tumor-targeted nanoparticle delivery of hur sirna inhibits ...ranganayaki muralidharan1,2, anish...

Small Molecule Therapeutics

Tumor-targeted Nanoparticle Delivery of HuRsiRNA Inhibits Lung Tumor Growth In Vitro andInVivoByDisrupting theOncogenicActivityof theRNA-binding Protein HuRRanganayaki Muralidharan1,2, Anish Babu1,2, Narsireddy Amreddy1,2, Akhil Srivastava1,2,Allshine Chen3, Yan Daniel Zhao2,3, Uday B. Kompella4, Anupama Munshi2,5, andRajagopal Ramesh1,2,6

Abstract

Selective downregulation of the human antigen R (HuR) pro-tein by siRNAmay provide a powerful approach for treating lungcancer. To this end, we investigated the efficacy of transferrinreceptor-targeted liposomal nanoparticle-based HuR siRNA(HuR-TfNP) therapy and compared with control siRNA (C)-TfNPtherapy both, in vitro and in vivo using lung cancer models. In vitrostudies showed HuR-TfNP, but not C-TfNP, efficiently down-regulatedHuR andHuR-regulated proteins in A549, andHCC827lung cancer cells, resulting in reduced cell viability, inhibition ofcell migration and invasion, and induction of G1 cell-cycle arrestculminating in apoptosis. However, HuR-TfNP activity in normalMRC-9 lung fibroblasts was negligible. In vivo biodistributionstudy demonstrated that fluorescently labeled HuR-siRNA or ICGdye–loaded TfNP localized in tumor tissues. Efficacy studiesshowed intratumoral or intravenous administration ofHuR-TfNP

significantly inhibited A549 (>55% inhibition) and HCC827(>45% inhibition) subcutaneous tumor growth compared withC-TfNP. Furthermore, HuR-TfNP treatment reduced HuR, Ki67,and CD31 expression and increased caspase-9 and PARP cleavageand TUNEL-positive staining indicative of apoptotic cell death intumor tissues compared with C-TfNP treatment. The antitumoractivity of HuR-TfNP was also observed in an A549-luc lungmetastatic model, as significantly fewer tumor nodules (9.5 �3.1; P < 0.001; 88% inhibition) were observed in HuR-TfNP–treated group compared with the C-TfNP–treated group (77.7 �20.1). Significant reduction in HuR, Ki67, and CD31 expressionwas also observed in the tumor tissues of HuR-TfNP-treatmentcompared with C-TfNP treatment. Our findings highlight HuR-TfNP as a promising nanotherapeutic system for lung cancertreatment. Mol Cancer Ther; 16(8); 1470–86. �2017 AACR.

IntroductionThe effectiveness of conventional therapies for lung cancer

has been challenged by ever increasing tumor heterogeneityand undesired side effects (1). This scenario emphasizes theneed for newer therapeutic strategies that overcome theselimitations. The therapeutic potential of anticancer agents canbe tremendously improved by means of targeted delivery

toward cancer cells that overexpress surface molecules (2, 3).Such molecules often act as receptors for ligands or antibodieswith high specificity.

The iron-binding serum protein transferrin (Tf) has specificaffinity towards transferrin receptors (TfR) expressed in the cellmembrane, which is a basic requirement for iron transport in cells(4). A significant number of lung cancers overexpress transferrinreceptors; moreover, their expression in patients with non–smallcell lung cancer (NSCLC) has shown significant prognostic value(5). Therefore, TfR-targeted drug delivery may be a promisingstrategy to improve the efficiency of lung cancer therapy.

Recent studies have shown that high levels of the humanantigen R (HuR), an RNA binding protein, is expressed in manycancer types (6–8), and regulates the expression of severalpathologically important mRNAs, particularly in lung cancer(9, 10). Furthermore, HuR regulates cancer cell proliferation,migration, angiogenesis, and metastasis (7–9, 11, 12). HuR hasalso been associated to play a role in chemotherapeutic resis-tance (13, 14). Recent studies from our laboratory demonstrat-ed that HuR downregulation inhibited cell growth in NSCLC invitro and altered the cells' ability to undergo migration andinvasion (12, 15). Furthermore, we recently showed HuRinhibition radiosensitized human breast cancer cells (16). Allof these studies indicate HuR is a molecular target for cancertherapy and advancing preclinical testing of HuR-targeted ther-apies could ultimately lead to clinical testing.

1Department of Pathology, University of Oklahoma Health Sciences Center,Oklahoma City, Oklahoma. 2Stephenson Cancer Center, University of OklahomaHealth Sciences Center, Oklahoma City, Oklahoma. 3Department of Biostatisticsand Epidemiology, University of Oklahoma Health Sciences Center, OklahomaCity, Oklahoma. 4Department of Pharmaceutical Sciences and Ophthalmology,University of Colorado, Denver, Colorado. 5Department of Radiation Oncology,University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma.6Graduate Program in Biomedical Sciences, University of Oklahoma HealthSciences Center, Oklahoma City, Oklahoma.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Rajagopal Ramesh, Department of Pathology, StantonL. Young Biomedical Research Center, Suite 1403, 975 N.E., 10th Street, Okla-homa City, OK 73104. Phone: 405-271-6101; Fax: 405-271-2472; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-17-0134

�2017 American Association for Cancer Research.

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RNA interference (RNAi)-based gene silencing provides apromising strategy to knockdown target genes involved in cancerpathology. Downregulation of the HuR gene using compli-mentary siRNA is an effective method that has demonstratedtherapeutic efficiency in cancer cells (8, 12). Because of its poorcell uptake and low serum stability, delivery of naked siRNA oftenutilizes nanocarriers, such as liposomes/lipid nanoparticles(8, 15, 17) polymer nanoparticles (18, 19), or nanoparticlesmade from inorganic materials (20, 21). In this study, we haveused a lipid-based nanoformulation usingDOTAP (1, 2-dioleoyl-3-trimethylammonium-propane chloride (DOTAP) and choles-terol as the carrier components for HuR siRNA delivery in vitroand in vivo (22–24).

For effective in vivo lung cancer therapy using HuR siRNA, wecreated a targeted delivery system bymodifying the DOTAP:Cholnanoparticle platform with transferrin as a targeting ligand(DSPE-PEG-Tf). The targeting moiety, Tf, was chosen based onthe expression levels of its receptors (TfR or CD71) in solid andmetastatic lung tumormodels. Initially, we tested the efficiency ofHuR-TfNP versus HuR-NP (nontargeted) in vitro. On the basis ofthe results obtained, we hypothesized that TfNP-based targeteddelivery of HuR siRNA would reduce the tumor burden in mousemodels of lung cancer. We studied TfNP's biodistribution inmouse models and, relying on this data, we evaluated the targetspecificity and efficacy of HuR-TfNP versus C-TfNP (controlsiRNA) in solid tumor models. Finally, we used in vivo liveimaging, tumor nodule counts, and IHC of specific molecularmarkers to investigate tumor growth inhibition and the antimeta-static activity of HuR-TfNP in a mouse model of metastatic A549-luc lung cancer.

Materials and MethodsChemicals

DOTAP, cholesterol, and 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[(polyethylene glycol)-2000] (DSPE PEG2000)were purchased from Avanti Polar Lipids. Transferrin and desfer-rioximine were purchased from Sigma-Aldrich Chemicals.RPMI1640medium, Eagleminimum essential medium (EMEM),and FBS were purchased from Gibco BRL Life Technologies.siGLO, control siRNA (50 UAA GGC UAU GAA GAGAUA C 30),and HuR siRNA (50 UCA AAG ACG CCA ACU UGU A 30) werepurchased from Dharmacon.

Cell lines and cell cultureThe human NSCLC A549 and HCC827 cell lines and normal

MRC-9 lung fibroblasts were purchased from ATCC and wereauthenticated via single tandem repeat (STR; IDEXX Laboratories)profiling before the experiments. Mycoplasma testing by PCRwasroutinely performed using specific oligonucleotides (IDT). Thepassage number for tumor cells and normal lung fibroblasts usedin the study was from 8 to 35 and from 4 to 12, respectively.Tumor cells and normal cells were cultured in RPMI1640 andEMEM, respectively, supplemented with 10% FBS and 1% pen-icillin/streptomycin.

Synthesis of Tf-NPPreparation of DSPE-PEG-Tf. Briefly, transferrin (Tf; 150 nmol)was converted to its thiolated form by reacting Tf with Trautreagent (2-iminothiolane) in sodium phosphate-EDTA buffer(pH 8.0). The crude product (Tf-SH) was then purified from

unreacted reagents using a PD-10 (Sephadex-G25) desaltingcolumn with sodium phosphate-EDTA eluent buffer (pH 7.1).The purified Tf-SH (150 nmol) was allowed to react withDSPE-PEG-Maleimide (300 nmol) in sodium phosphate buffer(pH 7.1) for 24 hours, reacting at 40�C to form a stable conjugate,DSPE-PEG-Tf. The product was then purified by dialysis againstultra-pure water overnight.

Preparation of TfNP. Liposomes (20 mmol/L DOTAP:Chol)were synthesized and extruded stepwise through polycarbonatefilters of different pore sizes (1.0, 0.45, 0.2, and 0.1 nm), asdescribed previously (12, 25). For preparation of siRNA:lipo-some complexes, DOTAP:Chol (20 mmol/L) stock solutionand siRNA solution diluted in 5% dextrose in water (D5W)were mixed in equal volumes to give a final concentrationof 4 mmol/L DOTAP:Chol-siRNA in a 300-mL final volume.DSPE-PEG-Tf ligands were inserted into preformed liposomesusing the post insertion technique (26). Briefly, DOTAP:Chol-siRNA was mixed with an aqueous dispersion of Tf-PEG-DSPE(from 0.01%, 0.03%, 0.05%, and 0.1 mol% of total lipid) andwas vigorously rinsed up and down in a pipette tip. Thiscomplex was incubated at room temperature for 60 minutes.In the case of unmodified DOTAP:Chol, D5W was addedinstead of Tf-PEG-DSPE solution. Both modified and unmod-ified liposomes were dialyzed against distilled water overnightat 4�C. Modified liposomes containing control siRNA and HuRsiRNA will henceforth be designated as C-TfNP and HuR-TfNP,respectively.

Nanoparticle characterizationConfirmation of Tf binding to NP. Conjugation of Tf into DSPE-PEG and DSPE-PEG-Tf into DOTAP:Chol was confirmed by dotblot, as described previously (27).

Nanoparticle size, zeta potential, and morphology. The averagehydrodynamic radius and zeta potential of NP and TfNP insolution was determined by dynamic light scattering (DLS) usinga Zeta PALS Zeta potential and particle size analyzer (BrookhavenInstruments). The shape and structure was analyzed using trans-mission electron microscopy (TEM) at the Oklahoma MedicalResearch Foundation (OMRF) core facility. The shape and sizedistribution of HuR-TfNP was observed under a Hitachi H600transmission electron microscope (Hitachi).

Protection of siRNA. The stability of encapsulated siRNA in thepresence of serum was monitored by incubating TfNP loadedwith siRNA in 50% FBS at 37�C. Aliquots of 20 mL werewithdrawn at 0 and 60 minutes, and subjected to gel electro-phoresis using agarose (1.2%) gel at 100 V for 20 minutes inTAE buffer (pH 8.0). The efficiency of siRNA condensation bynanoparticles was determined against the free siRNA control(18). The electrophoresis gel stained with ethidium bromide(Sigma) was visualized and imaged using a Syngene gel doc-umentation system.

siRNA release profile. TfNP loaded with siGLO were suspend-ed in tissue culture medium (pH 7.4), acetate buffer (ABS,pH 5.5), or tissue culture medium with 10% FBS and wereincubated at 37�C with gentle shaking at 120 rpm. The releaseof siRNA was monitored at several time intervals, as describedpreviously (21).

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Optimization of Tf-PEG2000-DSPE on DOTAP:Chol. We evaluat-ed the cellular uptake of TfNP in A549 cells with respect tomodification ratio. A549 (2 � 105/well) cells seeded in a 6-wellplate were transfected with a siGLO–TfNP complex containing0.01%, 0.03%, 0.05%, or 0.1 mol% of Tf-PEG2000-DSPE to thetotal lipid in serum-free medium for 6 hours. After transfection,cells were replenished with RPMI1640 containing 10% FBS,and incubation was continued. Cells were harvested at 24 hoursafter transfection, and fluorescence expression was determinedusing an Envision plate reader (Perkin Elmer). Cells that werenot transfected or transfected without free ligand served ascontrols.

Receptor-mediated endocytosis. A549 (2 � 105 cells/well) wereseeded in a 6-well plate and were treated with siGLO-TfNP.Untreated cells served as controls. After nanoparticle treatment,theplateswith cellswere incubated in either 37�Cor4�C(24). Thecells were harvested after 1 and 4 hours of incubation and thefluorescence intensity determined using an Envision plate reader.

Specificity studiesReceptor stimulation studies.A549 cells (2�105/well)were seededin 6-well plates. After 24 hours of incubation, the medium wasreplaced with 1 mL of serum-free medium, with or without 100mmol/L of desferrioxamine (DFO). After 24 hours of incubation,the media were replaced with siGLO-TfNP–containing media.After 6 hours, siGLO-TfNP–containing media were replaced withfresh 2% serum containing media and the cells were incubatedovernight. Next day, the cells were harvested and the fluorescenceintensity of siGLO was measured using Envision plate reader.

Receptor blocking studies. A549 cells (2� 105 cells/well) seeded in6-well plates were pretreated with or without human transferrin(HTf; 1 mg) and were incubated at 37�Cwith 5% CO2 for 1 hour.The cells were subsequently treated with siGLO-TfNP in serum-freemedium. Incubation continued for an additional 1, 4, and 24hours. At the endof the incubationperiod, the cellswere subjectedto fluorescence intensity measurement.

In another set of experiments, A549 cells (2 � 105 cells/well)seeded in 6-well plates were pretreated with or without 10 mLof human transferrin antibody and were incubated at 37�Cwith 5% CO2 for 1 hour. The cells were subsequently treatedwith siGLO-TfNP in serum-free medium. Incubation continuedfor an additional 1 and 4 hours. At the end of the incubationperiod, the cells were subjected to fluorescence intensitymeasurement.

Cellular uptake studies. Cellular internalization of TfNP loadedwith fluorescently labeled siRNA was measured quantitativelywith an Envision plate reader. A549, HCC827, and MRC-9 cellswere separately seeded in 6-well plates at 2 � 105 cells/well.The next day, cells were transfected with either siGLO-TfNP orsiGLO-NP in serum-free medium. Cells were harvested after 1, 4,and 24 hours of incubation, and were analyzed for the meanfluorescence intensity.

Cell viability assay. A549, HCC827, and MRC-9 cells were seededin a 6-well plate at 2 � 105 in 2 mL of RPMI and EMEM mediacontaining 10%FBS. After 24 hours of incubation in a humidifiedatmosphere of 5% CO2 at 37�C, the medium was replaced with1 mL of serum-free medium and was treated with TfNP loaded

with control siRNA (C-TfNP) or HuR-siRNA (HuR-TfNP). Thefinal concentration of siRNA used in these experiments was100 nmol/L. After 6 hours of incubation, the medium wasreplaced with RPMI and EMEM medium containing 2% serumand was incubated for an additional 24 and 48 hours at 37�C.Cells were harvested and cell viability was determined withTrypan blue exclusion assay (28).

Western blotting. Total cell lysates prepared from cultured cells ortumor tissues receiving various treatments were prepared usingRIPA buffer and were subjected to Western blot analysis asdescribed previously (24, 29, 30). Primary antibodies againsthuman HuR, Bcl2, Cyclin D1, Cyclin E, and p27 (Santa CruzBiotechnology); caspase 9 and PARP (Cell Signaling Technology),and b-actin (Sigma Chemicals) were all purchased and used asrecommendedby themanufacturers. Proteinswere detected usingthe appropriate horseradish peroxidase–tagged secondary anti-bodies and differences in protein expression determined asdescribed previously (24, 29, 30).

qRT-PCR. qRT-PCR assay for HuR, Bcl2, and p27 was performedusing specific oligonucleotide primers (Supplementary Table S1)as described previously (24, 25). Briefly, first-strand complemen-tary (c)DNA was synthesized from 2 mg of total RNA using Quantscript cDNA synthesis kit (Bio-Rad). The cDNA was subsequentlyused to perform qRT-PCR (Bio-Rad CFX96 Touch Real-Time PCRDetection System) with SYBR chemistry using iQTM SYBR GreenSuper Mix (Bio-Rad). The relative gene expression values werequantified by the 2�DDCt method (30, 31).

Cell-cycle analysis. C-TfNP and HuR-TfNP–treated cells were col-lected at 24 and 48 hours after treatment and stained withpropidium iodide and were subjected to flow cytometric analysisas described previously (24, 25, 32). Untreated cells served ascontrols.

Migration and invasion assay. The cell migration and invasionassay was carried out as described previously (12, 24, 30). Briefly,C-TfNP and HuR-TfNP–treated A549 cells were seeded in the topchambers of themigration and invasion inserts. Cells receiving notreatment served as controls. The bottom chamber was filled with20% FBS-containing culture medium. After 24 and 48 hours ofincubation, the number of migrated and invaded cells werecounted and the results represented as described previously(24, 30).

Annexin V assay.A549 cells (2� 105) seeded in a 6-well plate weretreated with C-TfNP and HuR-TfNP and stained with Annexin Vconjugated to Alexa Fluor 488 dye and propidium iodide (PI)using Dead Cell Apoptosis Kit (Life Technologies) according tomanufacturer's protocol. Briefly, the cells were harvested at 48hours after treatment and suspended in Annexin V binding bufferat a concentration of 1 � 106 cells/mL. One-hundred microlitersof cell suspension was incubated with 5 mL of Annexin V and 1 mLof 100 mg/mL of PI for 15minutes at room temperature. After theincubation period, the cells were analyzed in FACSCalibur flowcytometer using BD Cell Quest software (BD Biosciences) atexcitation 488 nm and emission 530 nm. The number of of cellsundergoing apoptosis was determined using the dual staining.Three subsets of cells were distinguished: viable cells (Annexin Vand PI negative, early apoptotic cells; Annexin V positive and PI

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negative), and dead cells (Annexin V and PI positive). The resultsthus obtained were plotted as percentage of cells undergoingapoptosis after 48 hours of treatment.

In vivo studiesSubcutaneous tumor xenograft and experimental lung metastasismodels. Four-to-six-week-old female nude mice (Nu-Nu) werepurchased from Charles River Laboratories. The studies wereconductedwith the approval of and in accordancewith guidelinesof the Institutional Animal Care and Use Committee at theUniversity of OklahomaHealth Sciences Center (Oklahoma City,OK). For establishing subcutaneous (s.c.) tumors, mice wereanesthetized with 2% isoflurane and injected with A549 andHCC827 tumor cells (5 � 106 cells in 100 mL of PBS) subcuta-neously in the lower right flank of mice. For studies involvingbioluminescence imaging, A549 stably expressing luciferase (luc;A549-luc) was used.When the tumors reached a size of about 50–100mm3, themicewere randomized andused for biodistributionand efficacy studies as described below.

For establishing experimental lung metastasis, A549-luc cells(5� 106 cells in 100 mL of PBS) were injected intravenously (i.v.)via tail-vein. Tumor formation in the lungs was detected byintraperitoneally (i.p.) injecting D-luciferin (150mg/kg) andmea-suring bioluminescence using the IVIS Spectrum Imaging System(Perkin Elmer). When the tumor bioluminescence signals reach-ed approximately average radiance of 5,000 p/s/cm2/sr,micewererandomly assigned to treatment groups and efficacy studiesconducted.

Biodistribution study. For determining the TfNP biodistributionin vivo, indocyanine green (ICG; an amphiphilic carbocyaninegreen dye; Sigma-Aldrich) that fluoresces at the near infra-red(NIR) regionwas encapsulated in TfNPs. TheseNPswere preparedfollowing the same method for encapsulating the siRNA as de-scribed above for in vitro studieswithminormodifications. Briefly,an equal volume of DOTAP:Chol (20 mmol/L) stock solutionand ICG solution (1 mg/mL in water) was mixed, vortexed, andincubated at room temperature for 20 minutes. DSPE-PEG-Tfligands were inserted into preformed liposomes using the postinsertion technique. The solutions were then dialyzed againstwater overnight using a 10 kDa MW cut-off membrane, and werecollected and labeled as ICG-TfNP.

ICG-TfNP (30 mg of ICG) was administered intravenously viatail vein tomice bearing subcutaneous A549 andHCC827 tumorsor to non-tumor–bearing na€�ve mice. Free ICG-injected subcuta-neous tumor-bearing mice were used as controls. Mice wereimaged (excitation: 785 nm; emission, 820 nm) at 10-minute,30-minute, 4-hour, and24-hour intervals postinjection using IVISSpectrum Imaging System. Similarly, for determining siRNAbiodistribution in vivo, fluorescently labeled DY657-HuR-siRNAcontained in TfNP (DY647-HuR-TfNP, 100 mg siRNA) wasinjected intravenously via tail vein to HCC827 subcutaneoustumor-bearing mice and imaged (excitation, 785 nm; emission,820 nm).

For determining the biodistribution in mice bearing experi-mental lung metastasis, ICG-TfNP (30 mg ICG) was injectedintravenously via tail vein of mice bearing A549-luc–expressinglung tumors. Prior to injecting ICG-TfNP,mice were injected withD-Luciferin (150mg/Kg, i.p.) and after 10minutes were imaged tolocalize tumors in the lung. Subsequently ICG-TfNP was admin-istered to mice and imaged and the localization of ICG-TfNP in

the luc-expressing lung tumors determined by overlaying theimages.

siRNAdetection and quantitation inHuR-TfNP–treated tumors. Fordetermining HuR siRNA in HuR-TfNP–treated tumors, dropletdigital PCR (ddPCR) QX100 (Bio-Rad) assay using a custom-designed TaqMan siRNA assay (Applied Biosystems) wasemployed. A549 and HCC827 tumor tissues frommice that weretreated with a single dose of HuR-TfNP (n ¼ 2) or treated withC-TfNP (n ¼ 2) were harvested and total RNA was isolated. Foruse as positive control, a tumor lysate prepared from a mousethat did not receive any treatment was spiked with 100 mg of HuRsiRNA and total RNA was isolated from it. Total RNA samplesisolated from HuR-TfNP, C-TfNP, and untreated control sampleswere diluted to 10 ng before the samples were used for the assay.Reverse transcription was performed with the diluted total RNAusing specific stem-loop reverse transcription primers (AppliedBiosystems) for human HuR siRNA. ddPCR was used to detectand quantify the siRNA in each tumor, following the manufac-turer's protocol. Briefly, the single-strand cDNA, TaqMan probes,ddPCR buffer, and water were mixed to obtain a 20:l ratio of theddPCR reaction mix. Using a Bio-Rad droplet generator, dropletswere generated with the ddPCR reaction mix and droplet-makingoil. The droplets were transferred into a 96-well plate. Aftersealing the plate with foil at 180�C, 40 cycles of PCR amplifica-tion were carried out in a Bio-Rad T100 iCycler. Each droplet wasquantified for TaqMan probe fluorescence using the Bio-RadQX100 droplet counter. Absolute copies per microliter werecalculated using the Poisson distribution algorithm. The copynumber of spiked siRNA detected by ddPCR was considered as100% input siRNA, and relative percent intake was further cal-culated in the treated tumor tissues and results expressed aspercent intake of siHuR/100% input siHuR.

Efficacy studies in subcutaneous tumor model. Nude mice bearingA549 and HCC827 subcutaneous tumors were randomizedinto two groups (n ¼ 5/group/tumor type) when the tumorsreached a size of 50–100 mm3. Mice were treated intratumo-rally with C-TfNP and HuR-TfNP containing 100 mg of scram-bled siRNA (C-TfNP) or HuR siRNA (HuR-TfNP). The animalswere treated three times a week for two weeks. Tumor volumeswere measured at regular intervals as described previously(33, 34). The experiment was terminated on day 25 after ini-tiation of treatment and tumor tissues were harvested and usedfor molecular and immunohistochemistry (IHC) studies.

For determining the therapeutic efficacy of systemic HuR-TfNPtreatment, mice bearing A549-luc and HCC827 subcutaneoustumorswere divided into two groups (n¼ 8/group/tumormodel)when the tumors reached a size of 50–100 mm3. The mice weretreated with C-TfNP and HuR-TfNP by intravenous tail-veinadministration. All other experimental conditions, includingnumber of treatments and tumor measurement, except the exper-iment duration, remained the same as described above for intra-tumoral treatments. For determining the therapeutic effect ofHuR-TfNP, twomice from each treatment group were euthanizedafter two treatments and the tumor tissues harvested formolecularanalysis. At the end of the study, tumor tissues from all of theanimals from the two treatment groups were harvested and usedfor molecular and IHC studies.

For determining growthof A549-luc tumors,we also conductedbioluminescence imaging and tumor growth calculated by






measuring luminescence ondays 1, 15, and33. Luminescencewasdetermined by injecting D-Luciferin (150 mg/Kg, i.p.) and 10minutes later subjecting the mice to imaging using IVIS SpectrumImaging System. The results were calculated and expressed asaverage radiance (photons/sec/cm2/sr) and subjected to statisticalanalysis. Imaging beyond day 33 could not be performed as themice in the C-TfNP treatment were euthanized due to large tumorburden and observance of signs of necrosis.

Efficacy studies in experimental lung metastasis model. A549-luccells (5 � 106 suspended in 100 mL of PBS) were injectedintravenously into the tail veins of nude mice. Once biolumines-cence signals in the lungs reached approximately 3,000p/s/cm2/sraverage photon radiance, they were randomly divided into twogroups (n ¼ 10/group) and treated with C-TfNP and HuR-TfNPcontaining 100 mg of siRNA. TfNPs were injected intravenous viathe lateral tail vein three days aweek for twoweeks for a total of sixtreatments. Tumor growth was measured by evaluating Lucexpression by imaging. Mice were imaged on day 0 (beforetreatment) using an IVIS Spectrum Imaging System within 10minutes after the injection of D-Luciferin (150 mg/kg). Theimaging was performed every two weeks for a total period of 6weeks. Living Image 2.5 software was used to determine theregions of interest (ROI), and average radiance (p/s/cm2/sr) wasmeasured for each mouse. The average ROI for each treatmentgroup was measured, and the difference in the ROI between thegroups was noted. All animals were sacrificed at week 6 and lungsfrom five animals in each group were collected and used formolecular and IHC studies.

The remaining 5 mice per treatment group were used forcounting extrapulmonary lung nodules as described previously(25, 33, 35). Briefly, 10 mL of Bouin fluid was intratracheallyinjected into the lungs of sacrificed mice. The inflated lungs wereremoved and washed with PBS. The extrapulmonary tumornodules appearing as white nodules in each lobe of the lungwere counted under the dissection microscope. The difference inthe average number of nodules per lung from each treatmentgroup was calculated and subjected to statistical analysis.

IHC. IHC staining on tumor tissues harvested from C-TfNP andHuR-TfNP–treated mice was performed using an automatedimmunostainer (Leica, Bond-III, Leica) with the following pri-mary polyclonal antibodies: HuR (Santa Cruz Biotechnology),Ki67, and mouse CD31 (Abcam). Appropriate positive and neg-ative controls were included. The stained tissue sections wereanalyzed using the Aperio ScanScope Image Analysis System(Aperio; ref. 36). HuR and Ki-67–stained tumor tissues wereanalyzed using a positive nuclear count algorithmwith the AperioImageScope viewer. The number of positive nuclei fromROIs wasdetermined for each animal from C-TfNP and HuR-TfNP–treatedgroups. For theCD31marker staining,we determined the numberofmicrovessels permm2of the tissue using theAperiomicrovesselanalysis algorithm. The results obtained for each of the marker inthe two treatment groups were subjected to statistical analysis.

TUNEL assay. TUNEL assay was performed using In Situ CellDeath Detection Kit from Roche Diagnostics, according to themanufacturer's protocol. Briefly, tumor tissue sections were incu-bated with proteinase K for 10 minutes, blocked, and incubatedwith TUNEL reaction mixture for 60 minutes at 37�C. Apoptoticnuclei were labeled by the addition of anti-fluorescein alkaline

phosphatase conjugate. Stained tumor sections were analyzedusing the Aperio Scanscope Image and the number of TUNEL-positive nuclei counted using the positive nuclei count algorithmwith the Aperio ImageScope viewer.

Statistical analysisThe SAS 9.2 software was used for statistical analyses. All

data were provided as mean � SD. Univariate statistical signifi-cancewasdeterminedbyone-wayANOVAwithTukey adjustmentfor pairwise comparisons. Differences between groups wereobtained using a linear mixed-effects model with Tukey adjust-ment. A P value <0.05 was considered statistically significant.

Supplementary DataSupplementary material is available for the physicochemical

characteristics of nanoparticles, cell uptake and ligand optimiza-tion studies, mRNA expression analysis, western blots quantifi-cation data, imaging data for bio-distribution and anti-tumorefficacy in mouse models, and IHC analysis of tumor tissues.

ResultsPhysicochemical characterization of TfNP

Cationic lipid-based nanoparticles are the most commonlyused carriers for gene therapeutics, due to their effective genetransfection ability in vitro and in vivo. Hence, we chose acationic liposome platform, DOTAP:Chol, which demonstrat-ed gene delivery potential in our previous studies (12, 22–24),in formulating TfR-targeted nanoparticles (TfNP). HuR siRNAwas allowed to interact electrostatically with cationic DOTAP:Chol liposomes to form HuR-NP. Table 1 shows the averageparticle sizes and zeta potentials of all formulations used in thisstudy. The unmodified nanoparticles carrying control siRNA(C-NP) were near spherical in structure, as observed with TEM(Supplementary Fig. S1A).

The scheme of HuR-TfNP synthesis is shown in the Fig. 1A.The optimal Tf ligand density in nanoparticles that favored anincreased cell uptake of fluorescently labeled siRNA (siGLO)was determined to be 0.05% compared with 0.01%, 0.03%,and 0.1% Tf ligand and no ligand (Supplementary Fig. S2;P < 0.05). The observation that 0.01% and 0.03% Tf liganddensity on nanoparticles showed relatively lower fluorescenceand cell uptake compared with no ligand on nanoparticles wassomewhat unexpected and likely due to insufficient number ofligands attached to the nanoparticle. In addition, the presenceof neutral polymer PEG, which is postinserted in DOTAP:Chollipid bilayer as DSPE-PEG-Tf, might have influenced thestrength of electrostatic binding of the nanoparticle to the cellmembrane causing a reduced cell uptake (37). The insufficientTf ligand concentration and contributory effect of PEG arelikely overcome when 0.05% Tf ligand concentration is usedresulting in enhanced cell uptake. Our results clearly demon-strate that ligand concentration on nanoparticle surface is animportant determining factor for the optimal affinity of nano-particle to the cell surface receptors and uptake and concurswith previous report (38).

The particle size showed a gradual increase from 180.54 �9.6 nm (empty nanoparticle) to 313.43 � 6.5 nm (HuR-TfNP)due to siRNA loading and surface modification with Tf ligand(Table 1). The siRNA was well condensed by TfNP, resulting inmobility retardation, as shown in the gel electrophoretogram

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(Supplementary Fig. S1B). The siRNA encapsulation efficiencyexpressed as percent (% EE) was calculated using the followingequation:

% EE ¼ [(siRNA added to the liposome-free "unentrappedsiRNA")/siRNA added to the liposome] � 100.

The siRNA EE was found to be 80.07 � 0.35% for HuR-TfNP.The in vitro release profiles of siRNA-Tf-NP were investigated

in three different media conditions such as acetate buffersolution (ABS; pH 5.5), tissue culture medium (pH 7.4), and10% serum-containing culture medium (pH 7.4) at 37�C over aperiod of 24 hours (Supplementary Fig. S1C). A burst siRNArelease pattern was observed for the first 6 hours (�25%–40%siRNA released) in culture medium and 10% serum-containingculture medium (pH 7.4). The observed siRNA release char-acteristics are typical of a lipid nanoparticle delivery system,and concur with previous studies (39, 40). However, the siRNA

release rate was higher when siRNA-TfNP was incubated inculture medium alone (�46% siRNA released) compared with10% serum-containing medium (�27% siRNA released). ThesiRNA release was noticeably slower after 6 hours until themeasurement at 24 hours, indicating a sustained release pat-tern. However, at low pH (5.5), siRNA release was rapid,reaching 64% in 6 hours and 93% in 24 hours. This result isconsistent with our previous report that shows enhanced siRNArelease rate at low pH (5.5; ref. 24).

In vitro evaluation of siGLO-TfNPAs the purpose of ligand (Tf) modification of nanoparticles

was to render specificity in cellular uptake (27), and to achieveenhanced siRNA delivery efficiency in cancer cells, we investi-gated the cell uptake mechanism of Tf-NP. In one of our studiesusing desferrioxamine (DFO), an iron chelator, TfR (known asCD71) expression was upregulated in A549 lung cancer cellsthat were subjected to siGLO-TfNP treatment (SupplementaryFig. S3A, top). We observed a clear difference in fluorescenceintensity of TfNP-delivered siGLO in DFO-treated and untreat-ed A549 cells (Supplementary Fig. S3A). The increased fluo-rescence intensity in the DFO-treated group indicates enhanceduptake of siGLO-TfNP compared with cells that were notpretreated with DFO.

Table 1. Particle size and zeta potential of siRNA containing nanoparticles

ComponentsDiameter(Mean � SD, nm)

Zeta potential(Mean � SD, mV)

NP 180.54 � 9.6 33.98 � 3.9HuR-NP 202.59 � 3.3 �0.57 � 1.9C-TfNP 295.74� 16.4 9.53 � 2.5HuR-TfNP 313.43 � 6.5 10.28 � 1.4

SH

A

C D

B

Transferrin

Transferrin-maleimide-PEG-DSPE

HuR-TfNP

HuR-NP

0

0

5

10

15

20

Cel

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wth

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un

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ntr

ol (

%)

25

30 A549

HuR-NP HuR-TfNP 24 h

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6 ControlHuR-NPHuR-TfNP

HuR BCL2 Cycin D1 p27

Rat

io: v

s. A

ctin

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p27

Cyclin D1

Bcl2

HuR

Control HuR-NP HuR-TfNP

1 h 4 h 24 h

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350,000

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l up

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ntr

ol (

a.u

.)

Actin

TfR

A549 HCC827 MRC-9 A549

HCC827

MRC-9Maleimide-PEG-DSPEO

O

O

SO

O

O

N

N

H

H

[(CH2)2O]n

[(CH2)2O]n

DSPE

DSPE

Figure 1.

Tumor-targeted HuR silencing suppresses lung tumor cell growth. A, Schematic illustration of HuR-siRNA–loaded, Tf-modified liposome (HuR-TfNP) preparation.B, Assessment of transfection efficiency of fluorescently labeled siRNA (siGLO-) TfNP in lung cancer cell lines (A549, HCC827) and normal lung fibroblasts(MRC-9) showed a time-dependent increase in uptake of the NPs by tumor cells compared with normal cells. Inset shows TfR expression is higher in tumorcompared with normal cells. C, Cell growth inhibition induced by HuR-TfNP (targeted delivery) compared with HuR-NP (nontargeted delivery) 24 hours aftertreatment of A549 cells. D, Western blot evaluation of HuR and HuR-regulated proteins 24 hours after HuR-TfNP or HuR-NP treatment in A549 cells. Thequantification of protein bands (Test protein/Actin ratio) is shown as a bar graph at the bottom. � , P < 0.05; �� , P < 0.001.






To further understand the specificity of TfNP, we conducted aseparate study in which human transferrin (HTf) and TfNP wereallowed to compete against each other (27), in interactingwith TfR-expressing A549 cells. The results showed that HTf(1 mg/well) significantly inhibited the uptake of siGLO-TfNP, asmeasured by fluorescence intensity, at all time points (1, 4, and24 hours; Supplementary Fig. S3B). To confirm the TfR-basedspecificity of TfNP, A549 cells were pretreated with humantransferrin receptor-antibody (TfRAb). Significant reductionin siGLO-TfNP fluorescence intensity was observed at 1 and4 hours with the TfRAb-added group, indicating receptor-mediated siGLO-TfNP uptake, is abrogated in the presence ofTfRAb (Supplementary Fig. S3C). Furthermore, the involve-ment of a TfR-based endocytosis mechanism in TfNP uptakewas demonstrated by incubating the cells at 4�C or 37�C whileadministering siGLO-TfNP treatment. Receptor-mediatedendocytosis is known to be affected by low temperature (41).The results showed a marked decrease, especially at 4 hours, incell uptake of siGLO-TfNP at 4�C compared with 37�C, sug-gesting that the predominant mechanism of cell uptake wasvia receptor-mediated endocytosis (Supplementary Fig. S3D).

Having made it clear that the TfNP internalization dependson TfR expression levels in the target cell, we wanted to verifywhether TfR expression levels contribute to TfNP's selectivitytoward lung cancer cells over normal lung fibroblast cells. Wefound that both A549 and HCC827 tumor cells expressedsignificantly higher levels of TfR than did normal MRC-9 cells(Fig. 1B; inset). As expected, the cell uptake response variedamong cancer cell lines and normal cells at 1-, 4-, and 24-hourtreatment, with siGLO-TfNP uptake corresponding to theirTfR expression levels (Fig. 1B). In A549 cells, the uptake ofsiGLO-TfNP gradually increased as measured by fluorescenceintensity (a.u.) from 40758.6 (1 hour) to 109446.6 (2 hours),and later to 268930.6 (24 hours; P < 0.01), compared withuntreated control. Although HCC827 cells also showed a simi-lar trend in cell uptake of siGLO-TfNP, the fluorescence inten-sity values at all time points were significantly less than A549cells. Importantly, MRC-9 cells showed negligible cell uptakeof siGLO-TfNP at all time points and was significantly lessthan the cancer cell lines studied (P < 0.05).

Efficiency of TfNP versus NP in siHuR delivery inTf-overexpressing lung cancer cells

As the transfection efficiency of siGLO-TfNP (targeted) overuntreated control was confirmed by cell uptake experiments, wereplaced siGLOwith siRNAagainstHuR to test it inA549 cells. TheHuR gene silencing effect induced by HuR-NP (nontargeted) andHuR-TfNP in A549 cells was compared by analyzing the HuR andassociated protein expressions and by cell growth inhibition. Asshown in Fig. 1C, growth inhibition induced by HuR-TfNP was24.5% comparedwith 18.1% forHuR-NP for a 24-hour treatmentperiod (P < 0.01), demonstrating the superior efficacy of HuR-TfNP over HuR-NP.

Western blot analysis showed a difference in HuR expres-sion between HuR-NP–treated (HuR/Actin ¼ 0.33) and HuR-TfNP–treated (HuR/Actin ¼ 0.19; P < 0.01) groups (Fig. 1D).This finding suggests that the TfNP facilitated enhanced andmore efficient knockdown of HuR compared with NP. Thedata also showed the effect of HuR knockdown in HuR-regulated proteins, such as BCl2, Cyclin D1, and P27. HuRstabilizes BCl2 mRNA, and thereby the BCl2 family of apo-

ptotic regulatory proteins, by binding to its 30 untranslatedregion (42). Knockdown of HuR deregulates BCl2, reducing itsexpression (Fig. 1D). Previous studies have shown that down-regulation of HuR resulted in reduced expression of cyclins,which are key cell-cycle proteins (12, 15, 43, 44).The reductionof HuR-regulated cyclin D1 as a consequence of HuR knock-down has been established by our studies (Fig. 1D). siRNA-mediated knockdown is reported to induce endogenous levelsof p27 protein, which is a cyclin-dependent kinase inhibitor(45, 46).Consistent with these reports, we observed that p27was upregulated when HuR was knocked down using eitherHuR-NP or HuR-TfNP, while the latter significantly increasedp27 expression (Fig. 1D). On the basis of the above results, weused TfNP as the targeted nanodelivery system for siHuR inlater experiments.

The possibility that the observed knockdown of HuR inHuR-NP- or HuR-TfNP treatment being nonspecific was elim-inated by testing a control scrambled siRNA and a siRNA usedfor initial HuR screening (HuRsi21). As shown in Supple-mentary Fig. S4, treatment of A549 cells with nanoparticlescontaining control siRNA (C-NP) or siRNA used for HuR screen(HuRsi21-NP) did not reduce HuR protein expression com-pared with untreated control cells. In contrast, treatment withHuRsi4 siRNA contained in nanoparticles greatly reducedHuR expression in A549 cells. These results show that HuRsi4siRNA–mediated inhibitory effect on HuR is specific and thatinclusion of any siRNA did not produce a similar inhibitoryactivity on HuR. On the basis of these results, we choseHuRsi4siRNA hereafter referred to as "HuR siRNA" and encap-sulated in TfNP (HuR-TfNP) for conducting the in vitro andin vivo studies and the results of which are described herein.

The target specificity of HuR-TfNP was confined toTf-expressing cancer cells

After achieving specific targeting and efficiency in HuR geneknockdown, we compared the efficiency of HuR-TfNP in A549and HCC827 lung cancer cells with varied levels of TfR expres-sion and MRC-9 lung fibroblasts with negligible TfR expres-sion. The downregulation of HuR and corresponding cellgrowth patterns were analyzed for 24 and 48 hours afterHuR-TfNP or C-TfNP treatments and compared with untreatedcontrols (Fig. 2A and B). At both 24 and 48 hours, HuR-TfNPsignificantly suppressed the expression of HuR mRNA (Sup-plementary Fig. S5), and protein (Fig. 2A; Supplementary Figs.S6 and S7) accompanied by cell growth inhibition (Fig. 2B; P <0.01) in A549 and HCC827 cells compared with C-TfNP. InHCC827 cells, an unexpected increase in HuR-mRNA expres-sion in C-TfNP–treated cells was observed at 48 hours aftertreatment compared with untreated control cells. The unex-pected increase in HuR-mRNA at the present time is not clearand needs further investigation. Nevertheless, the outcome ofneither the study nor the conclusions drawn is impacted.Interestingly, HuR-TfNP treatment resulted in negligible cellgrowth inhibition and minimal reduction in HuR and HuR-regulated target proteins in MRC-9 cells (Fig. 2A and B; Sup-plementary Fig. S8). This is probably due to low TfR expressionand inefficient HuR-TfNP uptake by MRC-9 cells.

Western blot analysis revealed the impact of HuR knock-down in HuR-associated markers, such as BCl2, Cyclin D1 andE, and p27 (Fig. 2A; Supplementary Figs. S6–S8). Reducedexpression of BCl2 and Cyclin D1 and E were observed in A549

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cells at 24 and 48 hours, consistent with HuR knockdown,whereas p27 levels were upregulated. The after-effect of HuRknockdown in HuR-associated markers was also consistentin HCC827 cells, although some observed variations in CyclinD1 and BCl2 expression at 24 hours may be due to cell-typeeffects (Fig. 2A). As with the poor HuR knockdown in MRC-9cells, HuR-regulated proteins were less affected at both timepoints.

Figure 2B shows the percentage difference in cell growth inhi-bition over untreated A549, HCC827, and MRC-9 cells upontreatment with HuR-TfNP or C-TfNP. At 24 hours, significantlygreater growth inhibition was observed in HuR-TfNP–treatedA549 cells (22.2% � 2.3%) than in C-TfNP–treated cells(�2.1% � 2.15%; P < 0.05). At 48 hours, this difference wasgreater (HuR-TfNP, 32.3% � 3.5%; C-TfNP, 2.4% � 1.2%; P <0.05), indicating increased cell growth inhibition over time. Asimilar trend was observed in HCC827 cells, in which the cellgrowth in the HuR-TfNP–treated group (20.7% � 3.7%) wassignificantly inhibited in comparison with the C-TfNP–treatedgroup (6.7% � 0.01%; P < 0.05) at 24 hours. At 48 hours, thisdifference was greater in HCC827 cells (HuR-TfNP, 26.3% �1.1%; C-TfNP, �1.7% � 1.5%). Conversely, no significant dif-

ference was observed between C-TfNP- and HuR-TfNP–treatedMRC-9 cells at either 24 or 48 hours, indicating less efficienttargetedHuR-TfNP–based cell growth inhibition. Hence, Tf-mod-ified nanoparticles carrying siHuR showed selectivity towardTfR-overexpressing lung cancer cells, leading to effective geneknockdown and cell growth inhibition.

HuR-TfNP induces G1 cell-cycle arrest and apoptosisin lung cancer cells

The progression of the cell cycle in A549, HCC827, andMRC-9 cells after treatment with either HuR-TfNP or C-TfNPfor 24 and 48 hours was evaluated. Compared with cellstransfected with C-TfNP, a large increase in the G1 populationwas observed in HuR-TfNP–treated A549 and HCC827 cells at24 and 48 hours, indicating a pronounced cell-cycle arrest atthe G1 phase (Fig. 2C; P < 0.05). Interestingly, no significant cellgrowth arrest at the G1 phase was observed in MRC-9 cellsupon HuR-TfNP treatment compared with C-TfNP treatment(Fig. 2C). Hence, the possible underlying mechanism ofHuR-TfNP–induced cell growth inhibition in lung cancer cellsoccurs via G1 cell-cycle arrest.

Actin

24 h

A549

A549 A549

HCC827

HCC827 HCC827

MRC-9

MRC-9 MRC-9

48 h 24 h 48 h 24 h 48 h

24 h 48 h 24 h 24 h48 h 48 h24 h

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)

48 h 24 h 48 h 24 h 48 h

Cyclin E

Cyclin D1

Bcl2

HuR

A

B C

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ntr

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C-T

fNP

C-T

fNP

Hu

R-T

fNP

Hu

R-T

fNP

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Hu

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p27

Figure 2.

HuR knockdown reduces HuR-target proteins in tumor cells but not normal cells. A, HuR and HuR-regulated protein expressions in A549, HCC827, and MRC-9 cellstreated with C-TfNP or HuR-TFNP at 24 and 48 hours after treatment. Among A549 and HCC 827 groups, HuR-TfNP treatment resulted in better knockdownof HuR protein expression in A549, whereas in MRC-9 when compared with both A549 and HCC827 cancer cells HuR-TfNP induced negligible HuR proteininhibition. HuR knockdown also altered the expression of downstream proteins like BCl2, cyclin D1, P27, and cyclin E in tumor cells but not in normal cells. B,Successful knockdownofHuR resulted in significant growth inhibition in lung cancer cells but not in normal cells, at both 24 and48 hours after treatment.C,HuR-TfNPinduces G1 phase arrest in A549 and HCC827 cells, but not in MRC-9 cells. � , P < 0.05; NS, not significant.






One question that arises is whether siRNA-targetedHuR knock-down induces only growth arrest or subsequently proceeds toapoptotic cell death. To answer this question, we examined forcaspase-9 and PARP by Western blotting, and for Annexin V–positive cells byflow cytometry, respectively. Amarked increase incleaved caspase-9 and PARP was observed in HuR-TfNP–treatedA549 cells compared with C-TfNP–treated and untreated controlcells (Supplementary Fig. S9A). Similarly, Annexin V assayshowed a significant increase in apoptotic cells in HuR-TfNP–

treated cells compared with C-TfNP-treated and untreated controlcells (Supplementary Fig. S9B; P < 0.001). Our results show thatHuR-TfNP treatment produces growth arrest that culminates inapoptotic cell death.

HuR-TfNP inhibits cancer cell migration and invasionAs we observed that the effectiveness of HuR-TfNP was more

prominent in A549 cells, we then evaluated the ability of HuR-TfNP to control migration and invasion in this cell line. Figure 3A

24 h

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Figure 3.

HuR-TfNP inhibits tumor cell migration andinvasion. Representative images of migration(A) and invasion assay (B) for A549 tumorcells are shown for C-TfNP and HuR-TfNPtreatment groups. Cells that did not receive anytreatment served as controls. Quantification ofmigration and invasion assays expressedby cell counting of respective assays is alsopresented. � , P < 0.05; �� , P < 0.001; NS, notsignificant.

Muralidharan et al.





shows themicroscopic images from the transwellmigration assay;the number of cells migrated per field is represented by bardiagram. The migration rate of A549 cells in HuR-TfNP was

significantly reduced compared with C-TfNP and untreated con-trol groups at 24 and 48 hours (P < 0.001). HuR-TfNP treatmentalso resulted in poor invasion of A549 cells, as evident from the

0 mins

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A549-SQICG-TfNP

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#2

10 mins 30 mins 5 h 24 h

Trans-fluorescence

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Color scaleMin = 3.31e7Max = 6.36e8

Radiance (photons)(p/sec/cm2/sr)

142546912

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uR

Figure 4.

In vivo biodistribution studies. A, IVIS Spectrum live images ofA549 (a) and HCC827 tumor bearing mice (b) obtained atdifferent time intervals after intravenous injection of indocyaningreen (ICG)-TfNP showed accumulation in the tumor by24 hours located on the lower right flank. Intravenousadministration of fluorescent HuR siRNA loaded nanoparticle(DY-647HuR-TfNP) showed nanoparticle (NP) accumulation inthe tumor by 24 hours (c). B, Graphical representation of siRNAaccumulation in tumor tissues ofA549 andHCC827micemodelsas measured by ddPCR at 24 hours after treatment. Sample # 1and # 2 represent tumor tissue from twomice of each treatmentgroup analyzed. C, Individual luminescent (A549 luc),fluorescent (Dy647HuR-TfNP), and overlaid images showinglocalization of DY647HuR siRNA in A549 lung tumors.� , P < 0.05; �� , P < 0.001.






microscopy images, compared with C-TfNP and untreated con-trols (Fig. 3B; P < 0.001). Significantly fewer cells invaded in theHuR-TfNP group, as shown in the bar diagram, at both 24 and48 hours (P < 0.001).

In vivo biodistribution and quantification ofsiRNA carrying TfNP

Having demonstrated the in vitro efficacy ofHuR-TfNP, our nextstep was to study the distribution behavior of TfNP in vivo. Thebiodistribution of TfNP was monitored in subcutaneous A549and HCC827 tumor models with ICG-TfNP. Figure 4A shows theIVIS spectrum live images from tumor-bearing mice that wereintravenously injected with ICG-TfNP and monitored for 24hours after injection. Ten minutes after ICG-TfNP injection, thenear infra-red signal was observed throughout the body in micewith both the tumormodels. Over time, the fluorescence intensitywas confined to the tumor site and the intensity of the signal wasdiminished throughout the body. Interestingly, the fluorescentsignal persisted in the tumors even 24 hours after injection.Intravenous injection of free ICG did not show preferential tumoraccumulation at any time points (Supplementary Fig. S10).Fluorescent signals obtained over 5 hours in tumor-bearing micesuggest the rapid clearance of free ICG. However, intravenousinjection of ICG-TfNP into healthy non-tumor–bearing miceshowed a prolonged signal for up to 24 hours, and the accumu-lation was mostly confined to the abdominal region, wherereticuloendothelial organs are located. This finding indicates thatTfNP could delay the process of ICG clearance in the circulationand, if no target tissues with high TfR expression, such as TfRexpressing cancer cells are present, ICG-TfNP undergoes normalbioclearance from the body.

Furthermore, we investigated the distribution of fluores-cently labeled siRNA upon in vivo administration in micebearing HCC827 tumors (Fig. 4A, c). Intravenous injection ofDy647HuR-TfNP into HCC827 subcutaneous tumor-bearinganimals demonstrated accumulation and localization of theparticles in the tumor site over time with maximum accu-mulation as evidenced by highest fluorescence signal occur-ring at 24 hours after injection.

Next, we determined the percent intake of siRNA by thetumor after intravenous administration of HuR-TfNP in micebearing subcutaneous A549 and HCC827 tumors by TaqManassay using ddPCR. HuR siRNA was detected in tumor tissuesharvested from the HuR-TfNP–treated animals, while tumortissues from C-TfNP–treated animals produced negligible sig-nals for control siRNA, confirming the specificity of siHuRdetection. Notably, individual tumor samples accumulated1.98% and 0.23% of administrated doses of siHuR, respective-ly, in A549, and 1.02% and 0.90%, respectively, in HCC827(Fig. 4B; P < 0.0001). Although siRNA levels detected amongthe HuR-TfNP–treated samples varied, nevertheless, the levelswere significantly higher compared with C-TfNP. Hence, it isevident that HuR-TfNP was capable of successfully deliveringits siRNA payload to the target tissue in vivo.

Our next step was to understand the fate of TfNP in a mousemodel of metastatic lung tumors. ICG-TfNP was administeredintravenously into mice bearing A549-luc tumors, and we mon-itored the accumulation at tumormetastatic site. Figure 4C showsthe overlaid image of luciferase-expressingmouse lungmetastasesand ICG (red fluorescence), suggesting the accumulation ofICG-TfNP in A549 lung-metastases over time. These results show

that our TfNP targets both subcutaneous and metastatic lungtumors when administered intravenously and thus HuR-TfNPcould be a therapeutic for lung cancer treatment.

Efficacy of HuR-TfNP in mouse lung tumor xenograftsAlthough intravenous administration of HuR-TfNP resulted

in its accumulation at the tumor site, we wanted to examine theefficacy of our formulation in subcutaneous lung tumor modelsupon direct intratumoral administration. Figure 5A and B showthe tumor growth patterns in animals bearing subcutaneousA549 and HCC827 tumors for a period of 25 days from theday of first treatment with either C-TfNP or HuR-TfNP. Micereceiving C-TfNP treatments showed uncontrolled tumorgrowth compared with the significantly reduced tumor volumesobserved in HuR-TfNP–treated mice bearing A549 tumors(Fig. 5A). The HuR-TfNP–treated tumors, which were harvestedafter measurement on the 25th day, showed a clear reduction(56% inhibition) in tumor volumes (153.7 mm3; P < 0.05) overC-TfNP–treated tumors (351.6 mm3). Western blotting andIHC studies showed reduction in the HuR expression thatcorrelated with reduced tumor volume in HuR-TfNP–treatedtumors compared with C-TfNP–treated tumors (Fig. 5A).

Likewise, the reductions in HCC827 tumor volumes were alsohighly pronounced upon HuR-TfNP treatment. The averagetumor volumes at the termination of the study were 548.0 mm3

for C-TfNP–treated tumors versus 195.1 mm3 (64% inhibition;P < 0.05) for HuR-TfNP–treated tumors (Fig. 5B). Tumor growthwas clearly inhibited upon intratumoral treatment, indicatingthe efficacy of HuR-TfNP. Moreover, the HuR protein expressionpattern in the isolated A549 (Fig. 5A) and HCC827 (Fig. 5B)tumor tissues frommice treatedwithHuR-TfNP showed amarkedreduction after two treatments compared with mice treated withC-TfNP. This finding suggests that the antitumor activity wascaused by HuR knockdown in vivo.

As it can be argued that intratumoral treatments are notclinically relevant for lung cancer treatment, we next assessedthe delivery efficiency and the therapeutic effect of targetedHuR-TfNP upon intravenous administration in mice bearingA549-luc SQ tumors. As shown in Fig. 5C, tumor growth wassignificantly inhibited upon HuR-TfNP treatment (P < 0.05).The difference in tumor volumes in the C-TfNP and HuR-TfNPtreatment groups was observed as early as the seventh day ofmeasurement (73% inhibition). The tumors in the C-TfNP–treated group grew uncontrollably, while a significant slowergrowth pattern was observed in the HuR-TfNP–treated group.On day 35, the tumor volume in the C-TfNP–treated groupreached 600 mm3, and all of the mice in that group wereeuthanized and the tumors were isolated and displayed asshown in Fig. 5D. The tumor volume in the HuR-TfNP–treatedgroup was significantly less and exhibited 75% growth inhibi-tion compared with C-TfNP treatment on day 35. Also shownare representative mice from each treatment group from whichthe tumors are harvested. Interestingly, we observed that thetumor progression in the HuR-TfNP–treated group was signif-icantly delayed and the tumor volume equivalent to C-TfNP–treated group was not reached till day 91 at which time thestudy was terminated (Fig. 5D). The tumor volume in HuR-TfNP–treated group on day 91 was only 315.4 mm3.

In parallel to measuring tumor volume by calipers, the efficacyof HuR-TfNP treatment was also assessed by bioluminescenceimaging at select time points (day 1, 15, and 33) in the

Muralidharan et al.





subcutaneous A549-luc tumor-bearing mice and compared withC-TfNP treatment (Supplementary Fig. S11). The average radianceemitted from tumors in each group were averaged and plottedagainst the days of measurement. A significant reduction in theaverage radiancewas observed inHuR-TfNP–treatedmice on days15 (2.6 � 104 p/sec/cm2/sr) and 33 (1.14 � 104 p/sec/cm2/sr)compared with the radiance in C-TfNP–treated mice on day 15(1.89 � 105 p/sec/cm2/sr) and day 33 (3.72 � 105 p/sec/cm2/sr;Supplementary Fig. S11; P < 0.05). Bioluminescent imaging wasdiscontinued after day 33 as the control animals were euthanizedon day 35 (Fig. 5C).

To further investigate molecular changes indicative of HuR-TfNP–mediated antitumor efficacy, tumors harvested at the endofthe study were subjected to molecular analysis. Western blottingand RT-PCR analysis of the tumor samples revealed a reduction inthe protein and mRNA expression of HuR and HuR-regulatedBCL2, whereas P27 showed an upregulation in its expression inHuR-TfNP–treated tumors compared with C-TfNP–treatedtumors (Fig. 5D; Supplementary Fig. S12; P < 0.05). Furthermore,HuR-TfNP–treated tumors showed increased activation of cas-pase-9 and PARP compared with C-TfNP–treated tumors (Sup-plementary Fig. S12).

A similar study conducted with mice bearing subcutaneousHCC827 tumors demonstrated the efficiency of intravenouslyadministered HuR-TfNP in targeted antitumor activity, mediatedby siHuR gene knockdown. The intravenous administration ofHuR-TfNP resulted in sluggish tumor growth compared with theactive growth observed in C-TfNP–treated tumors (Fig. 5E). Thetumor volumes were 522.5mm3 for theHuR-TfNP–treated groupand 958.9 mm3 for the C-TfNP–treated group (45% inhibition;P < 0.05) on the 37th day of measurement. Because of enhancedtumor burden in the control groups, we discontinued the studyper the IACUC recommended guidelines. Compared with A549tumors, the tumor inhibition in HCC827 tumors was slow,possibly because of the aggressive nature of HCC827 growth andrelatively low Tf receptor levels. Nevertheless, the HCC827 tumorgrowth was significantly inhibited with HuR-TfNP treatment,compared with the large tumors observed in the C-TfNP–treatedgroup (Fig. 5F). The Western blot image shows the HuR knock-down obtained in representative HuR-TfNP–treated tumors com-pared with C-TfNP–treated tumors (Fig. 5F).

IHC analysis of HuR revealed that the HuR expression wasreduced in the subcutaneous A549 and HCC827 tumors fromanimals that received HuR-TfNP treatment (Supplementary Figs.

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HuR-TfNP treatment suppresses lung tumor growth inhibition in vivo. A549 (A) and HCC827 (B) subcutaneous tumor-bearing mice were treated intratumorallywith C-TfNP or HuR-TfNP for 6 doses over two weeks of time period and tumor growth measured. Inset shows HuR expression in tumors after two treatments.Arrows denote treatments. In vivo tumor growth inhibition profile, representative tumors harvested from mice sacrificed on final day of experiment, IHC, andhematoxylin and eosin–stained tumor tissue sections from sacrificed mice on final day of the experiment are shown. C, Mice bearing subcutaneous A549-luctumors were treated intravenously with C-TfNP or HuR-TfNP for 6 doses over two weeks of time period and tumor growth measured by calipers and bybioluminescent imaging. A significant inhibition in tumor growth was observed in HuR-TfNP–treated mice compared with C-TfNP treatment. D, Representativegross tumor size harvested fromC-TfNP- andHuR-TfNP–treatedA549mice sacrificed on final dayof experiment, andWestern blotting for HuR in two tumor samples(# 1 and # 2) representative of each treatment group analyzed after two treatments is shown. E, Mice bearing subcutaneous HCC287 tumors were treatedintravenously with -TfNP or HuR-TfNP for 6 doses over two weeks of time period. A significant inhibition in tumor growth was observed in HuR-TfNP–treatedmice comparedwith C-TfNP treatment. F, Representative tumors harvested from C-TfNP and HuR-TfNP–treated HCC827mice sacrificed on final day of experiment,and Western blotting for HuR in tumors harvested from treated mice are shown. Arrows denote treatments. �, P < 0.05; �� , P < 0.001.






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S13 and S14). These results are consistent with Western blottingand RT-PCR results from the same tumor samples (Fig. 5A and B).To determine the mechanism involved in the efficacy of HuR-TfNP treatment, the harvested tumors were analyzed for Ki67 andCD31 expression. Ki67 IHC can be used to assess the proliferativeindex of the tumor (47). In this study, Ki67-positive cells weresignificantly higher in C-TfNP–treated tumors (A549 andHCC827) than in the HuR-TfNP–treated tumors (SupplementaryFigs. S13 and S14). These observations suggest that HuR expres-sion is closely associated with cellular proliferation. It confirmsour earlier data that inhibition of HuR expression abrogates thetumor proliferation rate. A rapidly growing tumor often expressesCD31, a microvessel density (MVD) marker, which is detectableby IHC (29, 48). We detected significant decrease in CD31-positive microvessels in tissue sections from HuR-TfNP–treatedtumors compared with C-TfNP–treated tumors in both A549 andHCC827 mice models (Supplementary Figs. S13 and S14). Astumor vasculature is characterized by increased number of tumorvessels, theCD31 IHCdata suggests less active vasculature inHuR-TfNP–treated groups, specifying the neovascularization inhibito-ry activity of HuR-TfNP in A549 and HCC827 tumors. Finally, asignificant increase in apoptotic cell death as determined byTUNEL staining was also observed in HuR-TfNP–treated A549tumors that were collected at the end of the study (SupplementaryFig. S13; P < 0.05). Although, we did not perform TUNEL stainingin HCC827 tumor tissues, we speculate that increased apoptoticcell death also occurs in HuR-TfNP–treated tumors resulting indelayed tumor growth.

Systemic delivery of HuR-TfNP inhibits experimental lungmetastasis in vivo

To investigate the antitumor activity of HuR-TfNP on lungmetastasis, we monitored the tumor growth in mice bearingA549-luc lung metastasis following HuR-TfNP treatment. Asshown in Fig. 6A, the decay of bioluminescence signals fromHuR-TfNP–treated A549-luc lung metastases were distinctly vis-ible, especially at the fourth and sixth weeks, indicating a signif-icantly reduced tumor burden compared with C-TfNP–treatedgroups (93% inhibition at week 6; P < 0.05). The average radianceof the HuR-TfNP–treated groups was reduced or unchanged,compared with the enhanced radiance observed in theC-TfNP–treated groups. Analysis of sixth week images showedan average radiance of 41,007.7 (p/sec/cm2/sr) for C-TfNP, whichis a 13-fold increase over HuR-TfNP–treated tumor signals of3,025 (p/sec/cm2/sr; P < 0.0001). As expected, there was lessHuR protein expression in the excised lung tissue from the HuR-TfNP–treated group than in lung tissue from the C-TfNP–treatedgroup (Fig. 6B). As shown in Fig. 6B, the metastatic nodules inHuR-TfNP–treated lungs (9.57; 88% inhibition) were significant-ly reduced, whereas tumor nodules occupied a major portion of

the C-TfNP–treated lungs (77.7; P < 0.0001). IHC analysis of thelung lobes revealed a significant, up to3-fold reduction in theHuRprotein levels in HuR-TfNP–treated groups compared with C-TfNP–treated groups. (Fig. 6C; P < 0.0001).

Correspondingly, there was a 2-fold reduction in microvesseldensity (MVD; indicated by CD31 expression) in sections oflung lobes from HuR-TfNP–treated animals (P < 0.0001).Similarly, Ki-67–positive cells were reduced 3-fold in HuR-TfNP–treated groups, indicating significantly less proliferationcompared with the C-TfNP–treated group (P < 0.0001). Therewere significantly fewer nodules in HuR-TfNP–treated tumorsthan in C-TfNP–treated tumors, as observed in the H&E-stainedsections (Fig. 6C). Thus, systemic delivery of HuR siRNA usingTfNP effectively suppressed HuR expression, and significantlyreduced MVD and cell proliferation resulting in inhibition ofexperimental metastasis.

DiscussionAlthough the potential of RNAi-based gene therapy has been

realized over the last decade, the absence of a reliable targeteddelivery system limits their efficient use in cancer therapy.Recently, the RNA binding protein HuR overexpressed inNSCLC has been recognized as a potential molecular targetfor RNAi therapy (11, 12, 14–16). As many of the NSCLCtumors are characterized by the overexpression of TfR (4, 5),our goal was to explore the efficacy of a rationally designedliposomal nanocarrier system targeting TfR and specificallydeliver HuR siRNA into the tumor cells. Our Tf-NP nanofor-mulation also helped in understanding the function, fate,biological action, and therapeutic effect of HuR siRNA specif-ically delivered in tumor site.

In this study, the postinsertion technique was employed formodifying HuR-NPwith Tf ligand (26).We employed sulfhydryl-reactive crosslinking chemistry to synthesize Tf-PEG-DSPE fornanoparticle modification purposes. The target specific celluptake studies revealed that it is the level of receptor expressionwhich influences the cellular uptake of receptor-targeted nano-particles (49), and thereby the delivery of its siRNA payload (50).Increased siRNA delivery by HuR-TfNP in TfR-overexpressingcancer cells resulted in the efficient knockdown of HuR andaltered the expression levels of its associated proteins (12, 15,42–46). Also, suppression of HuR using siRNA induced cell-cyclearrest in the G1 phase (46). Multistep processes, such as celladhesion, migration, and invasion, are involved in cancer metas-tasis (51), and HuR is known to regulate metastasis-relatedmRNAs (7, 52).In this study, the in vitro anticancer efficacy ofHuR-TfNP was demonstrated by growth inhibition, induction ofG1 cell-cycle arrest leading to apoptosis, and reduced migrationand invasion ability of lung cancer cells. That the observed

Figure 6.HuR-TfNP treatment suppresses experimental lungmetastasis. Mice bearingA549-luc lung tumorswere treated intravenouslywith C-TfNP or HuR-TfNP for a total ofsix treatments. Mice were imaged at regular intervals to monitor and measure tumor growth. A, Representative bioluminescence images showing inhibition ofexperimental lung metastasis in HuR-TfNP–treated mice compared with C-TfNP–treated mice. Bar graph represents the quantitation of bioluminescenceintensity as average radiance [p/s/cm2/sr]. The gradual decrease in the bioluminescence signals in HuR-TfNP compared with C-TfNP groups suggests significantreduction in tumor growth over the period of treatment. B, Western blot image shows reduced HuR expression in HuR-TfNP–treated A549 lung tumors (top).Sample # 1 and # 2 are representative samples for each treatment group analyzed at end of the study. Lung tumor nodules of a representative animal fromeach treatment group are also shown (middle panel). Animals were euthanized after six weeks; lungs were inflated with bovine solution and the number ofextrapulmonary tumor nodules counted (bottom). A significant reduction in the number of lung nodules were observed in HuR-TfNP–treated animals comparedwith control. C, Images represent the IHC staining of lung sections for HuR, CD31, and Ki67. Hematoxylin and eosin (H&E) stain showing the tissue pathologywith dashed circle marking tumors in the lung. Bar graph represents the quantitation of IHC stains. �, P < 0.05; �� , P < 0.001.






antitumor activitywas specific toHuR silencingwasdemonstratedby comparing HuR-specific siRNA (HuRsi4) and comparing withcontrol siRNA and a siRNA (HuRsi21) that was initially used forscreening HuR-inhibitory effects (Supplementary Fig. S4). Fur-thermore, it is known that the therapeutic HuR siRNA, if notspecifically targeted, may cause deleterious effects in normal cellstoo. However, HuR-TfNP caused no significant effect on normallung tumor fibroblasts, demonstrating safe and selective activityagainst lung cancer cells.

In vivo biodistribution study demonstrated preferential accu-mulation of Dy647HuR-TfNP in tumor milieu and this resultcorrelated with ICG-TfNP distribution in tumors. As neurons andbrain capillary endothelial cells in human andmice are known toexpress TfR in significant levels, one may expect a high accumu-lation of TfNP in mice brain tissue (53, 54). However, theaccumulation of ICG-TfNP in the brain was minimal and notprolonged as evident in the biodistribution images obtained (Fig.4; Supplementary Fig. S10). The fluorescence from ICG-TfNPfaded away frommice head and neck area in less than 30minutesafter injection, suggesting that the specific accumulation of TfNPin brain area is nonsignificant. One possibility for negligible TfNPaccumulation in the brain is that the TfR expression level in themice brain is relatively low compared with TfR expression in thetumor resulting in ICG-TfNP fluorescence level in mice brainfalling below the detection level of the instrument (Fig. 4A and B).This observation in our study was consistent with a previousreport indicating the TfR-targeted nanoparticle accumulation inbrain was the lowest amongmajor organs studied, in comparisonwith the tumor tissues (55). Another possibility is that the size ofthe HuR-TfNP (>200 nm) are large thereby limiting the particlesfrom crossing the blood–brain barrier (BBB) and enter the braintissue. As TfR targeting is one of the successful strategies employedto deliver nanoparticle drug carriers across BBB (56–58), the brainaccumulation potential of TfNP, if any, will be investigated infuture studies.

Systemic delivery of HuR-TfNP showed that it can effectivelyinhibit the growth of solid tumors of lung origin. The tumorinhibition in subcutaneous models was specific to HuR siRNAtreatment, as we accurately detected the presence ofHuR siRNA intumor tissues treated with HuR-TfNP in comparison with C-TfNPtreatments by ddPCR technique. The effect of HuR-TfNP treat-ment in subcutaneous lung tumors via systemic route adminis-trationwas significant, as tumor volume inhibitionwas correlatedwith HuR knockdown. The proliferative nature and active vascu-lature formation were significantly less in HuR-TfNP–treatedtumors as analyzed by IHC staining. The mechanism by whichHuR-TfNP reduces tumor vasculature is not known and is beyondof the scope of this study. One possibility is the direct inhibitoryactivity of HuR-TfNP on VEGF as a consequence of HuR knock-down. VEGF is amolecular target of HuR (7)However, additionalmechanisms for suppressing tumor vasculature may exist thatneed to be explored.

Metastatic spread of cancer cells from the primary tumors tothe distant organs is a major reason for high mortality amonglung cancer patients (59).To this end, the antimetastaticeffect of HuR-TfNP was assessed in a lung metastatic tumormodel in mice. Imaging studies revealed preferential accumu-lation of TfNP in lung metastatic tumor site. Furthermore, HuR-TfNP treatment resulted in efficacious targeted sequence-spe-cific knockdown of HuR and in triggering molecular events thatsuccessfully inhibited lung cancer metastasis that represents the

clinical relevance of this study. While in this study, we have notinvestigated the contribution of HuR-TfNP on the chemokineCXCR4 receptor, studies from our laboratory and othershave previously shown inhibition of HuR suppresses CXCR4(12, 60). Thus, it is possible that the observed inhibition ofexperimental metastasis could partly be due to CXCR4 inhibi-tion. Should HuR-TfNP treatment contribute to suppression ofCXCR4 in vivo and concur with our previous in vitro reports,then combining HuR-TfNP with CXCR4 inhibitors wouldbecome a viable therapy for lung cancer (12). Similarly, con-ducting combinatorial therapy studies in the future that incor-porate chemotherapy or radiotherapy with HuR-TfNP will be ofclinical relevance for tackling metastatic disease. Nevertheless,our results convincingly demonstrate that systemic delivery ofHuR-TfNP effectively suppresses lung metastasis.

It is important to realize that our studies were conducted intumor models using immunodeficient mice. It will be of interestto test the efficacy of HuR-TfNP in a tumor model setting thatutilizes immunocompetent host to determine the effects of HuRinhibition on host–immune system as HuR has previously beenreported to play a role in inflammation (61, 62). Furthermore,recent reports on the role of PDL1-PD1 in suppressing hostimmune T cells beg the question whether HuR-TfNP treatmentwill impact the PDL1-PD1 signaling and alter the host immunesystem to enhance the antitumor immune activity. These ques-tions while of interest are beyond of the scope of this study butwarrant investigation in the future (63, 64). In conclusion, wehave demonstrated HuR-TfNP treatment effectively inhibits lungcancer growth both in vitro and in vivo.

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

Authors' ContributionsConception and design: R. Muralidharan, A. Babu, N. Amreddy, A. Srivastava,U.B. Kompella, A. Munshi, R. RameshDevelopment of methodology: R. Muralidharan, A. Babu, N. Amreddy,A. Srivastava, R. RameshAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Muralidharan, A. Babu, N. Amreddy, A. SrivastavaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R.Muralidharan, A. Babu, N. Amreddy, A. Srivastava,A. Chen, Y.D. Zhao, U.B. Kompella, A. Munshi, R. RameshWriting, review, and/or revision of themanuscript: R. Muralidharan, A. Babu,N. Amreddy, A. Srivastava, Y.D. Zhao, U.B. Kompella, A. Munshi, R. RameshAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R. Muralidharan, N. Amreddy, R. RameshStudy supervision: R. Ramesh

AcknowledgmentsWe thank the Stephenson Cancer Center at the University of Oklahoma

Health Sciences Center for the use of the Functional Genomics Core,Biostatistics Core, and Small Animal Bioluminescent Imaging Core facilities.Assistance received from the institutional molecular imaging core facility forconducting flow cytometry studies is appreciated. The authors thank Ms.Kathy Kyler at the office of Vice President of Research, OUHSC, for editorialassistance.

Grant SupportThe study was supported in part by a grant received from the NIH R01

CA167516 (to R. Ramesh), an Institutional Development Award (IDeA) fromthe National Institute of General Medical Sciences (P20 GM103639; toA. Munshi and R. Ramesh) of the NIH, and by funds received from thePresbyterian Health Foundation Seed Grant (C5094701 and C5095801; to

Muralidharan et al.





R. Ramesh), Presbyterian Health Foundation Bridge Grant (C5098101; toR. Ramesh), Stephenson Cancer Center Seed Grant (to R. Ramesh), and Jimand Christy Everest Endowed Chair in Cancer Developmental Therapeutics(to R. Ramesh), The University of Oklahoma Health Sciences Center.

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 February 9, 2017; revised May 8, 2017; accepted May 19, 2017;published OnlineFirst June 1, 2017.

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Muralidharan et al.




2017;16:1470-1486. Published OnlineFirst June 1, 2017.Mol Cancer Ther Ranganayaki Muralidharan, Anish Babu, Narsireddy Amreddy, et al. Activity of the RNA-binding Protein HuR

By Disrupting the OncogenicIn Vivo and In VitroTumor Growth Tumor-targeted Nanoparticle Delivery of HuR siRNA Inhibits Lung

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