*3.2. Cytoplasmic Gal-3 Expression Is Increased in Invasive EFVPTC*

Gal-3 IHC analysis was carried out to determine its expression at the subcellular level in thyroid benign nodules, NIFTPs, invasive EFVPTCs and LT subgroup (Figure 1A–D and Figure 2A,B). Gal-3 positive control and the negative control with an isotype specific IgG were stained respectively using thyroid cancer tissue in each batch of immunostaining (Figure 1E,F). Gal-3 expression was mainly observed in tumor cells, whereas the stromal component of tumor did not show positive Gal-3 staining. Cytoplasmic Gal-3 expression (mean ± SD) was significantly increased in invasive EFVPTCs subgroup (4.80 ± 1.60) as

compared to NIFTPs (2.75 ± 1.58, *p* < 0.001) and benign nodules (2.09 ± 1.19, *p* < 0.001, Tables 2 and 3); and there was no significant difference between NIFTPs and benign lesions (*p* = 0.064). The presence of LT also enhanced cytoplasmic Gal-3 expression (3.80 ± 1.32) compared to NIFTPs (*p* = 0.016) and benign nodules (*p* < 0.001). Nuclear Gal-3 expression in EFVPTC (1.84 ± 1.30) was significantly higher than in NIFTPs (1.04 ± 0.72, *p* = 0.001).

**Figure 1.** Histological IHC of Gal-3 expression in the tissue sections of Benign Nodules, NIFTP, invasive EFVPTC and LT tissues. (**A**). Lower detectable cytoplasmic Gal-3 expression was observed in the Benign Nodules tissue section. (**B**). Faint cytoplasmic Gal-3 immunostaining was detected in the NIFTP tissue section. (**C**). Moderate to strong cytoplasmic Gal-3 expression was observed in EFVPTC with capsular invasion. (**D**). Mild to moderate cytoplasmic Gal-3 expression was observed in the stained tissue section of LT. (**E**). Positive control, a classic aggressive PTC stained with anti-Gal-3 antibody showed moderate to strong immunostaining. (**F**). Negative control, a classic PTC incubated with isotype specific IgG in place of the anti-Gal-3 antibody did not show detectable immunostaining. All images were shown at original magnification ×200. All images were shown at original magnification ×200.

**Figure 2.** Distribution of Gal-3 expressions in Benign Nodules, NIFTP, EFVPTC and LT tissues. (**A**). Cytoplasmic Gal-3 expression; (**B**). Nuclear Gal-3 expression. The box-and-whisker plot showed the minimum, first quartile, median, third quartile and maximum of each group of data. Double stars (\*\*) denoted a highly significant difference in means between groups at *p* < 0.01 (one-way ANOVA).



**Table 3.** Comparison of cytoplasmic Gal-3 expression among groups of Benign, NIFTP, EFVPTC and Lymphocytic thyroiditis (LT).


\* The mean difference is significant at *p* < 0.05 level (F(3162) = 29.395, *p* < 0.001 one-way ANOVA).

However, no significant difference was observed between invasive EFVPTC and benign nodules (1.44 ± 1.07, *p* = 0.215, Tables 2 and 4), which negated the utility of nuclear Gal-3 expression as a reliable diagnostic aid in detecting invasive EFVPTC.



\* The mean difference is significant at *p* < 0.05 level (F(3162) = 5.166, *p* = 0.002 one-way ANOVA).
