Antibodies

mAbs against CCR1 (clone 141-2, reference D063-3) and CCR3 (clone 444-11, reference D085-3) obtained from MBL International (Woburn, MA, USA) were used for flow cytometry (20 μg ml−1) and as blocking antibodies (10 μg ml−1) in the migration assays. Anti-CCR2 (clone E68, reference ab32144), used for flow cytometry (20 μg ml−1), and anti-CCR3 (clone Y31, reference ab32512), used for immunohistochemistry (15 μg ml−1), mAbs were purchased from Abcam (Cambridge, MA, USA). Monoclonal Abs against CXCR1 (Clone 42705, reference MAB330), CXCR2 (clone 48311, reference MAB331) and CXCR4 (clone 44716, reference MAB172) were used for flow cytometry (20 μg ml−1) and as blocking antibodies in the migration assays (10 μg ml−1), and were obtained from R&D Systems (Minneapolis, MN, USA). mAbs against human CCL7 (Clone 36320, reference MAB282) and pAbs against murine CCL7 (reference AF-456) were used as blocking antibodies in the migration assays (10 μg ml−1) were obtained from R&D Systems (Minneapolis, MN, USA). CXCL12 blocking mAbs (Clone 79014) directed against murine CXCL12 were obtained from R&D Systems (Minneapolis, MN, USA) and used for migration assay (10 μg ml−1).

Inhibitors and recombinant chemokines

Recombinant CCL7 protein was obtained from Peprotech (Rocky hill, CT, USA). The antagonist of CXCR1 and CXCR2, SB225002 (ref. 19) was purchased from Tocris (Bristol, UK) and used at final concentrations from 1 to 100 nM. SB225002 inhibits neutrophil chemotaxis in response to CXCL1 and IL8 in vitro (IC 50 =20 nM)19. The CXCR4 antagonist, AMD3100, which inhibits CXCL12-stimulated chemotaxis (IC 50 =50 nM)59, was purchased from Sigma-Aldrich and used at final concentrations from 1 to 100 nM. The inhibitor targeting CCR2, sc-202525, which inhibits CCL2-induced chemotaxis (IC 50 =10 nM)20 was purchased from Santa Cruz Biotechnology (Dallas, TE, USA) and used at final concentrations from 1 to 25 nM. The inhibitor targeting CCR1 and CCR3, UCB35625 was purchased from Tocris (Bristol, UK) and used at final concentrations from 1 to 200 nM. This molecule inhibits CCL11-induced chemotaxis in cells transfected with CCR3 (with an IC 50 value of 93.8 nM)21. The maximal doses (between two- and 2.5-fold the IC 50 ) do not exhibit toxicity on prostate cancer cells as determined by 3-(4,5-diMethylThiazol-2-yl)-2,5-diphenylTetrazolium bromide (MTT) assays after 24 h exposure (<15% decrease in the number of treated cells compared with untreated control). All the molecules were dissolved in dimethylsulfoxide (DMSO). The final DMSO concentration of each dilution was used in all corresponding controls.

Cell lines and culture

The human prostate tumour cell lines LNCaP (ATCCCRL-1740), C4-2B (from DSMZ, Braunschweig, Germany), Du-145 (ATCCHTB-81) and PC-3 (ATCCCRL-1435; provided by Dr Olivier Cuvillier, IPBS, Toulouse, France) were used in this study. LNCaP, Du-145 and PC-3 are derived from human prostate carcinoma metastases (lymphonodal, brain and bone lesions, respectively60,61,62). C4-2B cell line was derived from a bone metastasis after orthotopic transplantation of C4-2 cells (subclone of LNCaP) in nude mice63.

The human colon carcinoma cell lines sw480 (ATCCCCL-228) and sw620 (ATCCCCL-227; provided by Dr A. Ferrand, CRCT UMR 1037, Toulouse, France), the pancreatic cancer cell line CAPAN (ATCC HTB-79) and PANC- I (ATCCCRL-2547) (provided by Dr C. Bousquet, CRCT UMR1037, Toulouse, France), the breast cancer cell lines T-47D (ATCC HTB-133) and MDA-MB321 (ATCCCRM-HTB-26; provided by Dr K. Bistricky, LBME), the melanoma cell lines 501mel and Lu1205 (gifts from Dr L. Larrue, Institut Curie, Orsay, France ). All cell lines were cultured in RPMI medium (Invitrogen, Auckland, NZ) supplemented with 10% FCS, 125 mg ml−1 streptomycin and 125 UI ml−1 penicillin, in a humidified atmosphere of 5% CO 2 .

The murine prostate cancer cell line TRAMP-C1P3 (ATCCCRL-2730)39 was kindly provided by Dr Richard P. Ciavarra (Eastern Virginia Medical School, Norfolk, VA) and cultured in DMEM medium (Invitrogen, Auckland, NZ) supplemented with 10% FCS, 5 mg ml−1 insulin (Sigma-Aldrich, St Louis, MO, USA), 10 nM dihydrotestosterone (Sigma-Aldrich) and antibiotics. The TRAMP-C1P3 was obtained using an in vivo selection scheme of intraprostatic implantation of TRAMP-C1 cells, derived from a prostate adenocarcinoma in transgenic C57BL/6 mice expressing the antigen SV40 under the control of the prostate specific probasin promoter39. All the cell lines were used within 2 months after resuscitation of frozen aliquots. To obtain TRAMP-C1P3 cells with stable downregulation of CCR3 expression, cells were transduced with the lentiviral vector PLVTMH containing either control or shRNA coding sequences for murine CCR3 (NM_009914). Note that cells transduced with lentiviral vectors also expressed GFP. Interference sequences were generated with the ‘siDesign’ Dharmacon tool. The sequences of the sense strands of the shRNAs generated are:

m4CCR3 5′- cgcgtccccAGACCACACCCTATGAATAttcaagagaTATTCATAGGGTGTGGTCTtttttggaaat -3′, m5CCR3 5′- cgcgtccccGACCACACCCTATGAATATttcaagagaATATTCATAGGGTGTGGTCtttttggaaat -3′ m6CCR3 5′- cgcgtccccGGTGAGAGGTTCCGGAAACttcaagagaGTTTCCGGAACCTCTCACCtttttggaaat -3′ under the control of the H1 promoter. Nucleotide sequences targeting CCR3 are shown in capital letters, whereas the sequence responsible for the hairpin structure and sequences necessary for the directional cloning are shown in lowercase letters. 293T cells were kindly provided by Genethon (France) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS. Generation of 293T-LVTHM-shCCR3 and preparation of high-titer lentiviral vector pseudotyped with VSV-G protein was performed as followed. Subconfluent 293T cells were cotransfected with 20 μg of a plasmid vector, 15 μg of pCMV-ΔR8.91, and 5 μg of pMD2G-VSVG by calcium phosphate precipitation. After 16 h medium was changed, and recombinant lentivirus vectors were harvested 24 h later. Cells (50 × 103) were plated on 35-mm dishes 24 h before transduction with viral vectors at a multiplicity of infection of 10:1 (refs 64, 65). One week after transduction and amplification, sterile fluorescence-activated cell sorting was used to select a population consisting of >90% GFP-expressing cells. The cells were kept in culture for a maximum of 1 month and the expression of GFP was checked every week.

The murine 3T3-F442A pre-adipocyte cell line was cultured in DMEM (Invitrogen, Auckland, NZ) supplemented with 10% FCS, 2 mM glutamine and antibiotics. Differentiation was induced by incubating confluent cells in differentiation medium (DMEM supplemented with 10% FCS and50 nM insulin) for up to 14 days after which more than 90% of the cells had accumulated fat droplets.

Ad-CM was obtained by incubating the murine adipocytes in DMEM medium containing no FCS and 1% BSA (Sigma-Aldrich) and, after 12 h, the medium was harvested and immediately stored at −80 °C. Media from a six-well plate in which the cells were differentiated was pooled to create one sample of Ad-CM. The frozen aliquots were only used once, as for all the conditioned media used in our study. For migration assays, the conditioned medium was diluted to one half in DMEM medium containing 1% BSA. Cell lines were authenticated on the basis of viability, recovery, growth rate and morphology. Cell lines were regularly tested for mycoplasma contamination.

Mice

Mice were handled in accordance with National Institute of Medical Research (INSERM) principles and guidelines. C57Bl6/J male mice were obtained from Janvier (Le Genest St Isle, France). Mice were housed conventionally in an animal room at constant temperature (20–22 °C) and humidity (50–60%), and with a 12 h light–dark cycle. All the mice had free access to food and water throughout the experiment. The C57Bl6/J mice were assigned to a normal diet (PicoLab Rodent Diet 20, Purina Mills, Inc., Brentwood, MO, USA) or HFD (Research Diets, Inc., New Brunswick, New Jersey, USA). The energy contents of the diets were as follows: 16% protein, 81% carbohydrate and 4% fat for the normal diet; 20% protein, 20% carbohydrate and 60% fat for the HFD. C57Bl6/J mice (initially 10 weeks old) were fed a normal diet or a HFD for 8–10 weeks. A 10-week period of HFD is used to mimic the development of obesity in humans associated with the emergence of insulin resistance and low-grade inflammation66. To collect visceral adipose tissue (mu-VAT), mice were killed at 20 weeks. For intraprostatic injection of TRAMP-C1P3 cells expressing control (Ctrl) or anti-CCR3 shRNA, we used mice fed either a normal diet or a HFD at 18 weeks. The mice continued to be fed either a normal diet or HFD and were killed 3 weeks later (21 weeks old). The Midi-Pyrénées animal ethics committee approved all procedures.

Conditioned medium from mu-VAT

Mu-VAT was dissected from lean or obese mice immediately post mortem. Mu-VAT was weighed and 1 g of tissue was placed immediately in 8 ml of DMEM medium supplemented with 1% BSA. Mu-VAT was incubated overnight at 37 °C in a humidified incubator with 5% CO 2 , the medium was collected (mu-VAT-CM) and stored in small aliquots at −80 °C. Mu-VAT-CM was obtained from three animals from each group (lean or obese).

Conditioned medium from isolated adipocytes and SVF

In particular experiments (see text), mature adipocytes and SVF cells were isolated from whole mu-VAT. Briefly, mu-VAT (1 g of tissue) was incubated in 8 ml of DMEM, 1% BSA in the presence of liberase at a final concentration of 25 μg ml−1 (Roche Applied Science, Meylan, France) for 30 min at 37 °C under shaking. After digestion, FCS at a final 5% concentration was added to inhibit the liberase. The samples were then centrifuged (20 min, room temperature, 100 g) to separate adipocytes (floating cells) from the SVF (pellet). Six animals from each group (lean or obese) were used for one experiment. The pooled isolated primary adipocytes and SVF cells were then cultured (5 × 106 cells) in 5 ml of DMEM (without serum) containing 1% BSA for 24 h and the CM was collected at the end of the incubation period and immediately stored at −80 °C.

Hu-PPAT samples

hu-PPAT samples were collected from radical prostatectomy in accordance with the recommendations of the ethics committee of the Rangueil Hospital (Toulouse, France). All patients gave their informed consent to participate to this study, which was conducted in accordance with the Declaration of Helsinki Principles as revised in 2000. PPAT samples were collected at a distance from the tumours and were macroscopically devoid of fibrosis. These samples were collected and treated within 15 min after the surgery to limit the delay between devascularization and freezing, thus ensuring the preservation of labile molecules. PPAT samples were weighed and 1 g of tissue was placed immediately in 5 ml of DMEM supplemented with 1% BSA. Hu-PPAT was incubated overnight at 37 °C in a humidified incubator with 5% CO 2 and the medium was collected (hu-PPAT-CM). For each patient (n=5), hu-PPAT-CM was separated into small aliquots and immediately stored at −80 °C. In specified experiments, staged biopsies were performed in three independent prostatectomy pieces (punch biopsy with 4 mm trocard). These biopsies were performed in PPAT, at the same level as the prostate capsule and inside the prostate gland (at 2 and 4 cm from the prostate capsule, for biopsy 2 and 3, respectively). After sampling, the tissues were frozen in liquid nitrogen and stored at −80 °C. For CCL7 ELISA assays, tissues were dissociated with a tissue dissociator, which enables the release of secreted substances from the tissue (GentleMACS, Miltenyi Biotec, Inc., Auburn, CA, USA), in 1 ml of buffer containing PBS 1 × and 1% BSA.

Ex vivo differentiation of progenitors cells from the SVF of hu-PPAT

PPAT pieces were submitted to collagenase digestion as previously described in Methods, then centrifuged to separate adipocytes from SVF (pellet). The SVF was then used for ex vivo differentiation. Briefly, confluent cells (day 0) were differentiated in DMEM containing 1 μM insulin, 10 μM dexamethasone, 0.5 mM IBMX, supplemented with 10% FCS, 2 mM glutamine and antibiotics. Two days post induction, medium was changed to DMEM containing only 1 μM insulin, 10% FBS with glutamine and antibiotics. After 10–14 days of culture in lipogenic medium, Ad-CM was attained by incubating the obtained mature adipocytes (obtained from human PPAT) in DMEM medium containing no FCS and 1% BSA (Sigma-Aldrich) for 12 h, after which, the medium was harvested, and immediately stored at −80 °C. The media from each six-well plate of differentiated cells was pooled to create one sample of Ad-CM. The frozen aliquots were only used once, as for all the conditioned media used in our study. For migration assays, the conditioned medium was diluted to one half in DMEM medium containing 1% BSA.

Human visceral adipose tissue

Human visceral adipose tissue (hu-VAT) was collected according to the guidelines of and the full ethical approval from the Ethics Committee of Toulouse Rangueil and Nancy Jeanne d’Arc Hospitals. All patients gave their informed consent to participate in the study and research was conducted in accordance with the Declaration of Helsinki Principles as revised in 2000 (http://www.wma.net/en/30publications/10policies/b3/). Hu-VAT samples from normal weight individuals were obtained from eight patients undergoing intra-abdominal surgery (aged 42.7±4.5 years, BMI 23.1±3.3 kg m−2). Hu-VAT from obese individuals was obtained from 18 patients suffering from morbid obesity grade III undergoing bariatric surgery (aged 44.5±1.8 years, BMI 47.6±1.3 kg m−2). Tissue samples were immediately frozen in liquid nitrogen after removal and stored at −80 °C. Samples were then used for mRNA extraction and quantitative reverse transcription PCR (RT-qPCR).

Boyden chamber migration assays

For cell migration, 2 × 105 serum-deprived cells were seeded in the upper chamber of Transwell plates (obtained from Greiner Bio-One, Frickenhausen, Germany, 8 μM pores). The lower chamber was filled with either DMEM without serum, DMEM containing 10% FCS or Ad-CM. Similar experiments were also performed with lower chambers filled with mu-VAT-CM, primary Ad-CM, SVF-CM (from lean or obese mice) or hu-PPAT-CM. After 12 h incubation at 37 °C, non-migrated cells were removed by wiping the upper side of the membranes with a cotton swab. This step was omitted for a control assay to assess the total number of viable cells at the end of the experiment. The transmigrated cells present in the undersurface of the inserts were stained with Toluidine blue 1% supplemented with 0.1 M borax (Sigma, St Louis, MO, USA). Membranes were then cut and incubated in a lysis buffer (Tris-HCl 6.25mm (pH 6.8), 10% glycerol, 2% SDS, 10% β-mercaptoethanol). Absorbance at 570 nm was measured with a 100 μl aliquot in duplicate transferred to a 96-well plate (μQUANT from Biotek Instrument Inc, Winooski, VT, USA). In particular experiments, cells were pre-incubated for 30 min at 37 °C with blocking mAbs directed against CCR3, CCL7 or control IgG at a final concentration of 10 μg ml−1 or with pharmacological inhibitors against CXCR1/2, CXCR4, CCR1/CCR3 or CCR2. Experiments with recombinant chemokine CCL7 were also performed. In this case, the chemokine was added to the culture medium containing 0.1% BSA without serum in the lower chamber.

Flow cytometry and ELISA

For flow cytometry analyses, 1 × 106 prostate tumour cells were fixed using paraformaldehyde solution (3.7% diluted in PBS) during 20 min at 4 °C. After washing with PBS, cells are incubated for 2 h at 4 °C with 10–20 μg ml−1 of mAbs (as recommended by the manufacturer) or matched control isotypes at similar concentrations. After washing with PBS containing 0.5% BSA and 2% FCS, cells were incubated for 30 min at 4 °C with secondary fluorescein-labelled IgG. The cells were analysed in a FACScan flow cytometer (Becton Dickinson, Franklin Lanes, NJ). Secreted murine and human CCL7 were quantified with an ELISA kit from Peprotech and R&D Systems, respectively, according to the protocols provided by the manufacturers. Secreted murine CXCL12 was quantified with an ELISA kit from R&D systems, according to the protocols provided by the manufacturers.

RNA extraction and RTq-PCR

Total RNAs were extracted using the RNeasy mini kit (Qiagen GmbH, Hilden, Germany). Gene expression was analysed using RT-qPCR. Total RNAs (1 μg) were reverse transcribed for 60 min at 37 °C using Superscript II reverse transcriptase (Invitrogen, Auckland, NZ) in the presence of a random hexamer. A minus reverse transcriptase reaction was performed in parallel to ensure the absence of genomic DNA contamination. RT-qPCR was performed starting with 25 ng of cDNA and 300, 500 or 900 nM concentration of a mix of sense/antisense primers (depending on genes) in a final volume of 25 μl using the SYBR Green Universal PCR master mix (Applied Biosystems, Foster City, CA). Fluorescence was monitored and analysed in a GeneAmp 7300 detection system instrument (Applied Biosystems, Foster City, CA). Analysis of housekeeping genes HPRT and GAPDH (500 nM) was performed in parallel to normalize for gene expression. Data were analysed by the 2−ΔΔCt method and presented as fold change relative to a control sample after normalization against the expression of housekeeping genes. Here Ct corresponds to the number of cycles needed to generate a fluorescent signal above a predefined threshold. All primers used in this study have been validated for PCR efficiency and are presented in Table 2.

Table 2 Primers used for RT-qPCR analysis. Full size table

Analysis of the adipocyte secretome by mass spectrometry

Ad-CM (serum- and phenol red-free) was collected on ice, centrifuged and filtered to remove cell debris and supplemented with complete protease inhibitor cocktail (Sigma-Aldrich). A total of 5 ml of Ad-CM was concentrated with StrataClean resin (Stevens, CA, USA) according to the manufacturer’s instructions. After reduction and alkylation of cysteine, the sample was separated by 12% acrylamide SDS–PAGE. Proteins were visualized by Coomassie Blue staining and each lane was cut into 13 homogenous slices and subjected to in-gel tryptic digestion. The tryptic digest was analysed by nano liquid chromatography coupled with tandem-mass spectrometry with an Ultimate3000 system (Dionex, Amsterdam, The Netherlands) coupled to an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany)67, except that peptides were eluted with a 5–50% gradient of solvent B for 60 min at a 300 nl min−1 flow rate. Mascot was used to automatically extract peak lists from raw files. MS/MS data were searched against all entries in the Mus musculus database and identified peptides were validated with in-house software67, except that 1% false discovery rate was used for validation.

Surgical orthotopic implantation of TRAMP-C1P3 cells

Intraprostatic grafts of either TRAMP-C1P3 shCtrl or shm6CCCR3 were established in 18 week-old C57BL/6 mice fed either a normal diet or HFD by surgical orthotopic implantation. Mice were anaesthetized by isoflurane inhalation and placed in the supine position. A lower midline abdominal incision was made and 30 μl of tumour cell suspension (2 × 106 cells) was injected into the dorsal lobe of the prostate with a 30-gauge needle and glass syringe. The surgical wound was closed in two layers with 4–0 Dexon interrupted sutures. All procedures were performed with a dissecting microscope. The local Institutional Animal Care and Use Committee approved the experimental protocols described in the study.

Autopsy and histology

All mice were killed 21 days later. A midline incision was made to access to the abdominal cavity. The tumours were removed en bloc with the seminal vesicles and the surrounding adipose tissue, weighed, and a picture of the tumours was taken. Tumours were then fixed in 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin to confirm the nature of the disease and to assess extraprostatic extension and the aspect of adipose tissue. CCR3 expression was determined with the protocol described for human samples (see below).

Patients and prostate cancer TMA

The first TMA, previously developed by the Pathology Department of Toulouse Rangueil Hospital, containing specimens of prostate cancer (n=91 in duplicates) and normal epithelium (n=10 in duplicates) was used to report CCR3 expression in relation to cancer differentiation68. The sum of tumour Gleason scores was recorded for each microarray. The second fully annotated TMA was engineered from prostatectomy pieces of 101 patients that underwent surgery between 1 February 2010 and 1 December 2011 in the Department of Urology of the University Hospital of Toulouse (directed by Professor P. Rischmann). The clinical and biochemical (PSA measurement) follow-up of the patients was updated in March 2015. Surgical treatment consisted of a robot-assisted, open-retropubic radical prostatectomy. Prostatectomy was associated in some cases with ilio-obturator bilateral lymphadenectomy (standard or extended). All patients included in the study had localized disease without metastasis (as assessed by clinical and radiological examinations) at the time of the surgery. All patients signed a consent form for the use of their tissue samples for scientific purposes before surgical intervention. The surgery specimen was treated within 15 min of its removal to limit the delay between devascularization and freezing, thus ensuring the preservation of labile molecules. Tissue samples were placed in cryovials, frozen in liquid nitrogen and stored at −80 °C. Two pathologists (Dr Catherine Mazerolles and Dr Youri Socrier), who were blind to clinical data, independently selected the areas of interest used for the preparation of TMA. Selected tumour areas had a morphology and histological differentiation similar to the tumour Gleason score of the whole tumour or the worst Gleason score found in the tumour in the case of multifocal tumours. After selecting the sample block, 0.2 mm diameter core samples were included according to a predetermined pattern in a recipient block. Gleason scores, pathological and clinical stage, surgical margins (a positive surgical margin was defined as cancer cells in contact with the inked specimen surface), follow-up time and BCR, as defined by the European Association of Urology guidelines by two PSA readings >0.2 ng ml−1 (ref. 69), were available for all patients. Surgical treatment failure was defined by patients exhibiting either BCR, locoregional recurrence or distant metastases or by the use of adjuvant radiation or hormonal deprivation therapy, as previously defined38.

Analysis of the expression of CCR3 by immunohistochemistry

Immunohistochemical experiments were performed to detect the expression of CCR3 in prostate cancer TMA. Immunostaining was performed with the EnVision FLEX Mini Kit, High pH (Dako Autostainer/Autostainer Plus; Dako France, Trappes, France). The TMA were immersed in xylene to remove paraffin and then rehydrated by successive baths of graded alcohol (100–70% and then distilled water), followed by treatment with the antigen unmasking solution for antigen retrieving (Citrate Target Retrieval Solution from Dako) in a water bath at 95 °C. After saturation of endogenous peroxidases (Peroxidase blocking solution from Dako), the samples were incubated with the primary anti-CCR3 antibody (diluted to 1/100). TMAs were then incubated with FLEX/HRP conjugate secondary antibody (EnVision FLEX+ Mouse and Rabbit from Dako). The samples were rinsed, then treated with liquid DAB (BioGenex, San Ramon, CA) and washed with distilled water. Finally, the counter-staining was carried out with hematoxylin (Dako) and the various TMA were mounted with Eukitt reagent. Two pathologists, who were blind to clinical data, independently scored CCR3 expression in human tumours as negative, low, moderate (mid) or high (manual scoring). None of the tumours were negative for CCR3 expression. The slides were then digitally scanned via Hamamatsu Nanozoomer 2.0RS and analysed with the device software provided by the manufacturer. The intensity of CCR3 staining has been quantified by computer through the use of ImageJ software, with tumour staining separated using deconvolution plug-in35,36. As a first step, we used a color deconvolution technique to separate the pure DAB and hematoxylin stained areas leaving a complimentary image. The pixel intensities of separated DAB or hematoxylin images range from 0 to 255. Value 0 represents the lightest shade of the color while 255 represent the darkest shade of the color in the image. To assign an automated score by judging the pure DAB staining pattern, a histogram profile of every image, that is, the number of pixels of a specific intensity value versus their respective intensity was raised using ImageJ standard programme feature. Note that we excluded the pixel intensity values corresponding to the unspecific staining. Intensity for each tumour gland was quantified after manual selection of the area of interest under the supervision of pathologists. Finally, we verified that the results obtained were in accordance to the three groups defined by the manual scoring (median signal±s.e.m., low expression: 28.5±4.1, moderate: 46.2±2.0, high: 56.0±3.1 arbitrary units).

Expression of CCR3 in pT3 human tumours

Frozen tissues obtained after radical prostatectomy of eight patients with pT3a or pT3b prostate cancer, defined by two pathologists (Dr Catherine Mazerolles and Dr Youri Socrier), were analysed for CCR3 expression. Four of these patients presented BMI<25 and four BMI>25. All these patients underwent surgery between 1 February 2010 and 1 December 2011 in the Department of Urology of the University Hospital of Toulouse (directed by Professor P. Rischmann) and signed a consent form for the use of their tissue samples for scientific purposes before surgical intervention. The staining of CCR3 and slides digitalization has been performed as previously. Note that some blood vessels also express CCR3 but that quantification was only performed in tumour glands under the supervision of two pathologists.

Analysis of cell number by thiazolyl blue tetrazolium bromide (MTT) assay

PC-3 or TRAMP-C1P3 cells transfected with either a control vector (shCtrl) or an sh directed against CCR3 (shm6CCR3) were plated (3 × 103 cells) in quadruplicate for each time point in a 96-well plate. Following the attachment of all cells after 6 h, the number of viable cells was measured by an MTT assay. Briefly, 100 μl of MTT (50 mg ml−1) was added to the wells and incubated for 2 h. After aspiration, 100 μl of DMSO was added to each well and incubated for 15 min at 37 °C to solubilize the bio-reduced coloured MTT-formazan and to lyse the cells. The optical density was read at 570 nm in a microplate reader.

Statistical analysis

The statistical significance of differences between means was evaluated with unpaired Student’s t-tests. All statistical tests were two-sided. P values below 0.05 (*), <0.01 (**) and <0.001 (***) were deemed as significant and ‘NS’ was used to denote not significant. For the analysis of TMA, we first performed a descriptive analysis with the clinical and biological features of patients. We then examined the clinical and biological characteristics of patients to identify patients with aggressive and extended tumours. For the correlation between CCR3 expression and quantitative data (for example, prostate weight) or variables with more than two ordered classes regarding disease severity (for example, Gleason score or tumour stage), we used a Spearman’s rank correlation test assuming a monotonic relation between considered variables. We used the Student’s t-test to correlate CCR3 expression to non-ordered variables represented by a category (for example, presence or absence of lymphatic emboli). Non-parametric tests are more conservative than parametric tests. In some cases, we also expected a lack of statistical power given the limited size of some sub-populations of variables. Leptin and CCL7 expression in hu-VAT were correlated using Pearson’s correlation analysis.