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The intravessel microenvironment has significant effects on cancer metastasis. The aim of the present study was to determine how the morphologic and immunophenotypic features of cancer cells and infiltrating stromal cells within the permeated lymphatic vessels are associated with lymphogenic metastasis. A total of 137 primary lung adenocarcinoma patients with extratumoral lymphatic permeations were examined. Morphologically, the floating cancer nests within the permeated lymphatic vessels were divided into two types: Type A, consisting of a single large cancer nest; and Type B, consisting of multiple small cancer nests. We compared the clinicopathologic characteristics and the immunophenotypes of the cancer cells and infiltrating stromal cells between the Type A and Type B nests. Eleven of 54 Type A patients (20%) had intrapulmonary metastases, compared with 36 of 83 Type B patients (43%; P = 0.006). Immunohistochemically, Type B cancer cells expressed significantly higher levels of CD44 than Type A cancer cells (mean scoresAUTHOR: Scores - what is this score? Is it the number of cells expressing CD44 or the concentration of CD44 or some other type of scoring system? 43.0 vs 20.5, respectively) and E-cadherin (60.5 vs 31.5, respectively), but lower levels of Geminin (11.9% vs 20.3%, respectively) and cleaved caspase 3 (2.4% vs 7.8%AUTHOR: 11.9% vs 20.3%, respectively) and cleaved caspase 3 (2.4% vs 7.8%, – what do the percentages here refer to? The number of cells expressing geminin and caspase 3? The levels of these factors? Please clarify., respectively). Moreover, a significantly larger number of CD204-positive macrophages were present within the cancer-permeated lymphatic vessels in Type B patients than in Type A patients (mean number 9.5 vs 4.6, respectively). The present study reveals that intralymphatic cancer cell and stromal cell phenotypes are susceptible to lymphogenic metastasis, suggesting that lymphogenic metastasis may be affected by the intralymphatic microenvironment they create. (Cancer Sci 2012; 103: 1342–1347)
The cause of death in most cancer patients is the development of metastases from the primary tumor. The metastatic process includes various complex steps. The process starts with the separation of cancer cells from the primary lesion, followed by the permeation of these cells into vessels. In permeated vessels, the cancer cells survive apoptosis (a process known as anoikis), proliferate, and then transmigrate to the metastatic site. Thereafter, the tumor cells adhere to the endothelial cells and extravasate to the connective tissues surrounding the vessels. By interacting with the stromal cells present in that location, they finally invade the target organ parenchyma, followed by the development of metastatic lesions. We recently reported that, after extravasation from lymphatic vessels, cancer cells undergo a dynamic phenotypic change associated with the epithelial–mesenchymal transition (EMT) within the connective tissue of the vessel wall. The cellular and molecular mechanisms involved in each process have been the topic of constant debate and have inspired extensive research.
One study focusing on the location of lymphatic permeation in resected non-small cell lung cancers found that extratumoral lymphatic permeation was an independent prognostic factor. That study report implied that extratumoral lymphatic vessels are an important metastatic route. Sakuma et al., using lung adenocarcinoma cell lines, reported that tumor cells floating in lymphatic vessels resist anoikis by expressing phosphorylated (p-) Src. Conversely, recent reports have revealed the presence of circulating stromal cells that associate with cancer cells and play a role in cancer cell survival, proliferation, and invasion. Ishii et al. advocated that the blood in the vicinity of human lung cancers contains fibroblast progenitor cells that have the capacity to migrate into the cancer stroma and differentiate into stromal fibroblasts. Duda et al. also revealed that tumor-associated stromal cells shed from the primary tumor together with accompanying cancer cells survive in the blood circulation, as well as at secondary sites, and proliferate within the metastatic nodules. Together, these results suggest that the metastatic process may be affected not only by the characteristics of floating cancer cells, but also by those of circulating stromal cells.
We hypothesized that the intralymphatic microenvironment created by tumor cells and stromal cells has a considerable influence on the metastatic process and that the phenotypes of the tumor cells and infiltrating stromal cells in extratumoral lymphatic vessels may be informative for research into the mechanism of cancer metastasis, including intrapulmonary metastasis. We noted the high prevalence of intrapulmonary metastasis among patients with extratumoral lymphatic permeation of lung adenocarcinoma. The aim of the present study was to determine how the morphologic and immunophenotypic features of tumor cells and infiltrating stromal cells within permeated lymphatic vessels are associated with intrapulmonary metastasis.
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During the early phase of metastatic tumor development, floating cancer cells proliferate within the vessel lumen, adhere to the endothelium, extravasate from the vessel lumen, migrate into the connective tissue surrounding the vessels, and invade the target organ parenchyma. During each of these processes, cancer cells, accompanied by the surrounding stromal cells, organize their peculiar microenvironment. The present study is the first to show that pulmonary metastasis is correlated with the morphologic and immunophenotypic characteristics of intralymphatic cancer cells and infiltrating stromal cells.
E-Cadherin is now regarded as a key marker for EMT. In the field of carcinogenesis, EMT has been recognized as a phenomenon during which tumor cells in primary lesions invade the surrounding stroma, repressing the transcription of the adherens junction protein E-cadherin.[9-11] Recently, many studies have postulated that the loss or repression of E-cadherin is strongly linked with cancer invasiveness, metastasis, and patient prognosis.[9, 12] In contrast with these reports on the repression of E-cadherin and tumor invasiveness, the expression E-cadherin in the present study was elevated in the Type B group, even though this group exhibited more PM than the Type A group. Wells et al. discussed the role of E-cadherin in the cancer-associated mesenchymal-epithelial reverting transition (MErT) at distant metastatic sites and speculated that micrometastases may undergo a transition to become E-cadherin positive, just as the critical EMT event is the downregulation or silencing of E-cadherin. Others[13, 14] have also reported that the expression of E-cadherin at the metastatic site is higher than that at the primary site, consistent with the results of the present study.
Epithelial growth factor receptor is a receptor tyrosine kinase that plays essential roles under both normal physiological and cancerous conditions. Ligand binding to the EGFR leads to epithelial cell migration away from cohesive masses secondary to E-cadherin downregulation. Some studies[15, 16] have reported that inhibition of the autocrine EGFR loop results in E cadherin re-expression and cell–cell cohesion. These reports are in contrast with our results, in which the expression of EGFR was higher in the Type B group than in the Type A group, although the expression of E-cadherin was also higher in the Type B group. These findings suggest that these morphologic and immunophenotypic differences in the tumor cells within lymphatic vessels are not associated with EMT.
CD44 is an integral membrane glycoprotein that functions as a receptor for the extracellular matrix glycan hyaluronan. The expression of CD44 in the Type B group was higher than that in the Type A group, similar to the results for E-cadherin. These findings indicate that an adhesive ability may be deeply associated with the intrapulmonary metastatic process. Some studies[17-20] have also reported that the expression of CD44 is associated with cancer metastasis. Tomlinson et al. examined cell lines and human cancers and reported that, as a result of homophilic and homodimeric interactions among E-cadherin, the E-cadherin/α,β-catenin axis-associated adhesive network mediates tumor cell–tumor cell aggregates that are characteristic of the lymphovascular emboli observed during lymphovascular invasion.
The lower expression of Geminin and cleaved caspase 3 in the Type B group indicates that lower proliferative and apoptotic activities are present in this group. In a review, Wells et al. reported that metastatic seed cancer cells may inertly become part of the ectopic tissue and therefore surmount the metastatic inefficiencies to which most disseminated cancer cells succumb. Many other studies have also suggested that tumor cells with metastatic potential have a repressed proliferative activity and the ability to resist apoptosis,[3, 10, 22] also known as anoikis.
The tyrosine kinase Src is one of the key molecules that play a critical role in the development of resistance to apoptosis, known as anoikis.[23, 24] However, in present study no significant differences in the expression of p-Src were observed between the Type A and Type B groups. This result does not coincide with the results for cleaved caspase 3, which indicated a lower level of apoptosis in the Type B group. The environment within the lymphatic vessels was inappropriate for tumor cells, so the anoikis resistance process via p-Src may be fully activated in both the Type A and Type B groups. Thus, another mechanism underlying apoptotic resistance within lymphatic vessels may exist.
Regarding stromal cells around cancer nests, Ohtaki et al. recently suggested that CD204-positive macrophages clearly reflect the tumor-promoting phenotype of tumor-associated macrophages in lung adenocarcinoma. In the present study, the number of infiltrating CD204-positive macrophages around and within the cancer nests was significantly higher in the Type B group than in the Type A group. This implies that not only tumor cells, but also stromal cells (such as CD204-positive macrophages) may contribute to changing the intralymphatic microenvironment. Zhang et al. also advocated that the intravascular microenvironment is a critical staging area for the development of metastases that subsequently invade the parenchyma.
We hypothesized that there may be hypoxic area in the center of the Type A cluster, so we performed CA-IX immunostaining, However, there were no significant differences in CA-IX staining scores between the Type A and Type B groups.
Conversely, in the primary tumors, no significant differences were observed in immunohistochemical staining scores between Type A and Type B groups, except for that of cleaved caspase 3. These differences in staining scores between the intralymphatic cancer nests and primary tumors may be explained by the possibility that primary tumors are composed of a strongly heterogeneous cell population phenotypically and functionally. Conversely, intralymphatic tumors may be comprised of a relatively less heterogeneous cell population. Alternatively, cancer cells that have already permeated into lymphatic vessels perceive signals from circulating stromal cells, which lead to phenotypic changes to the cancer cells. However, we could not find any apparent differences in lymphatic vessel morphology and characteristics between Type A and Type B groups in the present study.
In conclusion, the present study clearly shows an association between the morphological and immunophenotypic features of tumor cells and stromal cells within lymphatics and pulmonary metastasis. From this finding, metastasis is predicted to proceed through dynamic changes in the intralymphatic microenvironment, demonstrating that the lymphatic vessels are far from merely a conduit connecting the primary site with the metastatic site. A more mechanistically oriented experimental approach is required to elucidate the key regulator(s) of the intralymphatic microenvironment, possibly leading to useful therapeutic options in the years to come.