The lymphatic system serves as the primary route for metastatic spread of gastric carcinomas, and the presence and extent of lymph node involvement are the principal parameters affecting survival [30
]. Growth factors (e.g., VEGF-C and VEGF-D) associated with lymphangiogenesis increase in malignant melanoma and breast, lung, and colon carcinoma [30
]. Many studies have suggested that lymphangiogenesis is a more reliable parameter than angiogenesis in predicting tumor progression [30
]. Despite all these data, how gastric tumor cells reach the potential to metastasize via lymphatic vessels and the biological role of tumor-associated lymphangiogenesis in the development of nodal metastases remain unclear.
Various methods are used to determine lymphangiogenic activity. Some studies have supported that the rate of LEC proliferation reflects lymphangiogenic activity [34
]. In most studies, LVD detection using lymphatic vessel endothelium-specific monoclonal antibodies (mAbs) is a reliable indicator of lymphangiogenesis [30
]. Therefore, we considered lymphatic endothelial cells to be independent of their proliferative activity. Using lymphatic endothelium-specific markers is a reliable method in the determination of lymphatic vessels and real LI in tumor tissue. For this purpose, many lymphatic endothelium-specific mAbs (e.g., anti-VEGFR-3, anti-LYVE-1, anti-Prox-1, anti-LyP-1, and anti-Nrp2) have been produced. D2-40 is the preferred mAb for investigating intratumoral and peritumoral lymphatics because it is more convenient and more sensitive than other lymphatic markers [40
]. In our study, we used podoplanin (clone D2-40) to determine LI and LVD; its reliability has been proven in previous studies [33
The microvascular density (MVD) method used by Weidner et al. for the determination of tumor angiogenesis is the most commonly used for LVD determination [22
]. It has been reported that the mean vessel number in three areas where vascular structures are most dense (hot spot) is sufficient to determine tumor angiogenesis [23
]. However, tumor-associated lymphatic vessels are fewer and more dispersed than blood vessels. Thus, assessment of an area as large as possible for the determination of LVD will be a more reliable marker of lymphangiogenic activity [24
]. Thus, the mean number of lymphatic vessels in 10 hot spots was used as the LVD in this study.
In our study, varying numbers of lymphatic endothelium/vessels were present in both intratumoral and peritumoral areas in all cases with gastric carcinoma. Lymphatic vessels in intratumoral areas were of immature appearance and collapsed. Lymphatic vessels were concentrated in peritumoral areas. Peritumoral lymphatic vessels had thin walls, irregular shapes, and dilated appearances. It has been argued that compression by tumor cells and oncotic pressure increase in intratumoral areas, causing lymphatic vessels to be few in number and have a narrow lumen [41
]. The morphological findings of our study support this view. In agreement with previous studies, we found that P-LVD was significantly higher than I-LVD. A positive correlation was found between I-LVD and P-LVD [26
]. This finding suggests that lymphangiogenic growth factors expressed from the tumor microenvironment affect intratumoral and peritumoral areas to different extents; however, the lymphangiogenic activity at both localizations is closely interrelated.
Lymphatic vessels are highly dynamic structures that intimately interact with their surrounding microenvironment. They have a profound influence on the immune system and therefore can manipulate inflammatory processes. Inflammation is a major cause of adulthood lymphangiogenesis and lymphovascular remodeling [41
]. Our study demonstrated that lymphatic vessel density in tumor tissue (I-LVD and P-LVD) was positively correlated with the extent of the local inflammatory response. Our findings suggest that the immune response to the tumor is a lymphangiogenesis-inducing factor and may play a role in the formation of tumor lymphatic metastases. Cytokines and the metabolic load increase during inflammation may cause this phenomenon [41
P-LVD has been shown to be associated with poor prognosis and short survival in breast, colon, and lung cancers [26
]. However, the effect of I-LVD on tumor progression remains a controversial issue [26
]. Our study demonstrated that both I-LVD and P-LVD were positively correlated with LI, LID, the presence of lymph node metastasis, and the number of metastatic lymph nodes. Those findings show that an increase in lymphangiogenic activity accelerates the development of nodal metastases by facilitating the transport of tumor cells to lymphatic vessels. Furthermore, our findings suggest that both I-LVD and P-LVD can be used for the prediction of lymphatic spread of the tumor.
The genesis of lymph node metastasis in tumors is a complex and multifactorial process. Invasion of lymphatic vessels at the primary tumor focus by tumor cells is the first and primary step. However, LI cannot be regarded as the sole indicator of lymph node metastasis because of the antitumor defense mechanisms of the host and tumor-associated lymphatic vessels containing anatomically/functionally abnormal features. An increased LI number in tumor tissue will increase the likelihood of metastasis by causing more tumor cells to enter the circulation [16
]. Therefore, we assessed tumor cell entry into the lymphatic circulation using two parameters: nLI and LID (nLI/mm2
). We detected a significant positive relationship between nLI, and LID and the presence of lymph node metastasis, and the number of metastatic lymph nodes. Our results suggest that the presence of multiple lymphatic invasion sites at the primary tumor focus increases the chance of tumor cells metastasizing to lymph nodes. In addition, LID may be a more reliable marker for predicting lymph node metastasis than LVI or LI.
It has been demonstrated in vitro
and in vivo
that NO molecules exert significant effects on tumor lymphangiogenesis and lymphatic spread in addition to tumor angiogenesis. NO induces the proliferation of lymphatic endothelial cells and prolongs their survival by increasing lymphangiogenic growth factors (e.g., VEGF-C/VEGF-D) and VEGFR-2/VEGFR-3 expression in tumor-associated lymphatic vessels [8
]. It has been determined that the increase in NOS activity in tumor tissue is positively correlated with lymphatic metastasis in head and neck, breast, thyroid, and gall bladder cancers and malignant melanomas [3
]. Lahdenranta et al. reported that blocking NOS activity in fibrosarcomas prevented peritumoral lymphatic hyperplasia and tumor cell spread to lymph nodes [6
Studies of iNOS expression in gastric carcinomas frequently indicate a relationship between NO and angiogenesis. Few studies have examined the relationship between NOS and lymphangiogenesis in gastric carcinomas. Wang et al. determined that, compared with normal gastric tissue, gastric carcinomas exhibited greater iNOS expression. In that study, it was determined that iNOS expression in tumor cells is a reliable marker of the iNOS mRNA level [11
]. Koh et al. reported that iNOS expression was closely related to the loss of differentiation in tumor cells and elevated levels of pro-inflammatory cytokines (e.g., TNFα) [19
]. Li et al. identified moderate-to-high iNOS expression in 62% of gastric carcinomas. These researchers found a significant correlation between iNOS expression in tumor cells and tumor size, invasion depth, lymph node involvement, and TNM stage [18
]. Zhang et al. reported that iNOS expression was an independently associated with survival [5
We did not identify iNOS expression in non-neoplastic gastric epithelial cells. We found varying degrees of iNOS expression in tumor cells in 87.8% of gastric carcinoma cases. We also identified a significant correlation between iNOS expression in tumor cells and inflammation density, loss of differentiation, and parameters related to lymphatic tumor spread/lymphangiogenesis. We found that iNOS expression was more prominent, particularly in EMT-like dedifferentiation areas with loss of cohesion and an invasive phenotype. Our results suggest that tumor cells are a principal source of iNOS, and an increase in iNOS activity has the potential to modulate lymphangiogenic activity in gastric carcinomas. Furthermore, our results indicate that tumor cells with a less differentiated/invasive phenotype express more iNOS, and that this increased iNOS expression contributes to metastatic spread. Similar to Koh et al. [19
], we showed that iNOS expression and the inflammatory response had a positive correlation. Our findings suggest that the increase in iNOS expression in tumor cells is a common factor that induces both a tumor-associated inflammatory response and lymphangiogenesis.
iNOS is expressed primarily by macrophages and neutrophils [2
]. In neoplastic tissues, tumor cells are a primary source of iNOS. Thomsen et al. showed that stromal fibroblasts and endothelial cells expressed iNOS, and stromal iNOS expression was correlated with tumor grade in breast carcinomas [29
]. We assessed iNOS expression in various cellular components of gastric carcinomas separately, and found that iNOS expression in tumor cells and iNOS expression in stromal cells were positively correlated. Our findings indicate that the NO level in the microenvironment of the tumor may be elevated by iNOS originating from various cellular components. In addition, we also found that iNOS expression in the tumor stroma was related to tumor lymphangiogenesis and lymphatic spread. However, compared with that originating from tumor cells, iNOS expression from tumor-associated stroma appears to play a less important biological role.