Jagged1-Notch1-deployed tumor perivascular niche promotes breast cancer stem cell phenotype through Zeb1

Zinc finger E-box binding homeobox 1 (Zeb1) has been demonstrated to participate in the acquisition of the properties of cancer stem cells (CSCs). However, it is largely unknown how signals from the tumor microenvironment (TME) contribute to aberrant Zeb1 expression. Here, we show that Zeb1 depletion suppresses stemness, colonization and the phenotypic plasticity of breast cancer. Moreover, we demonstrate that, with direct cell-cell contact, TME-derived endothelial cells provide the Notch ligand Jagged1 (Jag1) to neighboring breast CSCs, leading to Notch1-dependent upregulation of Zeb1. In turn, ectopic Zeb1 in tumor cells increases VEGFA production and reciprocally induces endothelial Jag1 in a paracrine manner. Depletion of Zeb1 disrupts this positive feedback loop in the tumor perivascular niche, which eventually lessens tumor initiation and progression in vivo and in vitro. In this work, we highlight that targeting the angiocrine Jag1-Notch1-Zeb1-VEGFA loop decreases breast cancer aggressiveness and thus enhances the efficacy of antiangiogenic therapy.


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

Data analysis
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 Shuang Yang Sep 2, 2020 BD FACSArial ‫ﮫ‬ was used to collect flow cytometric data. qPCR data was collected using Roche LightCycler 480. Immunofluorescence images were acquired with FV1000 confocal microscope (Olympus). Immunohistochemical images were taken using BX53 microscope (Olympus). Densitometric analysis of western blot imaging was performed by G:Box Chemi XRQ gel doc system (Syngene).
The tumor-initiating cell frequency was determined using the ELDA webtool.
RNA-seq data that support the findings of this study have been deposited in NCBI_SRA with accession number PRJNA511636 (https://www.ncbi.nlm.nih.gov/sra/? term=PRJNA511636). All the other data supporting the findings of this study are available within the article and its Supplementary Information files and from the corresponding author upon reasonable request. Data used to generate the figures are available in the Source Data file provided with this paper. Source data are provided with this paper.

nature research | reporting summary
October 2018 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 was chosen based on our prior studies using the same types of assays, as well as published literature, to ensure statistically significant results. For student's t test, at least 3 biological repeats was used to calculate p value. The sample size is commonly acceptable in biological experiment. All sample size was revealed in the sections of Materials and Methods or Figure Legends.
No data were excluded from the analyses.
Experiments were repeated with at least three biologically independent studies for all results presented in the main manuscript. All replication attempts were successful.
Allocation was random.
No blinding, as the same investigator performed most experiments and analyzed the data. For the IHC, both staining and analysis was completed in a blinded manner.
Descriptions of antibodies used (including concentration, supplier name, clone name and catalog number) can be found in supplementary information section 'Antibodies'.
All used antibodies were validated commercially. Certificated of analysis for the approved applications by the manufacturer are available on the company websites.
Cell line authentication was not performed as cells were not listed in the commonly misidentified category.
All cell lines used in this study tested negative for mycoplasma before their use.
No commonly misidentified cell line were used in the study.