Fig 2: Reconstruction of the neurovascular unit in the Brain-Chip(A) Schematic illustration of the Brain-Chip, a two-channel microengineered chip including iPSC-derived brain endothelial-like cells cultured on all surfaces of the bottom channel, and iPSC-derived Glutamatergic and GABAergic neurons, primary human brain astrocytes, pericytes, and microglia on the surface of the top channel.(B) Confocal images of the cell coverage in the brain channel on day 7 of culture. Top image: Immunofluorescence staining of the brain channel including MAP2 (green), GFAP (magenta), NG2 (red), and DAPI (blue). Bottom images: Representative merged confocal image of the brain channel on culture day 7, stained for neurons (MAP2, green), astrocytes (GFAP, magenta, IBA1, yellow), and pericytes (aSMA, red) (bar, 50 µm).(C) Representative immunofluorescent staining for s100ß (red) and GLAST (green) (bar,100 µm).(D) FACS analysis of cell-specific markers of microglia: Total population of microglia within the brain channel (gray), CD11b-positive population (magenta), CD45-positive population (magenta), quantification of CD11b:CD45-positive cells.(E) Representative merged confocal image of the brain channel co-stained with VGAT (green) for GABAergic neurons and VGlut1 (red) for Glutamatergic neurons (bar, 100 µm).(F) Immunofluorescence staining of the brain channel including MAP2 (green) and SYP (red) (bar, 100 µm).(G) Levels of secreted glutamate in the brain channel on culture days 5, 6, and 7 (n = four to six independent chips, ****p < 0.0001, NS: not significant compared to the transwells group n = 3-4). Data are represented as mean ± SEM, statistical analysis by two-way ANOVA with Tukey’s multiple comparisons test.

Fig 3: Representative images of immunohistochemically stained murine gastrocnemius muscle for CD31 (green) and CD11b (red) (n = 6). A DAPI was used to visualize nuclei staining (blue). B Quantification of CD11b- and C CD31-stained area of histological sections of muscle tissue derived from mice in the MSCs and BDACs treatment and vehicle groups

Fig 4: OG inhibits tumor growth, M2-type macrophages, and serum HSP90a levels in Panc 02 + EndoMT tumor model. (A) Measurement of the superficial tumor volumes from the C57BL/6 mice subcutaneously inoculated with Panc 02 + EndoMT cell grafts and treated with OG or control 40% PEG400 (CTRL). The measurement was started on Day 13 post-inoculation and continued every 3 days using Vernier caliper with the formula 1/2 × length × width2. (B) Tumors removed from the mice described in (A) on Day 32 post-inoculation. OG treatment significantly inhibited the tumor growth of Panc 02 + EndoMT cell grafts. (C) Representative H&E-stained tumor tissue sections of control and OG-treated mice. OG treatment did not obviously cause loosened cell arrangement and decreased chromatin basophilia which were observed in the Panc 02 tumor model. (D) Immunohistochemical staining of CK18 and a-SMA from the tumor tissues of control or OG-treated mice. OG treatment did not significantly change the CK18+ and a-SMA+ cell levels of tumor tissues. (E) Immunohistochemical staining of CD11b+, F4/80+, and CD163+ cells from the tumor tissues of control or OG-treated mice. (F) Quantitation of the levels of CD11b+, F4/80+, and CD163+ cells from the tumor tissues of control or OG-treated mice. OG treatment caused a significant increase of F4/80+ macrophages but decreases of CD11b+ myeloid cells and CD163+ macrophages in the tumor tissues. (G) Serum HSP90a levels of the mice described in (A). Mouse sera were collected every week and serum HSP90a levels were measured using ELISA assay. OG treatment resulted in a significant reduction of the serum HSP90a level. Data are the mean ± SD of 6 mice. # p < 0.001 when compared with “CTRL” group.