Co-Culture of Myeloma and Endothelial Cells: Defibrotide Downregulates Heparanase and Pro-Angiogenic Growth Factors.
Authors: Echart Cinara , Distaso Maria , Ferro Laura , Boccadoro Mario , Palumbo Antonio , Mitsiades Constantine , Anderson Kenneth , Richardson Paul , Iacobelli Massimo 
Published: April 2020

Abstract
Introduction: Heparanase (HPSE) expression in humans has been associated with advanced progression and metastasis of many tumor types, including multiple myeloma (MM), where its activity is correlated with altered gene expression that may promote an aggressive tumor phenotype with high microvessel density (ref). These findings indicate an important role of HPSE in regulating metastasis, angiogenesis and progression of MM. Defibrotide (DF) is an orally bio-available polydisperse oligonucleotide with anti-thrombotic, pro-fibrinolytic, anti-adhesive and anti-angiogenic properties. Recently, we have shown that DF is able to downregulate HPSE gene expression and activity in MM cell lines (International myeloma workshop, Greece, 2007).
Methods: We investigated whether the expression of HPSE and angiogenic growth factors (FGF-2 and VEGF) in human microvascular endothelial cells (HMEC) are modified by co-culture with MM cells or by growing in the media of MM cells. In addition, we evaluated whether DF has activity in regulating the expression of HPSE, FGF-2 and VEGF in HMEC co-cultured with MM cells. We then tested the effect of DF on the invasiveness of MM cells activated with HPSE. HMEC cells were co-cultured with RPMI 8226 MM cells for 48h in the presence and absence of DF (at dose of 150μg/ml). HMEC was also grown alone for 48h in MM cell-conditioned media. The expression of HPSE and angiogenic growth factors (FGF-2 and VEGF) present in endothelial cells were examined through real time polymerase chain reaction (RT-PCR) of cDNA prepared from HMEC. Tumor invasion was evaluated using the BD BioCoat MatrigelTM invasion system (BD Bioscience).
Results: Coculture with RPMI 8226 cells substantially induced the expression of HPSE (7.2 fold) and angiogenic growth factors (3-5 fold) in HMEC compared with endothelial cells growing alone. DF was able to downregulate HPSE, FGF-2 and VEGF gene expression in HMEC co-cultured with RPMI 8226 cells (4.0; 6.0-8.0 fold, respectively). Surprisingly, addition of conditioned media in absence of MM cells resulted in 6.5 fold of elevation of HPSE but not growth factors expression in HMEC. These results show that components released by MM cells are sufficient to modulate HPSE. However, to alter growth factor expression, HMEC needed to be culture in the presence of RPMI 8226 cells. Additionally, HPSE increased the invasiveness of RPMI 8226 cells and DF was able to significantly decrease MM invasiveness in the Matrigel assay by 50% (p&lt;0.05).
Conclusion: Taken together, these results suggest that cross-talk between MM and endothelial cells leads to enhanced angiogenesis. HPSE is a key factor in this process, correlating with both angiogenic stimulus and MM progression. Moreover, DF is able to downregulate HSPE and growth factor expression in activated HMEC induced by MM, as well as reducing the invasiveness of MM in this system. These observations suggest a potent potential anti-tumor effect of DF and support further preclinical evaluation in MM models as well as ongoing clinical studies in this setting.