Establishment of a Three-Dimensional In Vitro Model of Equine Papillomavirus Type 2 Infection

Abstract

There is growing evidence that equine papillomavirus type 2 (EcPV2) infection is etiologically associated with the development of genital squamous cell carcinoma (SCC) and precursor lesions in equids. However, the precise mechanisms underlying neoplastic progression remain unknown. To allow the study of EcPV2-induced carcinogenesis, we aimed to establish a primary equine cell culture model of EcPV2 infection. Three-dimensional (3D) raft cultures were generated from equine penile perilesional skin, plaques and SCCs. Using histological, molecular biological and immunohistochemical methods, rafts versus corresponding natural tissue sections were compared with regard to morphology, presence of EcPV2 DNA, presence and location of EcPV2 gene transcripts and expression of epithelial, mesenchymal and tumor/proliferation markers. Raft cultures from perilesional skin harboring only a few EcPV2-positive (EcPV2+) cells accurately recapitulated the differentiation process of normal skin, whilst rafts from EcPV2+ penile plaques were structurally organized but showed early hyperplasia. Rafts from EcPV2+ SCCs exhibited pronounced hyperplasia and marked dysplasia. Raft levels of EcPV2 oncogene transcription (E6/E7) and expression of tumor/proliferation markers p53, Ki67 and MCM7 expression positively correlated with neoplastic progression, again reflecting the natural situation. Three-dimensional raft cultures accurately reflected major features of corresponding ex vivo material, thus constituting a valuable new research model to study EcPV2-induced carcinogenesis.

Keywords: 3D model; EcPV2; horse; hyperplasia; papillomavirus; plaque; raft culture; skin; squamous cell carcinoma.

Figure A1

Pan-cytokeratin and vimentin immunostaining of raft cultures. Pan-cytokeratin and vimentin immunostaining of representative areas of perilesional skin-derived (A), plaque/hyperplasia-derived (B), squamous cell carcinoma (SCC)-derived (C), raft cultures and equine skin as control (D). All cells of the raft cultures are positive for the epithelial marker pan-cytokeratin, while the mesenchymal marker vimentin is not expressed within the raft cultures, except in a few cells within basaloid clusters in the upper layers of the SCC-derived raft ((C)—arrow). The equine skin control shows pan-cytokeratin positive epithelial cells in the epidermis and vimentin-positive mesenchymal cells in the dermis. No vimentin-positive cells are present in the epidermis. All photographs taken using 10× objective.

Figure 1

H&E staining of raft cultures and corresponding tissue sections. Representative areas of perilesional (A), plaque/hyperplasia (B) and squamous cell carcinoma (SCC) (C) raft cultures and tissue sections. Raft cultures derived from the three different ex vivo tissue sources correspond to their respective tissue of origin in terms of proliferation and differentiation. (A) Raft cultures derived from perilesional skin accurately reflect the keratinocyte differentiation process of normal skin. (B) Raft cultures derived from penile plaques are structurally organized but show early hyperplasia and crowding of basal cells (arrow) and hypertrophy of keratinocytes with occasional perinuclear halos (arrowhead). This is similar to the foci of hyperplasia (arrow) and koilocyte-like cells (arrowhead) present in the corresponding tissue section. (C) Raft cultures derived from penile SCCs exhibit marked hyperplasia and dysplasia. Of note are clusters of anucleate eosinophilic cells, suggestive of early keratinization (arrow), and clusters of basaloid cells (arrowhead) within the stratum spinosum, which likely mimic the keratin pearls (arrow) and infiltrative islands of epithelial cells (arrowhead) that characterize SCCs. All photographs taken using 10× objective. H&E stain.

Figure 2

Equine papillomavirus type 2 (EcPV2) signal distribution patterns in raft cultures and corresponding tissue sections using RNA in situ hybridization (RISH). EcPV2 RISH of representative areas of perilesional (A), plaque/hyperplasia (B) and squamous cell carcinoma (SCC) (C) raft cultures and tissue sections. (A) Perilesional skin-derived rafts and tissue sections contain just a few cells with weak granular signal (GS). (B) In plaque-derived rafts and tissue sections, moderate GS and scattered diffuse nuclear signal (DNS) (arrow) is present. (C) In SCC-derived rafts and tissue sections, strong GS (arrowhead) is present, while rafts also display rare DNS. All photographs taken using 10× objective.

Figure 3

p53, Ki67 and MCM7 immunostaining of raft cultures and corresponding tissue sections. Immunostaining using p53, Ki67 and MCM7 antibodies of representative areas of perilesional (A), plaque/hyperplasia (B) and squamous cell carcinoma (SCC) (C) raft cultures and tissue sections. Expression of p53, Ki67 and MCM7 increases with increasing neoplastic phenotype within both rafts and tissue sections of EcPV2-associated lesions. All photographs taken using 10× objective.