Extended Data Fig 1. Increased meningeal lymphatic vasculature confers protection against intracranial glioblastoma challenge in a draining lymph node and T cell dependent manner and provides long-term protection without BBB perturbance.

a-b Mice inoculated with 50,000 GL261-Luc cells were imaged every 7 days and showed consistent and reliable tumor growth (n = 4). c GL261-Luc shows lethality in mice in a cell number dependent way (500 cells, n = 5; 5000 cells, n = 5; 50,000 cells, n = 9). d Mice were injected I.V. with 70k MW Dextran-fluorescein and euthanized after 2 hours. Brains were collected and cryosectioned (n = 4) Experiment was repeated independently with similar results. e Mice were injected I.V. with 0.5% Evans Blue. After 2 hours mice were perfused intraventricularly and EB was extracted from brain tissue using DMF (WT, LPS, AAV-VEGF-C, VEGF-C-mRNA, n = 4; Tumor, Tumor + VEGF-C-mRNA, n = 5). f Representative image of AAV-CTRL and AAV-VEGF-C treated mice after implantation of 5,000 cells. Experiment was repeated independently with similar results. g Long term survival monitoring of mice after AAV-VEGF-C and AAV-CTRL injections into the cisterna magna (n = 5). h-i C57BL/6 mice received injection of AAV-CTRL or AAV-VEGF-C intra-cisternally (icm) 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 injected with CTRL-AAV or AAV-VEGF-C icm two months prior were implanted with 50,000 GL261-Luc cells in the striatum and monitored for survival (AAV-CTRL, n = 4; AAV-VEGF-C, n = 5) or. 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 tumor inoculation (GL261) and re-dosed every four days after (k; AAV-CTRL, n = 4; AAV-VEGF-C, n = 5; AAV-VEGF-C + αCD8, n = 4; AAV-VEGF-C + αCD4, n = 5). l muMT, B cell deficient mice were injected with AAV-CTRL or AAV-VEGF-C and challenged with 50,000 GL261-Luc cells two months after (AAV-CTRL, n = 5; muMT AAV-CTRL, n = 3; muMT AAV-VEGF-C, n = 5). m Schematic of mice procedure schedule in Fig 1f and Extended Data Fig 1m. Mice injected with AAV-CTRL or AAV-VEGF-C that survived over 100 days after 5,000 GL261-Luc challenge were re-challenged with 500,000 GL261-Luc in the flank. m IVIS imaging of mice ten days after flank re-challenge, and measurement of tumors at day 7 and 15 (n = 3). Data are pooled from two independent experiments (h-m). Data are mean ± S.D. *P < 0.05; **P < 0.01; ***P <0.001; ****P<0.0001 (two-tailed unpaired Student’s t-test, two-sided Log-rank Mantel-Cox test)