Collagen type 1

T cells in complete X-Vivo media supplemented with 100 IU/ml IL2 were suspended in a 2 mg/ml type I collagen (Corning) solution. For density-dependent migration studies, T cells in Immunocult media were activated with Immunocult CD2/CD3/CD28 expansion solution (STEMCELL) supplemented with 100 IU/ml IL2. T cells were then suspended in 2 mg/ml type I collagen (Corning) and a 0.22 μm-filtered reconstitution buffer that consisted of HEPES, HBSS, and sodium bicarbonate in milliQ-purified water. The T cell-collagen solution was solidified in a 37 °C and 5% CO₂ incubator for 1 hr. Immunocult media supplemented with 100 IU/ml IL2 was then added on top of the T cell-collagen gels. The gels were then incubated for 48 hrs and imaged for 1 hr on a Nikon Ti2 microscope equipped with a live-cell system maintained at 37 °C and 5% CO₂. Time-lapse images were constructed into time-lapse movies using Nikon Elements, and cell positions were tracked frame-by-frame to generate cell trajectories using Metamorph. Analysis of cell trajectories to pull out multiple parameters to characterize cellular motility was performed in Matlab (MathWorks) following the principles and equations previously reported^{13 (link),66 (link)}.

The aortic ring sprouting assay was performed as previously (20 (link)). Briefly, aortas were harvested from 8 to 10 weeks old mice and washed with DMEM/F14 medium (Gibco, Carlsbad, USA) supplemented with 100 U/mL penicillin, and 100 μg/mL streptomycin. The dissected aortas were subsequently cleaned, sectioned in 12–16 rings of 1 mm length, and embedded in collagen type 1 (Corning, New York, USA). After polymerization of the collagen gel, microvascular endothelial cell growth medium (PeloBiotech, Planegg, Germany) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 2% murine serum (BD) was added into the well. Tube-like structures were allowed to develop over 7 days. Thereafter, samples were fixed in 4% PFA and endothelial cells were visualized using antibodies against CD31 (Dianova, Hamburg, Germany) and VE-Cadherin (R&D), while NG2 (Merck, Darmstadt, USA) staining was employed to detect pericytes. The total volume of vascular and perivascular sprouting in each explant was calculated trough the IMARIS-BITPLANE 9.3 software. Additionally, total sprout length was measured with ImageJ.

The basis of the collagen gels used was collagen type 1 (3 mg/mL, Corning) containing 10% 10× M199 medium (Sigma-Aldrich) and 0.22 M NaOH mixed with methylcellulose (Sigma-Aldrich) (MC) media (70% methylcellulose (1.5% w/v in DMEM and 2 mM I-glutamine), 20% DMEM and 10% FCS). 5*10⁵ fibroblasts were suspended in 500 µL consisting of equal amounts of collagen suspension and MC media and poured into 24-well plates. Depending on the experimental setup, rSPARC was added to the gels as well. Empty collagen gels were casted as controls. Collagen gels were allowed to solidify in a humidified chamber at 37 °C for 30 min and then supplied with 1 mL DMEM containing 10% FCS. After 4 days, 5 × 10⁵ cancer cells were added on top of both fibroblast containing as well as empty gels and allowed to attach for 24 h. Afterwards, gels were washed, supplied with DMEM containing 10% FCS and incubated at 37 °C in a humidified atmosphere with 7.5% CO₂, renewing the media every 48 h. After additional 6 days, gels were washed with phosphate buffered saline (PBS), fixed with 1mL 4% formaldehyde (Histofix, PanReac AppliChem, Darmstadt, Germany) for 24 h and then processed for IHC.

143B control and WNT5B KO spheres were grown for 3 days in 96‐well low attachment plates, and then, using wide‐based pipet tips, spheres were diluted and plated onto grid plates to select for droplets with single spheres. These single spheres were subsequently plated onto collagen type 1 (Corning)‐coated 96‐well plates and treated with 10 ng/mL recombinant WNT5B or 20 µM LGK‐974. Spheres were allowed to settle to the collagen and were imaged at intervals of 0, 6, 12, and 24 h. The sphere migration area was measured using ImageJ.