Assistant Professor University of Oklahoma Norman, Oklahoma, United States
Introduction: One common hindrance in the fight against cancer is the lack of high resolution and cost-effective imaging systems for diagnosis and repeated monitoring. Current imaging modalities, including CT, MRI, SPECT, and optical imaging, are limited by their resolution, depth, and mobility for tracking. High resolution ultrasound with targeting contrast agents may help to address these issues. Gas vesicles (GVs) are naturally found in archaea and bacteria. These nanometer scale gas pockets provide contrast for ultrasound imaging. These gas vesicles can be chemically conjugated with antibodies for targeted imaging and therapy. Human epidermal growth factor receptor 2 (HER2) and programmed death ligand 1 (PD-L1) are common proteins for cancer therapy and were therefore chosen for this study. HER2 proteins assist in healthy cell growth but can be elevated in cancer cells necessitating the use of suppressive antibodies. PD-L1 proteins are also often elevated in cancer cells as an evasion from the immune system, but the antibody is able to thwart the evasion, restoring the ability of the immune system to defeating the cancer. We tested targeting efficiency of triple conjugation complexes between antibodies, GVs, and fluorescence dyes (GV-mAb-647), which were imaged using epi-fluorescent microscopy and ultrasound biomicroscopy (UBM) after targeting HER2+ and PD-L1+ cells.
Materials and
Methods: HER2 and PD-L1 antibodies were labelled with fluorescent tags using click chemistry for preliminary antibody targeting ability testing and measured by fluorescent microscopy. HER2 and PD-L1 were overexpressed in HeLa cells by homology-directed repair (HDR) process using a CRISPR-Cas9 system we developed [1]. We compared the brightness of captured fluorescent images of cells. Preliminary protein expression testing was completed through Western blot and immunofluorescence (IF) imaging. Gas vesicles were grown and purified from Anabaena flos-aquae [Ana]. For the final experiment, GVs were separately labelled with both fluorescent dyes and antibodies by chemical conjugation and dialysis. The triple conjugated gas vesicle complex (GV-mAb-647) was then added to the cells for targeting. Following a wash step and fluorescent imaging, targeted cells went through trypsinization, were mixed with agarose gel, and were loaded in cylindrical gel holders for ultrasound imaging by UBM system with in-house developed ultrasonic transducers.
Results, Conclusions, and Discussions: Antibody fluorescent labeling was shown successful by a calculation estimating each antibody to have an average of 2.44 fluorescent labels. Western blot testing and IF imaging show protein expression of the transfected cells (Fig. 1A-B). Preliminary antibody targeting shows statistical significance for both protein expression and antibody targeting (Fig. 1C). The middle images with green cells show the GFP being expressed either from HER2 (top) or PD-L1 (bottom). The right images with red cells show the fluorescently labeled antibodies targeting the protein of interest. These preliminary tests confirm the HER2 and PD-L1 protein expression and the antibody binding ability needed for the ultrasound imaging of gas vesicles targeting cancer cells. The next step is to optimize the GV-mAb-647 to target cells for ultrasound imaging and then to image after cross-targeting to prove exclusivity. This study will increase our understanding of gas vesicles as contrast agents and to deliver therapy.
Acknowledgements (Optional): This work is partially supported by the National Institute of Health (NIH) grant No. GM152704 and the National Science Foundation (NSF) grant No. CBET1943852.
[1] Yoon, S., et al., Acoustic-transfection for genomic manipulation of single-cells using high frequency ultrasound. Sci Rep, 2017. 7(1): p. 5275
