In the recent years, there has been increasing interest in identification of CRC with the use of noninvasive biomarkers [8]. The expression of ROBO4 and CLEC14A proteins in tumor neovasculature makes these molecules a potential target for use as a diagnostic and prognostic indicators of cancer, including CRC [17, 23, 24].
To the best of our knowladge, the present study investigated the serum level of ROBO4 and CLEC14A in colorectal cancer (CRC) patients for the first time. We found that the mean ROBO4 and CLEC14A concentrations in the serum of CRC patients were significantly higher than in the non-cancer controls. Previous literature reports based on immunohistochemical methods evidenced specific endothelial expression of ROBO4 and CLEC14A in various cell lines, i.e. in MCF-7 breast carcinoma and SY-SH-5Y-neuroblastoma cells [15, 17, 19]. Up-regulation of these biomarkers was also proved in human tissues, i.e. in vessels of colorectal liver metastases, bladder and breast carcinoma, and liver and kidney cancer [15, 19, 26]. Moreover, the expression of ROBO4 and CLEC14A proteins was dominant at sites of active angiogenesis and in regions exposed to hypoxia [19, 27, 28]. In CRC, up-regulation of ROBO4 mRNA was detected in more than 70% of carcinoma tissues and this protein was exclusively present in the endothelium of cancer vessels [29].
In our study, the ROBO4 and CLEC14A serum levels increased already in early-stage CRC, in comparison to the control samples. Moreover, we found that ROBO4 and CLEC14 had high power to discriminate between CRC patients and cancer-free individuals. Interestingly, the diagnostic sensitivity and specificity of serum CLEC14 reached 100% at the level of 23.98 ng/ml, which is higher than values noted for CEA (sensitivity: 62.5% and specificity: 77.0%) and Ca 19 − 9 (sensitivity: 81.3% and specificity: 91.4%), i.e. biomarkers that are currently commonly used in clinical practice. The high predictive ability of CLEC14A was previously described by Robinson et al., who performed ROC curve analysis of CLEC14A staining scores in various tumor tissues and evidenced their high sensitivity (75%) and specificity (85%) in distinguishing between cancer and non-cancer tissue status [30]. The results of our study, together with literature data evidencing that ROBO4 and CLEC14A molecules dominate in tumor endothelial cells, suggest that these biomolecules have diagnostic potential in cancers, presumably including CRC [15, 17, 19, 30, 31].
Further, we analyzed the association between the ROBO4 and CLEC14A serum concentrations and clinicopathological features of the CRC patients. In our study, the increased ROBO4 levels were related to the depth of tumor invasion as well as lymph node and distant metastases. In contrast, the high concentration of CLEC14A was not associated with the presence of lymph node and distant metastases. There is scarce information on the association between ROBO4 or CLEC14A expression and cancer advancement and prognosis. In prostate cancer, a higher histological tumor (Gleason) score was related to overexpression of ROBO4 [32]. In acute myeloid leukemia patients, overexpresion of ROBO4 was a poor prognostic factor and was corelated with shorter disease-free survival and overall survival [33]. Contrasting results were reported by Zhao et al., who evidenced that endothelial overexpression of ROBO4 suppressed breast cancer angiogenesis and reduced the speed of tumor growth [34]. Simmilary, in non-small lung cancer, high ROBO4 tissue expression was related to good prognosis and was connected with normalization of endothelial cells and reduction of cancer spread [16]. Considering CLEC14A, recent reports indicate that elevated levels of this molecule can inhibit carcinogenesis and progression of lung adenocarcinoma [35]. The expression of ROBO4 or CLEC14A molecules in various cancers tissues (up- or down-regulation) suggests that these proteins may act as important modulators of tumorgenesis and tumor progression. Indeed, ROBO4 and CLEC14A are known as angiogenic factors with an essential role in tumor growth. It was revealed that blocking anti-ROBO4/CLEC14 antibodies induced reduction of the formation of new vessels and led to inhibition of cancer mass [25, 31]. Currently, the pro-angiogenic properties of CLEC14A and its involvement in tumor growth are well documented [24, 25]. For example, the CLEC14A protein promotes filopodia formation and activates cell migration, which is detrimental for tumor cell proliferation [15]. Furthermore, the inhibition of the interaction between CLEC14A and multimerin 2 (MMRN2) by a blocking antibody reduces tumor vessel sprouting and hinders the growth of the tumor mass [25].
As a novel observation, we found that the ROBO4 serum concentrations decreased significantly within 3 months after the surgical removal of CRC. In the case of CLEC14A, we documented a tendency of the serum concentration to decline after the operation. Therefore, we hypothesized that the level of circulating forms of ROBO4 and CLEC14A is associated with the tumor mass. However, we did not find any literatre data to support this hypothesis. We can only speculate that resection of solid tumor mass and removal of existing new vessels that are known to express ROBO4 and CLEC14A proteins result in a decline in the concentrations of these biomarkers in blood. Prevoiusly, Krishna et al. observed reduction of tumor microvessel CLEC14A expresion after preoperative chemotherapy administered to patients with epithelial ovarian cancer [36]. It is accepted that chemotherapy performed prior to surgical cancer excision contributes to reduction of tumor mass, down staging, and a decrease in the expression of cancer-specific molecules, including tumor endothelial markers [37, 38].