HPV-transformed cancer cell lines have so far been intensively studied and characterized in monolayer cultures. To develop novel medical treatment options a three-dimensional (3D) model of HPV-transformed cells in their natural context is of superior importance when analyzing drug effects on these cells. We intended to create a 3D organotypic epithelial raft culture resembling HPV-induced (pre-)cancerous lesions. This will allow better evaluation and understanding of the effects of novel therapeutic options on HPV-transformed cancer cells in their natural context as well as on surrounding healthy keratinocytes.
A dermal equivalent comprising a fibrin gel containing human fibroblasts embedded in a tissue scaffold was created in a deep-well plate. A combination of primary human keratinocytes and cervical HPV-transformed cancer cell lines was then seeded on the dermal equivalent and grown to full confluency over the course of 24 hours. Epithelial differentiation of the keratinocytes occurred over the course of 2 weeks. The cultures were subsequently harvested. Histological slices were prepared and stained with hematoxylin & eosin. Furthermore, combined p16INK4a/Ki67 immunohistochemical staining as well as combined Keratin 14/Keratin 7 immunofluorescence were performed.
After two weeks of culture we observed a fully differentiated epithelium comprising healthy keratinocytes and clearly distinguishable lesions consisting of HPV-transformed cancer cells. p16INK4a/Ki67 immunohistochemical staining allowed for a clear distinction between normal keratinocytes and HPV-transformed cells with only HPV-transformed cells staining positive for both markers. Likewise, Keratin 14/Keratin 7 immunofluorescence granted a specific identification of the HPV-transformed cervical cancer cells. These transformed lesions, established with various HPV-transformed cell lines, resemble actual lesions found in a natural environment.
A 3D organotypic raft culture growing HPV-transformed cells in a more natural setting was established, providing an ideal model to study effects of future therapeutic approaches.