In Situ Immunofluorescence Imaging of Vital Human Pancreatic Tissue Using Fiber-Optic Microscopy

TL;DR

This study explores using fiber-optic microscopy to detect cancer cells in human pancreatic tissue during surgery, aiming to improve resection accuracy.

Contribution

The study introduces fiber-optic microscopy as a novel method for in situ immunofluorescence imaging of vital human pancreatic tissues.

Findings

Fiber-optic microscopy successfully detected epitope-based fluorescence in whole-mount tissues.

Antibody clones WM59, AY13, and 9C4 showed strong performance for imaging endothelial, tumor, and epithelial cells.

The method enables visualization of microvascular and malignant cells in real-time during surgery.

Abstract

Surgical resection is the only curative option for pancreatic carcinoma, but disease-free and overall survival times after surgery are limited due to early tumor recurrence, most often originating from local microscopic tumor residues (R1 resection). The intraoperative identification of microscopic tumor residues within the resection margin in situ could improve surgical performance. The aim of this study was to evaluate the effectiveness of fiber-optic microscopy for detecting microscopic residues in vital pancreatic cancer tissues. Experimental Design. Fresh whole-mount human pancreatic tissues, histological tissue slides, cell culture, and chorioallantoic membrane xenografts were analyzed. Specimens were stained with selected fluorophore-conjugated antibodies and studied using conventional wide-field and self-designed multicolor fiber-optic fluorescence microscopy instruments.

Whole-mount vital human tissues and xenografts were stained and imaged using an in situ immunofluorescence protocol. Fiber-optic microscopy enabled the detection of epitope-based fluorescence in vital whole-mount tissue using fluorophore-conjugated antibodies and enabled visualization of microvascular, epithelial, and malignant tumor cells. Among the selected antigen-antibody pairs, antibody clones WM59, AY13, and 9C4 were the most promising for fiber-optic imaging in human tissue samples and for endothelial, tumor and epithelial cell detection.

Fresh dissected whole-mount tissue can be stained using direct exposure to selected antibody clones. Several antibody clones were identified that provided excellent immunofluorescence imaging of labeled structures, such as endothelial, epithelial, or EGFR-expressing cells. The combination of in situ immunofluorescence staining and fiber-optic microscopy visualizes structures in vital tissues and could be proposed as an useful tool for the in situ identification of residual tumor mass in patients with a high operative risk for incomplete resection.

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