Pericyte FAK negatively regulates Gas6/Axl signalling to suppress tumour angiogenesis and tumour growth

The overexpression of the protein tyrosine kinase, Focal adhesion kinase (FAK), in endothelial cells has implicated its requirement in angiogenesis and tumour growth, but how pericyte FAK regulates tumour angiogenesis is unknown. We show that pericyte FAK regulates tumour growth and angiogenesis in multiple mouse models of melanoma, lung carcinoma and pancreatic B-cell insulinoma and provide evidence that loss of pericyte FAK enhances Gas6-stimulated phosphorylation of the receptor tyrosine kinase, Axl with an upregulation of Cyr61, driving enhanced tumour growth. We further show that pericyte derived Cyr61 instructs tumour cells to elevate expression of the proangiogenic/protumourigenic transmembrane receptor Tissue Factor. Finally, in human melanoma we show that when 50% or more tumour blood vessels are pericyte-FAK negative, melanoma patients are stratified into those with increased tumour size, enhanced blood vessel density and metastasis. Overall our data uncover a previously unknown mechanism of tumour growth by pericytes that is controlled by pericyte FAK.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection ImageJ software (V1.51) was used to quantify Western blot images, analyse HUVEC bead sprouting, Gas6 immunostaining, p-Axl immunostaining and single cell migration for the Dunn chamber assay Images obtained from spinning disc were processed and analysed using Fiji (V1.51) Axiovision Rel (V4.9.1) software was used to capture IF/IHC images on the Zeiss Axioplan microscope qRT-PCR data was analysed using StepOne Real Time PCR machine and software ELISA and adhesion assay data was analysed using the Tecan plate reader Prism V8.3.0 software was used for statistical analysis of data IncuCyte Zoom system and software (v2018B) was used for the proliferation assay Micro Manager acquisition software (v2.0 gamma) was used to collect images for the Dunn chamber assay Motion analysis of single cell migration was analysed using Wolfram Mathematica v7.0 software Data analysis sgRNAs were designed using the CRISPOR algorithm (http://crispor.tefor.net) For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability A Data availability statement has been included at the end of the manuscript, before the refs.

nature research | reporting summary
October 2018 "Data availability statement All the relevant data that support the findings of this study are available from the corresponding author on request."

Field-specific reporting
Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
Samples sizes were determined according to power calculations. In consultation with our in-house statisticians, Prof Duffy and Dr North, we have done pilot studies to determine the numbers of animals required to provide statistical significance in our results. Prof Duffy has done the power calculations to estimate that we will require 10 mice/cohort. Using a two-sided test with a 5% significance level, his calculations predict 85-90% power to reject the null hypothesis of no difference assuming the two genotypes differ by the magnitudes that we have observed in similar experiments. For tumour growth and tumour response studies two-way ANOVA or Student's t-test will be used.
Data exclusions Inclusion criteria: within each experiment animal groups were the same age, sex, genetic strain and maintained under the same conditions.
Exclusion criteria: mice were excluded from the experiment only if unexpected adverse effects were observed.

Replication
All experimental data are given including replicates. Details of experimental replicates are given in the figure legends. All reported attempts at replication were successful.
Randomization Within each experiment, mice were randomly assigned to different groups to avoid bias. In other experiments cells were randomly assigned to groups to avoid bias. All histological analyses were done in a blinded fashion.

Blinding
All data collection and analysis was blinded.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. For animals bred in-house-health screens (quarterly) were conducted in accordance with FELASA guidelines for health monitoring of rodent colonies, to confirm their free statues of known pathogens in accordance with FELASA screens. No clinical signs were detected. Animals were housed in groups of 4-6 mice per individually ventilated cage in a 12 h light dark cycle (06:30-18:30 light; 18:30-06:30 dark), with controlled room temperature (21 ± 1 °C) and relative humidity (40-60 %).

Ethics oversight
All procedures were approved by our local animal ethics committee, Queen Mary University of London, and were executed in accordance with United Kingdom Home Office regulations. All animal work was carried out in accordance with ARRIVE Guidelines.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants Note that full information on the approval of the study protocol must also be provided in the manuscript.

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.

Methodology Sample preparation
Pericytes and B16F0 tumour cells were grown in culture to the relevant confluency, then trypsinised prior to transfection with CrisprCas lentivirus. Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.