Extended Data Fig. 9: Macrophage screens for genes required for enhanced uptake of APMAP^KO cancer cells.

a, Phagocytosis assay for uptake of pHrodo-labelled Karpas-299-Cas9 cells expressing indicated sgRNAs, incubated with J774 macrophages expressing indicated sgRNAs, in the presence of anti-CD30 antibodies. Phagocytosis index (arbitrary units) corresponds to the total pHrodo Red signal per well, normalized to Safe^KO Karpas-299 cells fed to Safe^KO macrophages. Data represent mean +/− s.d. (n = 4 cell culture wells). Two-way ANOVA with Bonferroni correction. b, Phagocytosis assay for uptake of pHrodo-labelled Ramos cells expressing indicated sgRNAs, incubated with U937 macrophages expressing indicated sgRNAs (three independent sgRNAs per gene), in the presence of anti-CD20 antibodies. Phagocytosis index (arbitrary units) corresponds to the total pHrodo Red signal per well, normalized to Safe^KO Ramos cells fed to Safe^KO-1 macrophages. Data represent mean +/− s.d. (n = 4 cell culture wells). Two-way ANOVA with Bonferroni correction, P values for comparisons to Safe^KO Ramos/Safe^KO-1 U937 macrophages shown. c, Gating strategy for collecting single-positive and double-negative macrophage populations, corresponding to macrophages that phagocytosed calcein⁺ Safe^KO or Far-red⁺ APMAP^KO cells. d, Volcano plot for macrophage screen for genes required for uptake of Safe^KO Ramos cells, using 2,208-gene sublibrary (enriched for phagocytosis genes, but lacking GPR84), conducted in J774 macrophages. Dotted line indicates 5% FDR. e, Volcano plot for macrophage screen for genes required for uptake of APMAP^KO Ramos cells, using 2,208-gene sublibrary (enriched for phagocytosis genes, but lacking GPR84) in J774 macrophages. Dotted line indicates 5% FDR. f, Volcano plot for macrophage screen for genes required selectively for uptake of APMAP^KO cells, following screen design in Fig. 4a (comparison 2), but using 2,208-gene sublibrary (enriched for phagocytosis genes, but lacking GPR84) in J774 macrophages, for uptake of calcein⁺ Safe^KO cells and far-red⁺ APMAP^KO Ramos cells. Dotted line indicates 5% FDR. g, Phagocytosis assay for uptake of pHrodo-labelled Ramos cells expressing indicated sgRNAs, incubated with J774 macrophages expressing indicated sgRNAs, in the presence of anti-CD20 antibodies. Phagocytosis index (arbitrary units) corresponds to the total pHrodo Red signal per well, normalized to Safe^KO Ramos cells fed to Safe^KO macrophages. Data represent mean +/− s.d. (n = 3 cell culture wells). Two-way ANOVA with Bonferroni correction. P-values correspond to comparisons to Safe^KO-1. h, FACS-based phagocytosis assay for uptake of CellTrace Far-Red-labelled APMAP^KO cells and calcein-labelled CD47^KO Ramos cells by J774-Cas9 macrophages expressing indicated sgRNAs. Ratio of macrophages that phagocytosed APMAP^KO Ramos cells to macrophages that phagocytosed CD47^KO Ramos cells, normalized to Safe^KO/Safe^KO J774 macrophages, following 24 h co-incubation with anti-CD20 antibodies is plotted. Data represent mean +/− s.d. (n = 3 cell culture wells). One-way ANOVA with Bonferroni correction, P-values for comparisons to Safe^KO/Safe^KO J774 macrophages shown.