Figure 1.
Gene and protein expression analysis of CD4, CD25, Foxp3, IL-10, and TGF-β from patients with CRC (n = 65) by RT-qPCR and immunohistochemical analysis of cancer cells in early (UICC I/II) and late stage (UICC III/IV) of the disease.
(A) Significantly increased gene expression of CD4 and CD25 at stage UICC I/II compared to tumors at stage UICC III/IV. Gene expression of Foxp3, IL-10, and TGF-β was significantly decreased at stage I/II as compared with those at UICC III/IV. The normalization was performed with normal tissue. The relative quantification value, fold difference, is expressed as 2−ΔΔCt. *p<0.001. (B) Foxp3+, IL-10+, and TGF-β+ expressing cancer cells increased from UICC I/II to UICC III/IV compared to normal tissue. The result of the staining was expressed in percentages (%) positivity. All values were expressed as mean ± SD; all pairwise tests (Tukey) result in p<0.001 with exception of control vs. UICC I/II in Foxp3+ (p<0.050).
Figure 2.
Immunohistochemical analysis of CD4+, CD25+, Foxp3+, IL-10+, and TGF-β+ expression in Treg from patients with CRC (n = 65) in early (UICC I/II) and late stage (UICC III/IV) of the disease.
(A) Increased CD4+, CD25+, Foxp3+, IL-10+, and TGF-β+ expression at stage UICC I/II as compared with those at UICC III/IV. The result of the staining was expressed in percentages (%) positivity. All values were expressed as mean ± SD. All pairwise tests result in p<0.001 with three exceptions: Foxp3+, control vs. UICC III/IV, p = 0.091; IL-10+, UICC I/II vs. UICC III/IV, p = 0.021; TGF-ß+, UICC I/II vs. UICC III/IV, p = 0.020. (B) Representative example of an immunofluorescence double staining of Foxp3+ and CD4+ in Treg. Foxp3 expression was mainly observed on CD4+ Treg (arrow) (×400 magnification). FITC, green Fluoresceinisothiocyanate, Cy3, indocarbocyanin red, and DAPI 4′,6-Diamidino-2- phenylindoldihydrochlorid blue – nuclear counterstaining.
Figure 3.
Immunofluorescence double staining of Foxp3 and EPCAM in cancer cells from patients with CRC.
Representative example of an immunofluorescence double staining, showing Foxp3 expression and EPCAM costaining in cancer cells of patients with CRC (×100 magnification above; ×400 magnification below). FITC, green Fluoresceinisothiocyanate, Cy3, indocarbocyanin red and DAPI 4′,6-Diamidino-2- phenylindoldihydrochlorid blue – nuclear counterstaining.
Figure 4.
Protein expression of Foxp3 in colon cancer cell lines by flow cytometry and immunofluorecence double staining analysis.
(A) Flow cytometry assay of Foxp3 expression in SW480, SW620, and HCT-116 colon cancer cell lines compared to isotype control. 3.8% to 6.1% of colon cancer cells express Foxp3; PE: phycoerythrin; FS: forward scatter linear. (B) Representative examples of immunofluorescence double staining of Foxp3+ expression in SW480, SW620, and HCT-116 cancer cells. Cy3, indocarbocyanin red and DAPI 4′,6-Diamidino-2- phenylindoldihydrochlorid blue – nuclear counterstaining (×400 magnification).
Table 1.
Quantitative Real Time PCR analysis of Foxp3 expression in colon cancer cell lines.
Figure 5.
Correlation of Foxp3+ cancer cell expression with immunosuppressive cytokines IL-10+ and TGF-β+ and immunofluorescence double staining.
(A/B) Significant correlation of Foxp3+ cancer cell expression with the expression of IL-10 (A) and TGF-β (B). Regression analysis; R2, coefficient of determination. (C/D) Representative example of an immunofluorescence double staining of IL-10+ (C) and TGF-β+ (D) in Foxp3+ cancer cells (arrows). FITC green Fluoresceinisothiocyanate, Cy3 red and DAPI 4′,6-Diamidino-2- phenylindoldihydrochlorid blue – nuclear counterstaining.
Figure 6.
Correlation of Foxp3+ Treg with Foxp3+ cancer cell expression and immunohistochemical analysis of Foxp3+ Treg and Foxp3+ cancer cells.
(A) Significant inverse correlation of Foxp3+ cancer cell expression with the Foxp3 Treg expression. Regression analysis; R2, coefficient of determination. (B) Increased numbers of Foxp3+ Treg in Foxp3 negative cancer tissue (arrow). (C) Only occasionally or no Foxp3+ Treg in Foxp3 positive cancer tissue (arrow). DAB brown color, Haemalaun blue color – nuclear counterstaining. Representative example demonstrates area of magnifications x100 (left) and x200 (right).
Table 2.
Multivariate analysis of prognostic factors of the study population.
Figure 7.
Overall survival of cancer patients with Foxp3+ cancer cell expression in their CRCs compared with the overall survival of those with infiltrated Foxp3+ Treg in their tumors.
(A) Patients with high Foxp3+ cancer cell expression (>16%, as mean cut-off) had a poorer prognosis than those with low Foxp3+ cancer cell expression profiles (<16%; mean cut-off: 16%), (p<0.001, Log-Rank test). (B) No significant difference in the overall survival comparing patients with low and high Foxp3+ Treg expression profiles (mean cut-off: 12%), (p = 0.202, Log-Rank test). (C) Patients with lymph node metastasis had a poorer prognosis than those without lymph node metastasis (p<0.001, Log-Rank test). The times of the censored data are indicated by short vertical lines.
Table 3.
Clinicopathological characteristics of the study population and discrimination of Foxp3 expression profiles.