Megakaryocyte Reprogramming in Breast Cancer
Authors: Roweth Harvey G. , Malloy Michael , Goreczny Gregory , Johnson Kelly E. , McAllister Sandra S. , Italiano Joseph E. , Battinelli Elisabeth 
Published: February 2020

The first association between platelets and cancer dates back to the 19th century, where unexpected blood clots were perceived as a potential forewarning of occult malignancy. More recently, platelets have been shown to promote the progression of solid tumors through diverse mechanisms, including stimulation of angiogenesis, protection from immune surveillance, and facilitating cancer cell intravasation and arrest in the microvasculature.
While several have studied the interplay between platelets and cancer, how cancer affects megakaryocytes (MKs) remains uncharted. The number of platelets and their content is predominantly determined by MKs, which are responsible for platelet production and protein packaging. Despite this, very little is known about how the bone marrow environment regulates platelet production by MKs, or how such processes are altered in disease states. We hypothesize that in cancer, tumor-derived factors reprogram MKs to upregulate pro-metastatic and pro-neovascularization proteins for delivery to subsequent platelets, ultimately producing platelets with a more "pro-malignant" phenotype.
We first interrogated this question by exposing fetal liver-derived murine MKs (FLMKs) to the releasate of a triple-negative human breast cancer line (MDA-MB-231) for 24 hours. MKs were then lysed and processed for liquid chromatography-mass spectrometry (LC-MS) and bulk proteomic analysis. Several pro-metastatic proteins were upregulated in these MKs, most notably metalloproteinases (MMP2, MMP3), which play important roles in cell invasion and are secreted by platelet alpha granules. We further interrogated MMP expression by exposing MKs expanded from human placental cord-derived hematopoetic stem cells to MDA-MB-231 releasate, followed by specific probing for MMP and tissue inhibitor of metalloproteinase (TIMP) expression. Our findings show marked increases in MMP1 (3-fold), MMP2 (3.75-fold), MMP3 (3-fold), MMP9 (two-fold), MMP13 (2.2-fold) and TIMP1 (7.8-fold) compared to MKs exposed to control media. These initial experiments demonstrate that factors secreted by tumor cells can alter levels of cancer-promoting proteins in MKs. Whether such changes arise from direct endocytosis, or through altered protein synthesis by the MK remains an important question.
Our next aim was to assess if cancer affects MK protein content in vivo. To test this hypothesis, we isolated bone marrow-derived MKs from mice bearing mammary tumors following orthotopic injection of the triple-negative murine breast cancer cell line, Met-1. MKs were sorted from other bone marrow cells based on size (10-50 µm) and integrin ⍺IIbβ3 (glycoprotein IIb/IIIa, CD41/61) surface expression. Samples were then subject to LC-MS and bulk proteomic analysis. Blinded hierarchal clustering clearly segregated samples of tumor-bearing and non-tumor-bearing mice, with statistically significant differences observed for 182/1046 identified proteins. Ingenuity Pathway Analysis releveled that 41 of these significant proteins whose expression was increased are directly associated with cancer, most notably S100A8, Cathepsin G and Lipocalin-2. These data highlight clear alterations of MK protein expression in cancer. Subsequent experiments will assess if these factors are also increased in the platelet progeny and can be released following tumor-mediated platelet activation to augment disease progression.
Finally, we wanted to determine what tumor-derived factors are responsible for altered MK protein expression in breast cancer. We found that plasma levels of several proinflammatory cytokines were increased in our tumor-bearing model, including G-CSF (14-fold) and IL-6 (1.6-fold), which have previously been reported to increase myeloid expansion and thrombopoietin production, respectively. Further studies will be taken to determine if these and/or other factors can alter MK protein content in isolation and if changes in the MK can be reversed through inhibition of these cytokines.
In summary, our findings demonstrate that soluble factors released from a primary tumor are able to modulate the bone marrow niche and reprogram MKs, resulting in the increased expression of several proteins known to promote cancer. Future work will explore if agents that affect platelet production and/or function can reverse the MK reprogramming in cancer.

Disclosures
Italiano: Ionis Research Funding: Research Funding; Platelet Biogenesis: Employment, Equity Ownership.