The team, led by Dr. Frank Attenello, neurological surgeon with Keck Medicine of USC, developed and leveraged a CRISPR interference (CRISPRi) library to transduce glioblastoma cells before in vivo implantation.
Glioblastoma (GB) is the most common primary brain tumor in adults, with a mean survival rate of just over one year.1 Although advancements in glioblastoma treatment have prolonged the quality of survival for GB patients, existing therapies have limited efficacy. Studies have attempted to identify genes with alterations in mutation and gene expression that can serve as novel diagnostic and therapeutic targets. However, numerous barriers exist to identifying novel treatment targets, including the complex nature of cancer development, tumor cell heterogeneity, marker-based differential treatment response, and limited knowledge about novel new gene classes, such as long-noncoding RNAs (lncRNAs) and their phenotypes.[1]
CRISPR (clustered regularly interspaced short palindromic repeat)-based genetic screens are powerful tools for systematic and specific evaluation of phenotypes associated with specific target genes. CRISPR has been used in vitro to identify functional coding and noncoding genes in various cell types, including glioblastoma; these genes are ultimately validated in vivo. However, no studies have described the use of CRISPR screening in GB in vivo.
A multidisciplinary team of physician-scientists at Keck Medicine of USC utilized an in vivo CRISPR screening methodology to identify noncoding genes affecting GB growth within the mouse cerebral cortex. The team, led by Dr. Frank Attenello, Department of Neurological Surgery, Keck School of Medicine, developed and leveraged a CRISPR interference (CRISPRi) library to transduce glioblastoma cells before in vivo implantation. Then, the team systematically evaluated whether repression of certain GB- specific noncoding RNA candidates decreased the proliferation of GB cells implanted with the mouse cortex. Future investigations can leverage their CRISPRi protocol to systematically evaluate additional GB candidates as they engraft and proliferate within their native cortical microenvironment.
In vivo CRISPR screening for novel noncoding RNA functional targets in glioblastoma models
Various studies have implemented CRISPR screens across various tumors to identify novel genes with functional relevance in phenotypes ranging from tumor growth and invasion to chemoresistance. Recent studies have employed CRISPR screens to evaluate GB phenotypes, including growth and invasion, as well as chemotherapy resistance via CRISPR and CRISPRi screening in vitro. While these GB CRISPR screening studies have been successful in vitro, similar advancements have not been made in vivo.
Attenello, who is also a member of the USC Norris Comprehensive Cancer Center, and his team published a manuscript outlining their protocol for targeting and transcriptionally repressing GB-specific lncRNAs by CRISPR interference in vivo with tumor growth in the mouse cerebral cortex. They detail the transduction of a pool of CRISPRi screening plasmids into GB tumor limes, maintenance of adequate coverage during cortical injection, adequate in vivo maintenance, and subsequent isolation of transduced screen tumor cell populations for analysis. Furthermore, they describe their straightforward method of fluorescence-guided tumor resection, gentle dissociation, and collection of viable, tumor-positive, library-positive cells for bioinformatic analysis.
After successfully screening for candidate lncRNAs, Dr. Attenello's team investigated whether the in vivo screen could discern between essential and nonessential lncRNAs. To do this, they injected a 1:1 (HOTAIR essential knockdown: control nonessential lncRNA knockdown) mixture of fluorescently tagged U87 GB cells into the cortex of eight mice. Then, and they evaluated the selective depletion of HOTAIR-tagged cells at 14 days.1 Attenello and his collaborators used flow cytometry to analyze the fluorescently tagged populations for hiBFP (control knockdown) and green fluorescent protein (HOTAIR knockdown), revealing a 17% (p = 0.007) decrease in fluorescence associated with HOTAIR knockdown relative to control.1 These results suggest that the in vivo CRISPR screening methodology used by Dr. Attenello and his team can effectively identify and discern between coding and noncoding genes affecting GB growth within the mouse cortex. Future applications of Attenello's in vivo screening methodology will allow for systematic and high-throughput evaluation of functional GB phenotypes, which can serve as diagnostic and therapeutic targets.
About USC Neurological Surgery at Keck Medicine of USC
USC Neurological Surgery at Keck Medicine of USC has a long tradition of scientific innovation and excellence. The program combines clinical expertise, compassionate care, and state-of-the-art techniques to deliver outstanding neurosurgical services to its patients. Located in an academic medical center, USC Neurological Surgery pursues robust clinical and basic evidencebased research, ensuring that Keck Medicine contributes to advances in the field of neurological surgery.
Keck Medicine values its relationships with referring physicians and collaborates to deliver responsive and compassionate care to a diverse patient population in Los Angeles and beyond.
[1] Attenello, FJ, Tsung, K, Bishara, I, Loh, Y-1-IE, Chen, TC. In vivo CRISPR screening for novel noncoding RNA functional targets in glioblastoma models. J Neurosci Res. 2021; 99: 2029-2045. https://doi.org/10.1002/jnr.24850
