Although the major cause of mortality in breast cancer is hematogenous metastasis, there are currently no reliable methodologies to predict the risk for metastatic disease. Animal studies, however, may provide some insight. Intravital imaging of invasive tumor cell behavior in mammary tumors in rats and mice has revealed a direct role for macrophages in tumor cell invasion and intravasation (3
). High-resolution two-photon imaging of the interactions between perivascular macrophages and tumor cells during intravasation in mouse models of metastatic mammary carcinoma has revealed the presence of a microanatomic compartment that defines the site where intravasation by motile carcinoma cells occurs. We refer to this compartment as TMEM. The constituent cells of TMEM are an endothelial cell, a perivascular macrophage, and an invasive Mena-expressing tumor cell. Mena, a member of the Ena/VASP protein family, has been identified as an up-regulated gene in the invasive migratory subpopulation of tumor cells in animal models of breast cancer (5
). Mena has been shown to regulate actin-driven cellular protrusions and cell motility in a variety of cell types (4
), to be up-regulated in circulating tumor cells (7
) and primary human breast cancers (11
), and to sensitize tumor cells to epidermal growth factor signals and increase metastasis (14
). Hence, Mena has been postulated to be a marker for invasive, migratory tumor cells and metastatic potential (7
In our initial studies, we used tumor grade as a surrogate for prognosis (including metastasis) and asked if the individual components of TMEM alone could be predictive of poor outcome. To evaluate these components, we looked at invasive tumor cells expressing Mena, blood vessel density, and macrophage density. In our study, Mena expression was higher in tumors compared with benign epithelial cells but did not differ among the three grades of invasive carcinoma. With regard to macrophage and blood vessel density, although there are studies on human breast cancer suggesting an association between increased densities and poor outcome (3
), the significance of these counts as independent predictors has not been clearly shown. A link between macrophage density and poor prognosis, for example, may be evident only in the context of other currently accepted and more frequently used prognostic markers, such as lymph node metastasis, tumor grade, and hormone receptor status (21
). Similarly, when microvessel density is associated with prognosis, it is also in the context of tumor grade and lymphovascular invasion (22
). Our study found that microvessel density was generally elevated in the carcinomas compared with benign breast tissue, but this observation was not statistically significant, perhaps reflecting the relatively small sample size. Additionally, increasing blood vessel density was not significantly associated with increasing tumor grade. Macrophage density also tended to increase with tumor grade, but this trend again was not statistically significant.
Because tumor cells in in vivo
animal models show greatest motility and intravasation in association with perivascular macrophages (3
), we next assessed whether perivascular macrophage density might correlate better with tumor grade. What we found, however, was that the number of macrophages along a blood vessel, at least in invasive carcinomas, appears to be relatively constant regardless of grade.
Given the above results, then, we concluded that the individual components of our proposed microenvironment (microvessel density, macrophage density, and Mena expression) and one relational component (perivascular macrophage density) were not associated with tumor grade and therefore not likely to be of prognostic benefit.
When we looked at all the components together, however, the results were more suggestive of an association with risk of tumor progression. Defining TMEM as a perivascular macrophage in direct apposition to a Mena-expressing tumor cell, we found that TMEM counts differed significantly between well versus moderately and poorly differentiated tumors. In well-differentiated tumors, where the outcome is generally good, the TMEM count was low. In moderately and poorly differentiated tumors, there was a wide range in TMEM counts, but overall the TMEM counts were much higher than in the well-differentiated tumors. Because grade is only a surrogate for metastatic risk, finding a range is not surprising. Some moderately and poorly differentiated tumors metastasize, whereas others do not. However, very few well-differentiated tumors metastasize. In this group, not only were the overall TMEM counts low, but also the range of counts was small.
For completeness, we also looked at the staining patterns for Mena, macrophages, and endothelial cells in DCIS and nonneoplastic breast tissue. Not surprisingly, we found no TMEM in either tissue type. In benign breast tissue, there are no cancer cells, and in DCIS, the cells are noninvasive. The epithelial cells of DCIS and benign breast tissue are physically unable to appose perivascular macrophages due to an intact basement membrane as well as a layer of myoepithelial cells.
In the animal models, intravasation of tumor cells observed by direct imaging requires the direct interaction between invasive tumor cells and perivascular macrophages through a paracrine signaling loop (3
), and the presence of this microanatomic compartment is associated with the presence of intravascular tumor cell burden and metastasis (3
). In our study of human breast cancer samples, then, the next step was to evaluate TMEM in a case-control study of metastatic and nonmetastatic breast cancers. We identified 30 breast cancer patients with known distant metastases and 30 individually matched patients with breast cancer who had not developed metastasis from hematogenous spread. Using the same immunohistochemical triple stain, TMEM were quantified in the same fashion as for the case series of well, moderate, and poorly differentiated tumors. Well-differentiated tumors were not included due to lack of availability of metastatic samples (only two cases identified). For the series of matched pairs of moderately and poorly differentiated tumors, the difference in TMEM counts between metastatic and nonmetastatic tumors was substantial (median, 105 versus 50, respectively; P
= 0.00006). Our results indicate that TMEM density at initial cancer resection was associated with risk of metastasis. Specifically, for an increase in the TMEM count of 10, the odds of metastasis nearly doubled. The ability of TMEM density to predict systemic spread of carcinoma cells was independent of other currently used prognosticators, including lymph node metastasis, tumor size, presence of lymphovascular invasion, and tumor grade, although the sample size for this study precluded simultaneous adjustment for all of these factors.
Both the approach and the results in this study are novel. To date, no method for predicting metastatic risk exists that draws on the in vivo observation of hematogenous metastasis in animal models. These results suggest that the mechanism of hematogenous dissemination in humans is likely similar to that seen in rodent models where tumor cells intravasate in association with perivascular macrophages. A next step will be to validate these findings in a larger, independent population-based patient series with known outcome. If our results are substantiated, TMEM may be a powerful addition to the current approach for assessing metastatic risk and the need for systemic chemotherapy. If patients can be better classified as either low-risk or high-risk for metastasis, customized (patient-tailored) therapies can be designed to prevent overtreatment and undertreatment of patients, respectively.