Whole‐Tumor Clearing and Imaging of Intratumor Microbiota in Three Dimensions with miCDaL Strategy

Acquiring detailed spatial information about intratumor microbiota in situ is challenging, which leaves 3D distributions of microbiota within entire tumors largely unexplored. Here, a modified iDISCO-CUBIC tissue clearing and D-amino acid microbiome labeling-based (miCDaL) strategy are proposed, that integrates microbiota in situ labeling, tissue clearing, and whole-mount tissue imaging to enable 3D visualization of indigenous intratumor microbiota. Leveraging whole-mount spatial resolution and centimeter-scale imaging depth, the 3D biogeography of microbiota is successfully charted across various tumors at different developmental stages, providing quantitative spatial insights in relation to host tumors. By incorporating an immunostaining protocol, 3D imaging of the immunologic microenvironment is achieved in both murine and human mammary tumors that is previously assumed to be bacteria-free. Notably, immune infiltrates, including T cells and NK cells, and tertiary lymphoid structures are conspicuously absent in bacteria-colonized regions. This 3D imaging strategy for mapping Indigenous intratumor microbiota offers valuable insights into host-microbiota interactions.