Type 1 collagen

Inserts containing 8-μm pores in 24-transwell plates (Corning incorporated-Life Sciences, Tewksbury, MA, USA) were coated with type I collagen (Nitta Gelatin Inc, Osaka, Japan) (45 μg/30 μl/well) and hardened for 24 h. OSCC cells (1.5 × 10⁴) were placed in the inserts coated with type I collagen. NOFs or CAFs (1.5 × 10⁴) were added to the lower chamber of the well. A CXCL1-neutralizing antibody was added to evaluate whether CXCL1 induced the invasive growth of cancer cells. After 48 h, the penetrated cells through the pores of inserts were fixed, stained with 0.25% crystal violet and counted by light microscopy (Olympus, Tokyo, Japan).

Ninety‐six well plates were precoated with 0.3 mg/mL collagen type I (Nitta Gelatin, Osaka, Japan), 0.3 mg/mL collagen type IV (Nitta Gelatin), 50 μg/mL fibronectin (Sigma), FBS (BioWest), or 20 μg/mL laminin (Sigma) and then blocked with DMEM containing 0.5% BSA for 1 h. Thereafter, 2 × 10⁴ cells were seeded onto the coated plates and incubated for 30 min. Unadhered cells were removed by washing with DMEM containing 0.1% BSA. Adherent cells were fixed with 4% paraformaldehyde (PFA), stained with 0.5% crystal violet, and washed. Finally, crystal violet was solubilized with 2% SDS, and absorbance of 595 nm (A₅₉₅) was determined by a microplate reader.

Primary cultures of keratinocytes were prepared from the epidermis of newborn ICR mice as described previously [27 (link)]. Briefly, the epidermis was separated from the dermis and incubated in 0.25% (w/v) trypsin solution (Gibco BRL, Tokyo, Japan) overnight at 4 °C. The dispersed cells were plated in dishes coated with collagen type I (Nitta Gelatin, Osaka, Japan), and cultured in minimal essential medium (MEM) supplemented with 4% (v/v) Chelex (Bio-Rad, Hercules, CA, USA)-treated fetal calf serum (FCS), epidermal growth factor (10 ng/mL; Gibco BRL), and 0.05 mM CaCl2. Under these conditions, keratinocytes were maintained in an immature and proliferating state. The cells were used for experiments 1 week after plating.
To induce differentiation of keratinocytes, CaCl₂ concentration in the medium was increased to 0.2 mM. Calcium treatment induced morphological changes in cells as observed through a phase-contrast microscope and also induced expression of filaggrin protein, a marker of keratinocytes differentiation, as detected by immunoblot (Supplemental Figure S9). The cells were incubated in a medium containing 10 μM BrdU for 5 h, permeabilized with 0.1% (v/v) NP-40, and denatured in 50 mM NaOH. BrdU incorporation was detected using an anti-BrdU antibody (BD Biosciences, Franklin Lakes, NJ, USA). The nuclei were stained with DAPI (Invitrogen, Carlsbad, CA, USA).