Abstract 658: Establishment of a novel ex vivo bone metastasis culture platform for triple-negative breast cancer.

Abstract Drug development for bone metastasis has been hindered by the lack of appropriate in vitro models in early testing. Typical in vitro models lack the crucial bone microenvironment, leading to wrong conclusions about efficacy on bone metastases in vivo. We have established a novel bone metastasis ex vivo culture platform including bone discs from tibiae of mice with 4T1 triple-negative breast cancer (TNBC) tumors. Female Balb/c mice were injected intratibially with 10,000 (10k) or 20,000 (20k) 4T1 mouse TNBC cells or PBS (control). Tumor-induced bone changes were monitored by X-ray imaging. 4T1 tumor and control bones were collected and cut into 2-3 mm thick discs, rinsed with antibiotic-containing medium, and cultured in basal medium (RPMI-1640 medium supplemented with 10% FBS) in a humidified incubator. Osteoblastic medium included ascorbic acid and beta-glycerophosphate, and osteoclastic medium included M-CSF and RANKL supplements. The culture medium was partially replenished every 72 hours. Culture lengths from 7-21 days were tested and analyzed for cell viability (trypan blue), proliferation (CCK-8 assay), bone resorption (TRACP5b ELISA) and bone formation (PINP EIA and Alizarin S Red staining). Intratibial inoculation of 10k and 20k 4T1 cells induced progressive osteolytic lesions that were monitored for 13 days by X-ray imaging. Based on the extent of osteolytic lesions, days 3-5 were selected to model early bone metastasis growth with visible tumor-induced effects on bone, and 20k 4T1 cells were selected for future studies due to more uniform tumor growth. In ex vivo cultures of tibia bone discs in basic medium, cell viability remained high at days 7 and 10 (88 and 91%, respectively) but dropped to 47% at day 14. Therefore, 10 days was considered maximum culture length. In discs with 4T1 tumors, cell doubling time was higher than in control discs. Addition of osteoblastic supplements increased PINP levels in control discs and in discs including 4T1 tumors at day 10, after which the values decreased. Increased Alizarin Red staining was observed at day 10. Addition of osteoclastic supplements increased TRACP5b levels at days 10 and 14, after which the values decreased. Similar increase was not observed when adding both osteoblastic and osteoclastic supplements. Discs with 4T1 tumors had higher baseline TRACP5b levels than control discs, and the values were further increased with osteoclastic and both osteoclastic and osteoblastic supplements. We have established a novel ex vivo bone metastasis culture platform where cancer cell, osteoclast and osteoblast activities were maintained after in vivo extraction of bone discs, indicating that the ex vivo cultures were able to maintain in vivo properties of the tumor and its bone microenvironment. We conclude that this platform provides a miniaturized, labor and cost-effective way for preliminary testing of effects of experimental therapies on bone metastases. Citation Format: Tiina E. Kähkönen, Saif Wahid, Sangita Kushary, Debabani Roy Chowdhury, Arnab Roy Chowdhury, Jussi M. Halleen. Establishment of a novel ex vivo bone metastasis culture platform for triple-negative breast cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 658.