Multiplex Three-Dimensional Mapping of Macromolecular Drug Distribution in the Tumor Microenvironment

Macromolecular cancer drugs such as therapeutic antibodies and nanoparticles are well known to display slow extravasation and incomplete penetration into tumors, potentially protecting cancer cells from therapeutic effects. Conventional assays to track macromolecular drug delivery are poorly matched to the heterogeneous tumor microenvironment, but recent progress on optical tissue clearing and three-dimensional (3D) tumor imaging offers a path to quantitative assays with cellular resolution. Here, we apply transparent tissue tomography (T3) as a tool to track perfusion and delivery in the tumor and to evaluate target binding and vascular permeability. Using T3, we mapped anti-programmed cell death protein-ligand 1 (PD-L1) antibody distribution in whole mouse tumors. By measuring 3D penetration distances of the antibody drug out from the blood vessel boundaries into the tumor parenchyma, we determined spatial pharmacokinetics of anti-PD-L1 antibody drugs in mouse tumors. With multiplex imaging of tumor components, we determined the distinct distribution of anti-PDL1 antibody drug in the tumor microenvironment with different PD-L1 expression patterns. T3 imaging revealed CD31(+) capillaries are more permeable to anti-PD-L1 antibody transport compared with the blood vessels composed of endothelium supported by vascular fibroblasts and smooth muscle cells. T3 analysis also confirmed that isotype IgG antibody penetrates more deeply into tumor parenchyma than anti-Her2 or anti-EGFR antibody, which were restrained by binding to their respective antigens on tumor cells. Thus, T3 offers simple and rapid access to 3D, quantitative maps of macromolecular drug distribution in the tumor microenvironment, offering a new tool for development of macromolecular cancer therapeutics.