In vitro development of KRAS^G12D-driven CD11c⁺F4/80⁺CD207⁺cells. (A) BrdU incorporation of freshly isolated AdCre/KRAS^G12D lung cells enriched for CD11c. Freshly harvested/enriched CD11c⁺ cells were cultured for 18 hours with BrdU, and analyzed by flow cytometry. Most CD11⁺ cells are BrdU⁻, though substantial BrdU uptake was detected in CD11c^low cells (indicated in blue). (B) In vitro development of KRAS^G12D-expressing floating cell clusters in culture of AdCre/KRAS^G12D lung cells depleted for CD11c⁺ cells. Top, phase-contrast images of the culture at 2 weeks (left), 4 weeks (middle), and 7 weeks (right), showing floating cell cluster development during the culture. Original magnification ×200. Bottom, analyses of the culture at 7 weeks. Morphology of floating cells (left, Giemsa staining; scale bar, 10 μm), CD31⁺ endothelial network formation (middle, CD31 IHC; scale bar, 50 μm), and recombination of the KRAS^G12D allele in floating and adherent cells detected by PCR (left, with no-template/KRAS^WT controls) are indicated. The recombined KRAS^G12D allele (Lox-G12D) is clearly detected in the floating cells with much higher recombination rates than that in the adherent cells. (C) Flow cytometry analysis of the floating cells developing in CD11c-depleted AdCre/KRAS^G12D lung cell cultures at 7 weeks. Representative contour plots for cell surface CD45/CD11b (left), BrdU incorporation (18-hour labeling; second left) and surface CD11c/F4/80 (middle) are indicated. Cell surface CD207 expression in CD11c^highF4/80^high and CD11c^lowF4/80^low populations (shown in the middle CD11c/F4/80 plot in green and blue, respectively) are indicated in histogram plots in the right (green for CD11c^highF4/80^high and blue for CD11c^lowF4/80^low cells) with isotype (fluorescence minus one [FMO]) controls. (D) Representative phase-contrast (left, original magnification ×200) and CD207/MAC2 immunofluorescence (middle to right, by confocal laser-scanning microscopy) images of floating cluster cells replated at 7 weeks in culture. More than half of the cells show LC-like dendritic morphology, whereas some macrophage-like large round cells spreading on the culture plate are also seen (phase-contrast image in the left) at 48 hours following replating. LC-like cells are positive for CD207 at the plasma membrane with nuclear MAC2 staining (middle), but neither membrane CD207 nor nuclear MAC2 staining is detectable in macrophage-like cells (right). Scale bar, 10 μm (confocal images). MΦ, macrophage.