Extracellular matrix regulation of cell spheroid invasion in a 3D bioprinted solid tumor-on-a-chip

Abstract

Tumor organoids and tumors-on-chips can be built by placing patient-derived cells within an engineered extracellular matrix (ECM) for personalized medicine. The engineered ECM influences the tumor response, and understanding the ECM-tumor relationship accelerates translating tumors-on-chips into drug discovery and development. In this work, we tuned the physical and structural characteristics of ECM in a 3D bioprinted soft-tissue sarcoma microtissue. We formed cell spheroids at a controlled size and encapsulated them into our gelatin methacryloyl (GelMA)-based bioink to make perfusable hydrogel-based microfluidic chips. We then demonstrated the scalability and customization flexibility of our hydrogel-based chip via engineering tools. A multiscale physical and structural data analysis suggested a relationship between cell invasion response and bioink characteristics. Tumor cell invasive behavior and focal adhesion properties were observed in response to varying polymer network densities of the GelMA-based bioink. Immunostaining assays and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) helped assess the bioactivity of the microtissue and measure the cell invasion. The RT-qPCR data showed higher expressions of HIF-1α, CD44, and MMP2 genes in a lower polymer density, highlighting the correlation between bioink structural porosity, ECM stiffness, and tumor spheroid response. This work is the first step in modeling STS tumor invasiveness in hydrogel-based microfluidic chips. STATEMENT OF SIGNIFICANCE: We optimized an engineering protocol for making tumor spheroids at a controlled size, embedding spheroids into a gelatin-based matrix, and constructing a perfusable microfluidic device. A higher tumor invasion was observed in a low-stiffness matrix than a high-stiffness matrix. The physical characterizations revealed how the stiffness is controlled by the density of polymer chain networks and porosity. The biological assays revealed how the structural properties of the gelatin matrix and hypoxia in tumor progression impact cell invasion. This work can contribute to personalized medicine by making more effective, tailored cancer models.

Keywords: Bioprinting; Gelatin; Mechanobiology; Microstructure; Solid tumor spheroid.

Figure 1.

The fabrication process: A. Soft tissue sarcoma (STS) spheroid formation and encapsulation in low-attachment well-plates; B. Multi-material bioprinter used to extrude the 3D hydrogel construct; C. Chemistry of gelatin methacryloyl (GelMA) functionalized with methacrylic anhydride; D. Bioprinting strategy for our 3D hydrogel microfluidic chip using spheroid embedded GelMA and sacrificial ink showing the details and parts and more sophisticated perfusable channel patterns created through our customized g-code tool; E. Two bioprinted chips and the experimental setup to run fluid over time; F. Bioprinted spheroid-laden hydrogel chip and spheroid location at Day 0.

Figure 2.

A. Average stress vs. strain curves for each GelMA variation (i), average Young’s modulus for each GelMA permutation (ii); B. Swelling results of the three different concentrations (5, 7, and 10% w/v) showing the time history (i), and percentages (ii), along with biodegradation results (iii); C. Porosity imaging for the three different concentrations (5, 7, and 10% w/v) through SEM (scale bar is 100 μm); D. Comparative illustration demonstrating the impact of increased GelMA concentration on the biophysical properties of crosslinked GelMA. The data sets are presented as mean ± s.d., n = 4.

Figure 3.

Cell behavior in our bioprinted STS model: A. Smallest enclosing circle diameters of HT1080 spheroids embedded in GelMA at Day 5 of observation in three different GelMA concentrations; B. The bright field image of cell invasion behavior over time in 5% GelMA at Day 5 for an example (~ 400 μm at Day 1); C. Processed images in MATLAB using convex hull and smallest enclosing circle algorithms of spheroids; D. HIF-1α, CD44, MMP2, and MMP9 mRNA expression values normalized to β-actin expression (the data sets are presented as mean ± s.e., n = 3).

Figure 4.

Selected cell biomarkers in a bioprinted soft tissue sarcoma (STS) model. A. At Day 5, sarcoma cells have attached to the GelMA and migrated away from the 3D spheroids. This migration highlights certain cell components: Phalloidin is stained in red, and vinculin is stained green. DAPI is stained in blue. The scale bar is 50 μm; B. Images focus on tumor cell proliferation within the GelMA environment. Ki-67 is stained red, and the cell nuclei are stained with DAPI in blue. The accumulations of proliferative cells at the periphery of the migrated area in the 5% and 7% GelMA concentrations are shown; C. Relative intensity plot of focal adhesion markers; D. Relative intensity plot of Ki-67 staining.