Targeting of 3D oral cancer spheroids by αVβ6 integrin using near-infrared peptide-conjugated IRDye 680

Sep 22, 2026

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Oral cavity cancer outcomes depend heavily on achieving microscopically clear surgical margins, yet positive margins increase local recurrence and reduce survival. Near-infrared fluorescence–guided surgery may improve resection using fluorescent probes. αVβ6 integrin is promising because it is overexpressed in oral cancers. This study evaluates IRDye-A20 as a selective imaging agent for 3D tongue cancer spheroid models and determines whether it enables targeted labeling of tumor cells.

LanguageEnglish

CategoryResearch & Report

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Pages17

Reading Time43 min

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Abstract

Background

Methods

Results

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Dirheimer etal. Cancer Cell International (2024) 24:228 Cancer Cell International [https://doi.org/10.1186/s12935-024-03417-y](https://doi.org/10.1186/s12935-024-03417-y) RESEARCH Open Access Targeting of 3D oral cancer spheroids by αVβ6 integrin using near-infrared peptideconjugated IRDye 680 L. Dirheimer 1, T. Pons2, A. François 1,3, L. Lamy 1,3, S. Cortese4, F. Marchal 1,4 and L. Bezdetnaya 1,3* Abstract Background In the treatment of oral cavity cancer, margin status is one of the most critical prognostic factors. Positive margins are associated with higher local recurrence and lower survival rates. Therefore, the universal goal of oral surgical oncology is to achieve microscopically clear margins. Near-infrared fluorescence guided surgery (FGS) could improve surgical resection using fluorescent probes. αVβ6 integrin has shown great potential for cancer targeting due to its overexpression in oral cancers. Red fluorescent contrast agent IRDye 680 coupled with anti-αVβ6 peptide (IRDye-A20) represents an asset to improve FGS of oral cancer. This study investigates the potential of IRDye-A20 as a selective imaging agent in 3D three-dimensional tongue cancer cells. Methods αVβ6 integrin expression was evaluated by RT-qPCR and Western Blotting in 2D HSC-3 human tongue cancer cells and MRC-5 human fibroblasts. Targeting ability of IRDye-A20 was studied in both cell lines by flow cytometry technique. 3D tumor spheroid models, homotypic (HSC-3) and stroma-enriched heterotypic (HSC-3/ MRC-5) spheroids were produced by liquid overlay procedure and further characterized using (immuno)histological and fluorescence-based techniques. IRDye-A20 selectivity was evaluated in each type of spheroids and each cell population. Results αVβ6 integrin was overexpressed in 2D HSC-3 cancer cells but not in MRC-5 fibroblasts and consistently, only HSC-3 were labelled with IRDye-A20 . Round shaped spheroids with an average diameter of 400 μm were produced with a final ratio of 55%/45% between HSC-3 and MRC-5 cells, respectively. Immunofluorescence experiments demonstrated an uniform expression of αVβ6 integrin in homotypic spheroid, while its expression was restricted to cancer cells only in heterotypic spheroid. In stroma-enriched 3D model, Cytokeratin 19 and E-cadherin were expressed only by cancer cells while vimentin and fibronectin were expressed by fibroblasts. Using flow cytometry, we demonstrated that IRDye-A20 labeled the whole homotypic spheroid, while in the heterotypic model all cancer cells were highly fluorescent, with a negligible fluorescence in fibroblasts. Conclusions The present study demonstrated an efficient selective targeting of A20FMDV2-conjugated IRDye 680 in 3D tongue cancer cells stroma-enriched spheroids. Thus, IRDye-A20 could be a promising candidate for the future development of the fluorescence-guided surgery of oral cancers. *Correspondence: L. Bezdetnaya [L.bolotine@nancy.unicancer.fr](L.bolotine@nancy.unicancer.fr) Full list of author information is available at the end of the article © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit [http://creativecommons.org/l](http://creativecommons.org/l)icenses/by/4.0/. The Creative Commons

FAQs

Why are microscopically clear margins crucial in oral cancer surgery?

Margin status is a key prognostic factor. Positive margins correlate with higher local recurrence and lower survival, so achieving microscopically clear margins is a universal goal.

What is IRDye-A20 and what target does it engage?

IRDye 680 is coupled with an anti-αVβ6 peptide (IRDye-A20). The study focuses on αVβ6 integrin, which is overexpressed in oral cancers, as the targeting mechanism.

How was integrin expression and targeting ability evaluated?

αVβ6 integrin expression was assessed using RT-qPCR and Western blotting in 2D cell models. Targeting ability of IRDye-A20 was studied by flow cytometry, and 3D spheroids were characterized using (immuno)histological and fluorescence-based techniques.

Sep 22, 2026

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