The tumor microenvironment of colorectal cancer: stromal TLR-4 expression as a potential prognostic marker

Abstract

Background: Colorectal cancer can be efficiently treated when found at early stages, thus the search for novel markers is of paramount importance. Since inflammation is associated with cancer progression and angiogenesis, we investigated expression of cytokines like IL-6 and other mediators that play a key role in the innate immune system, in particular toll like receptor 4 (TLR4), in the microenvironment of lesions from different stages of colon disease progression, from ulcerative colitis to adenoma and adenocarcinoma to find useful markers.

Methods: The presence of inflammatory cells and expression of key cytokines involved in the inflammation process were quantified by immunohistochemistry in specific tissue compartments (epithelial, stromal, endothelial) by immunohistochemistry. A murine azoxymethane/dextran sulfate model in which Tir8, a negative regulator of the inflammatory response, was ablated was used to confirm the clinical observations. 116 Archival tissue samples from patients with different stages of colorectal disease: 13 cases of ulcerative colitis (UC), 34 tubular or tubulo-villous adenomas (AD), and 53 infiltrating adenocarcinomas. 16 specimens of healthy mucosa surgically removed with the cancerous tissue were used as a control.

Results: The differences between healthy tissues and the diverse lesions was characterized by a marked inflammatory-angiogenic reaction, with significantly (P < 0.05) higher numbers of CD68, CD15, and CD31 expressing cells in all diseased tissues that correlated with increasing grade of malignancy. We noted down-regulation of a potential modulator molecule, Hepatocyte Growth Factor, in all diseased tissues (P < 0.05). TLR-4 and IL6 expression in the tumor microenvironment were associated with adenocarcinoma in human samples and in the murine model. We found that adenocarcinoma patients (pT1-4) with higher TLR-4 expression in stromal compartment had a significantly increased risk in disease progression. In those patients with a diagnosis of pT3 (33 cases) colon cancer, those with very high levels of TLR-4 in the tumor stroma relapsed significantly earlier than those with lower expression levels.

Conclusions: These data suggest that high TLR-4 expression in the tumor microenvironment represents a possible marker of disease progression in colon cancer.

Figure 1

Hematoxylin and Eosin staining. Examples of hematoxylin and eosin (H&E) staining of healthy tissues (A), ulcerative colitis (B), adenomas (C) and adenocarcinomas (D) (magnification ×100).

Figure 2

IL-6 expression in human colon tissues. 2a Expression of IL-6 in normal healthy tissues (A), ulcerative colitis (B), adenomas (C) and adenocarcinomas (D); some scattered epithelial and stromal cells are stained with weak intensity. In the dysplastic conditions there is an increased staining (magnification ×400). 2b Different expression of IL-6 in endothelial, epithelial and stromal compartments show that in all groups this marker is significantly increased respect to healthy tissues (mean ± SEM; **P < 0.01, *** P < 0.001). HT = healthy tissues (N = 16); UC = ulcerative colitis (N = 13), AD = adenomas (N = 34; 29 low and 5 high grade), AC = adenocarcinomas (N = 53; 7 T1, 10 T2, 33 T3, 3 T4).

Figure 3

Expression of TLR-4 in human colon tissues. 3a TLR-4 immunohistochemistry analysis. Different expression of TLR-4 in healthy tissues (A), ulcerative colitis (B), adenomas (C) and adenocarcinomas (D) show that increasing grade of dysplasia directly correlates with higher expression of this marker (magnification ×400). 3b Different expression of TLR-4 in endothelial, epithelial area and stromal department shows that in all groups TLR-4 is significantly increased respect to healthy tissues (mean ± SEM; **P < 0.01, *** P < 0.001). HT = healthy tissues (N = 16); UC = ulcerative colitis (N = 13), AD = adenomas (N = 34; 29 low and 5 high grade), AC = adenocarcinomas (N = 53; 7 T1, 10 T2, 33 T3, 3 T4).

Figure 4

Expression of HGF in human colon tissues. 4a Different expression of HGF (present only in epithelial compartment) in healthy tissues (A), ulcerative colitis (B), adenomas (C) and adenocarcinomas (D). The peak of immunoreactivity is in the healthy tissue. In contrast, in the dysplastic lesions, there is a drop in expression as the grade of dysplasia increases. The lowest expression is in UC cases (magnification ×400). 4b Expression of HGF in healthy tissues (HT), UC, AD and AC. In all groups HGF is significantly reduced respect to healthy tissues (mean ± SEM; **P < 0.01, *** P < 0.001). HT = healthy tissues; UC = ulcerative colitis, AD = adenomas, AC = adenocarcinomas.

Figure 5

Staining in murine models of colorectal cancer. Comparison of H&E, TLR-4 and IL-6 immunostaining in mice wild type and knock-out for Tir8. Tir8 -/- mice had a higher grade of dysplasia and an increased expression of TLR-4 and IL-6 than wt mice (magnification ×400).

Figure 6

Disease Free Survival (DFS) curves of adenocarcinoma patients associated with TLR-4 expression in the tumor stroma. Disease free survival (time from diagnosis to relapse, progression or death of disease) were estimated for each marker by means of Kaplan Meier method for patients with CRC using the Survival Analysis System Excel addin by SG Shering, Univ College of Dublin. 6a DFS curve of all adenocarcinoma patients (pT1-4) (53 cases). CRC patients with a low percentage of TLR-4+ cells in the tumor stromal compartment (less than the median value corresponding to 20% of the cells positive) relapsed with a greater time interval and several showed survival of over 100 months, while those patients with a percentage of TLR-4+ cells in the stromal compartment higher than the median value relapsed earlier and fewer showed long term survival (RR 2.36; log rank chi-square 4.25, p < 0.05). 6b DFS curve of patients with adenocarcinoma at the pT3 (33 cases) stage. Patients with a percentage of TLR-4+ cells in the tumor stromal compartment more than 50% relapsed early (within 14 months), while those with a percentage of TLR-4+ cells expression less than 50% relapsed much later (within 40 months, RR 3.15; log rank chi-square 4.03, p < 0.05).

Figure 7

CD68 immunostaining in human colon tissues. 7a CD68 immunostaining in healthy tissues (A), ulcerative colitis (B), adenomas (C) and adenocarcinomas (D) shows a growing expression of intensity, percentage of positive cells and density in stromal compartment (magnification ×400). 7b Expression of CD68 in the stromal compartment of the different groups. In all groups CD68 is significantly increased respect to healthy tissues (mean ± SEM; **P < 0.01, *** P < 0.001). HT = healthy tissues (N = 16); UC = ulcerative colitis (N = 13), AD = adenomas (N = 34; 29 low and 5 high grade), AC = adenocarcinomas (N = 53; 7 T1, 10 T2, 33 T3, 3 T4).

Figure 8

CD15 expression in human colon tissues. 8a CD15 immunostaining in healthy tissues (A), ulcerative colitis (B), adenomas (C) and adenocarcinomas (D). The figure shows an increasing expression in all the compartments: endothelial, epithelial and stromal. In the AC tissues there is a wide distribution and a strong intensity of the marker (magnification ×400). 8b Expression of CD15 in three different compartment shows that in all groups CD15 is significantly increased respect to healthy tissues (mean ± SEM; **P < 0.01, *** P < 0.001). HT = healthy tissues (N = 16); UC = ulcerative colitis (N = 13), AD = adenomas (N = 34; 29 low and 5 high grade), AC = adenocarcinomas (N = 53; 7 T1, 10 T2, 33 T3, 3 T4).

Figure 9

CD31 staining in human colon tissues. 9a Immunohistochemistry for CD-31 in healthy tissue (A), ulcerative colitis (B), adenomas (C) and adenocarcinomas (D) showed a correlation between dysplastic condition and expression of the marker. From ulcerative colitis to adenocarcinoma there is an increase in vascular density and intensity of marker expression (magnification ×400). 9b Different expression of CD-31 (in endothelial compartment) in healthy tissues, ulcerative colitis (UC), adenomas (AD) and adenocarcinomas (AC) show that the increasing grade of dysplasia directly correlates increased vascularization. In all groups CD31 is significantly increased with respect to healthy tissues (mean ± SEM; **P < 0.01, *** P < 0.001). HT = healthy tissues (N = 16); UC = ulcerative colitis (N = 13), AD = adenomas (N = 34; 29 low and 5 high grade), AC = adenocarcinomas (N = 53; 7 T1, 10 T2, 33 T3, 3 T4).