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Background:
Complete surgical resection is a major determinant of local control in head and neck cancers (HNCs), yet intraoperative identification of tumor-involved margins remains difficult because of complex anatomy, occult microscopic extension, and the limited spatial sampling of frozen section analysis1,2. A rapid, specimen-based optical imaging could improve margin assessment immediately after resection3. Nectin-4 is a cell-surface adhesion protein with an accessible extracellular domain and restricted expression within normal tissues, making it a promising imaging target4,5. Recent evidence support nectin-4 as a biologically relevant and clinically tractable target in HNCs6,7, however, its relevance to surgical margin evaluation in HNCs has not been established.

 

Hypothesis/Objective:
To develop an optical strategy for margin assessment, we first characterized the transcriptomic landscape of nectin-4 in HNC specimens, focusing on cell-type specificity and margin-associated expression. Then we developed N188-ICG, a nectin-4-targeted fluorescent probe, and evaluated its performance in xenograft mouse models and ex vivo mapping of freshly resected human specimens. These studies focus on establishing a pathology-integrated, clinically translatable strategy to support tumor delineation and surgical margin assessment in patients with HNCs

 

Methods:
A discovery cohort integrating bulk RNA-seq data from TCGA-HNSC tumors (n = 504) and single-cell RNA-seq datasets from tumors and matched margin samples (n = 17) was analyzed to define the transcriptomic landscape of PVRL4/nectin-4 in HNCs and to assess its relevance to margin status. An independent validation cohort (n = 228) was used to evaluate nectin-4 expression and clinical significance across primary tumors, recurrences, and metastases by immunohistochemistry (IHC) and Kaplan-Meier survival analysis (Fig. 1A). Based on these findings, a nectin-4-targeted bicyclic peptide fluorescent probe (N188-ICG) was developed and evaluated in subcutaneous and metastatic xenograft models by in vivo and ex vivo fluorescence imaging, including receptor-blocking controls and nodal fluorescence analysis. Translational feasibility was further assessed in an ex vivo fresh-specimen workflow using surgical specimens from 21 patients with HNCs (Fig. 1E). Freshly resected tissues were incubated with N188-ICG for 15 min, washed for 3 min, imaged using a closed-field system, and correlated with matched hematoxylin and eosin (H&E) staining and nectin-4 IHC.

 

Results:
Transcriptomic screening identified PVRL4 as a leading candidate imaging target in HNCs, showing among the highest overall expression levels in the TCGA-HNSC cohort (Supplementary Fig. 1A). Analysis of margin-derived epithelial cells further supported its clinical relevance, with the positive-margin sample showing markedly higher PVRL4 expression and a greater fraction of PVRL4-positive epithelial cells than negative-margin samples (35.1% vs 5.0% and 8.7%; Supplementary Fig. 1B). Clinicopathologic validation confirmed frequent nectin-4 expression across primary tumors, recurrences, and metastases, with moderate-to-high expression observed in 74.6% of tumors, higher staining in tumor-positive than tumor-free margins, and an association between high nectin-4 expression and shorter progression-free and overall survival (Fig. 1B–D). In xenograft models, N188-ICG showed receptor-dependent tumor targeting and enabled clear tumor delineation in vivo, with higher tumor-to-background ratios in nectin-4-high tumors than in low-expressing or blocked controls and a sustained high-contrast imaging window from 24 to 72 h (Supplementary Fig. 1C–D). In metastatic models, N188-ICG also enabled in vivo and ex vivo visualization of metastatic lymph nodes, outperforming free ICG and showing preferential accumulation in ipsilateral nodal metastases, which was further supported by fluorescence microscopy and histological confirmation (Supplementary Fig. 1E–I). In a clinically practical ex vivo workflow, fresh surgical specimens were stained with N188-ICG for 15 min followed by a 3-min wash, generating fluorescence maps that spatially matched tumor-containing regions on histopathology and nectin-4 IHC, thereby enabling rapid discrimination of tumor-positive from tumor-free margins without systemic probe administration (Fig. 1E–F).

 

Conclusion:
Nectin-4 is a clinically relevant, tumor-enriched imaging target in head and neck cancers. The nectin-4–targeted probe N188-ICG enables rapid, non-destructive, and pathology-compatible assessment of freshly resected specimens, and may serve as a practical adjunct to frozen section analysis by highlighting suspicious regions for prioritized sampling. These findings support further clinical translation of nectin-4–targeted optical imaging for pathology-assisted surgery in head and neck cancers.

 

Image/Figure:
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Image/Figure Caption:
Figure 1. Integrated validation of nectin-4 as an imaging target and ex vivo N188-ICG-based margin assessment in head and neck cancer.

(A) Study overview. Discovery analyses integrated TCGA-HNSC bulk RNA-seq data from tumors (n = 504) and single-cell RNA-seq data from tumors (n = 14) and matched margin tissues (n = 3), followed by clinicopathologic validation in an independent cohort (n = 228) using immunohistochemistry (IHC) and survival analysis.

(B) Representative nectin-4 IHC in tumor-free and tumor-positive surgical margins, shown at low magnification and in corresponding higher-magnification views of the boxed regions.

(C) Distribution of semiquantitative nectin-4 IHC scores in primary tumors (n = 102), recurrences (n = 66), and metastases (n = 59).

(D) Kaplan–Meier analyses showing that high Nectin-4 expression was associated with shorter progression-free survival (PFS; left, P = 0.017, HR = 2.736) and overall survival (OS; right, P = 0.018, HR = 2.727).

(E) Ex vivo workflow for rapid fluorescence-based margin assessment. Freshly resected specimens were incubated with N188-ICG for 15 min, washed for 3 min, imaged using a closed-field fluorescence imaging system, and then processed for H&E staining and nectin-4 IHC.

(F) Ex vivo fluorescence imaging of human margin specimens. Top, representative pseudo-colored fluorescence maps of tumor-free and tumor-positive margins. Bottom, matched white-light (WL), fluorescence (FL), H&E, and nectin-4 IHC images from representative specimens showing concordance between fluorescence signal, histopathology, and target expression.

Scale bars: 500 μm and 100 μm in (B); 1000 μm and 100 μm in (F). HNSCC, head and neck squamous cell carcinoma; IHC, immunohistochemistry; H&E, hematoxylin and eosin; WL, white light; FL, fluorescence; PFS, progression-free survival; OS, overall survival; HR, hazard ratio.

 

Author
Wenhui Huang
Jinan University