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. Author manuscript; available in PMC: 2014 Aug 8.
Published in final edited form as: Sci Transl Med. 2010 Dec 22;2(63):63ra94. doi: 10.1126/scitranslmed.3001375

Figure 3. Cell Surface Calreticulin is the Dominant Pro-Phagocytic Signal on Several Human Cancers and is Required for Anti-CD47 Antibody-Mediated Phagocytosis.

Figure 3

(A) Primary human AML cells were fluorescently-labeled with CFSE and incubated with human macrophages in the presence of the indicated antibodies/peptides for 2 hours, after which phagocytosis was analyzed by fluorescence microscopy. Arrows indicate phagocytosis. (B) Cells from several normal human tissue types were incubated with human macrophages in the presence of the indicated antibodies and monitored for phagocytosis. No difference in phagocytosis was detected between IgG1 isotype control and anti-CD47 antibody incubation (p=0.77). (C) Primary human cancer cells were incubated with human macrophages in the presence of the indicated antibodies/peptides for two hours and monitored for phagocytosis. Each data point represents a different patient sample. Compared to IgG1 isotype control, incubation with anti-CD47 antibody enabled phagocytosis of cancer cells (p<0.0001) while incubation with calreticulin blocking peptide (p=0.37) or RAP, an LRP inhibitor (p=0.67), did not enable phagocytosis. In the presence of anti-CD47 antibody, incubation of cancer cells with either calreticulin blocking peptide or RAP completely abrogated anti-CD47 antibody-mediated phagocytosis (p=0.77 and p=0.16, respectively compared to IgG1 isotype control). *****p<0.00001 (2-sided Student’s t-test). (D) A positive correlation was observed between cell surface CRT expression and degree of anti-CD47 antibody mediated phagocytosis (Pearson’s correlation coefficient is shown). Each point represents a distinct patient sample that was incubated in the same in vitro phagocytosis assay. (E) Human NBM cells were incubated with human macrophages in the presence of the indicated antibodies or protein. Exogenous CRT enabled increased phagocytosis of NBM cells compared to vehicle control (p=0.05). No difference in phagocytosis was observed between IgG1 isotype control and anti-CD47 antibody (p=0.49). Conditions were performed in triplicate; data presented as mean ± SD.