Position-Dependent Diversification of Glioblastoma Tumor Cells Governed by Adjacency to Blood Vessels

Abstract

Introduction

Uneven tumor vascularization and a resultant differential exposure to blood-borne substances and angiocrine factors renders proximity to blood vessels (BVs) an important yet underappreciated factor in non-genetic intratumor diversification. Unveiling the whole spectrum of genes, pathways and phenotypes impacted by adjacency to BVs has been hampered by the lack of a technology to sort tumor cells according to their relative distance from BVs.

Materials and methods

Here we developed a general methodology for retrieving stroma-free Glioblastoma (GBM) tumor cell subpopulations situated at progressively increasing distances from BVs based on differential uptake of the i.v. injected, freely-diffusing fluorescent dye Hoechst33342. This method could retrieve fully functional tumor subpopulations on the basis of their relative distance from blood vessels amenable to multiple –OMICs (transcriptomics, metabolomics) and functional analyses.

Results and Discussion

We show that GBM cells immediately juxtaposed to BVs are distinguished by a unique `anti-Warburg` metabolic profile. Exemplified for mTOR, we show that its activity is confined to tumor cells residing no further than a few cell layers away from the nearest BV, a region also marked by downregulated expression of mTOR inhibitors. A similar pattern was also demonstrated in surgically removed human GBM. Adjacency to BVs endows GBM cells with mTOR-dependent increased migration/invasion, tumor-initiating capabilities and higher chemo- and radioresistance.

Conclusion

The novel methodology proved instrumental for uncovering unappreciated level of metabolic compartmentalization and distinctive aggressive properties of perivascular tumor cells. These results uncover a previously unrecognized `privileged` narrow tier of perivascular cells distinguished by exceptional aggressiveness.