Correlation between biological and mechanical properties of extracellular matrix from colorectal peritoneal metastases in human tissues

Abstract

Peritoneal metastases (PM) are common routes of dissemination for colorectal cancer (CRC) and remain a lethal disease with a poor prognosis. The properties of the extracellular matrix (ECM) are important in cancer development; studying their changes is crucial to understand CRC-PM development. We studied the elastic properties of ECMs derived from human samples of normal and neoplastic PM by atomic force microscopy (AFM); results were correlated with patient clinical data and expression of ECM components related to metastatic spread. We show that PM progression is accompanied by stiffening of the ECM, increased cancer associated fibroblasts (CAF) activity and increased deposition and crosslinking in neoplastic matrices; on the other hand, softer regions are also found in neoplastic ECMs on the same scales. Our results support the hypothesis that local changes in the normal ECM can create the ground for growth and spread from the tumour of invading metastatic cells. We have found correlations between the mechanical properties (relative stiffening between normal and neoplastic ECM) of the ECM and patients' clinical data, like age, sex, presence of protein activating mutations in BRAF and KRAS genes and tumour grade. Our findings suggest that the mechanical phenotyping of PM-ECM has the potential to predict tumour development.

Conflict of interest statement

The authors declare no competing interests.

© 2023. The Author(s).

Figures

Figure 1

YM distributions for the normal (green) and neoplastic (red) conditions from peritoneal ECMs for the 14 patients considered in the study. (A) Violin plots obtained by pooling all YM values from all FCs acquired in all regions of interest (ROIs) for a specific condition. The median value is represented by a white dot and black thick lines represent upper and lower quartiles. (B) Plots showing the distribution of median YM values measured from all force volumes (FVs) collected in different ROIs for each specific condition (green and black dots). Black bars represent the mean of the median values and the corresponding standard deviation of the mean, respectively (see “Statistics” section). The asterisk indicates statistical significance of the difference (p < 0.05).

Figure 2

Images of tissue samples from patients 2, 6, 8 and 12–14, for visualisation of cell nuclei (DAPI staining) and αSMA, magnification 10x; scale bar length is 50 µm.

Figure 3

Images of tissue samples from patients 2, 6, 8 and 12–14, for visualization of collagen and control staining (Picrosirius Red and haematoxylin and eosin—H&E—staining, respectively), magnification ×4; scale bar length is 100 µm.

Figure 4

(A) YM values (mean median value ± std of the mean) of healthy ECMs of the 13 patients affected by adenocarcinoma versus their age (circles and crosses represent men and females, respectively). (B) YM values as in (A) for patients who did not (−) and did (+) undergo chemotherapy. (C–H) Relative stiffening of the neoplastic ECMs from the same patients, versus: (C) chemotherapic treatment; (D) the patients’ age; (E) the presence of protein activating mutations in KRAS and BRAF genes; (F) the patients’ sex; (G) histology—MA mucinous adenocarcinoma, AS adenocarcinoma of the sigma, A adenocarcinoma, H tumour grade.

Figure 5

The figure summarises the main molecular and physical events that contribute to peritoneal metastatic niche development and ECM remodelling. Cancer cells exert their effect on the peritoneal metastasis microenvironment through three main modes: (1) the release of specific growth factors, such as TGFβ, leading to the recruitment of resident fibroblasts and their activation into CAF. (2) The production of exosomes that trigger the microenvironment and mediate the activation of CAFs and the polarisation of macrophages into M2 phenotype. (3) The production of pro-inflammatory cytokines, which contribute to enhancing the activity of CAFs and M2 macrophages by promoting the TGFβ pathway in a paracrine manner. Together, these events contribute to the production and/or deposition of naïve ECM and the concomitant increase in ECM stiffness, both around the site of injury and at distant sites in the peritoneal cavity, which may be a sign of disease progression.

Figure 6

Schematic representation of the nanomechanical measurement. (A) Optical image of an ECM (normal peritoneum-derived from patient 8), with the AFM cantilever and the selected region of interest for the indentation experiment. Slices with thickness between 100 and 200 μm are semi-transparent, which allows to select regions for the analysis that look sufficiently uniform and smooth. The red grid represents the locations where force curves (FCs) are acquired (scale bar length—50 µm). In the inset, the experimental setup for indentation measurements and the optical beam deflection system are shown. (B) Typical rescaled approaching force vs indentation curve. The red circle highlights the contact point. Only the portion of the curve characterized by positive indentation is considered for the Hertzian fit (Eq. (1) and also shown in the inset). (C) The map of Young’s modulus values extracted by the FCs acquired in the region of interest shown in (A). (D) Histogram representing the distribution of YM values represented in the mechanical map in (C). Under the hypothesis of a log-normal distribution, a Gaussian fit in semi-log scale allows to identify the median YM value, as the centre of the Gaussian curve.