Metastatic Breast-Bone Carcinoma Model Obtained through 3D Bioprinting

Breast cancer is the most frequently diagnosed cancer in the female population with about 50,000 new cases/year in Italy. Despite the significant improvement in survival rate following diagnosis of this type of cancer, there is still a clinically significant number of patients who develop distant metastases (about 12,000 new cases of metastatic breast cancer/year in Italy, with a prevalence of about 36,000 cases). Bone is the most frequent site of metastatization given the elective osteotropism of breast cancer. To recreate this pathology, we develop a cell culture system, using a sophisticated tissue engineering and 3D bioprinting approach, to recreate a model in vitro of human bone tissue and human breast tumor, which was connected in order to mimic a metastatic environment. First we develop the two models independently. The human breast cancer model was established using a commercial alginate ink enrich with laminin mixed with MDA-MB-231 cells, a human breast cancer cell line in metastatic phase, MCF-10A cells, a non-tumorigenic mammary epithelial cell line, and decellularized human breast tissue powder to better recreate a tumor-specific extracellular matrix microenviroment. This model has been cultured for 3 weeks, during which we observed that a part of the cells migrated toward the decellularized matrix in the ink, forming clusters around and inside of the matrix fragments. Additionally, some cells migrated out of the ink, showing attraction for the bone model construct. Human bone model was developed by using two distinct approaches. The first involved seeding human mesenchymal stem cells (hMSCs) onto beta-tricalcium-phosphate (bTCP) granules aggregated with TissueCol hydrogel. The second bone model was fabricated using 3D bioprinting to create a scaffold composed of an alginate-based ink enriched with tricalcium-phosphate and hydroxyapatite, combined with hMSCs. Both bone models were cultured for 8 weeks in an osteogenic medium, and we obtained a tissue-like structure, characterized by organized cellular networks and active extracellular matrix production (with collagen deposition and calcium mineralization). The final part of the study consist on modelling metastasis. A co-culture system was established using a transwell with the bioprinted bone model placed in the lower chamber and the breast pag. 6 cancer model in the upper chamber. GFP-labeled MDA-MB-231 cells were used to track the migration of cancer cells toward the bone model. The results demonstrated a preferential attraction of breast cancer cells toward osteogenically differentiated bone constructs, supporting the concept of bone-specific tropism and highlighting the active role of the bone microenvironment in metastatic priming. Overall, this work demonstrates that a modular, humanized 3D bioprinted system can successfully recapitulate key structural, biochemical, and functional aspects of breast cancer bone metastasis. This platform represents a reproducible and ethically sustainable alternative to animal models and provides a promising tool for studying metastatic mechanisms and for future applications in anti-metastatic drug testing and translational cancer research.