Figure 1.
Total CD68+ TAMs and their M1 and M2 subsets in the ovarian cancer tissue.
(A) Representative MUC2 and CD68 single-immunostained and CD68/HLA-DR, CD68/iNOS, CD68/CD163, CD68/VEGF double-immunostained sections from the high MUC2 expression group. (B) Representative MUC2 and CD68 single-immunostained and CD68/HLA-DR, CD68/iNOS, CD68/CD163, CD68/VEGF double-immunostained sections from the low MUC2 expression group. For VEGF- and iNOS-immunostaining, cancer cells (in red) that independently expressed these two proteins can be found in the cancer tissue sections from both groups (arrowhead). Asterisk, TAMs (in brown) that expressed the CD68 protein. Arrow, CD68+ TAMs (in purple) that co-expressed the indicated proteins (HLA-DR, iNOS, CD163 or VEGF). Scale bar (black), 100 μm. Inset scale bar (white), 10 μm. (C) Comparison of the percentages of different M1 and M2 cell subsets (i.e., M1/M2 distribution patterns) among all the TAMs. It could be noted that the percentages of M1 macrophages were significantly higher than those of M2 macrophages in the high MUC2 expression group; but on the contrary, the percentages of M1 macrophages were significantly lower than those of M2 macrophages in the low MUC2 expression group. *, p<0.05, ANOVA.
Figure 2.
Distribution characteristics of CD68+COX-2+ TAMs in the ovarian cancer tissue.
(A) CD68+COX-2+ TAMs in a representative cancer tissue section from the high MUC2 expression group were shown. (B) CD68+COX-2+ TAMs in a representative cancer tissue section from the low MUC2 expression group were shown. Nuclei were stained with SYTO 40, shown in purple. Arrowhead, CD68+COX-2+ TAMs. Arrow, CD68+COX-2- TAMs. Asterisk, COX-2+ cancer cells (CD68-) surrounding the CD68+COX-2+ TAMs. (C) The intratumoral densities of CD68+ TAMs and CD68+COX-2+ TAMs and the CD68+COX-2+/CD68+ TAM ratios of the high and low MUC2 expression groups were compared, as were the corresponding values for the cancer islet and stromal regions. It could be found that the ratio of CD68+COX-2+/CD68+ TAMs was significantly higher in the high MUC2 expression group than in the low MUC2 expression group *, p<0.05, Student’s t-test. **, p<0.05, ANOVA.
Figure 3.
M1 and M2 distribution patterns of the COX-2+ TAMs in the high and low MUC2 expression groups.
(A) Representative CD68, COX-2 and M1/M2 signature triple-immunostained sections are shown. Arrowhead, COX-2+ TAMs that expressed the M1 or M2 signatures. Yellow arrow, COX-2+ TAMs that did not express the M1 or M2 signature indices. Blue arrow, COX-2- M1 or M2 TAMs. Asterisk, COX-2+ cancer cells. Scale bar, 10 μm. (B) The percentages of M1- and M2-polarized TAMs in the CD68+COX-2+ TAM populations that infiltrated the cancer tissue in the two MUC2 expression-level groups were compared. (C) The percentages of COX-2+ cells in the M1 and M2 populations of the intratumoral TAMs in the two MUC2 expression-level groups were compared. It could be noted that most of the COX-2+ TAMs in both the high and low MUC2 expression groups exhibited M2 signatures (i.e., CD163+ and VEGF+). *, p<0.05, ANOVA. NS, no significance, ANOVA.
Figure 4.
Correlation of the COX-2 expression statuses of the cancer cells and TAMs.
(A) Representative microscopic fields in cancer tissue sections from the high and low MUC2 expression groups. In the cancer tissue sections from the high MUC2 expression group, COX-2+ cancer cells and COX-2+ TAMs (with cytoplasm in red) were frequently distributed throughout the entire islet region, whereas in the cancer tissue from the low MUC2 expression group, COX-2+ cancer cells and COX-2+ TAMs were both observed less frequently. Arrow, CD68+ TAMs (left, CD68+COX-2+ TAMs in purple; right, CD68+COX-2- TAMs in brown). Scale bar, 50 μm. (B) Comparison of the COX-2+ cancer cell densities between the high and low MUC2 expression groups. *, p<0.05, Student’s t-test. (C) Scatter plots of the COX-2+ cancer cell density versus the COX-2+ TAM density in specimens from the high (23 cases) and low MUC2 expression (79 cases) groups. r, Pearson’s product-moment correlation coefficient.
Figure 5.
Kaplan–Meier 5-year PFS and OS curves for ovarian cancer patients stratified by four pathological parameters of interest.
(A) MUC2 expression level, low (immunostaining -/+) vs. high (immunostaining ++/+++). (B) M1/M2 ratio, lower vs. higher than the mean value. (C) COX-2+ TAM density, lower vs. higher than the mean value. (D) COX-2+ cancer cell density, lower vs. higher than the mean value. PFS, progression-free survival. OS, overall survival.