Cell lines
Human BC (HT-1376 and UM-UC3) and the leukemic (K562) cell lines (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI 1640 medium (Gibco, Scotland, UK) supplemented with 10 % heat inactivated fetal bovine serum (FBS), 200 mM of L-glutamine (Sigma, St. Louis, USA), and penicillin (100 IU/mL)-streptomycin (100 mg/mL) (Gibco, Scotland, UK), at 37 °C in a 5 % CO2 incubator. CSCs were isolated from the BC cell lines as described previously [5].
Isolation of NK cells from healthy donors and bladder cancer patients
Polyclonal NK cells were isolated from healthy donor (HD, n = 30, mean age: 45 years old) buffy coats provided by the Portuguese Blood and Transplantation Institute or from the blood of BC patients after receiving informed consent and approval by the Institutional Review Board of Coimbra University Hospital (Approved ID: 018-CE-2016). BC patients’ blood was collected from a cohort of 10 male patients (mean age of 70 years) classified as Ta high-grade NMIBC before surgical treatment. Peripheral blood mononuclear cells were separated by density gradient centrifugation on Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden). NK cells were subsequently isolated by negative selection using the NK-cell isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions. Purified NK cells were cultured in complete RPMI-1640 medium (106/mL) supplemented with 10 % heat inactivated FBS, 200 mM of L-glutamine (Sigma), penicillin (100 IU/mL), and streptomycin (100 mg/mL). For activation and expansion, NK cells were incubated with the interleukins IL-2 (250 IU/mL) and IL-15 (0.1 mg/mL) (Peprotech, Rocky Hill, NJ, USA) for 24 and 48 h. The purity of the isolated CD3−CD56+ NK cell populations was > 95 % in all experiments.
Immunophenotyping of NK cells isolated from healthy donors and bladder cancer patients
NK cells were stained with fluorochrome-conjugated monoclonal antibodies against the following human surface antigens: CD56-PE-Cy7, CD16-APC-H7, CD3/CD14/CD19-PerCP-CY5.5, CD94/CD27/CD62L-FITC, NKG2C/NKp30/NKp46/NKG2D-APC, CD11b-PB, and NKG2A/NKp44/NKp80-PE (all purchased from Biolegend, San Diego, CA, USA). For intracellular staining, cells were washed, fixed, and permeabilized with Fix & Perm cell fixation and permeabilization kit (Invitrogen, Carlsbad, CA, USA) and stained with IL-4/TGF-β-FITC, TNF-α-PE, IL-10-APC, and IFN-γ-PB. Appropriate isotype controls were used. A minimum of 100,000 events were acquired using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed with the FlowJo analysis software (Tree Star, Inc., Ashland, USA). Results were expressed as the percentage of positively stained cells in the NK cell gate.
Immunophenotyping of BC cells
Single-cell suspensions of parental and corresponding sphere-forming cells were stained for 30 min at 4 °C with fluorescent conjugated monoclonal antibodies against HLA-ABC (clone w6/32, BioLegend), MICA/B (clone 6D4, BioLegend), ULBP1 (clone 170818, R&D Systems, Minneapolis, MN, USA), CD48 (clone 394607, R&D Systems), Nectin-2/CD112 (clone 610603, R&D Systems), CD155/PVR (clone 300907, R&D Systems), and Fas/CD95 (clone 2R2, eBiosciences, San Jose, CA, USA). For experiments with the supernatant of NK cells (NK-SN), spheres were previously incubated for 4 h with the supernatants of IL-2- and IL-15-activated NK cells before phenotyping. Appropriate isotype-matched controls were run with each experiment. Samples were analyzed using a FACSCanto II cytometer. A minimum of 100,000 events were collected and analyzed using the FlowJo software.
CD107a degranulation and cytokine production
Freshly and IL-2/IL-15-activated NK cells (106 cells) collected from HDs were co-cultured with target cells at an effector-to-target (E:T) ratio of 3:1 in U-bottomed 96-well plates for 4 h in a 5 % CO2 incubator with PE-conjugated anti-CD107a (H4A3, BioLegend) and Brefeldin A (Golgistop, BD). Stimulus with 25 ng/mL PMA plus 250 ng/mL ionomycin was used as a positive control and NK cells alone were used as a negative control. Cultured cells were then stained with fluorochrome-conjugated monoclonal antibodies against human blood surface antigens: CD3 PerCP/Cy5.5 (clone HIT3a), CD14 PerCP/Cy5.5 (clone M5E2), CD19 PerCP/Cy5.5 (clone HIB19), CD16 FITC (clone 3G8), and CD56 APC (clone HCD56), all purchased from BioLegend. The percentage of CD3−CD56+ NK cells positive for CD107a was calculated. All analyses were performed in duplicate using BD FACSCanto II and FlowJo analysis software.
Cytokines produced by 48 h IL-2/IL-15-activated NK cells co-cultured with tumor cells at an E:T ratio of 10:1 were measured using ELISA kits according to the manufacturer’s instructions (granzyme B and IFN-γ: Abcam, Cambridge, UK; and TNF-α: R&D Systems, MN, USA).
Chromium-51 (51Cr)-release assay
Target cells were loaded for 1 h with 50 μCi of 51Cr (PerkinElmer, Massachusetts), washed twice and incubated with fresh or activated NK cells at different E:T ratios (1:1, 3:1 and 10:1) in 200 μL of complete RPMI in 96-well U-bottom tissue culture plates at 37 °C in a 5 % CO2.
After a 4-h incubation period, the supernatants were harvested and counted for released radioactivity in a gamma counter (CRC-55tW Capintec), within a 51Cr sensitivity energy window (300–400 keV). The specific lysis of target cells was calculated as follows: Percentage of specific lysis = (experimental release – spontaneous release)/(maximum release – spontaneous release) × 100. Spontaneous release was calculated from target cells without effector cells. Maximum release was determined by incubating target cells with 4 % SDS detergent. In all experiments, the spontaneous release was < 20 % of maximum release.
For NK cells blocking receptor experiments, activated NK cells were pre-incubated with 10 μg/mL of anti-NKG2D (clone 149810, R&D Systems), 10 μg/mL of anti-DNAM-1 (clone 102511, R&D Systems), and 0.5 μg/mL of anti-FasL (clone ZB4, Merck Millipore, Germany), individually or in combination, before co-culture with tumor target cells.
NK cell supernatant assays
Both parental and CSCs were cultured for 4 h with the supernatant harvested from 48-h IL-2/IL-15-activated NK cells from HDs or BC patients. Thereafter, tumor cells were assayed for aldehyde dehydrogenase (ALDH) activity, expression of stemness-related markers and cell surface ligands for NK receptors and chemosensitivity to cisplatin.
Aldefluor assay
The activity of ALDH in tumor cells was measured using the Aldefluor kit (Stem Cell Technologies, Vancouver, BC, USA), according to the manufacturer’s instructions. FACS was performed on a BD FACSCanto II flow cytometer. Data was analyzed with the FlowJo software.
Gene expression by real-time quantitative PCR analysis (RT-qPCR)
Total RNA from sphere-forming and parental cells was extracted using the ReliaPrep RNA Cell Miniprep System (Promega) following the manufacturer’s instructions. The quantity and quality of isolated RNA was measured by the ND-1000 spectrophotometer (NanoDrop Technologies). Reverse transcription from 1 μg of total RNA was performed using NZY First-Strand cDNA Synthesis kit (Nzytech) and subsequent RT-qPCR for SOX2, ABCG2, ABCB1, ALDH1A1, ALDH2, CD44, CD47, and KRT14 was performed as previously described [5]. Primers used on RT-qPCR reaction are listed in Additional file 1: Table S1. mRNA expression was normalized to three housekeeping genes: 18S, GAPDH, and HRPT-1 using the ΔΔCt method and Bio-Rad CFX Manager™ 3.0 software.
Chemosensitivity to cisplatin
Cells were treated with increasing concentrations of cisplatin (Teva Pharma, Portugal) ranging from 1 to 100 μM over 48 h. Cell viability was analyzed using the standard MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma) assay as previously described [5]. Cell viability was expressed as the percentage of absorbance values of the treated cells related to the untreated control wells considered as 100 %.
Bladder tumor specimens and immunohistochemistry
Bladder tumor samples were obtained from 25 patients (19 males and 6 females) by transurethral resection at Coimbra University Hospital, following appropriate informed consent and ethical regulatory approval (Approved ID: 018-CE-2016). Tumors at initial diagnosis were stratified into non-muscle-invasive low (n = 15) and high (n = 7) grade and muscle-invasive tumors (n = 3) by a pathologist, according to the 2004 WHO criteria [20]. Formalin-fixed paraffin-embedded tissue blocks were sectioned at 3-μm thickness and incubated in a BenchMark Ultra Ventana, with a primary antibody against CD56, a surface marker for NK cells, clone 123C3 (1:50, Roche), for 30 min at 37 °C, and reaction signal was developed with 3-3′-diaminobenzidine tetrahydrochloride chromogen. Standard procedures were used for visualization and the intensity and percentage of positive staining was registered. Two investigators blinded to the data reviewed all slides independently.
Animal studies
Animal studies were approved by the Organization Responsible for Animal Welfare of the Faculty of Medicine of Coimbra (Approved ID: ORBEA/91/2015/08) and were performed according to National and International guidelines on animal experimentation. Female nude mice (Swiss nu/nu), 6–8 weeks old (Charles River Laboratories, Barcelona, Spain) were housed under pathogen-free conditions in individual ventilated cages. The subcutaneous tumor model was induced by subcutaneous injection into the lower flank of 1 × 106 of Luc+ HT-1376 cells suspended in 100 μL of a 1:1 PBS/Matrigel mixture. The orthotopic model that more closely resembles the clinical and histopathological features of primary MIBC was developed by intravesical instillation of Luc+ HT-1376 cells as previously described [5]. Bioluminescent images were taken 24 h post-implantation and every 3 days to monitor engraftment and growth of tumor cells using an IVIS Lumina XR (Caliper Life-Sciences, Hopkinton, MA, USA) after intraperitoneal injection with D-luciferin (150 mg/kg, Synchem, BHg, Germany) with the animals under anesthesia (100 mg/kg ketamine and 2.5 % of chlorpromazine solution). Quantification of bioluminescent signals was performed using the living image software version 4.10 (Xenogen). Values are expressed as photons/sec/cm2/sr. Subcutaneous tumors started the treatment on day 6 post-implantation by intratumoral inoculation of NK cells activated for 48 h (5 × 106/50 μL) from HDs twice a week over 2 weeks.
Animals bearing subcutaneous or orthotopic tumors were treated twice a week with healthy 48-h activated-NK cells (5 × 106/mouse) via intratumoral and intravesical instillation, respectively, over 2 weeks. NK cells were washed prior to administration and resuspended in PBS. Tumor progression was monitored by bioluminescent images 3 days after each treatment. Animals were sacrificed after treatments or when presenting hematuria or lost 20 % of initial body weight. Residual tumors were excised and processed into paraffin blocks for immunohistochemistry analysis of CD56 clone 123C3 (1:50, Roche) and for two CSC-related markers, SOX-2 (clone D6D9, 1:100, Cell Signaling) and ALDH2 (clone EPR4493, 1:100, Abcam) as described above for clinical samples.
Statistical analysis
Data are reported as the means ± SEM of the indicated number of experiments. Statistical analysis and graphic illustrations were performed using GraphPad Prism 6.0 software (San Diego, CA). Paired two-tailed Student’s t-tests, ANOVA, and Tukey’s tests were used to calculate P values. A P value of less than 0.05 was considered significant.