Figure 2

Protein composition of ECMs.

ECM was purified from pair-wised colon resection margins (healthy, H), perilesional areas (P) and colorectal carcinomas (CRC) from 5 patients listed in Supplementary Material 1 and proteomic analysis was performed as described in Methods. Comparison of purified ECMs (purple ellipse in the Venn’s diagrams) with the matrisome database¹⁸ revealed identification of 128 matrisome proteins; 76 structural components (12 proteoglycans, 18 collagens and 46 ECM glycoproteins, as highlighted by red circles in panel (A) and 52 matrix-associated components (16 ECM affiliated, 24 ECM regulators and 12 Secreted factors, as highlighted by red circles in panel (B) such as i) ECM-affiliated, as annexin, mucin, complement, calcium binding proteins S100; ii) ECM-regulators, as metallo-proteases, metallo-protease inhibitors; iii) Secreted factors bound to ECM, as latent TGF-b, growth factors, chemokines. Unsupervised hierarchical cluster analysis of LFQ intensities as derived from MaxQuant elaboration (Suppl. file 2.1) was performed upon parametric ANOVA test: the heatmap shows significantly differentially expressed ECM proteins with p-value < 0.01 (C). The ratio between collagen XII and the sum of the two forms of collagen VI reported in the above heat-map was estimated in all 5 ECMs and fold of expression was calculated vs healthy ECM (D). Western blot analysis for Matrilin-2 expression in ECMs and tissues from 3 surgical specimens; level of actin expression in tissue was used as loading control (E). Expression of Matrilin-2 was further evaluated by IHC, in pair-wised ECMs (one representative patient is shown) (F). Expression of Tenascin was evaluated by IHC (pair-wised tissues from one representative patient are shown) (G). (H) P and T in panel C and panel E indicate healthy, perilesional and tumor area, respectively. P value in panel C and asterisks in panel D indicate statistical significance.