Liquid Biopsy Proteomics in Ophthalmology

Abstract

Minimally invasive liquid biopsies from the eye capture locally enriched fluids that contain thousands of proteins from highly specialized ocular cell types, presenting a promising alternative to solid tissue biopsies. The advantages of liquid biopsies include sampling the eye without causing irreversible functional damage, potentially better reflecting tissue heterogeneity, collecting samples in an outpatient setting, monitoring therapeutic response with sequential sampling, and even allowing examination of disease mechanisms at the cell level in living humans, an approach that we refer to as TEMPO (Tracing Expression of Multiple Protein Origins). Liquid biopsy proteomics has the potential to transform molecular diagnostics and prognostics and to assess disease mechanisms and personalized therapeutic strategies in individual patients. This review addresses opportunities, challenges, and future directions of high-resolution liquid biopsy proteomics in ophthalmology, with particular emphasis on the large-scale collection of high-quality samples, cutting edge proteomics technology, and artificial intelligence-supported data analysis.

Keywords: aqueous humor; artificial intelligence; eye; liquid biopsy proteomics; precision medicine; vitreous.

Figure 1

Bioinformatic analysis of ocular liquid biopsy proteomics data. (A) Principal component analysis (PCA) graphically clusters samples based on two principal components in which their protein profiles differ most. Each point is one sample. NIV: neovascular inflammatory vitreoretinopathy. Panel adapted from Velez et al., Sci. Rep., 2019. (B) A heatmap visualizes protein levels of 55 proteins in the vitreous of patients with uveal melanoma with high metastatic risk (Class 2), low metastatic risk (Class 1) and control samples. Each row is one protein, and each column is one sample. The z-score represents a protein’s abundance level in relation to its mean level in all samples by standard deviation units. Panel adapted from Velez et al., Mol. Cancer, 2021. (C) TEMPO (Tracing Expression of Multiple Protein Origins) allows one to trace the cellular origin of thousands of aqueous humor proteins, including specific proteins from individual retinal cell types, allowing cell level analyses in nonregenerative tissues such as the retina in living humans. TEMPO is based on the integration of high-resolution liquid biopsy proteomics with single-cell transcriptomics from all known cell types in the human eye. Panel adapted from Wolf et al., Cell, 2023.

Figure 2

Artificial intelligence proteomic clocks assess age of the human eye. An artificial intelligence (AI) proteomic clock based on the aqueous humor proteome can predict a healthy person’s eye age. By integrating TEMPO, cell type-specific marker proteins were used to develop AI proteomic clocks to predict the age of a specific cell type, such as immune cells, vascular cells, and retinal cells. Adapted from Wolf et al., Cell, 2023.

Figure 3

Sample collection for eye proteomics. A variety of fluids can be collected from the human eye for proteomic analyses, including liquid biopsies from the aqueous humor (collected during intraocular surgery or using a slit lamp), vitrectomy (collected during vitrectomy), or tear samples. Immediately after the collection, the specimens are snap-frozen on dry ice and then stored at −80 °C until further analysis. A thorough sample annotation with links to the electronic health records of the patient is essential to obtain clinically meaningful insights. MORLI (mobile operating room lab interface), consisting of a cart on wheels with a flat lab bench surface, a computer with a barcode scanner, lab supplies, and a box with dry ice, helps with the processing of samples in the operating room.

Figure 4

Analytical instruments for eye proteomics. Mass spectrometry-based, aptamer-based, and proximity extension assay-based proteomics allow the analysis of hundreds to thousands of different proteins in a single sample, allowing us to perform pathway and proteome characterization as well as biomarker discovery studies. An enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation pull-down represent affinity-based methods that detect a single protein with high accuracy. A multiplex ELISA allows to detect tens to hundreds of different proteins in the same sample. These technologies are valuable for selective and targeted validation studies.