Abstract 4305: Dissecting the requirements for astrocytoma and invasion using genetically-engineered mouse models
Authors: Vitucci Mark , Huff Byron , Bash Ryan E. , Karpinich Natalie O. , Schmid Ralf S. , Miller C Ryan 
Published: August 2012

Astrocytomas are characterized by diffuse invasion, precluding their complete surgical resection. PTEN, a negative PI3 kinase (PI3K) pathway regulator, is altered in 40-80% of high-grade astrocytomas (HGA), including glioblastoma (GBM). However, its role in astrocytoma invasion remains unclear. Primary astrocytes from six genetically-engineered mouse (GEM) models, with conditional alleles that inactivate Rb (T) and/or Pten (P) and/or constitutively activate Kras (R, KrasG12D) upon Cre recombination, were used to analyze PI3K pathway signaling, proliferation, migration, and invasion in vitro by immunoblot, cell counting, wound healing and time-lapse video microscopy, and collagen invasion, respectively. Gene expression microarrays were used to compare the transcriptomes of GEM astrocytes to human HGA. Tumorigenicity and survival were determined in vivo in orthotopic allograft models. Invasion was assessed by morphometric analysis. Pten ablation increased levels of phospho-Akt and phospho-S6. In cells with both Rb inactivation and Kras activation (TR), complete inactivation of Pten shortened doubling time (DT) by 42% (P&lt;0.01), increased single cell migration velocity and wound healing closure 42% and 84%, respectively (P&lt;0.0001), and increased collagen invasion 68-fold (Pα0.0001). Both the proliferation and migration effects were rescued by transient transfection with wild-type murine Pten. In cells lacking both Rb and Pten (TP), activated Kras shortened DT 73% (P&lt;0.01), increased velocity 44% and wound closure 94% (P&lt;0.0005), and had no significant effect on invasion. Unsupervised gene expression analysis showed that transformed astrocytes (nα3 unique isolates per genotype) primarily cluster into three groups consisting of TR; TRP+/− and TRP-/-; and T, TP+/− and TP-/-. TRP+/− - TRP-/- astrocytes were similar to human mesenchymal HGA based on gene set analysis (P=0.043). In vivo, 105 TRP-/-, TRP+/−, TP-/-, and TR cells produced HGA in 9/19, 6/10, 14/18, and 6/10 animals, respectively. Median survivals were 31, 78, 75, and 207 d, respectively. TRP-/- tumors were 202% more invasive than TR tumors (P=0.0062) and 42% more invasive than TP-/- tumors (P=0.315). We conclude that Pten ablation significantly affects proliferation and migration of astrocytoma cells, both in vitro and in vivo. Tumor incidence and survival in the allograft is correlated with in vitro invasiveness. In future studies, the orthotopic allograft model system and morphometric analyses described herein can facilitate analysis of the genetics of mesenchymal HGA invasion in vivo. Use of alternative initiating genetic events in this model system well help define the requirements for development of other HGA human subtypes and provide a facile platform for use in preclinical studies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4305. doi:1538-7445.AM2012-4305