3D bioprinting: improving in vitro models of metastasis with heterogeneous tumor microenvironments

Abstract

Even with many advances in treatment over the past decades, cancer still remains a leading cause of death worldwide. Despite the recognized relationship between metastasis and increased mortality rate, surprisingly little is known about the exact mechanism of metastatic progression. Currently available in vitro models cannot replicate the three-dimensionality and heterogeneity of the tumor microenvironment sufficiently to recapitulate many of the known characteristics of tumors in vivo Our understanding of metastatic progression would thus be boosted by the development of in vitro models that could more completely capture the salient features of cancer biology. Bioengineering groups have been working for over two decades to create in vitro microenvironments for application in regenerative medicine and tissue engineering. Over this time, advances in 3D printing technology and biomaterials research have jointly led to the creation of 3D bioprinting, which has improved our ability to develop in vitro models with complexity approaching that of the in vivo tumor microenvironment. In this Review, we give an overview of 3D bioprinting methods developed for tissue engineering, which can be directly applied to constructing in vitro models of heterogeneous tumor microenvironments. We discuss considerations and limitations associated with 3D printing and highlight how these advances could be harnessed to better model metastasis and potentially guide the development of anti-cancer strategies.

Keywords: 3D bioprinting; Cancer; In vitro model; Metastasis; Tumor microenvironment.

Fig. 1.

Progression of events during metastatic disease. Cancer cells follow a series of steps during the course of metastatic disease, potentially invading as individuals or as clusters of cells. At the start of metastatic progression, tumor cells dissociate and locally invade tissue surrounding a primary tumor. Invasive tumor cells can eventually intravasate across the endothelial barrier and circulate through the bloodstream. Rarely, a small subset of circulating tumor cells will extravasate back across the endothelial barrier into distant tissue. At these secondary sites, another small subset of colonies will further adapt to the new secondary site and proliferate to form new macroscopic tumor sites.

Fig. 2.

Tumor microenvironment features that affect metastatic progression. Features of the tumor microenvironment are thought to play a role in facilitating or promoting tumorigenic behavior. These features include adhesive signals from extracellular matrix components such as collagen and fibrin; soluble signals like growth factors and cytokines; extracellular matrix mechanical features including stiffness and local tension or compression; and cell–cell interactions with intra- and extra-tumoral stromal cells. Adapted with permission from Hubbell (2008) and Lutolf and Hubbell (2005).

Fig. 3.

Layer-by-layer 3D printing. A common strategy for constructing three-dimensional objects is layer-by-layer construction, whereby a 3D structure is formed by stacking several layers of flat materials into a 3D pattern. Each layer can be thought of as a 2D pattern that has been expanded slightly into a thin 3D volume. An easy, illustrative example is provided by the formation of a pyramid shape. Each layer in a pyramid is a square 2D pattern with limited volume. (A) A low-resolution 3D object refers to an object formed from thick layers, which for a pyramid results in an object with thick, prominent steps. (B) By increasing the number of layers and decreasing thickness, the resolution of the pyramid is increased to give the appearance of a smooth surface. (C) For 3D bioprinting, complex structures such as vasculature can be constructed layer-by-layer with feature resolution dependent on layer thickness. Left panel shows an example 3D object representing a branching vascular structure is depicted. The vascular object can be constructed through iterative addition of 2D patterns. Right panel examples 1, 2 and 3 show top-down views of select 2D patterns at differing layers heights in the object.

Fig. 4.

Material extrusion-based 3D bioprinting. (A) For extrusion-based bioprinting, material is selectively guided onto a platform via pressurized emission through a nozzle. The material, or ‘bioink’, is composed of an ECM-like biomaterial, cells and soluble factors. (B) For inkjet-based bioprinting, droplets of bioink are distributed across a surface to form a patterned layer. (C) For support bath hydrogel 3DP, biomaterial is extruded into a support hydrogel material. At 22°C, the hydrogel bath is stable enough to support the extruded print material, but at 37°C, the hydrogel bath transitions into a more liquid state to release the 3D printed object. The support bath allows formation of complex structures with overhanging regions such as the 3D ‘S’ structure, which is not possible with regular extrusion 3DP. Additionally, support bath hydrogel 3DP enables fabrication of structures without the need for layer-by-layer production; material can be extruded along any linear path within the enclosed gel bath volume. Reproduced with permission from Hinton et al. (2015).

Fig. 5.

Light-based 3D bioprinting. (A) In laser patterning, a laser is focused onto singular points to locally photopolymerize material. The laser beam can be rastered across the surface to create 2D patterns of material. In a similar technique, selective laser sintering (SLS, not shown), a laser is used to fuse powder material together to form 2D patterns of material. SLS is particularly important because each layer is fully supported by the sintered or un-sintered powder of the previous layers, which permits freeform 3D printing of structures. (B) With projection stereolithography, a 2D pattern of light is directly projected onto a photopolymerizable material to form entire layers in singular steps. Projection stereolithography is notable in that each layer is formed with constant time, regardless of pattern complexity or shape.

Fig. 6.

Sacrificial template 3D bioprinting. An alternative method to ‘positive-space’ 3D printing is sacrificial template 3DP. For this method, a template material is formed into a 3D scaffold by a standard 3DP method. The product scaffold is cast with a biomaterial containing cells and/or soluble factors, and then the template material is removed by chemical dissolution or physical dislocation. In this example, a carbohydrate glass lattice (green) is fabricated via extrusion-based 3DP then encapsulated in ECM (gray) containing live cells (yellow). After the ECM solidifies, the sacrificial lattice is then dissolved, and the revealed vasculature can be perfused with media (red) to keep encapsulated cells alive. Reproduced with permission from Miller et al. (2012).