Type 1 collagen gel

The aortic ring assay was performed as described previously22 (link). Briefly, thoracic aortae were excised from Flk1-Nano-lantern BAC Tg mice, and peri-aortic tissues, such as the fat layer and adventitia, were removed using fine microdissecting forceps and iridectomy scissors. Rings of approximately 1 mm in length were prepared in serum-free Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, St. Louis, MO, USA). Individual rings were embedded in type I collagen gel (Nitta Gelatin, Osaka, Japan) on glass-bottom dishes and cultured in DMEM containing 50 ng/ml human recombinant VEGF-A (R&D systems). After 1 week of culture at 37 °C in a humidified environment, the collagen gels containing the aortic rings were fixed in 4% paraformaldehyde for 30 min.

We modified the previously described aortic ring assay^{13 (link),23 (link)} to observe EC behaviors during sprouting and branching morphogenesis mimicking angiogenesis. Instead of ring-shaped aortic sections, rectangle-shaped aortic explants (~3 × 4 mm) excised from the mouse thoracic aortae were embedded in type I collagen gel (Nitta Gelatin) with the inner lumen side down on glass-bottom culture dishes (Matsunami Glass) and were cultured in medium-199 containing 5% FCS, 10 μg/ml streptomycin, 100 units/ml penicillin and 50 ng/ml human recombinant VEGF (R&D). Medium replacement was performed every other day.

CAFs were transfected with siRNAs or infected with lentiviral vectors as described above and incubated for 24 h. Three-dimensional (3D) culture was performed as described [32 (link)]. Samples consisting of a mixture of 2.5 × 10⁵ CAFs and 1 mL of type I collagen gel (Nitta Gelatin) were placed in 12-well plates and incubated for 30 min. OSCC cells (1 × 10⁶ cells) were seeded onto the gel and cultured in DMEM supplemented with 20% FBS, after which the gels were transferred to 6-well plates. After 6 days, the gels were fixed with 10% formaldehyde and stained with hematoxylin and eosin. Invasion areas were assessed using ImageJ software ver. 1.52 (NIH) in five randomly selected fields per gel.