Bevacizumab-induced tumor calcifications can be elicited in glioblastoma microspheroid culture and represent massive calcium accumulation death (MCAD) of tumor endothelial cells

Bähr and colleagues reported that 22 of 36 glioblastoma patients treated with bevacizumab showed tumor calcifications on 8 week post therapy follow up with MRI. Early tumor calcification strongly predicted for response, time to progression, and overall survival. The authors didn’t understand the mechanism, but speculated that it was vascular in nature. At the 13th International Anti-Angiogenic Symposium (2011), we presented our discovery of the phenomenon of massive calcium accumulation death, wherein MCAD occurred in endothelial cells (tumor, circulating, and HUVEC), in response to VEGF depletion by bevacizumab and other putative anti-angiogenic agents, but not in response to non-specific cytotoxins. In subsequent work, we have documented marked MCAD to occur in primary microcluster cultures from 6 fresh human glioblastoma biopsies, following 96 hours of VEGF depletion in vitro by bevacizumab. The presence and degree of MCAD is strikingly dependent on the type of serum in the culture medium (RPMI-1640 + 25% serum) -- typically most striking in (very low VEGF) fetal calf serum, but inhibited (often) or enhanced (rarely) by 25% human serum from different patients or normal donors containing variable quantities of VEGF. There was not a linear relationship between VEGF concentration and MCAD inhibition (or enhancement), suggesting that other pro-angiogenic (or anti-angiogenic) serum factors may play a role. In epithelial metastatic tumors, circulating peripheral blood endothelial cells may be easily tested, using our methods, and the serum inhibition (or, rarely, enhancement) is faithfully reproduced on circulating endothelial cells, in comparison with the tumor cluster-associated endothelial cells. We propose MCAD as the mechanism of glioblastoma calcification following bevacizumab and further propose that testing tumor microclusters and/or circulating endothelial cells, in the presence of autologous serum, could be a useful predictive biomarker and research tool