Developing an Engineered In Vitro Bone Niche for Rapid Cell Adhesion and Early-Stage Interactions of Osteoblasts and Metastatic Breast Cancer Cells

Introduction:: Up to 70% of breast cancer patients develop metastases in bone, causing severe morbidities¹ and reducing 5-year survival rates to < 10%². Osteoclasts can be directly and/or indirectly activated by breast cancer cells, inducing the progression of breast cancer bone metastasis (BCBM) through tumor growth-bone absorption-tumor growth.³ The role of osteoblasts in BCBM is not fully understood. Therefore, it is critical to reconstruct an in vitro platform that mimics the surface chemistry, mineral phase ratio, and hydrophilicity of native bone and explore the interactions between breast cancer cells and pre-osteoblasts of the bone metastatic niche, toward developing new approaches for preventing and treating BCBM.

Materials and Methods:: To establish the engineered bone niche, polylactide (PL) pellets were first completely dissolved in dichloromethane (w:v 1:5). Varied amounts (0 wt% to 60 wt% polymer with 5 wt% increments) of the ceramic powder, nano-β-tricalcium phosphate (nTCP), was then added to the dissolved PL and stirred at 300rpm for 60 min to form a homogenous composite slurry. Meanwhile, the solvent was evaporated from the slurry during stirring. Using a Carver press, the dried slurry was pressed into a thin film at 190 °C under 6800 kg pressure and laser cut into disks (10 mm diameter). Subsequently, the disks were treated with sodium hydroxide (pH = 12) for 30 min to expose the nTCP particles on the surface and evaluated by hydrophilicity assay. The processed disks were eventually used as the engineered bone niche and cleaned for cell culture. MC3T3-E1 preosteoblasts (1×10³) were stained with CellTracker Green were seeded on the disks of group 1 (n = 8) and incubated at 37°C, 5% CO₂. Likewise, CellTracker Red stained MCF-7 breast cancer cells (1×10³) were seeded on group 2 (n = 4) and as well as on half of the MC3T3-E1 (n = 4, dynamically seeding on half of group 1). All the unadhered cells were removed by phosphate-buffered saline rinse (PBS, non-calcium and -magnesium) 1 hour after seeding. The cell numbers were determined with microscope images and processed via ImageJ. Differences in water contact angle (WCA, obtained via hydrophilicity assay) and cell number were assessed by one-way ANOVA with pairwise comparison.

Results, Conclusions, and Discussions:: The engineered bone niche was successfully fabricated. The colors of the disk gradually changed from near transparent to pure white as TCP content increased from 0% to 60% (Fig.1 black banner). The added nTCP and surface treatment significantly improved the hydrophilicity (p < 0.001). The 55%-60% TCP disks yielded the lowest WCA (21.8 ± 4.2° and 18.7 ± 1.8°, no statistical difference between 55% and 60%) (Fig.1. dashed blue line) compared to the rest of the groups (i.e. 57.7 ± 7° and 54.7 ± 3.9° for 0% and 5%, respectively) and the pure non-treated PL (116.4° ± 1.8°)⁴. Similarly, the cell adhesion number and rate were positively correlated to the nTCP content. The 60% disk produced the highest cell adhesion number and rate 1 hour after seeding (1051 ± 113; 105% and 778 ± 36; 78% for MC3T3 and MCF-7 cells, respectively, p < 0.001) in comparison to the lower nTCP content (Fig.1. green and red bars). The cell adhesion time of MC3T3 and MCF-7 cells took 3 days and 24 hours.⁵ Therefore, the disks with 60% nTCP significantly shortened the cell adhesion time for both cell lines, which was clearly caused by the addition of osteoconductive nTCP particles and improved hydrophilicity. More importantly, the 60% nTCP in the composite was similar to the native bone which comprises 60-65% mineral phase⁶. Additionally, the number of MCF-7 cells dynamically captured on the MC3T3-conditioned disks (134 ± 33) was significantly higher than the non-MC3T3 conditioned disks (44 ± 8) (p < 0.001) (Fig.2.), which aligned with a study suggesting osteoblasts-rich areas might serve as mediators of circulating breast cancer cells homing in trabecular bone⁷.

In this study, the engineered metastatic bone niche (PL with 60% nTCP) provided a feasible and effective in vitro testing platform as a model of combined bone tissue and tumor for in-depth BCBM evaluations, e.g. early detection, intervention prevention, and treatment. Investigations of the modified MC3T3-conditioned environment, 3D cell culture in a printed scaffold with the same material, surface treatment, and controlled release of therapeutic drugs are ongoing to clarify the interactions of osteoblasts and breast cancer cells.


Acknowledgements (Optional): : Funding for this work was provided by the Harbor Lights Endowment.

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