• 3D cell culture
• drug screening
• ovarian cancer
• polymeric biomaterials

Cancer is the second leading cause of death in the world. The complexity, heterogeneity, and dynamic nature of this pathology pose many challenges for its study and treatment. Furthermore, conventional anticancer therapies may have low specificity and are often accompanied by multiple and severe side effects. Therefore, current trends in oncology focus on increasing the selectivity and safety of developed therapies.
The discovery and development of new drugs is a very lengthy and costly process, characterized by a high failure rate. One of the major obstacles is represented by the in vitro models currently employed, which fail to adequately reproduce microenvironmental signals found in vivo. Although traditional 2D cultures represent an important tool for the study of cancer biology and development of anticancer therapies, it is becoming increasingly clear that such models are often poorly predictive of drug response in humans.
In the last decade, attention has shifted to the development of three-dimensional (3D) models that are able to reproduce the complex architecture and properties of native tissues, thereby overcoming the limitations of conventional cultures. 3D culture models can be a reliable alternative in the screening of new drugs, as they allow for more realistic cell-cell and cell-matrix interactions, as well as gene and protein expression levels that more closely mimic in vivo conditions.
Among 3D culture systems, scaffold-based technologies provide a physical support that mimics the native extracellular matrix on which cells can adhere, aggregate, proliferate, and migrate.
This work aims to develop a 3D in vitro model of ovarian cancer, which represents the gynecological tumor with the highest lethality rate worldwide and is characterized by late diagnosis, recurrence, and metastasis.
The polymeric materials of natural origin selected for this study are chitosan and alginate, polysaccharides with proven biocompatibility. In particular, they were employed for the fabrication of 3D polyelectrolyte complex (PEC) hydrogels using Computer-Aided Wet-Spinning (CAWS), an additive manufacturing technique that involves the layer-by-layer deposition of a polymeric solution/suspension into a coagulation bath.
These hydrogels were used to evaluate the cytotoxicity of cisplatin, eugenol, and gallic acid, as well as a cisplatin-eugenol synergistic combination, by investigating the different cell sensitivities in 2D and 3D cultures. Two human tumor cell lines were used: A2780, sensitive to cisplatin, and A2780cis, resistant to cisplatin.
Cytotoxicity analysis was performed using a colorimetric assay based on WST-1 tetrazolium salt. In the case of cisplatin and eugenol, higher half-maximal inhibitory concentration (〖IC〗_50) values were recorded in 3D cultures than in 2D cultures. Conversely, in combination therapy, we observed that cells grown on PEC scaffolds were more sensitive and showed a greater synergistic response than 2D cultures.
Regarding gallic acid, the 3D model for cytotoxicity studies has not yet been optimized, owing to it oxidation and/or interactions of the bioactive agent with the polymeric matrix that influence its bioactivity. Thermogravimetric analysis (TGA) and infrared spectroscopy (FTIR-ATR) analysis suggested the occurrence of a transesterification reaction between gallic acid and the polysaccharide.
The ability of PEC scaffolds to maintain the tumor aggressiveness phenotype was evaluated by Confocal Laser Scanning Microscopy (CLSM). This was performed by analyzing the expression of integrin β-1 and metalloproteinase-2, and qualitatively comparing their expression in the two culture methods, demonstrating the ability of these scaffolds to offer a suitable microenvironment for studying the pathophysiological processes involved in carcinogenesis.