A novel bone and cancer cells co-culture methodology to model breast cancer metastasis to bone ex vivo

Abstract

One of the major challenges in late-stage cancer research is studying the direct interactions between cancer cells and the metastatic tissue. Metastasis is an inefficient multistep process in which only a small subset of cancer cells successfully colonizes the new microenvironment. Bone is the most common tissue for breast and prostate cancer metastasis and the third most common site across all cancer types. We present an ex vivo co-culture protocol for modeling bone metastasis that enables direct investigation of cancer cell interactions with the native bone and marrow microenvironment. The procedure involves the extraction and preparation of mouse long bones (femurs and tibiae), injection of cancer cells into the bone marrow cavity, maintenance of the co-culture, and sample processing for downstream analysis. The protocol supports functional readouts, including bioluminescence imaging of tumor cell viability, conditioned media sampling, and downstream tissue analysis, such as immunohistochemistry or micro-computed tomography (µCT). The culture setup procedure can be completed in a single day, and daily media maintenance thereafter requires minimal time. Final sample processing times vary, depending on the analysis completed. The system maintains viable and metabolically active bone, marrow, and cancer cells for up to four weeks and preserves the heterogeneity of the bone microenvironment. It is highly adaptable for various cancer types and bone genotypes and enables high-throughput functional interrogation of cancer colonization in a complex microenvironment. The protocol requires basic expertise in mammalian cell culture and mouse dissection and is suitable for researchers studying metastasis, tumor–stroma interactions, or preclinical therapeutic screening in the bone microenvironment. Not applicable.