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Multiplex Fluorescence Immunohistochemistry for Early-Stage Melanoma Patient Stratification and Therapy Selection

In the era of precision oncology, clinicians must choose the optimal therapy for each patient from a growing armamentarium that includes chemotherapy, radiotherapy, immunotherapy and targeted agents. Consequently, reliable prediction of recurrence risk and assessment of therapeutic outcome have become central concerns for the medical community.

References:

On March 17, 2025, the Wang-Peng research group at the School of Life Science and Technology, ShanghaiTech University, in collaboration with the team of Professor Kong Yan at Peking University Cancer Hospital and other institutions, published a landmark study in Cell delineating the activation mechanism of the LAG-3 immune-checkpoint receptor. The work demonstrates, for the first time, that ubiquitination of LAG-3 upon ligand engagement exerts a positive regulatory effect on its inhibitory function, offering novel precision-oncology strategies and candidate biomarkers for tumor immunotherapy.

1.Research Highlights

Rapid, non-proteolytic poly-ubiquitination of LAG3 upon ligand binding

Within minutes of ligand engagement, lysine-498 in the LAG3 cytoplasmic tail undergoes robust poly-ubiquitination that persists ≥30 min without inducing protein degradation, thereby stabilizing the activated receptor.

Ubiquitination releases the membrane-sequestered FSALE motif and licenses inhibitory signaling

In resting T cells the FSALE helix is embedded in the plasma membrane; ubiquitin-dependent conformational un tethering exposes signaling residues required for recruitment of SHP-1/2 phosphatases and initiation of negative regulation.

LAG3 ubiquitination is indispensable for suppression of anti-tumor immunity in vivo

CRISPR-engineered ubiquitination-deficient LAG3-K498R mutants fail to restrain T-cell expansion, accelerate tumor rejection in syngeneic mouse models, and correlate with diminished ubiquitin signal in human tumor-infiltrating lymphocytes.

LAG3–CBL co-expression predicts response to LAG3 blockade

Multiplex immunofluorescence of melanoma biopsies revealed that patients whose tumors co-express LAG3 and CBL-family E3 ligases exhibit significantly higher objective response rates and prolonged progression-free survival upon anti-LAG3 therapy, establishing a companion diagnostic biomarker for precision immunotherapy.

2.Applications of mLF/mIHC Technology in LAG3 Research

In this study, multiplex immunofluorescence (Pheno 7-color kit, PVB2002) was employed to map LAG3 and CBL family proteins in tumor tissue, confirming their differential distribution among patients with PR, SD and PD and allowing direct visualization of the findings.

Tumor microenvironment dissection 

mIF/mIHC simultaneously quantified LAG3 and CBL expression and delineated their spatial topography within the TME, enabling identification of patients with high LAG3/CBL co-expression who are most likely to benefit from LAG3-targeted therapy.

Immune-cell infiltration analysis 

By co-staining LAG3, CBL and lineage markers for CD8⁺ T cells, CD4⁺ T cells and macrophages, mIF/mIHC revealed the geographic overlap between LAG3/CBL-high regions and immune infiltrates, providing spatial evidence for LAG3-mediated immunosuppression.

Efficacy-prediction model construction 

Integrating mIF/mIHC-derived LAG3/CBL co-expression data with clinical outcomes, we constructed a predictive model. Expression changes in LAG3 and CBL before and after therapy were tracked by mIF/mIHC, supplying direct evidence of response and facilitating multidimensional treatment optimization.

References:

Colorectal cancer (CRC) has long been a focal point in the quest for precision oncology. This common gastrointestinal malignancy carries one of the highest global incidences and mortality rates. For patients with stage III microsatellite-stable (MSS) disease, the ever-present threat of relapse looms like a sword of Damocles; accurately predicting recurrence and tailoring individualized treatment and surveillance strategies are therefore pivotal to improving outcomes.

Background

This study retrospectively analyzed archived FFPE samples from 17 patients with stage III MSS CRC, including 11 samples from relapsed patients and 6 samples from non-relapsed patients, aiming to explore their immune cell characteristics and their correlation with post-chemotherapy relapse. A PN 6-Plex detection kit (PhenoVision Bio Co., Ltd) was used for multiplex immunofluorescence (mIF) staining targeting macrophages, T cells, B cells, as well as panCK, PD-1, and PD-L1. After scanning the 17 stained slides, a pathologist identified the tumor areas based on H&E staining of consecutive sections from each sample. The PhenoVision mIF AI analysis system (trained on the Oncotopix Discovery system (Visiopharm)) was used to analyze non-tumor areas, tumor invasive margins (IM, defined as the range of 360μm inside and outside the tumor boundary, totaling 720μm), tumor parenchyma, and stromal regions.

Experimental Procedures

1.Objective 

Colorectal cancer (CRC) is a highly prevalent and lethal gastrointestinal malignancy. Stage III microsatellite-stable (MSS) CRC still carries a substantial post-chemotherapy recurrence rate. Although the immune microenvironment is intimately linked to relapse, conventional assays cannot resolve the fine-grained topography of immune-cell subpopulations, precluding accurate early-risk stratification and therapy tailoring. 

Prior studies have centered on tumor-intrinsic genomic alterations and isolated biomarkers; the spatial distribution of immune-cell subtypes within the tumor core and invasive margin remains insufficiently explored. Here, we focused on PD-1⁻/CD20⁺, CD3⁺, and PD-1⁻/CD3⁺ subsets to identify novel recurrence predictors and refine clinical decision-making.

2.Procedures 

The Wang–Hu team at the Second Affiliated Hospital of Harbin Medical University, in collaboration with PhenoVision, employed the PVB-2003 multiplex immunofluorescence kit and the PhenoVision mIF AI platform to simultaneously enumerate and map T cells, macrophages, B cells, tumor cells, PD-1⁺, and PD-L1⁺ cells on single FFPE sections, quantifying their spatial neighborhood relationships.

This study rigorously selected and enrolled archived FFPE samples from 17 stage III MSS colon cancer patients, stratified by recurrence status into 11 recurrent and 6 non-recurrent cases. Utilizing state-of-the-art multiplex immunofluorescence (mIF) coupled with the PhenoVision mIF AI analytical platform, we precisely mapped and quantified target immune-cell subtypes within tumor islands, stromal compartments, and the tumor-invasive margin. By comparing density and proportional differences of these subsets between recurrent and non-recurrent cohorts, we delineated immune signatures potentially associated with disease relapse.

3.Results 

The tumor microenvironment differed significantly between recurrent and non-recurrent patients. 

– Within the tumor bed, PD-1⁻/CD20⁺ cells in the stroma were markedly higher in the non-recurrent cohort (6.64 % vs 2.58 %, p = 0.026). 

– PD-L1⁺/CD3⁺ cells also showed a significant inter-group difference (p = 0.01). 

In the invasive-margin (IM) analysis, the PD-1⁻/CD20⁺ fraction rose from 0.68 % in the tumor core to 2.21 % at the IM in recurrent cases, whereas CD3⁺ and PD-1⁻/CD3⁺ subsets displayed the converse, IM-enriched pattern in non-recurrent cases. 

These data indicate that the topography of PD-1⁻/CD20⁺ cells is tightly linked to relapse risk and may serve as a predictive biomarker. The selective accumulation of this subset in the IM of non-recurrent patients suggests that enhancing its infiltration into the tumor nest could improve outcomes, although this therapeutic concept requires further investigation.

4.Conclusions & Outlook 

This study provides a comprehensive, high-resolution map of immune-cell topography in stage III MSS colon cancer and establishes a robust association between PD-1⁻/CD20⁺ distribution and recurrence. The identified signatures are readily translatable into clinical assays for risk stratification and tailored surveillance. Moreover, therapeutic strategies aimed at increasing PD-1⁻/CD20⁺ and related immune subsets within tumor tissue warrant evaluation in larger, multicentric trials to refine precision management and prolong survival in this high-risk population.