Quantitative Imaging of Hypoxic CAIX-Positive Tumor Areas with Low Immune Cell Infiltration in Syngeneic Mouse Tumor Models

[Image: see text] Limited diffusion of oxygen in combination with increased oxygen consumption leads to chronic hypoxia in most solid malignancies. This scarcity of oxygen is known to induce radioresistance and leads to an immunosuppressive microenvironment. Carbonic anhydrase IX (CAIX) is an enzyme functioning as a catalyzer for acid export in hypoxic cells and is an endogenous biomarker for chronic hypoxia. The aim of this study is to develop a radiolabeled antibody that recognizes murine CAIX to visualize chronic hypoxia in syngeneic tumor models and to study the immune cell population in these hypoxic areas. An anti-mCAIX antibody (MSC3) was conjugated to diethylenetriaminepentaacetic acid (DTPA) and radiolabeled with indium-111 ((111)In). CAIX expression on murine tumor cells was determined using flow cytometry, and in vitro affinity of [(111)In]In-MSC3 was analyzed in a competitive binding assay. Ex vivo biodistribution studies were performed to determine in vivo radiotracer distribution. CAIX(+) tumor fractions were determined by mCAIX microSPECT/CT, and the tumor microenvironment was analyzed using immunohistochemistry and autoradiography. We showed that [(111)In]In-MSC3 binds to CAIX-expressing (CAIX(+)) murine cells in vitro and accumulates in CAIX(+) areas in vivo. We optimized the use of [(111)In]In-MSC3 for preclinical imaging such that it can be applied in syngeneic mouse models and showed that we can quantitatively distinguish between tumor models with varying CAIX(+) fractions by ex vivo analyses and in vivo mCAIX microSPECT/CT. Analysis of the tumor microenvironment identified these CAIX(+) areas as less infiltrated by immune cells. Together these data demonstrate that mCAIX microSPECT/CT is a sensitive technique to visualize hypoxic CAIX(+) tumor areas that exhibit reduced infiltration of immune cells in syngeneic mouse models. In the future, this technique may enable visualization of CAIX expression before or during hypoxia-targeted or hypoxia-reducing treatments. Thereby, it will help optimize immuno- and radiotherapy efficacy in translationally relevant syngeneic mouse tumor models.