Abstract 2039: Direct interactions between multiple myeloma cells and osteocytes in the hypoxic myeloma microenvironment increase vegf-a production by osteocytes
Authors: Mulcrone Patrick L. , Petrusca Daniela N. , Condon Keith W. , Delgado-Calle Jesus , Roodman Garson D. 
Published: August 2019

Abstract
About 80% of multiple myeloma (MM) patients develop bone disease (MMBD), characterized by severe bone-destructive lesions, decreased bone formation, and increased marrow angiogenesis. Osteocytes, the most abundant cells in bone, regulate bone homeostasis and play a major role in MMBD. We previously found that osteocytes directly interact with MM cells, which induces bidirectional Notch signaling that fuels tumor growth, increases osteoclast resorption, and suppresses osteoblast activity. In addition, the MMBD tumor microenvironment is very hypoxic and facilitates angiogenesis to increase MM growth. Although osteocytes produce pro-angiogenic factors during mechanical load stress and in pro-inflammatory environments, it is unclear if osteocyte:MM interactions also contribute to enhanced bone marrow vascularization associated with MMBD. We hypothesize that hypoxia and direct interactions of osteocytes with MM cells increase pro-angiogenic signaling in osteocytes, which in turn may contribute to the enhanced vascularization in MMBD. We found that MLOA5 murine osteocyte-like cells cultured in 1% O2 (hypoxic) had increased expression of pro-angiogenic genes by qPCR array. Validation studies showed that Vascular endothelial growth factor-a (Vegf-a), a key protein for blood vessel formation, was increased at the mRNA and protein levels in hypoxic MLOA5s compared to normoxic cells. MLOA5s were then co-cultured with different MM cells for 24hrs in either normoxia or hypoxia, and murine Vegf-a levels were analyzed in the conditioned media (CM) from these co-cultures by ELISA. Co-culture of MM:MLOA5 in hypoxia induced greater than two-fold production of Vegf-a when compared to normoxic mono-culture and a 42-55% increase compared to normoxic co-cultures. In vivo, we detected an increased number of Vegf-positive osteocytes in 5TGM1 MM tumor-bearing mice compared to controls 8 weeks after inoculation. To test if elevated secretion of Vegf-a promoted blood vessel formation, CM from normoxic or hypoxic MLOA5s were collected and used to treat HUVECs grown on growth factor-reduced matrigel. Hypoxic CM increased HUVEC network branching by 56% at 8hrs when compared to normoxic CM. This increase was abrogated when a neutralizing antibody targeting murine Vegf-a was added to the hypoxic CM. Together, these results demonstrate that hypoxia and MM cells increase osteocyte production of Vegf-a, which supports angiogenesis in vitro. Thus, angiogenic signals from osteocytes may contribute to MM progression and resistance to therapy, and may be potential targets for the treatment of MM.
Citation Format: Patrick L. Mulcrone, Daniela N. Petrusca, Keith W. Condon, Jesus Delgado-Calle, Garson D. Roodman. Direct interactions between multiple myeloma cells and osteocytes in the hypoxic myeloma microenvironment increase vegf-a production by osteocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2039.