Enhancing Tissue Integrity for Improved Robustness of Multiplexed Fluorescent Imaging

Abstract

Tissue-based cyclic immunofluorescence, or t-CyCIF (hereafter referred to as “CyCIF”), accomplishes iterative multiplexing of fluorescent markers by diminishing active fluorophores with an oxidizing solution capable of bleaching most antibody-conjugated dyes, allowing for sample re-staining. CyCIF provides a powerful spatial-omics tool with the ability to predict treatment response and inform therapeutic strategies, and can currently generate up to 60-plex images from formalin-fixed, paraffin embedded (FFPE) tissue using widely available and cost effective reagents and instrumentation (Lin et al., 2018)(Mehta et al., 2021) (Färkkilä et al., 2020). Currently, the most significant limitation to profiling additional biomarkers is tissue degradation. Each cycle reduces tissue integrity through physical and chemical forces, cumulatively leading to tissue loss, preventing further sample processing. The goal of this research was to test and implement CyCIF protocol developments to increase the maximum number of markers able to be profiled in a single specimen. These developments include: (i) implementation of a spacer to prevent mechanical forces between sample and slide coverslip, (ii) assessment and utilization of alternative blocking buffers to mitigate chemical damage, and (iii) use of alternative DNA markers to ameliorate the loss of nuclear signal seen at high cycle numbers. After initial testing, these protocol developments were implemented together to accomplish profiling of 120+ antibodies and dyes over 35 CyCIF cycles in multiple tissue types, a significant improvement over the standard protocol. We then preformed image processing and preliminary data analysis to assess for dataset utility and to identify challenges to working with such a large dataset. We predict that augmenting CyCIF’s profiling capability in this way will expand the number of cell types and states able to be identified, and to better produce high dimensional data allowing for the characterization of spatial interactions between different cell types.