Figure 5.
Reducing cancer cell adhesion promotes phagocytosis. (A) SKOV3 cells with a membrane tethered mCherry (mCh-CAAX) and a nuclear iRFP (H2B-iRFP) were plated untreated 8 h prior (adherent), with 50 μg/ml RGD peptide 8 h prior (RGD Peptide), or untreated at the time of macrophage addition (suspended). (B) Her2 CAR GFP or control GFP-CAAX macrophages were incubated with adherent SKOV3 cells, RGD peptide–treated SKOV3 cells, or suspended SKOV3 cells. The percent of macrophages that phagocytosed (right) and trogocytosed (left) was measured during an 8-h time-lapse. (C) The same experimental conditions were recapitulated with GFP-CAAX macrophages in either the presence or absence of opsonization with anti-CD47. (D) SKOV3 were infected with CRISPR-Cas9 and an sgRNA-targeting integrin β1, β5, αV, or a NT control guide. Infected cells were selected with puromycin. Knockout cells lines were stained for paxillin (magenta) and phalloidin (actin, green) to validate change in the amount of focal adhesions and structural morphology. Nuclei were labeled with DAPI (blue). (E) Paxillin was used to determine the total area of focal adhesion per cell (left) and the total count of focal adhesions per cell (right) in the knockout lines. (F) SKOV3 cells expressing mCherry-CAAX and H2B-iRFP were infected with the same CRISPR-Cas9 and sgRNA. Infected cells were selected with puromycin, and knockdown was confirmed by flow cytometry (Fig. S4). The resulting polyclonal cell lines were incubated with Her2 CAR GFP or control GFP-CAAX macrophages, and the percent of macrophages that phagocytosed (left) and trogocytosed (right) was measured during an 8-h time-lapse. Data in E and F were compared using one-way ANOVA with Holm–Sidak multiple comparison correction. N = 4 independent experiments in B, C, and F, N = 6 in E. For B, C, E, and F, bars represent the mean ± SEM. Data collected on the same day are annotated with the same shape point. * denotes P < 0.05; ** denotes P < 0.005; *** denotes P < 0.0005; **** denotes P < 0.00005. Scale bars denote 20 μm. Refer to the image caption for details.

Reducing cancer cell adhesion promotes phagocytosis. (A) SKOV3 cells with a membrane tethered mCherry (mCh-CAAX) and a nuclear iRFP (H2B-iRFP) were plated untreated 8 h prior (adherent), with 50 μg/ml RGD peptide 8 h prior (RGD Peptide), or untreated at the time of macrophage addition (suspended). (B) Her2 CAR GFP or control GFP-CAAX macrophages were incubated with adherent SKOV3 cells, RGD peptide–treated SKOV3 cells, or suspended SKOV3 cells. The percent of macrophages that phagocytosed (right) and trogocytosed (left) was measured during an 8-h time-lapse. (C) The same experimental conditions were recapitulated with GFP-CAAX macrophages in either the presence or absence of opsonization with anti-CD47. (D) SKOV3 were infected with CRISPR-Cas9 and an sgRNA-targeting integrin β1, β5, αV, or a NT control guide. Infected cells were selected with puromycin. Knockout cells lines were stained for paxillin (magenta) and phalloidin (actin, green) to validate change in the amount of focal adhesions and structural morphology. Nuclei were labeled with DAPI (blue). (E) Paxillin was used to determine the total area of focal adhesion per cell (left) and the total count of focal adhesions per cell (right) in the knockout lines. (F) SKOV3 cells expressing mCherry-CAAX and H2B-iRFP were infected with the same CRISPR-Cas9 and sgRNA. Infected cells were selected with puromycin, and knockdown was confirmed by flow cytometry (Fig. S4). The resulting polyclonal cell lines were incubated with Her2 CAR GFP or control GFP-CAAX macrophages, and the percent of macrophages that phagocytosed (left) and trogocytosed (right) was measured during an 8-h time-lapse. Data in E and F were compared using one-way ANOVA with Holm–Sidak multiple comparison correction. N = 4 independent experiments in B, C, and F, N = 6 in E. For B, C, E, and F, bars represent the mean ± SEM. Data collected on the same day are annotated with the same shape point. * denotes P < 0.05; ** denotes P < 0.005; *** denotes P < 0.0005; **** denotes P < 0.00005. Scale bars denote 20 μm.

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