Uncovering genomic rearrangements in Glioblastoma Multiforme (GBM) using super-resolution imaging
Introduction
Glioblastoma Multiforme (GBM) is the most common type of malignant primary brain tumor as well as one of the most aggressive, with an average patient survival time of around 15 months. Typical cancer therapeutics have not shown much success in the treatment of GBM, which may in part be due to the high degree of inter- and intra-tumor heterogeneity associated with this type of cancer. With recent studies beginning to highlight the important role genome organization plays in cancers, it is unknown how this organization plays a role in the large degree of heterogeneity seen in GBM. Here, we aim to use super-resolution microscopy to elucidate rearrangements in the 3D organization of oncogenic loci in the genome of patient-derived glioma stem cells (GSCs) and how this may be contributing to tumor heterogeneity as well as the increased expression of these oncogenes.
Methods
A DNA FISH library of Oligopaint probes was designed specific to the epidermal growth factor receptor (EGFR) locus for use in SMLM imaging. The library spans a 2 Mb region with EGFR being located roughly in the center of the library. Additionally, an RNA FISH Oligopaints library for EGFR mRNA transcripts was designed to visualize EGFR expression in the same single cells as DNA FISH. This will be done in normal microglia (HMC3) and astrocyte (HA) cells to serve as a controls, and will then be done in GSCs derived from patient GBM samples to identify structural changes that may be contributing to higher expression levels of EGFR. Eventually this methodology will be expanded to other oncogenes that we expect will be upregulated in our specific patient cell lines.
Results
Preliminary images have been acquired for the entirety of the DNA FISH library in the control HMC3 cell line and cluster analysis has begun. Cluster analysis was run on Vutara SRX software using DBSCAN to identify the regions of interest. We have been able to image both RNA and DNA FISH successfully on the same cells in widefield, and have been able to image a single step of the DNA FISH library in super-resolution. Experiments that combine RNA FISH widefield imaging with our protocol for tracing of the entire 2 Mb DNA FISH region in super-resolution are currently in progress.
Conclusion
Our protocols for library design/amplification and SMLM imaging are working well in our control cell line (HMC3). This process will then be repeated on the patient-derived GSC lines and these results will be compared to the controls to identify any significant changes seen in the cancer cells. We hypothesize that the changes seen in the GSCs will not only correlate to the expression patterns of oncogenes, but will also allow us to identify tumor cell subpopulations that are known to occur in GBM.