Moreover, we found that neuroblastoma cells expressing CXCR5 receptor migrate toward a source of recombinant CXCL13. Lastly, neuroblastoma cells induced to glial cell differentiation expressed CXCL13 mRNA and protein. The chemokine released in the culture medium was able to stimulate chemotaxis of LA1–5S neuroblastoma cells. Collectively, our data suggest that CXCL13 produced by stromal cells may contribute to the generation of an environment in which the malignant neuroblasts are retained, thus limiting the possible development of metastases in patients with SR tumor. Mol Cancer Res; 9(7); 815–23. ’2011 AACR.

Introduction with a 3-years OS ranging between 30% and 35%. This dramatic behavior of the metastatic disease is mainly due to Neuroblastic tumors are a group of pediatric cancers that the capacity of neuroblastoma cells to metastasize at bone, onset as localized or disseminated disease (1). Patients with bone marrow, liver, and lymph nodes. Particularly, in localized tumor have good prognosis with a 3-years overall patients with disseminated disease, the normal bone marrow survival (OS) of 90% to 95%, whereas patients older than cell population is almost completely substituted by malig- one year with a disseminated disease have a worse prognosis nant neuroblasts that find a favorable environment for their proliferation. Neuroblastic tumors show a quite heterogeneous histol- Authors' Affiliation: 1Translational Oncopathology, 2Department of ogy with different tumor histotypes including: neuroblas- Immunology, 3Department of Diagnostic Technologies, 4Laboratory of toma predominantly composed of small round Immunological Therapy, 5Italian Neuroblastoma Foundation, National Cancer Research Institute; and 6Department of Oncology and Genetics, undifferentiated or poorly differentiated neuroblastic cells, University of Genoa, Genoa, Italy with few or absent Schwannian stromal cells, ganglioneur- Notes: Supplementary data for this article are available at Molecular oblastoma intermixed (GNB), and ganglioneuroma (GN) Cancer Research Online (http://mcr.aacrjournals.org/). mostly composed of Schwannian stromal cells in which some Current address for P. Scaruffi: Center of Physiopathology of Human nests or very few neuroblastic cells are present (2). The Reproduction, Dept. Obstetrics and Gynecology, "San Martino" Hospital, absence of stromal cells makes the tumor more aggressive and Genoa, 16132, Italy. patients have a worse outcome. On the contrary, the abun- Corresponding Author: Gian Paolo Tonini, Translational Oncopathol- ogy, National Cancer Research Institute (IST), L.go R. Benzi, 10-16132, dance of Schwannian stromal cells, as observed in GNB and Genoa, Italy. Phone: 39-010-5737487/381; Fax: 39-010-5737487; GN, is associated with a less aggressive tumor and a localized E-mail: [email protected] disease with a more favorable prognosis (1, 3). doi: 10.1158/1541-7786.MCR-10-0367 Several evidences support a nonmalignant origin of ’2011 American Association for Cancer Research. Schwannian stromal cells (4, 5), which are supposed to

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control tumor growth by secreting soluble factors that phocytic cell line were cultured at 37 C and 5% CO2 in influence cell proliferation, differentiation, and angiogen- RPMI 1640, supplemented with L-glutamine, penicilline/ esis. Indeed, various molecules have been found expressed streptomycin, nonessential amino acids, and 10% FBS by Schwannian stromal cells (6, 7), although their role in (Lonza). Cells were removed from substrate with PBS/ controlling neuroblastic cells growth and dissemination is EDTA. All cell lines were tested for mycoplasma contam- not completely understood. In several tumors apart neuro- ination and authenticated (Supplementary Table 2). Total blastic tumors, the presence of stromal cells is associated RNA was isolated from neuroblastoma cell lines by Per- with a better disease outcome and the contiguity between fectPure RNA Cell Kit (5Prime), and from tumor tissues stroma and tumor cells has an important role in cancer by PerfectPure Tissue RNA Cell Kit (5Prime), and treated growth and invasion. with RNase-free DNase I. RNA integrity and quantifica- Recently, we studied a series of neuroblastic tumors by tion were checked by RNA 6000 Nano LabChip kit and gene-expression profiling analysis and we observed different 2100 BioAnalyzer instrument (Agilent Technologies). levels of CXCL13 transcripts in microdissected neuroblastic cells with respect to Schwannian stromal cells (5, 8). Our Isolation of neuroblastic and Schwannian stromal cells results showed that this chemokine was more expressed in by laser capture microdissection and total RNA stromal cells than in malignant neuroblasts, suggesting a isolation functional role of CXCL13 in the interaction between About 800 cells were laser microdissected from 3 SP and stroma and neuroblastic cells. 3 SR frozen tumors to obtain pure cell populations of Chemokines are a large family of molecules that, together neuroblastic cells and Schwannian stromal cells, as pre- with their receptors, are involved in the regulation of viously described (8). Total RNA from laser capture micro- chemotaxis, cell proliferation, cell migration, and several dissection-derived material was extracted by PicoPure RNA other crucial cell–cell interactions (9). Moreover, chemo- isolation kit (Arcturus Engineering), including a DNase kines play a major role in tumor growth, angiogenesis, and treatment, RNA quality control and quantification whereas invasion (10, 11) in several cancers and neoplastic cells may conducted by RNA 6000 Pico LabChip kit and the 2100 express chemokine receptors (12). Over the last years, BioAnalyzer instrument (Agilent Technologies). CCR1, CCR5, CCR6, CCR9, CXCR1, CXCR2, CXCR4, CXCR5, and CXCR6 were found expressed in neuroblas- Reverse-transcription quantitative real-time PCR toma and it has been shown that CXCR4 is virtually RNA (1 mg) from neuroblastoma cell lines and tumors expressed in all human neuroblastoma cell lines (13–16). was reverse transcribed using 20 pmoles of random hex- Starting from our observation we further investigated the amers (Eppendorf) and 200 U of SuperScript II enzyme expression of CXCL13 and its receptor CXCR5 in neuro- (Invitrogen Life Technologies). RNA (50 ng) from micro- blastic primary tumors and human neuroblastoma cell lines. dissected cells was amplified and reverse transcribed by WT- CXCL13 and CXCR5 mRNAs were differently expressed Ovation RNA Amplification System kit (NuGEN Tech- between Schwannian stromal cells and neuroblastic cells nologies). CXCL13, CXCR5, CXCR5a, S100A6, and 18S suggesting a cell–cell crosstalk via CXCL13-CXCR5 axis. rRNA transcripts were quantified by TaqMan Gene Expres- This crosstalk may contribute to retain neuroblastic cells sion Assays (Applied Biosystems). An assay specific for within stroma-rich tumors and possibly to inhibit the malig- CXCR5b isoform was designed by PrimerDesign Ltd. nant cells dissemination. Our results add new information 18S rRNA was used as reference gene. Any amplified about the CXCL13–CXCR5 axis in neuroblastic tumors and product with a quantification cycle (Cq) higher than 36 in the crosstalk between neuroblastic and stromal cells. cycle was considered undetectable. According to the Mini- mum Information for Publication of Quantitative Real- Materials and Methods Time PCR Experiments (MIQE) (17, 18), a checklist including technical details is submitted as Supplementary Tumor samples, neuroblastoma cell lines, and total data (MIQE checklist). The relative amount of each tran- RNA isolation script was determined using the equation 2 dCq, where dCq ¼ – Tumor specimens were collected from 26 patients at (Cqtarget gene Cq18SrRNA). onset of disease, who were diagnosed with a primary neuroblastic tumor and referred to the Gaslini Children's CXCR5 detection in neuroblastoma cells by flow Hospital. The study was approved by Ethics committee of cytometry the Gaslini Children's Hospital and informed consent was Neuroblastoma cell suspensions (3 105 cells/tube) and obtained by all children's legal guardians. According to the Raji cell line suspension (1.5 105 cells/tube) were washed International Neuroblastoma Pathology Committee (2), 13 with PBS and incubated with 2.5 mg/ml monoclonal mouse samples were from SP tumors with at least 80% of neuro- anti-human CXCR5/Blr-1 primary antibody (R&D Sys- blastic cells, and 13 were from SR with Schwannian stromal tems) in 2% BSA/PBS for 30 minutes on ice. After cells ranging from 80% to 90% (Supplementary Table 1). incubation, cells were washed twice with 2% BSA/PBS Eleven neuroblastoma cell lines (ACN, GI-CA-N, GI-LI- and 50 ml of goat anti-mouse IgG2b-PE secondary antibody N, GI-ME-N, IMR-5, IMR-32, LA1–5S, SH-SY5Y, SK- (Becton Dickinson) was added. Cell suspensions were N-BE(2), SK-N-BE(2)c, SK-N-SH) and the Raji B-lym- incubated for 30 minutes on ice. Percentage of positive

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cells and mean fluorescence intensity of stained cells were LA1–5S) were incubated for 30 minutes at 37C with analyzed using a FACScan (Becton Dickinson). The back- monoclonal anti-human CXCR5/BLR1 mouse antibody ground levels were set using cells stained with secondary (20 mg/ml for neuroblastoma cell lines; 10 mg/ml for tumor antibody alone and viable cells were discriminated by sections) and anti-human CXCL13/BLC/BCA-1 goat anti- evaluating the level of propidium incorporation. body (0.8 mg/ml for tumor sections) (R&D Systems, Inc) and anti-human GFAP (Glial fibrillary acidic protein) poli- CXCR5 and CXCL13 detection in neuroblastoma cells clonal rabbit antibody (Ventana Medical System). Anti- by immunofluorescence CXCR5 antibody was detected by UltraViewRed detection Neuroblastoma cells were directly fixed in a methanol kit (Ventana Medical System) based on polymeric alkaline fixing solution and were dropped on microscope glass slides phosphatase detection system. Staining with anti-CXCL13 and dried at room temperature (RT). Slides were incubated antibody was detected by LSABþSystem HRP Kit (DAKO 30 minutes with 10% goat serum in PBS. Primary mono- Cytomation, Glostrup, Denmark). Fixed cells and sections clonal anti-human CXCR5/Blr-1 mouse antibody was were counterstained with Gill modified hematoxylin diluted 1:40 with PBS 1.5% normal goat serum, applied (Ventana Medical System), dehydrated, and mounted. on slides at a concentration of 12 mg/ml and incubated 1 Staining without primary antibody was carried out as hour at RT in a humidified chamber. After washing, slides negative control. Staining of Raji cells and frozen or were incubated 40 minutes at RT in a humidified chamber paraffin-embedded from lymph nodes and spleen samples with goat anti-mouse-IgG2b-FITC conjugated secondary was carried out as positive control. antibody (Santa Cruz Biotechnology, Inc.) diluted to a concentration of 1.5 mg/ml with PBS 3% goat normal Glial differentiation assays serum. To identify the expression of CXCL13, acetone N-type (SH-SY5Y), I-type (SK-N-BE(2)c) and S-type permeabilized cells were incubated 1 hour at room tem- (LA1–5S) neuroblastoma cell lines were grown by adding perature in a humidified chamber with primary anti-human 10 mmol/L 50-Bromo-20-deoxyuridine (BrdU) (Sigma CXCL13 goat antibody diluted 1:50 with PBS 1.5% Aldrich, Inc.) to cell culture medium to induce glial differ- normal donkey serum, applied on slides at 2 mg/ml. After entiation (19). Treatment was maintained for 3 weeks by washing, slides were incubated 45 minutes at RT in a renewing media three times per week. During the first and humidified chamber with donkey anti-goat IgG(HþL) the second week of treatment, cell proliferation was not TRITC conjugated secondary antibody (Jackson Immu- affected by the treatment and cells were split twice. Culture noresearch Europe Ltd) diluted 1:50 with PBS 3% donkey media of treated cells were collected every three days for normal serum. Staining without primary antibodies was 3 weeks and then concentrated from 40 ml to 200 ml with carried out as negative control. Slides were mounted with centrifugal filter devices Centricon Plus-70 (Millipore, mounting medium and inspected under the microscope Inc.). Total RNA was extracted from the cells after 1, 2, with a fluorescence lamp. and 3 weeks of treatment with BrdU. Pictures of differentiated cells have been taken after 3 weeks of treatment. CXCR5 detection in primary tumor by immunofluorescence CXCL13 detection in neuroblastoma cell line Frozen sections of 4 mm from 5 SP and 5 SR tumors were conditioned medium by ELISA fixed in 4% paraformaldehyde for 2 minutes. Sections were CXCL13 protein concentration was detected in neuro- incubated with Ultra V Block (Thermo Fisher Scientific, blastoma cell line conditioned medium by Quantikine LabVision Inc.) for 8 minutes at RT to block unspecific Human CXCL13 Immunoassay (R&D Systems, Inc.), binding of the antibody. Primary monoclonal anti-human following the manufacturer's recommendations. The assay CXCR5/Blr-1 mouse antibody was diluted 1:40 with PBS employs the quantitative sandwich enzyme immunoassay and incubated 1 hour at RT in a humidified chamber. After technique. A monoclonal antibody for CXCL13 was washes three-time, slides were incubated for 45 minutes at precoated onto a microplate. Human recombinant RT in a humidified chamber with goat anti-mouse IgG CXCL13 (hrCXCL13) standards, saliva, untreated, and (HþL) Alexa Fluor 488 Conjugate (Molecular Probe, Invi- BrdU differentiated neuroblastoma cells conditioned trogen, Inc.) diluted 1:100 in PBS and counterstained with mediawerepipettedintothewellsandCXCL13was 40, 6 diamidino 2 phenylindole (DAPI). Staining without bound by the immobilized antibody. After washing, an primary antibody was carried out as negative control. enzyme-linked monoclonal antibody specific for CXCL13 was added. After incubation and extensive washing, a CXCR5 and CXCL13 detection in primary tumors and substrate solution was added and the amount of CXCL13 neuroblastoma cells by immunohistochemistry was quantified by a colorimetric measurement on a stan- Immunohistochemistry analysis was conducted using dard curve of serial dilution of hrCXCL13. polymeric complex technique and automated immunostai- ner Benchmark XT (Ventana Medical System). Sections of 5 Neuroblastoma cell migration assay mm from the same tumor samples were air dried and fixed (10 Cell migration assays were conducted on SH-SY5Y, ACN, minutes with acetone and 5–10 seconds in methanol at RT) and LA1–5S cells in microchambers (NeuroProbes Gaithers- and methanol fixed neuroblastoma cell lines (SH-SY5Y and burg) as previously described (20). Cells were extensively

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washed with PBS, resuspended in serum-free medium, and Results placed in the upper compartment. The two compartments of the chamber were separated by 5 mm pore size polycarbonate CXCL13 and CXCR5b mRNA expression in whole filters coated with 0.01% gelatine. The hrCXCL13 (100 or tumor, isolated neuroblastic and Schwannian stromal 200 ng/ml) and concentrated serum-free conditioned med- cells, and neuroblastoma cell lines ium from BrdU-differentiated neuroblastoma cells were used We carried out RT-qPCR of 26 tumor samples to assess as chemoattractants in the lower chamber. To obtain specific the expression of CXCL13 and CXCR5b mRNA in neuro- neutralization of hrCXCL13 bioactivity a blocking antihu- blastic tumors. We firstly observed that SP tumors showed man CXCL13/BLC/BCA-1 goat antibody (R&D Systems, higher levels of CXCR5b mRNA than SR tumors. Con- Inc) was added to hrCXCL13 and to BrdU differentiated versely, CXCL13 was more expressed in SR tumors (Fig. 1). neuroblastoma cells conditioned medium in the lower Noteworthy, RT-qPCR analysis showed CXCR5b transcript chamber at 3.5 mg/ml and 0.5 mg/ml, respectively. To variant (accession number 032966; ref. 21) in neuroblastic specifically neutralize CXCR5 receptor on neuroblastoma tumors. In our analysis we used primers for both CXCR5a cell surface, cells were previously incubated 30 minutes with and CXCR5b variants but only the CXCRb transcript was 15 mg/ml blocking monoclonal antihuman CXCR5/BLR1 detected whereas CXCR5a was not detectable in any sam- mouse antibody (R&D Systems, Inc). After 6 hours of ples (data not shown). incubation at 37 Cin5%CO2, the filters were recovered, Because neuroblastic tumors show tissue heterogeneity the cells on the upper surface were mechanically removed, and nonmalignant cells may infiltrate the tumor, we isolated and the cells on the lower surface were fixed and stained. The either neuroblastic or Schwannian stromal cells from 6 migration was measured by counting the remaining cells on neuroblastic tumors (3 SP and 3 SR) by laser capture the filter lower surface. Experiments were done in six microdissection. Next, we extracted total RNA from both replicates and repeated three times. purified cell populations and we carried out RT-qPCR for

5,000 4,500 4,000 CXCL13 10,000 CXCL13 3,500 1,000 3,000 100 2,500 10 MNE P = 0.1 2,000 1 1,500 0.1 1,000 0.01 500 0.001 0.0001 0 SP SR _SP _SR 4_SP 1_SR 8_SR9_SR 20_SP 2036_SP2216_SP2277_SP1636_SP22 1548_SP1678_SP1618_SP1958_SP2122_SP16442497_SP163 1630_SR1533 1644_SR176 1689_SR1811_SR1797_SR1590_SR1525_SR208 166 1627_SR1589_SR

1,600

1,400 CXCR5b CXCR5b 1,200 10,000 1,000

800 1,000 MNE 600 100 400 10 200 P = 0.002 0 1 R SP SR _SR

2036_SP2216_SP2277_SP1636_SP2220_SP1548_SP1678_SP1618_SP1958_SP2122_SP1644_SP2497_SP1634_SP 1630_SR1533_SR1644_SR1761_SR1689_SR1811_SR1797_SR1590_S15252088_SR1669_SR1627_SR1589_SR

Figure 1. CXCL13 and CXCR5b expression in tumors. CXCL13 and CXCR5b expression by RT-qPCR in 26 Neuroblastic tumors: 13 SP and 13 SR tumors. Left, the level of CXCL13 and CXCR5 in SP and SR are shown, whereas right, shows the box plot representation; P value is reported. Most of SR tumors express CXCL13 and very few express CXCR5; on the contrary, SP tumors express CXCR5 at various amount. CXCL13 is very low or undetectable expressed in SP tumors. (MNE, Mean normalized expression).

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Next, we analyzed SH-SY5Y and LA1–5S neuroblastoma 3.5E-05 CXCL13 cell lines by either immunofluorescence (Fig. 3G and 3H) or 3.0E-05 immunohistochemistry (Fig. 3I and Fig. 3J) and we observed 2.5E-05 a mild surface expression of CXCR5 receptor in about 20% 2.0E-05 of SH-SY5Y and 30% of LA1–5S neuroblastoma cells, NME 1.5E-05 respectively (Fig. 3G and Fig. 3H). Immunofluorescence 1.0E-05 analysis also showed some cytoplasmic staining, possibly 5.0E-06 related to a CXCR5 internalization. Immunofluorescence 0.0E+00 1589_SS 1591_SS 1913_SS 2570_Nb 1919_Nb 2182_Nb analysis was also carried out in the other neuroblastoma cell lines with similar results and we confirmed CXCR5 expression in SK-N-BE(2), SK-N-BE(2)c and SK-N-SH cells by 6.0E-06 CXCR5b FACS analysis (data not shown). Although IMR-5, SK-N- 5.0E-06 SH and GI-LI-N, expressed CXCL13 mRNA, only IMR-5 4.0E-06 showed CXCL13 cytoplasmatic expression but the secreted

3.0E-06 protein was not detected (data not shown).

NME 2.0E-06 Induction of neuroblastoma cells to glial differentiation 1.0E-06 and CXCL13 production 0.0E+00 1589_SS 1591_SS 1913_SS 2570_Nb 1919_Nb 2182_Nb Human neuroblastoma cell lines retain a bilineage potential and may differentiate toward a neuroblast or stromal/glial phenotype on different conditions (19). Because secreted Figure 2. CXCL13 and CXCR5b expression in isolated neuroblastic and CXCL13 was not detectable by ELISA in the conditionated Schwannian stromal cells. CXCL13 and CXCR5b expression by RT-qPCR in neuroblastic (Nb) and Schwannian stromal (SS) cell populations isolated medium of any neuroblastoma cell line (data not shown), we by Laser Capture Microdissection. Isolated SS express CXCL13 that is treated SH-SY5Y, SK-N-BE(2)c and LA1–5S cells with almost undetectable in Nb cells. The receptor was found expressed in BrdU for 3 weeks to evaluate whether neuroblastoma cells isolated neuroblastic cells of 2750_Nb sample and at very low levels in the produce CXCL13 after glial differentiation induction. Fol- remaining Nb samples but not detected in SS cells. (MNE, Mean normalized expression). lowing BrdU treatment most of SH-SY5Y and SK-N-BE(2)c cells acquired an evident flat, large and more adherent cell morphology resembling Schwannian stromal cells (Fig. 4A). CXCL13 and CXCR5b. We observed expression of CXCL13 A morphologic change was also observed in LA1–5S cells at a in the stromal cell components, as opposed to very low or less extent. The induction towards glial cell lineage was undetectable levels observed in isolated neuroblastic cells shown by increase of S100A6 mRNA expression in all 3 cell (Fig. 2), supporting our results obtained by whole tumors lines (Fig. 4A). In addition, a variable de novo CXCL13 analysis. On the contrary, we definitely detected CXCR5b mRNA induction was detected in neuroblastoma cell lines expression in the isolated neuroblastic cells but not in reaching a maximum at 3 weeks in BrdU-differentiated SK- stromal cells (Fig. 2). Afterward, we investigated the expres- N-BE(2)c cells (Fig. 4B). The secretion of CXCL13 protein sion of CXCL13 and CXCR5b mRNA in 11 neuroblastoma in the culture medium was remarkable in differentiated SK- cell lines and we detected a variable expression of CXCL13 N-BE(2)c and LA1–5S (Fig. 4C). and CXCR5b. Among neuroblastoma cell lines, 3/11 (27%) express CXCL13 (GI-LI-N, IMR5, SK-N-SH) and 9/11 Triggering of neuroblastoma cells migration by (82%) express CXCR5b (GI-LI-N, GI-ME-N, IMR-32, CXCL13 IMR5, LA-1–5S, SH-SY5Y, SK-N-BE(2), SK-N-BE(2)c, We tested whether the expression of CXCR5 could be SK-N-SH). GI-LI-N, IMR5, SK-N-SH cells express both sufficient to trigger neuroblastoma cell migration in response CXCL13 and CXCR5b and ACN and GI-CA-N cells do not to CXCL13 stimulation by chemotaxis assays. Figure 5A express either CXCL13 or CXCR5b (data not shown). shows that the CXCR5-positive SH-SY5Y and LA1–5S cells significantly migrated in presence of hrCXCL13 chemokine, CXCR5 and CXCL13 protein expression in whereas the CXRC5-negative ACN cells failed to migrate neuroblastic tumors and neuroblastoma cell lines (P ¼ not significant, data not shown). LA1–5S cell migration To show that Schwannian stromal cells of SR tumors was inhibited by either blocking the receptor or the chemo- express CXCL13 but not CXCR5 we carried out immu- kine activity, following addition of specific antibodies against nohistochemistry analysis on tumor tissue. As shown in CXCR5 or hrCXCL13 (Fig. 5B). LA1–5S cell migration Fig. 3C, Schwannian stromal cells strongly express was also observed when the conditioned medium of neuro- CXCL13, whereas CXCR5 is not expressed in the same blastoma cells differentiated to the glial cell lineage were used tissue (Fig. 3D). Because CXCR5b mRNA was found as chemoattractants, thus further substantiating the possi- variable expressed we employed immunofluorescence tech- bility that cell-released CXCL13 induces neuroblastoma cell nique to have a better sensibility to detect CXCR5 expres- migration (Fig. 5C). This result shows that neuroblastoma sion. CXCR5 protein was observed in neuroblastic cells cells induced to glial cell lineage and acquiring a Schwannian- (Fig. 3E and 3F), but not in SR tumors (data not shown). like morphology secrete CXCL13 that stimulates chemotaxis

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Figure 3. CXCL13 and CXCR5 protein expression in tumors and neuroblastoma cell lines. CXCR5 CD and CXCL13 expression in GNB intermixed stroma-rich and neuroblastoma stroma-poor tumors and in neuroblastoma cells detected by immunohistochemistry (A–D and I–L) and immunofluorescence (E–H). A shows hematoxylin and eosin staining of GNB intermixed stroma-rich; in B Schwannian stromal cells are markedly stained by GFAP (Glial fibrillary acidic EF protein) antibody (Cell Marque Inc.); the same cells are positive for CXCL13 (C) but negative for CXCR5 (D). Detectable CXCR5 protein expression is observed in neuroblastoma stroma-poor by immunofluorescence (E and F; magnification 40 and 100). CXCR5 expression is clearly detected in SH-SY-5Y and LA1– 5S cells by immunofluorescence (G and H) and GH immunohistochemistry (I and L). Fluorescent positive signals are evident on the cell membrane and in the cytoplasm of some cells, indicating the internalization of CXCR5 (G and H). The arrows in I and L indicate the CXCR5 positive edge of SH-SY-5Y and LA1–5S cells. An AxioImager M1 microscope with light and fluorescence lamp (Zeiss Inc.) was used for cells and tumor tissue IL observation. Images were captured by Image capture system AxioVision Release 4.6.

in neuroblasts expressing CXCR5. Moreover, migration more limited migration than LA1–5S in response to the of LA1–5S neuroblastoma cells was significantly suppressed conditioned medium of Schwannian-like cells and their by the addition of a neutralizing anti-CXCL13 antibody. migration was not inhibited by anti-CXCL13 antibody On the contrary, CXCR5-negative ACN cells showed a (Fig. 5C).

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A B

CXCL13 3.0E-07 2.5E-07 2.0E-07 SH-SY5Y SK-N-BE(2)c LA1-5S 1.5E-07

MNE 1.0E-07 5.0E-08 0.0E+00

W )c 1W LA1-5S -5S 2W SH-SY5Y E(2 E(2)c 3W LA1-5S LA11W LA1-5S 3W SH-SY5YSH-SY5Y 1 SH-SY5Y 2W SK-N-BE(2)c 3W N-B SH-SY5Y SK-N-BE(2)c LA1-5S SK- SK-N-BE(2)cSK-N-B 2W + 10 µmol/L BrdU + 10 µmol/L BrdU + 10 µmol/L BrdU

C S100A6 1.0E-04 CXCL13 in NB conditioned medium 8.0E-05 6.0E-05 100.0 75.0 MNE 4.0E-05 50.0 2.0E-05 25.0 0.0E+00 0.0

CXCL13 (pg/mL) U U U saliva Brd LA1-5S LA1-5S SH-SY5Y E(2)c 3W SH-SY5Y -5S Brd LA1-5S LA1-5S1W 2WLA1-5S 3W E(2)c SH-SY5YSH-SY5Y 1W SH-SY5Y 2W SK-N-BE(2)c 3W SK-N-BE(2)c LA1 SH-SY5Y Brd SK-N-BE(2)cSK-N-BE(2)c 1WSK-N-B 2W SK-N-B

Figure 4. Effects of treatment with 50-bromo-20-deoxyuridine (BrdU) on morphologic features and S100A6 mRNA expression (A) and CXCL13 mRNA expression (B) and detection of secreted CXCL13 protein (C) in different neuroblastoma (NB) cell lines. SH-SY5Y, SK-N-BE(2)c, and LA1–5S cells belonging to Neuroblastic, Intermediate, and Substrate-adherent neuroblastoma subtypes, respectively, were grown in presence of 10 mmol/L BrdU to induce glial differentiation and obtain a model for Schwannian-like cell. A shows the effect of BrdU on morphological features after 3 weeks of treatment: large flattening and subadherent cell shape is clearly shown in SH-SY5Y and SK-N-BE(2)c cells but less evident in LA1–5S cells. Morphologic changes correlate with the induction of S100A6 (calcyclin) by RT-qPCR in SH-SY5Y, SK-N-BE(2)c, and LA1–5S cells after treatment with 10 mmol/L BrdU for 1, 2, or 3 weeks, indicating the induction of cells toward glial-cell lineage. A corresponding CXCL13 mRNA expression is observed in SK-N-BE(2)c and LA1–5S cell lines but not in SH- SY5Y (B). C shows the amount of secreted CXCL13 protein in cell conditioned media collected for 3 weeks. Detectable secretion of the chemokine in LA1–5S and SK-N-BE(2)c conditioned media is depending on treatment with BrdU. (W, week; MNE, Mean normalized expression).

Discussion Neuroblastic tumors stroma-rich tumors are usually composed of abundant stroma and less amount of neuro- Chemokines play several roles in both physiologic and blastic cells; these tumors onset as a localized mass and rarely pathologicconditions.Hereby,weshowthat CXCL13andits show metastatic disease. Patients with stroma-rich tumor receptor CXCR5 are involved in the relation between neuro- are at low risk of relapse after complete surgical tumor blastic and Schwannian stromal cells of neuroblastic tumors, a resection and they usually have a good outcome. Because we heterogeneous group of pediatric cancers. Several chemokines observed that CXCL13 is expressed by Schwannian stromal have been found expressed in neuroblastoma; in particular, cells, and neuroblastic cells express CXCR5 we hypothesize the role of the CXCR4-CXCL12 axis in neuroblastoma that CXCL13-CXCR5 axis may contribute to retain malig- cells proliferation, survival, and bone marrow dissemination nant CXCR5-positive neuroblasts in stroma-rich tumors is still controversial (13, 14, 22). Airoldi and colleagues (15) limiting their metastatic spreading. Such hypothesis is showed that neuroblastoma cells express CXCR5 and they strongly supported by the ability of neuroblastoma cells hypothesized that malignant cells are able to migrate even in to migrate toward a source of hrCXCL13. Moreover, our the bone marrow in response to CXCL13 stimulation. data indicate that the CXCR5b mRNA variant encodes for a We confirm that neuroblastoma cells express CXCR5 functionally active CXCR5b protein, which is capable to receptor and we add a detailed analysis of CXCL13 and specifically respond to the CXCL13 stimulus. CXCR5 expression in neuroblastic tumors. Our results show The role of Schwannian stromal cells in neuroblastic that only malignant neuroblasts express CXCR5 protein and tumors has been widely debated. Ambros and colleagues we show, for the first time, that CXCL13 is also secreted by (4) suggested that these cells secrete factors that limit the Schwannian stromal cells of neuroblastic tumors. proliferation of malignant neuroblasts and Chienski and

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A SH-SY5Y LA1-5S

700 60 P < 0.0001 P < 0.0003 600 P < 0.0001 50 500 40 P < 0.007 400 30 300 20 200 Migrated cells/field Migrated 100 cells/field Migrated 10 0 0 SFM CXCL13 CXCL13 SFM CXCL13 CXCL13 100 ng/mL 200 ng/mL 100 ng/mL 200 ng/mL BC LA1-5S P < 0.0001 LA1-5S ACN 60 100 50 80 P < 0.0001 40 60 30 P < 0.0001 P < 0.0001 P = ns 20 40 10 20

Migrated cells/field Migrated 0 cells/field Migrated 0 SFM CXCL13 + anti- + anti- CM + anti- CM + anti- 200 ng/mL CXCR5 CXCL13 CXCL13 CXCL13

Figure 5. CXCL-13 mediates neuroblastoma cells migration. A shows the number of migrating SH-SY5Y and LA1–5S neuroblastoma cells after 6 hours of incubation at two different doses (100 ng/ml, 200 ng/ml) of hrCXCL13 added in the lower chamber. A low number of cells migrate in the presence of serum free medium (SFM). B shows that CXCR5 positive LA1–5S migration is specifically mediated by the CXCR5/CXCL13 interaction by inhibiting both the receptor and the chemokine with anti-CXCR5 (15 ng/ml) and anti-CXCL13 (3.5 ng/ml) blocking antibody, respectively. C shows CXCR5-positive LA1–5S and CXCR5-negative ACN neuroblastoma cells chemotaxis in the presence of concentrated serum-free conditioned-medium of BrdU-differentiated neuroblastoma cells secreting CXCL13. Migration of LA1–5S cells is significantly reduced by anti-CXCL13 (0.5 ng/ml) blocking antibody added to the conditioned medium. ACN cells showed limited migration that was not influenced by anti-CXCL13 antibody. Statistical analysis was carried out by unpaired two-tailed t-test. (X-axis shows the different chemoattractants, y-axis the number of cells migrated/field. ns: not significant).

colleagues (7) indicated that Schwannian stromal cells medium of BrdU differentiated neuroblastoma cells and that release other factors that modulate angiogenesis. Here, this effect is blocked by CXCL13-neutralizing antibodies. we show that CXCL13 is produced by Schwannian stromal Overall data indicate that differentiated neuroblasts pheno- cells adding new information about the factors released by typically similar to Schwannian stromal cell are able to secrete stroma of neuroblastic tumors and we suggest a relevant role CXCL13 and to exert a chemotactic effect on CXCR5- of the CXCL13/CXCR5b axis in the crosstalk between positive neuroblastoma cells, whereas CXCR5-negative neu- neuroblasts and stromal cells. The novel discovery that roblastoma cells showed limited migration possible due to a Schwannian stromal cells express CXCL13 supports the slight effect of other chemotactic factors produced by Schwan- previous findings that cells of neural origin are able to nian-like cells. Collectively, our results strongly indicate a produce CXCL13 (23, 24). close relation between Schwannian stromal cells and neuro- As an in vitro model to test the possible involvement of blastic cells mediated via aCXCL13–CXCR5 interaction. CXCL13 in the crosstalk between malignant neuroblasts and This observation is enforced by the finding that anti-CXCL13 Schwannian stromal cells we induced glial cell differentiation antibody significantly inhibits the migration of CXCR5- of neuroblastoma cells. Neuroblastoma cell lines treated with positive neuroblastoma cells in response to the conditioned BrdU induce immature neuroblasts to a more mature phe- medium. Hence, in addition to the other factors that exert notype with a large and subadherent Schwannian-like mor- their effects on neuroblast survival and differentiation, and on phology (19). After BrdU treatment, neuroblastoma cells tumor angiogenesis (4, 6, 7), our finding that Schwannian- stop to proliferate and produce calcyclin (25), a glial, and like cells are able to secrete a chemotactic factor acting on Schwannian cells marker. We show for the first time, that neuroblastic cell migration adds insight on a broad role of neuroblastoma cells, which possess a bilineage potential (19), Schwannian stromal cells in stroma-rich neuroblastic tumors. express CXCL13 only after induction to a glial phenotype. In conclusion, our model suggests that CXCL13 produced Furthermore, we showed that LA1–5S neuroblastoma cells by Schwannian stroma may contribute to limit dissemina- are able to migrate under the effect of the conditioned tion of CXCR5-expressing neuroblasts outside the SR

822 Mol Cancer Res; 9(7) July 2011 Molecular Cancer Research

CXCL13-CXCR5 Crosstalk in Neuroblastoma

tumors, whereas CXCR5-positive neuroblastic cells of Acknowledgments stroma-poor tumors more easily disseminate to CXCL13- producing distal sites, such as the bone marrow. Although we We are grateful to surgeons, clinicians, pathologists of the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) for providing tumor samples, to cannot exclude the influence of CXCL13 released from distal Dr. Alessandro De Ambrosis for the helpful discussions and to Silvia De Luca bone marrow sites on neuroblastona cells, this effect appears (Fondazione Italiana per la Lotta al Neuroblastoma) for language revision. unlikely in the case of localized tumors. Indeed, very low concentrations of CXCL13 are usually present in the sys- Grant Support temic circulation, whereas the Schwannian stromal cells and malignant neuroblasts are in close contact thus facilitating The work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC, local CXCL13-CXCR5 interaction. Obviously, this model Project IG 5331, 2008), Fondazione Italiana per la Lotta al Neuroblastoma, cannot be the only reason for the different biological and Ministero dell’Universita, Ricerca Scientifica e Tecnologica, Italy. M. Croce and L. Longo are supported by F. Italiana per la Lotta al Neuroblastoma, F. Valdora is a clinical behavior of SR and SP tumors, which is predomi- fellow of the University of Genoa. nantly related to the different genetic and molecular char- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance acteristics observed in localized and metastatic tumors (26). with 18 U.S.C. Section 1734 solely to indicate this fact. Disclosure of Potential Conflicts of Interest Received August 11, 2010; revised April 26, 2011; accepted May 25, 2011; No potential conflicts of interest were disclosed. published OnlineFirst June 3, 2011.

References 1. Maris JM, Hogarty MD, Bagatell R, Cohn SL. Neuroblastoma. Lancet 14. Raffaghello L, Cocco C, Corrias MV, Airoldi I, Pistoia V. Chemokines in 2007;369:2106–20. neuroectodermal tumour progression and metastasis. Semin Cancer 2. Shimada H, Ambros IM, Dehner LP, Hata J, Joshi VV, Roald B. Biol 2009;19:97–102. Terminology and morphologic criteria of neuroblastic tumors: recom- 15. Airoldi I, Cocco C, Morandi F, Prigione I, Pistoia V. CXCR5 may be mendations by the International Neuroblastoma Pathology Commit- involved in the attraction of human metastatic neuroblastoma cells to tee. Cancer 1999;86:349–63. the bone marrow. Cancer Immunol Immunother 2008;57:541–8. 3. Shimada H, Umehara S, Monobe Y, Hachitanda Y, Nakagawa A, Goto 16. Meier R, Muhlethaler-Mottet€ A, Flahaut M, Coulon A, Fusco C, S, et al. International neuroblastoma pathology classification for Louache F, et al. The chemokine receptor CXCR4 strongly promotes prognostic evaluation of patients with peripheral neuroblastic tumors. neuroblastoma primary tumour and metastatic growth, but not inva- Cancer 2001;92:2451–61. sion. PLoS ONE 2007;2:e1016. 4. Ambros IM, Zellner A, Roald B, Amann G, Ladenstein R, Printz D, et al. 17. Lefever S, Hellemans J, Pattyn F, Przybylski DR, Taylor C, Geurts R, Role of ploidy, chromosome 1p, and Schwann cells in the maturation et al. RDML: structured language and reporting guidelines for real- of neuroblastoma. N Engl J Med 1996;334:1505–11. time quantitative PCR data. Nucleic Acids Res 2009;37:2065–9. 5. Coco S, Defferrari R, Scaruffi P, Cavazzana A, Di Cristofano C, 18. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Longo L, et al. Genome analysis and gene expression profiling of et al. The MIQE Guidelines: Minimum Information for publication of neuroblastoma and ganglioneuroblastoma reveal differences Quantitative Real-Time PCR experiments. Clin Chem 2009;55:611–22. between neuroblastic and Schwannian stromal cells. J Pathol 2005; 19. Acosta S, Lavarino C, Paris R, Garcia I, de Torres C, Rodríguez E, et al. 207:346–57. Comprehensive characterization of neuroblastoma cell lines subtypes 6. Huang D, Rutkowski JL, Brodeur GM, Chou PM, Kwiatkowski JL, reveals bilineage potential similar to neural crest stem cells. BMC Dev Babbo A, et al. Schwann cell-conditioned medium inhibits angiogen- Biol 2009;9:12. esis. Cancer Res 2000;60:5966–71. 20. Albini A, Benelli R, Giunciuglio D, Cai T, Mariani G, Ferrini S, et al. 7. Chlenski A, Liu S, Crawford SE, Volpert OV, DeVries GH, Evange- Identification of a novel domain of HIV Tat involved in monocyte lista A, et al. SPARC is a key schwannian-derived inhibitor control- chemotaxis. Biol Chem 1998;273:15895–900. ling neuroblastoma tumor angiogenesis. Cancer Res 2002;62: 21. Barella L, Loetscher M, Tobler A, Baggiolini M, Moser B. Sequence 7357–63. variation of a novel heptahelical leucocyte receptor through alternative 8. Albino D, Scaruffi P, Moretti S, Coco S, Truini M, Di Cristofano C, et al. transcript formation. Biochem J 1995;309:773–9. Identification of low intratumoral gene expression heterogeneity in 22. Airoldi I, Raffaghello L, Piovan E, Cocco C, Carlini B, Amadori A, et al. neuroblastic tumors by genome-wide expression analysis and game CXCL12 does not attract CXCR4þ human metastatic neuroblastoma theory. Cancer 2008;113:1412–22. cells: clinical implications. Clin Cancer Res 2006;12:77–82. 9. Bonecchi R, Galliera E, Borroni EM, Corsi MM, Locati M, Mantovani A. 23. van de Pavert SA, Olivier BJ, Goverse G, Vondenhoff MF, Greuter M, Chemokines and chemokine receptors: an overview [review]. Front Beke P, et al. Chemokine CXCL13 is essential for lymph node initiation Biosci 2009;14:540–51. and is induced by retinoic acid and neuronal stimulation Nat Immunol 10. Balkwill F. Cancer and the chemokine network. Nat Rev Cancer 2009;10:1193–9. 2004;4:540–50. 24. Ramesh G, Borda JT, Gill A, Ribka EP, Morici LA, Mottram P, et al. 11. Savino Mantovani A, Locati M, Zammataro L, Allavena P, Bonecchi R. Possible role of glial cells in the onset and progression of Lyme The chemokine system in cancer biology and therapy [review]. Cyto- neuroborreliosis. J Neuroinflammation 2009;6:23. kine Growth Factor Rev 2010;21:27–39. 25. Tonini GP, Casalaro A, Cara A, Di Martino D. Inducible expression of 12. Slettenaar VIF, Wilson JL. The chemokine network: a target in cancer calcyclin, a gene with strong homology to S-100 protein, during biology? Adv Drug Deliv Rev 2006;58:962–74. neuroblastoma cell differentiation and its prevalent expression in 13. Gross N, Meier R. Chemokines in neuroectodermal cancers: The Schwann-like cell lines. Cancer Res 1991;51:1733–7. crucial growth signal from the soil. Semin Cancer Biol 2009; 26. Maris JM. Recent Advances in Neuroblastoma. N Engl J Med 2010; 19:103–10. 362:2202–11.

www.aacrjournals.org Mol Cancer Res; 9(7) July 2011 823

Role of CXCL13-CXCR5 Crosstalk Between Malignant Neuroblastoma Cells and Schwannian Stromal Cells in Neuroblastic Tumors

Federica Del Grosso, Simona Coco, Paola Scaruffi, et al.

Mol Cancer Res 2011;9:815-823. Published OnlineFirst June 3, 2011.

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