Interaction between lung cancer cells and astrocytes via specific inflammatory cytokines in the microenvironment of brain metastasis

The study was approved by the Animal Care and Use Committee at Kyushu University and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male 5-week-old nude mice (BALB/c nu/nu) (Kyudo, Kumamoto, Japan), kept in a specific pathogen-free environment, were used. A single suspension of human lung squamous cell carcinoma-derived cells (HARA-B) (2 × 10⁵ cells/0.2 ml PBS) was inoculated into the left ventricle of the heart in nude mice according to the method described previously [12]. After 4–6 weeks, brains were subjected for immunohistochemical staining.

A total of 6 paraffin-embedded samples from patients with lung tumor brain metastasis were used. All sections were obtained from the National Hospital Organization Shikoku Cancer Center. Use of the human specimens was in accordance with the University Ethics Commission. The formalin-fixed, paraffin-embedded archival tissue blocks were retrieved, and matching hematoxylin and eosin (H & E)-stained slides were reviewed and screened for representative tumor regions by a neuropathologist.

Nude mice were perfused transcardially with 50 ml of 10 U/ml heparin and 0.5% procaine in PBS and 4% paraformaldehyde (PFA) in PBS prior to excision of the brain. Then the brain was removed, post-fixed for 3 h, and cryoprotected for 24 h in PBS containing 20% sucrose. The brain was cut into slices (30 μm thick) using a cryostat and the sections were placed on glass slides. In order to acquire the better immunoreactive images, sections were autoclaved with 0.01 M citrate buffer solution (pH 6.0), permeabilized with 0.3% TritonX-100 in PBS for 15 min, and then blocked in BlockAce (Dainippon Pharmaceutical, Japan) for 1 h at room temperature. Sections were incubated with mouse anti-human cytokeratin monoclonal antibody (AE1/AE3 pool of cytokeratin) (Dako, Glostrup, Denmark, 1:100) at 4°C overnight. Biotinylated anti-mouse IgG (Jackson, 1:200) were incubated for 2 h at room temperature, followed by the incubation with streptavidin Alexa488 (Molecular Probes, 1:500) for 2 h at room temperature. For double-staining of cytokeratin and GFAP, sections were incubated with Cy3-conjugated anti-GFAP antibody (Sigma, USA) (1:1000) at 4°C overnight after staining of cytokeratin. Every treatment was followed by washing three times with PBS containing 0.3% TritonX-100 for 5 min. The sections were mounted in the Perma Fluor Aqueous Mounting Medium (Thermo Shandon, Pittsburgh, PA, USA) and analyzed with a confocal microscope (LSM510 META, Carl Zeiss, Co. Ltd. Germany). Z-stack images were obtained from each section by LSM 510 META and total intensity were calculated by LSM image browser.

As for human tissue samples, after removal of paraffin in xylene and rehydration in a graded of alcohols (100%, 90%, 80%, 70%, 60%), sections were incubated for 30 min in 0.05 M phosphate buffer pH 7.6 containing tripsin and KCl for antigen retrieval. Then, the sections were incubated for 1 h in 0.3% H₂O₂, and blocked in PBS containing 1% BSA and 5% normal donkey serum (Jackson Immuno Research Laboratories Inc., West Grove, PA, USA) for 1 h at room temperature. Then, the sections were incubated with anti-GFAP antibody (ImmunoStar) (1:15) at 4°C overnight, goat anti-rabbit IgG Alexa 568 (Molecular Probes) (1:500) for 3 h at room temperature and FITC-conjugated anti-human cytokeratin antibody (CAM5.2) (Becton–Dickinson Biosciences, New Jersey, USA) (undiluted solution) for 1 h at room temperature. Every treatment was followed by washing three times with PBS containing 0.3% TritonX-100 for 5 min. The sections were mounted in the Perma Fluor Aqueous Mounting Medium (Thermo Shandon, Pittsburgh, PA, USA) and analyzed with a confocal microscope (LSM510 META, Carl Zeiss, Co. Ltd. Germany).

Primary glial cell cultures were performed according to the method described previously [13]. Briefly, the cerebral cortex obtained from 1-day-old C57BL/6 mice (Kyudo, Kumamoto, Japan) were isolated under a dissecting microscope and carefully separated from the choroid plexus and meninges. The isolated cerebral cortex were minced and treated with trypsin–EDTA solution (0.25% trypsin, 1 mM EDTA) and 1500 U DNase in Dulbecco’s modified Egle medium (DMEM; Nissui, Tokyo, Japan) at 37°C for 10 min. Cell suspensions were filtered through 70 μm pore size mesh (BD Falcon, Bedford, MA, USA) into DMEM containing 10% fetal calf serum (FCS; Hyclone, UT, USA), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.37% NaHCO₃, and 110 μg/ml pyruvic acid. After centrifugation, cells were filtered through 40 μm pore size-mesh (BD Falcon), plated into poly-L-lysine coated 75 cm² cell culture flask at the density of two brains per flask in 10 ml of DMEM, and maintained at 37°C in 10% CO₂–90% air with a change of the medium twice per week. Astrocytes were obtained after 28 days of mixed glial cell cultures as follows. After removing other glial cells by shaking the flasks, the astroglial layer was removed from the flasks by the treatment with trypsin–EDTA solution (0.06% trypsin, 0.25 mM EDTA in serum free DMEM) at 37°C for 45 min. Suspended astrocytes were filtered through 40 μm pore size-mesh and seeded. Astrocyte purity ranged from 90 to 95% as determined by immunostaining with anti-GFAP antibody (Sigma, St. Louis, MO, USA) (data not shown). Astrocytes were maintained in the same medium used for cell suspension from cerebral cortex at 37°C in 10% CO₂–90% air. HARA-B cells and other lung cancer cell lines (QG56, EBC-1; squamous cell carcinoma) and PC-9 (non-small cell lung cancer cell) were maintained under the same condition. Cells were grown in 25 cm² cell culture flask (Nalge Nunc International), and single-cell suspension of cells were obtained by trypsin treatment.

In the co-culture experiment, HARA-B cells (0.5 × 10³ cells/well) and astrocytes (2.5 × 10³ or 5 × 10³ cells/well) were seeded into 8-well cell culture slide (BD Falcon) in DMEM for 24 h. Then, cells were rinsed twice with PBS and incubated in serum free DMEM. After 72 h of co-culture, cells were fixed with 4% PFA for 30 min at room temperature and permeabilized with 0.3% TritonX-100 in PBS for 15 min, followed with blocking solution containing 1% BSA and 5% normal donkey serum (Jackson) in PBS for 1 h at room temperature. Then cells were incubated with mouse anti-human cytokeratin monoclonal antibody (AE1/AE3 pool of cytokeratin) (Dako, Glostrup, Denmark) (1:100) at 4°C overnight, followed by the incubation with the secondary antibody (FITC-conjugated anti-mouse IgG; Sigma, 1:500) for 5 h at room temperature, and then, incubated with 300 nM 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI, Sigma) for 30 min at room temperature. The number of HARA-B cells in each well, which were positively stained with an anti-human cytokeratin antibody, was counted using a digital camera system (Axio Cam, Carl Zeiss) mounted on a light and fluorescent microscope (Axopscope2 plus, Carl Zeiss). The results were expressed as the percentage of control (single cell culture of HARA-B cells).

Insert culture-medium was obtained as follows. Astrocytes (5 × 10⁴ cells/well) were plated in 6-well cell culture plates (Falcon). Tumor cells derived from each lung tumor (EBC-1, PC9, QG56 and HARA-B cells) (5 × 10³ cells/insert) were plated in cell culture-inserts (membrane pore size 0.4 μm; Becton–Dickinson), and then, placed in the well of astrocyte cultures. After 24 h of the insert-culture, cells were rinsed twice with PBS, incubated in serum-free DMEM for further 48 h. Then, the medium was collected. Each conditioned medium was centrifuged to remove debris (1500 rpm for 10 min at 4°C) before use. Tumor cells derived from each lung tumor (0.5 × 10³ cells/well) were seeded into 8-well cell culture slide (BD Falcon) in DMEM for 24 h. Then, cells were rinsed twice with PBS and incubated in each Insert culture-medium. After 72 h, cells were fixed with 4% PFA for 30 min at room temperature and incubated with 300 nM DAPI for 10 min at room temperature. The number of tumor cells in each well, which were positively stained with DAPI, was counted as mentioned above. The results were expressed as the percentage of control (each tumor cells cultured in serum-free DMEM for 72 h).

The astrocyte-conditioned medium (ACM) was obtained from the primary culture of astrocytes at a density of 10⁴ cells/well in serum free DMEM after 72 h-incubation. This ACM was centrifuged (1500 rpm for 10 min at 4°C), and then, added to HARA-B cells cultured for 1 day at a volume of 25% or 50%. The number of HARA-B cells were counted after the 72 h-incubation in the presence of ACM as described above.

HARA-B-stimulated astrocyte-conditioned medium (H-ACM) was obtained as follows. Culture medium of HARA-B cells (5 × 10³ cells/well) were added to astrocytes cultures (5 × 10⁴ cells/well) and incubated for 24 h. Then the medium was collected and centrifuged to remove debris (1500 rpm for 10 min at 4°C) before use.

In the proliferation assay using recombinant mouse cytokines, HARA-B cells 24 h after plating were rinsed twice with PBS and incubated in serum free DMEM with each cytokine (mIL-1β, 1 to 10 pg/ml; mTNF-α, 10 to 500 pg/ml; mIL-6, 10 to 500 pg/ml, Peprotech, Rocky Hill, NJ, USA). After 72 h, HARA-B cells were immunostained and counted as mentioned above.

In the experiments using neutralizing antibodies, cells were plated, rinsed twice with PBS after 24 h, and incubated in serum free DMEM with each specific neutralizing antibodies; mIL-1β antibody (ab), 0.2 to 2.0 μg/ml (R&D systems, Minneapolis, MN USA), mTNF-α ab, 0.2 to 2.0 μg/ml (R&D systems), mIL-6 ab, 2.0 to 20 ng/ml (Peprotech) for 48 h. As controls, rat IgG (Sigma) and rabbit IgG (R&D systems) were used. HARA-B cells were immunostained and counted as mentioned above.

HARA-B cells (1 × 10⁵ cells/insert) and astrocytes (1 × 10⁶ cells/well) were cultured in cell culture-insert systems as mentioned above. Cells were collected by treatment with trypsin after 24, 48 and 72 h of insert-culture. As controls, single cell culture of HARA-B cells or astrocytes was used. Total RNA was isolated from each cell type by an extraction procedure using the RNA blood mini kit (QIAGEN in Japan). Contaminating DNA was removed by RNase-free DNase (QIAGEN). Single-strand cDNA was synthesized from cellular mRNA by using random 9 mer and RNA PCR kit (AMV) (Takara Bio Inc., Ootsu, Japan). PCR amplification was undertaken for plain SYBR Green I detection in using Light Cycler system (Roche Diagnostics GmbH, Mannheim, Germany). Each reaction was carried out in a total volume of 20 μl in glass capillary, containing 1 μl of cDNA, 2, 3 or 4 mM MgCl₂, 10% LightCycler-DNA Master SYBR Green I buffer (Taq DNA polymerase, reaction buffer, deoxynucleotide triphosphate mix, 10 mM MgCl₂ and SYBR Green I dye) and 0.5 μM of each primer (Table 1). After the PCR reaction, we confirmed that there was no primer dimer and non specific product in each PCR product by agarose gel electrophoresis and staining with ethidium bromide. The expression of all target genes was normalized to β-actin. Analysis was carried out with Light Cycler 3.5 software (Roche) and Microsoft Excel.

To see the reconstituted effects of HARA-B-derived substances on astrocytic expression of TNF-α, IL-1β and IL-6, the mouse primary astrocyte cells were treated with human IL-8 (R&D systems, Minneapolis, MN, U.S.A), MIF (R&D systems), or PAI-1 (Peprotech, Rocky Hill, U.S.A) for 72 h in serum-free medium. Total RNA was extracted using an RNeasy Plus Mini kit (QIAGEN, Hilden, Germany) and QIA shredder (QIAGEN). cDNA was synthesized using a SuperScript VILO cDNA synthesis kit (Invitrogen) SYBR-Green real-time PCR (Applied Biosystems, Foster City, CA) was performed on cDNA prepared from each sample using Platinum SYBR-Green qPCR Super-Mix-UDG (Invitrogen) and 0.5 μM each primer (Table 1). Thermal cycling condition were 10 min at 95°C, 45 cycles at 95°C for 15 s, followed by 1 min at 60°C. Data Analysis was completed using the ABI PRISM 7500HT Sequence detection software (Applied Biosystems). β-actin was used for normalization.

Co-cultures of HARA-B cells (0.5 × 10³ cells/well) and astrocytes (5 × 10³ cells/well) or astrocytes alone (5 × 10³ cells/well) were seeded into 8-well cell culture slides in DMEM with FCS for 24 h. Then, cells were rinsed twice with PBS and incubated in serum free DMEM. After 48 or 72 h of culture, each conditioned medium was collected and centrifuged to remove debris (1500 rpm for 10 min at 4°C) before use. The amount of mouse IL-1β, TNF-α and IL-6 in each conditioned medium was measured with an ELISA kit for mouse cytokines (Biosource International). The absorbency at 450 nm was measured by a Microplate Reader (Immuno-Mini NJ-2300, Nalge Nunc International).

HARA-B cells (1 × 10⁶ cells) were grown in 10 cm dish (BD, Franklin Lake, NJ, U.S.A) for 24 h and culture medium was collected. Protein array analysis was performed according to the manufacture’s instruction. Positive controls were located in the upper left-hand corner (two spots), upper right-hand corner (two spots) and the lower left-hand corner (two spots) of each array kit. Medium and culture medium were measured using the human cytokine array Panel A (Proteome Profiler) (R&D systems, Minneapolis, MN, U.S.A). Horseradish peroxidase substrate (Thermo scientific, Rockford, IL, U.S.A) was used to detect protein expression and captured by exposure to X-ray Film (FUJIFILM, Tokyo, Japan).

HARA-B cells (0.5 × 10³ cells/well) with or without astrocytes (5 × 10³ cells/well) were seeded into 8-well cell culture slides in complete medium. Cells were fixed with 4% PFA for 30 min at room temperature and permeabilized with 0.3% TritonX-100 in PBS for 15 min and blocked in PBS containing 1% BSA and 5% normal donkey serum (Jackson) for 1 h at room temperature. Cells were incubated with primary antibodies, containing monoclonal mouse anti-human cytokeratin antibody (AE1/AE3 pool of cytokeratin) (Dako, 1:100), rabbit anti-human IL-6Rα antibody (Santa Cruz, CA, USA) (1:200), rabbit anti-human gp130 antibody (Santa Cruz, 1:500), rabbit anti-human IL-1RtI antibody (Santa Cruz, 1:200), and goat anti-human TNFRI (Santa Cruz, 1:100) overnight at 4°C. Control cells were incubated without primary antibody (PBS containing 1% BSA) to test non-specific staining. The cells were then incubated for 5 h at room temperature with secondary antibody, containing FITC-conjugated anti-mouse IgG (Sigma, 1:500), Cy3-conjugated anti-rabbit IgG (Jackson, 1:500) and Cy3-conjugated anti-goat IgG (Jackson, 1:500) and then for 30 min at room temperature with 300 nM 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI, Sigma). Slides were mounted in the Perma Fluor Aqueous Mounting Medium (Thermo) and were analyzed with a Zeiss LSM510 META confocal microscope.