Figure 1.
SPARC and vimentin staining from a single patient. Images demonstrate serial DAB-stained tissue sections for both (A) SPARC and (C) vimentin and computer analysed images of the respective DAB staining (B and C). Blue represents DAB-negative pixels (dark areas), orange DAB-positive pixels and red strongly DAB-positive pixels (light areas). SPARC expression was selectively measured only in the regions of the tissue that were also vimentin-positive.
Figure 2.
Correlation between SPARC expression and AJCC disease staging.
(A) Percentage of positively stained cells in AJCC stage I, II, III and IV CRCs compared to normal colonic tissue (Con). SPARC expression was significantly greater in CRC compared to control. There was a significantly greater number of SPARC positive cells in stage II, III and IV CRC compared to stage I (p = 0.092, p = 0.005 and p = 0.0007 respectively). Data presented as a scatter plot with the mean ± SEM. (B) P values comparing the percentage of SPARC positive cells in stage I, II, III and IV CRC. (C) P values comparing SPARC expression in stage II to III good and poor outcome CRC. No differences in SPARC levels between good and poor stage II CRC or good and poor stage III CRC. Data expressed as a percentage of positively stained cells ± SEM.
Figure 3.
SPARC expression in stage II and III CRC using TMAs.
(A) The SPARC to vimentin ratio was significantly greater in CRC tissue compared to normal colonic tissue. (B) No difference in SPARC expression was observed between stage II and III CRC. (C) SPARC was significantly greater in patients with a good disease outcome in stage II CRC, but not in (D) stage III CRC. Each dot represents one patient and the bars represent the median value ± SEM.
Figure 4.
FOXP3, CD8 and CD45RO expression in CRC and their effects on cancer stage and disease outcome.
(A) FOXP3 expression was significantly greater in CRC tissue compared to normal colonic tissue, whilst (B) CD8 and (C) CD45RO expression levels were significantly greater in normal colonic tissue compared to CRC tissue. When looking at T cell density and disease stage (D – FOXP3; E – CD8 and F – CD45RO), the expression of all three markers were significantly greater in stage II CRC compared to stage III CRC. Each dot represents one patient and the bars represent the median value ± SEM.
Figure 5.
Representative images of CD8, CD45RO and FOXP3 staining in normal colonic tissue, stage II and III CRC.
Images demonstrate DAB-stained tissue sections for CD8 positive cells, CD45RO-positive cells and FOXP3 positive cells. Images were taken at 20X scanning magnification.
Figure 6.
T cell density and disease outcome.
When dividing the patients into good and poor disease outcome, FOXP3 expression was significantly greater in patients with a good disease outcome in (A) stage II CRC but not in (B) stage III CRC. Similarly, CD8 was also significantly greater in patients with a good disease outcome in (C) stage II CRC but not in (D) stage III, as with CD45RO in (E) stage II and (F) stage III. Each dot represents one patient and the bars represent the median value ± SE.
Figure 7.
The mean SPARC and FOXP3 expression in the primary tumour was measured and correlated with disease recurrence. The number of patients with or without disease recurrence at each time point is displayed below each graph. (A) SPARC expression in stage II CRC. Patients without disease recurrence had significantly higher SPARC levels at all time point up to 60 months and for most points up to 138 months compared to those patients who suffered a disease recurrence. The overall SPARC expression was significantly different between the two groups (p<0.0001). (B) SPARC expression in stage III CRC. SPARC expression was significantly higher in patients without disease recurrence from 84 months and beyond. SPARC was also significantly different between the two groups (p<0.0001). (C) FOXP3 expression in stage II CRC. Higher FOXP3 levels was also significantly associated with less disease recurrence as with (D) FOXP3 expression in stage III CRC, however, the differences were not as marked as in stage II CRC. FOXP3 expression was significantly different between the two groups in both stage II and III (p<0.0001). Each data point represented the mean SPARC or FOXP3 expression level at each time point. (*) p<0.05, (**) p<0.01.
Figure 8.
Kaplan-Meier survival curves were derived to show high and low marker levels in relation to survival. (A) High SPARC levels significantly correlated with better long-term survival. (B) High FOXP3 levels also significantly correlated with better long-term survival. Although high (C) CD8 and (D) CD45RO expression levels were associated with better long-term survival, the separation and significance between the curves were not as great.
Table 1.
(A) Univariate survival analysis for SPARC, FOXP3, CD8 and CD45RO in CRC tissue from patients with stage II and III CRC.
Table 2.
Correlation of CRC pathology with T-cell density and SPARC expression.
Table 3.
Adjuvant chemotherapy.