A Co-culture Cell-Based Reporter Assay for Quantitative Measurement of Integrin αvβ8–Mediated Activation of Latent TGF-β1

Abstract Integrin αvβ8 is a major activator of latent transforming growth factor-β (TGF-β) and an emerging therapeutic target in cancer and immune regulation. However, functional assays that directly measure αvβ8-mediated activation of latent TGF-β in a physiologically relevant context remain limited. Here, we report a co-culture cell-based reporter assay for quantitative measurement of αvβ8-mediated activation of latent TGF-β1. NIH/3T3 reporter cells were engineered to express a SMAD-responsive NanoLuc reporter, constitutive firefly luciferase for internal normalization, and cell-surface GARP–latent TGF-β1. When co-cultured with αvβ8-expressing LN-229 cells, reporter cells produced a robust signal that directly reflected localized latent TGF-β1 activation. The assay demonstrated stable expression of the required biological components, reproducible signal-to-background performance, and sensitivity to benchmark αvβ8-blocking antibodies. Inhibition studies showed potent dose-dependent blockade by an anti-αvβ8 antibody. In contrast, pan-TGF-β neutralizing antibody displayed markedly weaker apparent potency, suggesting that targeting localized αvβ8-mediated activation is more effective than neutralizing released TGF-β in this assay context. The assay also enabled screening and ranking of anti-αvβ8 antibodies, identifying several high-potency clones, and detected αvβ8-mediated activation of a non-cleavable latent TGF-β1 mutant. This platform provides a sensitive, internally normalized, and scalable approach for mechanistic studies and therapeutic discovery targeting the αvβ8–TGF-β axis.