Research ArticleCancer

Calreticulin Is the Dominant Pro-Phagocytic Signal on Multiple Human Cancers and Is Counterbalanced by CD47

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Science Translational Medicine  22 Dec 2010:
Vol. 2, Issue 63, pp. 63ra94
DOI: 10.1126/scitranslmed.3001375
  • Fig. 1

    Cell surface calreticulin (CRT) is expressed on cancer, but not normal, stem and progenitor cells. (A) Cell surface CRT expression was determined by flow cytometry on primary human patient samples from several hematologic cancer types and normal cell counterparts, including normal bone marrow (NBM; n = 9), normal peripheral blood (NPB; n = 3), acute myeloid leukemia (AML; n = 8), acute lymphoblastic leukemia (ALL; n = 21), chronic myeloid leukemia (CML; n = 13), and non-Hodgkin’s lymphoma (NHL; n = 7). (B) A similar analysis as in (A) was performed for solid tumors (glioblastoma, n = 9; transitional cell bladder carcinoma, n = 8; serous papillary ovarian carcinoma, n = 9) and normal human fetal tissues (neurons, n = 3; astrocytes, n = 6; bladder cells, n = 6). ESA (epithelial-specific antigen)–positive urothelium was analyzed for normal fetal bladder. Primary human bladder cancer patient samples and samples that had been passaged once in mice were used for profiling. (C and D) Cell surface CRT expression was determined on normal stem and progenitor cells, lymphocytes, and cancer stem and progenitor cells. Each symbol represents a different patient sample. Patient samples tested: NBM = 10, AML = 8, CML = 13, bladder cancer = 8, glioblastoma = 8. NBM hematopoietic stem cell (HSC) = CD34+CD38CD90+Lin, AML LSC = CD34+CD38CD90Lin, GMP = CD34+CD38+IL3rα+CD45RA+, CMP = CD34+CD38+IL3rα+CD45RA. (E) CRT expression did not differ between bulk and cancer stem cell populations for either bladder cancer (P = 0.54) or glioblastoma (P = 0.14). Bladder cancer CSC = CD44+Lin (8), glioblastoma CSC = CD133+Lin (22, 23). Annexin V–positive cells were excluded in the analysis of all samples.

  • Fig. 2

    Increased CD47 expression on cancer cells protects them from CRT-mediated phagocytosis. (A) Correlation between cell surface CRT and CD47 expression was determined for human cancer cell lines (top left) and primary human normal and cancer samples (top right, bottom panels). Expression was calculated as mean fluorescence intensity (MFI) normalized over isotype control and for cell size. Pearson correlation (r) and P value are shown for each correlation. Top left panel: blue solid circle, HL60; blue open circle, Kasumi1; blue open inverted triangle, MOLM13; blue open diamond, KG-1; red triangle, Jurkat; red solid square, CCRF-CEM; red open square, CCRF-HSB2; red diamond, MOLT4; black star, Raji; black open diamond, SUDHL6; black open triangle, Daudi; black ×, U937; green plus, 639V; green open diamond, HT1197; green inverted triangle, UMUC3. (B) CD47 protein expression was determined by flow cytometry on Raji cells transduced with lentiviruses encoding shRNA CD47 knockdown constructs (shCD47) or controls. (C) Relative CD47 expression levels were quantified by comparing MFI to wild-type Raji cells. (D) Raji cell clones were incubated with human macrophages in media alone or with CRT-blocking peptide for 2 hours, after which phagocytosis was analyzed by fluorescence microscopy. Knockdown of CD47 in Raji cells (shCD47-1 and shCD47-2) resulted in increased phagocytosis compared to untransduced Raji cells. No difference in phagocytosis was observed between untransduced and GAPD control–transduced Raji cells (P = 0.45) Blockade of CRT on CD47 knockdown Raji cells completely abrogated phagocytosis. (E) MOLM13 cells, a CD47-deficient human AML cell line, were incubated with human macrophages for 2 hours with the indicated peptides and monitored for phagocytosis as above. High levels of phagocytosis were observed with IgG1 isotype control, whereas blockade of CRT or LRP reduced levels of phagocytosis (P = 0.03 and P = 0.01, respectively). Conditions were performed in triplicate; data are presented as means ± SD. *P < 0.05; **P < 0.005; ***P < 0.0005 (two-tailed Student’s t test).

  • Fig. 3

    Cell surface CRT is the dominant pro-phagocytic signal on several human cancers and is required for anti-CD47 antibody–mediated phagocytosis. (A) Primary human AML cells were fluorescently labeled with carboxyfluorescein diacetate succinimidyl ester (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 2 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), whereas incubation with CRT-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 CRT-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 (two-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 are presented as means ± SD.

  • Fig. 4

    Increased CRT expression confers a worse clinical prognosis in multiple human malignancies. (A to F) Stratification of clinical outcomes based on the level of expression of CRT mRNA is shown in previously described cohorts (5055) of patients with diverse malignancies including neuroblastoma (A and B), superficial or invasive bladder cancer (C and D), and mantle cell lymphoma (E and F). Patients were divided into CRT high- and low-expression groups based on median CRT expression, with Kaplan-Meier analyses of patient outcome shown. Hazard ratios (HR) and log-rank P values are shown for the relationship of outcomes to dichotomous expression of CRT. HR and log-rank P values for CRT expression as a continuous variable using a univariate Cox regression model are shown in table S1. All P values were <0.05 when CRT was considered as a continuous variable. Description of clinical data sets is shown in table S1.

Additional Files

  • Supplementary Material for:

    Calreticulin Is the Dominant Pro-Phagocytic Signal on Multiple Human Cancers and Is Counterbalanced by CD47

    Mark P. Chao,* Siddhartha Jaiswal, Rachel Weissman-Tsukamoto, Ash A. Alizadeh, Andrew J. Gentles, Jens Volkmer, Kipp Weiskopf, Stephen B. Willingham, Tal Raveh, Christopher Y. Park, Ravindra Majeti, Irving L. Weissman

    *To whom correspondence should be addressed. E-mail: mpchao{at}stanford.edu

    Published 22 December 2010, Sci. Transl. Med. 2, 63ra94 (2010)
    DOI: 10.1126/scitranslmed.3001375

    This PDF file includes:

    • Fig. S1. Live calreticulin-positive cancer cells form tumors in vivo.
    • Fig. S2. Cell surface CRT correlates with ERp57 expression on tumor cells.
    • Fig. S3. Live calreticulin-positive cells from normal human tissues have higher levels of CD47 compared to calreticulin-negative cells.
    • Fig. S4. Calreticulin expression is unaffected by CD47 shRNA knockdown in Raji cells.
    • Fig. S5. CD47 is expressed on normal human cells.
    • Fig. S6. Abrogation of anti-CD47 antibody–mediated phagocytosis is dosedependent on calreticulin blockade.
    • Fig. S7. Model for the integration of pro-phagocytic (CRT) and anti-phagocytic (CD47) signals on normal and tumor cells at steady state and during anti-CD47 antibody therapy.
    • Table S1. Analysis of the prognostic value of calreticulin in human malignancies.

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