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Tumor tissue sections were treated with 1% H₂O₂, blocked with 1% FBS (Gibco, CA, USA), and immunostained with anti-E-cadherin (1:1000; Cell Signaling Technology, MA, USA) or anti-Vimentin (1:500; Sigma-Aldrich, MO, USA) for 12 h at 4°C. After that, tumor tissue sections were incubated with a secondary antibody (1:500; Cell Signaling Technology) for 2 h at 25°C. All sections were developed by 3,3’-diaminobenzidine (DAB) kit (Thermo Scientific, CA, USA) [36 (link)].

Colon tissues from 15 weeks old mice (n = 4 per group) were fixed in 4% paraformaldehyde. Tumor sections were stained with hematoxylin and eosin and IHC antibodies: Ki-67 (Thermo Fisher Scientific, catalog no. MA5-14520, RRID: AB_10979488, 1:100), CD45 (Thermo Fisher Scientific, catalog no. 14-0451-82, RRID: AB_467251, 1:200), CD68 (Cell Signaling Technology, catalog no. 97778, RRID: AB_2928056, 1:200), F4/80 (Cell Signaling Technology, catalog no. 70076, RRID: AB_2799771, 1:200). All antibody staining was performed overnight at 4°C, followed by incubation with either anti-rabbit or anti-goat secondary antibody. Slides were developed using a 3,3′-Diaminobenzidine (DAB) kit (Thermo Fisher Scientific, catalog no. 34065), and nuclei were counterstained with hematoxylin. Images were captured using Olympus imaging software, cellSens standard version 2 (RRID: SCR_014551). The average number of Ki-67⁺ cells per mm² of tumor area was counted in individual tumors from at least five randomly selected microscopic field at high magnification (40×). The methods for assessing of tumor-infiltrating lymphocytes (TIL) were determined on the basis of the recommendation provided by the International TILs Working Group (11 (link)). The average number of CD45⁺, CD68⁺, and F4/80⁺ TILs was counted and analyzed separately in either intratumoral or tumor-surrounding stromal compartments.

HCC tissues were fixed in 4% formalin and embedded in paraffin. Sections were incubated with anti-CD68, anti-CD163, or anti-CD206 antibodies and then treated with immunoperoxidase by means of a 3,3′-diaminobenzidine (DAB) kit (Thermo, USA). The sections were examined under a light microscope at 400× magnification. A consensus reading was obtained for discordant cases.