Abstract
Abstract Bone microenvironment is an important factor in regulating bone metastasis. When cancer cells home to bone they secrete factors that stimulate osteoclast activity leading to increased bone resorption. Stimulatory factors released from the resorbed bone matrix in turn promote proliferation of cancer cells and this process, referred as the vicious cycle, often leads to osteolytic lesions in bone when osteoclastic bone resorption exceeds osteoblastic bone formation. The vicious cycle observed in osteolytic disease occurs also in osteoblastic metastases, and in addition cancer cells produce osteoblast-stimulating factors. Cancers with bone metastases often include both osteolytic and osteoblastic lesions. Our aim was to establish in vitro cell culture models to study the effects of cancer therapeutics on osteoblast and osteoclast differentiation and activity. In the osteoclast differentiation assay, human osteoclast precursor cells (Lonza) were cultured on bovine bone slices for 7 days. Denosumab was added in the cultures at day 0, and tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) activity was measured from the culture medium collected at day 7. Osteoclast activity was studied by allowing osteoclasts resorb bone after 7 days’ maturation period for additional 3 days in the presence of odanacatib. The amount of C-terminal cross-linked telopeptides of type I collagen (CTX-I) was measured in the culture medium collected at day 10 to quantitate osteoclast activity. Denosumab and odanacatib showed strong concentration dependent inhibition of osteoclast differentiation and activity, respectively. A mouse osteoblast progenitor cell line KS483 (Pharmatest Services) was used to study differentiation and activity of osteoblasts with test compounds bone morphogenetic protein 2 (BMP-2) and estradiol (E2). In the osteoblast differentiation assay, the activity of cellular alkaline phosphatase (ALP), a marker of osteoblast differentiation, was measured at day 8. In the osteoblast activity assay, the cells were cultured for 13 days. N-terminal propeptide of type I procollagen (PINP) secreted into the culture medium was determined at day 11 to indicate the effects on organic bone formation and calcium deposition was determined at the end of the study as a marker of inorganic bone formation. BMP-2 and E2 stimulated osteoblast differentiation and activity indicated by the increase in ALP activity, PINP and calcium levels. We conclude that these culture systems can be used for studying the effects of cancer therapeutics and identifying new potential compounds affecting the disease process of bone metastases with different mechanisms of action. Citation Format: Jenni H. Mäki-Jouppila, Mervi Ristola, Jukka Rissanen, Katja Fagerlund. In vitro assays for investigating the effects of cancer therapeutics on bone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2920.
Published Version
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