The increased expression of IL-6 receptor (IL-6Rα) in human lung squamous cell carcinoma-derived cells (HARA-B) has been shown immunocytochemically under co-culture with primary cultured mouse astrocytes [8]. To confirm the increase of IL-6Rα quantitatively, the protein level of IL-6Rα in HARA-B cells was tested by Western blotting after culturing with or without mouse astrocytes for 72 h using insert-culture to prevent physical contact (Figure 1a). The quantitative analyses showed a significant increase in IL-6Rα after 72 h when HARA-B cells were insert-cultured with astrocytes (Figure 1b).

Not only the in vitro conditions, but strong expression of the IL-6 receptor and receptor subunit (IL-6Rα and gp130) on HARA-B cells were observed in the brain slices obtained from metastasized brain at four-five weeks after inoculation of HARA-B cells into the left ventricle of the heart in nude mice (Figure 1c).

Finally, we checked the expression of IL-6R in postmortem human brain from lung cancer patients. It was shown that astrocytes accumulate around metastasized lung cancer cells in human brain [8]. At metastasized foci, IL-6R was strongly stained (Figure 4), suggesting that up-regulation of IL-6R also occurs in the brain metastases of lung cancer in human.

In our previous study [8], three other cell lines derived from human squamous cell carcinoma (QG56, EBC-1) and non-small cell lung cancer (PC-9) were examined in vitro, showing the similar responses to the medium from insert-culture with astrocytes. Those results suggest that mutual activation of astrocytes and lung tumor cells are common phenomena and IL-6 plays an important role in the progression of tumor cells in the microenvironment of the metastasized brain area.

Though brain metastases, especially with lung cancer, melanoma and breast cancer, are becoming a common clinical problem, limited treatment options have existed [23], such as gamma knife radiosurgeries [24,25], stereotactic radiosurgery [26] or irradiation [27], UVC irradiation [28] and whole brain radiotherapy [29–31]. Recently, a potential role of targeted agents, including antiangiogenic compounds and epithelial growth factor receptor inhibitors, have been implicated for the prevention of brain metastasis formation in breast cancer or non-small cell lung cancer [32]. MicroRNAs [33,34], targeting angiogenesis [35] and cytotoxic chemotherapy, in some specific situations [36], might be potential options. In addition to them, our findings may also contribute to a better control or inhibition of metastatic lung tumor growth in the brain.

Primary glial cell cultures were performed according to the method described previously [37,38]. Briefly, the cerebral corteces obtained from one-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 corteces were minced and treated with trypsin-EDTA solution (0.25% trypsin, 1 mM EDTA) and 1500 U DNase in Dulbecco’s modified Eagle 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 a 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 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 the cerebral cortex at 37 °C in 10% CO₂–90% air. HARA-B cells were maintained under the same condition. Cells were grown in a 25 cm² cell culture flask (Nalge Nunc International, Napersville, IL, USA), and single-cell suspensions of the cells were obtained by trypsin treatment.

The expression protein level of IL-6Rα in HARA-B cells was examined by Western blotting relative to β-actin. Though β-actin is frequently too abundant to serve as a suitable control for lower-abundance proteins, the signal was not saturated nor near-saturated in our case.

HARA-B cells cultured with or without insert-culture of astrocytes were collected and homogenized in a lysis buffer containing protease inhibitor cocktail (Sigma, St. Louis, MO, USA, 2%) and phosphatase inhibitor cocktail (Sigma, St. Louis, MO, USA, 1%). Twenty mg of proteins were loaded for each lane, separated by SDS-PAGE gel (10%) and transferred to PVDF membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% low-fat dried milk and incubated with the following for 1 h at room temperature: rabbit polyclonal anti-human IL-6Rα antibody (Santa Cruz, CA, USA) (1:100) and mouse monoclonal antibody anti-β-actin (1:200, Sigma, St. Louis, MO, USA). The membrane was washed and incubated with specific secondary antibodies (Amersham ECL anti-rabbit IgG and anti-mouse IgG horseradish peroxidase-linked species-specific whole antibody, 1:1000, GE Healthcare, Piscataway, NJ, USA). The blots were detected by use of an ECL Western blotting detection system (GE Healthcare) and LAS-4000 imaging system (Fujifilm, Tokyo, Japan). Bands were quantified using the software Multi Gauge (Fujifilm).

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 five-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.

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 IL-6R or gp130, sections were incubated with rabbit polyclonal anti-human IL-6Rα antibody (Santa Cruz, CA, USA) (1:200), rabbit anti-human gp130 antibody (Santa Cruz, 1:500) at 4 °C overnight after staining of cytokeratin. The cells were then incubated for 5 h at room temperature with secondary antibody, containing FITC-conjugated anti-mouse IgG (Sigma, St. Louis, MO, US, 1:500) and Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA, 1:500). 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 was calculated by a LSM image browser.

As for human tissue samples, after removal of paraffin in xylene and rehydration in a grade of alcohols (100%, 90%, 80%, 70% and 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-human IL-6Rα antibody (1:200) at 4 °C overnight, Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA, 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).

HARA-B-stimulated astrocyte-conditioned medium (H-ACM) was obtained as follows. The culture medium of HARA-B cells (5 × 10³ cells/well) was 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. For staining, 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 a blocking solution containing 1% BSA and 5% normal donkey serum (Jackson ImmunoResearch, West Grove, PA, USA) 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 incubation with the secondary antibody (FITC-conjugated anti-mouse IgG; Sigma, St. Louis, MO, USA, 1:500) for 5 h at room temperature and, then, incubated with 300 nM 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI, Sigma, St. Louis, MO, USA) 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).

After inoculation of HARA-B cells into the left ventricle of the heart of nude mice, the metastases of tumor cells were recognized at about three weeks. Therefore, after three weeks of inoculation, either tocilizumab (1.0 mg/100 μL) or human IgG (1.0 mg/100 μL) was injected intravenously twice a week during the following three weeks. The amount of tocilizumab was calculated from clinical dosage (8 mg/kg, every two weeks). Even though it has low permeability of the blood-brain barrier (1000–10,000 times lower concentration in the brain), it was estimated to be similar to the effective concentration in vitro. Then, the animals were anesthetized, perfused and fixed, as described above. The brain was cut into 30 μm in thickness using a cryostat. All sections were stained with DAPI (Sigma, St. Louis, MO, USA) for 10 min, then washed with PBS and coverslipped. The volume of tumor foci was calculated according to the following formula: tumor volume (mm³) = (major axis) × (minor axis)² × 0.52 [39,40]. The major axis is the longest diameter of the tumor, and the minor axis is the maximal line drawn perpendicular to the major axis [41,42].

All data are presented as mean ± SEM. One-way analysis of variance (ANOVA) and a post-hoc Bonferroni/Dunn test were used to examine the statistical differences. Differences were considered significant at p < 0.05.