Little is known about how megakaryocytes may affect metastasis beyond serving as a source of platelets. In this study, we explored the functional implications of megakaryocyte accumulation in the femurs of mice after injection of metastatic or nonmetastatic breast cancer cells in 4T1.2 BALB/cJ and MDA-MB-231 nude mouse models. At bone metastatic sites, but not primary growth sites, tumor growth was associated with increased megakaryopoiesis in both model systems. In the orthotopic BALB/cJ model, extramedullary hematopoiesis occurred in the spleen, resulting in a four-fold increase in megakaryocytes. In support of the hypothesis that reducing megakaryocytes may reduce metastasis, we found that thrombopoietin-deficient mice exhibited a 90% relative decrease in megakaryocytes, yet they developed more aggressive metastasis than wild-type hosts. In human clinical specimens, we observed an increase in megakaryocytes in the bone marrow of 6/8 patients with metastatic breast cancer compared with age-and gender-matched controls. Taken together, our results suggested that an increase in megakaryocytes occurring in response to metastatic cells entering the bone marrow confers some measure of protection against metastasis, challenging present views on the role of megakaryocytes in this setting.

Bone is one of the preferred sites for breast cancer cells to metastases. Once patients are diagnosed with skeletal metastases, the relative five-year survival rate falls from 90% to less than16%. Thrombocytosis is a sign of poor prognosis for survival, and thromboembolisms are one of the leading causes of death in all cancers patients. These conditions are due to high numbers and/or high platelet activity. Megakaryocytes (MKs) are responsible for platelet production, but also play a role in bone metabolism and skeletal homeostasis. We had determined in two mouse models of experimental metastasis (MDA-MB-231 in athymic mice, and 4T1.2 in Balb/c mice), that bone metastasis was associated with increased numbers of MKs. Under normal conditions MKs represent less than 1% of the total bone marrow cell population, but after analysis of the femurs of the mice with metastasis, we found at least a two-fold increase in the numbers of MKs present in the bone marrow in the MDA-MB-231mice. The Balb/c mice injected in the mammary glands with mouse 4T1.2 cells showed a four-fold increase in MKs in the spleen (extrameduallary hematopoiesis). Therefore we hypothesized that increased megakaryocytosis was linked to metastasis, and that decreasing the number of MKs would decrease metastasis. We transferred a thrombopoietin (TPO) expression deletion mutation to a Balb/c background and inoculated the mice with 4T1.2 cells. These TPO -/- mice showed approximately a 90% decrease in bone marrow MKs compared to the wild type mice. The numbers of blood platelets was also decreased by 90%. We tested our hypothesis by inoculation of 4T1.2LUC cells into the mammary gland, and then followed the course of the cancer and metastasis by luciferase imaging. Contrary to our hypothesis the TPO -/- mice developed metastasis to the bone more rapidly than the heterozygotes or wild type mice. Literature suggests that platelets interact with tumor cells to contribute to hematogenous metastasis and angiogenesis. In the TPO-/- mice with very few platelets, metastasis was more aggressive and developed more rapidly than in the wild type or the heterozygote mice. The next step will be to separate the role of the megakaryocytes from the platelets. This work was sponsored by the U.S. Army Medical Research and Materiel W81XWH-10-1-0253 Citation Format: Walter Jackson, Andrea M. Mastro, Donna M. Sosnoski. Thrombopoietin and megakaryocytes in breast cancer metastasis to bone. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3265. doi:10.1158/1538-7445.AM2015-3265

Breast cancer cells frequently metastasize to bone. Despite the clinical ramifications, very little is understood about the fundamental mechanisms responsible for this phenomenon. While analyzing femur sections from mice that had been inoculated in the left ventricle of the heart with human metastatic MDA-MB-231 breast cancer cells, we saw a several fold increase in the number of megakaryocytes (MKs) in the bone marrow. We hypothesized that either MKs aid in the growth of cancer cells in the bone by preparing a niche for the metastases or that the increase resulted from the altered microenvironment of the marrow due to the presence of the cancer. MKs are the pre-cursors to platelets and normally represent a small portion of all bone marrow cells. However, they can increase exponentially under stressful conditions such as primary thrombocythemia and in chronic myelogenous leukemia. Platelets are known to interact with tumor cells to contribute to hematogenous metastasis and angiogenesis. Thrombocytosis, high platelet count, is a poor prognostic factor for breast cancer metastasis; and moreover, many cancer patients die from thromboembolisms. MKs also play a crucial role in bone metabolism and skeletal homeostasis. Objectives: to determine (1) if the increase in MKs precedes the growth of cancer cells in the bone; (2) the role that cancer cell-osteoblast/stromal interactions play in the increase in megakaryopoiesis. For aim 1, MK numbers in the femurs of athymic mice inoculated with MDA-MB-231-luc intracardially (metastasis) were compared with those inoculated in the mammary gland (no metastasis). Serum was assayed for key MK maturation factors thrombopoietin (TPO) and stromal-derived factor-1(SDF-1). There was on average a two-fold increase in MK numbers in the femurs of mice with metastasic disease. However, this increase was not found in mice with localized growth but no metastasis. The increase correlated with the increase in metastatic tumor burden. There was no increase in platelet count, SDF-1, or TPO even when MK numbers were high. We concluded that the cancer cells preceded the increase of MKs in the bone marrow in a xenograft model. A syngeneic model of Balb/c mice with 4T1.2 cancer cells that metastasize from the mammary gland and 67NR cells that do not are also being compared. Thus far we have found that femurs of mice carrying 4t1.2 cells for 30 days also showed an increase in the numbers of MKs. Finally, Balb/c mice lacking TPO and thus having low MKs will be tested for metastasis. This work was supported by the U.S. Army Medical Research and Materiel Command under W81XWH-10-1-0253. Citation Format: Walter Jackson, Andrea M. Mastro. The role of megakaryocytes in breast cancer metastasis to bone. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3936. doi:10.1158/1538-7445.AM2013-3936