(L-471) MECHANO-BIOLOGICAL EFFECTS OF THE EXTRACELLULAR MATRIX ON BREAST TUMOR CELL INVASION

Introduction:: The stiffness of breast tumors increases gradually from core to the periphery of the tumor [1]. Increased breast density on mammography, a finding associated with greater content of collagens, correlates with increased risk of breast cancer progression [2]. However, increased mammographic density does not carry an increased risk for death. Instead, softer and more mechanically compliant tumors may actually pose a greater risk to patients [3]. Therefore, our objective was to develop a 3D hydrogel with tunable mechanical stiffness within a range relevant to human breast tumors (0.5-20 KPa) while maintaining the composition of the ECM constant. We used this hydrogel model to study effects of ECM stiffness on epithelial-to-mesenchymal transition (EMT) and local invasion of breast cancer cells.


Materials and Methods:: Hybrid collagen-alginate hydrogels were prepared by mixing Collagen Type I (3 mg/ml), low viscosity alginic acid sodium salt (5 mg/ml), 10X EMEM, and cell culture medium with or without cells. We adjusted the pH of the solution to neutral and added three different molar concentrations of CaCl₂ (3.75 mM, 7.5 mM and 15 mM) to prepare hydrogels of different stiffness values, labeled soft, middle, and stiff, respectively [4]. Microstructures of the hydrogels were examined by freeze drying them and imaging them with a scanning electron microscope (SEM). Permeability of the hydrogels were studied by measuring the diffusion of fluorescein isothiocyanate-labeled dextran (FITC-dextran) (M_w=70 kDa) through hydrogels over time. Metabolic activity of the VARI-068 (triple negative breast cancer) cells transfected with Click Beetle Green (CBG) dispersed in hydrogels of different stiffness values was measured using a luciferin–luciferase bioluminescence assay over a 7-day period. Spheroids of VARI-068 cells were prepared using an aqueous two-phase system (ATPS) technology [5]. Spheroids were encapsulated in hybrid hydrogels of three different stiffness values and harvested on day 4 for gene expression analysis of EMT markers using qPCR. The fold change in mRNA expression was determined as 2^-ΔΔCt and normalized to β-actin and GAPDH.


Results, Conclusions, and Discussions:: SEM images showed changes in microstructures of the hydrogels at different calcium crosslinking concentrations, interconnections of collagen fibrils, and alginate flakes (fig 1a). All hydrogels had well-organized pore structures. Gels crosslinked with the two lower concentrations of CaCl₂ (3.75 and 7.5 mM) contained more round pores. At the highest concentration of CaCl₂ of 15 mM, calcium ions crosslinked the alginate monomers and resulted in more compressed, flat pores. Diffusive transport of a macromolecule (FITC dextran) through the hydrogels indicates the permeability of hydrogel. The fluorescence intensity of the FITC dextran released through the hydrogel increased over time. We found no significant effect of calcium ion concentration on FITC-dextran diffusion from the hybrid hydrogels (fig 1b). This suggest that all three hydrogels have a similar porosity to facilitate transport of reagents.

The luminescent signal from cells gradually increased over the 7-day culture (fig 2a). Cells in the stiff hydrogels gave significantly greater signal, suggesting higher proliferation of cells in the stiff matrix. qPCR results showed that the stiff hydrogels significantly upregulated EMT gene markers SNAIL and ZEB2 by 4-fold and E47 by 3-fold (fig 2b). Considering that cancer cells adopt an epithelial-to-mesenchymal transition (EMT) to invade through ECM and metastasize [6], upregulation of several EMT markers in the stiff ECM suggests that a stiffer tumor stroma promotes local invasion of TNBC cells. We are currently performing studies to validate the role of ECM stiffness on TNBC invasiveness.

In conclusion, we developed porous and biocompatible collagen-alginate hybrid hydrogels with adjustable stiffness by varying the cross-linker concentration while keeping the collagen content constant. The hydrogels facilitated transport of nutrients and oxygen to the encapsulated cancer cell spheroids to promote cell viability and proliferation. Increased ECM stiffness elevated the expression of prominent EMT markers in TNBC cells. Overall, the hybrid hydrogels will enable mechanistic studies of effects on ECM stiffness on cancer cell invasiveness and the underlying signaling mechanisms.


Acknowledgements (Optional): : Funding was provided by NSF grant 2140104 and NIH grant CA225549.


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