As
a first approach, standard 2D cell culture techniques are usually
employed for the screening of drugs and nanomaterials. Despite the
easy handling, findings achieved on 2D cultures are often not efficiently
translatable to
in vivo
preclinical investigations.
Furthermore, although animal models are pivotal in preclinical studies,
more strict directives have been implemented to promote the use of
alternative biological systems. In this context, the development and
integration into preclinical research workflow of 3D neoplasm models
is particularly appealing to promote the advancement and success of
therapeutics in clinical trials while reducing the number of
in vivo
models. Indeed, 3D tumor models bridge several discrepancies
between 2D cell culture and
in vivo
models, among
which are morphology, polarity, drug penetration, osmolality, and
gene expressions. Here, we comprehensively describe a robust and high-throughput
hanging drop protocol for the production of 3D models of both Human
Papillomavirus (HPV)-positive and HPV-negative head and neck squamous
cell carcinomas (HNSCCs). We also report the standard cascade assays
for their characterization and demonstrate their significance in investigations
on these aggressive neoplasms. The employment of relevant 3D cancer
models is pivotal to produce more reliable and robust findings in
terms of biosafety, theranostic efficacy, and biokinetics as well
as to promote further knowledge on HNSCC pathophysiology.