Fluorescence lifetime FRET imaging reveals spatial heterogeneity of antibody-HER2 engagement in breast cancer models

Effective engagement of therapeutic antibodies with their molecular targets in solid tumors is strongly influenced by the tumor microenvironment. In particular, collagen-rich stroma can impede antibody transport and limit receptor binding. Using near-infrared macroscopic fluorescence lifetime imaging–based Förster resonance energy transfer (NIR MFLI-FRET) in vivo, we previously demonstrated reduced binding of the anti-HER2 antibody trastuzumab (TZM) in HER2-positive SKOV-3 ovarian tumor xenografts compared with HER2+ AU565/XTM breast tumor xenografts, despite comparable receptor expression, and showed that these differences correlated with increased collagen content. In the present study, we extend lifetime-based FRET imaging to the mesoscale using time-domain mesoscopic fluorescence molecular tomography (TD-MFMT) to spatially map TZM–HER2 engagement across whole excised tumors. Ex vivo TD-MFMT imaging of AU565-XTM and SKOV-3 tumors was performed to generate fluorescence intensity and FRET-derived donor fraction (FD Fraction) maps, which report functional antibody–receptor binding. Quantitative histological analyses, including Masson’s trichrome staining and immunohistochemistry, were used to assess collagen distribution and validate imaging findings. TD-MFMT revealed significantly lower FD Fraction values in SKOV-3 tumors relative to AU565-XTM tumors, indicating reduced TZM–HER2 engagement. Pixel-wise analysis across tumor diameters showed relatively uniform FD Fraction distributions from periphery to center in AU565-XTM tumors, whereas FD Fraction was consistently suppressed throughout SKOV-3 tumors. These spatial binding patterns were inversely correlated with collagen abundance. Together, these results demonstrate that TD-MFMT-based mesoscale FLI-FRET imaging enables whole-tumor mapping of therapeutic antibody–receptor engagement and provides spatial insight into how collagen-rich tumor microenvironments may restrict TZM binding in HER2-positive tumors.