Extranodal dissemination of non-Hodgkin lymphoma requires CD47 and is inhibited by anti-CD47 antibody therapy

Lymphocyte trafficking is essential for the regulation of systemic immune processes, as well as lymphocyte differentiation and development. Most mature lymphocytes recirculate continuously from blood to tissue and back to the blood again.¹  This recirculation is not random but rather is guided by lymphocyte-endothelial interactions mediated by adhesion molecules (L-selectin, CD44, integrin α₄β₇, VLA-4, and LFA-1) and select chemokines.^2,3 

Malignant transformation of normal lymphocytes results in lymphoma, many subtypes of which migrate and disseminate. Unlike the metastasis of other cancers, lymphoma dissemination probably reflects conserved physiologic behavior, rather than acquisition of metastatic potential. Indeed, mechanisms of normal lymphocyte homing and recirculation have been implicated in lymphoma dissemination and invasion. For example, adhesion molecules involved in normal lymphocyte trafficking have been shown to play a role in lymphoma dissemination, including LFA-1, α₃β₃, and other homing-associated integrins.^4-6  Furthermore, several of these adhesion molecules have been therapeutically exploited, as antibodies targeting the adhesion receptors LFA-1, integrin α_vβ₃, and CD44 can inhibit dissemination of lymphoma in experimental models.^5,7-9 

CD47, also known as integrin-associated protein, has been implicated in the migration and mobilization of normal leukocytes.^10-14  In cancer, we recently demonstrated that CD47 regulates lymphoma pathogenesis by enabling evasion of phagocytosis through binding of the inhibitory receptor SIRPα on phagocytes.¹⁵  Furthermore, a blocking monoclonal antibody targeting CD47 eliminated human lymphoma in xenotransplant models through phagocytosis of tumor cells, and synergized with rituximab, a therapeutic antibody commonly used in non-Hodgkin lymphoma (NHL) therapy.¹⁵  Given the roles of CD47 in normal cell migration and lymphoma pathogenesis, we investigated the function of CD47 in NHL dissemination and whether therapeutic targeting of CD47 could inhibit such spread.

NHL samples were obtained as previously described¹⁵  from patients at the Stanford University Medical Center with informed consent according to an Institutional Review Board–approved protocol (Stanford IRB #13500) or with informed consent from the Norwegian Radium Hospital (Oslo, Norway) according to a Regional Ethic Committee–approved protocol (REK #2.2007.2949) following the Declaration of Helsinki. For all in vivo experiments, anti–human CD47 (clone B6H12.2) was used and obtained as previously described.¹⁵  The nonblocking anti–human CD47 antibody clone 2D3, mouse IgG1 isotype control, and anti–human CD45 antibodies were obtained from eBioscience.

For analysis of primary and xenografted NHL cells, human CD19, human CD45, mouse Terr19, mouse CD45, and mouse F4/80 were used (Invitrogen and BD Biosciences). Analysis of human CD47 expression was performed with an anti–human CD47 FITC antibody (clone B6H12.2, BD Biosciences).

A luciferase-positive Raji cell line was generated and analyzed by luciferase imaging as previously described.¹⁵ 

For the localized model, 3 × 10⁶ luciferase-labeled Raji cells were injected subcutaneously into the right flank of 6- to 10-week-old NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. After 1 week, mice were treated with daily 200 μg intraperitoneal injections of either mouse control IgG or anti-CD47 antibody. These mice were then followed weekly by luciferase imaging for the presence of dissemination of luciferase-positive disease. Tumor volume was measured weekly with calipers and determined by (length × width)/2. Appearance of metastatic luciferase-positive disease was present at ∼ 2-3 weeks for control IgG-treated mice. For the disseminated model, 1.5 × 10⁶ luciferase-labeled Raji cells were injected intravenously into the retro-orbital sinus of adult NSG mice. Antibody treatment consisted of coating Raji cells ex vivo with 30 μg/mL of IgG1 isotype control antibody, anti-CD45 antibody, anti-CD47 antibody clone B6H12.2, or anti-CD47 antibody clone 2D3 before intravenous transplantation. Tumor engraftment was analyzed 5 days after transplantation by luciferase imaging.

A total of 2-3 × 10⁶ bulk diffuse large B-cell lymphoma (DLBCL) cells were transplanted into sublethally irradiated (200 cGy) NSG mice. Three weeks later, mice were treated with daily 400 μg intraperitoneal injections of either control mouse IgG or anti-CD47 antibody. The presence of DLBCL disease was analyzed by flow cytometry for the presence of human CD45⁺CD19⁺ cells in the peripheral blood or bone marrow.

shRNA constructs targeting knockdown of human CD47 or a GFP control were used and transduced into Raji cells as previously described.¹⁵  Knockdown of CD47 protein levels was determined by staining with CD47 antibody (clone B6H12.2) with fold knockdown calculated by reduction of mean fluorescence intensity normalized over isotype control.

NSG mice engrafted subcutaneously with luciferase-labeled Raji cells were killed after control IgG or anti-CD47 antibody treatment, and organs harvested and analyzed for human lymphoma dissemination by H&E staining according to standard protocols. Hematoxylin and eosin sections were analyzed in a double-blinded fashion by a pathologist who determined whether dissemination of human lymphoma was present in each mouse organ. Micrographs were obtained using an Olympus BX41 microscope with a 40× objective, and images were acquired with a Canon G9 digital camera and processed using Axiovision 4.7 software.

Depletion of macrophages in NSG mice was performed using liposomal clodronate or liposomal control as previously described.¹⁵  Clodronate or liposomal control was administered to human DLBCL-engrafted mice for the duration of anti-CD47 antibody or control IgG therapy. Macrophage depletion was assessed by flow cytometric analysis of the percentage of F4/80⁺ macrophages in the bone marrow.

For in vivo MRI acquisition, mice were anesthetized with isoflurane (IsoFlo; Abbott Laboratories; 1%-1.75%). Gadolinium-based contrast, gadopentate dimeglumine (Berlex Laboratories), was injected intraperitoneally 20-30 minutes before image acquisition (2.5 mL/kg in PBS, 100 μL). Temperature and respiration were monitored continuously. Body temperature was maintained at 37 ± 1°C using a warm-air circulation system. The mice lay supine with the liver region positioned at the coil center. Anatomic images were obtained through the upper abdomen. MRI experiments were performed on Magnex/Varian self-shielded 30-cm bore 7-Tesla magnet, a Research Resonance Instruments 9-cm bore gradient insert, and the GEHC “Micro”-Signa software environment. Acquisition parameters are as follows: T1-weighted fast spin echo, TE 5.7 ms, TR 800, thickness 0.5 mm, FOV 6 × 6, NEX 1.0. For image analysis, an off-line workstation was used (Osirix).

The chemotaxis assay across polycarbonate transwell inserts were performed as previously described.¹⁶  Raji cells were incubated with IgG1 isotype control, anti-CD45, anti-CD47 (B6H12), or anti-CD47 (2D3) antibody for 20 minutes at 4°C and washed before chemotaxis. A total of 1 × 10⁶ Raji cells were then suspended in 100 μL of serum-free IMDM and added to the top chamber of a 24 well transwell plate with 5 μm pore-size filters (Corning/Costar). In the bottom chamber, there was 600 μL of media with either chemokine 10nM SDF-1α or 1 μg/mL CXCL13 (PeproTech). SDF-1α and CXCL13 were chosen as chemokines previously shown to enable migration of lymphoma cells.^17,18  Chemokine dosing was determined by dose titration for maximal migration of Raji cells (data not shown). Cells were incubated for 3-3.5 hours at 37°C. In chemotaxis assays involving antibodies, Raji cells were preincubated with 10 μg/mL of IgG1 isotype control or anti-CD47 antibodies for 20 minutes at 4°C before subjecting them to the transwell assay. Upper chamber inserts were removed, and migrated cells in the lower chamber were standardized using 100-μL microbeads per sample and counted on a flow cytometer (LSRII, BD Biosciences). Percentage migration was calculated using the number of starting and migrated cells.

To evaluate static adhesion, Raji cells were washed and resuspended at 0.5 to 1 × 10⁶ cells/mL in adhesion buffer (DMEM, 1% BSA, 2mM Ca²⁺/Mg²⁺). For antibody blocking experiments, cells were incubated for 20 minutes at 4°C with antibodies at indicated concentrations before their use. The 96-well plates were coated with recombinant human VCAM (R&D Systems) at 1 μg/mL overnight at 4°C. Plates were then blocked for 1-3 hours with PBS + 0.1% BSA at room temperature; 100-μL cells were plated per well in triplicate. Cells were incubated at 37°C for 1-2 hours and washed gently more than 4 times with PBS + Ca²⁺/Mg²⁺. To quantify cells, 20 μL CellTiter 96 (Invitrogen) was added to each well containing adherent cells in 100 μL PBS and incubated at 37°C for 1-3 hours. Absorbance at 490 nm was read on a plate reader and calculated relative to Raji cells incubated with BSA-coated plates.

To investigate the role of CD47 in NHL dissemination, we first determined the effect of knocking down CD47 on NHL spread in vivo. We previously developed a human xenotransplant model of NHL whereby Raji cells, a human Burkitt lymphoma cell line, transplanted subcutaneously into the flanks of NOD/SCID/IL2R-γ null (NSG) immunodeficient mice developed nodular disease.¹⁵  In this model, engrafted lymphoma disseminated to extranodal secondary sites, including the liver (Figure 1E). Lentiviral shRNA vectors were used to knock down CD47 expression in Raji cells (Figure 1A-B) as previously described.¹⁵  Raji cells with > 50% reduction in CD47 expression (shCD47-1 and shCD47-2) exhibited no difference in proliferation rate compared with control-transduced cells in vitro (supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). However, CD47 knockdown Raji cells (shCD47-1,2) grew at a slower rate in vivo (Figure 1C-D). Transplanted mice were killed 28 days later and analyzed for secondary liver lesions. Although mice transplanted with control cells developed numerous liver masses, the number of lesions was dramatically reduced with CD47 knockdown cells (Figure 1E-F). Liver involvement did not occur by direct extension as the primary subcutaneous tumor did not directly invade into the vasculature or adjacent organs (Figure 1E). CD47 knockdown Raji cells developed an average 4-fold fewer liver lesions, compared with control, with several mice showing complete absence of secondary lesions (Figure 1F). Thus, dissemination of Raji cells in vivo is dependent on the level of CD47 expression.

Next, we investigated whether extranodal NHL dissemination could be prevented by targeting CD47 with a blocking antibody that was previously shown to induce phagocytosis and elimination of several human tumors.^15,19-22  Luciferase-labeled Raji cells were transplanted subcutaneously into NSG mice. One week later, mice were administered daily treatment with 200 μg of either anti-CD47 antibody or control mouse IgG. Compared with control, anti-CD47 antibody therapy decreased the rate of tumor growth (Figure 2A) but did not eliminate the primary disease as previously reported.¹⁵  However, anti-CD47 antibody prevented lymphoma dissemination as assessed by 3 independent modalities. First, serial luciferase imaging demonstrated dissemination to secondary sites 3 weeks after transplantation in controls, whereas no spread was observed in the anti-CD47 antibody-treated mice (Figure 2B). Second, MRI with gadolinium-based contrast at 3 weeks after transplantation showed that mice treated with control IgG developed multiple liver tumor nodules, whereas anti-CD47 antibody inhibited liver dissemination (Figure 2C). Lastly, mice were killed and analyzed for extranodal disease by gross tissue analysis. Lastly, multiple liver tumor nodules were grossly observed at necropsy in controls, whereas no tumor nodules were detected in anti-CD47 antibody-treated mice (Figure 2D).

Although liver dissemination is observed in patients with NHL, extranodal spread has been observed in almost every organ, particularly the gastrointestinal tract and CNS. Therefore, control and anti-CD47 antibody-treated mice were analyzed for lymphoma invasion in major organs by tissue histopathology. IgG control-treated mice displayed evidence of diffuse micro-dissemination, including invasion into liver, pancreas, kidney, nasal cavity, and bone (Figure 3A,C). Hematogenous dissemination and invasion into the brain were also observed as evidenced by disease in the bone marrow and cerebral cortex (and pituitary gland), respectively (Figure 3A,C). In contrast, mice treated with anti-CD47 antibody exhibited no evidence of disease in any major organ (Figure 3B-C).

We extended these studies with Raji cells to next investigate the effect of anti-CD47 antibody on tumor dissemination of primary human lymphoma cells. We developed a novel localized DLBCL xenotransplant model by subcutaneous transplantation of cells from a patient with DLBCL (previously described as sample NHL17¹⁵ ) into sublethally irradiated NSG mice. Three weeks after transplantation, mice developed a palpable mass and were treated daily with 400 μg of either control mouse IgG or anti-CD47 antibody. Similar to Raji-engrafted mice, anti-CD47 antibody decreased the rate of tumor growth compared with control; however, this effect was not statistically significant (Figure 4A). Antibody treatment was administered for only 11 days, at which point mice were killed because of large tumor masses. Tumor dissemination to secondary sites was first investigated by MRI and gross pathology, which showed no disease outside of the primary subcutaneous transplant site (data not shown). However, analysis of the bone marrow and peripheral blood demonstrated hematogenous spread of human CD45⁺CD19⁺ DLBCL cells in controls, which was completely inhibited in anti-CD47 antibody-treated mice (Figure 4B-D).

CD47 binds several ligands, including integrins, thrombospondin-1, and SIRPα, which regulates functions of cell adhesion, migration, immune modulation, and phagocytic evasion.¹⁴  We previously showed that blockade of the CD47-SIRPα interaction eliminates tumors through induction of phagocytosis.^19,20  From this, we postulated that anti-CD47 antibody eliminated circulating lymphoma cells by inducing their phagocytosis, leading to the testable hypothesis that higher levels of CD47 are required for lymphoma cells to evade perivascular macrophages to disseminate and invade secondary sites.

To test this hypothesis, we analyzed CD47 expression on lymphoma cells from both the primary subcutaneous and secondary bone marrow sites in NSG mice engrafted subcutaneously with primary DLBCL. Average CD47 expression was increased on disseminated cells in the bone marrow ∼ 2-fold compared with cells from the primary site (Figure 5A-B). To determine whether this finding was recapitulated in human lymphoma patients, CD47 expression was examined on a panel of patient samples from various NHL subtypes (supplemental Table 1), with either primary nodal disease or hematogenous dissemination to the peripheral blood. Peripheral blood lymphoma cells exhibited > 2-fold increased expression of CD47 protein compared with cells isolated from lymph nodes (Figure 5C), suggesting that lymphoma dissemination is associated with increased CD47 expression. Although higher CD47 expression was observed on peripheral blood compared with lymph node-resident lymphoma cells, this difference may not only be the result of dissemination but the result of intrinsic differences between NHL disease subtypes, as lymph node and peripheral blood lymphoma cells isolated from the same patient were not available for analysis.

In addition to phagocytosis, CD47 has been implicated in integrin-mediated adhesion and cell migration,¹⁴  which we investigated as potential mechanisms for regulating lymphoma dissemination. First, control and CD47 knockdown Raji cells were assessed for their ability to migrate toward known lymphoma chemoattractants (SDF-1α^17,18  and CXCL13^17,18 ) in a transwell migration assay in vitro. Knockdown Raji cells exhibited decreased migration in response to both SDF-1α and CXCL13 compared with control cells (Figure 6A). Migration differences between Raji cell lines were not the result of differential expression of SDF-1α and CXCL13 receptors (CXCR4 and CXCR5, respectively) as all cell lines expressed similar levels of both (supplemental Figure 2). We next investigated the effect of anti-CD47 antibody blockade on Raji cell migration. Compared with isotype control, blocking anti-CD47 antibody reduced the migration of Raji cells to both SDF-1α and CXCL13 (Figure 6B) in a dose-dependent manner (supplemental Figure 3).

Regulation of lymphoma adhesion by CD47 was investigated using an in vitro static adhesion assay of Raji cells on VCAM, which plays an essential role in integrin-mediated adhesion, mobilization of hematopoietic cells, and tumor metastasis.^23,24  Indeed, Raji cells express the VCAM receptor, VLA-4 (also known as α₄β₁ integrin; supplemental Figure 2), suggesting that the VCAM/VLA-4 pathway could play a role in mediating Raji cell adhesion. Whereas Raji cells adhered to human VCAM-coated plates in vitro, no differences were observed in adhesion of CD74 knockdown Raji cells compared with controls (Figure 6C). In addition, wild-type Raji cells incubated with anti-CD47 antibody did not modulate VCAM-mediated adhesion compared with isotype control (Figure 6D).

We previously demonstrated that macrophages are the principal immune effector cell type mediating anti-CD47 antibody-induced phagocytosis of human NHL cells in vitro and in vivo.¹⁵  Therefore, we investigated whether macrophages also mediated anti-CD47 antibody inhibition of lymphoma dissemination. Primary DLBCL cells were transplanted subcutaneously into NSG mice and divided into control IgG and anti-CD47 antibody treatment groups that were further separated into cohorts receiving either liposomal clodronate, a bisphosphonate that depletes phagocytes, or a liposomal control. Clodronate was used as previously described,¹⁵  resulting in an average reduction of F4/80⁺ macrophages to 11% of liposomal control-treated mice (data not shown). Mice were followed serially for tumor volume and the presence of lymphoma in the bone marrow and peripheral blood. Macrophage depletion partially abrogated the minimal effect of anti-CD47 antibody in reducing primary tumor volume; however, this effect was not statistically significant (Figure 7A). No difference in tumor volume was observed with or without macrophage depletion in control IgG-treated mice (Figure 7A). In contrast, macrophage depletion completely abrogated anti-CD47 antibody-mediated inhibition of hematogenous lymphoma dissemination (Figure 7B-C), as evidenced by lymphoma cells in the bone marrow and peripheral blood, but had no effect on dissemination in mice treated with control IgG (supplemental Figure 4).

Lastly, we determined whether anti-CD47 antibody-mediated inhibition of NHL dissemination was specifically dependent on blockade of the CD47-SIRPα interaction. Luciferase-labeled Raji cells were incubated ex vivo with antibody controls or an anti-CD47 antibody capable or not capable of blocking the CD47-SIRPα interaction (clones B6H12.2 and 2D3, respectively). Both anti-CD47 antibodies bound Raji cells similarly (Figure 7D), consistent with prior results.¹⁵  Antibody-coated Raji cells were then transplanted intravenously into adult NSG mice and analyzed for lymphoma engraftment by bioluminescent imaging to determine the effect of antibody coating on disease dissemination. Consistent with our prior report,¹⁵  the blocking anti-CD47 antibody (B6H12.2) inhibited tumor dissemination in 100% of mice compared with IgG1 isotype and anti-CD45 antibody controls (Figure 7E-F). In contrast, the nonblocking anti-CD47 antibody (2D3) had a minimal effect on lymphoma dissemination compared with antibody controls, as 66% (4 of 6) mice developed detectable engraftment (Figure 7E-F).

We report here that extranodal dissemination of lymphoma requires CD47 and is inhibited by blockade of the CD47-SIRPα. We developed 2 in vivo models of human NHL (Burkitt lymphoma and DLBCL), whereby disease from primary subcutaneous sites disseminated to multiple secondary sites. Although treatment of these mice with a blocking anti-CD47 antibody had a minimal effect on reducing disease at the primary site, anti-CD47 antibody robustly inhibited dissemination of disease to secondary sites in both models. CD47 expression was required for NHL cells to spread in vivo as knockdown of CD47 decreased the invasive potential of NHL cells; furthermore, increased CD47 expression was observed on disseminated NHL cells compared with cells at the primary site. These results demonstrate that CD47 is required for NHL dissemination and that this secondary spread can be therapeutically targeted with a blocking anti-CD47 antibody.

We hypothesize that dissemination of lymphoma cells regulated by CD47 resembles the physiologic role of CD47 in the normal migration of hematopoietic cells. Indeed, CD47 plays a migratory role in multiple hematopoietic cell types. For example, CD47 is required for granulocyte and T-cell recruitment to sites of infection^25,26  and for dendritic cell trafficking to secondary lymphoid organs.²⁷  In mouse melanoma, CD47 has been implicated in tumor metastasis to bone.²⁸  The physiologic and tumorigenic role of CD47 in migration may be the result of several distinct mechanisms given that CD47 interacts with several proteins, including SIRPα, integrins, and thrombospondin-1.^14,29  First, CD47-mediated regulation of cell migration may be integrin- or chemokine-dependent as CD47 has been shown to bind several integrins, including α_vβ₃, α_iibβ₃, and α₂β₁.^14,30  In this way, CD47 may mediate normal and tumor cell migration through integrin activation and cell attachment. We show here (Figure 6A-B) that down-regulation of CD47 expression as well as administration of a blocking anti-CD47 antibody inhibited chemotaxis of lymphoma cells to 2 chemokines (SDF-1α and CXCL13) that have been shown to trigger integrin activation of LFA-1, α4β1, and α_vβ₃.^31-33  In another study, antibodies to CD47 and α_vβ₃ inhibited in vitro chemotaxis of melanoma and prostate cancer cell lines.³⁴  Although CD47 plays a role in chemokine-mediated migration of lymphoma cells, we did not observe a role for CD47 in integrin-mediated adhesion. Modulation of CD47 did not impact VCAM/VLA-4 adhesion (Figure 6B-C); however, CD47 may play a role in other integrin-mediated adhesion pathways.

Apart from integrins, the function of CD47 in regulating cell migration may be mediated by inhibition of phagocytosis through ligation of SIRPα on phagocytes. We have previously shown that mobilization of mouse HSCs from the bone marrow to the periphery requires transient up-regulation of CD47.³⁵  During mobilization, HSCs must navigate through and past perviascular and sinusoidal macrophages in blood vessels, lymph nodes, and other tissues, raising the possibility that increased CD47 expression is necessary to evade phagocytosis during the migratory process. We show that this potential mechanism is probably conserved in tumor cell migration based on 5 lines of evidence. First, lymphoma cells engineered with reduced CD47 expression exhibited decreased dissemination compared with wild-type cells (Figure 1). Second, antibody blockade of CD47 inhibited lymphoma dissemination to secondary sites in 2 different models (Figures 2,Figure 3–4). Third, lymphoma cells that disseminated to secondary sites expressed higher levels of CD47 compared with cells at the primary site (Figure 5), and this increased expression was required for dissemination (Figure 1). Fourth, macrophages played an essential role in lymphoma dissemination in a CD47-dependent manner. When macrophages were depleted, the anti-CD47 antibody-mediated inhibition of lymphoma dissemination was completely abrogated (Figure 7A-C). Fifth, inhibition of dissemination was observed only with an anti-CD47 antibody capable of blocking the CD47-SIRPα interaction (clone B6H12.2), whereas a nonblocking anti-CD47 antibody (clone 2D3) had no effect (Figure 7D-F).

Human lymphomas display a wide spectrum of growth patterns, from limited to widespread invasion. In NHL, subtypes are partly classified in terms of anatomic distribution: nodal versus extranodal lymphomas. These differences often correlate with clinical prognosis as extranodal lymphomas resident in the brain, testicles, and eye generally confer a worse prognosis and are less responsive to standard treatment.³⁶  In addition, advanced stage, which factors in degree of both anatomic involvement and extranodal involvement, confers a worse clinical prognosis across multiple NHL subtypes.³⁷  Many of the aggressive NHL subtypes (DLBCL, Burkitt lymphoma, and peripheral T-cell lymphoma) that confer a poor prognosis also have a high tendency to disseminate.

Of specific clinical concern with dissemination is CNS involvement, which in NHL can occur in up to 30% of some lymphoma subtypes³⁸  and carries an extremely poor prognosis despite the administration of aggressive therapies. Although combination chemotherapy with the anti-CD20 antibody rituximab has significantly improved outcomes in NHL,³⁹  it is well known that rituximab and many standard chemotherapeutic agents (cyclophosphamide, doxorubicin, vincristine, and prednisone) do not effectively penetrate the blood-brain barrier and reach the CNS.⁴⁰  Moreover, the addition of rituximab to standard therapy has not shown a definitive reduction in CNS involvement.^41-44  Thus, intravenous and/or intrathecal administration of cytotoxic agents is routinely used for prophylaxis against secondary CNS disease and is often associated with significant systemic toxicity.

In the present study, we demonstrate that anti-CD47 antibody is able to prevent the dissemination of NHL from its primary site in 2 xenograft models. Although anti-CD47 antibody treatment had only a partial effect on debulking the primary solid tumor site (Figures 2A and 4A), antibody therapy was completely effective at inhibiting or eliminating invasion of lymphoma cells to major organs, including the CNS (Figures 2 and 3). These preclinical results suggest that anti-CD47 antibody may be an effective therapy in the prophylaxis of CNS lymphoma in contrast to standard antibody therapy with rituximab. In addition, we have previously shown that anti-CD47 antibody has a minimal preclinical toxicity profile^15,19,22  and thus could also be an attractive alternative to CNS prophylaxis with toxic chemotherapies. Lastly, anti-CD47 antibody could be used as an adjunct in combination with standard therapy. For example, cytoreduction of bulky lymph node disease could be achieved with standard chemotherapy or radiation, whereas administration of anti-CD47 antibody could be used to either prevent disease spread in early-stage NHL or for the treatment of late-stage NHL where lymphoma has already disseminated to other organs.

In conclusion, we find that CD47 plays an essential role in the dissemination of human lymphoma and that a blocking anti-CD47 antibody may have clinical utility in preventing or eliminating disease spread. Given the shared role of CD47 in the biology and pathogenesis of many cancers, including solid tumors, targeting of CD47 may be a potential therapeutic strategy to prevent dissemination and metastasis of multiple tumor types.

The authors thank Jonathan Irish and June Helen Myklebust for providing NHL samples and corresponding clinical information, Adriane Mosley for animal handling, and Theresa Storm and Feifei Zhao for excellent laboratory management.

This work was supported by the National Institutes of Health (grant P01CA139490, I.L.W.) and the Ludwig Foundation. M.P.C. and C.T. are supported by a medical fellowship from the Howard Hughes Medical Institute. M.P.C. is supported by the National Cancer Institute, DHHS (PHS grant CA09302). R.K.P. is supported by an ASCO Young Investigator Award and California Breast Cancer Research Program Fellowship. R.M. holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund.

National Institutes of Health

Contribution: M.P.C., C.T., R.K.P., and R.C. performed the experiments and analyzed results; and M.P.C., R.M., and I.L.W. designed experiments and wrote the manuscript.

Conflict-of-interest disclosure: I.L.W., M.P.C., and R.M. have filed US Patent Application Serial No. 12/321,215 entitled “Methods For Manipulating Phagocytosis Mediated by CD47.” The remaining authors declare no competing financial interests.

Correspondence: Mark P. Chao, Stanford Institute for Stem Cell Biology and Regenerative Medicine, Lorry Lokey Research Bldg, 256 Campus Dr, Rm G3005, Stanford, CA 94305; e-mail: mpchao@stanford.edu.