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Additionally, RAFT cultures are a more reliable and cheaper solution to study antiviral drugs respect to
xenografts in SCID mice, since they are more suitable to be analysed by in situ hybridization and RT-qPCR
[55]
techniques .
Moreover, they can allow the study of host-virus interactions in absence of other cell types or tissues that
could hide fundamental pathways promoted by the pathogen, especially when the studies require miRNA
or EMT analysis.

As previously described, HR-HPVs E6 and E7 proteins interact and inhibit host p53 and pRb onco-
suppressors respectively. Recently, the idea that E6 and E7 can also bind host miRNA or miRNA regulators
[67]
has been evaluated . Indeed, despite HPV inability to encode for miRNAs, HPV mediated indirect
regulation of both onco-suppressive or tumorigenic miRNAs may offer the opportunity to identify novel
therapeutic targets.

RaFT CULTRes, DRUG ResIsTaNCe aND emT ReseaRCH
Regarding drug resistance and EMT markers expression analysis, RAFT cultures can overcome both 2D
[16]
and spheroid cultures limitations. Melissaridou et al. , working with 5 HNSCC-derived cell lines grown
in both 2D and spheroids assays, observed that, while 2D eukaryotic cells and spheroid conformation
changes the receptor topography and gene expression, and erases the drug resistance pathways that occur
in vivo, 3D cultures can better mimic what happens during in vivo therapies and allow to study EMT
expression markers such as CDH1, NANOG, SOX and EGFR.

However, when epithelial models are necessarily to be used, spheroids utility is limited, because of the less
reliability of the data they produce respect to those obtained with the organotypic models. In fact, although
they are both made up of epithelial cells, spheroids morphology and structure are too much simplified
and do not reproduce the mucosal epithelium in such a representative way as the 3D epithelial RAFTs do.
Therefore, compared to 3D RAFTs, the spheroids are less performant for studies on the specific interactions
that occur in a well-defined in vivo environment.

On the other hand, RAFT cultures can be built with both isolated healthy or tumour/cancer associated
keratinocytes and fibroblasts, able to better elucidate the interactions between stromal and epithelial
[56]
compartments that are at the basis of the initial steps of the carcinogenetic process . Moreover, they
recapitulate all the epithelial layers, the physiological cytokeratin differentiation and the carcinogenic
process.

[57]
By using in vitro reconstructed epithelial systems, Hogervorst et al. highlighted the importance of the
papillary-reticular fibroblasts switch. In fact, when epithelial cultures are grown in presence of switched
fibroblasts, the expression of CAF associated markers (α-SMA and vimentin) and EMT markers (SNAIL2,
N-cadherin and ZEB1) increase, thus suggesting that the fibroblasts switch could be considered an indicator

[58]
of squamous cell carcinoma (SCC) progression. Similarly, Fullar et al. proved that HPV16 induces the
modification of connective matrix that, by the action of epithelial Matrix Metalloproteinase MMP-7 and
fibroblast-produced MMP-2, loses collagen and fibronectin in favour of laminin, thus facilitating EMT and
carcinogenic events. Comparable effects on MMP super-families were also observed in other HR-HPV
[68]
positive models .
In agreement with the above, the last trend is to reproduce tissue models with the whole organ
[69]
complexity ; in fact, the models have been implemented with the vascular component, with macrophages
and T-cells, thus allowing to further improve the knowledge in the microbiology field [59-61] .