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

Abstract

The incidence of brain metastasis is increasing, however, little is known about molecular mechanism responsible for lung cancer-derived brain metastasis and their development in the brain. In the present study, brain pathology was examined in an experimental model system of brain metastasis as well as in human brain with lung cancer metastasis. In an experimental model, after 3-6 weeks of intracardiac inoculation of human lung cancer-derived (HARA-B) cells in nude mice, wide range of brain metastases were observed. The brain sections showed significant increase in glial fibrillary acidic protein (GFAP)-positive astrocytes around metastatic lesions. To elucidate the role of astrocytes in lung cancer proliferation, the interaction between primary cultured mouse astrocytes and HARA-B cells was analyzed in vitro. Co-cultures and insert-cultures demonstrated that astrocytes were activated by tumor cell-oriented factors; macrophage migration inhibitory factor (MIF), interleukin-8 (IL-8) and plasminogen activator inhibitor-1 (PAI-1). Activated astrocytes produced interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin-1 β (IL-1β), which in turn promoted tumor cell proliferation. Semi-quantitative immunocytochemistry showed that increased expression of receptors for IL-6 and its subunits gp130 on HARA-B cells. Receptors for TNF-α and IL-1β were also detected on HARA-B cells but down-regulated after co-culture with astrocytes. Insert-culture with astrocytes also stimulated the proliferation of other lung cancer-derived cell lines (PC-9, QG56, and EBC-1). These results suggest that tumor cells and astrocytes stimulate each other and these mutual relationships may be important to understand how lung cancer cells metastasize and develop in the brain.

Fig. 1

Astrocyte accumulates around HARA-B cells in vivo. a Typical examples of immunostaining of astrogliosis (GFAP) around invaded tumor cells (human cytokeratin, CK). Accumulation of GFAP-positive astrocytes has relation to the size of the tumors. b Correlation between tumor size and astrogliosis. Accumulation of astrocytes was indicated as an intensity of GFAP fluorescence. c Typical examples of immunostaining indicating more accumulation of astrocytes in hippocampus than in cerebral cortex. d Correlation curve between tumor size and GFAP intensity in cortex (closed circle) and hippocampus (open circle). In hippocampus, astrogliosis around metastatic tumor foci increased logarithmically with correlation factor (R ²) of 0.72

Fig. 2

Astrocyte stimulated the proliferation of tumor cells via soluble factors. a The normalized number of HARA-B cells increased according to the ratio of astrocytes to HARA-B cells and incubation time in co-culture treatment (HARA-B cells : astrocytes = 1:5, A5; HARA-B cells : astrocytes = 1:10, A10). b Culture medium from insert-culture of astrocytes with HARA-B cells (HARA-B cells : astrocytes = 1:10, A10) significantly increased the proliferation of tumor cells compared to the one without insert-culture medium (control). c H-ACM (HARA-B-stimulated astrocyte-conditioned medium), but not HCM (HARA-B-conditioned medium) nor ACM (astrocyte-conditioned medium) significantly increased the proliferation of tumor cells. The incubation time was 48 h (gray bars) and 72 h (black bars). Each value represents the mean ± SEM (n = 6). ** P < 0.01

Fig. 3

Expression of mRNA and release of cytokines and growth factors from activated astrocytes. (a) Quantitative RT–PCR of IL-1β, IL-6, TNF-α, transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and platelet-derived growth factor-B (PDGF-B) in astrocytes insert-cultured with HARA-B cells. The expression level of each cytokine or growth factor mRNA was normalized to the level of each cytokine in astrocytes cultured alone. The relative values of each cytokine mRNA in insert-cultured astrocytes for 72 h are shown. Each value represents the mean ± SEM (n = 3). Release of IL-1β (b), TNF-α (c) and IL-6 (d) into the culture medium of single-culture of astrocytes (Astro), insert-culture or co-culture of astrocytes and HARA-B cells for 48 and 72 h were detected by ELISA. Each value represents the mean ± SEM (n = 6). ** P < 0.01, ^## P < 0.01

Fig. 4

Effects of recombinant cytokines on HARA-B cell proliferation and inhibitory effects of neutralizing antibodies. a Effects of recombinant mouse (m) IL-1β (1–50 pg/ml), mTNF-α (10–500 pg/ml), and mIL-6 (10–500 pg/ml). Data were given as the percentage of tumor cell proliferation without cytokines (without recombinant cytokines; 100%). HARA-B cells were cultured for 24 h in DMEM and then for 48 h in serum free DMEM with each cytokine. Each value represents the mean ± SEM (n = 6). b Effects of neutralizing antibodies. Anti-mIL-1β (1 μg/ml), anti-mTNF-α (1 μg/ml), anti-mIL-6 (10 ng/ml) neutralizing antibodies, all three antibodies (all ab) and corresponding control IgG were added to co-culture of HARA-B cells and astrocytes. Antibodies were added after 24 h of co-culture of HARA-B cells and astrocytes, and then maintained for 48 h with neutralizing antibodies or control IgG. Data were given as the percentage of control (single-culture of HARA-B cells; 100%) under the same condition without adding antibodies. Each value represents the mean ± SEM (n = 5). ** P < 0.01, ^# P < 0.05

Fig. 5

HARA-B-derived factors which stimulate astrocytes and their effects on expression of mRNA of inflammatory cytokines in astrocytes. a Cytokine expression in HARA-B cells culture medium using the proteome profiler. The cytokine expression in medium (10% FBS DMEM) as negative control (upper panel) and in HARA-B conditioned medium (lower panel), showing the expression of IL-1ra, IL-2, IL-8, MIF, and SERPINE1 (PAI-1). b–d Expression of mRNA of inflammatory cytokines (TNF-α, IL-1β, IL-6) in astrocytes treated with each recombinant cytokines (IL-8, MIF, PAI-1). Quantitative RT–PCR of TNF-α (b), IL-1β (c), and IL-6 (d) in astrocytes treated with each cytokine released from HARA-B cells IL-8, MIF, and PAI-1. The expression level of each cytokine was normalized to the level of each cytokine in non-treated astrocytes. The relative values of each cytokine mRNA in astrocyte treated with each cytokine for 72 h are shown. Each value represents the mean ± SEM (n = 3). Data of PAI-1-treatment was not shown in TNF-α mRNA

Fig. 6

Time-dependent expression of cytokine receptors on HARA-B cells. a Immunostaining of cytokine receptor and receptor subunit (IL-6Rα, gp130, TNFRtI and IL-1RtI) on HARA-B cells with or without co-culture with astrocytes for 24, 48, and 72 h. HARA-B cells were also immunostained with anti-cytokeratin (CK) antibody. b Quantification of fluorescent intensity for each receptor or receptor subunit per area of single cell. Data were given as the percentage of intensity in control HARA-B cells without co-culture (100%). Each value represents the mean ± SEM (n = 8). ** P < 0.01, ^## P < 0.01

Fig. 7

Increased proliferation of different lung cancer cell lines by astrocytes in vitro. The proliferation of HARA-B cells (a), QG56 (b), EBC-1 (c), PC9 (d) were enhanced when they were incubated with insert-culture medium of astrocytes for 72 h. Each value represents the mean ± S.E.M (n = 4). *** P < 0.005 (significance from control)

Fig. 8

Astrocytes accumulate around metastasized lung cancer cells in human brain. a Hematoxylin and eosin (H & E)-staining of lung cancer cell metastasis (dark color) in the human brain section. b Immunostaining of astrocytes and tumor cells from human brain section. GFAP-positive astrocytes aggregated around cancer cells (CAM5.2), which looked similar to the brain metastasis of model mice