3D Bioprinting as a Powerful Technique for Recreating the Tumor Microenvironment

Abstract

In vitro three-dimensional models aim to reduce and replace animal testing and establish new tools for oncology research and the development and testing of new anticancer therapies. Among the various techniques to produce more complex and realistic cancer models is bioprinting, which allows the realization of spatially controlled hydrogel-based scaffolds, easily incorporating different types of cells in order to recreate the crosstalk between cancer and stromal components. Bioprinting exhibits other advantages, such as the production of large constructs, the repeatability and high resolution of the process, as well as the possibility of vascularization of the models through different approaches. Moreover, bioprinting allows the incorporation of multiple biomaterials and the creation of gradient structures to mimic the heterogeneity of the tumor microenvironment. The aim of this review is to report the main strategies and biomaterials used in cancer bioprinting. Moreover, the review discusses several bioprinted models of the most diffused and/or malignant tumors, highlighting the importance of this technique in establishing reliable biomimetic tissues aimed at improving disease biology understanding and high-throughput drug screening.

Keywords: bioprinting; hydrogels; tumor microenvironment.

Figure 1

Schematic representation of the tumor microenvironment (TME): legend with the cellular and extracellular components of the TME (left); TME structure (center); some biological phenomena occurring within the TME (right). Created with BioRender.com.

Figure 2

Schematic representation of the most common bioprinting methods. (a) Stereolithography with scanner mirrors that crosslink each layer point-by-point. (b) Inkjet-based bioprinting produces droplets through thermal or piezoelectric actuators. (c) Laser-based bioprinting employs a laser beam to create droplets starting from a bioink ribbon. (d) Extrusion-based bioprinting realizes continuous bioink filaments. Created with BioRender.com.

Figure 3

Bioink requirements. (a) Schematic representation of the biofabrication window. (b) Bioink general properties. Reproduced/adapted with the permission of [43].

Figure 4

Vascularized breast-to-bone metastatic cancer model. (a) Laser-based stereolithographic 3D bioprinting scheme (b) Tri-culture model scheme, with breast cancer-laden region, endothelialized vessel, and bone region. (c) Morphology of hFobs in the bone region. (d) Morphology of ECs in the vessel region. (e) Morphology of MDA-MB-231 cells in the tumor region. (f) Morphology of MDA-MB-231 cells at days 1, 3, and 7. (g) Morphology of MCF-7 cells at days 1, 3 and 7. Reproduced/adapted with the permission of [84].

Figure 5

Ductal carcinoma model. (a) The main steps of sacrificial bioprinting: an agarose microfiber was extruded onto a GelMA layer previously crosslinked and covered with another GelMA layer, followed by the photo-crosslinking of the entire construct and the removal of the agarose microfiber to induce the formation of a duct-like structure; finally, cells were seeded into the microchannel. (b) Confocal microscopy 3D image of the ductal carcinoma model after 14 days of culture. Nuclei staining in blue. (c) Dead/alive staining of MCF-7 cells after 24 days of culture. Reproduced/adapted with the permission of [85].

Figure 6

Glioblastoma-on-a-chip model. (a) Photographs of the GBM-on-a-chip model, including the dECM bioink laden with GBM cells (blue) or HUVECs (magenta), printed within a gas-permeable silicone on a glass slide and covered by a glass slip. (b) Compartmental subdivision of the bioprinted model in core, intermediate, and peripheral areas with GBM cells and external layer with HUVECs. (c) Oxygen level simulation within the bioprinted hydrogel, carried out with COMSOL Multiphysics. (d) Immunostaining images of core, intermediate, and peripheral zones employing pimonidazole (PM) for the hypoxic cells, Ki67 for the oxygenated proliferating cells, and DAPI for the cell nuclei. Scale bar, 200 μm. Reproduced/adapted with the permission of [68].

Figure 7

Heterogeneous stiffness liver cancer model. (a) Schematic representation of digital light processing-based bioprinting employed to fabricate the liver cancer model. (b) Bright-field images showing bioprinted cell-free and cell-laden hydrogels. Scale bar: 500 μm. (c) Plot showing the percent area of cell invasion deriving from the three different hydrogels over 7 days; ** p ≤ 0.01, *** p ≤ 0.001. (d) Merged fluorescence (top) and bright-field (bottom) images showing the HepG2 cell locations relative to their regions at day 0 and day 7; red, green, and yellow represent the soft, medium, and stiff matrices, respectively. Scale bar, 500 μm. Reproduced/adapted with the permission of [103].

Figure 8

Intestinal model with finger-like villus structure. (a) 3D coaxial bioprinting process for producing a core–shell intestinal model with epithelial Caco-2 cells and endothelial HUVECs using collagen/SIS bioinks. (b) Cell tracker image of the epithelium region (red) and capillary region (green) on day 1. (c) DAPI (blue)/MUC17 (green)/CD31 (magenta) image of the epithelium and capillary regions on day 28. (d) Optical (i) and (ii) SEM images of the villi and crypt regions for the bioprinted model. Reproduced/adapted with the permission of [115,116].