Rapid extravasation and establishment of breast cancer micrometastases in the liver microenvironment.

To examine the interplay between tumor cells and the microenvironment during early breast cancer metastasis, we developed a technique for ex vivo imaging of murine tissue explants using two-photon microscopy. Cancer cells in the liver and the lung were compared by imaging both organs at specific time points after the injection of the same polyomavirus middle T-initiated murine mammary tumor cell line. Extravasation was greatly reduced in the lung compared with the liver, with 56% of tumor cells in the liver having extravasated by 24 hours, compared with only 22% of tumor cells in the lung that have extravasated. In the liver, imaged cells continually transitioned from an intravascular location to an extravascular site, whereas in the lung, extravasation rates slowed after 6 hours. Within the liver microenvironment, the average size of the imaged micrometastatic lesions increased 4-fold between days 5 and 12. Histologic analysis of these lesions determined that by day 12, the micrometastases were heterogeneous, consisting of both tumor cells and von Willebrand factor-positive endothelial cells. Further analysis with intravenously administered lectin indicated that vessels within the micrometastatic tumor foci were patent by day 12. These data present the use of two-photon microscopy to directly compare extravasation times in metastatic sites using the same tumor cell line and highlight the differences in early events and metastatic patterns between two important secondary sites of breast cancer progression with implications for future therapy.

Establishment and quantitative imaging of a 3D lung organotypic model of mammary tumor outgrowth.

The lung is the second most common site of metastatic spread in breast cancer and experimental evidence has been provided in many systems for the importance of an organ-specific microenvironment in the development of metastasis. To better understand the interaction between tumor and host cells in this important secondary site, we have developed a 3D in vitro organotypic model of breast tumor metastatic growth in the lung. In our model, cells isolated from mouse lungs are placed in a collagen sponge to serve as a scaffold and co-cultured with a green fluorescent protein-labeled polyoma virus middle T antigen (PyVT) mammary tumor cell line. Analysis of the co-culture system was performed using flow cytometry to determine the relative constitution of the co-cultures over time. This analysis determined that the cultures consisted of viable lung and breast cancer cells over a 5-day period. Confocal microscopy was then used to perform live cell imaging of the co-cultures over time. Our studies determined that host lung cells influence the ability of tumor cells to grow, as the presence of lung parenchyma positively affected the proliferation of the mammary tumor cells in culture. In summary, we have developed a novel in vitro model of breast tumor cells in a common metastatic site that can be used to study tumor/host interactions in an important microenvironment.

Effect of ablation or inhibition of stromal matrix metalloproteinase-9 on lung metastasis in a breast cancer model is dependent on genetic background.

Matrix metalloproteinases (MMP) are a family of enzymes with a myriad of functions. Lately, we have come to realize that broad-spectrum inhibition of these enzymes, as was tried unsuccessfully in multiple phase III trials in cancer patients, is likely unwise given the protumorigenic and antitumorigenic functions of various family members. Here, we used the multistage mammary tumor model MMTV-PyVT to investigate roles for either MMP7 or MMP9 in tumor progression. We found no effect of genetic ablation of MMP7 or MMP9 on the multifocal tumors that developed in the mammary glands. Lack of MMP7 also had no effect on the development of lung metastases, suggesting that MMP7 is irrelevant in this model. In contrast, MMP9 deficiency was associated with an 80% decrease in lung tumor burden. The predominant cellular source of MMP9 was myeloid cells, with neutrophils being the largest contributor in tumor-bearing lungs. Experimental metastasis assays corroborated the role of host-derived MMP9 in lung metastasis and also facilitated determination of a time frame most relevant for the MMP9-mediated effect. The lung tumors from MMP9-deficient mice showed decreased angiogenesis. Surprisingly, the antimetastatic outcome of MMP9 ablation seemed to be dependent on strain. Only mice that had genetic background derived from C57BL/6 showed reduced metastasis, whereas mice fully of the FVB/N background showed no significant effect. These strain-specific responses were also observed in a study using a highly selective pharmacologic inhibitor of MMP9 and thus suggest that responses to MMP inhibition are controlled by genetic differences.

The other side of MMPs: protective roles in tumor progression.

The matrix metalloproteinase (MMP) family of extracellular proteinases have long been associated with cancer invasion and metastasis by virtue of their ability to collectively degrade all components of the extracellular matrix (ECM). The general belief that overexpression of a specific MMP, either by tumor cells or the surrounding stroma, is pro-tumorigenic led to the development of synthetic MMP inhibitors for the treatment of cancer. However, there is an increasing amount of literature demonstrating that the expression of certain MMPs, either at the primary or the metastatic site, provides a beneficial and protective effect in multiple stages of cancer progression. Here, we review the evidence for protective effects of MMPs and contrast this with pro-tumorigenic effects of either the same enzyme, or a different MMP of the same family. These studies highlight the importance of targeting specific MMPs for cancer treatment, and point to a potential reason why clinical trials of pharmaceutical inhibitors for MMPs were disappointing. In order to effectively target MMPs in cancer progression, a better understanding of both their pro- and anti-tumorigenic effects is required.

Matrix metalloproteinase 7 mediates mammary epithelial cell tumorigenesis through the ErbB4 receptor.

To delineate the role of matrix metalloproteinase 7 (MMP7) in mammary tumorigenesis, MMP7 was expressed in the normal murine mammary gland cell line, c57MG. MMP7 markedly enhanced the growth rate of the c57MG cells in three-dimensional culture and promoted tumor formation in vivo. Subsequent investigation showed that MMP7 (a) up-regulated ErbB4 receptor levels, (b) solubilized the ErbB4 receptor cognate ligand heparin-bound epidermal growth factor, and (c) mediated the proteolytic processing of ErbB4 to yield a soluble intracellular domain (ICD) that localized to the cytoplasm and the nucleus. Furthermore, overexpression of the ErbB4 ICD in the c57MG cell line recapitulated the proliferative effects of MMP7 in vitro and in vivo. These data indicate a novel mechanism for mammary epithelial cell transformation by MMP7.

The application of a murine bone bioreactor as a model of tumor: bone interaction.

A limited number of in vivo models that rapidly assess bone development or allow for the study of tumor progression in a closed in vivo environment exist. To address this, we have used bone tissue engineering techniques to generate a murine in vivo bone bioreactor. The bioreactor was created by implanting an osteoconductive hydroxyapatite scaffold pre-loaded with saline as a control or with bone morphogenetic protein-2 (BMP-2) to the murine femoral artery. Control and BMP-2 bioreactors were harvested and histologically assessed for vascularization and bone formation at 6 and 12 weeks post implantation. BMP-2 significantly enhanced the formation of osteoid within the bioreactor in comparison to the controls. To test the in vivo bone bioreactor as a model of tumor: bone interaction, FVB mice were implanted with control or BMP-2 treated bioreactors. After 6 weeks, an osteolytic inducing mammary tumor cell line tagged with luciferase (PyMT-Luc) derived from the polyoma virus middle T (PyMT) model of mammary tumorigenesis was delivered to the bioreactor via the femoral artery. Analysis of luciferase expression over time demonstrated that the presence of osteoid in the BMP-2 treated bioreactors significantly enhanced the growth rate of the PyMT-Luc cells in comparison to the control group. These data present a unique in vivo model of ectopic bone formation that can be manipulated to address molecular questions that pertain to bone development and tumor progression in a bone environment.

Breast tumors that overexpress nuclear metastasis-associated 1 (MTA1) protein have high recurrence risks but enhanced responses to systemic therapies.

Nuclear metastasis-associated 1(MTA1) protein is an estrogen receptor co-repressor that regulates transcription via chromatin remodeling, and MTA1 messenger ribonucleic acid (mRNA) levels are elevated in several kinds of locally advanced and metastatic tumors relative to non-metastatic tumors. Previous studies in our laboratory mapped MTA1 into a region showing significantly lower LOH (loss of heterozygosity) in primary breast cancers with metastases compared to node-negative tumors, suggesting that epigenetic alterations of MTA1 affect metastatic potential. The present study examined immunohistochemical expression of the MTA1 protein in treated and untreated primary human breast cancers to study the relationship between MTA1 expression and clinical outcome. Node-negative tumors that overexpress MTA1 protein had recurrence risks similar to node-positive tumors. In multivariate analysis of untreated node-negative tumors, highest expression of MTA1 was associated with increased relapse risk (hazard ratio (HR)=2.72, p=0.0003 for multivariate analysis). Tamoxifen and/or anthracylcene-based chemotherapies eliminated all MTA1 associations with clinical outcome, suggesting MTA1 overexpression predicts early disease relapse, but sensitizes breast tumors to systemic therapies.

MMP-7 promotes prostate cancer-induced osteolysis via the solubilization of RANKL.

We developed a rodent model that mimics the osteoblastic and osteolytic changes associated with human metastatic prostate cancer. Microarray analysis identified MMP-7, cathepsin-K, and apolipoprotein D as being upregulated at the tumor-bone interface. MMP-7, which was produced by osteoclasts at the tumor-bone interface, was capable of processing RANKL to a soluble form that promoted osteoclast activation. MMP-7-deficient mice demonstrated reduced prostate tumor-induced osteolysis and RANKL processing. This study suggests that inhibition of MMP-7 will have therapeutic benefit in the treatment of prostate cancer-induced osteolysis.