Large Molecule Therapeutics

SapC–DOPSNanovesiclesasTargetedTherapy forLung CancerShuli Zhao1, Zhengtao Chu2,3, Victor M. Blanco2, Yunzhong Nie4, Yayi Hou4, andXiaoyang Qi2,3

Abstract

Lung cancer is the deadliest type of cancer for both men andwomen. In this study, we evaluate the in vitro and in vivo efficacyof a biotherapeutic agent composed of a lysosomal protein(Saposin C, SapC) and a phospholipid (dioleoylphosphatidyl-serine, DOPS), which can be assembled into nanovesicles(SapC–DOPS) with selective antitumor activity. SapC–DOPStargets phosphatidylserine, an anionic phospholipid preferen-tially exposed in the surface of cancer cells and tumor-associatedvasculature. Because binding of SapC to phosphatidylserine isfavored at acidic pHs, and the latter characterizes the milieu ofmany solid tumors, we tested the effect of pH on the bindingcapacity of SapC–DOPS to lung tumor cells. Results showed that

SapC–DOPS binding to cancer cells was more pronounced atlow pH. Viability assays on a panel of human lung tumor cellsshowed that SapC–DOPS cytotoxicity was positively correlatedwith cell surface phosphatidylserine levels, whereas mitochon-drial membrane potential measurements were consistent withapoptosis-related cell death. Using a fluorescence tracking meth-od in live mice, we show that SapC–DOPS specifically targetshuman lung cancer xenografts, and that systemic therapy withSapC–DOPS induces tumor apoptosis and significantly inhibitstumor growth. These results suggest that SapC–DOPS nanove-sicles are a promising treatment option for lung cancer. MolCancer Ther; 14(2); 491–8. �2015 AACR.

IntroductionLung cancer is the most common and the deadliest type of

cancer worldwide, accounting for 1.8 million new cases and 1.6million deaths in 2012 (1). Cigarette smoking is the single leadingcause of lung cancer, followed by environmental and occupa-tional exposure to pollutants such as radon, arsenic, asbestos,Bis(chloromethyl) ether, chromium, nickel, vinyl chloride, andpolycyclic aromatic compounds (2–4). Startlingly, a direct causalrelationship between atmospheric particulate matter (air pollu-tion) and lung and other cancers was recently established. Asreported by the International Agency for Research on Cancer,more than half of lung cancer–related deaths caused by airpollutants worldwide occurred in China and other East Asiancountries (5). Standard treatment options for lung cancer (che-motherapy, radiation, and surgery) have several undesirable sideeffects that affect the quality of life of the cancer patient.

Liposomal formulations as vehicles for drug delivery are thesubject of intense research. Comparedwith nonencapsulated, free

drugs, they provide improved biocompatibility, pharmacokinet-ics, biodistribution, and toxicologic profile. Despite promisingresults in preclinical models of lung cancer (6) and many othercancer types (7), only a fewnontargeted formulations (i.e., relyingon passive tumor accumulation through the enhanced perme-ability and retention effect) have been approved for cancer treat-ment by regulatory agencies (8, 9). Clinical trials are under way toevaluate novel liposomal formulations of the chemotherapydrugs paclitaxel (LEP-ETU; ref. 10) and cisplatin (Lipoplatin;ref. 11) in patients with lung cancer. Liposomes can deliver largedrug payloads but, as numerous clinical studies have shown, thisdoes not always translate into enhanced therapeutic efficacy;results so far show that liposomal drug delivery offers reducedtoxicity, but only modest survival benefits, compared with freedrug administration (12, 13). In this study we report a noveltargeted biotherapeutic agent, saposin C-dioleoylphosphatidyl-serine (SapC–DOPS) proteoliposomes, with selective antineo-plastic activity on lung cancer cells both in vitro and in vivo.

SapC is a small glycoprotein present in all tissues that acts asbiologic activator of lysosomal enzymes, such as acid b-glucosi-dase, acid sphingomyelinase, and acid b-galactosylceramide,which degrade fatty acid substrates resulting in the productionof ceramide, a key intermediate in the synthesis of sphingolipids,and a well-known apoptosis inducer (14, 15). SapC has a strongaffinity for negatively charged phospholipids such as phosphati-dylserine (PS), towhich it bindsmore avidly at acidic pH (16–18).Because the expression of PS is relatively abundant in the surfaceof tumor cells and tumor-associated vasculatures (19, 20), but notin normal cells, and in view of the characteristically acidic tumormicroenvironment that results from glycolytic conversion ofglucose into lactate, we hypothesized that SapC–DOPS nanove-sicles could be an effective antitumor agent.

Previouswork fromour group showed that SapC–DOPS targetsa variety of cancer cells in vitro and in vivo, and has anticancer

1State Key Laboratory of Reproductive Medicine, Central Laboratoryof Nanjing First Hospital, NanjingMedical University, Nanjing, Jiangsu,China. 2Division of Hematology/Oncology, Department of InternalMedicine, University of Cincinnati College of Medicine, Cincinnati,Ohio. 3Division of Human Genetics, Department of Pediatrics, Cincin-nati Children'sHospitalMedical Center,Cincinnati,Ohio. 4Immunologyand Reproductive Biology Laboratory, Medical School and State KeyLaboratory of Pharmaceutical Biotechnology, Nanjing University,Nanjing, Jiangsu, China.

Corresponding Author: Xiaoyang Qi, Division of Hematology and Oncology,Department of Internal Medicine, College of Medicine, University of Cincinnati,The Vontz Center for Molecular Studies, 3125 Eden Avenue, Cincinnati, OH45267. Phone: 513-558-4025; Fax: 513-558-6703; E-mail: xiaoyang.qi@uc.edu

doi: 10.1158/1535-7163.MCT-14-0661

�2015 American Association for Cancer Research.

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effects in animal models of human solid tumors (21–26). In thisstudy, we evaluate the tumor-targeting and cytotoxic potentialof SapC–DOPS on human lung cancer cells, and assess itseffectiveness in suppressing lung carcinoma growth in vivo. Tothis end, we first describe the pH dependence of SapC–DOPS cellbinding and assess its cytotoxicity in a panel of lung cancercells. The induction of apoptosis in cultured lung cancer cells isconfirmed through DNA fragmentation analysis. Next, we usein vivo fluorescence imaging to assess SapC–DOPS tumor-selective targeting in allogeneic lung tumor mouse models. Final-ly, the therapeutic efficacy of SapC–DOPS against tumor growthis studied in nude mice bearing human lung cancer xenografts.Our results show that SapC–DOPS effectively targets lung cancercells in vitro and in vivo, and possesses antitumor activity in apreclinical model of human lung cancer.

Materials and MethodsCell culture

The human lung carcinoma A549 and mouse Lewis lungcarcinoma (LLC) cell lines were from American Type CultureCollection (ATCC; 1998 and 2001). The human lung carcino-ma cell lines SPC-A-1, H1299, NCI-H661, SK-MES-1, NCI-H460, and 801-D were obtained from the Institute of Biochem-istry and Cell Biology, Chinese Academy of Sciences from 1999to 2010. Cells were routinely tested for mycoplasma contam-ination. No authentication of the cell lines was done by theauthors. Human cells were cultured in RPMI-1640 mediumsupplemented with 10% fetal bovine serum (FBS) and 100 mg/mL streptomycin and 100 U/mL penicillin. LLC cells weregrown in Dulbecco's Modified Eagle Medium supplementedwith 10% FBS, 100 mg/mL streptomycin, and 100 U/mL pen-icillin. Culture media and FBS were purchased from Life Tech-nologies. All cell lines were cultured at 37�C in a humidified 5%CO2 atmosphere.

Preparation of SapC–DOPS nanovesiclesDioleoylphosphatidylserine (DOPS; Avanti Polar Lipids Inc.)

was dried under a stream of N2 gas, combined with recombinantSapC protein (27) in citrate/phosphate buffer (pH 5.0), andbath sonicated in PBS as described in detail previously (21). Tofluorescently label SapC–DOPS nanovesicles, an aliquot ofCellVue Maroon (CVM; Molecular Targeting Technologies Inc.)in ethanol was dried together with DOPS before SapC additionand bath sonication. Free CVM was eliminated from this prep-aration by filtration through a Sephadex G25 column (PD-10;Amersham Pharmacia Biotech). For in vivo treatment, a lyoph-ilized formulation was used: dry DOPS was suspended in 80%tert-butanol. SapC and sucrose (10 mg/mL) were dissolved inwater. DOPS suspension and SapC plus sucrose (1:0.6, vol:vol)was lyophilized in a freeze dryer (VirTis Unitop 1000L linked toa Freezemobile 25XL; The VIRTIS). The stable powder cake wasresuspended in saline solution to form SapC–DOPS nanovesi-cles. Vesicles were monitored with a submicron particle sizeanalyzer (Coulter Model N4 Plus). The molar ratio of SapC:DOPS was 1:7.

SapC–DOPS binding in vitroTo evaluate the influence of pH on the binding of SapC–DOPS

to tumor cells, SapC–DOPS–CVM(containing 50mg/mLSapC)orfree, equimolar CVM were dissolved in complete RPMI-1640

mediumwith different pH values (adjusted by addition of NaOHorHCl). A549 cells were collected by trypsinization, washed twicewith PBS, and incubated in media adjusted to pH 5.5, 6.6, 7.5, or8.5 for 2 hours at 37�C. The cells were thenwashed twice with PBSand analyzed by flow cytometry (FACSCanto II; BD Biosciences),using excitation at 633 nmand a 760 nmband-pass filter to detectCVM emission.

Viability assaysThe colorimetric Cell Counting Kit-8 (CCK-8) assay

(Dojindo Laboratories) was used to monitor the cytotoxiceffects of SapC–DOPS. Lung carcinoma cells were plated at adensity of 5,000 cells per well in 96-well plates in triplicate.After 12 hours in culture, cells were treated with SapC–DOPS atfinal SapC concentrations of 0 (control), 0.5, 1, 2, 4, 8, 16, 32,64, or 128 mg/mL. After 72 hours, 10 mL CCK-8 was added toeach well. Optical density (OD) values were obtained fromabsorbance readings at 450 nm. Viability was calculated as thepercentage of viable cells compared with control using theformula: growth inhibition (%) ¼ (ODC � ODT)/ODC �100, where ODC and ODT are the OD values of the controland treated samples, respectively. IC50 was calculated by linearinterpolation using the formula: IC50 ¼ [(50 � A)/(B � A)] �(D� C)þ C, where A is the first point on the curve, expressed aspercent inhibition, that is less than 50%; B is the first point onthe curve, expressed as percent inhibition, that is greater than orequal to 50%; C is the concentration of inhibitor that gives A%inhibition; and D is the concentration of inhibitor that givesB% inhibition (28).

Phosphatidylserine levels, DNA fragmentation, andmitochondrial membrane potential measurements

Membrane phosphatidylserine (PS) levels were measuredusing Annexin V-FITC (Invitrogen) according to the manufac-turer's instructions. In brief, cells were trypsinized, washed withPBS, and resuspended at 2,500,000 cells/mL in Annexin Vbinding buffer. Aliquots of cells (0.2 mL/tube) were incubatedwith 10 mL of Annexin V-FITC for 15 minutes at room tem-perature in the dark. Then, 800 mL of binding buffer was addedto each tube. Samples were kept on ice and analyzed by flowcytometry within 1 hour.

Analysis of DNA fragmentation and mitochondrial mem-brane potential (Dcm) measurements were carried out in A549cells cultured at a density of 5 � 105 cells per well in 6-wellplates. Twenty-four hours after plating, cultures were treatedwith SapC–DOPS (containing 0, 10, 20, or 40 mg/mL of SapC)for 48 hours. To identify apoptotic cells by DNA fragmentationanalysis, cells were harvested, washed with PBS, and fixed in70% ice-cold ethanol overnight. Fixed cells were incubatedwith 20 U/mL RNase I and 50 mg/mL propidium iodide (PI) for30 minutes. PI signal was quantified by flow cytometry andcells in the sub-G0/G1 phase of the cell-cycle were identified asapoptotic.

Changes in Dcm were monitored using the lipophilic cationicprobe 5,50,6,60-tetrachloro-1,10,3,30- tetraethyl-benzimidazolcar-bocyanine iodide (JC-1). After treatment for 48 hours, the cellswere trypsinized,washedwithPBS, and incubatedwith JC-1 as perthemanufacturer's instructions at 37�C in the dark for 30minutes.Dcmwas measured by flow cytometry by exciting with a 488 nmlaser line and measuring emitted fluorescence with the FL1(green) and FL2 (red) channels.

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In vivo imaging of tumor targeting by SapC–DOPS–CVMAll procedures involving animals were approved by the

Ethics Committee for Animal Research of Nanjing University.Murine lung tumor cells (Lewis lung cells, LLC; 5 � 105 cells)were injected into 4 week-old BALB/c female nude micesubcutaneously or into FVB male mice through the tail vein.Sham animals received a PBS injection. At day 10, 200 mLSapC–DOPS–CMV, DOPS–CVM, CVM, or PBS in normalsaline were intravenously injected into tumor-bearing mice.Two to 150 hours later, mice were anesthetized to effect with2% isoflurane and imaged with an IVIS 200 imaging system(PerkinElmer).

In vivo SapC–DOPS treatmentHealthy female BALB/c nude mice (5–6 weeks old) were

obtained from Vital River Lab Animal Co., Ltd., Beijing Labo-ratory Animal Research Center (Beijing, China), and were

housed in specific pathogen-free conditions at Drum TowerHospital Animal Center, Nanjing, China with controlled tem-perature (21 � 2�C), humidity (55 � 5%), and light (12 hourlight/dark cycle). Water was available to mice ad libitum. Afterthe mice had acclimated for 2 weeks, 2 � 106 A549 cells in0.1 mL PBS were injected subcutaneously into their dorsalright flanks. Mice were monitored for solid tumor growth forapproximately 10 days, and when the tumor volume reached100 mm3, they were randomly assigned to 5 groups (n ¼6/group). The mice then received intravenous injections (0.2mL) every other day for 5 weeks as follows: group 1 (control,normal saline), group 2 (cisplatin 4 mg/kg), group 3 (high-dose, SapC–DOPS 11.8 mg/kg), group 4 (medium-dose, SapC–DOPS 5.9 mg/kg), and group 5 (low-dose, SapC–DOPS 2.9mg/kg). Tumor dimensions were measured every other dayduring treatment. After the treatment regimen was completed,the mice were sacrificed and the tumors were removed, mea-sured, and weighed. Tumor volume (in mm3) was calculatedusing the formula: L � W2 � 0.5. The inhibition of tumorgrowth was calculated as % inhibition ¼ [1 � (average tumorweight for each treatment group/average tumor weight for thecontrol group)] � 100%. Apoptosis in paraffin-embeddedtumor samples was evaluated using a TUNEL (terminal deox-ynucleotidyl transferase-mediated dUTP nick end-labeling)assay (Boster Bio).

Statistical analysisA t test analysis or two-way ANOVA/Tukey test was

used to determine statistical significance for experimentswith two or more groups, respectively. Analyses were donewith SPSS 12.0. Data are presented as the arithmetic mean �SEM.

Figure 1.SapC–DOPS binding to tumor cells is enhanced by acidic pH. A, flowcytometry analysis of A549 cells incubated with CMV (medium pH ¼ 7.5;purple-filled trace) or SapC–DOPS–CVM in media of various pH: 5.5(green); 6.5 (pink); 7.5 (blue); 8.5 (orange). B, quantification of data fromexperiments like the one shown in A. � , P < 0.05; �� , P < 0.0.1; ��� , P < 0.001with respect to control (one-way ANOVA followed by Tukey–Kramermultiple-comparison test); n ¼ 3.

Figure 2.Half-maximal cytotoxic dose of SapC–DOPS as a function of cellsurface membrane phosphatidylserine levels in lung tumor cells. MTTassays were performed to determine SapC–DOPS cytotoxicity in culturedhuman lung cancer cells. The concentration of SapC–DOPS required toreduce cell viability by 50% is significantly lower in cells with high PSexposure, suggesting that increased PS levels enhance SapC–DOPSantitumor activity. Cell lines were grouped on the basis of their surfacePS expression levels, as denoted by Annexin V-FITC binding (% signalincrease over unstained controls): SPC-A-1, 28.9%; H1299, 48.4%; NCI-H661, 51.1% (low PS group); A549, 195.8%; SK-MES-1, 336.8%; NCI-H460,108.9; 801-D, 81.9% (high PS group). � , P < 0.05.

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ResultsAcidic pH enhances the binding of SapC–DOPS to tumorcells

Acidosis, resulting from high glycolytic activity and impairedperfusion, is a prominent feature in the microenvironment ofsolid tumors. Because the binding of SapC to anionic phos-pholipids such as PS is favored by low pH, we tested whether aweakly acidic environment could promote the binding andinternalization of SapC–DOPS in lung cancer cells in vitro. Forthese experiments, we chose A549 lung adenocarcinoma cells,which in addition to expressing high levels of surface PS (seenext section), are among the best characterized and most widelyused lung tumor cell lines. A549 cells were incubated withSapC–DOPS–CMV nanovesicles in culture media with differentpH values (5.5, 6.6, 7.5, or 8.5), at 37�C for 2 hours. Cell-bound CVM fluorescence intensity was then assessed by flowcytometry (Ex 633 nm/Em 760 nm). The results depicted in Fig.1A and quantified in Fig. 1B show that SapC–DOPS–CMVtargeting was inversely correlated with pH, i.e., binding washigher as media pH decreased.

SapC–DOPS has cytotoxic activity against lung cancer cellsTo evaluate the cytotoxic effects of SapC–DOPS on lung tumor

cells, viability assays were performed in vitro in a panel of humanlung cancer cell lines. Cells were incubated with SapC–DOPS atconcentrations ranging from 0 to 128 mg/mL and 72 hours later,the effects on cell growth were measured using the CCK-8assay. Figure 2 shows the calculated IC50 values plotted as afunction of cell surface membrane PS levels (low PS vs. high PS;

cutoff ¼ 52%), as assessed with annexinV-FITC staining. Similarto previous observations in glioblastoma (23) and pancreaticcancer cell lines (24), the selectivity of SapC–DOPS toward PSwasevidenced by a significant, inverse correlation between IC50 valuesand surface PS in these lung cancer cell lines.

SapC–DOPS induces apoptosis in lung tumor cellsTo assess whether the loss in cell viability caused by SapC–

DOPS was associated with the induction of apoptosis, tworelevant apoptosis parameters, namely mitochondrial mem-brane potential (Dcm) and DNA fragmentation, were evaluat-ed by flow cytometry in A549 cells. Dcm was assessed with thefluorescent dye JC-1. When living cells are incubated with JC-1,the dye is incorporated into the membranes of healthy, polar-ized mitochondria, forming aggregates that emit red (�590nm) fluorescence upon excitation with a 488 nm laser. Duringearly apoptosis, the collapse of Dcm induces dissociation andrelease of JC-1 monomers to the cytosol, causing a fluorescenceemission shift to green (�529 nm) fluorescence. Treatment ofA549 cells with SapC–DOPS (0, 10, 20, or 40 mg/mL) for 48hours induced a dose-dependent shift from red to green fluo-rescence, reflecting a collapse of Dcm consistent with apoptoticcell death (Fig. 3A).

In parallel experiments, we analyzed the effects of SapC–DOPSon cell-cycle progression and apoptotic cell death by quantifyingthe amount of DNA-bound PI representing DNA content. AsDNA is fragmented in apoptotic cells, PI fluorescence intensityis lower than in intact cells in the G0–G1 cycle, giving raise to the"sub G0/G1" phase. As shown in Fig. 3B, SapC–DOPS incubation

Figure 3.SapC–DOPS induces apoptosis in human lung adenocarcinoma A549 cells. Cultured A549 cells were treated with SapC–DOPS (0, 10, 20, and 40 mg/mL) for 48hours. A, SapC–DOPS causes dose-dependent Dcm depolarization, as assessed by quantification of P2-gated populations. B, dose-dependent DNAfragmentation induced by SapC–DOPS. The percentage of cells with fragmented DNA is represented by the Sub G0/G1 (M1) population. The figures arerepresentative profiles of at least three experiments. C, quantification of results from experiments like those shown in A (P2 events; n ¼ 4) and B (M1 events;n ¼ 3). �� , P < 0.01; ��� , P < 0.001.

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caused a dose-dependent increase in the percentage of cells in subG0/G1 phase (M1). Figure 3C shows summary data for theseexperiments. Taken together, the dose-dependent loss of Dcmand the DNA fragmentation observed in these experiments dem-onstrate that SapC–DOPS induces apoptosis in A549 lung cancercells.

SapC–DOPS targets lung tumors in vivoThe tumor-targeting capability of SapC–DOPS was tested in

mouse allogeneic tumor models generated through subcutane-ous implantation or systemic injection of LLC cells into femaleBALB/c nude mice or male FVB mice, respectively. Ten days aftertumor implantation, animals were injected into the peritonealcavity or via tail vein with SapC–DOPS–CVM or DOPS-CVMnanovesicles, CVM alone, or PBS; in vivo imaging was performed24 hours later. As shown in Fig. 4A (intraperitoneal injections)and Fig 4B (intravenous injections), tumor-specific fluorescencein subcutaneous xenografts was detected only in SapC–DOPS–CVM–injected mice. SapC–DOPS' tumor-targeting capabilitywas also assessed in mice harboring allogeneic lung tumorsproduced by systemic injection of LLC cells. Figure 4C shows aLLC-tumor-bearing mouse and a sham (PBS-injected) mouseimaged 2 and 150 hours after intravenous injection with SapC–DOPS–CVM. As in the prior model, SapC–DOPS–CVM fluores-cence localized specifically to the tumor.

In vivo antitumor efficacy of SapC–DOPS nanovesiclesThe anticancer efficacy of SapC–DOPS against human lung

cancer cells was evaluated in nude mice bearing subcutaneousA549 lung tumors. About 10 days after implantation (when

tumors reached �100 mm3), animals (6 per group) were treatedintravenously with SapC–DOPS, PBS, or the chemotherapy drugcisplatin, and tumor growth was monitored over time. Quanti-fication of tumor burden revealed that SapC–DOPS treatment atall three different doses tested reduced tumor weight by approx-imately 50% at day 35 (Fig. 5). Cisplatin, an alkylating chemo-therapy drug used here as a positive control for tumor growthinhibition (29), was also effective in reducing tumor growth.

Consistent with our observations in lung tumor cells in vitro,TUNEL staining confirmed the presence of apoptotic cells inSapC–DOPS–treated subcutaneous A549 lung tumors (Fig. 5C).

DiscussionLung cancer is the most common and the deadliest form of

cancer worldwide. Because most lung tumors are diagnosedonly when they reach advanced stages, standard treatments,namely surgical resection, radio- and chemotherapy, are mostlyineffective. Hence, recent efforts, including the development ofimmunotherapy agents, reflect the urgent need for more effec-tive, specific therapeutic approaches (30–33). The present studyshows that SapC–DOPS, a liposomal biotherapeutic agentcomposed of a lysosomal hydrolase coactivator (SapC) and aphospholipid (DOPS), induces apoptosis in lung tumor cellsin vitro and delays the growth of lung cancer xenografts in vivo.

Unlike typical anticancer liposomal formulations, SapC–DOPS possesses intrinsic tumor-targeting and tumoricidalactivities, which result from the combined properties of bothits lipid phase and its targeting protein. In this sense, SapC–DOPS is not merely a platform for chemotherapy drug delivery,

Figure 4.SapC–DOPS targets lung tumor cells in vivo. Nude mice bearing subcutaneous LLC cell allografts were imaged 24 hours after SapC–DOPS–CVM, DOPS-CVM,CVM, or PBS administration via intraperitoneal (A) or tail vein (B) injection. C, a lung tumor–bearing mouse (LLC) and a sham-injected, control mousewere intravenously injected with SapC–DOPS–CVM and imaged after 2 hours (in vivo) and 150 hours (ex vivo).

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but an anticancer drug in itself. While multiple modificationsare being assayed to overcome some common drawbacks oftargeted liposomal formulations, such as limited cell internal-ization, incomplete drug release and lysosomal degradation(7), SapC–DOPS readily targets exposed PS residues on cancercells' membranes and exploits the cell's own lysosomalmachinery to induce tumor toxicity. Importantly, a key featureof liposomes, i.e., the ability to be endowed with enhancedtargeting and cytotoxic capabilities by functionalization withdifferent ligands and drugs, is also shared by SapC–DOPS. Thebasis of SapC–DOPS anticancer actions resides in the affinity ofSapC toward PS, a membrane phospholipid which is seques-tered in the inner side of the plasma membrane in healthy cells,but is aberrantly exposed on the surface of many different(nonapoptotic) tumor cells (19, 20). This peculiarity hasprompted the development of a number of PS-targeting, anti-body-based imaging and therapeutic probes, with recent stud-ies using liposomal formulations showing promising results(34, 35). Upon tumor cell binding, SapC–DOPS cytotoxicactivity is thought to arise from lysosomal destabilization andleakage, and/or activation of lysosomal fatty acid hydrolasesand ceramide accumulation leading to caspase-mediated apo-ptosis (16, 21, 22, 36).

In this study, we show that SapC–DOPS exerts cytotoxicactions against a variety of human lung carcinoma cells in vitro,and that higher levels of exposed PS correlate with higherkilling efficacy by SapC–DOPS (i.e., lower IC50). We reportedsimilar results in glioblastoma (23) and pancreatic cancer (24)cell lines. Conversely, SapC–DOPS' lack of toxicity towarduntransformed human cells in vitro has been demonstrated inmammary (MCF-10A) cells, fibroblasts (21), and pancreaticductal epithelial cells (24).

The binding of SapC to PS is pH dependent, with an estimatedpKa of 5.3 (17, 18). The present results show that SapC–DOPSbinding to A549 human lung cancer cells in media with differentpHs (5.5 to 8.5) was highest at the most acidic pH value tested.Because acidosis, secondary to hypoperfusion, hypoxia and aug-mented glycolytic lactate production, is a common feature of themicroenvironment of solid tumors, SapC–DOPS may be partic-ularly effective to counteract hypoxia-driven malignant progres-sion and to treat tumors that show resistance to antiangiogenictherapies (23).

The present data also indicate that treatment of A549 lungcancer cells with SapC–DOPS induces a dose-dependent decreasein Dcm paralleled by fragmentation of cellular DNA (sub G0/G1

peak), consistent with apoptotic cell death.

Figure 5.Effects of SapC–DOPS on the growth of human lung tumor xenografts. After tumor establishment (day 0), mice were intravenously injected with saline(control), SapC–DOPS (high, medium, or low dose), or cisplatin every 2 days for 5 weeks. A, subcutaneous A549 tumor volume measurements. B,tumor weight at sacrifice. By 5 weeks after treatment onset, all experimental regimens significantly reduced tumor growth. C, apoptosis detection byTUNEL staining in subcutaneous A549 tumors. Original magnification, �400. Bar graph, quantification of apoptotic cells (averaged from 10 microscopic fieldsper treatment). � , P < 0.05; ��, P < 0.01; ��� , P < 0.001.

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Using fluorescently labeled nanovesicles, we show that system-ically injected SapC–DOPS specifically targets subcutaneouslyimplanted mouse lung tumor allografts, as well as mouse pul-monary tumors in vivo. Finally, the therapeutic efficacy of SapC–DOPS was evidenced by its ability to significantly reduce thegrowth of xenografted human lung tumors. As shown by TUNELstaining, and consistent with the reported effects of SapC–DOPSon lung tumor cells in vitro, this effect can be attributed, at least inpart, to apoptosis induction.

Taken together, the present results suggest that SapC–DOPS is apromising compound with potential clinical applicability for thediagnosis and treatment of lung tumors. Aberrant PS exposure is acommon aspect of different cancer types, and we have shown thatSapC–DOPS exerts both in vitro and in vivo antitumor actions inmouse models of neuroblastoma (21), glioblastoma (23), squa-mous cell carcinoma (25), pancreatic cancer (24), and metastaticbrain tumors (26). Unlike chemotherapy drugs, SapC–DOPSnanovesicles are well tolerated and nontoxic in preclinical cancermodels (21).

In conclusion, our in vivo and in vitro experiments suggest thatSapC–DOPS nanovesicles are a promising treatment option forlung cancer, worthy of further clinical development.

Disclosure of Potential Conflicts of InterestX.Qi is a consultant/advisory boardmember for Bexion Pharmaceuticals. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: Y. Hou, X. QiDevelopment of methodology: S. Zhao, Z. Chu, Y. Nie, X. QiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Zhao, Z. Chu, X. QiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): V.M. Blanco, Y. Nie, X. QiWriting, review, and/or revision of the manuscript: V.M. Blanco, X. QiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Zhao, Z. ChuStudy supervision: Y. Hou, X. Qi

AcknowledgmentsThe authors thank Dr. S. Abu-Baker and A. Stevens for the editing help.

Grant SupportThis workwas supported in part byNewDrug State Key Project grant number

009ZX09102-205 (to X. Qi), 1R01CA158372 (to X. Qi), and Research Funds/Hematology-Oncology Programmatic Support from University of CincinnatiCollege of Medicine (to X. Qi).

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 August 7, 2014; revised October 30, 2014; accepted November 12,2014; published online February 10, 2015.

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2015;14:491-498. Mol Cancer Ther   Shuli Zhao, Zhengtao Chu, Victor M. Blanco, et al.   Cancer

DOPS Nanovesicles as Targeted Therapy for Lung−SapC

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