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Description
Prostate cancer is one of the most commonly diagnosed cancer in men, and the 5th leading cause of death worldwide1. The lack of the of robust and biologically relevant in vitro model systems that precisely recapitulate the pathophysiology of the human disease delay our understanding of the disease, and thus the discovery of the treatments. Owing the fact that tumor microenvironment plays an essential role in tumor progression and metastasis, functional materials mimicking the prostate extracellular matrix (ECM) properties and including stromal components will provide enhanced models 2. Bioprinting has emerged as a promising technique to fabricate 3D in vitro models with precise control of constructs’ architecture and cell spatial distribution3. This work describes the design and fabrication of prostate cancer 3D in vitro models using the combination of both advanced biomaterials and bioprinting. With this strategy we aim to better mimic properties of the native prostate tumour microenvironment, offering new models to study how prostate cancer progresses in vitro. Successful bioprinting methods rely on the selection of a suitable bioink that enables control of 1) the resolution of the printing process and 2) deposition of known bioactive materials and viable mammalian cells. Alginate-based bioinks were selected due to the biocompatibility, ease of functionalization, inherent shear thinning property, printability, and quick gelation with divalent ions (i.e.Ca2+). Alginate maintain stable 3D printed tumor models, with reduced degradation in vitro thanks to the absence of alginate degrading enzymes. To improve cell adhesion, oxidized alginate (OA), with target 50% oxidation degree, to control the amount of aldehyde groups able to covalently link active peptides. As laminin is one of the main proteins of prostate ECM and the main constituent of basement membrane, we have selected IKVAV and AG73, known to promote cell adhesion and mimic laminin, and conjugated with OA (i.e. OA-PEG-pep).
Furthermore, alginate allows control over mechanical properties of formed hydrogels and in the typical range of prostate tissue (~ 1-10 kPa). Prostate cancer cells (PC-3) were encapsulated at a concentration of 1 × 106 cells/mL in alginate-based hydrogels (e.g. 1% (w/v) OA-PEG-pep / 1% (w/v) alginate/ 3% (w/v) gelatin), with final PEG-Pep concentration per gel formulation of 0, 100, and 400 µM. Hydrogels were physically crosslinked with CaCl2 (0.1 M or 0.3M) for 10 min at 37°C. PC-3 cells adaption to the microenvironment, e.g. physico-chemical and mechanical properties, was evaluated by assessing cellular viability, proliferation, morphology, and expression of epithelial to mesenchymal transition markers. Finally, extrusion-based printing technology was used to print spatially defined system with spatial control over stiffness and cellular deposition of PC-3 with cellular viability (75-85%). Future works will involve the co-culture of PC-3 with cancer associated fibroblasts to evaluate the contribution of stromal cells. Bioprinted models measured approximately (2 mm x 2 mm x 1 mm), where cancer cells (PC-3) constrained to the central core of the printed constructs. Designed alginate based bioinks enables printing 3D prostate constructs with high cell viability providing by that biomimetic tissue to study prostate cancer progression and metastasis.
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