The lymphangiogenic properties of ECM1 have been reported elsewhere . However, whether this protein is involved in the formation of new lymphatic vessels in tumor progression is not fully understood. The present study highlighted a positive correlation between ECM1 protein expression and LMVD, in both the tumor specimens and the lymph nodes. In addition, VEGF-C expression in the lymph nodes was correlated with LMVD. These results indicate that ECM1 has a closer relationship with LMVD than VEGF-C. LMVD reflects the status of lymphangiogenesis and the incidence of lymphatic metastasis. Therefore, like VEGF-C, ECM1 appears to be a potent enhancer of tumor lymphangiogenesis and may contribute to an increased rate of metastatic spread of breast cancer cells to the lymph nodes. Furthermore, the LMVD in both ECM1- and VEGF-C-positive tumor specimens was statistically higher than that noted in the both of the ECM1- and VEGF-C-negative ones as well as the ECM1- or VEGF-C-positive ones; this suggests that ECM1 and VEGF-C might have a synergistic effect on lymphangiogenesis in breast cancer.
In the present study, cytoplasmic ECM1 was significantly elevated in breast cancer specimens, compared to the peritumoral normal counterparts from the same patients. Han et al.  and Wang et al.  also reported that ECM1 was overexpressed in breast cancer tissues. Moreover, the ECM1 mRNA and protein levels detected by real-time RT-PCR and IHC were consistent with each other, suggesting that the elevated ECM1 protein expression levels may derive from increased transcription of the gene.
A previous study demonstrated that breast cancers with lymph node metastasis were more likely to be ECM1-positive (10/13, 76.9%) than those without metastasis (3/9, 33.3%) . However, we failed to identify any statistically significant differences in ECM1-staining from different patients with regard to lymph node metastasis; this finding is in accordance with an analysis of a single hospital-based cohort of patients, in which ECM1 expression was not statistically associated with the status of lymph node metastasis . Differences observed between our findings and those reported by Wang et al.  and Lal et al.  could be related to a number of differences between the studies; for example, differences in the number of cases (i.e., sample size effects), use of antibodies from different suppliers, or the different compositions of the histological types. Participants in the Lal et al.  study, as well as our own, mainly presented with infiltrating breast cancer, but Wang et al.  did not specify the breast cancer stage. In our study, differences in the relative ECM1 mRNA expression levels between the metastasis and non-metastasis cases were not statistically significant. Additionally, we found VEGF-C expression had no statistically significant difference between the metastasis and non-metastasis cases, which is consistent with that reported by Kinoshita et al. . However, other studies have reported associations between VEGF-C expression and lymph node metastasis [31, 32]. Methodological variability, particularly with respect to the antibodies and specimens used, may cause inconsistencies in the results obtained in the different studies. Recent studies have demonstrated that the expansion of lymphatic networks within the lymph nodes occurred prior to the onset of metastasis . Therefore, it appears likely that the status of lymph node metastasis cannot be used as an early predictor of lymphatic invasion. These findings, as well as the absence of a correlation between ECM1 and VEGF-C expression in the tumor cells and lymph node metastasis may be explained by the fact that metastatic establishment in lymph nodes is a complex process in which multiple growth factors are involved. Lymphangiogenesis initiated by lymphangiogenic factors secreted from tumor cells occurs at the onset of metastasis. Hence, our finding that ECM1 and VEGF-C expression is not associated with lymph node metastasis is plausible. One possible explanation is that ECM1 or VEGF-C might be responsible for the early events relating to lymphatic spread prior to lymph node metastasis .
To explore whether ECM1 expression was associated with the metastatic character of the breast cancer cells, we analyzed the ECM1 expression profiles between the primary tumors and the metastatic foci in individuals with metastasis. Theoretically, the metastatic focus in the nodes and the primary nests are consanguine. Additionally, we found two cases that were ECM1-negative in the primary tumor, but ECM1-positive in the corresponding lymph node metastases. Han et al.  and Wang et al.  also found a case that was ECM1-negative in the primary tumor but exhibited ECM1-positive staining in its corresponding metastatic focus. This suggests that ECM1 is associated with breast cancer cells that have a potential for metastasis, although the exact mechanism remains unclear. Recently, it has been reported that ECM1 is selectively expressed in Type 2 helper T cells (TH2 lymphocytes), and regulates TH2 cell migration via expression of KLF2 and S1P1 . Whether ECM1 regulates metastasis associated genes, or interacts with other extracellular matrix proteins, or both, or is involved in tumor cells migration is currently unknown.
Recently published work suggests that overexpression of TFAP2α or TFAP2γ induced ECM1 expression in human mammary epithelial cells could result in modified ER responsiveness . In our study, ER positive tumors appeared more likely to be ECM1-positive. This finding indicates that ECM1 is associated with estrogen responsiveness in breast cancer. Further investigations are needed to prove this as well as the role of ECM1 in the breast estrogen-receptor-axle. It will be interesting to see whether ECM1 could become a new target for breast cancer hormonotherapy.
It is possible that ECM1 alone is not sufficient to facilitate lymphangiogenesis, which may require multiple lymphangiogenic factors. VEGF-C is the most extensively studied molecule for tumor lymphangiogenesis and we found that VEGF-C has potential synergy with ECM1 for facilitating lymphatic metastasis. One limitation of this study was the relatively small sample size. Nevertheless, our findings support a potential role for ECM1 in the lymphatic progression of breast cancer, an area that will require further study to explore the mechanisms involved.