Altered organization of collagen fibers in the uninvolved human colon mucosa 10 cm and 20 cm away from the malignant tumor

Abstract

Remodelling of collagen fibers has been described during every phase of cancer genesis and progression. Changes in morphology and organization of collagen fibers contribute to the formation of microenvironment that favors cancer progression and development of metastasis. However, there are only few data about remodelling of collagen fibers in healthy looking mucosa distant from the cancer. Using SHG imaging, electron microscopy and specialized softwares (CT-FIRE, CurveAlign and FiberFit), we objectively visualized and quantified changes in morphology and organization of collagen fibers and investigated possible causes of collagen remodelling (change in syntheses, degradation and collagen cross-linking) in the colon mucosa 10 cm and 20 cm away from the cancer in comparison with healthy mucosa. We showed that in the lamina propria this far from the colon cancer, there were changes in collagen architecture (width, straightness, alignment of collagen fibers and collagen molecules inside fibers), increased representation of myofibroblasts and increase expression of collagen-remodelling enzymes (LOX and MMP2). Thus, the changes in organization of collagen fibers, which were already described in the cancer microenvironment, also exist in the mucosa far from the cancer, but smaller in magnitude.

Figure 1

SHG images showing patterns of collagen fibers organization in the lamina propria of colon mucosa in the healthy patients and 10 cm and 20 cm away from the malignant tumor. Collagen fibers were wavy, orderly organized throughout lamina propria and around the crypts in the mucosa of healthy patients (a); In the lamina propria at the distance 10 cm (b,c) and 20 cm away from the cancer (d), proper arrangement of collagen fibers was partly lost: regions with parallel collagen fibers (b, arrows), thick and dense collagen fibers (c, arrow), regions with edema of lamina propria where collagen fibers were separated with large pores (d, arrow showing pore) were frequently observed.

Figure 2

CT FIRE and CURVE Align in analyzing whole SHG images of collagen fiber in the lamina propria of colon mucosa in the healthy patients and 10 cm and 20 cm away from the malignant tumor. Original SHG image of lamina propria of healthy patient. (a) Graphical output from CT FIRE showing automatic extraction of collagen fibers, same patient. (b) Graphical outputs from CURVE Align for calculating alignment of collagen fibers. (c,d) Graphs are showing increased width (e), straightness (f) and alignment (g) of collagen fibers in the lamina propria 10 cm and 20 cm away from the cancer, calculated using CT FIRE and CURVE Align. *p < 0.05, **p < 0.001, ***p < 0.0001 (n = 32 healthy patients/96 images and n = 35 cancer patients/105 images; Values are express as mean ± sd; ANOVA).

Figure 3

CT FIRE and CURVE Align in analyzing regions of interest (ROIs) on SHG images of collagen fiber in the lamina propria of colon mucosa in the healthy patients and 10 cm and 20 cm away from the malignant tumor. Original SHG image of lamina propria of healthy patient, with labeled rectangular ROIs which include collagen fibers near the Liberkün’s crypts (a) and example of CT FIRE collagen fiber extraction within ROIs (b–d). Graphs are showing increased width (e), straightness (f) and alignment (g) of collagen fibers in the lamina propria 10 cm and 20 cm away from the cancer, calculated using CT FIRE and CURVE Align. *p < 0.05, **p < 0.001, ***p < 0.0001 (n = 32 healthy patients and n = 35 cancer patients; Values are express as mean ± sd, ANOVA).

Figure 4

Representative SEM images of collagen fibers in the lamina propria of colon mucosa in the healthy patients (a) and 10 cm (c) and 20 (b) cm away from the malignant tumor. In the lamina propria of healthy patients (a) thin collagen fibers were forming relatively dense network. At the distance 10 cm and 20 cm away from the tumor, regions with thick (b, 20 cm away from tumor) and aligned collagen fibers (c, 10 cm away from tumor) were more frequently observed. Magnification x10 000.

Figure 5

Representative images of αSMA-positive cells in the lamina propria of colon mucosa in healthy patients and at the distance 10 cm and 20 cm away from the tumor. Arrows are showing αSMA-positive myofibroblasts (a); Graph is showing increased representation (in %) of αSMA-positive cells in the lamina propria 10 cm and 20 cm away from the cancer, compared with healthy patients. *p < 0.05, **p < 0.001, ***p < 0.0001 (n = 27 healthy patients and n = 30 cancer patients; Values are express as mean ± sd; ANOVA).

Figure 6

Representative images of LOX and MMP2 staining in the epithelium and lamina propria of colon mucosa in healthy patients and at the distance 10 cm and 20 cm away from the tumor (a); Graphs are showing increased representation (in%) of LOX (b) and MMP2-positive cells (c) in the lamina propria 10 cm and 20 cm away from the cancer, compared with healthy patients; *p < 0.05, **p < 0.001, ***p < 0.0001 (n = 27/28 healthy patients and n = 30 cancer patients; Values are express as mean ± sd; ANOVA).