Related Experiment Video
Updated: May 21, 2026
Multiplex Immunohistochemical Analysis of the Spatial Immune Cell Landscape of the Tumor Microenvironment
Published on: August 18, 2023
Multiplex profiling of 16 immune checkpoints identifies novel serum biomarker panels for breast cancer detection and
Mouna Stayoussef1, Azza Habel1, Weili Xu2
1University of Tunis El Manar (UTM), Faculty of Sciences of Tunis (FST), Laboratory of Mycology, Pathologies and Biomarkers (LR16ES05), Tunis, Tunisia.
Background:
Immune checkpoints (ICs) are key regulators of anti-tumor immunity, yet their diagnostic potential as blood-based biomarkers in breast cancer (BC) remains insufficiently characterized. Comprehensive serum profiling using multiplex immunoassays may enable minimally invasive detection and molecular stratification, particularly for triple-negative breast cancer (TNBC).
Methods:
Serum samples from 88 treatment-naïve BC patients and 49 age-matched controls were analyzed using a 16-analyte MILLIPLEX® immuno-oncology panel. Six co-inhibitory and ten co-stimulatory IC proteins were quantified. Diagnostic accuracy was assessed using ROC curves and logistic regression. Associations with TNBC subtype, chemotherapy response, and 6-month progression-free survival (PFS) were evaluated.
Results:
Seven immune checkpoint proteins (LAG-3, BTLA, CD80, GITRL, CTLA-4, GITR, TLR-2) showed significant differential expression between BC patients and controls. A seven-protein panel demonstrated high diagnostic accuracy (AUC = 0.89; sensitivity 83%; specificity 86%), surpassing CA15-3 and CEA. TNBC patients exhibited a distinct eight-protein signature, with TIM-3, CTLA-4, and CD28 independently associated with TNBC classification. Elevated baseline TIM-3 and PD-L1 were associated with chemotherapy resistance and shorter PFS.
Conclusions:
Comprehensive serum IC profiling identified biomarkers with strong diagnostic and subtype-discriminatory potential. These minimally invasive panels show potential for BC detection and TNBC stratification, pending validation in prospective and longitudinal studies. Validation in larger, multi-center cohorts is warranted.
