Recombinant LGR4 extracellular domain attenuates osteoclastogenesis and prevents bone metastasis via RANKL inhibition

Bone metastasis is a severe complication of advanced cancers, disrupting bone remodeling by hijacking the RANK/RANKL/OPG axis, which leads to excessive osteoclast activation and bone resorption. Current therapies, such as bisphosphonates and denosumab, offer limited survival benefits and carry significant safety risks, highlighting the need for novel agents that effectively inhibit osteolytic progression.

This study aimed to evaluate the therapeutic potential of a recombinant extracellular domain of leucine-rich repeat-containing G-protein coupled receptor 4 (rLGR4-ECD), produced in CHO cells, to inhibit RANKL signaling, suppress osteoclastogenesis, and prevent bone metastasis in preclinical models.

The human LGR4-ECD (residues 25-540) was cloned into the pcDNA4/TO vector and expressed in CHO-K1 cells. Stable monoclonal lines were established, and recombinant protein was purified via Ni-NTA affinity chromatography. Binding affinity to RANKL and RSPO4 was assessed by co-immunoprecipitation (co-IP). In vitro, bone marrow-derived monocytes were treated with RANKL and varying concentrations of rLGR4-ECD (0-80 ng/mL), and osteoclast formation was evaluated by TRAP staining, qPCR, and Western blotting for osteoclast markers. For in vivo evaluation, a breast cancer bone metastasis model was established by intracardiac injection of 4 T1-Luc cells into BALB/c mice. Mice were treated daily with 1 mg/kg rLGR4-ECD or PBS via tail vein injection. Metastatic burden was monitored by bioluminescence imaging, osteoclast activity by TRAP staining of calvaria, and gene expression in bone tissue by qPCR. Survival was analyzed using Kaplan-Meier curves.

rLGR4-ECD was successfully expressed and purified with over 95% purity and a yield of 2.38 mg/L. It bound specifically to RANKL and RSPO4 in co-IP assays. In vitro, rLGR4-ECD dose-dependently inhibited osteoclast formation, with near-complete suppression at 80 ng/mL (IC50 = 42.38 ng/mL). Expression of osteoclast genes Acp5, Ctsk, and Nfatc1 was reduced by 91.4% (***p < 0.001), 87.1% (***p < 0.001), and 81.2% (***p < 0.001), respectively, at 80 ng/mL. Protein levels of TRAP, Cathepsin K, and NFATc1 were also significantly suppressed. In vivo, rLGR4-ECD treatment reduced metastatic burden by 67.3% (***p < 0.001) and decreased osteoclast surface per bone surface by 56.2% (***p < 0.001). Gene expression in bone tissue showed reductions of 71.3% for Acp5 (***p < 0.001), 40.1% for Ctsk (***p < 0.001), and 86.5% for Nfatc1 (***p < 0.001). rLGR4-ECD median survival by 41.7% (12 vs. 17 days) without affecting lung metastasis.

rLGR4-ECD effectively inhibits RANKL-induced osteoclastogenesis and reduces bone metastasis burden through specific suppression of osteoclast activity, leading to improved survival in a preclinical model. Its excellent efficacy and manufacturability support its potential as a novel therapeutic agent for bone metastatic disease.