Poster N12 - Development of a 3D-printed microfluidic model to investigate endothelial crosstalk in breast cancer progression

Introduction: Crosstalk with neighboring vascular endothelial cells can influence the growth and progression of triple-negative breast cancer through the exchange of cellular signals (1). While previous studies have identified that the exchange of cytokines, growth factors, and extracellular vesicles can affect breast cancer and endothelial behavior and phenotype, few studies have investigated the impact of spatial relationships on the crosstalk between breast cancer and endothelium in a 3D environment (2,3). Bioprinting enables replication of key aspects of the 3D microenvironment through precise spatial placement of matrix and cells using bioinks, allowing for the study of cell-cell interactions in a more physiologically-relevant system. A tumor’s influence on endothelial phenotype can be assessed using a bioprinted microfluidic model which allows for the controlled placement of a tumor spheroid in relation to a perfusable vessel. To develop a bioprinted microfluidic model three gelatin/alginate blends (4%/1%, 4%/4%, and 1%/4%) and two GelMA-based bioinks (5% GelMA with and without 0.5% collagen) were investigated for their suitability for extrusion bioprinting and modeling breast cancer-endothelial crosstalk through rheological, mechanical, and biocompatibility characterization. The objective of this study is to determine the suitability of printable hydrogel bioink formulations to recapitulate native breast tissue mechanical properties, maintain triple negative breast cancer and endothelial cell viability, and enable the formation of a perfusable endothelialized channel. These characterization studies will further the development of a model system to study endothelial-cancer crosstalk in the 3D microenvironment.

Materials and

Methods: Bioink formulations were prepared consisting of three gelatin/alginate blends (4%/1%, 4%/4%, and 1%/4%) crosslinkable with 2% CaCl2, and GelMA-based bioinks with 0.5% LAP photoinitiator (5% GelMA with and without 0.5% collagen) crosslinkable with 405nm light. Shear thinning properties of uncrosslinked bioinks were assessed with rheological shear rate sweeps from 10 to 1,000s-1 performed on a 40mm parallel plate DHR-1 rheometer (TA Instruments). The stiffness of crosslinked bioinks was investigated utilizing compression testing by a Universal Testing Machine (MTS Criterion). Print fidelity was assessed by bioprinting the bioinks into lattice structures using the Cellink Bio6X extrusion bioprinter and comparing the bioprinted filaments’ width, length, and intersectional width to theoretical values derived from the CAD design. Cell viability of endothelial (HUVEC) and triple-negative breast cancer (MDA-MB-231) cells was performed by culturing HUVECs on the crosslinked bioink surface while MDA-MB-231s were embedded within the bioinks and then crosslinked. After 24hr, viability was assessed via calcein-AM and ethidium homodimer-1 live/dead staining. A bioprinted microfluidic model was created by plasma bonding PDMS to a glass slide, inserting a 26-30G needle through the PDMS, bioprinting and crosslinking the GelMA bioink, and removing the needle to form a hollow channel that was then seeded with endothelial cells. The fabricated channel was visualized by confocal fluorescence imaging of the matrix bioink stained with FITC and 2µm fluorescent beads (Fluospheres, Thermo Fisher) introduced in the hollow channel. To assess channel endothelization, HUVECs were seeded in the hollow channels and stained after 46hrs with calcein-AM and Hoechst nuclear stain.

Results, Conclusions, and Discussions: Rheological shear rate sweeps of uncrosslinked bioinks revealed that three bioinks (4%/1% and 4%/4% alginate/gelatin and 5%/0.5% GelMA/collagen) exhibited shear-thinning properties (Fig. 1A,B). Compression testing of crosslinked bioinks found that alginate/gelatin bioinks exhibited Young’s moduli of 16-38kPa, whereas GelMA bioinks exhibited moduli closer to normal breast tissue stiffness (2-4 kPa) (Fig. 1C). 1%/4% alginate/gelatin exhibited the highest print fidelity, out of the alginate/gelatin bioink formulations, with the closest filament width, length, and intersectional width to the CAD design values and showed the least bioink spreading. (Fig. 1D). HUVECs exhibited greater cell spreading in addition to high viability (>80%) on the GelMA bioink formulations, while MDA-MB-231 cells showed high cell viability (>80%) in both alginate/gelatin and GelMA bioinks (Fig. 1E, F). These bioink characterization studies suggest that the GelMA bioinks may be better suited for a bioprinted microfluidic model designed to investigate triple-negative breast cancer-endothelial interactions. Successful channel formation was accomplished using a GelMA matrix bioink, and confocal imaging revealed a hollow perfusable channel that allowed fluorescent microspheres to flow through (Fig. 1G). Formed GelMA channels were seeded with HUVEC endothelial cells and showed endothelialization and spreading on the GelMA channel surface, beginning to form the tubular structure of the model vessel (Fig. 1H).

This work lays the foundation to develop a bioprinted microfluidic model to study endothelial and breast cancer interactions. This study assessed multiple different alginate-gelatin and GelMA bioinks for their rheological, mechanical, and biocompatibility properties and suitability for forming endothelialized channels. GelMA bioinks had desirable shear thinning properties for bioiprinting, had similar stiffness to normal breast tissue, and maintained triple negative breast cancer and endothelial cell viability. Furthermore, GelMA-based inks showed successful channel formation that allowed for endothelialization. Future work will assess the endothelial phenotype through immunofluorescence staining of endothelial markers and utilize bioprinting techniques to place breast cancer spheroids at defined distances from the endothelialized channel. Through controlling breast cancer spheroid placement using 3D bioprinting, the bioprinted microfluidic model can be utilized to study how the tumor spatial arrangement can affect endothelial phenotypes that fuel breast cancer progression.

Acknowledgements (Optional): 1. Yin, L., Duan, J.-J., Bian, X.-W. & Yu, S. Triple-negative breast cancer molecular subtyping and treatment progress. Breast Cancer Res 22, 61 (2020).

2. Li, Z.-X. et al. TGF-β1 promotes human breast cancer angiogenesis and malignant behavior by regulating endothelial-mesenchymal transition. Frontiers in Oncology (2022).

3. Yeon, J. H. et al. Cancer-derived exosomes trigger endothelial to mesenchymal transition followed by the induction of cancer-associated fibroblasts. Acta Biomateria (2018).