Immunohistochemical Analysis of FFPE Tissues

The ISH, IHC and TUNEL staining of FFPE tissues were performed as described previously.^{14 (link)} The primary antibodies used in IHC were as follows: RHOF (1 : 200, ab101349, Abcam), TWIST1 (1 : 200, PA5-49688, Invitrogen), E-cadherin (1 : 400, 3195, CST), Vimentin (1 : 100, 5741, CST). Semiquantitative scoring of ISH and IHC was based upon the staining intensity (I: negative, 0; weak, 1; moderate, 2; intense, 3) and the percentage of positive-staining cells (P: 0–5%, scored 0; 6–35%, scored 1; 36–70%, scored 2; and >70%, scored 3) to obtain a final score (Q) defined as the product of I × P. Low expression was defined as Q<4 and high expression with Q ≥ 4. Two senior pathologists performed the scorings independently in a blinded manner.