Understanding Hematogenous Breast Cancer Metastasis: Tumor Cell Integrin Activation Matters.

Metastatic disease is the leading cause of morbidity and mortality in patients with breast cancer. Elucidating the mechanisms that govern the hematogenous spread of cancer cells would provide novel targets for the development of new therapeutic strategies. Our group has previously demonstrated that the activated form of integrin specifically promotes breast cancer metastasis. Here, we have investigated the mechanisms by which activated av33 directly impacts defined steps in hematogenous dissemination of cancer cells in vivo. The lung colonization assay was used as a model to analyze tumor cell arrest in the vasculature, extravasation and secondary tumor cell growth at the target site. To address a role of the activation state of adhesion receptor integrin av33 in these steps of the metastatic cascade, we genetically tagged variants of the MDA-MB 435 human breast cancer cell model with dsRed2 or luciferase, and followed cells that express either non-activated 33wild type (WT) or constitutively activated mutant 33D723R, upon injection into the circulation of SCID mice. The presence of the cells and metastatic burden was analyzed by fluorescence imaging of lung whole mounts, supported by real time PCR quantification of human Alu sequences, and by non-invasive whole body imaging based on bioluminescence detection. Within 24 hours after injecting 1 million tumor cells into the tail vein, 90% of 33D723R expressing cells and 99.5% of 33WT expressing cells were cleared from the lungs. In both cell variants, the cleared cells undergo apoptosis as shown by immunohistochemical analysis. However, significantly more 33D723R than 33WT expressing cells consistently arrested and survived in the pulmonary vascular bed. A hallmark of constitutively activated integrin av33 is the ability of the receptor to bind soluble plasma protein ligands and thereby promote tumor cell interaction with platelets, a mechanism that supports tumor cell arrest during blood flow in-vitro. The contribution of platelets to the initial steps of tumor cell target organ colonization was evaluated in mice with experimental thrombocytopenia compared to mice with normal platelet counts. Following initial tumor cell arrest, 33D723R expressing cells showed a significantly shorter lag phase and enhanced proliferation than 33WT expressing cells, and this further determined the development of metastatic foci in the lungs. Slower growth of 33D723R expressing cells in-vitro indicates a specific advantage that these cells have in the microenvironment of the lung tissue. For both cell variants, target organ colonization was restricted to the lungs, as demonstrated using imaging of bioluminescence signal. Thus, we demonstrated that the activated form of integrin av33 confers an advantage on breast cancer cells at two distinct steps within the metastatic cascade. The first step is specific arrest within the pulmonary vascular bed, followed by a critical second step where expression of activated av33 is associated with increased tumor cell survival and a faster transition from a lag phase to rapid proliferation leading to the successful generation of multicellular lesions in the lungs and the establishment of clinically relevant metastatic disease.