Bz x700 digital microscope

Paraffin‐embedded tumor sections were dewaxed in xylene and ethanol and autoclaved for 15 min in an antigen retrieval solution to retrieve their antigen epitopes, and endogenous peroxidase activity was blocked with 3% H₂O₂. Tissue sections were incubated overnight at 4°C with primary antibodies, including rabbit polyclonal anti‐CD3 (1:200 dilution; ab4055, Abcam, Cambridge, UK) for CD3⁺ T lymphocytes, mouse monoclonal anti‐CD8 (1:300 dilution; clone G10F5, BD Pharmingen, San Diego, CA, USA) for CD8⁺ T lymphocytes, anti‐CD4 (1:300 dilution; clone 10D6, Novocastra, Newcastle, UK) for CD4⁺ T lymphocytes, and anti‐FOXP3 (1:300 dilution; clone 10D6, Novocastra) for FOXP3⁺ T lymphocytes. The secondary antibody was incubated in a ready‐for‐use EnVision–peroxidase system (Dako Japan, Tokyo, Japan). Sections were incubated with horseradish peroxidase‐labeled polymer (EnVision1kit, Dako, Carpinteria, CA, USA) for 30 min at 25°C and incubated with 3,30‐diaminobenzidine tetrahydrochloride (applied as a 0.02% solution containing 0.005% H₂O₂ in 0.05 M Tris–HCl; pH 7.6) at 25°C for 5–15 min and counterstained with hematoxylin. We counted each positive lymphocyte in the invasive tumor margin using a BZ‐X700 digital microscope at a magnification of 200× (Keyence) using hybrid cell count software (BZ‐H3C; Keyence; Figure S1).

Frozen tissue sections were mounted on slides and allowed to dry. Slides were immersed in eosin (VWR, catalog #95057-848) and then rinsed 8 times in PBS. Slides were incubated in 3% acetic acid, pH 2.5, for 3 min and then placed in a solution of 1% w/v Alcian blue in 3% acetic acid, pH 2.5, for 30 min (EMD Millipore, catalog #TMS-010-C). Slides were then rinsed in 3% acetic acid and allowed to dry overnight. Slides were cleared in xylene and preserved with Entellan mounting medium (EMD Millipore, catalog #14802) and coverslips. Stained sections were imaged with a Keyence BZ-X700 digital microscope.

Frozen tissue sections were mounted on slides and allowed to dry. Mounted sections were dehydrated in 100% propylene glycol for 5 min and then stained with ORO for 10 min (Abcam, catalog #ab150678). The sections were then differentiated in 85% propylene glycol for 3 min and rinsed with distilled water 3 times. Coverslips were applied to all slides using an aqueous mounting medium (Vector Laboratories, catalog #H-1400). Stained sections were imaged with a Keyence BZ-X700 digital microscope.

Cells were fixed with 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd., Saitama, Japan) for 10 min at 24°C, and reacted with 0.1% TritonX-100 (Sigma-Aldrich) and 5% of goat normal serum (Agilent Technologies) in PBS (Wako Pure Chemical Industries, Ltd.) for 10 min. Cells were then incubated overnight with each primary antibody (S6 Table) in PBS at 4°C. They were then incubated at 24°C with the secondary antibody for each primary antibody conjugated with Alexa Fluorescent dye (1:300 dilution, Thermo Fisher Scientific Inc.). The nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) (Wako Pure Chemical Industries, Ltd.) for 45 min. To prevent fading, cells were then mounted in DakoCytomation Fluorescent Mounting Medium (Agilent Technologies). Samples were observed and images were captured with an Olympus IX71 inverted microscope (Tokyo, Japan) or a Keyence BZ-X700 digital microscope (Osaka, Japan). F-actin was discerned by staining with CytoPainter Phalloidin-iFluor 594 Reagent (ab176757, Abcam).