Extracellular matrix changes in colorectal carcinoma and correlation with lymph node metastasis

The process of proliferation and invasion of tumor cells depends on changes in the extracellular matrix (ECM) through the activation of enzymes and alterations in the profile of ECM components. We aimed to investigate the mRNA and protein expression of ECM components such as heparanase (HPSE), heparanase-2 (HPSE2), matrix metalloproteinase-9 (MMP-9), and syndecan-1 (SYND1) in neoplastic and non-neoplastic tissues of patients with colorectal carcinoma (CRC). It is a cross-sectional study in which twenty-four adult patients that had CRC were submitted to resection surgery. We analyzed the expression of HPSE, HPSE2, MMP-9, and SYND1 by quantitative RT-PCR and immunohistochemistry. Differing from most of the studies that compare the mRNA expression between tumor samples and non-neoplastic tissues, we decided to investigate whether variations exist in the expression of the ECM components between the affected tissue and nontumoral tissue collected from the same patient with CRC. We removed both tissue samples immediately after the surgical resection of CRC. The data showed higher mRNA and protein expression of HPSE2 (P = 0.0058), MMP-9 (P = 0.0268), and SYND1 (P = 0.0002) in tumor samples compared to the non-neoplastic tissues, while there was only an increase in the level of HPSE protein in tumor tissues. A greater expression of HPSE2 was observed in patients with lymph node metastasis (P = 0.048), suggesting that such protein can be a marker of lymph node metastasis in CRC.

It encodes heparanase-1 isoform (HPSE), whose action disassembles the subendothelial 83 basement membrane and facilitates the installation of metastatic cells disseminated by the 84 bloodstream in the tissues by cleavage of the proteoglycans' HS [8,10]. In normal tissue and 85 under physiological conditions, HPSE expression levels are low, and the protein is found in 86 keratinocytes, trophoblasts, and platelets, as well as mast cells and leukocytes [11]. HPSE also 87 stimulates tumor angiogenesis and vascularization [8,10] and is involved in cell surface HS and 88 ECM degradation in both neoplastic and nonneoplastic tissues. The Heparanase-2 (HPSE2) 89 gene is located on chromosome 10q24.2 [10] and encodes the heparanase-2 isoform (HPSE2), 90 a cell membrane protein. HPSE2 has no enzymatic activity and appears to regulate HPSE 91 activity [10]. The level of expression of HPSE2 is elevated in the brain, small intestine, breasts, 92 uterus, bladder, prostate, and testis [12]. 93 HPSE activity participates in disseminating neoplastic metastasis, and neoplasms that 94 exhibit high HPSE expression and activity also correlate with a more aggressive tumor 5 95 phenotype [7,8]. Researchers have emphasized the involvement of HPSE in exosome formation, 96 immune system activation, autophagy, and chemo-resistance, which further the tumor response 97 to host defense factors, which are influenced by the mediation of interference between tumor 98 cells and tumor microenvironment [7-9]. HPSE has a protumorigenic effect by facilitating cell 99 invasion and triggering the tumor microenvironment's installation mainly due to its degradation 100 activity of HS [8,13]

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Our group previous studies showed a direct correlation between HPSE and SYND1, 145 whereas we observed an inverse correlation between HPSE2 and HPSE and between HPSE2 146 and SYND1 in the colorectal adenomas [37]. We also observed an increased HPSE2 protein   subjects with missing data were excluded. Overall, 50 patients were operated on curative or 182 palliative basis for CRC, of which 26 of these patients were excluded, and 24 patients met the 183 above criteria (Fig 1), including 12 men (50%) and 12 women (50%), all participants were white.     Eclipse® light optical microscope to identify the areas that best represented the immunolabeling.

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For each case, microphotographs of 640x480 pixels were obtained from consecutive and non-248 coinciding fields, with magnification of 10X and 40X microscope objective lenses, through an 249 optical microscope using an Aperio CS2 (Leica Biosystems) scanner, generating digital files.

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All slides were digitalized using a TS100 Nikon Eclipse® optical microscope and divided 258 into two quadrants. For each quadrant, 10 equal and random areas were selected for a given 259 intensity of expression, as described (zero, mild, moderate, intense and very intense). The entire   Table 1.  The quantification of mRNA expression of HPSE, HPSE2, MMP-9, and SYND1 was  was obtained using the Student t-test. 301 We observed no difference regarding HPSE mRNA expression between neoplastic and 302 nonneoplastic tissues obtained from CRC patients. However, HPSE2, MMP-9 and the heparan 303 sulfate proteoglycan SYND1 have significantly increased mRNA expression in tumor tissues 304 (Neoplastic) compared to the nonneoplastic samples (Fig 2).

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There were significantly changes in the protein expression comparing tumor and 306 nonaffected tissues from CRC patients. Immunohistochemistry analyzes clearly demonstrated 307 higher protein level of HPSE, HPSE2, MMP-9 and SYND1 in neoplastic samples (Fig 3). Expression intensity (EIt) was obtained after immunohistochemistry using specific antibodies. The results of this study show that significant differences exist regarding the mRNA and 346 protein expression of HPSE2, MMP-9, and SYND1 between neoplastic and non-neoplastic 347 CRC tissues. The increased expression of such constituents has already been described in