Student Researcher Oregon Health and Sciences University, Northeastern University, Sunset High School Portland, Oregon, United States
Introduction: Early cancer diagnosis is crucial to improve patient prognosis. However, over 50% of cancers are detected at advanced stages, rendering treatments ineffective. This project aims to advance recent research regarding liquid biopsies - blood analyses for cancer detection.
Cancer tumors shed neoplastic cells into the bloodstream early in metastasis. These cells, called circulating tumor cells (CTCs), are found in the blood at a concentration of ~1-10 CTCs/mL. In 2019, a novel type of CTC called a circulating hybrid cell (CHC) was discovered in the blood at a concentration 10x higher than other CTCs. Both cell types overexpress the epithelial growth factor receptor (EGFR) and its ligand, the epithelial cell adhesion molecule (EpCAM). These proteins are overexpressed in over 30 types of cancer, highlighting their potential as a target for universal cancer detection assays. Although protein-specific antibodies are used to stain these targets, they are too large to be practically used in in vivo assays.
This project aims to explore the potential of EGFR/EpCAM as universal targets for in vivo CTC detection via small-molecule, near-infrared fluorescent probes. Secondly, this research aims to test external probe practicality to further develop diffuse in vivo flow cytometry (DiFC) - a novel non-invasive detection technique. Although current methods such as CellSearch can detect CTCs in 6mL blood samples, DiFC prevents blood degradation, analyzes a larger fractional blood volume, and does so on a continuous basis. The technology developed by this research will enable non-invasive metastasis detection at early stages, thus making existing treatments more effective.
Materials and
Methods: CHEMICAL SYNTHESIS: A novel fluorescent probe was synthesized by conjugating peptide SNFYMPL to fluorophore AlexaFluor 647 (AF647) via a polyglycine linker, creating molecule SNF-AF647 in which one end binds to EpCAM and one end fluoresces under near-infrared (NIR) light. Molecular docking in Schroedinger was used to better understand the nature of binding. Synthesis was confirmed using liquid-chromatography mass spectrometry and the compound was purified via high-performance liquid chromatography.
BIOLOGICAL VALIDATION: The molecule was tested for EpCAM specificity through fluorescence microscopy and flow cytometry. 6 cell lines (2 CHC, 4 CTC) of four cancer types - breast (4T1), pancreatic (KPC), colorectal (MC38), and skin (A431) - were cultured to evaluate the universality of the biomarkers. All cells were stained with contrast agent prior to experimentation.
Fluorescence microscopy: SNF-AF647’s efficacy was compared against established anti-EpCAM antibody, VU1D9. The breast, pancreatic, and colorectal cell lines were plated in high concentration and fluorescently imaged. Images were analyzed on ImageJ software for overlap between the antibody and the small molecule stain.
Flow Cytometry: A431 samples were run through a flow cytometer pre and post-DiFC, and analyzed using FlowJo software to quantify the probe’s EpCAM binding.
DiFC Development: A431 samples were tested through a block mimicking skin's optical properties (phantom block) on a two-probe, NIR DiFC laser. A preliminary in vivo test was conducted on the laser by injecting a sample of ~250k CTCs into a mouse’s bloodstream. The DiFC non-invasively returned fluorescence that were analyzed for true cancer cell detection on MATLAB.
Results, Conclusions, and Discussions: LCMS analysis identified the main peak at 698.1, matching SNF-AF647’s calculated mass and confirming synthesis. Anti-EpCAM antibodies have a mass of ~40 kDa whereas SNF-AF647’s mass is 40x smaller at ~698 Da ( < 1 kDa). This increases movement efficiency through the bloodstream, improving practicality for in vivo translations.
The microscopy data attached shows a near complete overlap between the antibody control and SNF-AF647. As VU1D9 has been well validated to stain EpCAM and associated ligands with high specificity, the overlap suggests that the synthesized compound can accurately locate and bind to the EpCAM/EGFR proteins in CTCs and CHCs.
FlowJo analysis of A431 pre-DiFC flow cytometry found that the probe bound to ~98.9% of cells in each sample and fluoresced at higher brightness than controls, showing high EpCAM/EGFR specificity and low autofluorescence. The DiFC device was successfully paired with this probe, detecting ~100 CTCs in each sample, matching post-DiFC flow cytometry. Although data suggests that only ~15% of cells were stained brightly enough to be detected on the DiFC system, further investigation regarding the nature/extent of NIR laser effects on cells is required to support this conclusion. Additionally, results of both DiFC and fluorescent staining also support cytometry conclusions of high brightness and low background.
DiFC data from the preliminary in vivo injection experiment identified ~5-10 cancer cells in the bloodstream. Repeated trials are required for conclusive analysis.
This study found that EpCAM and EGFR can be used as targets across multiple untested cancer types pointing to their potential as targets for comprehensive cancer tests. The probe was successful in detecting both CHCs and CTCs, allowing for the number of potential targets in the bloodstream to rise from ~5 to ~55 cells/mL. Future steps include testing the efficacy of the probe in a mouse with naturally occurring CTCs, developing a machine learning algorithm to optimize DiFC peak detection, and applying an image analysis algorithm to quantify overlap in fluorescent stains. The ultimate goal is to create a system where cancer patients receive an injection of the fluorescent probe and are noninvasively imaged for CTCs, revolutionizing early cancer diagnostics.
Acknowledgements (Optional): I would like to thank the research group at the Gibbs Lab at Oregon Health and Sciences University - Summer Gibbs for giving me the opportunity to intern in her lab, Nicole Rueb for mentoring me throughout this project, and Gourav Kumar for lending his expertise in molecular docking. I would also like to thank the research group at the Niedre Lab at Northeastern University for allowing me to further develop my project - Mark Niedre for giving me the opportunity to conduct research in his lab, as well as Joshua Pace and Grace Matheson for mentoring me and handling mice. Thank you also to the Kuni foundation for the grant to fund this research.
References (Optional): Ma, X., Li, M et al. (2019). Identification of tumor specific peptide as EPCAM ligand and its potential diagnostic and therapeutic clinical application. Molecular Pharmaceutics, 16(5), 2199–2213. https://doi.org/10.1021/acs.molpharmaceut.9b00185
Sutton, T. L., Wong, M. H., et al. (2022). Circulating cells with macrophage-like characteristics in cancer: The importance of circulating neoplastic-immune hybrid cells in cancer. Cancers, 14(16), 3871. https://doi.org/10.3390/cancers14163871
Wang, L. G., Gibbs, S. L., et al. (2023). Oregonfluor enables quantitative intracellular paired agent imaging to assess drug target availability in live cells and tissues. Nature Chemistry, 15(5), 729–739
Dietz, M. S., Wong, M. H, et al. (2021). Relevance of circulating hybrid cells as a non-invasive biomarker for myriad solid tumors. Scientific Reports, 11(1). https://doi.org/10.1038/s41598-021-93053-7
Liu, Y., Zhang, B, et al (2022a). Understanding the versatile roles and applications of epcam in cancers: From bench to bedside. Experimental Hematology & Oncology, 11(1). https://doi.org/10.1186/s40164-022-00352-4
Pace, J., Ma, J., Lee, J., Srinivasarao, M., Kallepu, S., Wang, L., Hubbell, G., Malankar, G., Whalen, R., Wong, M. H., Gibbs, S. L., Low, P. S., & Niedre, M. (2024). In vivo labeling and detection of circulating tumor cells with fluorescent molecular contrast agents. Optica Biophotonics Congress: Biomedical Optics 2024 (Translational, Microscopy, OCT, OTS, BRAIN). https://doi.org/10.1364/ots.2024.om1d.5
Pace, J., Lee, J. J., Srinivasarao, M., Kallepu, S., Low, P. S., & Niedre, M. (2024). In vivo labeling and detection of circulating tumor cells in mice using OTL38. Molecular Imaging and Biology, 26(4), 603–615. https://doi.org/10.1007/s11307-024-01914-0
