Extended Data Fig. 1: Increased meningeal lymphatic vasculature confers protection against intracranial glioblastoma challenge and provides long-term immunity without perturbance of the blood–brain barrier.
From: VEGF-C-driven lymphatic drainage enables immunosurveillance of brain tumours
a, b, Mice inoculated with 50,000 GL261-Luc cells were imaged every 7 days and showed consistent and reliable tumour growth (n = 4). c, GL261-Luc cells result in lethality in mice in a cell-number-dependent manner (500 cells, n = 5 mice; 5,000 cells, n = 5 mice; 50,000 cells, n = 9 mice). d, Mice were injected intravenously with dextran–fluorescein (molecular weight, 70,000 kDa) and euthanized after 2 h. Brains were collected and cryosectioned (n = 4) The experiment was repeated independently with similar results. e, Mice were injected intravenously with 0.5% Evans Blue. After 2 h mice were perfused intraventricularly and Evans Blue was extracted from brain tissue using dimethylformamide (wild type, LPS, AAV-VEGF-C, VEGFC mRNA, n = 4; tumour, tumour + VEGFC mRNA, n = 5). BBB, blood–brain barrier. f, Representative images of AAV-CTRL and AAV-VEGF-C-treated mice after implantation of 5,000 cells. The experiment was repeated independently with similar results. g, Monitoring of the long-term survival of mice after AAV-VEGF-C and AAV-CTRL injections into the cisterna magna (n = 5). h, i, C57BL/6 mice received an injection of AAV-CTRL or AAV-VEGF-C through the cisterna magna. Six to eight weeks later, mice were euthanized and the dura was collected to image the lymphatic vasculature (LYVE1+) in the superior sagittal sinus (AAV-CTRL, n = 4; AAV-VEGF-C, n = 5). j, C57BL/6 mice that had been injected with CTRL-AAV or AAV-VEGF-C two months previously were implanted with 50,000 GL261-Luc cells in the striatum and monitored for survival (AAV-CTRL, n = 4; AAV-VEGF-C, n = 5). k, AAV-CTRL- or AAV-VEGF-C-treated mice were depleted of CD4 or CD8 T cells using anti-CD4 (GK1.5) or anti-CD8 (YTS169.4) antibodies starting one day before tumour inoculation (GL261) and redosed every four days afterwards (AAV-CTRL, n = 4; AAV-VEGF-C, n = 5; AAV-VEGF-C + anti-CD8, n = 4; AAV-VEGF-C + anti-CD4, n = 5). l, µMT B-cell-deficient mice were injected with AAV-CTRL or AAV-VEGF-C and challenged with 50,000 GL261-Luc cells two months afterwards (AAV-CTRL, n = 5; µMT AAV-CTRL, n = 3; µMT AAV-VEGF-C, n = 5). m, Top, schematic of the schedule of procedures for the experiments below (bottom panel) and in Fig. 1f. Mice injected with AAV-CTRL or AAV-VEGF-C that survived over 100 days after challenge with 5,000 GL261-Luc cells were rechallenged with 500,000 GL261-Luc cells in the flank. Bottom, IVIS imaging of mice ten days after flank rechallenge. Data are pooled from two independent experiments (h–m) and are mean ± s.d. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (two-tailed unpaired Student’s t-test or two-sided log-rank Mantel–Cox test).
