Immunofluorescent (IF) and immunohistochemistry (IHC) staining

Tissues were fixed with 10% formaldehyde solution for two days. Subsequently, tissues are embedded in paraffin wax and transferred to the slice. IF staining was performed in the tyramide signal amplification method [32]. The Abs targeting PCNA (BM0104, Boster), Ki67 (ab16667, Abcam), CD8 (ab217344, Abcam), and CD4 (ab183685, Abcam) were used in the assays. Tunel (C1086, Beyotime) and picrosirius red staining (365,548, Sigma-Aldrich) assays were performed following manufacturers’ recommendations. Captured images were overviewed using Caseviewer or Hamamatsu Nanozoomer software, and two pathologists defined the regions of interest (ROIs).

The staining was quantitatively evaluated using ImageJ 1.53 (NIH). The mean infiltration depth of CD4⁺/CD8⁺ cells was assessed as previously described [19, 22]. Briefly, the infiltration depth was measured as the minimum distance of CD4/8⁺ cells to the border and scaled by tumor radius. The levels of Ki67, PCNA, and Tunel were measured by the proportions of positive pixels. The integral optical density (IOD) of picrosirius red staining was used to measure collagen expression.