Complex Tumor Spheroid Formation and One-Step Cancer-Associated Fibroblasts Purification from Hepatocellular Carcinoma Tissue Promoted by Inorganic Surface Topography

Abstract

In vitro cell models play important roles as testbeds for toxicity studies, drug development, or as replacements in animal experiments. In particular, complex tumor models such as hepatocellular carcinoma (HCC) are needed to predict drug efficacy and facilitate translation into clinical practice. In this work, topographical features of amorphous silicon dioxide (SiO2) are fabricated and tested for cell culture of primary HCC cells and cell lines. The topographies vary from pyramids to octahedrons to structures named fractals, with increased hierarchy and organized in periodic arrays (square or Hexagonal). The pyramids were found to promote complex 2D/3D tissue formation from primary HCC cells. It was found that the 2D layer was mainly composed of cancer-associated fibroblasts (CAFs), while the 3D spheroids were composed of tumor cells enwrapped by a CAF layer. Compared with conventional protocols for 3D cultures, this novel approach mimics the 2D/3D complexity of the original tumor by invading CAFs and a microtumor. Topographies such as octahedrons and fractals exclude tumor cells and allow one-step isolation of CAFs even directly from tumor tissue of patients as the CAFs migrate into the structured substrate. Cell lines form spheroids within a short time. The presented inorganic topographical surfaces stimulate complex spheroid formation while avoiding additional biological scaffolds and allowing direct visualization on the substrate.

3D topographies promote macrophage M2d-Subset differentiation

“…This is the case of inert silicon dioxide (SiO 2 ) substrates decorated with fractal-like structures of varied morphology, i.e., from pyramids and octahedrons to fractals of increased hierarchy organised in periodic arrays. These arrays enable surface topography, which can be arrayed, adopting a hexagonal (Hex) configuration [ 11 ], as shown in Fig. 1 , highlighted by the yellow arrow and hexagon.…”

“…Fractals are known for their high level of organisation, functional morphology, and similarity over a range of dimensional scales. These topographical architectures with increased hierarchy have successfully been used for the differentiation of intestinal tumour cells [ 10 ], as well as the formation of complex tumour spheroids [ 11 ]. Due to their multiscale geometry with a set of characteristic distances ranging from a few micrometers lattice spacing down to 500 nm features [ 12 ], fractal-like inorganic architectures arrayed over a substrate can serve as biomimetics, such as tissue and wound healing applications.…”

3D single-molecule super-resolution imaging of microfabricated multiscale fractal substrates for self-referenced cell imaging

“…As mentioned previously, the microstructured fractal substrates are of particular interest in cell biology due to their capacity to modulate the cell phenotype and behaviour. This was successfully used to isolate cell lines, promote tumor growth and drive cell differentiation in a fast and reliable way [14, 16]. More generally, 3D substrates open promising perspectives to produce complex in vitro models faithfully mimicking cell behaviours in tissues that can be subsequently used for a range of applications, particularly in the domain of drug screening and toxicity measurements.…”

The Fast Track for Intestinal Tumor Cell Differentiation and In Vitro Intestinal Models by Inorganic Topographic Surfaces

“…It is interesting that the differentiating HT29 colon cancer line forms a 2D cell layer. It has previously been observed that primary tumour cells with cancer-associated fibroblasts and hepatocellular carcinoma cell lines such as HLF form on the same G1 spheroids [45]. It seems that on the topographic substrates, the cell growth is supported in its natural form.…”