Therapy-induced modulation of tumor vasculature and oxygenation in a murine glioblastoma model quantified by deep learning-based feature extraction.

Glioblastoma presents characteristically with an exuberant, poorly functional vasculature that causes malperfusion, hypoxia and necrosis. Despite limited clinical efficacy, anti-angiogenesis resulting in vascular normalization remains a promising therapeutic approach. Yet, fundamental questions concerning anti-angiogenic therapy remain unanswered, partly due to the scale and resolution gap between microscopy and clinical imaging and a lack of quantitative data readouts. To what extend does treatment lead to vessel regression or vessel normalization and does it ameliorate or aggravate hypoxia? Clearly, a better understanding of the underlying mechanisms would greatly benefit the development of desperately needed improved treatment regimens. Here, using orthotopic transplantation of Gli36 cells, a widely used murine glioma model, we present a mesoscopic approach based on light sheet fluorescence microscopic imaging of wholemount stained tumors. Deep learning-based segmentation followed by automated feature extraction allowed quantitative analyses of the entire tumor vasculature and oxygenation statuses. Unexpectedly in this model, the response to both cytotoxic and anti-angiogenic therapy was dominated by vessel normalization with little evidence for vessel regression. Equally surprising, only cytotoxic therapy resulted in a significant alleviation of hypoxia. Taken together, we provide and evaluate a quantitative workflow that addresses some of the most urgent mechanistic questions in anti-angiogenic therapy.