Nature 2020 01 30 Part.02

Article

50,000 GL261 Luc cells

Day 7 post tumor inoculation

Day 14

Day 21-28

050 100 150 200

0

20

40

60

80

100

Days

Percent surviva

l C57BL/^6

50,000 cells5,000 cells

500 cells

d7 d14d 21 d28d 35 d42

50

3000

AAV-CTRL

AAV-VEGF-

C

a

c

0510 15 20 25

0.0

5.0 105

1.0 106

1.5 106

Days

RLU

b

d

(^00100200300)
20
40
60
80
100
Days
Percent survival
AAV-CTRL
N = 5
WT
AAV-VEGF-C
e
50,000 cells
No Tumor Injected
WT AAV-VEGF-CVEGF-C-mRNA Tumor
VEGF-C-mRNATumor
Dextran70k
WT
LPS 6h
AAV-VEGF-CVEGF-C-mRN
A
TumorTumor +
VEGF-C-mRNA
0.0
0.1
0.2
0.3
Relative
Ab
s
BBB Permeability (Evans Blue)

fg
P=0.0013 P=0.01
P=0.0002
GL261-Luc
GL261-Luc
fg
e
020406080100120
0
20
40
60
80
100
Days
Percent survival
muMT AAV-CTRL
muMT AAV-VEGF-C
AAV-CTRL
P = 0.02
P = 0.00 4
**

• Intracranial Tumor
  5,000 cells
  Flank Tumor
  500,000 cells
  AAV-VEGF-C/AAV-CTRL
  Intracisternal Injection
  AAV-VEGF-C IC Tumor D100
  10d post GL261 flank
  No tumor Naive
  020406080100120
  0
  20
  40
  60
  80
  100
  Days
  Percent survival
  GL261 50,000 Cells
  AAV-VEGF-C
  AAV-VEGF-C + CD8
  AAV-VEGF-C + CD4
  AAV-CTRL
  P = 0.015
  P = 0.39
  P = 0.12
  
  GL261
  GL261-Luc
  h
  (^0) AAV-CTRLAAV-VEGF-C
  1
  2
  3
  Superior Sagittal Sinus
  Relative
  Area
  P = 0.00^2
  020406080100120
  0
  20
  40
  60
  80
  100
  Days
  Percent surviv
  al
  AAV-CTRL
  AAV-VEGF-C
  50,000 Cells
  **
  P = 0.00 3
  400μm
  SagittalSinus
  i
  GL261-Luc
  j
  klm
  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.
  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–f luorescein (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, A AV-VEGF-C, VEGFC mRNA, n = 4; tumour,
  tumour + VEGFC mRNA, n = 5). BBB, blood–brain barrier. f, Representative
  images of A AV-CTRL and A AV-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 A AV-VEGF-C and A AV-
  CTRL injections into the cisterna magna (n = 5). h, i, C57BL/6 mice received an
  injection of A AV-CTRL or A AV-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 (A AV-CTRL, n = 4;
  A AV-V EG F- C , n = 5). j, C57BL/6 mice that had been injected with CTRL-A AV or
  A AV-VEGF-C two months previously were implanted with 50,000 GL261-Luc
  cells in the striatum and monitored for survival (A AV-CTRL, n = 4; A AV-VEGF-C,
  n = 5). k, A AV-CTRL- or A AV-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
  (A AV- C T R L , n = 4; A AV-VEGF-C, n = 5; A AV-VEGF-C + anti-CD8, n = 4; A AV-
  VEGF-C + anti-CD4, n = 5). l, μMT B-cell-deficient mice were injected with A AV-
  CTRL or A AV-VEGF-C and challenged with 50,000 GL261-Luc cells two months
  afterwards (A AV-CTRL, n = 5; μMT A AV-CTRL, n = 3; μMT A AV-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 A AV-CTRL or A AV-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 f lank. Bottom, IVIS imaging
  of mice ten days after f lank 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).