Fibronectin

Whenever necessary, cells were pre-treated with 100 U/mL IFN-γ for 24 h. Cells (1 × 10⁴) were seeded on 8 μm Boyden chamber inserts (Becton Dickinson Labware, Bedford, MA, USA) [24 (link)] coated with fibronectin (R&D Systems, Minneapolis, MN) (5 µg/mL) on both sides. Three hundred microliter of serum-free media (SFM) was added to the inserts and the bottom wells of 24-well plates with or without 100 U/mL IFN-γ. Cells were allowed to adhere for 3 h. The SFM in the bottom well was aspirated and 500 µL 20% FBS in DMEM was added to the bottom chamber. After 5 h, unmigrated cells were removed from the inside of the inserts with a cotton swab and the cells were fixed in ice-cold 100% methanol for 5 min and stained with 1% w/v crystal violet (ThermoFisher Scientifics) for 3 min. The entire membrane was then imaged at 4× on a Cytation 5 Imaging Multi-Mode Reader. All migrated cells were counted manually using ImageJ cell counter software.

FACS‐enriched iCD8α cells (CD45⁺CD103⁺CD8α^hi), CD8α^‐ IEL (CD45⁺CD103⁺CD8α⁻), and IEC (CD45⁻) were cultured in the presence or absence of recombinant IL‐12 (10–20 ng/mL). After 24 hours supernatants were recovered and immediately placed in a 96‐well plate coated with fibronectin or BSA (R&D systems, MD, USA) and incubated for four hours at 37°C with slight shaking. Some wells were incubated with plain media as control wells. Some wells were incubated with 1 mM of 3,4‐dichloroisocoumurin (Santa Cruz Biotechnology, Santa Cruz, CA). After incubation, supernatants were removed and calcein AM (Tocris Bioscience, Minneapolis, MN, USA)‐labeled B16 melanoma cells were added to the wells (1 × 10⁵/well). After brief centrifugation, cells were incubated for 30 min at 37°C. Then, “before wash” fluorescence was measured at excitation and emission wavelengths of 485 and 520 nm, respectively. Non‐adherent cells were washed and “after wash” fluorescence was measured. Specific adherence was calculated as follows: Adhesion = (after wash/before wash) ×100. Specific Adhesion = fibronectin adhesion—BSA adhesion. For normalization: (Sample SA × control SA)/100.

The in vitro permeability assay was performed as we previously described [34 (link)]. Briefly, hBMECs transfected with miR-24 mimic or miR scramble were grown on 0.4-mm fibronectin-coated (R&D Systems, Inc., Minneapolis, MN, USA) Transwell filters (Corning Inc., Corning, NY, USA). After 48 h, the medium in the upper well was replaced by FITC-dextran 70 kD (0.5 mg/mL in PB).
Cells were stimulated in the lower well with PBS alone or PBS containing 50 ng/mL VEGF-A₁₆₅ (R&D Systems). The entity of endothelial permeabilization was determined measuring at 520 nm the fluorescence of Dextran that passed in the bottom chamber through the cell monolayer.

We isolated antral lysates or human gastric smooth muscle cell lysates and measured conversion of PI(3,4,5) P3 to PI(4,5)P2 (PTEN activity ELISA, Echelon) after incubation with recombinant proteins (rMfge8, RGE, Fibronectin, or Vitronectin, R&D Systems, Inc. 10 μg/ml) or blocking antibodies against α8, β3, and β5 (10 g/ml). In this competitive ELISA, we incubated lysates on a PI(4,5)P2 coated microplate and then added a PI(4,5)P2 detector protein. PI(4,5)P2 produced by PTEN in lysate binds to the detector protein and thus prevents it from binding immobilized PI(4,5)P2 on the plate. We then used a peroxidase-linked secondary to measure PI(4,5)P2 detector protein binding to the plate in a colorimetric assay where the colorimetric signal is inversely proportional to the amount of PI(4,5)P2 produced by PTEN.

96-well plates were coated overnight at 4°C with vitronectin (1 μg/cm²; R&D Systems), fibronectin (10 μg/cm²; R&D Systems), Matrigel (5 μg/cm²; BD Biosicences) or collagen IV (10 μg/cm²; R&D Systems) and then blocked for 1 h with 0.5% bovine serum albumin. Cells were seeded at a density of 4 × 10⁴ cells/well and incubated at 37°C for 1.5 h. Non-adherent cells were removed by washing with PBS, and adherent cells were fixed with cold methanol and stained with 0.1% crystal violet for 25 min at room temperature. After removing the crystal violet solution, the stained cells were washed with water and 10% acetic acid was added to dissolve the crystal violet. Absorbances were measured at 590 nm using a microplate spectrophotometer.

E3 PA was dissolved in HEPES buffer (10 mM HEPES, 3 mM KCl, 150 mM NaCl) at pH 7.4 to a concentration of 10 mg/mL, transiently heated to 80 °C for 30 min and mixed with cells to obtain hydrogels (2.5 × 104 cells/cell type/5 µL hydrogel) as illustrated in Fig. 2a. For the assembly of the hydrogels, a custom ECM solution was prepared by diluting collagen type I (Advanced BioMatrix, USA) tenfold in HEPES buffer to a final concentration of 500 μg/mL, followed by dilutions of fibronectin (50 μg/mL final concentration) (R&D Systems, USA), laminin (50 μg/mL) (R&D Systems, USA), 10 kDa hyaluronan (500 μg/mL; Lifecore, UK) and 1 MDa hyaluronan (500 μg/mL; Lifecore, UK). These ECM macromolecules, in similar ratios (Supplementary Fig. 10), are known to be among the most abundant in the PDAC ECM^{39 (link)}. In 96-well plates, 5 μL PA cultures were co-assembled with 50 μL of ECM. Alternatively, when evaluating the effects of paracrine factors or physical parameters on PA cultures, 20 μL of 20 mM CaCl₂ were used as gelling solution to avoid the confounding biological effects of the custom ECM components.