Nature 2020 01 30 Part.02

3

nature research | reporting summary

October 2018

(17A2, biotin, 1:500), anti-CD31 (2H8, GeneTex, 1:500) and anti-LYVE1 (AF2125, R&D systems, 1:250) antibodies were used with

anti-streptavidin (FITC, BD biosciences, 4031801, 1:1000), anti-hamster (127-165-160, Cy3, Jackson ImmunoResearch, 128827,

1:500), and anti-goat (705-175-147, Cy5, Jackson ImmunoResearch , 138513, 1:500) secondary antibodies respectively.

Validation antibodies were all validated on their respective websites, and all are antibodies regularly used in our lab for other purposes.

Eukaryotic cell lines

Policy information about cell lines

Cell line source(s) GL261 parental cells were obtained from the NIH cancer cell repository. GL261-Luciferase cells were kind gifts from Dr.

Jiangbing Zhou (Yale Neurosurgery) and Dr. Carla Rothlin (Yale Immunobiology). YUMMER 1.7 cell lines are previously

reported50. CT2A and CT2A-BFP cells were a kind gift from Dr. Thomas Mathivet (PARCC, Paris). B16 cells were a kind gift

from Dr. Noah Palm (Yale Immunobiology). HEK293T cells were purchased from ATCC. HEK293T, CT2A, CT2A-BFP and B16

cells were cultured in complete DMEM (4.5g/L glucose, 10% FBS, 1% penicillin/streptomycin). YUMMER 1.7 cells were

cultured in DMEM/F12 media (10% FBS, 1% nonessential amino acids, 1% penicillin/streptomycin). GL261 and GL261-Luc

cells were cultured in RPMI (10% FBS, 1% penicillin/streptomycin).

Authentication No authentication was performed.

Mycoplasma contamination All cells were mycoplasma negative

Commonly misidentified lines

(See ICLAC register)

No commonly misidentified lines were used, and all cells were obtained from trusted sources (in material methods).

Animals and other organisms

Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals Four- to eight-week-old mixed sex C57BL/6 mice, B6.129S2-IghtmICgn/J (μMT) mice, B6.SJL-PtprcaPepcb/BoyCrl mice, were

purchased from the National Cancer Institute, Jackson Laboratory and Charles River respectively and subsequently bred and

housed at Yale University. All procedures used in this study (sex-matched, age-matched) complied with federal guidelines and

institutional policies by the Yale School of Medicine Animal Care and Use Committee.

Wild animals no wild animals were used

Field-collected samples No field-collected samples were used.

Ethics oversight All procedures used in this study (sex-matched, age-matched) complied with federal guidelines and institutional policies by the^

Yale School of Medicine Animal Care and Use Committee.

Note that full information on the approval of the study protocol must also be provided in the manuscript.

Clinical data

Policy information about clinical studies
All manuscripts should comply with the ICMJE guidelines for publication of clinical research and a completed CONSORT checklist must be included with all submissions.

Clinical trial registration No clinical studies were performed

Study protocol Note^ where^ the^ full^ trial^ protocol^ can^ be^ accessed^ OR^ if^ not^ available,^ explain^ why.

Data collection Describe^ the^ settings^ and^ locales^ of^ data^ collection,^ noting^ the^ time^ periods^ of^ recruitment^ and^ data^ collection.

Outcomes Describe how you pre-defined primary and secondary outcome measures and how you assessed these measures.

Flow Cytometry

Plots

Confirm that:

The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).

The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).

All plots are contour plots with outliers or pseudocolor plots.

A numerical value for number of cells or percentage (with statistics) is provided.