A novel signal amplification technique for multiplex immunofluorescence to detect low-expressed biomarkers in the tumor microenvironment

Clara     Andrade Sinthon Andrade Sinthon; Paula Juričić; Jade Nguyen; Cansaran Saygili Demir; Bastian Nicolai; Justyna Zaborowska; Isabelle Blanc; Logan Engle; Janis Taube; Saška Brajkovic

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A novel signal amplification technique for multiplex immunofluorescence to detect low-expressed biomarkers in the tumor microenvironment

Clara   Andrade Sinthon Andrade Sinthon

¹,

Paula Juričić

¹,

Jade Nguyen

¹,

Cansaran Saygili Demir

¹,

Bastian Nicolai

¹,

Justyna Zaborowska

^{*

1},

Isabelle Blanc

¹,

Logan Engle

²,

Janis Taube

²,

Saška Brajkovic

¹ Lunaphore, Lausanne, Switzerland
² Johns Hopkins University, Baltimore, USA

Academic Editor: Samuel Mok

Published: 05 November 2025 by MDPI in CancersScape: Spatial Biology of the Tumor Ecosystem session Spatial Intra-Tumoral Heterogeneity in the Tumor Microenvironment

Abstract:

Multiplex immunofluorescence is a crucial technique in identifying biomarkers (PMID: 31502166). Detecting markers at different expression levels carries biological meaning (PMID: 34112666), but it is challenging and requires improved detection (PMID: 25242720). We introduce seqLA™, an automated method for amplifying individual markers in multiplex panels for efficient staining cycles.

Using the sequential immunofluorescence (seqIF™, PMID: 37813886) assays on the COMET™ platform, the seqLA™ method augments the number of detection antibodies per primary antibody cyclically, leading to intensified signals. The amplification complex can be eluted, enabling successive staining cycles while upholding tissue integrity. Using formalin-fixed paraffin-embedded tissue sections from human colorectal carcinoma and breast cancer, a 26-plex panel comprising 20 standard seqIF™ markers for immune and stroma compartments, with 6 markers (LAG-3, ICOS, TCF-1, FOXP3, PD-1, and PD-L1) detected with the seqLA™ method. Staining was compared with single-plex chromogenic immunohistochemistry (IHC) for each amplified marker (PMID: 32414858). Lunaphore HORIZON™ software was used for the quantification of the dynamic ranges and image analysis.

We successfully incorporated 6 amplified markers into a 20-plex seqIF™ panel. Our amplification method yielded an adjustable signal intensity increase between low- and high-expressing cell populations, signifying a broader dynamic range compared to unamplified seqIF™ markers. Notably, amplification facilitated the detection of the full expression spectrum of PD-1 and PD-L1, delving into various low-expressing subtypes of regulatory immune cells like Treg and macrophages within the TME. The staining patterns of amplified markers exhibited a strong correlation with chromogenic IHC staining. Combining amplified markers within a larger panel enabled an enhanced analysis of the TME and complex immune cell classification within tertiary lymphoid structures. 

This novel amplification strategy will enable the identification of markers expressed at low levels, thereby capturing the complete expression spectrum of crucial immune checkpoint markers.

Keywords: spatial biology; tumor microenvironment; multiplexing; amplification

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