Journal: Brain

Article Title: CCL2 blockade combined with PD-1/P-selectin immunomodulators impedes breast cancer brain metastasis

doi: 10.1093/brain/awae347

Figure Lengend Snippet: Astrocytes and breast cancer cells engage in reciprocal interactions . ( A and B ) Proliferation rates after 96 h of ( A ) iRFP-labelled murine 4T1 cells in the presence or absence of murine astrocytes ( n = 3) and ( B ) iRFP-labelled human MDA-MB-231 cells in the presence or absence of human astrocytes ( n = 3). Data are represented as fold-change to time 0. Statistical significance was determined using one-way ANOVA and Dunnett’s multiple comparisons test. ( C and D ) Proliferation rates after 190 h of ( C ) murine astrocytes in the presence or absence of 4T1 conditioned medium ( n = 4) and ( D ) human astrocytes in the presence or absence of MDA-MB-231 conditioned medium ( n = 3). Data are represented as fold-change to time 0. ( E and F ) Quantification of tumour spheroids area after 48 h of ( E ) mCherry-labelled 4T1 cells in the presence or absence of murine astrocytes ( n = 5) and ( F ) mCherry-labelled MDA-MB-231 cells in the presaence or absence of human astrocytes ( n = 4). Data are presented as fold-change to time 0. ( G and H ) Representative images of tumour spheroids from E and F , respectively. Scale bar = 400 µm. ( I ) Quantification of mCherry-labelled MDA-MB-231 trans-endothelial cells and/or trans-astrocytic migration, presented by the MDA-MB-231 mCherry signal recorded on the surface level of the Transwell® membrane. Reduction in the fluorescent signal indicates the transmigration of the cells through the barrier ( n = 1). ( J ) Trans-epithelial electrical resistance (TEER) values of a barrier consisting of induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cells (BMEC) and human pericytes, with or without human astrocytes and mCherry-labelled MDA-MB-231 measured 20 h after the addition of MDA-MB-231 cells. Data represented as % of time 0. Statistical significance was determined using one-way ANOVA and Tukey’s multiple comparisons test. ( K – L ) Illustration of the blood–brain barrier (BBB) models used in I and J , respectively. This image was created with BioRender.com . All data are expressed as mean ± standard error; Unless otherwise stated, statistical significance was determined using an unpaired Student’s t -test. CM = conditioned medium; hAstro = human astrocytes; mAstro = murine astrocytes.

Article Snippet: Human primary brain vascular pericytes (ScienCell 1200-scl) were seeded on top of the BMEC layer at a ratio of 1:3 pericytes:BMEC.

Techniques: Migration, Membrane, Transmigration Assay, Derivative Assay

Journal: Brain

Article Title: CCL2 blockade combined with PD-1/P-selectin immunomodulators impedes breast cancer brain metastasis

doi: 10.1093/brain/awae347

Figure Lengend Snippet: The CCL2–CCR2/CCR4 axis is important for breast cancer proliferation and invasion . ( A and B ) Proliferation rates after 160 h of ( A ) mCherry-labelled 4T1 cells in the presence or absence of murine astrocytes and CCL2 inhibitor bindarit ( n = 3) and ( B ) mCherry-labelled MDA-MB-231 cells in the presence or absence of human astrocytes and CCL2 inhibitor bindarit ( n = 3). Data are represented as fold-change to time 0. ( C and D ) Proliferation rates of ( C ) iRFP-labelled 4T1, shCCR4 or negative control sequence (shNC) cells in the presence or absence of murine astrocytes after 94 h (one representative of n = 2; dots on the graph represent technical repeats) and ( D ) iRFP-labelled MDA-MB-231, shCCR4 or shNC cells in the presence or absence of human astrocytes after 72 h (one representative of n = 2; dots on the graph represent technical repeats). Data are represented as fold-change to time 0. ( E and F ) Illustrations of the blood–brain barrier (BBB) models used in G and in H and I (respectively). This image was created with BioRender.com . ( G ) Trans-epithelial electrical resistance (TEER) values of a barrier consisting of induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cells (BMEC) and mCherry-labelled MDA-MB-231 cells, with or without the addition of recombinant CCL2 protein, measured 16 h after the addition of MDA-MB-231 cells ( n = 3). Statistical significance was determined using an unpaired Student t -test. ( H ) TEER values of a barrier consisting of iPSC-derived BMEC, human pericytes, human astrocytes and mCherry-labelled MDA-MB-231 cells, with or without the addition of CCL2 inhibitor bindarit, measured 20 h after the addition of MDA-MB-231 cells ( n = 3). Statistical significance was determined using an unpaired Student t -test. ( I ) Immunostaining of a barrier consisting of iPSC-derived BMEC, human pericytes, human astrocytes and mCherry-labelled MDA-MB-231 cells, with or without the addition of CCL2 inhibitor bindarit or recombinant CCL2 protein, measured 20 h after the addition of MDA-MB-231 cells. DAPI (cyan), tight junction marker ZO1 (green) and mCherry-labelled MDA-MB-231 cells (red). Scale bar = 20 µm. ( J and K ) Quantification of tumour spheroids area after 48 h of ( J ) mCherry-labelled 4T1 cells in the presence or absence of murine astrocytes and CCL2 inhibitor bindarit (one representative of n = 2; dots on the graph represent technical repeats, data are presented as fold-change to time 0) and ( K ) mCherry-labelled MDA-MB-231 cells in the presence or absence of human astrocytes and CCL2 inhibitor bindarit ( n = 3) (data are presented as % of control). ( L and M ) Representative images of tumour spheroids from J and K (respectively). Scale bars = 500 µm. All data are expressed as mean ± standard deviation. Unless otherwise stated, statistical significance was determined using one-way ANOVA and Tukey’s multiple comparisons tests. mAstro = murine astrocytes; hAstro = human astrocytes.

Article Snippet: Human primary brain vascular pericytes (ScienCell 1200-scl) were seeded on top of the BMEC layer at a ratio of 1:3 pericytes:BMEC.

Techniques: Negative Control, Sequencing, Derivative Assay, Recombinant, Immunostaining, Marker, Control, Standard Deviation

Journal: The European Journal of Neuroscience

Article Title: VEGF‐E Attenuates Injury After Ischemic Stroke by Promoting Reparative Revascularization

doi: 10.1111/ejn.70114

Figure Lengend Snippet: VEGF‐E‐stimulated brain endothelial cells promote perivascular cell mobility under ischemia and reperfusion‐like condition. (a) Schematic illustration of the experimental design of iHBEC exposed to OGD and reperfusion followed by stimulation with vehicle (VEH) or VEGF‐E (100 ng/mL) for 24 h. (b) Representative Western blot images of phosphorylated (p) and total (t) ERK1/2 expression in iHBEC stimulated with vehicle (VEH) or VEGF‐E. Analysis of (c) p‐ERK1 and (d) t‐ERK1 expression as well as (e) p‐ERK1/t‐ERK1 ratio (i.e., pathway activation). Analysis of (f) p‐ERK2 and (g) t‐ERK2 expression as well as (h) p‐ERK2/t‐ERK2 ratio (i.e., pathway activation) in iHBEC stimulated with VEH and VEGF‐E after OGD. (i) Representative Western blot images of phosphorylated (p) and total (t) P38 expression. Analysis of (j) p‐P38 and (k) t‐P38 expression as well as (l) p‐P38/t‐P38 ratio (i.e., pathway activation) in iHBEC stimulated with VEH and VEGF‐E after OGD. (m) Schematic illustration of the experimental design of wound healing assay performed on HBVP after stimulation with VEH, VEGF‐E, or the conditioned medium (CM) of VEGF‐E‐treated iHBEC. (n) Representative brightfield images of the wound in VEH, VEGF‐E, or CM‐stimulated HBVP at baseline, 24 h, or 48 h. Analysis of wound closure of VEH, VEGF‐E, or CM‐treated HBVP at (o) 24 h and (p) 48 h, represented as percent of control (baseline, 0 h). Data are boxplot with min/max (n = 3–4 independent experiments/condition). * p < 0.05/** p < 0.01/*** p < 0.001 compared to control (c‐e, j, l, unpaired two‐tailed t ‐test; o, p, one‐way ANOVA). Abbreviations: H, hours; iHBEC, immortalized human brain endothelial cells; OGD, oxygen and glucose‐deprived; VEGF, vascular endothelial growth factor; VEH, vehicle.

Article Snippet: Moreover, primary human brain vascular pericytes (HBVP) (ScienCell; 1200; 33634) were used to investigate the action of VEGF‐E on factor release from iHBEC cells.

Techniques: Western Blot, Expressing, Activation Assay, Wound Healing Assay, Control, Two Tailed Test