Abstract 5020: Neutrophils in the bone marrow niche promote multiple myeloma chemoresistance
Authors: Ramachandran Indu , Lin Cindy , Condamine Thomas , Ozerova Maria , Garfall Alfred , Vogl Dan , Gabrilovich Dmitry , Nefedova Yulia 
Published: August 2015

Abstract
Purpose: The bone marrow (BM) niche provides a sanctuary for multiple myeloma (MM) cells, allowing them to escape chemotherapy. Previous in vitro studies have implicated some BM cell populations, including stromal cells, macrophages, osteoclasts and plasmacytoid dendritic cells, in MM chemoresistance. However, the role of the major cellular component of BM represented by immature and mature neutrophils (polymorphonuclear cells, PMNs) in MM survival from chemotherapy remains unknown. We therefore evaluated the role of PMNs in MM cell chemoresistance within the BM niche. Methods: We employed in vivo mouse models to modulate the number of myeloid cells in the BM of MM-bearing mice. To deplete CD11b+ myeloid cells, we generated CD11b-DTR BM chimeras and injected diphtheria toxin (DT) or a control toxin every 4 days after MM cell injection. To expand CD11b+ cells we overexpressed GM-CSF by hydrodynamic transfection of a GM-CSF expression plasmid 2 days before MM cell injection. Mice were then treated with doxorubicin or vehicle control and their survival was evaluated. For in vitro studies, Gr-1+CD11b+ mouse PMNs and PMN-myeloid derived suppressor cells (PMN-MDSCs) were isolated from the BM of tumor-free or MM-bearing mice respectively. CD33+CD14-CD11b+ human PMNs and PMN-MDSCs were FACS sorted from the BM of healthy individuals or MM patients, respectively. Results: Depletion of myeloid CD11b-expressing cells led to a significant reduction of MM tumor growth in these mice and improved antitumor effect of doxorubicin. Conversely, GM-CSF-induced expansion of myeloid cells in the BM of MM-bearing mice resulted in reduced survival and increased resistance to chemotherapy. Both mouse and human PMNs and PMN-MDSCs were able to protect mouse and human MM cells, respectively, from doxorubicin and melphalan-induced apoptosis in vitro. The addition of supernatant collected from these myeloid cells to MM cells was sufficient to provide tumor cell chemoprotection. In vitro, mouse MM cells induced PMNs to release DNA in the form of neutrophil extracellular traps (NETs). These NETs were efficiently taken up by tumor cells and contributed to PMN-induced chemoresistance in vitro, as addition of DNAse I cancelled this effect. No other myeloid cells, stromal cells or MM tumor cells were able to release DNA. In vivo, we observed a significant increase in circulating cell-free DNA in the blood plasma and BM plasma of MM-bearing mice, and in the BM plasma of MM patients compared to healthy individuals. Coculture of MM cells with PMNs or PMN-derived DNA induced the expression of the survival factor sXBP-1. Knockdown of sXBP-1 was sufficient to block the chemoprotective effect of DNA on MM cells. Conclusion: Our data demonstrate that PMNs contribute to MM chemoresistance by releasing their DNA, resulting in induction of sXBP-1 in tumor cells. Thus, targeting cell free DNA or sXBP-1 induction in conjunction with chemotherapy is a promising strategy for overcoming MM chemoresistance.
Citation Format: Indu Ramachandran, Cindy Lin, Thomas Condamine, Maria Ozerova, Alfred Garfall, Dan Vogl, Dmitry Gabrilovich, Yulia Nefedova. Neutrophils in the bone marrow niche promote multiple myeloma chemoresistance. [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 5020. doi:10.1158/1538-7445.AM2015-5020