The Role of Megakaryocytes in Breast Cancer Metastasis to Bone

Breast cancer frequently metastasizes to the skeleton. While examining sections of femurs from mice inoculated with metastatic breast cancer cells, we made the serendipitous observation that megakaryocytes were increased significantly compared with femurs of control mice. We propose to determine the role of megakaryocytes in the colonization of metastatic breast cancer cells in the bone. A search of the literature to uncover what was already known about the role that megakaryocytes may play in bone metastasis came up empty. However, we found various threads of information that when taken together indicate that megakaryocytes may be important. For example, megakaryocytes are the source of platelets which are known to contribute to the metastatic process; i.e., they release growth and angiogenic factors; they contribute to cancer cell escape from the primary tumor; they increase tumor cell adhesion. Also, an increase in the number of circulating platelets is a poor prognostic factor for metastatic breast cancer, and thromboembolism is a common cause of death for cancer patients. Megakaryocytes differentiate in the bone marrow near the osteoblasts that produce thrombopoietin, a differentiation factor. This niche is also where cancer cells home. In addition, megakaryocytes affect bone remodeling. They produce many characteristic molecules that regulate the osteoblasts and osteoclasts which deposit and degrade bone, respectively. Thus, the published information as well our observation led to the hypothesis that megakaryocytes contribute to growth of breast cancer cells in the bone either by preparing a niche and/or by responding to the cytokines that result from the interaction of the cancer cells with the cells of the bone microenvironment.

The specific aims are to (1) determine if the increase in megakaryocytes precedes the growth of cancer cells in the bone marrow, and (2) determine what role the osteoblast-cancer cell interactions play in the increase in megakaryocytosis. Regardless of the outcome of Aim 1, the cancer cell-modified microenvironment may lead to increased megakaryocyte differentiation and ultimately increased platelets. We will use two mouse models. In the first, human metastatic (or the control of low-metastatic) breast cancer cells will be inoculated in the heart of athymic mice to allow growth in the bone, or injected into the mammary gland, which allows growth but no metastasis. In the second model, an immunocompetent model, 4T1.2 (metastatic) or 67NR (non-metastatic) mouse mammary carcinoma cells will be injected into the mammary glands of Balb/c mice. The endpoints will be numbers of megakaryocytes over time in the femurs compared to the appearance of cancer cells, and/or growth of the primary tumors. Circulating levels of platelets, thrombopoietin, and other cytokines will be measured. In addition, some femurs will be crushed and cultured to release cytokines. Cytokines from the bone that may change prior to or in the presence of cancer cells include VEGF, IL-6, MCP-1, MIP-2, SDF-1, and KC. Finally, we will use a Balb/c thrombopoietin knockout mouse and 4T1.2 cells to ask if a reduction in megakaryocytes will block metastatic breast cancer. Depending on the results, we will also culture megakaryocytes with conditioned medium from breast cancer cells or from co-cultures of cancer cells and osteoblasts or other stromal cells to identify cytokines or growth factors that might be active in the bone microenvironment in the presence of breast cancer cells.

The result of this series of experiments will reveal the relationship between cancer cells and megakaryocytes in the bone niche. It may also point out new targets for therapy as well as suggest that current treatments for platelet reduction following chemotherapy be carefully considered.