124 Enhanced analysis of tumor microenvironment and immune regulation via an automated adjustable signal amplification technique for multiplex immunofluorescence

<h3>Background</h3> Multiplex immunofluorescence (mIF) has become fundamental for tumor microenvironment (TME) and biomarker research.¹ Therein, different marker expression levels often have biological meaning and the detection of markers expressed at low vs. high levels can provide crucial insights.² However, detecting antigens expressed at lower levels can be challenging and often benefits from signal amplification.³ Here, we demonstrate a new automated method enabling the amplification of individual markers within multiplex panels. This technique provides an adjustable level of amplification and an efficient elution for subsequent staining cycles. <h3>Methods</h3> COMET™ is an automated platform that performs sequential immunofluorescence (seqIF™) assays.⁴ Based on this, the novel amplification method increases the number of detection antibodies per primary antibody in a cyclic manner leading to a stronger signal. The amplification complex can be eluted, enabling subsequent staining cycles while preserving tissue integrity. Formalin-fixed paraffin-embedded (FFPE) tissue sections from human colorectal and breast carcinoma were stained using a 26-plex panel including 20 markers in standard seqIF™ and 6 amplified markers, together covering most basic immune cell types, functions, and stroma compartments. The performance was assessed by comparing marker expression with and without amplification using Lunaphore HORIZON™ image analysis software as well as comparing amplified staining to single-plex chromogenic immunohistochemistry (IHC) for each marker.⁵ Finally, Hematoxylin and Eosin (H&amp;E) staining was performed on slides retrieved from COMET™. <h3>Results</h3> Our study demonstrated the successful integration of 6 amplified markers within a 20-plex seqIF™ panel. Amplified low-expressed markers could be reliably detected and their amplification produced a controllable signal intensity increase between low and high expressing cell populations, providing a higher dynamic range when compared to the unamplified seqIF™. Specifically, amplification enabled the detection of the full spectrum of PD-1 an PD-L1 expression intensity and identification of several low-expressing subtypes of regulatory immune cells such as Treg and macrophages within the TME. Furthermore, the staining patterns of amplified markers shows good correlation with chromogenic IHC staining, and elution of the amplification complex did not damage the tissue as demonstrated by the post-COMET™ H&amp;E staining. <h3>Conclusions</h3> Hyperplex seqIF™ panels integrating this novel amplification technique will enable the detection of markers that are expressed at low levels, which could not be robustly captured by non-amplified mIF, such as the full expression range of critical immune checkpoint markers. Analyzing a high number of markers simultaneously while allowing selective amplification will improve the profiling of immune and tumoral cells within their environment. <h3>References</h3> Francisco-Cruz A, Parra ER, Tetzlaff MT, Wistuba II. Multiplex immunofluorescence assays. <i>Methods Mol Biol</i> 2020;<b>2055</b>:467-495. Berry S, Giraldo NA, Green BF, <i>et al</i>. Analysis of multispectral imaging with the AstroPath platform informs efficacy of PD-1 blockade. <i>Science</i> 2021;<b>372</b>(6547):eaba2609. Stack EC, Wang C, Roman KA, Hoyt CC. Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of tyramide signal amplification, multispectral imaging and multiplex analysis. <i>Methods</i> 2014;<b>70</b>(1):46-58. Rivest F, Eroglu D, Pelz B, <i>et al</i>. Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics. <i>Sci Rep</i> 2023;<b>13</b>(1):16994. Taube JM, Akturk G, Angelo M, <i>et al</i>. The society for immunotherapy of cancer statement on best practices for multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation. <i>J Immunother Cancer</i> 2020;<b>8</b>(1):e000155.