Rapid and Specific Diagnosis of Astrocytomas with Fluorescent Aptamers

Abstract

Introduction Neurosurgical tumor resections are routinely assisted by intraoperative histological assessment of biopsies. However, it is difficult to differentiate, via gross appearance or frozen section analysis, high-grade gliomas from non-operative lesions, such as primary central nervous system lymphoma (PCNSL). Aptamers are an emerging class of polynucleotides that function as “chemical antibodies” with high binding affinity to specific cellular targets. They can identify samples in minutes compared to the typical 24-to-48-hour timeframe via immunohistochemistry (IHC). We have previously developed a PCNSL-specific aptamer and aim to develop a fluorescent aptamer targeting glial fibrillary acid protein (GFAP), a common IHC target for gliomas.

Hypothesis: GFAP aptamers can accurately identify glioma cell lines in twenty minutes, a timeframe that can support intraoperative decision-making for best treatment practices.

Objective: Develop a GFAP aptamer to utilize as an intraoperative assay to diagnose glioma tumors.

Methods Our data was obtained from human glioma cells (U251) and human PCNSL (Ramos) cell lines. We demonstrate a CD20-specific aptamer labeling protocol to rapidly identify Ramos cells and preliminary data which demonstrates a GFAP aptamer’s ability to label U251 cells. Cell lines were cultured with their appropriate mediums and grown at 37C at 5% CO2. Aptamers had AF488 and Cy5 fluorophores for the CD20 (TD05) and GFAP aptamers, respectively. Annealed aptamers were incubated on ice for 20 min, treated with fixation/permeabilization, and tested at different concentrations. For comparison, we ran positive and negative control validation experiments for each aptamer individually and a mixed aptamer assay with analysis of fluorescence via flowcytometry.

Results We found a concentration of 400nM or higher of both the TD05 and the GFAP aptamers were highly sensitive and appropriate for labeling Ramos and U251 cells, respectively. When gating baseline fluorescence of non-stained cells, 100% of cells at all aptamer concentrations greater than 400nM showed AF488 fluorescence for Ramos cells stained with TD05 aptamer and Cy5 fluorescence for U251 cells stained with the GFAP aptamer. We unexpectedly found Cy5 fluorescence with 100% of Ramos stained with GFAP aptamers, AF488 fluorescence with 37.3% of U251 stained with TD05 aptamers. With a mixed aptamer assay, we found Cy5 fluorescence in 99.8% of U251 stained with GFAP aptamers and AF488 fluorescence in 97.6% of Ramos stained with TD05 aptamers. We found Cy5 fluorescence in 78.7% of Ramos stained with GFAP aptamers and AF488 fluorescence in 96.7% of U251 stained with TD05 aptamers.

Discussion/Conclusion Our current aptamer protocol to label Ramos and U251 cells are highly sensitive in labeling the appropriate cells. However, we found cells which should not be labeled with an aptamer are displaying fluorescence. Such results should not have occurred as CD20 is a surface receptor expressed only on lymphoma cells and GFAP is an intracellular protein only expressed in glioma cells. This could be due to non-specific binding. Thus, future refinement of our staining protocol, and testing different concentrations may reduce false positives. Our results could also potentially be explained by a known phenomenon that Cy5 fluorophore can be taken up in mitochondria.

Acknowledgment of Support We would like to acknowledge the Valley Research Partnership for their funding support via a 2021-2022 round 6 grant.