NANOVISION: Entwicklung von HNSCC-Markern für die Visualisierung und operative Tumorresektion in der Kopf-Hals-Onkologie

Introduction

This work shows the process to establish a workflow of removing tumor tissue from HNSCC during ENT surgery using tumor-specific surface molecules. Of particular interest are FAP (fibroblast-activating protein) and EphrinA2 to determine their suitability for intraoperative tumor imaging.

Material

Specimen collection took place during surgery in 17 ENT-patients. All were pre-diagnosed with HNSCC during panendoscopy. Samples were taken, stored in a medium at 5°C and washed with antimicrobial buffer solution. FACS and qRT-PCR were used for cell examination. Within 24h the tissue was dissected into its components using a Miltenyi kit to dissociate tissue into single cell suspensions and afterwards enzymatically digested with components of the kit and the gentleMACS™ dissociation device for the mechanical dissociation. After dissociation, cells were washed, counted and checked for viability using propidium iodide staining.

Results

Depending on sample quality, there was up to 90% viability. On average, 1xe7 cells/0.7g of tissue could be isolated, where 70% of cells from tumor and 83% from healthy tissue were viable. FAP was expressed in approximately 10% of tumor cells, whereas EphrinA2 was expressed at RNA but no longer detectable at protein level.

Discussion

We were able to establish a reproducible workflow between patient identification, sample collection, processing and cell analysis. Cell viability after dissociation is high. Single cell analysis showed that contrary to expectations, EphA2 is not expressed in adequate amounts as a surface protein, whereas FAP is. This finding will be utilized in future studies to develop a binding method with fluorescence molecules, enabling the detection of tumor cells.

Keywords:

Nanovision, FAP, EphprinA2, HNSCC

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