For AKT phosphorylation staining, surface markers were first stained
on ice for 30 min. Cells were then fixed and washed following the direc-
tions of the BD Phosflow kit. Samples were run on an Attune NxT flow
cytometer.
Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay (ELISA) was performed using
a mouse VEGF-C ELISA Kit (Cusabio LLC, E07361m-96) following the
manufacturer’s instructions.
mRNA tropism
Mice were injected in the cisterna magna with Cy5-labelled GFP mRNA
with JETPEI. A day (24 h) after injection, brains, meninges and lymph
nodes were collected for flow cytometry. Samples were run on an Attune
NxT flow cytometer.
Blood–brain barrier permeability
Mice were injected intravenously with fluorescein-labelled dextran
(molecular weight, 70,000 kDa) (Thermo Fisher Scientific) or 0.5%
Evans Blue. For microscopy, brains were collected 2 h later and flash-
frozen for sectioning. For Evans Blue quantification, mice were per-
fused with ice-cold PBS intraventricularly (heart) and Evans Blue was
extracted using dimethylformamide. The relative absorbance was
measured using a SpectraMax i3 platform (Molecular Devices) at
620-nm wavelength.
T cell proliferation
T cells were isolated using the EasySep Mouse T cell Isolation Kit
(StemCell Technologies). T cells were stained using CellTrace Violet,
and stimulated with CD3/CD28 antibodies from Bio X Cell for 24 h in
complete RPMI. After 24 h, cells were resuspended in medium contain-
ing IL-2 and VEGF-C for 4 days and FACS was performed at the end of
the 4 days.
Culture and stimulation of bone-marrow-derived dendritic cells
Bone marrow was collected from wild-type mice and cultured in com-
plete RPMI supplemented with granulocyte–macrophage colony-stim-
ulating factor for six days. After 6 days, cells were stimulated with LPS
and VEGF-C for 24 h and FACS was performed.
Statistical analysis
For revision experiments, power analysis was completed to estimate
sample size. A summary of key phenotypes that we observed through-
out multiple experiments were graphed. These experiments showed
response rates of around 70%, and the sample size calculation required
for an α value of 0.05 was n = 6 (power 95%) and n = 5 (power 90%).
For all tumour-related experiments with luciferase constructs, mice
were measured for tumour size at day 4–6, and randomized into groups
to ensure similar tumour size. For experiments in which mice were
pooled, those mice within each pool were also ensured to have tumours
of a similar size to minimize variation in downstream analysis.
Mice that underwent treatment were randomized after the measure-
ment of tumour size at day seven. Investigators were not blinded for
the majority of the study; however, all survival studies were monitored
with the help of veterinarians from the Yale animal facility who were
blinded to the studies and reported endpoints accordingly. In addition,
animals were in mixed cages and labelled with numbers that blinded
all involved with scoring mice health.
Survival curves were analysed using a log-rank (Mantel–Cox) test.
For other data, normally distributed continuous variable comparisons
used a two-tailed unpaired Student’s t-test or paired Student’s t-test
with Prism software.Graphical illustrations
Graphical illustrations were made using BioRender (https://biorender.
com/).Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.Data availability
No new sequencing data were generated for this study. All datasets
generated and/or analysed during the current study are available in the
Article, the Source Data files that accompany Figs. 1–4 and Extended
Data Fig. 1–10, or the Supplementary Information.Code availability
All of the code used for analysis is described in the methods. Detailed
files will be made available from the corresponding authors on request.- Wang, J. et al. UV-induced somatic mutations elicit a functional T cell response in the
YUMMER1.7 mouse melanoma model. Pigment Cell Melanoma Res. 30 , 428–435 (2017).
Acknowledgements We thank members of the A.I. laboratory for insightful discussions and
help with protocols; M. Saltzman (Yale Biomedical Engineering) for allowing us to use mice
stereotaxic equipment and for help with materials; S. Lee from the J.-L.T. laboratory for initial
help in breeding mice; and the ICM Vectorology platform for production of AAV material. This
study was supported by National Institutes of Health grants T32GM007205 (MSTP training
grant) and F30CA239444 (to E.S.); AI054359 and AI127429 (to A.I.); R01EB016629-01 and R01
EY025979-01 (to J.-L.T.); and CA196660, CA128814 and CA121974 (to M.B.). A.I. is an investigator
of the Howard Hughes Medical Institute. L.S.B.B. and J.-L.T. were supported by the Yale School
of Medicine. Work in the K.A. laboratory was funded by the iCAN Digital Precision Cancer
Medicine Flagship, Academy of Finland (grants 292816, 273817 and 307366), the Centre of
Excellence Program 2014–2019, the Cancer Foundation in Finland, the Sigrid Juselius
Foundation, the Hospital District of Helsinki, Uusimaa Research Grants, Helsinki Institute of Life
Sciences (HiLIFE) and Biocenter Finland.
Author contributions E.S., J.-L.T. and A.I. planned the project. E.S. and A.I. designed, analysed
and interpreted data and wrote the manuscript. E.S., L.S.B.B. and T.M. performed experiments
and analysed data. H.D. bred and cared for mice. J.-L.T. provided AAV material. S.A., M.B. and
K.A. provided expertise, materials and analysis of data.Competing interests A.I., E.S. and J.-L.T. have filed a patent related to the manuscript as
inventors (application number, US 62/768,390; status of application, provisional; specific
aspect of manuscript covered in patent application, manipulation of meningeal lymphatic
vasculature for brain and CNS tumour therapy. K.A. is an inventor on several patents that relate
to VEGF-C. He and his wife currently have no income, stock or other benefits from companies
that are related to the manuscript. Helsinki University is a shareholder in companies that are
related to the use of VEGF-C. M.B. is a consultant for Eli Lilly and Company.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41586-019-
1912-x.
Correspondence and requests for materials should be addressed to J.-L.T. or A.I.
Peer review information Nature thanks Jonathan Kipnis, Ingo K. Mellinghoff, Michelle Monje-
Deisseroth, Christine Moussion and Shannon Turley for their contribution to the peer review of
this work.
Reprints and permissions information is available at http://www.nature.com/reprints.