Fig 2: Flt2‐11 peptide binds to NRP‐1. (A) Solid‐phase binding assay of biotinylated Flt2‐11 peptide on a plate coated with NRP‐1, NRP‐2, VEGFR‐1 and BSA, as a negative control. (B) Solid‐phase binding assay in the presence of different biotinylated peptides (P7, Flt2‐11, P4, P12 and B3) derived from the second Ig‐like domain of VEGFR‐1 [34, 36], or in peptide absence (No add), on NRP‐1‐coated plates. Attached peptides were quantified by incubation with alkaline phosphatase‐conjugated streptavidin and a colorimetric assay. Experiments were performed in triplicate and repeated at least three times, with comparable results. Representative experiments are shown. Data are reported as mean ± SE of peptide adhesion with respect to control (peptide absence). Student’s t‐test: *P ≤ 0.05; **P ≤ 0.01.

Fig 3: Effect of Flt2‐8 and Flt2‐5 peptides on EC adhesion and migration. (A) SASA of the 3D structure of VEGFR‐1 Ig‐like domain II. Colour coding: green, residues comprised in the Flt2‐11 or Flt2‐8 peptide that are in contact with VEGF‐A165; yellow, residues comprised in the Flt2‐11, Flt2‐8 or Flt2‐5 peptide that are not in contact with VEGF‐A165; cyan and magenta, other VEGFR‐1 residues that are and are not in contact with VEGF‐A165, respectively. The images were generated using the insightii program (Accelrys Inc.). (B) EC adhesion to sVEGFR‐1 in the presence of peptide Flt2‐11, Flt2‐8, Flt2‐5 or scrambled (Scr) Flt2‐11, or in the absence of any peptide (No add). Results are expressed as percentage of basal EC adhesion to sVEGFR‐1. (C) Migration towards sVEGFR‐1 in the presence of peptide Flt2‐11, Flt2‐8, Flt2‐5 or Scr Flt2‐11, or in the absence of any peptide (No add). Results are expressed as percentage of basal EC migration in the absence of any stimulus. In B and C, representative experiments performed in triplicate are shown; data are reported as mean ± SE. Student’s t‐test: **P ≤ 0.01, comparing peptide treated to untreated (No add) controls. Experiments were repeated at least three times with comparable results.

Fig 4: sVEGFR‐1/α5β1 integrin interaction. (A) SPR analysis of the interaction between sVEGFR‐1 and α5β1 integrin. sVEGFR‐1 is immobilized on sensor chips and α5β1 integrin is injected at different concentrations (from bottom to top: 80 nm, cyan; 160 nm, red; 350 nm, green; 600 nm, blue; and 1 μm, black). Interaction parameters: k on = 1.5 × 103 M−1·s−1; k off = 2.9 × 10−4 s−1; K D = 195 ± 40 nm. (B) Secondary structure representation of the 3D structure of VEGFR‐1 Ig‐like domain II. β‐strands are represented by arrows and labelled from A to H; loops are shown as tubular ribbons; the N‐ and C‐terminal ends of the domain are indicated by labels. Colour coding: peptide 12 (interacting with α5β1 integrin) is blue; Flt2‐11 peptide (interacting with NRP‐1) is yellow in the five‐residue NITVT region corresponding to the Flt2‐5 peptide (see text), and green in the remaining six‐residues region; the rest of the domain is magenta. (C) Proposed mechanism of action of Flt2‐11 peptide. Left: Light blue arrows indicate the high‐affinity interactions: (i) between NRP‐1 (yellow) and α5β1 integrin (black) on the EC membrane; and (ii) between NRP‐1 on the EC membrane and the sVEGFR‐1 substrate (green). The dark blue arrow indicates the low‐affinity interaction between α5β1 integrin on the EC membrane and the sVEGFR‐1 substrate. The low‐affinity interaction is stabilized by the two high‐affinity interactions, and the angiogenic stimulus is triggered. Centre: The Flt2‐11 peptide inhibits the high‐affinity interaction between NRP‐1 on the EC membrane and the sVEGFR‐1 substrate. The low‐affinity interaction between α5β1 integrin on ECs and the sVEGFR‐1 substrate is not sufficiently stable to trigger the angiogenic stimulus that starts upon integrin engagement. Right: The affinity of the interaction between α5β1 integrin (black) on the EC membrane and the P12 peptide substrate (light blue arrow) is high enough that additional stabilizing interactions are not required. Consequently, the α5β1 integrin/P12 peptide interaction takes place both in the presence and in the absence of the Flt2‐11 peptide.

Fig 5: Endothelial Raptor/mTORC1 supports transendothelial delivery of fatty acids.(A) Schematic of transendothelial transport assay. (B) Representative images of BODIPY-C16 (green) in LLC tumor cells after 1 hour. VEGFR1-Fc was used in control samples to bind tumor cell–derived VEGF-B. Endothelial cells act as a physical barrier for BODIPY-C16 access, demonstrated in a representative image from an endothelial cell–free control (“No ECs”). A control performed without tumor cells (“No TCs”) is shown to confirm removal of endothelial cells prior to fluorescence imaging of basolateral tumor cells. Scale bar: 100 μm. (C) BODIPY-C16 fluorescence in LLC tumor cells, normalized to WT plus VEGFR1-Fc control (n = 3 per group). (D) Representative confocal images of WT or Rptor-KO endothelial cells treated with BODIPY-C16 for 5 minutes. Nuclear (Hoechst, blue) and actin (phalloidin, red) staining was used to detect perinuclear and cell boundaries, respectively. Total (all z-planes) and basolateral (bottom 10% of z-planes) BODIPY-C16 (green) staining are also shown, with the basolateral BODIPY presented at higher exposure (HE). Scale bar: 20 μm. BODIPY-C16 intensities at the basolateral surface were normalized to total signal in each cell. WT (n = 16) and Rptor-KO (n = 10) cells were analyzed from 2 independent experiments. (E and F) WT (n = 4) or RptorECKO (n = 5) mice were inoculated with LLC tumor cells as described in Figure 1A. One hour prior to tumor harvest, animals were injected with BODIPY-C16 (50 μg). Median fluorescence intensity (MFI) was determined by flow cytometry in (E) CD45–CD31+ endothelial cells and (F) CD45–EpCAM+FSChi tumor cell–enriched populations and normalized to littermate WT controls. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 by 2-way ANOVA with Tukey’s post hoc test (B) or unpaired, 2-tailed Student’s t test (D–F).