Figure 2.
Her2 CAR macrophages trogocytose more than phagocytose in 3D spheroid models. (A) SKOV3 cells (mCh-CAAX, magenta) were assembled into a 3D spheroid and embedded in Matrigel alone (top), with control GFP (green) macrophages (middle), or with Her2 CAR GFP (green) macrophages (bottom). Images are maximum projections of spinning disc confocal z-stacks. Dashed line highlights the spheroid boundary. Scale bars denote 500 μm. On days 5 and 10, multiple images were stitched together to capture the entire spheroid in the no macrophage and GFP macrophage conditions. Unstitched images are presented on a gray background so the image scale is consistent. (B) Graph depicts spheroid growth over time for the same conditions shown in A. Individual spheroids were tracked for 10 days, and the diameter was normalized to the starting diameter. The length and width of the spheroid were measured on the max projection of confocal z-stacks and averaged to calculate spheroid diameter. N = 6 spheroids acquired in four independent experiments. The growth curves from individual spheroids in the Her2 CAR condition is shown in Fig. S2. (C) Inset from A shows that the mCherry signal on day 10 is mostly contained within Her2 CAR GFP (green) macrophages. Arrowheads point to macrophages. Scale bars denote 20 μm. (D) The top image shows the H2B-iRFP (blue), mCherry-CAAX (magenta) SKOV3 cell line used to assemble spheroids. Images show examples of Her2 CAR GFP (green) macrophages that have phagocytosed (middle, internalized nuclei position is highlighted with a yellow arrowhead) or trogocytosed (bottom, yellow arrowhead) SKOV3 cells. Scale bars denote 20 μm. (E) The percent of Her2 CAR GFP macrophages that had phagocytosed (internalized H2B-iRFP) or trogocytosed (internalized mCh-CAAX) was quantified on each day. Only macrophages touching the spheroid were included in the analysis. N = 3 spheroids from three independent experiments. In B, data acquired on day 10 were compared with a one-way ANOVA with Holm–Sidak multiple comparison test. In E, data from each day were compared with a two-way ANOVA. Bars represent the mean ± SEM. * denotes P < 0.05; ** denotes P < 0.005; **** denotes P < 0.00005. Refer to the image caption for details.

Her2 CAR macrophages trogocytose more than phagocytose in 3D spheroid models. (A) SKOV3 cells (mCh-CAAX, magenta) were assembled into a 3D spheroid and embedded in Matrigel alone (top), with control GFP (green) macrophages (middle), or with Her2 CAR GFP (green) macrophages (bottom). Images are maximum projections of spinning disc confocal z-stacks. Dashed line highlights the spheroid boundary. Scale bars denote 500 μm. On days 5 and 10, multiple images were stitched together to capture the entire spheroid in the no macrophage and GFP macrophage conditions. Unstitched images are presented on a gray background so the image scale is consistent. (B) Graph depicts spheroid growth over time for the same conditions shown in A. Individual spheroids were tracked for 10 days, and the diameter was normalized to the starting diameter. The length and width of the spheroid were measured on the max projection of confocal z-stacks and averaged to calculate spheroid diameter. N = 6 spheroids acquired in four independent experiments. The growth curves from individual spheroids in the Her2 CAR condition is shown in Fig. S2. (C) Inset from A shows that the mCherry signal on day 10 is mostly contained within Her2 CAR GFP (green) macrophages. Arrowheads point to macrophages. Scale bars denote 20 μm. (D) The top image shows the H2B-iRFP (blue), mCherry-CAAX (magenta) SKOV3 cell line used to assemble spheroids. Images show examples of Her2 CAR GFP (green) macrophages that have phagocytosed (middle, internalized nuclei position is highlighted with a yellow arrowhead) or trogocytosed (bottom, yellow arrowhead) SKOV3 cells. Scale bars denote 20 μm. (E) The percent of Her2 CAR GFP macrophages that had phagocytosed (internalized H2B-iRFP) or trogocytosed (internalized mCh-CAAX) was quantified on each day. Only macrophages touching the spheroid were included in the analysis. N = 3 spheroids from three independent experiments. In B, data acquired on day 10 were compared with a one-way ANOVA with Holm–Sidak multiple comparison test. In E, data from each day were compared with a two-way ANOVA. Bars represent the mean ± SEM. * denotes P < 0.05; ** denotes P < 0.005; **** denotes P < 0.00005.

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