Christoph Jindra

Squamous cell carcinoma of the head and neck (HNSCC) is a malignant cancer disease in humans and animals. There is ample evidence that the high plasticity of cancer cells, i.e., their ability to switch from an epithelial to a mesenchymal, endothelial, and stem cell-like phenotype, chiefly contributes to progression, metastasis, and multidrug resistance of human HNSCCs. In feline HNSCC, the field of cancer cell plasticity is still unexplored. In this study, fourteen feline HNSCCs with a known feline papillomavirus (FPV) infection status were subjected to histopathological grading and subsequent screening for expression of epithelial, mesenchymal, and stem cell markers by immunohistochemistry (IHC) and immunofluorescence staining (IF). Irrespective of the FPV infection status, all tumors except one corresponded to high-grade, invasive lesions and concurrently expressed epithelial (keratins, E-cadherin, β-catenin) and mesenchymal (vimentin, N-cadherin, CD146) proteins. This finding is indicative for partial epithelial-mesenchymal transition (pEMT) events in the lesions, as similarly described for human HNSCCs. IF double staining revealed the presence of CD44/CD271 double-positive cells notably within the tumors’ invasive fronts that likely correspond to cancer stem cells. Taken together, the obtained findings suggest that feline HNSCCs closely resemble their human counterparts with respect to tumor cell plasticity.

Equine sarcoids (EqS) are fibroblast-derived skin tumors associated with bovine papillomavirus 1 and 2 (BPV-1 and -2). Based on Southern blotting, the BPV-1 genome was not found to be integrated in the host cell genome, suggesting that EqS pathogenesis does not result from insertional mutagenesis. Hence, CRISPR/Cas9 implies an interesting tool for selectively targeting BPV-1 episomes or genetically anchored suspected host factors. To address this in a proof-of-concept study, we confirmed the exclusive episomal persistence of BPV-1 in EqS using targeted locus amplification (TLA). To investigate the CRISPR/Cas9-mediated editing of BPV-1 episomes, primary equine fibroblast cultures were established and characterized. In the EqS fibroblast cultures, CRISPR-mediated targeting of the episomal E5 and E6 oncogenes as well as the BPV-1 long control region was successful and resulted in a pronounced reduction of the BPV-1 load. Moreover, the deletion of the equine Vimentin (VIM), which is highly expressed in EqS, considerably decreased the number of BPV-1 episomes. Our results suggest CRISPR/Cas9-based gene targeting may serve as a tool to help further unravel the biology of EqS pathogenesis.