Abstract 1176: Tumor derived peptide from a2 isoform of Vacuolar ATPase immunomodulates tumor associated neutrophils

Abstract

Abstract It has been recently discovered that tumor associated neutrophils (TAN) develop N2 polarization similar to tumor associated macrophages (TAM). It is believed that the tumor microenvironment can provoke the wound repair response from TAN and TAM which in turn promotes tumor progression. Transforming growth factor- β (TGF-β) plays a key role in N2 polarization, however, other regulatory mechanisms are still unclear. a2 isoform Vacuolar ATPase (a2V) are highly expressed on tumors and macrophages. Recent evidence showed that a cleaved peptide from the N-terminal domain of a2V (a2NTD) is secreted in the microvesicles collected from various tumor cell lines. This peptide promotes the pro-tumorigenic characteristics in macrophages. Moreover, priming neutrophils with tumor conditioned media (TCM) collected from breast cancer cell lines promotes the pro-tumorigenic properties in neutrophils. In this study, we are evaluating the in vitro immune-modulatory effect of a2NTD on polymorphnuclear leukocytes (PMN) in order to explore the role of the a2V in the sterile inflammation associated with tumor progression. We isolated PMN from heparin anti-coagulated blood collected from healthy adult volunteers. TCM were collected from MDA-MB-231 breast cancer cell line. PMN were stimulated with either recombinant a2NTD or with TCM to simulate the tumor microenvironment. In both a2NTD and TCM cultures, distinctive morphological changes were revealed by microscopical examination and significant elevation of ROS levels were detected by flow cytometry (p-value <0.05). Moreover, we have observed an upregulation in PMN gene expression of CCL2, CXCL-1, CXCL-2, TNFα, VEGF, MMP-9, neutrophil elastase as well as a2V when it is cultured with either a2NTD or TCM using quantitative real time PCR. In accordance with Queen M., et al, flow cytometry showed delayed PMN apoptosis in cultures with TCM while upon inhibiting V-ATPase, PMN survival decreased significantly (p-value <0.05).This suggests that the delayed apoptosis observed in TCM primed PMN is mediated at least partially by V-ATPase. In addition, a2NTD increased significantly the PMN survival but to a lesser extent compared with TCM. Interestingly, we observed significant surface over-expression of a2V on PMN in both cultures using flow cytometry (p-value <0.05) and confocal microscopy. Our study demonstrates the potential immune-modulatory effect of tumor associated a2NTD on PMN which may indicate its involvement in tumor progression. Citation Format: Safaa A. Ibrahim, Magdy Amin, kenneth D. Beaman. Tumor derived peptide from a2 isoform of Vacuolar ATPase immunomodulates tumor associated neutrophils. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1176. doi:10.1158/1538-7445.AM2014-1176