Calreticulin is the dominant pro-phagocytic signal on multiple human cancers and is counterbalanced by CD47 - PubMed (original) (raw)

. 2010 Dec 22;2(63):63ra94.

doi: 10.1126/scitranslmed.3001375.

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

Affiliations

Calreticulin is the dominant pro-phagocytic signal on multiple human cancers and is counterbalanced by CD47

Mark P Chao et al. Sci Transl Med. 2010.

Abstract

Under normal physiological conditions, cellular homeostasis is partly regulated by a balance of pro- and anti-phagocytic signals. CD47, which prevents cancer cell phagocytosis by the innate immune system, is highly expressed on several human cancers including acute myeloid leukemia, non-Hodgkin's lymphoma, and bladder cancer. Blocking CD47 with a monoclonal antibody results in phagocytosis of cancer cells and leads to in vivo tumor elimination, yet normal cells remain mostly unaffected. Thus, we postulated that cancer cells must also display a potent pro-phagocytic signal. Here, we identified calreticulin as a pro-phagocytic signal that was highly expressed on the surface of several human cancers, but was minimally expressed on most normal cells. Increased CD47 expression correlated with high amounts of calreticulin on cancer cells and was necessary for protection from calreticulin-mediated phagocytosis. Blocking the interaction of target cell calreticulin with its receptor, low-density lipoprotein receptor-related protein, on phagocytic cells prevented anti-CD47 antibody-mediated phagocytosis. Furthermore, increased calreticulin expression was an adverse prognostic factor in diverse tumors including neuroblastoma, bladder cancer, and non-Hodgkin's lymphoma. These findings identify calreticulin as the dominant pro-phagocytic signal on several human cancers, provide an explanation for the selective targeting of tumor cells by anti-CD47 antibody, and highlight the balance between pro- and anti-phagocytic signals in the immune evasion of cancer.

PubMed Disclaimer

Conflict of interest statement

The authors do not have any competing interests and fully endorse all contents of this study.

Figures

Figure 1

Figure 1. Cell surface calreticulin is expressed on cancer, but not normal, stem and progenitor cells

(A) Cell surface calreticulin 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 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+ 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 calreticulin 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 HSC=CD34+CD38-CD90+Lin-, AML LSC=CD34+CD38-CD90-Lin-, GMP=CD34+CD38+IL3rα+CD45RA+, CMP=CD34+CD38+IL3rα+CD45RA-. (E) Calreticulin 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.

Figure 2

Figure 2. Increased CD47 Expression on Cancer Cells Protects Them from Calreticulin- Mediated Phagocytosis

(A) Correlation between cell surface calreticulin 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 normalized over isotype control and for cell size. Pearson correlation (r) and p-value is 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 x=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,-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 calreticulin on CD47-knockdown Raji cells completely abrogated phagocytosis. (E) MOLM-13 cells, a CD47-deficient human AML cell line, were incubated with human macrophages for two hours with the indicated peptides and monitored for phagocytosis as above. Significant phagocytosis was observed with IgG1 isotype control, while blockade of calreticulin or LRP reduced levels of phagocytosis (p=0.03 and p=0.01, respectively). Conditions were performed in triplicate; data presented as mean ± SD. *p<0.05, **p<0.005, ***p<0.0005 (2-tailed Student’s t-test).

Figure 3

Figure 3. Cell Surface Calreticulin 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 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.

Figure 4

Figure 4. Increased calreticulin expression confers a worse clinical prognosis in multiple human malignancies

Stratification of clinical outcomes based on the level of expression of calreticulin mRNA is shown in previously described cohorts (–55) of patients with diverse malignancies including neuroblastoma (A,B), superficial or invasive bladder cancer (C,D), and mantle cell lymphoma (E,F). Patients were divided into calreticulin high and low expression groups based on median calreticulin 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 continuous expression of calreticulin using a univariate Cox regression model. HR, 95% confidence intervals, and log-rank p values for calreticulin expression as a dichotomous variable are shown in table S1. Description of clinical datasets is shown in table S1.

References

    1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100:57–70. - PubMed
    1. Stagg J, Johnstone RW, Smyth MJ. From cancer immunosurveillance to cancer immunotherapy. Immunol Rev. 2007;220:82–101. - PubMed
    1. Swann JB, Smyth MJ. Immune surveillance of tumors. J Clin Invest. 2007;117:1137–1146. - PMC - PubMed
    1. Zitvogel L, Tesniere A, Kroemer G. Cancer despite immunosurveillance: immunoselection and immunosubversion. Nat Rev Immunol. 2006;6:715–727. - PubMed
    1. Jaiswal S, Jamieson CH, Pang WW, Park CY, Chao MP, Majeti R, Traver D, van Rooijen N, Weissman IL. CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis. Cell. 2009;138:271–285. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources