Ultrasound-Responsive Biomaterials for Controlled Genetic Manipulation of 3D-Biofabricated Tumor Models with Vascular Structures

Introduction: : Crosstalk with nearby endothelial cells can influence breast cancer progression through the exchange of cellular signals. While studies have identified that cytokines and extracellular vesicles play a role in this crosstalk,[1,2] few have studied how the 3D environment and spatial relationships affect this crosstalk between early-stage breast cancer and the endothelium. To address this, 3D biofabricated in vitro models are needed that can control the placement of breast epithelial spheroids in relation to the endothelium and allow controlled activation of oncogenes to simulate early-stage disease. Spatially-controlled genetic manipulation of oncogenes in the spheroid cells is a critical tool to initiate the expression of cancer phenotypes in the model and to begin studying the resulting crosstalk between cell types in early-stage breast cancer. However, traditional transfection methods face diffusional barriers in the thick hydrogel matrices that are used in these 3D model systems. Our group has shown that ultrasound-responsive gene delivery microbubbles incorporated into hydrogel matrices can spatiotemporally control gene delivery in 3D bioprinted constructs[3]. However, it is unknown if the hydrogel material properties, which can impact printability and cellular behavior, are altered when ultrasound-responsive microbubbles are embedded in hydrogel matrices. The objectives for the study are to develop a 3D vascular model that allows for controlled placement of breast epithelial spheroids and enables ultrasound-activated oncogene delivery to the spheroids (Fig. 1A), and to characterize the responsive matrix material properties. 3D biofabricated vascular models with ultrasound-controlled oncogene delivery can facilitate studying early-stage breast cancer progression and the influence of endothelial crosstalk.

Materials and

Methods: : Hydrogel bioink formulations consisting of alginate (4%, 6%, 8% w/v), crosslinkable with 2% CaCl2, and photocrosslinkable gelatin methacryloyl (GelMA, 5%, 7.5%, 10% w/v) with 0.5% LAP photoinitiator were utilized in characterization studies. The material properties of hydrogels with and without embedded gas-core lipid-shell ultrasound-responsive microbubbles at increasing concentrations were assessed for shear-thinning properties, gelation rate, and stiffness. To assess shear-thinning properties of uncrosslinked bioinks, shear rate sweeps from 0.1 to 1,000 s-1 were performed on a DHR-1 rheometer. Gelation point was assessed by performing time sweeps and introducing a crosslinker, 2% CaCl2, for alginate bioinks or exposure to 5 mW 402 nm light for GelMA bioinks. The stiffness of crosslinked bioinks was assessed via compression testing with a Universal Testing Machine. To assemble an in vitro vascular model, a PDMS chip was fabricated, and a needle was inserted into the device, the reservoir was filled with GelMA bioink and photocrosslinked, and the needle was removed, revealing a hollow channel that was then seeded with human umbilical vein endothelial cells (HUVECs) to form the model vasculature. To control the spatial distance of breast epithelial spheroids and the vasculature, the CellCelector Flex aspiration-assisted bioprinting system was used to place MCF10A breast epithelial spheroids at controlled distances from the needle. Alginate bioinks were printed by support-bath mediated methods with HEK293T cells or MCF10A spheroids embedded with ultrasound-responsive microbubbles coupled with GFP or HER2 overexpression plasmid. Focused ultrasound (2.25 MHz) was applied centered within the bioprinted constructs.

Results, Conclusions, and Discussions:: Our platform design contains a biofabricated endothelialized vessel, with breast epithelial spheroids printed at controlled distances, and ultrasound-responsive microbubbles with electrostatically-bound DNA. Applied ultrasound activates microbubbles in the ultrasound focal zone resulting in the transfection of cells in the breast epithelial spheroid to stimulate early-stage breast cancer oncogenic overexpression (Fig. 1A). Fabricated microbubbles are 1-5 µm in diameter (Fig. 1B,C) which is in the optimal range for ultrasound-responsivity and can be incorporated into hydrogels. No changes in shear-thinning properties, crosslinking kinetics, or stiffness were observed in alginate and GelMA hydrogels with embedded microbubbles (Fig. 1D-G). A 3D printed mold was used to fabricate a PDMS device (Fig. 1J), a needle was inserted, and GelMA was crosslinked (Fig. 1K) with subsequent needle removal to form a hollow channel (Fig. 1L). The channel was endothelialized and exhibited high cell viability, endothelial spreading, and lumen formation (Fig. 1M). The model’s design allowed for breast epithelial spheroid placement at controlled distances from the channel (Fig. 1N). To demonstrate ultrasound activation, alginate filaments with HEK293T cells or MCF10A spheroids and ultrasound-responsive microbubbles were bioprinted into lattice structures (Fig. 1O,P). After exposure to focused ultrasound, microbubbles were activated resulting in their rupture within the focal zone (Fig. 1Q,R). After 48 hrs, GFP or HER2-expressing cells were observed in the ultrasound-exposed regions (Fig. 1S,T).

This work lays the foundation for a 3D vascular model that allows controlled placement of breast epithelial spheroids and the utilization of ultrasound-responsive gene delivery microbubbles for spatiotemporal control of oncogene expression. Ultrasound-responsive microbubbles had no significant influence on alginate and GelMA material properties. A 3D-printed vascular model was fabricated with GelMA hydrogels that enabled channel formation and allowed the controlled placement of breast epithelial spheroids. In bioprinted constructs, bioinks containing ultrasound-responsive microbubbles showed successful spatiotemporally-controlled transfection of cells and spheroids in the ultrasound focal zone. Ongoing work will utilize ultrasound-responsive gene delivery bioinks in the vascular model to spatiotemporally control oncogene overexpression in breast epithelial spheroids. This platform will enable studying early-stage breast cancer interactions with the endothelium, providing a new tool for modeling early-stage disease progression.

Acknowledgements and/or References (Optional):: 1. Li, Z.-X. et al. TGF-β1 promotes human breast cancer angiogenesis and malignant behavior by regulating endothelial-mesenchymal transition. Frontiers in Oncology (2022).
2. Yeon, J. H. et al. Cancer-derived exosomes trigger endothelial to mesenchymal transition followed by the induction of cancer-associated fibroblasts. Acta Biomateria (2018).
3. Lowrey MK, et al. Remote-Controlled Gene Delivery in Coaxial 3D-Bioprinted Constructs using Ultrasound-Responsive Bioinks. Cell Mol Bioeng. (2024).