Inhibition of CD47 Effectively Targets Pancreatic …...1Stem Cells and Cancer Group, Molecular Pathology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

Cancer Therapy: Preclinical

Inhibition of CD47 Effectively Targets PancreaticCancer Stem Cells via Dual MechanismsMichele Cioffi1, Sara Trabulo1,2, Manuel Hidalgo3, Eithne Costello4,William Greenhalf4,Mert Erkan5,6, Joerg Kleeff5, Bruno Sainz Jr1, and Christopher Heeschen1,2

Abstract

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is acancer of the exocrine pancreas with unmet medical need andis strongly promoted by tumor-associated macrophages(TAM). The presence of TAMs is associated with poor clinicaloutcome, and their overall role, therefore, appears to beprotumorigenic. The "don't eat me" signal CD47 on cancercells communicates to the signal regulatory protein-a onmacrophages and prevents their phagocytosis. Thus, inhibitionof CD47 may offer a new opportunity to turn TAMs againstPDAC cells, including cancer stem cells (CSC), as the exclu-sively tumorigenic population.

Experimental Design:We studied in vitro and in vivo the effectsofCD47 inhibition onCSCsusing a large set of primary pancreaticcancer (stem) cells as well as xenografts of primary human PDACtissue.

Results: CD47 was highly expressed on CSCs, but not on othernonmalignant cells in the pancreas. Targeting CD47 efficientlyenhanced phagocytosis of a representative set of primary humanpancreatic cancer (stem) cells and, even more intriguingly, alsodirectly induced their apoptosis in the absence of macrophagesduring long-term inhibition of CD47. In patient-derived xeno-graft models, CD47 targeting alone did not result in relevantslowing of tumor growth, but the addition of gemcitabine orAbraxane resulted in sustained tumor regression and preventionof disease relapse long after discontinuation of treatment.

Conclusions: These data are consistent with efficient in vivotargeting of CSCs, and strongly suggest that CD47 inhibitioncould be a novel adjuvant treatment strategy for PDAC indepen-dent of underlying and highly variable driver mutations. ClinCancer Res; 21(10); 2325–37. �2015 AACR.

IntroductionPancreatic ductal adenocarcinoma (PDAC) remains one of the

most devastating cancers with a 5-year survival rate of less than 5%(1). Despite expanding research activities, there has been littletherapeutic progress toward improving patients' long-term surviv-al. Gemcitabine (2), FOLFIRINOX (3), and more recently theaddition of nab-paclitaxel (Abraxane; ref. 4) are able tomoderatelyextend median survival, but eventually the vast majority ofpatients still succumb from progressive disease. Therefore, devel-oping new and more effective anti-PDAC treatments represent anurgent and unmetmedical need (5, 6). Because the cancer stem cell(CSC) hypothesis was functionally validated for leukemia in 1994

(7), convincing evidence has emerged for several solid tumorsindicating that like adult tissues, tumors are sustained and pro-moted by cells that exhibit features of stem cells, includingunlimited self-renewal (8). We and others have provided conclu-sive evidence for a hierarchical organization in human PDAC and,even more importantly, demonstrated that pancreatic CSCs, at theapex of the hierarchy, have exclusive tumorigenic and metastaticpotential and are inherently resistant to chemotherapy (9–11).Indeed, the survival of such resistant CSCs during chemotherapy,despite initial tumor regression, represents a plausible explanationfor the later fatal relapse of disease in most patients (3, 4).

Studies based on the inhibition of regulatory pathways that arecrucially relevant for the self-renewal capacity of CSCs are prom-ising (12, 13); however, the overly heterogeneous genetic back-ground of PDAC may render larger populations of cells resistantto the targeting of single pathways. Consequently, we askedwhether targeting pancreatic CSCs with broader immune-basedtherapeutic approaches could represent a more viable and potentalternative for eliminating these highly tumorigenic and chemore-sistant cells.Macrophages play crucial roles in adaptive and innateimmunity. In PDAC, tumor-associated macrophages (TAM) rep-resent the major immune cell type present in the PDAC tumormicroenvironment (14), and these cells are believed to drivecancer progression, presumably via promoting cancer cell prolif-eration, tumor angiogenesis, extracellular matrix breakdown, andsubsequently tumor invasion and metastasis (15, 16). In addi-tion, CD47, a transmembrane protein expressed on many cancercells, serves as a ligand to signal regulatory protein-a (SIRPa),a molecule expressed on macrophages (17), resulting in theinhibition of phagocytosis by macrophages through a signaling

1Stem Cells and Cancer Group, Molecular Pathology Programme,Spanish National Cancer Research Centre (CNIO), Madrid, Spain.2Centre for Stem Cells in Cancer and Ageing, Barts Cancer Institute,A CR-UK Centre of Excellence, Queen Mary University of London,UnitedKingdom. 3Gastrointestinal CancerClinical ResearchUnit,Clin-ical Research Programme, CNIO, Madrid, Spain. 4Liverpool CancerResearch UK Centre, University of Liverpool, Liverpool, United King-dom. 5Department of Surgery, Technical University Munich, Munich,Germany. 6Koc University School of Medicine, Instanbul, Turkey.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Christopher Heeschen, Centre for Stem Cells in Cancerand Ageing, Barts Cancer Institute, Queen Mary University of London, Charter-house Square, London EC1M 6BQ, United Kingdom. Phone: 44-0-20-7882-8201;Fax: 44-0-20-7882-3885; E-mail: [email protected].

doi: 10.1158/1078-0432.CCR-14-1399

�2015 American Association for Cancer Research.

ClinicalCancerResearch

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cascade mediated via phosphorylation of the immunoreceptortyrosine-based inhibitory motif present on the cytoplasmic tail ofSIRPa (18).

Previous work in preclinical models of bladder cancer, leuke-mia, and lymphoma demonstrated that inhibiting the interactionbetween CD47 and SIRPa using anti-CD47 mAbs allows forincreased phagocytosis of cancer cells in vitro and decreased tumorburden in vivo (19–21). Recently, CD47 was shown to be prefer-entially expressed in liver CSCs and inhibition of CD47 sup-pressed growth of hepatocellular carcinoma xenografts and had achemosensitizing effect (22), suggesting that CD47may also be apromising therapeutic target for hepatocellular CSCs. Here, wenow demonstrate that CD47 is also highly expressed on pancre-atic cancer (stem) cells and that anti-CD47 mAbs did not onlyenable phagocytosis of these cells by macrophages, but alsodirectly induced apoptosis of the cancer (stem) cells, whileexerting no effect on nonmalignant cells. Although CD47 target-ing as a single treatment strategy was not effective in vivo, thecombination with chemotherapy resulted in long-lasting tumorregression. Thus, our results demonstrate that targeting CD47 onPDAC cells changes the behavior of resident TAMs to inhibit,rather than promote tumor growth, and thus may evolve as apotent addition to our still sparse armamentarium against PDAC.

Materials and MethodsPrimary human and mouse pancreatic cancer cells andmacrophages

Human PDAC tissues were obtained with written informedconsent from all patients and expanded in vivo as patient-derivedxenografts (PDX), as previously described (12). For in vitro studies,PDX tissue fragments were minced, enzymatically digested withcollagenase (STEMCELL Technologies) for 90 minutes at 37�C(12) and after centrifugation for 5 minutes at 1,200 rpm cellpellets were resuspended and cultured in RPMI supplementedwith 10% FBS and 50 U/mL penicillin–streptomycin. Theseprimary cultures were used in vitro only until passage 10. MurinePDAC cells were derived from the K-Rasþ/LSL-G12D;Trp53LSL-R172H;PDX1-Cre mice (KPC) as a model of advanced PDAC(23). KPC-derived tumors were minced, mechanically (gentle-MACS Dissociator; Miltenyi) and enzymatically dissociated withcollagenase (STEMCELL Technologies), and subsequently cul-tured in vitro as previously detailed (24). Epithelial clones were

picked, pooled, and further expanded to heterogeneous primarycancer cell cultures (AAU77G and CHX6).

Human peripheral blood–derived mononuclear cells wereobtained from healthy donors with informed consent. Mono-cyte-derived macrophage cultures were established in IMDMsupplemented with 10% human AB serum as previouslydescribed (25). Sixty ng/mL GM-CSF or M-CSF (R&D Systems)was added to the cultures to generate M1 and M2 monocyte–derived macrophages, respectively (26). Murine monocytes wereisolated from mechanically disrupted spleens, passed through a40-mm mesh filter, and differentiated into macrophages underadherent conditions onnon-tissue culture-treated100-mmdishesin RPMI supplementedwith 10%FBS and 10ng/mLofmurineM-CSF (PeproTech). To generate M1- and M2-polarized murinemacrophages, 10 ng/mL of IFNg (PeproTech), and LPS (Sigma;M1) or 10 ng/mL IL4 (M2; PeproTech) were added to the cultures.

Sphere formation assaySpheres were generated by culturing 2 � 104 pancreatic cancer

cells in suspension in serum-free DMEM/F12 supplemented withB27 (1:50; Invitrogen), 20 ng/mL bFGF, and 50 U/mL penicillin–streptomycin for a total of 7 days, allowing spheres to reach a sizeof >75 mm. For serial passaging, 7-day-old spheres were retainedusing 40-mm cell strainers, dissociated into single cells, and thenrecultured for 7 additional days as previously described (13).

RNA preparation and quantitative real-time PCRTotal RNAs from human primary pancreatic cancer cells and

spheres were extracted with TRizol (Life Technologies) accordingto the manufacturer's instructions. One microgram of total RNAwas used for cDNA synthesis with SuperScript II reverse transcrip-tase (LifeTechnologies) and random hexamers. Quantitative real-time PCR was performed using SYBR Green PCR master mix(LifeTechnologies), according to the manufacturer's instructions.Primers sequences used are:

ACTIN: Forward—GCGAGCACAGAGCCTCGCCTTReverse—CATCATCCATGGTGAGCTGGCGG

CD47: Forward—GCGATTGGATTAACCTCCTTCGTCAReverse—CCATGCATTGGTATACACGCCGC

Flow cytometryCellswere adjusted to a concentration of 106 cells/mL in sorting

buffer [1X PBS; 3% FBS (v/v); 3 mmol/L EDTA (v/v)] beforeanalysis or sorting with a FACS Canto II or FACS Influx instru-ment, respectively (BD Biosciences). To identify distinct cancer(stem) cells, the following antibodies were used: anti–CD133/1-APC or PE (Miltenyi Biotec); CD47-APC, CXCR4-APC, SSEA-1-APC, or appropriate isotype-matched control antibodies (all fromBD Biosciences). DAPI was used for exclusion of dead cells. Datawere analyzed with FlowJo 9.2 software (Tree Star). For theassessment of apoptosis, cells were incubated with the DAPI andAnnexin V FITC Staining Kit (BD Biosciences) according to themanufacturer's instructions.

Antibody preparationThe anti-hCD47 (B6H12) hybridoma was obtained from the

ATCC. Hybridoma cells were cultured using previously describedconditions (27) and antibodies were purified by protein G.

Translational Relevance

Pancreatic ductal adenocarcinoma (PDAC) remains one ofthe most devastating cancers, and very few new treatmentshave revealed meaningful improvements in patient survivalover the past decades. On the basis of our previous workdemonstrating the existence of cancer stem cells (CSC) inpancreatic cancer and their strong resistance to standard che-motherapy, we now provide multiple lines of functional andmechanistic evidence for a treatment regimen, including inhi-bition of CD47 targeting both CSCs as well as their moredifferentiated progenies. Therefore, this new therapeutic strat-egy should be further explored in the clinical setting as itssuccess bears the potential to improve the poor prognosis ofpatients with PDAC.

Cioffi et al.

Clin Cancer Res; 21(10) May 15, 2015 Clinical Cancer Research2326




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Figure 1.CD47 is expressed in pancreatic cancer (stem) cells. A, quantification of CD47 expression in primary patient TMA–containing cores of normal pancreas, pancreatitis,PDAC, and metastases. Shown are the mean relative intensity values of CD47 staining within each core. B, representative pictures of CD47-stained TMAcores, including FFPE section of human-derived xenografts. C, RT-qPCR analysis of CD47 in normal pancreas samples, PSC cells, and several primary humanpancreatic cancer cultures. b-Actin was used as a normalization control. D, flow-cytometry analysis of CD47 cell surface expression comparing adherent cellsand sphere-derived cells. E, flow-cytometry analysis of CD47 and CD133 expressions on sphere-derived cells (left) and quantification of CD133þ cells also expressingCD47 (right).

CD47 Targets Pancreatic Cancer Stem Cells

www.aacrjournals.org Clin Cancer Res; 21(10) May 15, 2015 2327




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Cioffi et al.





In vitro phagocytosis assayFor in vitro phagocytosis analysis, 5 � 104 monocyte-derived

macrophages were plated in each well of 24-well tissue-cultureplates and labeled with PKH26 according to the manufacturer'sinstructions (Sigma). Macrophages were incubated in serum-freemedium for 2 hours before adding 2 � 105 GFP-labeled livecancer cells. Anti-CD47 (B6H12) antibody (10 mg/mL) or IgG1control antibody was added and incubated for 2 hours at 37�C.Macrophages were repeatedly washed and subsequently imagedusing an inverted microscope (Leica DMI6000B). The phagocyticindex was calculated as the number of phagocytosed GFPþ cellsper 100 macrophages.

ImmunohistochemistryFor histopathologic analysis, FFPE blocks were serially sec-

tioned (3-mm thick) and stained with hematoxylin and eosin(H&E). Additional serial sections were used for immunohisto-chemical (IHC) studies with anti-CD47 antibody (0.2 mg/mL;Abcam ab3283). Antigens were visualized using 3,3-diaminoben-zidine tetrahydrochloride plus (DABþ). Counterstaining wasperformed with hematoxylin. Histologic quantification ofdigitalized slides was performed using Pannoramic Viewer(3DHistech).

Tissue microarraysFour human tissue microarrays (TMA) containing quadru-

plicate 1-mm cores from selected areas of paraffin-embeddedpancreatic surgical specimens, including ducts, acini, pancrea-titis, PDAC, and PDAC metastasis were constructed. A total of42 tumors were included. Two xenograft TMAs containingquadruplicate 1-mm cores from selected tumor areas of 56paraffin-embedded human PDACs grafted in nude mice werealso constructed. The use of human tissue samples for theconstruction of the TMAs was approved by the Ethics Com-mittee of the Hospital de Madrid Norte Sanchinarro. All sec-tions were assessed and scored by an in-house pathologist(Maria Lozano).

In vivo tumorigenicity assayPrimary pancreatic cells were treated in vitro with anti-CD47

and serial dilutions of single-cells were resuspended in Matrigel(BD Biosciences) and s.c. injected into female 6- to 8-week-oldNU-Foxn1nu nude mice (Harlan Laboratories). In some experi-ments, macrophages were depleted with the following treatmentschedule: 200 ml of clodronate was injected i.v. twice a week.Tumor formation was evaluated after 2 months. Mice werehoused according to institutional guidelines and all experimentswere approved by the local Animal Experimental Ethics Commit-tee of the Instituto de Salud Carlos III (PA 34-2012) and per-formed in accordance with the guidelines for Ethical Conduct inthe Care and Use of Animals as stated in The InternationalGuiding Principles for Biomedical Research involving Animals,developed by the Council for International Organizations ofMedical Sciences.

In vivo treatmentPrimary tumor tissue pieces of approximately 2 mm3 were

implanted s.c. into the flanks of NU-Foxn1nu nudemice, and oncetumors were established mice were randomized to the respec-tive treatment groups. Gemcitabine was administered twice aweek (125 mg/kg i.p.), Abraxane was administered every4 days (50 mg/kg i.v.), and anti-CD47 was administered daily(500 mg/mouse i.p.; ref. 20).

Statistical analysesResults for continuous variables are presented as means � SD

unless stated otherwise and significancewas determined using theMann–Whitney test. All analyses were performed using SPSS 22.0(SPSS).

ResultsCD47 is expressed at higher levels in PDAC compared withnormal pancreatic tissue

We evaluated the level of CD47 expression by immunohisto-chemical analysis of paraffin sections of tissue microarrayscontaining primary human tissues from "normal" adjacentnon-tumor pancreatic tissue, pancreatitis, PDAC, and regionallymph, and liver metastases. CD47 expression was significantlyoverexpressed in primary PDAC tumors (P < 0.001) and metas-tasis (P < 0.05) versus pancreatitis and normal pancreatic tissue(Fig. 1A). Importantly, although CD47 was still detectable innormal (non-cancer) pancreatic tissue, the level of expressionwassignificantly lower compared with PDAC, patient-derived PDACxenograft and metastasis samples, where CD47 expression wasmarkedly stronger, but restricted to epithelial cancer cells andabsent in the stroma (Fig. 1B and Supplementary Fig. S1A). Wenext analyzed independent large collections of primary tissues forthe expression of CD47. The results indicated that CD47 expres-sion varies considerably within tissues (Supplementary Fig. S1B)and between patients with about one third of the patients bearinglow to undetectable levels of CD47. Patients represented on eachof the two independent sets of TMAs were dichotomized accord-ing to low to undetectable CD47 expression (CD47 negative) andintermediate to high CD47 expression (CD47 positive); however,no association between CD47 expression and outcome could beidentified (Supplementary Fig. S1C).

We next determined CD47 mRNA expression in a set of nineprimary patient-derived pancreatic cancer cell cultures, twonormal pancreas samples, and primary pancreatic stellate cells(PSC). We observed low to undetectable levels of CD47 mRNAexpression in normal tissue and in PSCs as compared withPDAC cells. The latter could be subdivided into three groupsbased on their CD47 mRNA expression: low (JH029 and 247),medium (198, 253, 215, and 354), and high (163, 185,and A6L; Fig. 1C). To confirm the expression of CD47 at theprotein level, flow-cytometry analysis was performed on bothadherent cells and sphere-derived cells, the latter of which areenriched in CSCs (13). We observed relatively homogenous

Figure 2.Pancreatic CSCs are mostly confined to CD47þ cells. A, representative flow-cytometry plots of CD47 staining showing the gating strategy for sorting. B,representative images of spheres (left) and quantification of spheres (right) in 185, 354, and 215 primary pancreatic cancer cells sorted for CD47. C, In vivotumorigenicity of FACSorted 185, 354, and 215 primary pancreatic tumor cells for CD47. D, sphere formation capacity for cells FACSorted for CD47 and CD133. E, invivo tumorigenicity of cells FACSorted for CD47 and CD133 injected in mice depleted for macrophages by treatment with clodronate (twice a week).






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Figure 3.Anti-CD47 enables phagocytosis of pancreatic CSCs. A, representative confocal images (top) and phagocytic index (bottom) of human peripheral blood (PB)–derivedmacrophages (red) phagocytosing patient–derived CSCs (green) in the presence of blocking anti-CD47mAb (B6H12) or IgG1 isotype control Ab. (Continuedon the following page.)

Cioffi et al.





expression of CD47 in differentiated cells (ranging from 40% to57%) whereas in sphere culture-enriched CSCs, the surfaceexpression of CD47 was higher, although more variable (rang-ing from 54% to 85%), suggesting an enrichment of CD47 inCSCs (Fig. 1D). We next assessed the percentage of CD47þ cellswithin the CD133þ [a well-established pancreatic CSC marker(9)] subpopulation, and as shown in Fig. 1E, the majority ofCD133þ cells expressed CD47 albeit with different percentages(ranging from 77% to 97%).

Pancreatic CSCs are mostly confined to CD47þ cellsBecause our data suggested that CD47 is preferentially

expressed in pancreatic CSC (i.e., CD133þ cells), we aimed to

assess whether CD47þ cells were more "stem-like." We firstFACSorted primary pancreatic cancer cells for CD47 (Fig. 2A)and then determined their self-renewal capacity using sphereformation as a readout. We observed that CD47þ cells isolatedfrom 185, 215, and 354 primary cells formed significantly moreand larger spheres compared with CD47� cells (Fig. 2B), suggest-ing that CD47þ cells are indeed enriched in CSCs. However, toobtain conclusive evidence for the latter, we performed in vivolimiting dilution tumorigenicity assays. Ten weeks after injection,CD47þ cells had formed more tumors, indicating that CSCs aremostly contained in the CD47þ cell population (Fig. 2C). Tofurther evaluate the function of CD47 in the CSCs context, wesorted four populations based on CD133 and CD47 expressions

(Continued.) B, the phagocytic index of macrophages phagocytosing human PDAC cells FACSorted for CD47 and CD133 in the presence of blockinganti-CD47 mAb or IgG1 isotype control Ab. C, phagocytic index of human unpolarized, M1, M2, and CSC media polarized macrophages. D, the phagocytic index ofmurine M1- and M2-polarized macrophages in the presence of blocking anti-CD47 mAb or IgG1 isotype control Ab. E, flow-cytometry analysis of CD133 cellsurface expression on surviving cells following incubation with primary human macrophages and treatment with anti-CD47 mAb (B6H12) or IgG1 isotype controlmAb. F, sphere formation quantification of cells after treatment with anti-CD47 mAbs, compared with IgG1 control treated cells.

AD

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Figure 4.Anti-CD47 treatment directly induces apoptosis of pancreatic CSCs. A, flow-cytometry analysis of apoptosis as determined by Annexin V/DAPI staining, innontransformed human cells; and B, several primary human pancreatic sphere–derived cell cultures after treatment for 12 hourswith anti-CD47mAb (B6H12) or IgG1isotype control mAb.






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GEM

GEM+Anti-CD47

Anti-CD47

HE

CK

19

* P < 0.01 vs. single treatment** P < 0.01 vs. control

* P < 0.05

Abraxane

Gemcitabine

Anti-CD47

Control

Abraxane+ Anti-CD47

Gemcitabine+ Anti-CD47

Tum

ori

gen

icit

y (t

um

or

take

rat

e in

%)

Tum

or

volu

me

(mm

3 )

IgG1-Iso Anti-CD47 IgG1-Iso Anti-CD47 IgG1-Iso Anti-CD47 in 354 Anti-CD47 in 215

Abraxane

Gemcitabine

Anti-CD47

Control

Abraxane+ Anti-CD47

Gemcitabine+ Anti-CD47

*

*

HE

CK

19

Control ABX+Anti-CD47

ABXGEM GEM+Anti-CD47

Anti-CD47

0 102 103 104 105

APC-A: CD133

0

50K

100K

150K

200K

250K

SS

C-A

48.2

0 102 103 104 1050

50K

100K

150K

200K

250K

SS

C-A

20

37.8%

48.2%

20.0%

Control

ABX+Anti-CD47

ABX

CD133

Time (days)

* *

*

* P < 0.05

* P < 0.05EPCAM

800

1,000


Cioffi et al.




and observed that CD47þCD133þ cells possessed the highestsphere formation capacity (Fig. 2D). Building upon the latter, wesorted cells for both CD133 and CD47 and injected them intonude mice depleted for macrophage by means of treatment withclodronate. Macrophage depletion was performed to more defin-itively demonstrate that the tumorigenic capacities observed invivowere indeed due to differences in functional CSC content andnot related to the inherent resistance of a cell to macrophagephagocytosis based on cell surface CD47 expression. Not only didwe confirm that in vivo tumorigenicity is indeed mostly confinedto theCD47þpopulation, as previously seen (Fig. 2C), butwe alsoshow that CD47þCD133þ cells are more highly enriched forCSCs, and thus more tumorigenic, whereas CD47�CD133� cellsbear the least tumorigenic potential (Fig. 2E). Taken together,these data suggest that targeting CD47 should achieve a majorreduction in CSC activity.

Anti-CD47 treatment enables phagocytosis of pancreatic CSCsIt has been previously demonstrated that blocking CD47-

mediated SIRPa signaling using targeted mAbs induces phago-cytosis of leukemia, lymphoma, and bladder cancer cells byhuman and mouse macrophages (19, 21, 28). Using primaryPDAC cells stably infected with a lentivirus-expressing GFP(green) and PKH26 dye (red)–labeled primary human mono-cyte-derived macrophages isolated from healthy donors (ratiocancer cells:macrophages 4:1), we show that in contrast with cellstreated with an isotype-matched mouse IgG control antibody,primary PDAC cells treated with the blocking anti-human CD47(hCD47) mAb B6H12.2 were efficiently phagocytosed by macro-phages. This effect was observed for adherent cells, which mainlycontain non-CSC, and sphere-derived cells or CD47þCD133þ

sorted cells, which are enriched for CSCs (Fig. 3A and B) and wasindependent of the method of macrophage polarization (Sup-plementary Fig. S2A).

We next attempted to mimic the tumor in vivo micro-environment conditions by polarizing macrophage culturestoward an "M1" phenotype with GM-CSF and an "M2" phe-notype with M-CSF, respectively (26), or by exposing them toCSC-conditioned media from primary cultures of PDACspheres (29). We first confirmed that GM-CSF–treated macro-phages possessed a classic M1 circular morphology, whereas M-CSF-treated and CSC-conditioned macrophages both showed amore elongated shape typical of M2-polarized macrophages(Fig. 3C, top). In the absence of anti–CD47-blocking antibo-dies, CSC-conditioned "M2" macrophages had the lowestphagocytic index levels compared with the other macrophagesubtypes (0.9 vs. 2.8 for M2-polarized macrophages), consis-tent with a truly protective and protumorigenic role for thesemacrophages. Treatment with the blocking mAb B6H12.2,however, significantly enhanced phagocytosis of cancer cellsacross all macrophage subtypes, with a more pronouncedincrease for M2 (6.7-fold increase) and CSC-conditioned

(13-fold increase) macrophages (Fig. 3C, bottom). Important-ly, primary monocyte–derived murine macrophages, regardlessof their initial polarization, were also capable of phagocytosinghuman PDAC cells when CD47 was blocked (Fig. 3D).

Importantly, we evaluated the percentage of CD133þ CSCsfollowing anti-CD47 treatment and observed a significantreduction compared with isotype-treated cells (Fig. 3E). More-over, we observed a consistent and significant reduction in thesphere formation capacity of surviving/nonphagocytosed cells,indicating that anti-hCD47 treatment indeed eliminated theCSCs pool (Fig. 3F). Using a more stringent ratio of macro-phage:cancer cells (1:1), we observed similar effects in terms ofphagocytosis as well as significant reduction in sphere forma-tion and CD133þ CSC content (Supplementary Fig. S2B–S2D).In contrast, for nontransformed cells no significant inductionof phagocytosis was found (Supplementary Fig. S2B, right),which might be attributed to the lack of "eat-me" signals onthese cells. Finally, to validate the specificity of anti–CD47-induced phagocytosis, we tested a different antibody that alsobinds a large fraction of PDAC cells (i.e., anti-CD44). How-ever, treatment with anti-CD44 did not induce phagocytosiswhereas in the same experiments treatment with anti-CD47showed strong induction of phagocytosis (Supplementary Fig.S2F). These data demonstrate that induction of phagocytosisby anti-CD47 is likely independent of FcR stimulation ofmacrophages. It is worth noting, however, that blocking CD47using a Fab molecule would be necessary to definitivelydemonstrate that Fc receptor is not required. Nonetheless, thedata suggest that CD47 is a legitimate therapeutic target forPDAC.

Anti-CD47 treatment induces apoptosis of pancreatic CSCsAntibodies directed against CD47 have also been shown to

directly induce apoptosis of several hematopoietic malignan-cies (30–32). We therefore incubated sphere-derived cells withanti-hCD47 mAb B6H12.2, but in the absence of macro-phages, and subsequently assessed apoptosis 2 and 12 hoursafter treatment by Annexin V staining. Although we observedno induction of apoptosis in nontransformed human cells (Fig.4A) or in primary murine PDAC tumor cells (SupplementaryFig. S3A), we did detect a significant increase in apoptotic cellsacross several primary human PDAC cell lines following treat-ment with the anti-CD47 antibody compared with IgG mAb-treated controls (Fig. 4B). Importantly, no apoptosis wasobserved in any of the samples tested following 2 hours oftreatment (Supplementary Fig. S3B). Thus, because phagocy-tosis of CSCs by macrophages was detected as early as 15minutes after incubation with the anti-CD47 antibody (datanot shown), we identify two distinct mechanisms of action, thefirst being phagocytosis whereas the second being an apparentPDAC-specific elimination of CSCs via direct induction ofapoptosis without involvement of macrophages.

Figure 5.Anti-CD47 treatment inhibits in vivo tumorigenicity and tumor progression, preventing relapse. A, in vivo tumorigenicity; B, tumor weight; and C, flow-cytometryanalysis for EPCAM, CD133, and SSEA1 for primary pancreatic sphere–derived cells after treatment with anti-CD47 mAb (B6H12) or IgG1 isotype control mAb. D,experimental setup for in vivo treatment (top left) and effects of allocated treatment regiments in 185 tissue xenografts transplanted in immunocompromised mice(bottom left). The mean tumor volume is given; n ¼ 6 tumors per group. Representative images of tumors extracted from mice at the end of the respectiveobservation period (right). E, H&E staining and IHC analysis of CK19 expression in paraffin section from the tumors. F, flow-cytometry analysis of CD133 cell surfaceexpression in cells isolated from tumors of mice treated as indicated.






Anti-CD47 treatment inhibits in vivo tumorigenicity and tumorprogression, preventing relapse

To test the efficiency of CSCs elimination in vitro, we testedthe ability of surviving/nonphagocytosed cells after anti-CD47treatment to form tumors in vivo. We observed a significantreduction in the tumorigenicity of anti–CD47-treated cellscompared with isotype-treated cells (Fig. 5A), and the fewtumors that formed from the anti-CD47–treated cultures weresignificantly smaller in size compared with isotype controltumors (Fig. 5B). In addition, although a similar amount ofEPCAM expression was observed across all samples regardlessof the treatment, anti–CD47-treated tumors contained a sig-nificantly lower percentage of cells expressing the CSC surfacemarkers CD133 and SSEA1 (Fig. 5C). Again, when we used amore stringent ratio of macrophage:cancer cells (1:1), weobserved that tumorigenicity following injection of survivingcells was essentially abrogated (Supplementary Fig. S2E).

Encouraged by these promising in vivo tumorigenicity data, wenext performed in vivo therapeutic intervention studies withhuman-derived PDAC xenografts expressing intermediate levelsof CD47. Once tumors had formed (�100 mm3), mice wererandomized to one of the following six treatment groups: Diluentcontrol; gemcitabine (biweekly 125mg/kg i.p.) fromday14 to 56;Abraxane (every 4 days 50 mg/kg i.v.) from day 14 to 28; anti-CD47 (daily 500mg/mouse i.p.) fromday14 to 35; gemcitabineþanti-CD47; and Abraxane þ anti-CD47. Interestingly, for bothused PDX models no significant differences were observed forchemotherapy and anti-CD47 single treatments; however, tumorstreated with a combination of chemotherapy þ anti-CD47 weresignificantly reduced compared with control tumors and singletreatment tumors. Specifically, for PDAC-185, treatment withAbraxane plus anti-CD47 significantly stalled tumor growth.Although mice previously treated with Abraxane alone showedsimilar initial response, tumors eventually relapsed. No relapse(i.e., de novo growth of tumors); however, was observed whenmice were treated with both Abraxane and anti-CD47 (Fig. 5D).PDAC-185 tumors in the gemcitabine þ anti-CD47 treatmentgroup had to be harvested early due to ulcerations. Of note,tumors did not differ in gross morphology, as assessed by H&Eand CK19 immunohistochemistry (Fig. 5E), and were alsosimilarly vascularized and contained M2 macrophages (Sup-plementary Fig. S4A). Importantly, flow-cytometry analysis ofdigested tumors showed a significant decrease in the percentageof cells expressing the CSCmarker CD133 only in mice that hadreceived anti-CD47 treatment (Fig. 5F), suggesting that anti-CD47 treatment effectively targeted the CSC population. In thePDAC-215 PDX model, similar treatment benefits wereobserved when anti-CD47 was combined with a chemothera-peutic, although in this PDX model gemcitabine was moreeffective than Abraxane when used in combination with anti-CD47 as evidenced by the lack of tumor relapse during long-term follow-up (Supplementary Fig. S4B and S4C).

DiscussionMacrophages can undergo specific differentiation/polarization

depending on the environment and surrounding cellular context.Two distinct states ofmacrophage polarization have been defined(33): (i) the classically activated (M1) macrophage that plays animportant proinflammatory effector role in TH1 cellular immuneresponses, including the secretion of cytokines and phagocytosis

of target cells or (ii) the alternatively activated (M2) macrophagethat is involved in type II helper T-cell processes, such as woundhealing and humoral immunity. In cancer, protumorigenic M2TAMs enhance neoplasia via matrix remodeling, angiogenesis andthe secretion of protumor growth factors, such as TGFb (34). Incontrast,M1macrophages are believed to inhibit tumor growth viaantitumor-adaptive immunity mechanisms that include phagocy-tosis. The latter, however, mainly depends on macrophage recog-nition of prophagocytic ("eat me") signals on target cells, but canbe inhibitedby simultaneous expressionof anti-phagocytic ("don'teat me") signals, such as CD47. In the context of cancer, CD47 hasbeen found to be strongly overexpressed on different tumor cells,conferring an anti-phagocytic benefit to these cells (35, 36).Importantly, inhibition of CD47 using mAbs efficiently inducesphagocytosis of cancer cells by macrophages in experimentalmodels of leukemia, lymphoma, and bladder carcinomas (19–21); however, the relevance of this molecule and its therapeutictargeting in PDAC (stem) cells had yet to be studied.

Herein, we show that CD47 is overexpressed in the majority,but not in all primary PDACpatient samples tested usingmultipletissue microarrays (>150 patients represented). Although CD47was significantly overexpressed in about two thirds of neoplastictissues, we did not observe a correlation between high CD47protein expression and poor clinical outcome (SupplementaryFig. S1C), which is in contrast with what has been shown forother cancers including, AML, HCC, glioma, and ovarian cancer(19–21, 36). The analysis of tissue microarrays may not besufficient to capture the general CD47 expression profile for eachindividual tumor. Indeed, we observed significant variationbetween different cores thatwere available from the same patients(Supplementary Fig. S1A and S1B). Thus, analysis of completesections of primary patient PDAC samples is likely needed beforea correlative connection can be definitively determined for PDAC.Notably, although CD47 was not "clinically predictive" in ourtissuemicroarrays, we did note that CD47 expression increased insphere-derived CSC-enriched cultures, was expressed at evenhigher levels in CD133þ cells, and CD47þ PDAC cells exhibitedhigher self-renewal and tumorigenic properties compared withCD47� cells. Thus, like leukemia, bladder cancer, and HCC (19–21), CD47 expression is strongly expressed on pancreatic CSCs;however, it is likely not a suitable surrogate CSC marker as itsstrong expression on a large fraction of non-CSCs limits the levelof enrichment for CSCs in CD47þ cells, and thus would requirethe use of other CSC markers (e.g., CD133) in combination.

Using a neutralizing antibody for CD47, we found that inhibit-ing the anti-phagocytic functionofCD47allowed for bothhumanand mouse macrophages to phagocytose CSCs cells in vitro, andthe non-phagocytosed surviving PDAC cells exhibited significant-ly reduced expression of CSCmarkers and functional phenotypes,such as self-renewal and in vivo tumorigenicity. Importantly, thisphenotype was independent of the polarization state of themacrophage, as M1 and M2 macrophages were equally capableof phagocytosing PDAC cells treated with anti-CD47 mAbs com-pared with unpolarizedmacrophages. In the context of the tumormicroenvironment, M1 macrophages infiltrate the tumor duringimmune surveillance, but once recruited into tumor sites, M1macrophages can differentiate into M2 macrophages upon expo-sure to cytokines released by tumor cells and tumor stromal cells(e.g., TGFb, IL4, IL13, and IL10; ref. 33). Therefore, our findingthat MCSF-polarized M2 macrophages as well as CSC-condi-tioned M2-like macrophages were able to phagocytose PDAC

Cioffi et al.





cells treated with anti-CD47 mAbs highlights the potential of M2macrophages, the predominantmacrophage subtypepresentwiththe PDAC tumor microenvironment, as biologic tools to targetCSCs and their more differentiated non-CSCs progenies.

From a therapeutic perspective, we additionally show in twoxenotransplantation models of PDAC that treatment with mAbsfor CD47 in combination with gemcitabine or Abraxane signif-icantly reduced primary tumor growth. Specifically, we found thatanti-CD47 therapy alone only marginally reduced the size andrate of tumor growth, which again contrasts with previous find-ings for other epithelial cancers (19–21), andmay be attributed tothe more aggressive growth nature of PDAC, which cannot becompletely controlled by phagocytic macrophages. Alternatively,untreated tumors with their dense stroma may represent toostrong a barrier for the CD47 antibodies to reach the cancercells. Importantly, however, in mice treated with gemcitabine orAbraxane, the addition of anti-CD47 therapy resulted in efficientgrowth control of tumors and prevented relapse after discontin-uation of treatment. The later was particularly apparent whenAbraxane was used in combination with anti-CD47. Specifically,tumors in mice treated with both Abraxane and anti-CD47 mAbsdiminished in size, such that one tumor was completely elimi-nated, and the remaining tumors failed to relapse as comparedwith mice treated with Abraxane alone where relapse was evidentin all mice by day 77. Regarding the tumor CSC content, weobserved that only anti-CD47 therapy was able to reduce thepercentage of CD133þ cells in the tumor, which confirms ourin vitro results and indicates that anti-CD47 mAbs preferentiallytarget CSCs. Taken together, these results strongly suggest thatanti-CD47 therapy could be an effectivemeans of treating primaryPDAC tumors, but combinationwith other anticancer therapeuticagents, such as Abraxane, is needed.

Although the main acute mechanism of action of anti-CD47therapy relies onmacrophage-mediated phagocytosis of CSCs, wedid observe that long-term treatment of PDAC cells with anti-CD47 mAbs induced a prominent and cancer cell–specific induc-tion of apoptosis. It has previously been shown that ligation ofCD47 triggers caspase-independent programmed cell death innormal and leukemic cells (31, 32, 37); thus, in addition toblocking the antiphagocytic CD47 ligand on PDAC cells, anti-CD47 mAbs may also function to eliminate CSCs via a separateapoptotic-specific mechanism of action. Additional studies arestill needed to resolve whether anti-CD47 therapy induces apo-ptosis of tumor cells in vivo. In addition, it is important to note thatduring cell death, prophagocytic ("eat me") signals such as calre-ticulin are shuttled to the cell membrane (38). Thus, it is alsological to hypothesize that anti-CD47 mAb-induced apoptosismay also facilitate macrophage-mediated apoptosis via upregula-tion of the prophagocytic ("eatme") signal calreticulin. Therefore,anti-CD47 therapymayhavemultiplemechanismsof action, eachof which likely facilitates macrophage phagocytosis of CSCs.

Is CD47 targeting in PDAC suitable and ready for furtherclinical exploration? Although our in vivo study provides proof-of-concept for CD47 targeting in PDAC, indeed several openquestions remain to be addressed in further preclinical studies,but could not be tackled in the present studies based on thecurrently prohibitive costs of low scale antibody production.Once high-affinity clinical grade antibodies or high-affinity SIRPamonomers are available in larger amounts (39), it should bedetermined whether the abundant stroma in PDAC tumorsrepresents a relevant physical barrier for the antibodies to reach

the cancer cells. Therefore, it should be tested whether coadmin-istration of a stroma targeting agents leads to better response ratesfor CD47 antibody treatment. However, a cautionary note comesfrom recent studies demonstrating that stroma targeting alonecould result in adverse outcomes. Specifically, mouse studiesdemonstrated that loss of stroma leads to dedifferentiation ofcancer cells rendering themmore aggressive (40, 41). In addition,a recent clinical trial on hedgehog pathway inhibition was pre-maturely stopped on the basis of excessive death rates in thetreatment group. Whether enhanced delivery of CD47 antibodiesto stroma-depleted tumors and subsequently enhanced treatmentresponse will outweigh these putative adverse effects of stroma-targeting remains to be determined in carefully designed preclin-ical studies. Second, we observed considerable variation in CD47expression across a large panel of primary PDAC samples. Spe-cifically, 10% of patients showed no detectable or very low levelsof CD47 staining. Those patients may not gain significant ther-apeutic benefit from anti-CD47 treatment. Such stratificationcould be based on CD47 expression on circulating tumor cellsas these have been shown to also express CD47 (42). Third, itseems reasonable to explore the possibility of combining anti-CD47mAb therapywith treatments that either target TAM recruit-ment (e.g., anti-CSF1 therapy) or their polarization toward M2macrophages (e.g., anti-TGFb therapy).

In conclusion, we have found that CD47 is expressed onprimary PDAC cells and we have demonstrated that inhibitingCD47 function using mAbs is an effective method of treatingPDAC in vitro and in vivo, thereby forming the rationale forevaluating the clinical efficacy of anti-CD47 therapy in morecomprehensive preclinical studies, which may eventually lead tofirst trials in human patients with PDAC. Although further mech-anistic studies are still needed to determine how anti-CD47treatment reduces tumor growth (i.e., phagocytosis and/or apo-ptosis), the data presented herein add to the growing repertoire oftumors that can be potentially treated with anti–CD47 mAb-based therapies.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Cioffi, M. Hidalgo, B. Sainz Jr, C. HeeschenDevelopment of methodology: M. Cioffi, B. Sainz JrAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Cioffi, S. Trabulo, M. Hidalgo, E. Costello, W.Greenhalf, M. Erkan, J. Kleeff, B. Sainz JrAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):M.Cioffi, S. Trabulo, E. Costello,M. Erkan, B. Sainz Jr,C. HeeschenWriting, review, and/or revision of the manuscript:M. Cioffi, M. Hidalgo, M.Erkan, J. Kleeff, B. Sainz Jr, C. HeeschenStudy supervision: B. Sainz Jr

AcknowledgmentsThe authors thank Magdalena Choda for excellent technical assistance. They

also thank Maria Lozano for assistance with the PDAC TMA analyses.

Grant SupportThe research was supported by the ERC Advanced Investigator Grant

(Pa-CSC 233460), European Community's Seventh Framework Programme(FP7/2007-2013) under grant agreement n�256974, the Subdirecci�on Generalde Evaluaci�on y Fomento de la Investigaci�on, Fondo de Investigaci�on Sanitaria






(PS09/02129 & PI12/02643), and the Programa Nacional de Inter-nacionalizaci�on de la IþD, Subprogramma: FCCI 2009 (PLE2009-0105; bothMinisterio de Ciencia e Innovaci�on, Spain), all to C. Heeschen. M. Cioffi issupported by the La Caixa Predoctoral Fellowship Program.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 4, 2014; revised February 3, 2015; accepted February 10, 2015;published OnlineFirst February 23, 2015.

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2015;21:2325-2337. Published OnlineFirst February 23, 2015.Clin Cancer Res Michele Cioffi, Sara Trabulo, Manuel Hidalgo, et al. via Dual MechanismsInhibition of CD47 Effectively Targets Pancreatic Cancer Stem Cells

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Inhibition of CD47 Effectively Targets Pancreatic …...1Stem Cells and Cancer Group, Molecular Pathology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

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