The Role of Megakaryocytes in Breast Cancer Metastasis to Bone.

Open Access
Author:
Jackson, Walter
Graduate Program:
Molecular, Cellular and Integrative Biosciences
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
April 08, 2015
Committee Members:
  • Andrea Marie Mastro, Dissertation Advisor
  • Andrea Marie Mastro, Committee Chair
  • Pamela Hankey Giblin, Committee Member
  • Mary J Kennett, Committee Member
  • Robert Paulson, Committee Member
  • Henry Joseph Donahue, Special Member
  • Melissa Rolls, Committee Member
Keywords:
  • Breast Cancer
  • Megakaryocytes
  • Bone metastasis
  • Thrombopoietin
  • Stromal derived factor-1
  • Platelets
  • Extramedullary Hematopoiesis
Abstract:
Megakaryocytes (MK) are large multinucleated cells found in the bone marrow and are the source of platelets. MKs represent less than one percentage of total bone marrow cells under normal conditions, but can increase several fold under stress. We had noted an increase in the number of MKs in the femurs of athymic mice with bone metastatic cancer four weeks following intracardiac inoculation of MDA-MB-231 human metastatic breast cancer cells. The increase in MKs was three to six fold higher than found in femurs of non-cancer bearing mice. In autopsy samples of bone from human metastatic breast cancer patients, we also saw an increase in MKs when age and gender matched patients without cancer were compared. Moreover, it has been reported that the largest cause of death among cancer patients is thromboembolism due to platelet aggregation. Thus, evidence suggested a relationship between MKs and metastatic cancer in the bone. MKs display an intimate relationship with many bone marrow cells especially osteoblasts (OB) and osteoclasts (OC). MKs differentiate from hematopoietic stem cells (HSC) that develop adjacent to OBs in the bone compartment known as the endosteal niche. The OBs serve as source for thrombopoietin (TPO), which is necessary for megakaryopoiesis. Once the MKs are mature, stromal derived factor-1 (SDF-1) guides them from the endosteal to the vascular niche to release platelets into the sinusoids. MKs also play a role in bone metabolism by inhibiting OC production and promoting OB proliferation. The interaction of metastatic breast cancer cells with OBs and OCs is key to the expansion of metastatic breast cancer cells in the bone and to bone degradation described in a series of events named the ‘vicious cycle’. Due to the lack of reports on MKs and bone metastasis, we asked if MKs played a role in the vicious cycle as well. This information taken together led us to propose that MKs are important contributors to the colonization of bone with metastatic breast cancer cells. We hypothesized that either the megakaryocytes precede the cancer cells and prepare a niche for them in the bone marrow, or that the metastatic cells in the bone marrow lead to an increase in cytokines in the microenvironment that bring about an increase in MKs. These two outcomes are not mutually exclusive. The objectives of this study were to determine if the increase in MKs precedes the growth of cancer cells in the bone; and determine the role that cancer cell-bone marrow cell interactions play in the up-regulation in production of MKs. Because MKs express von Willebrand factor (VWF), a primary antibody to VWF and an indirect immunohistochemistry (IHC) protocol were used to visualize the MKs. MK numbers in the femurs of athymic mice inoculated with MDA-MB-231-luc cells intracardiacally (metastastic model) were compared at several times following inoculation of the cancer cell. In addition mice with metastatic tumors were compared to those with the same cancer cells inoculated orthotopically (only primary tumor growth) to determine if the primary tumor systemically affected MKs in the absence of metastasis. Serum levels of TPO and SDF-1 and complete white blood counts were determined. There was a significant increase in MKs in the femurs of mice with metastatic disease, but not those with primary tumor growth only. The increase in MKs was not significant until about 30 to 40 days post cancer cell inoculation. No increase in platelet count, SDF-1, or TPO concentrations were detected at any time. The major conclusion was that the cancer cells preceded the increase of MKs in the bone marrow, and that primary tumor growth did not affect MK numbers. We next asked if this observation of increased megakaryopoiesis occurred in an immunocompetent mouse model. Immunocompetent BALB/c mice were injected in the mammary gland with mouse mammary carcinoma 4T1.2 (bone metastatic) or 67 NR (non-metastatic) cancer cells. There was no increase in MK numbers in the femurs of either group; however, a significant increase in number of MKs was seen in the spleens of the 4T1.2 injected mice indicative of extramedullary hematopoiesis. Again the data suggested that the cancer cells metastasized prior to the increase in MKs. For the third aim, we wanted to determine if metastasis decreased in mice with decreased numbers of MKs. We hypothesized that if MKs helped the tumor cells to colonize the bone, then bone metastases would be minimal. TPO KO mice had about a 90% reduction in MKs and platelets. Residual MKs were likely due to other cytokines involved in megakaryopoiesis. We inoculated TPO knock-out (KO) mice with 4T1.2 cancer cells. Contrary to our hypothesis, the TPO KO mice developed metastasis more quickly and at a higher rate than the wild-type and heterozygous geneotypes. In conclusion, it is likely that MKs are affected by an increase in cytokines and growth factors necessary for megakaryopoiesis. Those cytokines include: TPO, Interleukin-1 (IL-1), Interleukin-3 (IL-3), Interleukin-6 (IL-6), Interleukin-11 (IL-11), Granulocyte macrophage colony-stimulating factor (GM-CSF), Granulocyte colony-stimulating factor (G-CSF), and Stem cell factor (SCF). Many of these have been reported to increase in the bone microenvironment in the presence of cancer cells. The combination of IL-6, G-CSF, GM-CSF, and TPO are enough to manipulate an increase in MK numbers. We did not observe an increase in MKs in the TPO KO mice when injected with the 4T1.2, which suggests that TPO Is a major factor in megakaryopoiesis. Overall it appears that the presence of metastatic cancer cells in the bone marrow provokes megakaryopoiesis. The increase in MKs does not prevent metatastic colonization but a deficiency in MKs correlates with more aggressive metastatic colonization.