TargetingCD47andAutophagyElicitedEnhanced ... · horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG (MR Biotech, MR-R100, and MR-M100, respectively). Cell

Research Article

TargetingCD47andAutophagyElicitedEnhancedAntitumorEffects inNon–Small Cell LungCancerXuyao Zhang1, Jiajun Fan1, Shaofei Wang1, Yubin Li1,2, Yichen Wang1,Song Li3, Jingyun Luan1, Ziyu Wang1, Ping Song1, Qicheng Chen1,Wenzhi Tian3, and Dianwen Ju1

Abstract

CD47-specific antibodies and fusion proteins that blockCD47–SIRPa signaling are employed as antitumor agents forseveral cancers. Here, we investigated the synergistic antitumoreffect of simultaneously targeting CD47 and autophagy in non–small cell lung cancer (NSCLC). SIRPaD1-Fc, a novel CD47-targeting fusion protein, was generated and was found to increasethe phagocytic and cytotoxic activities of macrophages againstNSCLC cells. During this process, autophagy was markedly trig-gered, which was characterized by the three main stages ofautophagic flux, including formation and accumulation of autop-hagosomes, fusion of autophagosomes with lysosomes, anddegradation of autophagosomes in lysosomes. Meanwhile, reac-tive oxygen species and inactivation of mTOR were shown to be

involved in autophagy initiation in SIRPaD1-Fc–treated cells,indicating a probable mechanism for autophagy activation aftertargeting CD47 by SIRPaD1-Fc. Inhibition of autophagyenhanced macrophage-mediated phagocytosis and cytotoxicityagainst SIRPaD1-Fc–treated NSCLC cells. In addition, simulta-neously targeting both CD47 and autophagy in NSCLC xenograftmodels elicited enhanced antitumor effects, with recruitment ofmacrophages, activated caspase-3, and overproduction of ROS atthe tumor site. Our data elucidated the cytoprotective role ofautophagy in CD47-targeted therapy and highlighted the poten-tial approach for NSCLC treatment by simultaneously targetingCD47 and autophagy. Cancer Immunol Res; 5(5); 363–75.�2017 AACR.

See related Spotlight by Kaufman, p. 355

IntroductionLung cancer, with non–small cell lung cancer (NSCLC)

accounting for 85% of all diagnosed cases, has been the leadingcause of cancer-related death worldwide and is increasing inincidence and mortality rate (1, 2). A series of therapeutic inter-ventions, such as platinum-based chemotherapy (3), tyrosinekinase inhibitors (4), and antibodies (5), have been developedto benefit the treatment of NSCLC. However, for the overwhelm-ing majority of NSCLC patients, treatment approaches are scant,and the prognosis still remains poor (6). Therefore, novel andefficient therapeutics for NSCLC are urgently needed.

Clinical trials have revealed that immune checkpoint inhibitorscan induce robust antitumor effects and hold promise for treatingmalignant tumors (7–9). CD47, also known as integrin-associ-ated protein (IAP), is a multifunctional counterreceptor for

ligands, such as thrombospondin-1 and signal-regulatory pro-tein-alpha (SIRPa), and affects a range of cellular responses thatinclude cell proliferation, fusion, andmigration.Most important-ly, CD47 is also known as a key antiphagocytic moleculethat renders tumor cells resistant to host immune surveillance(10, 11). Through direct interaction with SIRPa, CD47 acts as afundamental "don't eat me" signal and prevents macrophage-mediated phagocytosis in a growing list ofmalignancy types (12).In this sense, blocking CD47–SIRPa signal transduction bymonoclonal antibodies or fusion proteins could increase macro-phage phagocytosis of cancer cells. Blockade of the CD47/SIRPaaxis by monoclonal antibody is a potential immunotherapeuticstrategy for acute myeloid leukemia (13), breast cancer (14), andsmall cell lung cancer (15). CD47 is also highly expressed onNSCLC cells (16, 17). In the present study, SIRPaD1-Fc, a CD47–SIRPa–blocking fusion protein comprising the first extracellulardomain of human SIRPa and the Fc fragment of human IgG1,wasengineered and its potential anti-NSCLC efficacy was evaluated.

Autophagy, a key player in microenvironment maintenance,can be induced bymany conditions, such as nutrient deprivation,oxidative stress, and drug treatment (18). Although autophagyacts as a "double-edged sword" in tumorigenesis (19), autophagyacts more as a cytoprotective mechanism in tumor therapy (20–22). Many anticancer agents, such as imatinib, cisplatin, vismo-degib, and asparaginase, can also induce autophagy in tumorcells, whereas inhibition of autophagy significantly enhances theantitumor efficacies of these agents, indicating that a combinationof autophagy inhibitors and antitumor agents could be an effi-cient therapeutic strategy for malignancy (23–25). Accumulatingevidence suggests that blocking CD47 with antibodies confersradioresistance to human normal tissues and cells through

1Department of Microbiological and Biochemical Pharmacy and The Key Lab-oratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy,Fudan University, Shanghai, P. R. China. 2Perelman School of Medicine, Univer-sity of Pennsylvania, Philadelphia, Pennsylvania. 3ImmuneOnco Biopharma(Shanghai) Co., Ltd., Shanghai, P. R. China.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

X. Zhang and J. Fan contributed equally to this article.

Corresponding Author: Dianwen Ju, Fudan University, 826 Zhangheng Road,Shanghai, 201203, China. Phone: 86-21-51980037; Fax: 86-21-51980036; E-mail:[email protected]

doi: 10.1158/2326-6066.CIR-16-0398

�2017 American Association for Cancer Research.

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inducing a protective autophagy (26–28). Therefore, we hypoth-esized that combining use of the SIRPaD1–Fc fusion protein totarget CD47 with autophagy inhibitors might elicit an enhancedantitumor efficacy.

In the present study, we investigated the therapeutic effects oftargeting CD47 with SIRPaD1–Fc fusion protein. Also, we inves-tigated the synergistic antitumor effect of SIRPaD1–Fc in com-bination with autophagy inhibitors in NSCLC both in vitro and invivo. Targeting CD47 with SIRPaD1–Fc could elicit potent mac-rophage-mediated phagocytosis and cytotoxicity against NSCLCcells. During this process, autophagy was dramatically triggeredand played a cytoprotective role in NSCLC cells. However, simul-taneously targeting CD47 and autophagy significantly increasedmacrophage-mediated phagocytosis and cytotoxicity againstNSCLC cells and showed enhanced antitumor effects in NSCLCxenograft models. Thus, our data demonstrated that simulta-neously targeting CD47 and autophagy could elicit enhancedinhibition or even complete elimination of NSCLC in vitro and invivo, which could be a promising therapeutic strategy for NSCLC.

Materials and MethodsConstruction, expression, and purification of SIRPaD1–Fc

SIRPaD1–Fc is a recombinant fusion protein based on the firstextracellular domain of human SIRPa and the Fc fragment ofhuman IgG1 (Supplementary Fig. S1). To construct the SIRPaD1–Fc expression vector, 57 nucleotides encoding the signal peptideof mouse IgG1 heavy chain were added to the 50 end of SIRPaD1-coding sequence, a Kozak sequence was added to the 50 end of thesignal peptide sequence, and cloning sites,HindIII andEcoRI,wereadded to the 50 and 30 ends of the resulting sequence, respectively.This designed SIRPaD1 expression cassette sequence was synthe-sized (Convenience Biotech) and subcloned into the HindIII andEcoRI sites of the pMac-Fc vector (Convenience Biotech, ID: P008-3). The recombinant fusion protein was expressed and purifiedfrom Chinese Hamster Ovary (CHO) cells (ATCC, Cat# CCL-61).The Purity of SIRPaD1–Fc fusion protein was above 95%, and thecontent of endotoxin was below 0.5 U/g.

Reagents and antibodiesReagents were purchased as follows: Rapamycin (Sangon Bio-

tech, A606203), chloroquine and ammonium chloride (NH4Cl)(Sigma-Aldrich, A9434 and C6628, respectively), N-acetyl-L-cys-teine (NAC) and carboxyfluorescein diacetate succinimidyl ester(CFDA SE; Beyotime Institute of Biotechnology, S0077 andC0051, respectively), Cyto-ID autophagy detection kit (Enzo LifeSciences, ENZ-51031-K200), LysoTracker Red dye and MitoSOXRed dye (Invitrogen, L7528 and M36008, respectively). Theprimary antibodies used forWestern blot analyseswere purchasedas follows: anti-SQSTM1 (Cell Signaling Technology, 8025), anti-LC3 (Cell Signaling Technology, 3868), anti-cytochrome C (CellSignaling Technology, 11940), anti-PARP (Cell Signaling Tech-nology, 9532), anti–b-actin (Cell Signaling Technology, 3700),anti–caspase-9 (Cell Signaling Technology, 9502), anti-caspase-3(Cell Signaling Technology, 9665), anti–phospho-Akt (Ser473)(Cell Signaling Technology, 4060), anti-Phospho-mTOR(Ser2448; Cell Signaling Technology, 2971), anti-–Phospho-p70S6 Kinase (Ser371; Cell Signaling Technology, 9208), anti–phospho-4E-BP1/2/3 (Thr45; Abcam, ab68187). The secondantibodies used for Western analyses were obtained as follows:horseradish peroxidase (HRP)-conjugated goat anti-rabbit and

anti-mouse IgG (MR Biotech, MR-R100, and MR-M100,respectively).

Cell lines and culture conditionsHuman NSCLC cell lines A549 and NCI-H1975, and mouse

macrophages Ana-1 cell line were obtained in 2015, from CellBank of Shanghai Institutes for Biological Sciences, ChineseAcademy of Sciences (Shanghai, China), authenticated under themethod of short tandem repeat (STR) fingerprinting by the cellbank. Cells were immediately expanded and stored in liquidnitrogen upon receipt. Each new aliquot was passaged inour laboratory for fewer than six months after resuscitation. Allcells were cultured in RPMI-1640 (Corning, 10-040-CVR) con-taining 10% fetal bovine serum (Capricorn Scientific, FBS-12A),100 U/mL of penicillin and 100 mg/mL of streptomycin (Beyo-time Institute of Biotechnology, C0222) at 37�C in a humidifiedatmosphere of 5 % CO2 incubator.

siRNA transfection assayHuman ATG7 (siB111124164552) siRNA, ATG5

(siG10726164423) siRNA, and nonsilencing scrambled control(SCR) siRNA (siNO581512211471-10) were purchased fromGuangzhou RiboBio Co., Ltd. Human BECN1 (Beclin 1) siRNAand control siRNA (SC-29797) were obtained from Santa CruzBiotechnology, Inc. A549 cells and NCI-H1975 cells were trans-fectedwith siRNAusing Lipofectamine 2000TransfectionReagent(Invitrogen, 11668) following the manufacturer's instructions.

In vitro phagocytosis assayIn vitro phagocytosis assay was performed as described previ-

ously (16). Briefly, 1� 105 macrophages were planted per well inglass bottom cell culture dishes (NEST Biotechnology, 801002).According to the manufacturer's protocol, A549 or NCI-H1975cells were labeled with CFDA SE. Macrophages were incubated inserum-free medium for 2 hours before the adding 2 � 105 ofCFDA SE-labeled tumor cells. SIRPaD1-Fc were added and incu-bated for 2 hours at 37�C. Macrophages were repeatedly washedand subsequently imaged with confocal microscope. The phago-cytic index was calculated as the number of phagocytosed CFSEþ

cells per 100 macrophages.

Cytotoxicity assayCytotoxicity against A549 and NCI-H1975 cells elicited by

macrophages was measured by a 6 hours lactate dehydrogenase(LDH) release assay. Pretreated with autophagy inhibitors orsiRNA for 12 hours, cells were treated by SIRPaD1-Fc and/orautophagy suppressor or siRNA in the presence of macrophageAna-1 at the indicated effector:target cell ratio. Then, LDHrelease was measured by the CytoTox 96 Non-Radio. Cytotox-icity Assay (Promega, G1781) following the manufacturer'sinstructions.

Transmission electron microscopyHuman NSCLC A549 and NCI-H1975 cells were incubated

with or without SIRPaD1-Fc for 24 hours. After being harvestedand processed as described (29), samples were sliced and detectedwith a JEM 1410 transmission electron microscope (TEM; JEOL,Inc.). The micrographs were taken at 7,000� or 20,000�magnification.

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Immunofluorescent stainingAfter treatment with SIRPaD1-Fc for 24 hours, A549 and NCI-

H1975 cells were rinsed briefly with phosphate-buffered saline(PBS) and fixed in 4%paraformaldehyde at room temperature for10 minutes. Then the cells were permeabilized with 0.2% TritonX-100 for 10 minutes and incubated with rabbit anti-LC3 anti-body overnight at 4�C. Subsequently, cells were incubated withFITC-conjugated goat anti-rabbit IgG (Thermo Fisher ScientificInc., A24532) and 40, 6-diamidino-2-phenylindole (DAPI) for 1hour. The images were obtained by confocal microscopy andrelative fluoresent intensity was quantified by ImageJ software.

Xenograft tumor modelsAll procedures involving animals were conducted in accor-

dance with the standards approved by Animal Ethical Committeeof School of Pharmacy at Fudan University. Six-week-old femaleBALB/c nude mice (19.5 � 1.1 g) were subcutaneously injectedwith 5 � 106 of A549 or NCI-H1975 cells suspended in PBScontaining 50% Matrigel Matrix (Coining, 354234) to establishNSCLC xenograft models. Tumor-bearing mice were randomizedinto 5 cohorts. SIRPaD1-Fc and chloroquine were intraperitonealinjected twice a week and once a day, respectively. The micetreatedwith cyclophosphamide once a daywere served as positivecontrols. Tumor volumewas calculated by the formula: volume¼length � width � width/2 (30).

Statistical analysisThe data were analyzed by GraphPad Prism 5 (GraphPad

Software Inc.) and the results were presented as mean � SD.Comparisons were performed using a two-tailed student t test.P value < 0.05 was considered statistically significant.

ResultsTargeting CD47 elicited potent phagocytosis and cytotoxicityagainst NSCLC cells

The effects of SIRPaD1–Fc on macrophage-mediated phago-cytosis of A549 cells and NCI-H1975 cells were detected withconfocal microscopy. After treatment with SIRPaD1–Fc for 2hours, A549 cells and NCI-H1975 cells were efficiently phagocy-tosed bymacrophages (Fig. 1A–D). When compared with isotypecontrol IgG1-Fc, the average phagocytic index in SIRPaD1-Fc–treated cells increased from 5.5 to 29.4 in A549 cells (Fig. 1B) andfrom 6.6 to 28.6 in NCI-H1975 cells (Fig. 1D). Cytotoxicityagainst A549 cells and NCI-H1975 cells mediated by macro-phages was also determined by LDH release assay. Our findingsshowed that SIRPaD1–Fc had a negligible direct effect on theviability of A549 cells and NCI-H1975 cells (Supplementary Fig.S2AandS2B), but could significantly induce cell lysis of A549 cellsandNCI-H1975 cells in the presence ofmacrophages (Fig. 1E andF and Supplementary Fig. S2C and S2D).

Taken together, our results indicated that blockade of CD47-SIRPa signaling by SIRPaD1-Fc could elicit potent macro-phage-mediated phagocytosis and cytotoxicity against NSCLCcells.

SIRPaD1-Fc–induced accumulation of autophagosomes andtriggered autophagy flux

Ultrastructural analysis by TEM was done to observe the for-mation and accumulation of autophagosomes. After NSCLC cellswere treated with SIRPaD1–Fc for 24 hours, an abnormal for-mation and accumulation of autophagosomes, characterized by

double-membrane vesicles, could be observed in the cytoplasmofA549 cells and NCI-H1975 cells (Fig. 2A). Autophagy inductionwas confirmed by Western blot analysis of the expression ofautophagy-related protein LC3 (microtubule-associated protein1 light chain 3) and SQSTM1 (sequestosome 1). Western blotsshowed a significant decrease in the expression of SQSTM1 andincrease of LC3-II protein in A549 cells and NCI-H1975 cells in atime-dependent manner after treatment with SIRPaD1–Fc (Fig.2B). Also, Cyto-ID, a dye specific for autophagy, was used to detectautophagic vacuoles, including pre-autophagosomes, autopha-gosomes, and autophagolysosomes inA549 cells andNCI-H1975cells after treatment with SIRPaD1–Fc for the indicated times.Similar to the positive control of rapamycin-treated cells, cellsexposed to SIRPaD1-Fc increased their punctate fluorescence in atime-dependent manner, with autophagic vacuoles localized incytoplasm, which confirmed the onset of autophagy (Fig. 2C andD). LC3 immunofluorescent stainingwas alsoperformed todetectthe autophagy induced by SIRPaD1–Fc. Our data (Supplemen-tary Fig. S3A and S3B) showed that treatment with SIRPaD1–Fcsignificantly (P < 0.01) increased the mean fluorescence intensityof LC3 in A549 cells and NCI-H1975 cells.

To further confirm whether complete autophagy was inducedby SIRPaD1–Fc, we examined autophagic flux in SIRPaD1-Fc–treated NSCLC cells. By combined staining of Cyto-ID and Lyso-Tracker, three stages of autophagic flux: formation and accumu-lationof autophagosomes at 12hours, fusionof autophagosomeswith lysosomes at 24 hours, and degradation of autophagosomesin lysosomes at 36 hours (Supplementary Fig. S3C and S3D)weredistinctly observed in SIRPaD1-Fc–treated A549 and NCI-H1975cells. In addition, chloroquine, a late-stage autophagy inhibitorthat could suppress the fusion of autophagosomes with lyso-somes and block LC3-II degradation, was applied to furtherconfirm SIRPaD1-Fc–induced autophagic flux. After cells wereexposed to SIRPaD1–Fc for various time periods, additionaltreatment with chloroquine caused a further increase of LC3-II(Fig. 2E and F and Supplementary Fig. S3E and S3F), indicatingthat SIRPaD1–Fc triggered complete autophagic fluxes that ulti-mately resulted in degradation of the increased LC3-II inlysosomes.

In summary, these data showed that SIRPaD1–Fc not onlyinduced the initiation of autophagy but also resulted in autop-hagic flux in A549 cells and NCI-H1975 cells.

Macrophage-mediated phagocytosis and cytotoxicity enhancedby autophagy inhibition

Subsequently, we investigated the role of autophagy in mac-rophage-dependent phagocytosis and cytotoxicity against tumorcells induced by SIRPaD1-Fc. Western blot assay showed that theprotein level of LC3-II in the cells cotreated with SIRPaD1–Fc andautophagy inhibitors was higher than that in the cells treated withSIRPaD1–Fc alone (Fig. 3A and B), indicating that both chloro-quine and NH4Cl successfully inhibited SIRPaD1-Fc–inducedautophagy in A549 cells and NCI-H1975 cells. Chloroquine orNH4Cl alone did not significantly increase the phagocytosisand cytotoxicity of macrophages against A549 cells and NCI-H1975 cells (Fig. 3C and Supplementary Fig. S4). Meanwhile,treatment of SIRPaD1–Fc with chloroquine or with NH4Cl sig-nificantly increased SIRPaD1–Fc–inducedmacrophage-mediatedphagocytosis of A549 cells and NCI-H1975 cells (Fig. 3C andSupplementary Fig. S4). LDH release assays showed thatSIRPaD1-Fc–induced cytotoxicity against the NSCLC lines

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A549 and NCI-H1975 mediated by macrophages was greaterwhen autophagy was inhibited (Fig. 3D). Rapamycin, an autop-hagy inducer, was also used to determine the effect of activation ofautophagy in macrophage-mediated cytotoxicity against NSCLCcells (Supplementary Fig. S5A and S5B). Activation of autophagyexhibited negligible effect on the cytotoxicity mediated bymacro-phages against NSCLC cells.

To further identify the role of autophagy in SIRPaD1-Fc–induced macrophage-mediated phagocytosis and cytotoxicity,we knocked down ATG5 and ATG7, two core autophagy mole-cules that are necessary for the formation of autophagosomes.Western blot analysis showed that both the siRNA-ATG5 andsiRNA-ATG7 selectively reduced the protein expression of ATG5

and ATG7 in A549 cells and NCI-H1975 cells when comparedwith the nonsilencing scrambled control siRNA-SCR (Fig. 4Aand B). After suppression of ATG5 and ATG7 in SIRPaD1–Fc–treated A549 cells and NCI-H1975 cells, macrophage phago-cytosis and cytotoxicity against NSCLC cells were significantlyenhanced (Fig. 4C and D). We knocked down Beclin 1, aprincipal regulator in autophagosomes formation, to confirmthe role of autophagy in SIRPaD1–Fc–induced macrophage-mediated phagocytosis and cytotoxicity. Western blot analysisshowed the selective knockdown of Beclin 1 in siRNA-BECN1–-treated cells (Fig. 4E and F), which significantly increasedmacrophage-mediated phagocytosis and cytotoxicity (Fig. 4Eand F).

Figure 1.

Macrophage-mediated phagocytosis and cytotoxicity against NSCLC cells after treatment with SIRPaD1–Fc. A–D, Representative images of macrophagesAna-1 phagocytosing NSCLC cells following treatment with SIRPaD1-Fc for 2 hours. Arrows point to phagocytosed tumor cells. Phagocytic index indicatedthe number of NSCLC cells phagocytosed per 100macrophages The datawere presented asmean� SD of five independent experiments (Student t test, �� , P <0.01).E and F, LDH release represented SIRPaD1–Fc–induced cell cytotoxicity against NSCLC cells in the presence of macrophages at the indicated effector: targetcell ratio of 5:1, 10:1, 20:1 for 6 hours. The data were presented as mean � SD of three independent experiments (Student t test, � , P < 0.05; �� , P < 0.01 vs. IgG1-Fcwas used as isotype control).

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Figure 2.

Treatment with SIRPaD1–Fc induced accumulation of autophagosomes and triggered autophagy flux in NSCLC cells. A, Ultrastructural analysis of A549cells and NCI-H1975 cells after exposed to SIRPaD1-Fc (10 mg/mL) for 24 hours. B, Western blot analysis of autophagy marker protein, LC3-II andSQSTM1, in total cell lysates after treatment with SIRPaD1–Fc (10 mg/mL) for 24 hours. b-Actin was provided as a loading control. Densitometric values werequantified by Image J software and normalized to control. The values of control were set to 1.0. The data were presented as means � SD of threeindependent experiments. C and D, Cyto-ID staining was applied to detect extensive accumlation of autophagosomes in A549 cells and NCI-H1975 cells afterexposure to SIRPaD1–Fc (10 mg/mL) for the indicated time. Rapa presented the positive control rapamycin. Results were presented as means � SDof three independent experiments. E and F, Inhibition of autophagic flux by chloroquine resulted in further accumulation of SIRPaD1–Fc–inducedLC3-II. b-Actin was used as a loading control. The experiment was repeated three times, and the statistical data were shown inSupplementary Fig. S3.






Therefore, these data indicated that SIRPaD1-Fc–-inducedautophagy played a cytoprotective role in A549 cells and NCI-H1975 cells. Simultaneously targeting CD47 and autophagycould significantly increase macrophage-mediated phagocyto-sis and cytotoxicity against NSCLC cells.

SIRPaD1–Fc treatment inactivated Akt/mTOR signaling andactivated ROS

To investigate the intracellular mechanism of SIRPaD1–Fc–induced autophagy in A549 cells and NCI-H1975 cells, theautophagy-related Akt/mTOR signaling pathway was explored in

this study. SIRPaD1–Fc decreased the amount of phosphorylatedmTOR in a dose- and time-dependent manner (Fig. 5 and Sup-plementary Fig. S6). Next, we examined the expression of phos-phorylated Akt, an upstream inducer ofmTOR, and observed thatthe phosphorylation of Akt was efficiently inhibited. Further-more, phosphorylation of p70S6K and 4E-BP1, two downstreamsubstrates of mTOR, was significantly decreased after SIRPaD1-Fctreatment (as shown in Fig. 5A and B,). The autophagy-relatedAkt/mTOR signaling pathway in xenograft tumors was also exam-ined, and the amount of phosphorylated Akt and mTOR wassignificantly decreased after SIRPaD1–Fc treatment (Fig. 5C and

Figure 3.

Autophagy inhibition enhanced macrophage-mediated phagocytosis and cytotoxicity against NSCLC cells after treatment with SIRPaD1-Fc. A and B,Western blot analysis showed the amount of LC3-II in A549 cells and NCI-H1975 cells treated with SIRPaD1–Fc, with or without chloroquine or NH4Cl. b-actinwas provided as a loading control. Densitometric values were quantified by ImageJ software and presented as means � SD of three independentexperiments (Student t test, � , P < 0.05; �� , P < 0.01). C, Phagocytic index indicated the number of NSCLC cells phagocytosed per 100 macrophages and thedata were presented as means � SD of five independent experiments (Student t test, �� , P < 0.01). D, LDH release was a measure of cell cytotoxicity againstNSCLC cells mediated by macrophages after treatment with SIRPaD1–Fc, with or without autophagy inhibitors, at the indicated effector: target cell ratio of 5:1,10:1, and 20:1 for 6 hours. The data were presented as means � SD of three independent experiments (Student t test, � , P < 0.05; �� , P < 0.01).

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Supplementary Fig. S7). Thus, SIRPaD1–Fc could downregulateAkt/mTOR signaling and the protein synthesis, which finallyactivated autophagy in NSCLC cells.

In addition,weexaminedwhether reactiveoxygen species (ROS)were involved in SIRPaD1-Fc–induced autophagy in A549 cellsand NCI-H1975 cells (Fig. 5D and Supplementary Fig. S8). ROSformation (red fluorescence) was detected approximately 4 hoursearlier than the induction of autophagy (green fluorescence) in

A549 cells and NCI-H1975 cells after SIRPaD1-Fc exposure. Toinvestigatewhether SIRPaD1–Fc–induced autophagy inA549 cellsand NCI-H1975 cells was mediated by ROS generation, NAC, apotent antioxidant, was employed to scavenge ROS (Fig. 5E).Pretreatment with NAC (5 mmol/L) was able to counteractSIRPaD1–Fc–induced ROS formation and could partially inhibitSIRPaD1-Fc–induced autophagy. Collectively, these data showedthat SIRPaD1-Fc–induced autophagy in NSCLC cells by

Figure 4.

Knockdown of ATG5, ATG7, and Beclin 1 enhanced macrophage-mediated phagocytosis and cytotoxicity against NSCLC cells after treatment with SIRPaD1-Fc.A and B, A549 cells and NCI-H1975 cells were transiently transfected with ATG5 or ATG7 siRNAs for 24 hours, and the protein levels of ATG5, ATG7 andGAPDH were detected by Western blot assays. GAPDH was used as a loading control. Densitometric values were quantified by Image J software andpresented as means � SD of three independent experiments (Student t test, �� , P < 0.01). C and D, LDH release was a measure of cell cytotoxicity againstNSCLC cells mediated by macrophages following treatment with indicated SIRPaD1-Fc with or without ATG5 and ATG7 knockdown. The data were presentedas means � SD of three independent experiments (Student t test, � , P < 0.05; �� , P < 0.01). E and F, A549 cells and NCI-H1975 cells were transiently transfectedwith BECN1 siRNAs for 24 hours, and the protein levels of Beclin1 and b-actin were detected by Western blot assays. b-Actin was used as a loading control.Densitometric values were quantified by Image J software. LDH release represented cell cytotoxicity against NSCLC cells mediated by macrophagesfollowing treatment with indicated SIRPaD1-Fc with or without Beclin 1 knockdown. The data were presented as means � SD of three independent experiments(Student t test, � , P < 0.05; �� , P < 0.01).






inactivation of the Akt/mTOR signaling pathway and intracellularROS formation was involved in the induction of autophagy.

Autophagy targeting plus SIRPaD1–Fc suppressed growth ofNSCLC xenografts

To reveal whether targeting CD47 and autophagy could be apotential therapeutic approach for NSCLC in vivo, A549 xenograftmodels were established to investigate the synergistic antitumorefficacy of SIRPaD1–Fc and autophagy inhibitor (Fig. 6A). Tumorvolume was reduced significantly from day 8 of combinedSIRPaD1–Fc and chloroquine therapy (P < 0.001 vs. vehicle).When mice were sacrificed on day 22 and the tumors wereresected, the tumor weight in mice treated with SIRPaD1–Fcalone was 357.10 � 137.81 mg versus 901.43 � 135.21 mg ofthe vehicle control (P < 0.0001; Fig. 6A and Supplementary Fig.S9A), and the tumor weight in mice treated with SIRPaD1–Fc incombination with chloroquine was 78.57� 49.81 mg (P < 0.001vs. the cohort treated with SIRPaD1–Fc alone). However, chlo-roquine did not show antitumor effects in A549 xenograftmodelswhen compared with vehicle control. Similarly, in NCI-H1975xenograft models, tumor weight in mice treated with bothSIRPaD1–Fc and chloroquine was 51.43 � 52.42 mg (P <0.005 vs. the cohort treated with SIRPaD1–Fc alone), whereasthe tumor weight in mice treated with SIRPaD1–Fc alone and thevehicle control were 190.00 � 76.81 mg (P < 0.001 vs. vehiclecontrol) and 612.00 � 211.71 mg, respectively (Fig. 6B). One

tumor-bearing mouse was tumor free after 24 days of combinedtreatment of SIRPaD1–Fc and chloroquine (Supplementary Fig.S9B). Taken together, these data indicated that targeting CD47 bySIRPaD1–Fc elicited potent antitumor effects inNSCLC xenograftmodels and simultaneously targeting CD47 and autophagy couldelicit synergistic anti-NSCLC effects.

Targeting of autophagy and CD47 increased apoptosis andmacrophage recruitment in vivo

To investigate the mechanisms of the potent anti-NSCLCefficacy induced by SIRPaD1–Fc and chloroquine, necrosis andapoptosis were thus determined in tumor mass from the tumor-bearing mice. To verify whether blocking the CD47–SIRPa path-way by SIRPaD1–Fc could recruit macrophages to tumor site,CD68, a macrophage specific marker, was used to detect theinfiltration of macrophages in transplanted NSCLC (Fig. 6C andD). Histopathologic analysis showed prominent macrophageinfiltration of SIRPaD1–Fc–treated tumors. In tumor-bearingmice treated with both SIRPaD1–Fc and chloroquine, macro-phage infiltration and tumor cell necrosis increased, indicatingthat the combination of SIRPaD1–Fc and chloroquine couldincrease the recruitment of macrophages to the tumor site andthis might directly contribute to the inhibition of the developingtumor (Fig. 6 and Supplementary Fig. S10). Inhibiting the CD47–SIRPa pathway also induced autophagy in NSCLC cells in vivo.Ultrastructural analysis and Western blot assays of tumor tissue

Figure 5.

Inactivation of the Akt/mTOR signaling pathway and production of ROS after NSCLC cells treated with SIRPaD1–Fc. A, A549 cells were exposed to SIRPaD1–Fc (10 mg/mL) for the indicated time and whole-cell lysates were analyzed by Western blot to examine the expression of SQSTM1, LC3, p-Akt, p-mTOR, p-p70S6K, and p-4E-BP1. Densitometric values were quantified by ImageJ software and normalized to control. The values of control were set to 1.0. The datawere presented as means � SD of three independent experiments (Student t test, �� , P < 0.01). B, NCI-H1975 cells were incubated with SIRPaD1-Fc for theindicated time and whole-cell lysates were analyzed by Western blot to examine the expression of SQSTM1, LC3, p-Akt, p-mTOR, p-p70S6K, and p-4E-BP1.b-Actin was provided as a loading control. Densitometric values were quantified as described inA. C, Expression of phosphorylated Akt and mTOR from the whole-cell lysate was detected by Western blot in A549 xenograft tumors and NCI-H1975 xenograft tumors after SIRPaD1–Fc treatment. The experiment wasrepeated three times and the statistical data were shown in Supplementary Fig. S7. D, Representative fluorescence images of A549 cells and NCI-H1975 cellscostained with Cyto-ID green dye and MitoSox red dye after exposure to SIRPaD1-Fc for the indicated time. The data were presented as means � SD ofthree independent experiments. E, Inhibiting effects of treatment with NAC on SIRPaD1–Fc–induced autophagy in A549 cells and NCI-H1975 cells.

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showed that blocking the CD47–SIRPa pathway by SIRPaD1–Fcinduced the accumulation of autophagosomes (Fig. 6E),decreased SQSTM1, and increased LC3-II in transplanted NSCLC

cells (Fig. 7A and B). Chloroquine could not only inhibit theautophagy activated by SIRPaD1–Fc, but also significantlyincreased necrosis in the transplanted tumor tissues (Fig. 7A and

Figure 6.

Targeting autophagy enhanced growthinhibition of NSCLCs xenograft tumorafter treatment with SIRPaD1-Fc. A,BABL/c nude mice were transplantedsubcutaneously with A549 cells. Sevendays later, random allocation was takento divide tumor-bearing BABL/c nudemice into 5groups, and xenograft tumorvolume was evaluated every other dayby direct caliper measurements. Thedata were presented as means � SD.After treatment with SIRPaD1–Fc (10mg/kg) twice a week in combinationwith or without autophagy inhibitorchloroquine (50 mg/kg) once a day for22 days, tumor-bearing BABL/c nudemicewere sacrificed. Tumorweightwaspresented asmean� SD and each pointrepresented a value from anindependent mouse. B, NCI-H1975xenograft models were established asdescribed inA. Thedatawere presentedas means � SD. After treatment withSIRPaD1–Fc (10 mg/kg) twice a week incombinationwith or without autophagyinhibitor chloroquine (50 mg/kg) oncea day for 28 days, mice were sacrificed.Tumor weight was presented asdescribed in A. C, Representative H&Eimages of NCI-H1975 xenograft tumorstreated with SIRPaD1-Fc and/orautophagy inhibitor chloroquine.D, Representative images ofimmunohistochemistry CD68 stainingof NCI-H1975 xenograft tumors treatedwith SIRPaD1–Fc and/or autophagyinhibitor chloroquine. E, Ultrastructuralanalysis of SIRPaD1–Fc–treated NCI-H1975 xenograft tumor tissues by TEM.






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B). We then investigated whether ROS and apoptosis wereinvolved in vivo in the killing of tumors after SIRPaD1–Fc andchloroquine treatment (Supplementary Fig. S11). ROS formation(red fluorescence) in NCI-H1975 xenograft tissues was inducedafter treatment with SIRPaD1-Fc and chloroquine. Western blotanalysis of the xenograft tumors showed cleavage of caspase-9,caspase-3 and its substrate PARP, and the release of cytochrome Cinto the cytosol (Fig. 7 and Supplementary Fig. S12), suggestingthat treatment with SIRPaD1–Fc and chloroquine activated theapoptosis pathway.

In brief, these results indicated that simultaneously targeting ofboth CD47 and autophagy recruited more macrophages to thetumor sites, promoted ROS generation, activated apoptosis-relat-ed pathways, and increased necrosis in tumor cells.

DiscussionTo date, immune checkpoint inhibitors that target CD47 fall

into three main categories: antibodies to CD47, SIRPa–Fc fusionprotein, and SIRPa–antibody fusion protein. Anti-CD47 antibo-dies, such as B6H12 and Bric126, showed dramatic inhibition ofthe tumor growth, including ovarian cancer, breast cancer, coloncancer, glioblastoma, and small cell lung cancer, by blocking thetransmitted ability of the CD47–SIRPa axis (10, 15, 16). The fullextracellular domain of human SIRPa and the Fc fragment ofhuman IgG1-based SIRPa–Fc fusion protein were reported toimpair human AML engraftment and dissemination throughdisruption of SIRPa–CD47 (31). Another study reported that abispecific fusion protein-based on SIRPa and antibody to CD20elicited potent elimination of lymphoma cells and caused nosignificant toxicity in nonhuman primates (11). In this study, wedeveloped a fusion protein SIRPaD1–Fc, consisting of the firstextracellular domain of human SIRPa and the Fc fragment ofhuman IgG1, and demonstrated its potent antitumor effects forNSLCL in vitro and in vivo.

A series of immunotherapeutic approaches, including anti–PD-1 therapy and anti–PD-L1 therapy, had previously been estab-lished for NSCLC (7, 32). In this study, our results showed thatSIRPaD1–Fc therapy could elicit potent anti-NSCLC efficacy invitro and in vivo. SIRPaD1–Fc could directly target theNSCLC cellsand activate macrophage phagocytosis of tumor cells by disrupt-ing the CD47/SIRPa axis "don't eat me" signal in NSCLC xeno-graft models. CD47 is also expressed on normal tissues, whichcould act as antigenic sinks, a potential limitation of treatingpatients by blocking CD47. To get around this issue, much efforthas gone into producing low-affinity/high-affinity bispecific anti-bodies and SIRPabodies (11). Meanwhile, new data from thephase I trial of TTI-621 (a SIRPaFc fusion protein) suggest thatrepeat dosing of TTI-621 overcame the antigen sink while main-taining clinically acceptable platelet levels (33),which could be aneffective strategy to circumvent antigenic sinks in other studies.Although many studies have revealed that the therapeutic benefitof blocking CD47–SIRPa interactions in immune competenthosts depends on the myeloid immune subsets, including neu-

trophils, NK cells, T cells, and dendritic cells (34–36), the primarycell type contributing to effectiveness ismacrophages (10, 37–39).Our data here also showed that targeting CD47 by SIRPaD1–Fcelicited potent macrophage-mediated antitumor efficacy inNSCLC. Whether other myeloid immune subsets were activatedby this therapy still needs further investigation.

Although increasing evidence shows that blockade of CD47has potent antitumor efficacy in some solid malignancies,additional approaches to increase efficacy are still urgentlyneeded (40). To further increase the antitumor effects of target-ing CD47 by SIRPaD1–Fc in NSCLC, we examined probablemechanisms of resistance to SIRPaD1–Fc treatment. Autop-hagy, an important mechanism of tumor therapy resistance, isincreasingly regarded as a target for synergistic antitumor ther-apy (41–43). We report here that targeting CD47 could triggerautophagy in NSCLC cells. CD47 deficiency regulates theexpression of LC3-II, BECN1, ATG5, and ATG7 to stimulateautophagy and autophagic flux, and conferring radioprotectionto normal cells and tissues, which suggests that CD47 blockadecould serve as a pharmacologic route to protect normal tissuefrom radiation injury by modulating autophagy (28). In thepresent study, our data showed that autophagy was inducedthrough inactivation of the Akt/mTOR signaling pathway, and acomplete autophagic flux was also triggered during the treat-ment, further supporting a crucial role for autophagy in CD47targeting-based NSCLC therapy.

Up to now, assessments of the role of autophagy in immunecheckpoint inhibitors-based tumor therapies were scarce. In thisstudy, we addressed whether autophagy participated in CD47blockade–basedNSCLC therapy. Although autophagy plays a keyrole in both cell survival and cell death (44), our results stronglysupport autophagy as a cytoprotective mechanism in this system.We found that inhibition of autophagy while simultaneouslyproviding SIRPaD1–Fc–based NSCLC therapy, by either phar-maceutical inhibitors or genetic approaches, resulted in approx-imately 90% inhibition or even complete elimination of NSCLCxenograft tumors, indicating that targeting CD47 and autophagytogether couldbe amore effective therapy strategy forNSCLC thantargeting CD47 alone.

Besides its regular function in cell microenvironment mainte-nance, autophagy is associated with the innate and adoptiveimmune response (45). Autophagy can stimulate the productionof immune inhibitory cytokines, such as IL10, TGFb, and IL27,whereas inhibition of autophagy can be beneficial to the phago-cytosis of bacteria bymacrophages (46).Our results also indicatedthat autophagy played a role in resistance to immunotherapy andwas involved in immune responses mediated by CD47 blockadein NSCLCs. After suppression of SIRPaD1–Fc–induced autop-hagy in NSCLC, we observed an increased presence of macro-phage infiltration in xenograft tumors, which might lead toapoptosis and growth disadvantage in NSCLC tumors throughrelease of cytochrome C and activation of caspase-9 and caspase-3. ROS generation was also observed in the xenograft tumor cellstreated with both SIRPaD1-Fc and chloroquine. A previous study

Figure 7.Simultaneously targeting CD47 and autophagy activated apoptosis-related pathways in vivo. A, After treatment with SIRPaD1–Fc in combination with or withoutchloroquine for 28 days, NCI-H1975 xenograft mice were sacrificed. Total tissue lysates of independent tumor tissue samples were analyzed by Western blot toexamine the expression of SQSTM1, LC3, PARP, caspase-9, and caspase-3. The level of cytochrome C in the cytosol was also determined. b-Actin was providedas a loading control. B, Densitometric values of protein expression of all independent tumor tissue samples in each group were quantified by ImageJ softwareand normalized to control. The values of control were set to 1.0. The datawere presented asmeans� SDof three independent experiments (Student t test, � ,P <0.05;�� , P < 0.01). C, A graphical description of how simultaneously targeting CD47 and autophagy could elicit enhanced antitumor effects in NSCLC.






demonstrated that focal regions of macrophage recruitment over-lap with regions of enhanced ROS formation (47). Our dataindicated that ROS induced by increasedmacrophage recruitmentmight partly contribute to NSCLC cell death in the cohort treatedwith SIRPaD1-Fc and chloroquine. Knockdown of CD47, orbinding of CD47 by its ligands, can result in a series of cellularresponses, including loss of stem cell characteristics, inhibition ofcell proliferation, and spheroid formation, which might promote"eatme" signals that are recognized bymacrophages and enhancemacrophage phagocytosis (48–50). Here, our data indicated thatSIRPaD1–Fc induced signals via CD47 in NSCLC cells and thencaused autophagy, a response against cellular stress. Thus, autop-hagy is probably a mechanism to suppress the "eat me" signals.Our results suggested a probablemechanismof abolishing autop-hagy to increase the elimination ofNSCLC cells by SIRPaD1–Fc invivo and indicated a novel strategy for NSCLC treatment based ontargeting CD47 and autophagy.

Taken together, in the present study, our results demonstratedthat targeting CD47 by SIRPaD1–Fc in NSCLC could elicit potentantitumor efficacy.During the treatment, autophagywas triggeredvia inactivation of the Akt/mTOR signaling pathway and played acytoprotective role in NSCLC cells. Simultaneously targetingCD47 and autophagy could elicit enhanced macrophage-medi-ated phagocytosis and cytotoxicity against NSCLC cells andshowed enhanced inhibition, or even complete elimination, ofNSCLC. A schematic of themechanisms of the potent anti-NSCLCefficacy induced by simultaneously targeting CD47 and autop-hagy is presented in Fig. 7C. These data revealed that CD47 was apotential target for use in NSCLC therapy and highlighted thesynergistic antitumor effect of simultaneously targetingCD47 andautophagy, providing a scientific basis for further enhancing theantitumor efficacy of immune checkpoint inhibitors.

Disclosure of Potential Conflicts of InterestS. Li is employed at ImmuneOnco Biopharma (Shanghai) Co., Ltd.W. Tian is

the founder of ImmuneOnco Biopharma (Shanghai) Co., Ltd. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: X. Zhang, S. Wang, W. Tian, D. JuDevelopment of methodology: X. Zhang, J. Fan, S. Li, Q. Chen, D. JuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. Zhang, J. Fan, S. Wang, Y. Li, Y. Wang, J. Luan,P. Song, Q. Chen, D. JuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. Zhang, J. Fan, S. Wang, Y. Li, Y. Wang, Z. Wang,D. JuWriting, review, and/or revision of the manuscript: X. Zhang, J. Fan, S. Wang,Y. Li, D. JuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X. Zhang, J. Luan, P. Song, D. JuStudy supervision: W. Tian, D. Ju

AcknowledgmentsThis work was supported by the National Key Basic Research Program of

China under grants 2015CB931800 and 2013CB932502, the National NaturalScience Foundation of China under grant 81573332, the Shanghai Science andTechnology Funds under grant 14431900200, and Special Research Foundationof State Key Laboratory of Medical Genomics and Collaborative InnovationCenter of Systems Biomedicine.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 28, 2016; revised February 10, 2017; accepted March 24,2017; published OnlineFirst March 28, 2017.

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2017;5:363-375. Published OnlineFirst March 28, 2017.Cancer Immunol Res Xuyao Zhang, Jiajun Fan, Shaofei Wang, et al.

Small Cell Lung Cancer−Effects in Non Targeting CD47 and Autophagy Elicited Enhanced Antitumor

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