The human tumor cell lines, A549 (lung adenocarcinoma), DLD-1 (colorectal adenocarcinoma), and MDA-MB-231 (breast adenocarcinoma) were purchased from ATCC. The long-term human glioma cultures, A767 and A772 [16, 17], have been generously provided by Dr. Rolf Mentlein (Kiel, Germany). All cells were propagated in either DMEM (4.5 g/l glucose; Gibco, Life Technologies, Carlsbad, CA; A767, A772, MDA-MB-231) or RPMI 1640 (Gibco; A549, DLD-1), supplemented with 0.05% gentamycin (Gibco) and 10% fetal bovine serum (FBS; Gibco). Cells were plated on 10 cm culture dishes (TPP, Trasadingen, Switzerland). Subconfluent cultures were used for experiments. For immunofluorescent labeling, cells were seeded on glass coverslips (Thermo Fisher Scientific, Dreieich, Germany) previously coated with poly-L-ornithine (0.4 μg/ml; Sigma, St. Louis, MO) for 24 h.
Western blot analysis
For Western blot analysis, cultured cells were lysed in 2% SDS and protein content was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher) according to the manufacturer’s instructions. Proteins (10–20 μg/lane) were separated by SDS-(10%) polyacrylamide gel electrophoresis and transferred to nitrocellulose by electroblotting. If required, activated sodium orthovanadate (10%) was added to the lysis buffer to avoid protein dephosphorylation. After blocking non-specific binding sites with 5% bovine serum albumin or 5% skimmed milk for 60 min, blots were incubated overnight at 4 °C with the respective primary antibody (see additional file 1), following a 1-h-incubation with peroxidase-conjugated secondary antibodies (see additional file 1). Antibody-labeling was visualized with Pierce™ ECL Western Blotting Substrate (Thermo Fisher). To control for protein loading, membranes were reblotted with anti-GAPDH antibodies (see additional file 1). Chemiluminescence was captured on a Biostep Celvin S Imager (Biostep, Burkardtsdorf, Germany) and immunoreactive protein bands were quantified using the TotalLab 1D software (TotalLab, Newcastle-Upon-Tyne, UK).
Migratory responses of cancer cells to CXCL11 and/or CXCL12 were analyzed using a modified 12-well Boyden chamber (Neuro Probe, Cabin John, MD) in which the upper and lower wells were separated by polyornithine coated Nucleopore® PVP-free polycarbonate filter (Whatman; Maidstone, UK; 8 μm pore size). Prior to analysis, cells were harvested and incubated for 1 h in serum-free medium, supplemented with one of the following chemokine receptor antagonists or (downstream) pathway inhibitors: AMG487 (CXCR3 antagonist; 10 μM; Tocris, Wiesbaden, Germany; dissolved in DMSO), CCX771 (CXCR7 antagonist; 100 nM; ChemoCentryx; Mountain View, CA; dissolved in DMSO), AMD3100 (CXCR4 antagonist; 10 μM; Sigma; dissolved in double-distilled water), PD98059 (MEK1-inhibitor; 20 μM; Cell Signaling Technology, Danvers, MA; dissolved in DMSO), LY294002 (PI3-kinase inhibitor; 20 μM; Cell Signaling Technology; dissolved in DMSO), SB203580 (p38 MAP kinase inhibitor; 10 μM; Cell Signaling Technology; dissolved in DMSO). For control purposes, untreated cultures were additionally supplemented with adequate concentrations of DMSO. For seeding, cells were counted using an improved Neubauer chamber and 10,000 cells were placed into the upper well of the Boyden chamber. The lower well received 150 μl of serum-free medium supplemented with CXCL11 (1 ng/ml to 100 ng/ml; Cell Guidance Systems, Cambridge, UK), and/or CXCL12 (1 ng/ml to 100 ng/ml; ALMAC, Craigavon, UK). In selected experiments, the lower well received a constant concentration (10 ng/ml) of either CXCL12 or CXCL11 and in addition varying concentrations (1 ng/ml to 100 ng/ml) of the respective other chemokine. As a positive control, 10% FBS was added to the lower wells. Chamber was incubated at 37 °C in a water- saturated atmosphere of 95% air and 5% CO2 for 4 h. After incubation, non-migrated cells, attached to the upper part of the membrane, were wiped off and migrated cells, attached to lower part of the membrane, were fixed with methanol, stained with DAPI (AAT Bioquest, Sunnyvale, CA), and counted on an Olympus BX40 microscope at 100x magnification using the Olympus cellSens Dimension software (Olympus, Shinjuku, Japan). Number of cells migrating in the absence of chemoattractants was set to 1. Migration index was calculated as the ratio of cells migrating in the presence and absence of chemokines.
To assess invasion behavior of tumor cells, Boyden chamber assay was performed with polycarbonate filters, previously coated with Matrigel (Corning, Acton, MA; 30 μl, further diluted 1:3 with culture medium) at 37 °C for 1 h. For seeding, cells were resuspended in either DMEM or RPMI containing 1% FBS and 0.05% gentamycin. Number of migrated cells was determined after 72 h as delineated above. Number of cells invading the membrane in the absence of chemokines was set to 1. Invasion index was calculated as the ratio of cells invading the membrane in the presence and absence of chemokines. To assess the role CXCR4, CXCR7, and CXCR3 in CXCL11- and/or CXCL12-dependent cell invasion, invasion assay was performed with cells preincubated with AMG487 (10 μM), CCX771 (100 nM), or AMD3100 (10 μM) for 1 h as described above.
Efficacy of different cytostatic agents to induce death of cancer cells was initially assessed by immunocytochemistry. For this purpose, cells were plated on glass cover slips and treated with different concentrations of doxorubicin (0.1 μM to 10 μM), cisplatin (10 μM to 40 μM) or temozolomide (10 μM to 200 μM; all obtained from the University of Leipzig Medical Center Pharmacy) for 3 h to 24 h. Cells were subsequently fixed and stained with DAPI and antibodies against cleaved-caspase-3 as delineated below. Apoptotic cells were counted on a Zeiss confocal laser scan microscope and expressed as per cent of total number of cells present in the same observation fields. To assess effects of chemokines on apoptotic cell death, cells were treated with 100 ng/ml of CXCL11 and/or CXCL12 24 h prior to the addition of cytostatics. After another 6 - 12 h, apoptotic cells were identified by the BD FITC Annexin V Apoptosis Detection Kit (Becton Dickinson, Franklin Lakes, NJ) and 10.000 events were quantified on a Becton Dickinson LSRFortessa™ flow cytometer. Data were analyzed using the FlowJo version 10 software (FlowJo; Ashland, Oregon). Apoptotic cell numbers present in cytostatic-treated cultures were set to 1.
For evaluation of gelatinase activity, cells were grown in serum-containing culture medium on 6-well plates with a change to serum-free medium 24 hours prior to treatment with either CXCL11 (100 ng/ml), CXCL12 (100 ng/ml), or both. Cell supernatant was collected after 48 h and concentrated with Amicon Ultra-2 Centrifugal filter units (Merck Millipore, Burlington, MA). Proteins were loaded (20–30 μg per lane) on polyacrylamide gels containing gelatin (1 mg/ml) and separated by electrophoresis under non-reducing conditions. Gels were subsequently incubated in activation buffer (2.5% Triton X-100, 50 mM Tris HCl, 5 mM CaCl2, 1 μM ZnCl2) for 24 h and stained with Coomassie-Blue for 1 h. Following destaining with methanol/acetic acid, gels were analyzed on a Biostep Celvin S Bioluminescence Detector. Recombinant activated matrix metalloproteinase (MMP)-2 (#550502, Biolegend, San Diego, CA) and recombinant activated MMP-9 (ab81550, Abcam, Cambridge, UK) were used as controls.
Quantitative real-time-PCR (qRT-PCR)
Total RNA was extracted by TRI Reagent™ solution (Invitrogen, Carlsbad, CA), followed by reverse transcription of 1 μg RNA using Protoscript First Strand cDNA Synthesis Kit (Biolabs, Frankfurt, Germany) as specified by the manufacturer. For quantification of gene expression, qRT-PCR analysis was performed with Maxima SYBR® Green/ROX qPCR Master Mix (Thermo Fisher, Waltham, MA) on a CFX 96 Thermal Cycler system (Bio-Rad, Munich, Germany). Gene expression was calculated by the ΔΔCT method and normalized to β-actin. The following primers were used:
MMP-2, forward 5′- CTCAGATCCGTGGTGAGATCT-3′, reverse 5′- CTTTGGTTCTCCAGCTTCAGG-3′.
MMP-9, forward 5′-ATCCAGTTTGGTGTCGCGGAGC-3′, reverse 5′-GAAGGGGAAGACGCACAGCT-3′.
Beta-Actin, forward 5′-GGCCTCGCTGTCCACCTT-3′, reverse 5′-TGTCACCTTCACCGTTCCAGTTTT-3′.
For immunostaining, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min. To block unspecific binding sites and at the same time permeabilize cells, cultures were incubated with PBS containing 5% bovine serum albumin and 0.05% saponin for 1 h. Primary antibodies (see additional file 1) were applied overnight at 4 °C, followed by incubation with an appropriate Alexa Fluor 488- or Alexa Fluor 555-labelled secondary antibody (see additional file 1) at 37 °C for 1 h. Cultures in which the primary antibody was omitted served as controls. Following staining of cell nuclei with DAPI, cultures were mounted with Dako Glycergel Mounting Medium (Dako Inc., Carpinteria, CA) and analyzed on a Zeiss confocal laser scan microscope, using ZEN software (Carl Zeiss, Oberkochen, Germany).
To assess effects of chemokines on cell proliferation, cells were seeded into 6-well culture plates and maintained with serum-free medium, supplemented with either CXCL11 (100 ng/ml) and/or CXCL12 (100 ng/ml). Medium, containing the respective chemokine(s) was renewed on day 2 and additionally supplemented with bromodeoxyuridine (10 μM; Becton Dickinson; Franklin Lakes, NJ). After a 2-h-incubation step, cells were harvested, fixed, and stained for BrdU using the BD FITC BrdU Flow Kit (Becton Dickinson, Franklin Lakes, NJ) according to the manufacturer’s instructions. Following labeling DNA with 7-AAD (BD, Franklin Lakes, NJ), cells were analyzed by flow cytometry using a Becton Dickinson LSR Fortessa.
Data, obtained from at least three experiments, are given as mean ± SD. One way analysis of variance (ANOVA) followed by pairwise multiple comparison procedures (Tukey post-hoc test) was used for statistical analysis (GraphPad). Differences with p < 0.05 were considered significant.