Peripheral blood lymphocytes were obtained from women with breast cancer and women who were not affected by cancer (healthy women). Peripheral blood samples (5 mL) were collected using ethylenediaminetetraacetic acid (EDTA). Peripheral blood mononuclear cells (PBMCs) were obtained after centrifugation (3000×g for 30 min) in the presence of Ficoll Histopaque (Ficoll Hypaque; Organon Teknika®, Durham, NC, USA). PBMCs were counted in a Neubauer chamber and adjusted to a final concentration of 1 × 106 cells/mL for all assays.
The breast cancer cell line (MCF-7 cells) or lymphocytes collected from breast cancer patients or healthy women were maintained at 5% CO2 atmosphere and 37 °C in DMEM (Dulbecco’s Modified Eagle Medium) (Life Technologies®, Carlsbad, California, USA), containing 10% fetal bovine serum (FBS) (Invitrogen by Life Technologies®, Carlsbad, California, USA), 50 U/mL penicillin G (Invitrogen) and 50 mg/mL streptomycin sulfate (Invitrogen). For each assay, lymphocytes and plasma samples were obtained from different healthy donors or cancer patients.
The cells analyzed by flow cytometry (FACSCalibur®, BD Biosciences, New Jersey, USA) were previously permeabilized with 0.01% saponin in 0.1 M sodium phosphate buffer for 15 min, followed by specific antibody labeling. To determine the percentage of T-lymphocytes, B-lymphocytes and NK (natural killer) cells in the PBMC fraction, the following antibodies were used: anti-CD3 (human anti-mouse FITC clone HIT3a), anti-CD4 (PE mouse anti-human clone RPA-T4), anti-CD19 (PE mouse anti-human clone 4G7) and anti-CD56 (PE CyTM mouse anti-human clone B159). All antibodies were obtained from BD Bioscience Pharmingen®, Inc. (California, USA) and used at a final dilution of 1:500. To analyze the heparanase isoform samples, anti-HPA1 C-20 and anti-HPA2 C-17 were used (Santa Cruz Biotechnology Inc., California, USA) for HPSE and HPSE2, respectively.
The lymphocytes (1 × 106 cells) were co-cultured for 18 h with 1 × 106 MCF-7 cells maintained in DMEM, 5% CO2 and 37 °C. The co-culture medium was collected for other assays.
Lymphocyte activation in vitro
Lymphocytes were incubated with conditioned medium from MCF-7 cells, MCF-7 cells (co-culture), plasma collected from healthy women or plasma obtained from breast cancer patients for 4 h at 37 °C with constant stirring (100 rpm). Lymphocyte activation assays were also performed in the presence of anti-syndecan-1 (clone CD138 BB4 MCA681) diluted 1:50 (AbD Serotec®, Bio-Rad Company Co., Oxford, UK), or the co-culture medium was previously treated with heparitinase II (HTase II from Flavobacterium heparinum). The digestion with HTase II was performed in 20 mM Tris–HCl buffer containing 50 mM NaCl and 4 mM CaCl2, pH 7.5. The activation of lymphocytes was also performed in the presence of bovine lung heparan sulfate (50 µg/mL) prepared in the Molecular Biology Laboratory at Universidade Federal de São Paulo as described .
Heparanase immunostaining was performed using anti-HPA1 C-20 and anti-HPA2 C-17 antibodies (Santa Cruz Biotechnology®, Santa Cruz, CA, USA). A biotin-avidin-peroxidase complex was used to develop the reaction with 3,3′-diaminobenzidine used as the chromogen. Two independent observers scored 300 cells/slide as positive or negative according to the presence of staining for each of the above mentioned antibodies. The immunocytochemistry staining was analyzed by digital quantification. The slides were examined under a light microscope (Nikon Eclipse® TS100) to identify the areas that best represented typical immunostaining (hot spots). In each case, the quantitation was performed by digital analysis, and the values are expressed as the expression index (EI), following the methodology described by Matos and colleagues . Photomicrographs of 640 × 480 pixels were obtained from consecutive nonoverlapping fields at 400× magnification with a digital camera (Nikon Coolpix® 4300) using the same parameters. The images were analyzed by a processing system and image analysis was performed using ImageLab (Softium Informática®, São Paulo, Brazil) using a micrometer scale (µm).
Profile of sulfated glycosaminoglycans (GAGs)
Lymphocytes in culture were labeled using 100 µCi/mL of sodium sulfate [Na35SO4] for 18 h at 37 °C and 5% CO2. After metabolic labeling, the medium was collected, and lymphocytes were lysed using 3.5 M urea. The cellular fraction and culture medium were subjected to proteolysis with protease P126, also known as maxatase, at 4 mg/mL (Biocon do Brasil Indústria Ltda, RJ, Brazil) for 24 h. An aliquot of the cell fraction that did not undergo proteolysis was used to quantify the total protein using 117 µM Coomassie™ Brilliant Blue G (Sigma-Aldrich®, Co. St. Louis, MO, USA). The identification of GAGs was performed by gel electrophoresis using 0.55% agarose with acetate buffer and 0.05 M 1,3-diaminopropane (PDA), pH 9.0, at 100 volts for 1 h at 4 °C. The gel was dried, stained and subjected to autoradiography by exposure to X-ray film. The film was imaged using the scanning apparatus Cyclone® (Packard Instrument Company, Meriden, CT, USA). For quantification of 35S-GAGs, the radioactive bands were excised from the agarose gels and were counted in 5 ml of Ultima Gold (PerkinElmer) using a liquid scintillation spectrometer.
Enzymatic degradation of sulfated glycosaminoglycans
Approximately 10,000 cpm of [35S]-glycosaminoglycans, synthesized by lymphocytes, was incubated with 0.1 U of the specific enzymes chondroitinase AC from Flavobacterium heparinum, chondroitinase ABC from Proteus vulgaris  and heparitinase II from Flavobacterium heparinum .
Quantitative RT-PCR (qRT-PCR)
Total RNA extraction was obtained using the TRIzol® reagent (Life Technologies™ by Ambion, CA, USA), following the manufacturer’s instructions. Reverse transcription was performed using the reverse transcriptase enzyme ImPromII® (Promega Co.®, WI, USA) according to the manufacturer’s instructions to obtain complementary DNA (cDNA). The mRNA expression of heparanase isoforms (HPSE and HPSE2) and Syn-1 were analyzed using the following primers: HPSE forward, 5′TGGCAAGAAGGTCTGGTTAGGAGA3′ and reverse, 5′GCAAAGGTGTCGGATAGCAAGGG3′; HPSE2 forward, 5′AGACAGAG CTGCAGGTTTGAAGGA3′ and reverse, 5′AGCTTAGGAAATCGAGCCAGCCAT3′; Syn-1 forward, 5′AGGGCTCCTGCACTTACTTGCTTA3′ and reverse, 5′ATGTGCA GTCATACACTCCAGGCA3′. The expressions of the endogenous genes 60S ribosomal protein L13A (RPL13a) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were analyzed using the following primers: RPL13a forward, 5′TTGAGGACCTCTGTGTATTTGTCAA3′ and RPL13a reverse, 5′CCTGGAGGAGAAGAGGAAAGAGA3′; GAPDH forward, 5′TCGACAGTCAGCCGCATCTTCTTT3′ and GAPDH reverse, 5′GCCCAA TACGACCAAATCCGTTGA3′. The values are expressed as −ΔCt. Amplification was performed using Maxima® reagent SYBR® Green qPCR Master Mix (2x) (Applied Biosystems®, CA, USA) using a 7500 Real Time PCR Cycler® (Applied Biosystems®, CA, USA).
Total exosome purification was performed from overnight conditioned culture medium collected from MCF-7 cells, non-activated lymphocytes (obtained from women who were not affected by cancer) and activated lymphocytes (obtained by previous co-culture with MCF-7 cells). The supernatant containing the cell-free media was used to obtain exosomes using a Total Exosome Isolation kit (Invitrogen® by Life Technologies®), following the manufacturer’s protocol. Exosome preparation samples underwent total RNA extraction with TRIzol® (Life Technologies®, CA, USA). Reverse transcription and quantitative RT-PCR were performed as previously described.
A sample of 100 µL of each exosome preparation was submitted to cytocentrifugation at 250×g for 2 min. The exosome samples were fixed with methanol for 5 min on glass slides. The exosomes were permeabilized with 0.01% saponin and incubated with primary antibodies in the presence of 1% BSA (bovine serum albumin) for 2 h at room temperature. The slides were incubated overnight at 4 °C with the primary antibody anti-CD63 GTX44174 conjugated with FITC (CLBGran/12 with FITC GeneTex®) at a 1:20 dilution. For the detection of HS, the exosomes were incubated with an anti-HS primary antibody (MAB204; Millipore, Massachusetts, USA) diluted with PBS (1:250) for 2 h at room temperature. The exosomes were then incubated with Alexa Fluor® 647 (1:250) in PBS for 1 h at room temperature. Coverslips were mounted on glass microscope slides using Fluoromount-G. The images were captured with a confocal laser scanning microscope (Leica TCS SP8) equipped with a Plan-Apochromat 63x objective (numerical aperture 1.4) under oil immersion. The pinhole device was adjusted to capture the fluorescence of one Airy unit. The images represented the maximum intensity projections that corresponded to the z-series of confocal stacks. The fluorescence of exosomes (anti-HS-Alexa Fluor® 647 and anti-CD63-FITC) was adjusted to the mean threshold, and exosomes were counted using a particle analysis plugin in the ImageJ software (NIH-ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA). The exosomes were counted in five different fields, and the assay was carried out in triplicate.
The quantitative variables are described as the median and range (difference between maximum and minimum values). Absolute and relative frequencies are used for the qualitative variables. The distributions were defined as nonparametric by the Shapiro–Wilk test. The Mann–Whitney test was used for comparisons between two groups; for comparisons among three or more groups, the Kruskal–Wallis test, with the Dunn auxiliary test in comparisons of subgroups, was used. For all analyses, a statistical significance level of 5% was adopted (P ≤ 0.05), and Prism® software version 5.0 (GraphPad Prism Software Inc.®, California, USA) was used.