Reply to “On the Mechanism and Benefit of siRNA-mediated Targeting of CD47 in Cancer”
Authors: Yuhua Wang, Zhenghong Xu, Leaf Huang
Journal: Molecular Therapy (2013)

To the editor: In this issue we report effective RNA interference (RNAi)–based antitumor therapy targeting CD47, a "self-marker" often overexpressed on the surface of cancer cells.1Wang Y Xu Z Guo S Zhang L Sharma A Robertson GP et al.Intravenous delivery of siRNA targeting CD47 effectively inhibits melanoma tumor growth and lung metastasis.Mol Ther. 2013; 21: 1919-1929Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar We achieved efficient silencing of CD47 expression in B16F10 melanoma cells via systemic delivery of short interfering RNA (siRNA) against CD47 using a tumor-targeted liposomal nanoparticle formulation. Loss of CD47 promoted macrophage-mediated phagocytosis of B16F10 cells in culture and inhibited solid-tumor growth and metastasis in vivo. The effects of treatment were abolished when macrophages were depleted by pretreatment of mice with liposomal clodronate. In their letter, Zhao et al. raise several issues regarding our interpretation of the data.2Zhao X Matlung H Kuijpers TW van den Berg TK On the mechanism and benefit of siRNA-mediated targeting of CD47 in cancer.Mol Ther. 2013; 21: 1811-1812Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar The authors recently reported that injection of B16F10 cells overexpressing CD47 into wild-type and SIRPα-mutant mice gave rise to similar patterns of tumor formation in each strain.3Zhao XW van Beek EM Schornagel K Van der Maaden H Van Houdt M Otten MA et al.CD47–signal regulatory protein-α (SIRPα) interactions form a barrier for antibody-mediated tumor cell destruction.Proc Natl Acad Sci USA. 2011; 108: 18342-18347Crossref PubMed Scopus (218) Google Scholar They concluded that SIRPα signaling was dispensable for the inhibition of tumor growth and metastasis, and therefore inhibition of binding between CD47 and SIRPα would be predicted to have no significant therapeutic effect. Similarly, Ishikawa-Sekigami et al.4Ishikawa-Sekigami T Kaneko Y Saito Y Murata Y Okazawa H Ohnishi H et al.Enhanced phagocytosis of CD47-deficient red blood cells by splenic macrophages requires SHPS-1.Biochem Biophys Res Commun. 2006; 343: 1197-1200Crossref PubMed Scopus (32) Google Scholar reported that clearance of CD47+/+ red blood cells (RBCs) from the peripheral circulation was not accelerated in SIRPα-mutant mice relative to wild-type mice. However, CD47−/− RBCs, obtained from CD47-null mice, were more quickly eliminated from the peripheral circulation of wild-type mice than were CD47+/+ RBCs, in a manner conceptually consistent with our findings. Targeting of CD47 appears to be a more effective way to induce macrophage-mediated phagocytosis. It was not surprising to see the lack of tumor growth inhibition when CD47-expressing B16F10 melanoma cells were transfused into the SIRPα-mutant mice. Our results, however, showed that siRNA silencing of CD47 promoted phagocytosis in vitro and inhibited tumor growth and metastasis in vivo. Our results are more consistent with initial findings that first unveiled CD47's role in removal of damaged RBCs5Oldenborg PA Zheleznyak A Fang YF Lagenaur CF Gresham HD Lindberg FP Role of CD47 as a marker of self on red blood cells.Science. 2000; 288: 2051-2054Crossref PubMed Scopus (1265) Google Scholar and platelets6Olsson M Bruhns P Frazier WA Ravetch JV Oldenborg PA Platelet homeostasis is regulated by platelet expression of CD47 under normal conditions and in passive immune thrombocytopenia.Blood. 2005; 105: 3577-3582Crossref PubMed Scopus (128) Google Scholar from circulation. Zhao et al. identified a perceived discrepancy between in vitro antibody-dependent, cell-mediated cytotoxicity (ADCC) studies3Zhao XW van Beek EM Schornagel K Van der Maaden H Van Houdt M Otten MA et al.CD47–signal regulatory protein-α (SIRPα) interactions form a barrier for antibody-mediated tumor cell destruction.Proc Natl Acad Sci USA. 2011; 108: 18342-18347Crossref PubMed Scopus (218) Google Scholar conducted in their laboratory and our findings.1Wang Y Xu Z Guo S Zhang L Sharma A Robertson GP et al.Intravenous delivery of siRNA targeting CD47 effectively inhibits melanoma tumor growth and lung metastasis.Mol Ther. 2013; 21: 1919-1929Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar Direct comparison between the two studies may not be meaningful, however, because two completely different experimental setups were used to measure distinct cellular activities. Zhao et al. reduced CD47 expression in a human breast cancer cell line, SKBR3, and then incubated cells with human neutrophils and monocytes to measure ADCC. We reduced CD47 expression in murine B16F10 cells and then incubated cells with mouse macrophages obtained from C57 mice after the induction of inflammation. Weiskopf et al.7Weiskopf K Ring AM Ho CC Volkmer JP Levin AM Volkmer AK et al.Engineered SIRPalpha variants as immunotherapeutic adjuvants to anticancer antibodies.Science. 2013; 341: 88-91Crossref PubMed Scopus (320) Google Scholar report engineered SIRPα variants with high affinity for CD47. The mechanism of antitumor effects for this class of anti-CD47 antagonist was enhancement of ADCC by blocking of CD47. However, as described by Chao et al.,8Chao MP Majeti R Weissman IL Response: mechanisms of targeting CD47–SIRPα in hematologic malignancies.Blood. 2012; 119: 4334-4335Crossref Scopus (3) Google Scholar F(ab)" fragments of some CD47-specific antibodies can induce macrophage-mediated phagocytosis without the ligation of Fc fragment and Fc receptor on macrophages. This supports the existence of an ADCC-independent cell-killing pathway induced by CD47-specific antibody. Together these results show the mechanistic complexity of antibody-based therapies: different CD47-specific antibodies will result in different physiological responses with different mechanisms of action. Zhao et al. provide an alternative explanation for our results, suggesting that nanoparticles "directly" opsonize tumor cells for macrophage-mediated phagocytosis.2Zhao X Matlung H Kuijpers TW van den Berg TK On the mechanism and benefit of siRNA-mediated targeting of CD47 in cancer.Mol Ther. 2013; 21: 1811-1812Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar We consider this unlikely because the nanoparticles were surface-functionalized with anisamide to facilitate specific uptake by sigma receptor–overexpressing cancer cells. Nanoparticles were internalized as soon as the anisamide was recognized by the sigma receptors. Furthermore, the macrophage-mediated phagocytosis assays were performed in vitro, 24 hours post-transfection after nanoparticle internalization. Heat inactivation of serum used in the assays ensured that complement proteins were not involved in the interaction. Finally, nanoparticles loaded with control siRNA did not inhibit tumor growth in vivo, consistent with the nanoparticles themselves being biologically inert. T cell–mediated immunity has also been suggested as a mechanism of action,9Tseng D Volkmer JP Willingham SB Contreras-Trujillo H Fathman JW Fernhoff NB et al.Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response.Proc Natl Acad Sci USA. 2013; 110: 11103-11108Crossref PubMed Scopus (386) Google Scholar but the authors of that paper report the induction of phagocytosis by blocking of CD47 and SIRPα. T cell–mediated immunity is probably a factor in the inhibition of tumor cell growth but as an event that would occur after phagocytosis. Macrophage-mediated antigen presentation is a prerequisite for T cell–mediated immunity. We respectfully disagree with the correspondents for three reasons. First, our study did not involve the experimental use of antibodies. Our in vitro phagocytosis assay was conducted with cocultured B16F10 and macrophages. The B16F10 tumor-bearing animal model would not be expected to generate antitumor antibodies because of the highly immunosuppressive microenvironment. Second, RNAi depleted >90% of CD47 surface marker expression, potentially more efficient in disrupting self-recognition. The lack of CD47 explains why we observed effective inhibition of tumor growth and metastasis, as well as the accelerated clearance of CD47-deficient RBCs in the blood circulation.4Ishikawa-Sekigami T Kaneko Y Saito Y Murata Y Okazawa H Ohnishi H et al.Enhanced phagocytosis of CD47-deficient red blood cells by splenic macrophages requires SHPS-1.Biochem Biophys Res Commun. 2006; 343: 1197-1200Crossref PubMed Scopus (32) Google Scholar This macrophage-mediated clearance is independent of complement and antibodies, and is driven only by interaction between CD47 and SIRPα. Third, macrophage depletion by liposomal clodronate abolished the antitumor effect. This confirms that macrophages were the key player in the in vivo antitumor effect observed in our experiments. We share the concern regarding enhanced proliferation of cancer cells when CD47 is silenced. However, this activity was not observed in the immunocompetent mice, indicating that cell killing mediated by the immunosurveillance outweighed the proliferation induced by CD47 deficiency. In conclusion, our studies demonstrate that CD47 expression was elevated in the melanoma cells and that this served as a mechanism to avoid immunosurveillance. The depletion of CD47 with nanoparticle-delivered siRNA efficiently silenced the target gene and inhibited tumor growth and metastasis as a result of enhanced phagocytosis. The nanoparticle-based RNAi therapy, which targeted CD47, proved to be an alternative and efficacious approach to the antibody-based therapy.