Mark P. Chao,1Chad Tang,1Russell K. Pachynski,2,3Robert Chin,1*Ravindra Majeti,1,4and *Irving L. Weissman1
1Institute for Stem Cell Biology and Regenerative Medicine, Stanford Cancer Center, and Ludwig Center at Stanford, Stanford, CA;2Department of Internal
Medicine, Division of Oncology,3Department of Pathology, and4Division of Hematology, Stanford University, Stanford, CA
Non-Hodgkin lymphoma (NHL) presents
as both localized and disseminated dis-
ing a worse prognosis. Although path-
ways driving NHL dissemination have
been identified, there are few therapies
capable of inhibiting them. Here, we re-
port a novel role for the immunomodula-
tory protein CD47 in NHL dissemination,
and we demonstrate that therapeutic tar-
geting of CD47 can prevent such spread.
We developed 2 in vivo lymphoma metas-
tasis models using Raji cells, a human
NHL cell line, and primary cells from a
lymphoma patient. CD47 expression was
required for Raji cell dissemination to the
liver in mouse xenotransplants. Targeting
of CD47 with a blocking antibody inhib-
ited Raji cell dissemination to major
tem, and inhibited hematogenous dis-
semination of primary lymphoma cells.
mediated elimination of circulating tumor
cells occurred through phagocytosis, a
previously described mechanism for
blocking anti-CD47 antibodies. As pre-
ade of phagocyte SIRP? and required
macrophage effector cells. These results
demonstrate that CD47 is required for
NHL dissemination, which can be thera-
peutically targeted with a blocking anti-
CD47 antibody. Ultimately, these findings
are potentially applicable to the dissemi-
nation and metastasis of other solid tu-
mors. (Blood. 2011;118(18):4890-4901)
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.1This recirculation
is not random but rather is guided by lymphocyte-endothelial
interactions mediated by adhesion molecules (L-selectin, CD44,
integrin ?4?7, 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,
?3?3, and other homing-associated integrins.4-6Furthermore, sev-
eral of these adhesion molecules have been therapeutically ex-
ploited, as antibodies targeting the adhesion receptors LFA-1,
integrin ?v?3, and CD44 can inhibit dissemination of lymphoma in
CD47, also known as integrin-associated protein, has been
implicated in the migration and mobilization of normal leuko-
cytes.10-14In cancer, we recently demonstrated that CD47 regulates
lymphoma pathogenesis by enabling evasion of phagocytosis
through binding of the inhibitory receptor SIRP? 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.15Given 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.
Human samples and antibodies
NHL samples were obtained as previously described15from 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.15The nonblocking anti–human CD47 antibody clone
2D3, mouse IgG1 isotype control, and anti–human CD45 antibodies were
obtained from eBioscience.
Flow cytometric analysis
For analysis of primary and xenografted NHL cells, human CD19, human
CD45, mouse Terr19, mouse CD45, and mouse F4/80 were used (Invitro-
gen and BD Biosciences). Analysis of human CD47 expression was
Submitted February 17, 2011; accepted July 24, 2011. Prepublished online as
Blood First Edition paper,August 9, 2011; DOI 10.1182/blood-2011-02-338020.
*R.M. and I.L.W. contributed equally to this study.
An Inside Blood analysis of this article appears at the front of this issue.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2011 by TheAmerican Society of Hematology
4890 BLOOD, 3 NOVEMBER 2011?VOLUME 118, NUMBER 18
For personal use only.on November 5, 2015. by guest
performed with an anti–human CD47 FITC antibody (clone B6H12.2, BD
Generation of luciferase-positive Raji cells and luciferase
Aluciferase-positive Raji cell line was generated and analyzed by luciferase
imaging as previously described.15
In vivo anti-CD47 antibody treatment in a localized and
disseminated lymphoma xenograft model
For the localized model, 3 ? 106luciferase-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 ?g 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 ? 106luciferase-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 ?g/mL of IgG1
isotype control antibody, anti-CD45 antibody, anti-CD47 antibody clone
B6H12.2, or anti-CD47 antibody clone 2D3 before intravenous transplanta-
tion. Tumor engraftment was analyzed 5 days after transplantation by
In vivo anti-CD47 antibody treatment in a primary localized
DLBCL xenograft model
A total of 2-3 ? 106bulk 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 ?g intraperitoneal injections
of either control mouse IgG or anti-CD47 antibody. The presence of
DLBCLdisease was analyzed by flow cytometry for the presence of human
CD45?CD19?cells in the peripheral blood or bone marrow.
Lentiviral-encoded shRNA knockdown of Raji cells
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.
Histopathologic analysis of lymphoma-engrafted mice
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 usingAxiovision 4.7 software.
In vivo macrophage depletion
Depletion of macrophages in NSG mice was performed using liposomal
clodronate or liposomal control as previously described.15Clodronate 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.
MRI of NHL-engrafted mice
For in vivo MRI acquisition, mice were anesthetized with isoflurane
(IsoFlo; Abbott Laboratories; 1%-1.75%). Gadolinium-based contrast,
ally 20-30 minutes before image acquisition (2.5 mL/kg in PBS, 100 ?L).
Temperature and respiration were monitored continuously. Body tempera-
ture 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 fol-
lows: 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
The chemotaxis assay across polycarbonate transwell inserts were per-
formed as previously described.16Raji 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 ? 106Raji cells were then suspended in 100 ?Lof serum-free IMDM and
added to the top chamber of a 24 well transwell plate with 5 ?m pore-size
filters (Corning/Costar). In the bottom chamber, there was 600 ?Lof media
with either chemokine 10nM SDF-1? or 1 ?g/mL CXCL13 (PeproTech).
SDF-1? and CXCL13 were chosen as chemokines previously shown to
enable migration of lymphoma cells.17,18Chemokine dosing was deter-
mined 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 ?g/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-?L microbeads per sample and counted on a flow cytometer (LSRII,
BD Biosciences). Percentage migration was calculated using the number of
starting and migrated cells.
Static adhesion assay
To evaluate static adhesion, Raji cells were washed and resuspended at
0.5 to 1 ? 106cells/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 ?g/mL overnight at 4°C. Plates were then blocked for
1-3 hours with PBS ? 0.1% BSA at room temperature; 100-?L 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 ?L CellTiter 96 (Invitrogen) was added to each well containing
adherent cells in 100 ?L 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.
Dissemination of NHL is dependent on CD47
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-?
null (NSG) immunodeficient mice developed nodular disease.15In
ary sites, including the liver (Figure 1E). Lentiviral shRNAvectors
were used to knock down CD47 expression in Raji cells (Figure
1A-B) as previously described.15Raji 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
ROLE OF CD47 IN DISSEMINATION OF NHL 4891 BLOOD, 3 NOVEMBER 2011?VOLUME 118, NUMBER 18
For personal use only. on November 5, 2015. by guest
and cell adhesion. Cancer Lett. 2009;283(1):29-
33. Parmo-Cabanas M, Bartolome RA, Wright N,
HidalgoA, DragerAM, Teixido J. Integrin
alpha4beta1 involvement in stromal cell-derived
factor-1alpha-promoted myeloma cell transendo-
thelial migration and adhesion: role of cAMP and
the actin cytoskeleton in adhesion. Exp Cell Res.
34. Shahan TA, FawziA, Bellon G, Monboisse JC,
Kefalides NA. Regulation of tumor cell che-
motaxis by type IV collagen is mediated by a
Ca(2?)-dependent mechanism requiring CD47
and the integrin alpha(V)beta(3). J Biol Chem.
35. Jaiswal S, Jamieson CH, Pang WW, et al. CD47
is upregulated on circulating hematopoietic stem
cells and leukemia cells to avoid phagocytosis.
36. Gospodarowicz MK, Sutcliffe SB. The extranodal
lymphomas. Semin Radiat Oncol. 1995;5(4):281-
37. Armitage JO. Treatment of non-Hodgkin’s lym-
phoma. N Engl J Med. 1993;328(14):1023-1030.
38. van Besien K, Ha CS, Murphy S, et al. Risk fac-
tors, treatment, and outcome of central nervous
system recurrence in adults with intermediate-
grade and immunoblastic lymphoma. Blood.
39. Coiffier B, Lepage E, Briere J, et al. CHOP che-
motherapy plus rituximab compared with CHOP
alone in elderly patients with diffuse large-B-cell
lymphoma. N Engl J Med. 2002;346(4):235-242.
40. KikuchiA, Kawada H, Iwaki Y, et al. [Measure-
ment of rituximab concentration in the cerebrospi-
nal fluid in CNS lymphoma]. Rinsho Ketsueki.
41. Yamamoto W, Tomita N, Watanabe R, et al. Cen-
tral nervous system involvement in diffuse large
B-cell lymphoma. Eur J Haematol. 2010;85(1):6-
42. Feugier P, Virion JM, Tilly H, et al. Incidence and
risk factors for central nervous system occur-
rence in elderly patients with diffuse large-B-cell
lymphoma: influence of rituximab. Ann Oncol.
43. Boehme V, Schmitz N, Zeynalova S, Loeffler M,
Pfreundschuh M. CNS events in elderly patients
with aggressive lymphoma treated with modern
chemotherapy (CHOP-14) with or without ritux-
imab: an analysis of patients treated in the RI-
COVER-60 trial of the German High-Grade Non-
Hodgkin Lymphoma Study Group (DSHNHL).
44. Shimazu Y, Notohara K, Ueda Y. Diffuse large
B-cell lymphoma with central nervous system re-
lapse: prognosis and risk factors according to ret-
rospective analysis from a single-center experi-
ence. Int J Hematol. 2009;89(5):577-583.
ROLE OF CD47 IN DISSEMINATION OF NHL4901 BLOOD, 3 NOVEMBER 2011?VOLUME 118, NUMBER 18
For personal use only.on November 5, 2015. by guest
online August 9, 2011
2011 118: 4890-4901
Mark P. Chao, Chad Tang, Russell K. Pachynski, Robert Chin, Ravindra Majeti and Irving L.
is inhibited by anti-CD47 antibody therapy
Extranodal dissemination of non-Hodgkin lymphoma requires CD47 and
Articles on similar topics can be found in the following Blood collections
Updated information and services can be found at:
(2152 articles) Lymphoid Neoplasia
Information about reproducing this article in parts or in its entirety may be found online at:
Information about ordering reprints may be found online at:
Information about subscriptions and ASH membership may be found online at:
Copyright 2011 by The American Society of Hematology; all rights reserved.
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
For personal use only.on November 5, 2015. by guest