Engineering an advanced in vitro endochondral ossification model to study cancer metastasis

Metastasis to the bone significantly impacts patient morbidity and mortality for multiple cancers. The lack of in vitro models able to recapitulate bone formation limits the study of bone metastasis in vitro, representing a major obstacle to developing effective therapies. This study aimed to develop a tissue engineered multicellular model of endochondral ossification in vitro, which then incorporated cancer cells under fluid flow to mimic specific aspects of cancer metastasis at a physiological scale. To set a benchmark, we initially evaluated the effect of ex vivo human bone fragments on cancer cell proliferation and migration in vitro. A375 luc/GFP+ melanoma cells were co-cultured with patient-derived ex vivo bone, which significantly stimulated cancer cell proliferation but not migration. Then, as an initial validation to the tissue-engineered approach, mineralised pellets derived from chondrogenically differentiated paediatric human marrow stromal cells (pMSCs) were co-cultured with both A375 luc/GFP+ melanoma and MDA-MB-231 luc/cop-GFP+ breast cancer cells. The mineralised pellets supported in situ proliferation of the cancer cells in a similar range to that observed with human bone, and additionally supported cancer cell migration. Breast cancer cells exhibited a stronger response compared to melanoma cells. Next, we investigated the development of a novel in vitro model of endochondral ossification. Mineralised pMSC pellets were seeded with primary human CD14 + monocytes and osteoclast formation was induced. Then, using a novel in-house designed 3D-culture system with flow, pellets seeded with CD14 + monocytes/osteoclasts were cultured in a fibrin gel containing alginate microparticles and human umbilical vein endothelial cells (HUVECs) and adipose-derived stromal cells (ASCs), as vessel forming cells. MDA-MB-231 luc/cop-GFP+ cancer cells were perfused through a central channel within the construct. Finally, the ability of cancer cells to invade and proliferate within the tissue-engineered in vitro model were measured at different time-points, revealing the potential of this system for mimicking critical aspects of bone cancer metastasis in vitro. This research contributes to the development of new in vitro strategies to recapitulate cancer cell migration to the bone, laying groundwork for the use of tissue-engineered constructs that can mimic different aspects of the bone microenvironment.