Abstract 4956: PrPc depletion in glioblastoma patient derived xenograft cells by CRISPR/Cas9

Abstract

Abstract Glioblastoma (GBM), the most common primary malignant brain tumor, exhibits a highly proliferative cell subpopulation called Glioblastoma stem-like cells (GSCs), presumed to contribute to the aggressive behavior of GBM. Our group has shown that the Prion Protein (PrPC), a cell surface glycoprotein and regulator of several stem cell functions, is enriched in GSCs and co-expresses with conventional GSCs markers. PrPC has been proposed as a scaffold protein, able to govern the assembly of multi-component complexes in the cell membrane. Integrin α6 (ITGA6), a cell surface laminin receptor, is enriched in GSCs as well, and exhibits relevant effects on GSCs biology, related to self-renewal and migration. To investigate a potential association between PrPC and ITGA6, we will perform loss-of-function experiments in GSCs via siRNA and CRISPR/Cas9 to characterize these cell surface molecules expression and their effect on GSCs proliferation, migration and invasion. Preliminary results show that depletion of PrPC perturbates ITGA6 labeling on plasma membrane of GSC. Thus far, cells from patient-derived xenografts (GBM33-PDX2) were transfected a construct for PrPC knockout by CRISPR/Cas9 via Nucleofector Technology. We found around 60% GFP-positive cells via flow cytometry for the CRISPR pool and we are currently isolating clones to confirm PrPC expression. Remarkably, the colonies exhibited distinct morphologies such as neural-like, epithelial-like and stem cell-like, indicating the heterogenous nature of this tumor. Western blotting assays will be performed to assess PrPC and ITGA6 expression in both commercial cell line U87 and GBM33-PDX2. The characterization of PrPC-ITGA6 association might provide insight into GSCs behavior and new anti-GBM therapeutic strategies. Citation Format: Maria Isabel Melo Escobar, Barbara Paranhos Coelho, Mariana Brandão Prado, Rebeca Iglesia, Marilene Hohmuth Lopes. PrPc depletion in glioblastoma patient derived xenograft cells by CRISPR/Cas9 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4956.