Abstract C12: Platelets promote breast cancer metastasis by reprogramming tumor cells to produce IL-8
Authors: Johnson Kelly E. , Machlus Kellie R. , Forward Jodi A. , Tippy Mason D. , El-Husayni Saleh A. , Italiano Joseph E. , Battinelli Elisabeth M. 
Published: October 2016

Abstract
Platelets, primarily known for their role in hemostasis, are now recognized to play an integral role in cancer progression and metastasis. Recent evidence has established that platelets are activated by tumor cells, including breast cancer cells, leading to the release of hundreds of growth factors, cytokines, chemokines and angiogenesis mediators that could influence tumor growth and metastasis. Indeed, work from our group has demonstrated that factors released from activated platelets promote both metastasis and angiogenesis. However, little is known about the specific factors and signaling pathways that mediate this critical platelet-tumor cell crosstalk. To address this question, we performed an angiogenesis array (Ray Biotech) to identify specific pro-angiogenic and pro-metastatic factors released by tumor cells during platelet-tumor cell interactions. We identified several factors that were secreted by MCF-7 breast tumor cells in response to activated platelet releasate, including high levels of interleukin 8 (IL-8, CXCL8). IL-8 is a cytokine known to play a critical role in metastasis and angiogenesis and is elevated in the serum and tumor tissue of breast cancer patients. We confirmed that exposure to platelets strongly induced the production of IL-8 in several human breast cancer cell lines (MDA-MB-231, BT-20, SKBR-3 and MCF-7) by ELISA and found that platelets themselves do not contain detectable levels of IL-8. Furthermore, IL-8 production was highest in the more aggressive, triple negative MDA-MB-231 and BT-20 lines, suggesting a link between platelet-induced IL-8 and tumor subtype. Next we sought to determine the role of platelet-induced IL-8 in metastasis. We performed standard invasion assays using MDA-MB-231 cells transfected with IL-8shRNA or control cells. Platelets were able to increase the invasion of control MDA-MD-231 cells by 5 fold, while IL-8 knockdown reduced the effect by 50%. Furthermore, the ability of platelets to promote tumor cell migration across an endothelialized membrane was reduced 87% in IL-8 knockdown MDA-MB-231s compared to controls in standard transendothelial migration assays. These results suggest that platelets promote metastasis, in part, by driving tumor cell IL-8. To identify the specific component or components of platelet releasate responsible for driving tumor cell IL-8, we first characterized the contents of activated platelet releasate by array (Ray Biotech) and found an abundance of both chemokine (C-C motif) ligand 5 (CCL5, RANTES) and epidermal growth factor (EGF). Next, we treated breast tumor cell lines directly with recombinant CCL5 or EGF and observed an increase in IL-8 production; however, sensitivity to CCL5, EGF or the combination varied among the cell lines tested. We found that cell lines MCF-7 and MDA-MB-231, which express the CCL5 receptor CCR5, produced IL-8 in response to CCL5 while BT-20 and SKBR-3 cells produce IL-8 in response to EGF and express high levels of EGFR. To determine if platelet-derived CCL5 drives tumor cell IL-8 in MDA-MD-231 and MCF-7 cells, tumor cells were pretreated with the CCR5 blocker maraviroc and then exposed to platelets. CCR5 blockade abrogated the induction of IL-8 in response to platelets and decreased platelet-induced invasion. Similarly EGFR blockage with AG-1478 reduced IL-8 production in platelet-treated BT-20 and SKBR-3 tumor cells. Furthermore, pre-treatment of platelets with aspirin, an irreversible platelet inhibitor, diminished their ability to drive tumor cell IL-8 and to enhance invasion. Taken together, these results suggest that platelets, through release of soluble factors, drive tumor cells to produce IL-8 and that blocking this communication can disrupt the pro-metastatic potential of platelets. Ultimately, these studies support targeting specific platelet-tumor cell interactions as a novel means of limiting disease progression in breast cancer.
Citation Format: Kelly E. Johnson, Kellie R. Machlus, Jodi A. Forward, Mason D. Tippy, Saleh A. El-Husayni, Joseph E. Italiano, Jr., Elisabeth M. Battinelli. Platelets promote breast cancer metastasis by reprogramming tumor cells to produce IL-8. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C12.