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

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

Preparation of asymmetric phospholipid vesicles for use as cell membrane models

© 2018, The Author(s). Freely suspended liposomes are widely used as model membranes for studying lipid–lipid and protein–lipid interactions. Liposomes prepared by conventional methods have chemically identical bilayer leaflets. By contrast, living cells actively maintain different lipid compositions in the two leaflets of the plasma membrane, resulting in asymmetric membrane properties that are critical for normal cell function. Here, we present a protocol for the preparation of unilamellar asymmetric phospholipid vesicles that better mimic biological membranes. Asymmetry is generated by methyl-β-cyclodextrin-catalyzed exchange of the outer leaflet lipids between vesicle pools of differing lipid composition. Lipid destined for the outer leaflet of the asymmetric vesicles is provided by heavy-donor multilamellar vesicles containing a dense sucrose core. Donor lipid is exchanged into extruded unilamellar acceptor vesicles that lack the sucrose core, facilitating the post-exchange separation of the donor and acceptor pools by centrifugation because of differences in vesicle size and density. We present two complementary assays allowing quantification of each leaflet’s lipid composition: the overall lipid composition is determined by gas chromatography–mass spectrometry, whereas the lipid distribution between the two leaflets is determined by NMR, using the lanthanide shift reagent Pr 3+ . The preparation protocol and the chromatographic assay can be applied to any type of phospholipid bilayer, whereas the NMR assay is specific to lipids with choline-containing headgroups, such as phosphatidylcholine and sphingomyelin. In ~12 h, the protocol can produce a large yield of asymmetric vesicles (up to 20 mg) suitable for a wide range of biophysical studies