Activating KRAS mutations expressed in 3D endothelial spheroids induce blebbing morphologies associated with amoeboid-like migration

Highlights: What are the main findings? Endothelial spheroids expressing KRAS^G12V exhibit characteristic features associated with abnormal vessel development in arteriovenous malformations as well as novel phenotypes not previously observed in 2D monolayers. Expression of KRAS^G12V can induce blebbing morphologies associated with mesenchymal-to-amoeboid transitions (MAT) and amoeboid-like migration in brain endothelial spheroids after extended culture. What are the implications of the main findings? Amoeboid migration may play a role in the aberrant angiogenesis of KRAS-driven arteriovenous malformations or in resistance to inhibitors targeting mesenchymal migration alone. 3D brain endothelial spheroids transduced with adeno-associated viral constructs offer a novel platform to investigate the plasticity of driver mutations associated with arteriovenous malformations. 

Introduction: A 3D endothelial spheroid model expressing mosaic gain-of-function KRAS mutations was established to further understand the molecular changes associated with sporadic brain arteriovenous malformations (AVMs). Methods: Repellent 96-well U-bottom plates were seeded with human cerebral microvascular endothelial cells and resultant spheroids transduced with recombinant adeno-associated virus expressing KRAS^G12V. Spheroids were monitored using live-cell imaging for extended culture periods. Results: In the early growth period, KRAS^G12V expression increased spheroid growth rates and enhanced spheroid sprouting on gel matrices consistent with known AVM characteristics. With extended culture, novel endothelial characteristics were observed. KRAS^G12V-expressing spheroids displayed dynamic blebbing associated with the formation of rounded, hypertrophic cells disposed to engage in spheroid escape. These cells displayed reduced cell–cell adherence with rapid plasma membrane blebbing characteristic of amoeboid-like migration and mesenchymal-to-amoeboid transition. Spheroid growth and blebbing were reversed with MEK and mTOR inhibitors; Rho/ROCK inhibition specifically targeted the blebbing phenotype. Conclusions: Endothelial spheroids expressing KRAS^G12V exhibit characteristic features associated with abnormal vessel development in brain AVMs as well as novel phenotypes not previously observed in 2D monolayers. The ability to extend culture periods in this simple 3D model may allow further phenotypic exploration of important AVM driver mutations.