Mesoscale visualization of three-dimensional microvascular architecture and immunocyte distribution in intact mouse...

Rational: The complex vascular architecture and diverse immune cells of the liver are critical for maintaining liver homeostasis. However, quantification of the network of liver vasculature and immunocytes at different scales from a single hepatic lobule to an intact liver lobe remains challenging. Methods: Here, we developed a fast and fluorescence-preserving transparency method, denoted liver-CUBIC, for systematic and integrated analysis of the microcirculation and the three-dimensional distribution of dendritic cells (DCs)/macrophages in intact liver lobes. Results: Whole-mount imaging at mesoscale revealed that the hepatic classical lobule preferred the oblate ellipsoid morphology in the mouse liver and exhibited hepatic sinusoids with heterogeneous arrangement and intricate loop structure. Liver fibrosis not only induces sinusoidal density increase but also promotes sinusoidal arrangement with increased sinusoidal branch and loop structure. Meanwhile, we found that CD11c⁺ DCs followed a lognormal distribution in the hepatic lobules, skewing toward lobular boundary in steady state. CCl₄-induced chronic liver injury promoted CD11c⁺ DC rearrangement at the lobular border before the formation of liver fibrosis. Furthermore, through whole-mount imaging of tumor-immune cell-vascular crosstalk in intact lobes based on liver-CUBIC, we characterized an accumulation of CX3CR1⁺CCR2⁺F4/80⁺ macrophages at metastatic foci in early colorectal liver metastases. Importantly, colorectal cells secrete CCL2 to mobilize CX3CR1⁺CCR2⁺F4/80⁺ macrophages to accumulate at liver micrometastases, and the interruption of CCL2-induced macrophage accumulation inhibits early colonization of metastasis in the liver. Conclusions: Our investigation of the sinusoidal network and DC/macrophage arrangements through the liver-CUBIC approach and whole-mount imaging provide a powerful platform for understanding hepatic circulatory properties and immune surveillance in the liver.