Cancer Stem Cells Using RSPO-ConjugatedDoxorubicin LiposomesJing Cao1, Chong Li1, Xiaohui Wei1, Meiqing Tu1, Yan Zhang1, Fengwei Xu1,and Yuhong Xu1,2
Abstract
Cancer stem cells (CSC) that may account for only a smallfraction of tumor mass were found to play crucial roles duringtumor initiating, progression, and metastasis. However, theyare usually difficult to be treated and notoriously resilient todrug eradication. In this study, we aimed at the Wnt signalingcharacteristic of CSCs and designed a liposomal drug deliverysystem to target CSCs. Liposomes decorated with RSPO1on the surface were constructed for specific interactions withthe Wnt pathway coreceptor LGR5. Doxorubicin carried by theRSPO1-liposomes was more effective at lower concentrationsthan the same drug loaded in PEG-liposomes. More impor-
tantly, we showed using a patient-derived xenograft tumormodel where LGR5þ CSCs coexisted with LGR5– cells, theRSPO1-liposomes were able to access more CSC cells anddeliver the drug specifically and efficiently. Such a focusedeffect in eradicating LGR5þ cells led to massive tumor tissuenecrosis and growth inhibition even when only a fraction ofthe conventional drug dose was used. These data clearlydemonstrated the advantages of CSC-targeted drug deliveryand would support the development of such approaches as anew cancer treatment strategy. Mol Cancer Ther; 17(7); 1475–85.�2018 AACR.
IntroductionTargeted drug therapies to specific cancer-related signal trans-
duction pathways have been quite successful in clinical treat-ment. But there are also growing evidences suggesting thecritical roles of cancer stem cells (CSC) resulting in drugresistances and escape mutations (1–5). CSCs were initiallydefined based on the observation that a small fraction of drug-treated cells could escape growth arrest and result in regrowth ofthe entire tumor cell population. Although serial transplanta-tion experiments in rodent models represent the gold standardfor defining CSCs, various surface markers including PROM1(CD133), CD44, and CD24 are also commonly used. It wasonly recently that more mechanistic studies were reported thatidentified key players in various development pathways aspotential CSC targets to curb cancer progression and metastasis(6–7).
The leucine-rich repeat-containing G protein–coupled receptor5 (LGR5) was found to be an important coreceptor of the Wnt/b-catenin signaling pathway (8–12) and overexpressed in manytypes of tumors, including hepatocellular, colorectal, ovarian,basal cell, and breast cancer (13–17). Several studies used LGR5expression to characterize CSCs and demonstrated their tumor-initiating properties (18–19). Most recently, Shimokawa and
colleagues demonstrated that selective ablation of LGR5þ CSCscould lead to tumor regression (20). Junttila and colleaguesreported the development of a LGR5 antibody–drug conjugate(ADC) for targeted therapy toward CSCs with potent antitumorefficacy (21).
Compared with the ADC format of target drug delivery, wereasoned that a nanoparticle system may be able to load moredrug and enable better drug accumulate in the tumor tissue basedon the EPR effect. Therefore, in this study, we prepared nanolipo-somes encapsulating doxorubicin (Dox) and containing surfaceconjugation of the LGR5 natural ligand RSPO1. We used both aLGR5þ cancer cell model and a patient-derived xenograft (PDX)model containing only some LGR5þ CSCs and demonstratedimproved drug delivery and efficacy by the RSPO1-liposomes.Most importantly, we showed that dramatic antitumor activitiescan be achieved by focusing and eradicating only the CSCs using avery small drug dose. Such CSC-targeted treatment strategy maybehighly efficient and should be included in various combinationtherapy for cancer.
Materials and MethodsCell lines and PDX samples
A549, Caco2, and TCF/LEF reporter-HEK293 cells were grownin DMEME supplemented with 10% FBS. HCT116, AsPC1, andRAW264.7 cells were grown in RPMI 1640 medium supplemen-ted with 10% FBS. LoVo cells were cultured in DMEM/F12medium supplemented with 20% FBS. The TCF/LEF reporter-HEK293 cell line was provided by Curegenix Inc. Other cell lineswere originally fromTypeCulture Collection of Chinese Academyof Sciences. All cell lines were authenticated by short tandemrepeat analysis at the time of receipt and determined to bemycoplasma free (MycoProbe, R&D). Cells were used within
1Shanghai Jiaotong University, School of Pharmacy, Shanghai, China. 2College ofPharmacy and Chemistry, Dali University, Dali, China.
15 passages of their initial freeze down. The PDX model waspurchased from Shanghai LIDE Biotech. The tumor tissues wereimplanted into the subcutaneous flank of nude mice to generateheterotopic PDX model.
RSPO1-PEG-DSPE synthesisRSPO1 protein (StemRD) was incubated with 50-fold
molar excess of tris(2-carboxyethyl) phosphine for 1 hourat room temperature to maintain the free cysteine residuein reduced form. 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[maleimide (polyethylene glycol)�2000] (DSPE-PEG2000-Mal; Pharmaron) was dissolved in 30 mmol/LHEPES buffer (pH 6.5) saturated with nitrogen. The reducedprotein was immediately added to DSPE-PEG2000-Malmicelle solution at 1:40 to 1:60 molar ratio while maintainingmixing under nitrogen at 10�C overnight. Protein conjugationto the lipid was confirmed by gel electrophoresis. Sampleswere loaded in a 10% SDS-PAGE gel under reducing condi-tions and stained with Coomassie brilliant blue G250. Quanti-fication of lipid-conjugated protein molecules was done bydetermination of gray level intensity of protein bands on thegels using ImageJ software. Known concentrations of protein(1 mg) were used for calibration.
In vitro characterization of RSPO1 binding and activitiesBiological activity of RSPO1 protein was detected using
the TCF/LEF-Luciferase reporter gene assay. TCF cells cultured in96-well plates were treated with RSPO1/RSPO1-PEG-DSPE andincubated in a humidified incubator at 37�C for 18 hours.Luciferase assay was performed using the Bright-Glo LuciferaseAssay Kit (Promega) according to themanufacturer's instructions.
Preparation and characterization of RSPO1-conjugatedliposomes
The lipid composition of empty liposomes was Egg phos-phatidylcholine (EPC, NOF) and Cholesterol (Avanti PolarLipids) at the molar ratio of 2:1. The fluorescence-labeled lipo-somes with the same formulation of empty liposomes weremade by incorporating 1% (molar percentage) of eitherN-(fluorescein-5-thiocarbamo-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (fluorescein-DHPE; Invitrogen) or DiI(Sigma). Drug-loaded liposomes were composed of hydrogenat-ed soybean phosphatidylcholine (HSPC, NOF), cholesterol, and1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000, NOF) at themolar ratio of 55:40:2. The lipid concentration was about20 mg/mL. Dox was loaded into the liposomes using the ammo-nium sulfate gradient method. The loaded drug concentrationwas 2 mg/mL for Dox HCl. A postinsertion method was used todecorate liposomes with RSPO1-PEG2000-DSPE. The liposomeswere added to the RSPO1-PEG-DSPE or PEG-DSPE micellesolution and stirred at 37�C for 24 hours. The liposomal sizedistribution and zeta potential were measured by ZetaSizer3000HSA (Malven). Liposome morphology was confirmed byJEM-2010HT transmission electron microscopy and cryogenictransmission electron microscopy (FEI).
Western blot analysis of LGR5 expressionThe LGR5 expression levels of various cell lines were deter-
mined by Western blotting. Cells (A549, Caco2, HCT116,AsPC1, and LoVo) were washed with ice-cold PBS (pH 7.4)
and lysed with ice-cold RIPA Lysis Buffer (Thermo Scientific)supplemented with protease inhibitors at 4�C for 1 hour.Samples were centrifuged at 12,000 g for 10 minutes at 4�C.Equal amount of proteins was subjected to SDS-PAGE andtransferred to a nitrocellulose (NC) membrane (Applygen). TheNC membrane was blocked with blocking buffer (5% skimmilk in TBST buffer) and incubated with primary antibodyagainst lgr5 (ab75850; Abcam) followed by IRDye 800CWDonkey Anti-Rabbit IgG (LI-COR). The blotting signals weredetected using Odyssey scanner (LI-COR).
FACS analysisLoVo and RAW264.7 cells were seeded in 24-well cell culture
plates at 5� 105 cells per well and allowed to grow for 48 hours.They were then incubated with serum-free medium containingeither FITC-labeled RSPO1-PEG-liposomes or FITC-labeledPEG-liposome at lipid concentration of 200 mmol/L for 1 hourat 37�C. After an ice-cold PBS wash, cells were collected byadding 0.25% trypsin-EDTA followed by suspension in 500 mLPBS. They were analyzed using a flow cytometer (BD FACSCaliber). For the analysis, the signals were gated to exclude celldebris and dead cells and collected for 10,000 cells counts.Liposomal uptake was calculated by dividing the mean log ofFITC fluorescence from liposome-bound viable cells by themean log of FITC fluorescence of control cells.
For the analysis of LGR5þ cells in PDX007 tumor, the explantedtumor was digested for 2 hours in the mixture of 2 U/mL ofdispase (Yisen Bio) and 2,000 U/mL of collagenase VI (Yisen Bio)at 37�C with vigorous shaking. The resulted cell suspension wasfiltered (40mmpore size) andwashed in PBS. Cells (2� 106)werecollected in 300 mL of PBS containing diluted primary antibodyLGR5 (1:40) and incubated on ice for 30 minutes. The cells wererinsed and labeled with diluted fluorescent dye–conjugatedsecondary antibody (1:100; CWBio) for 30 minutes on ice. Flowcytometric analysis was performed using a Beckman CoulterMoFloXDP Sorter.
Confocal analysisLoVo cells were seeded on 16 mm circular cover glasses placed
in 12-well cell culture plate and allowed to grow for 48 hours untilreaching 60% confluence. The regular culture medium wasremoved and replaced with serum-free medium containing theDiI-labeled liposomes at 100 mmol/L lipid concentration. After15, 30, and 60 minutes of incubation at 37�C or 4�C with orwithout free RSPO1 protein, cells were washed with ice-cold PBSand fixed with 4% paraformaldehyde for 10 minutes at roomtemperature. The cover slips were washed thoroughly with PBSandmounted cell-side down onmicroscope slides with Anti-fadeMounting Medium (Vector Laboratories). The slides wereobserved using a Leica SP2 confocal microscope (LeicaMicrosystems).
Cell viability assayLoVo cells were seeded in 96-well cell culture plates at 2 � 104
cells per well and allowed to grow for 48 hours. The cellswere incubated with lipo-Dox, PEG-lipo-Dox, or RSPO1-PEG-lipo-Dox at Dox concentration of 7.5 to 30 mg/mL for 4 hours,the medium was removed, and the cells were incubated foradditional 24 and 48 hours in fresh complete medium. Afterincubation, cell viability assay was performed using a Cell Count-ing Kit-8 (Sigma) according to the manufacturer's protocol.
Cao et al.
Mol Cancer Ther; 17(7) July 2018 Molecular Cancer Therapeutics1476
Tumor tissue staining and analysisFor immunohistology analysis of the tumor tissue, explanted
tumors were fixed with 4% paraformaldehyde and embedded inparaffin blocks. Note that 5-mm-thick sections were cut andmounted on slides. Sections were blocked with 1% BSA (SangonBiotech) and incubated overnight at 4�C with primary antibodyagainst LGR5 (ab75850; Abcam) after dewaxing and rehydrationand horseradish peroxidase–conjugated secondary antibody at37�C for 1 hour. Hematoxylin and eosin staining was done bysoaking the tissue sections on slides in hematoxylin solution for10 minutes, washing in running water for 5 minutes, rinsing in95% ethanol, and counterstaining in eosin solution for 1minute.The Alcian Blue staining of the paraffin sections was done byimmersing of tissue slides in 3% (vol/vol) acetic acid solution for3 minutes and staining with 1% Alcian Blue (Sigma) in 3% aceticacid solution at pH 1.0 for 30 minutes.
For fluorescence-labeled liposome distribution studies, tumorsamples were fast frozen after embedding. Tumor sections (7 mm)were mounted on microscope slides, fixed in 4% paraformalde-hyde for 10 minutes at room temperature. Terminal deoxynu-cleotidyl transferase–mediated dUTP nick end labeling (TUNEL)staining was performed following themanufacturer's instructions(Roche Laboratories). Immunohistologic staining using CD34antibodies and DAPI were also done. Images were taken using ascanning confocal microscope (Leica TCS SP2).
Animal studiesFemale BALB/c nudemice at the age of 6 weeks were purchased
from SLAC Laboratory Animal Center and housed in groups of 5with free access to food and water. All animal procedures wereapproved by Shanghai Jiao Tong University Animal Care and UseCommittee. For LoVo tumor model, mice were injected subcu-taneously with LoVo cells (1 � 106 per animal, suspended inserum-free medium) over the left flank. Thirty-five mice wererandomly divided into five groups and treated by i.v. injections ofPBS, free Dox, Doxil, PEG-Lipo-Dox, and RSPO1-PEG-Lipo-Dox(n¼ 7). Treatment started when the tumor volume reached 50 to100 mm3 at Dox doses of 0.5 mg/kg. The doses were repeatedevery 3 days for 8 times. ForGA007PDXmodel, theGA007 tumortissue was diced into 2 � 2 � 3 mm pieces with scissor, placed inDMEM medium, and implanted subcutaneously into mice overthe left flank. Intravenous injection of PBS, PEG-Lipo-Dox, orRSPO1-PEG-Lipo-Dox atDoxdoses of 0.5mg/kg startedwhen thetumor volume reached 50 to 100 mm3. The doses were repeatedevery 3 days for 6 times. Tumor volume and body weight wererecorded twice aweek for all tumor-bearingmice. Tumor volumeswere calculated using the formula: V ¼ (length)2 � width � 0.5.Mice were euthanized when the tumor size of any group reached1,500 mm3.
Statistical analysisData are expressed as mean � SD. Statistical analysis were
performed using t test or two-way ANOVA using GraphPad prism5 software (GraphPad Software).
ResultsThe design and construction of RSPO1-decorated liposomescontaining Dox
The RSPO protein family contained four members that wereexpressed by different cells but with similar functions (22). Weexamined their structure models and found there was a free
cysteine residue in the N-terminal signaling sequence of RSPO1that may be exposed on the surface (Fig. 1A). We used thiscysteine residue to react with DSPE-PEG2000 containing amaleimide functional group (DSPE-PEG2000-Mal; Fig. 1B).The reactant was analyzed using SDS-PAGE (Fig. 1C) to locatethe DSPE-PEG2000-RSPO1 band which was roughly 3 kDahigher in molecular weight. The band was smeared because ofthe molecular weight variation of the PEG segment. We canestimate based on the intensities that roughly more than 95%of the RSPO proteins were reacted. The biological activitiesof RSPO1 after conjugation were evaluated using a luciferasereporter cell line driven by a promoter containing multipleTCF/LEF-binding sites. As shown in Fig. 1D, both RSPO1 andRSPO1-PEG2000-DSPE were able to potentiate Wnt pathwaysignaling with similar concentration dependency. The EC50
values were 7.37 and 24.47 ng/mL, respectively, indicating thatRSPO1-PEG2000-DSPE retained most of the binding capabilityof RSPO1 after chemical conjugation.
RSPO1-decorated liposomes were prepared by incubationand insertion of RSPO1-PEG2000-DSPE into preformed anddrug-preloaded liposomes (23–24). It is an established methodto ensure liposome surface decoration of RSPO1 and unbound-ed RSPO1 was removed with a membrane dialysis step. ThePEG molecules and RSPO1 proteins were estimated to accountfor about 5% mol. and 0.1% mol. of the surface molecules,respectively. In the binding experiment that compared differentRSPO1 densities (Fig. 2B), the liposomes contained 0.025%,0.05%, or 0.1% mol. of RSPO1 protein.
The cytotoxic drug Dox was loaded using the method devel-oped by Mayer and Bally with high encapsulation efficiency upto 100% (25–26). Any unencapsulated drug was also removedby membrane dialysis. The Cyro-TEM images of the liposomes(Fig. 1E) confirmed they are small unilamellar vesicles withaverage diameters of 90 to 110 nm.
FACS analysis of RSPO1-PEG-liposome binding to LGR5þ cellsWe screened various cell lines for the target molecule LGR5
expression and found the LoVo cell as a great LGR5þ cell model(Fig. 2A). RAW264.7 is a macrophage cell line and was used as aLGR5– cell model. RSPO1-PEG-liposomes containing differentnumbers of RSPO1 but same fluorescence probes on the sur-face were prepared and compared for binding based on FACSanalysis. The cell-associated fluorescence intensity increasedwith the numbers of RSPO1 proteins per liposome surface(Fig. 2B). Assuming all the liposomes were 100 nm in diameterand complete RSPO1-PEG-DSPE incorporation, we estimatedthat there were about 25, 50, or 100 RSPO1 molecules perliposome in those samples. In the presence of 2-fold or 5-foldof free RSPO1, a dose-dependent decreases of bound fluores-cence were observed (Fig. 2C). In contrast, for the LGR5–
RAW264.7 cells, the bound fluorescence intensities were min-imal and independent of the presence of anti-RSPO1 on lipo-some surface (Fig. 2D).
Confocal fluorescence microscopy studies of RSPO1-PEG-liposome distribution in vitro and in vivo
DiI-labeled liposomes were first examined for their bindingand uptake by LoVo cells in vitro. The RSPO1-PEG-liposomeswere shown to be taken up by the cells quickly upon binding at37�C, whereas the PEG-liposomes remained to be minimumassociated (Fig. 3A). But the binding- and energy-dependent
Liposomes Targeting to LGR5þ CSCs
www.aacrjournals.org Mol Cancer Ther; 17(7) July 2018 1477
endocytosis processes were inhibited either when the cells wereincubated at 4�C or in medium containing 5-fold excess of freeRSPO1 (Fig. 3B). The in vivo distribution and binding offluorescence-labeled liposomes were also examined under con-focal microscopy. We took tissue section samples of LOVOtumor–bearing mice at 12 hours after i.v. injection. Figure 3Cshowed the colon and tumor tissue images costained withFITC-labeled CD34 antibodies for the vascular endotheliumcells and DAPI for the nucleus. Most DiI-labeled liposomeswere found still circulating in the microvessels in colon. On theother hand, they were more diffused throughout the perivas-cular region in the tumor tissue.
Drug delivery and activities of RSPO1-PEG-liposomes in theLOVO colon cancer model
The RSPO1-liposomes were loaded with drugs and examinedfor drug activities in vitro and in vivo. For the in vitro cytotoxi-city, Dox-loaded liposomes with or without the RSPO1 surfaceligand were incubated with LOVO cells for 4 hours at differ-ent concentrations, and cell viabilities were evaluated after24 hours or 48 hours. The results shown in Fig. 4A indicated thatRSPO1-PEG-Liposome-Dox were more effective in killing cancercells than PEG-Liposome-Dox. The cytotoxicity test of emptyRSPO1-PEG-liposomes on LoVo cells shows that the presenceof RSPO1-conjugated liposomes does not affect the cell viabilityup to 500 mg/mL lipid.
The cytotoxicity of Dox-loaded liposomes in vivo was alsoexamined. RSPO1-PEG-liposome-Dox and PEG-liposome-Doxwere injected intravenously at 2.5 mg/kg Dox dose. Twenty-four or 48 hours after the injection, tumor tissues were explant-ed and stained for TUNEL activities (Fig. 4B). The numbers ofapoptotic cells were counted and summarized in Fig. 4C.Clearly, one single injection of RSPO1-PEG-liposome-Doxresulted in significantly more apoptotic cells than that of thePEG-liposome-Dox. In addition, cell apoptosis resulted fromPEG-liposome-Dox was mostly detected after 48 hours ofinjection, indicating a gradual drug release effect, whereas theresults from RSPO1-PEG-liposome-Dox were more prompt anddramatic. The comparison of overall antitumor activities afterrepeated injections (every 3 days for 8 times) in the LOVOxenograft model was plotted in Fig. 4D. The entire study wasdone using a very low Dox dose (0.5 mg/kg) in each injection.At this dose, the drug by itself had almost no antitumor activ-ity. The commercial Dox liposome product Doxil and PEG-lipo-Dox were also not too much different. But the RSPO1-PEG-liposomes were much more effective and delayed tumorgrowth at almost all time points after the first injection.
Drug delivery and activities of RSPO1-PEG-liposomes in aPDX tumor model
We then used a clinically more relevant Patient Derived Xeno-graft (PDX) model to analyze the targeted drug delivery effect of
Figure 1.
Preparation of RSPO1-conjugated liposomes. A, The human RSPO1 protein sequence containing the Cysteine residue labeled in red. B, Conjugation reactionof DSPE-PEG2000-maleimide to RSPO1. C, SDS-PAGE analysis of RSPO1-PEG2000-DSPE. One microgram of RSPO1 protein was loaded on each band.D, Biological activities of RSPO1 and RSPO1-PEG-DSPE in a TCF/LEF Luciferase reporter cell line. E, Cryogenic transmission electron microscope(Cryo-TEM) micrograph of RSPO1-PEG-liposomes.
Cao et al.
Mol Cancer Ther; 17(7) July 2018 Molecular Cancer Therapeutics1478
cells and LGR5– cells.A, LGR5 protein expressionwas determined by Western blotting in cancercell lines, and b-actin was used as a loadingcontrol. B, Binding of FITC-labeled liposomescontaining different numbers of RSPO1 to LGR5þ
LoVo cells. C, Liposomes binding in the presenceof 2-fold or 5-fold of free RSPO1 protein. D,Liposomes binding to LGR5– RAW264.7 cells.Left plot, representative histogram plot of theFACS analysis. Right plot, mean fluorescenceintensities of all the cells. The data were plottedas mean � SD, n ¼ 3. The differences betweenthe means were analyzed based on unpairedStudent t test: �� , P < 0.01 and ��� , P < 0.001.
Liposomes Targeting to LGR5þ CSCs
www.aacrjournals.org Mol Cancer Ther; 17(7) July 2018 1479
RSPO1-PEG-liposomes. GA007 is a human gastric tumor PDXmodel obtained from Shanghai Lide Biotech. The tumor tissuewas found to consist of heterogeneous cell populations with only
some LGR5þ cells (Fig. 5A). We also did FACS analysis of singlecell suspensions of the tissue and sorted out the LGR5þ andLGR5- cells (Fig. 5B).
Figure 3.
The distribution and uptake of RSPO1-decorated liposomes in vitro and in vivo. A, DiI-labeled liposomes binding and uptake by LoVo cells in vitro.B, DiI-labeled liposomes binding to LoVo cells. Scale bars, 20 mm. C, DiI-labeled liposomes extravasation and distribution in vivo.
Cao et al.
Mol Cancer Ther; 17(7) July 2018 Molecular Cancer Therapeutics1480
All the drug dosing studies were done in vivo. After a singledose (2.5 mg/kg) of the liposomal Dox, the tumor tissues werecollected 24 or 48 hours afterward and stained for TUNEL(Fig. 5C and D). Treatment with RSPO1-conjugated Dox lipo-somes yielded 3.9- and 4.4-fold greater number of apoptosiscells than treatment with nontargeted liposomes after 24- and48-hour incubation. The number of apoptotic cells keptincreasing from 24 to 48 hours after RSPO1-conjugated Doxliposome injection, suggesting the continuing activity of thetargeted liposomes.
Figure 6 summarized the antitumor activities in the PDXmodel after repeated doses (every 3 days for 6 times). Becauseonly part of the tumor mass in the PDX tissue containedLGR5þ cells, the partial eradication effect of RSPO1-PEG-lipo-dox did not immediately affect the tumor growth curve(Fig. 6A). But after 4 to 5 times injections, the differences intumor volume became more clear (Table 1). The real differ-ences were shown concerning the internal structures inside thetumor tissues (Fig. 6B). The tissues sections of tumors in theRSPO-PEG-Lipo-Dox–treated group contained massive area of
necrotic cells, whereas the PEG-lipo-Dox–treated tumorsshowed only some toxicity.
DiscussionsLGR5 was initially found in crypt base columnar cells of the
small intestine and colon (27–28). It belongs to the leucine-rich repeat-containing G-protein–coupled receptor (LGR) fam-ily and structurally homologous to LGR4 and LGR6 (29–31).LGR5þ cells were identified as stem cells in the stomach, smallintestine, colon, and hair follicles. They were also proposed tobe the cells of origin of various gastrointestinal cancers and playimportant roles in maintaining and promoting tumor growthand metastasis (19, 32). Shimokawa and colleagues in a veryrecent report demonstrated that selective ablation of LGR5þ
CSCs could lead to tumor regression (20).R-spondin family proteins (RSPO1–4) are natural ligands of
LGR5 (29–31). They can all bind to LGR5 with high affinity(IC50 < 10 nmol/L) and participate in the regulation of Wntsignaling (11). Structurally, they all contain a N-terminal signal
Figure 4.
Cytotoxicity of Dox-loaded liposomes in LoVo cells and mouse xenografts. A, LoVo cell viability after treatment of empty RSPO1-PEG-liposomes.B, LoVo cell viability after treatment of Dox-loaded liposomes. The percentage cell viability was calculated by considering untreated cells to be 100%viable. C, TUNEL assays of LoVo xenograft tissues treated with one injection of RSPO1-PEG-Lipo-Dox and PEG-Lipo-Dox at 2.5 mg/kg Dox dose.D, Quantitative summary of the TUNEL images. E, Effects on tumor growth of different Dox formulations. The LoVo tumor–bearing mice wereintravenously treated with Dox and Dox-loaded liposomes at the Dox dose of 0.5 mg/kg every 3 days after tumor volume reached approximately 100 mm3
(total eight injections). The data are mean � SD, n ¼ 7 (�� , P < 0.01 and ��� , P < 0.001).
Liposomes Targeting to LGR5þ CSCs
www.aacrjournals.org Mol Cancer Ther; 17(7) July 2018 1481
peptide, independent of the two adjacent cysteine-rich furin-like (CR) domains that are essential and sufficient for LGR5binding (33–35). We chose RSPO1 because there is a freecysteine residue in the signal peptide that is easily accessibleon the surface and can be used for site-specific conjugation.Indeed, we were able to achieve higher than 95% conjugationefficacy and almost complete preservation of its binding affinity(Fig. 1). The RSPO1-conjugated DSPE-PEG were incorporatedinto preformed (drug loaded) liposomes by incubation at 37�Cfor 24 hours. The conventional method requires incubation at55�C (23, 24, 36). But in order to preserve RSPO1 stability, weused a lower incubation temperature but longer incubationtime. After the RSPO1-PEG-DSPE incorporation, the liposomeswere able to bind to LGR5þ cells efficiently and specifically(Figs. 2 and 3A). The more RSPO1 on the surface, the moreliposomes were bound (Fig. 2B).
In vivo, after i.v. injection, we showed that the liposomes wereable to extravasate through tumor vasculatures into the tumortissues but at the same time stay inside the microvessels innormal tissues (Fig. 3C). Such an EPR effect is consideredcharacteristic of nanodrug carriers irrespective of the surfacetargeting ligands. It was initially demonstrated in the develop-ment of FDA-approved Dox formulation Doxil/Caelyx whichcontains no surface ligands (37). Later studies incorporatingpeptides and antibodies on these liposomes reported no effecton tissue distribution, but there was great improvement ontargeted cell uptake (38–39). In our study, we compared thetargeted drug delivery effects in one tumor model (LOVO)consisted of all LGR5þ cells and another (GA007) containingboth LGR5þ and LGR5– cells. By comparing the distribution ofapoptotic cells resulted from a single dose of RSPO1-lipo-doxand PEG-lipo-dox, we were able to provide a more in-depth
Figure 5.
Targeted drug delivery and efficacy toward LGR5þ cells in PDX tumor models. A, The identification of LGR5þ cells in GA007 PDX tissues. B, The coexistenceof LGR5þ and LGR5– cells in the GA007 tumor tissues. C, TUNEL assays of PDX tumor tissues treated with one injection of RSPO1-PEG-Lipo-Dox andPEG-Lipo-Dox at 2.5 mg/kg Dox dose. D, Quantitative summary of the TUNEL images. The data are mean �SD, n ¼ 3 (��� , P < 0.001).
Cao et al.
Mol Cancer Ther; 17(7) July 2018 Molecular Cancer Therapeutics1482
analysis of the LGR5þ cell targeting effect in vivo inside thetumor tissue. In Figs. 4B and 5C, apoptotic cells were labeledand counted at 24 and 48 hours after a single injection. Therewere very limited numbers of apoptotic cells in PEG-lipo-dox–treated tumor sections at 24 hours and a little bit more at 48hours. Because there were limited interactions between PEG-lipo-dox and cells, these apoptotic cells were the results of drugsgradually released from adjacent liposomes. For the RSPO1-lipo-dox, however, the liposomes could be actively taken up byLGR5þ cells and drugs released inside the cells, so many moreapoptotic cells were observed. More interestingly, the numberof apoptotic cells peaked at 24 hours in the LOVO tumormodel, whereas there was a big increase from 24 to 48 hoursin the GA007 model. Such a difference implies the interplaybetween liposome diffusion and their affinity to the targetedcells in vivo. In the LOVO model, the liposomes were sur-rounded by LGR5þ cells, and their diffusion may be limitedby an "affinity barrier." In the GA007 model, there was lessbarrier so the liposomes could travel further. But diffusion takestime so we saw a gradual increase of apoptotic cells from 24 to48 hours after injection. Here, we have to emphasize that thesedifferences could only be observed when using a small lipo-dox
dose (subtherapeutic in terms of PEG-lipo-dox). Most previousstudies dosed Dox liposomes at a higher dose (10 mg/kg;refs. 40–41) when the drugs released may overflow to theneighboring cells to result in a by-stander effect.
For the repeated dose experiments, we also tried to limitthe drug dose to enable specific eradication of LGR5þ cellsonly. Most CSC-targeted therapeutic effects using ADCs(21, 42), Bite (bispecific T-cell engager; ref. 43), or nanopar-ticles (44–46) were supported by xenotransplantation of iso-lated CSCs. But it has been difficult to take into considerationthe CSC plasticity and pinpoint the real on-target effect.Shimokawa and colleagues had to use a genetic knockin modelto recapitulation the stem cell hierarchy. They also used agenetic tool to eradicate LGR5þ cells and LGR5þ cells only. Inour study, we used a PDX model that is considered morerelevant to real human tumor. We showed that there wereLGR5þ and LGR5– cells coexisting in PDX tissue, but only theLGR5þ cells behaved like CSCs. When a small dose of RSPO1-lipo-dox was applied, they would mostly interact with LGR5þ
cells to insert the cytotoxic effect, whereas limited by-standereffects were seen in LGR5– cells (Fig. 5C). The dose was furtherreduced to 0.5 mg/kg in the repeated dose efficacy study tominimize the amount of drugs leaked before liposome inter-acting with LGR5þ cells. As shown in Fig. 6, the RSPO1-lipo-dox treatment resulted in massive tissue necrosis in areasbeyond LGR5þ cell existence. In comparison, the effects ofrepeated injections of PEG-lipo-Dox were still patchy andlocalized. Apparently, the death of LGR5þ CSCs in the targetedtreatment had affected the growth of LGR5– cells as well toresult in extensive tumor tissue damage. This agrees withreports that destruction of CSCs and their functions might besufficient for tumor regression (20, 47, 48). Because the PDXmodels are more relevant to real tumor scenarios, our datasupport the further development of RSPO1-PEG-liposomes inCSC-targeted drug delivery and treatment plans.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
Authors' ContributionsConception and design: J. Cao, C. Li, Y. XuDevelopment of methodology: J. Cao, C. Li, X. Wei, Y. XuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Cao, F. Xu, Y. XuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Cao, C. Li, Y. XuWriting, review, and/or revision of the manuscript: J. Cao, Y. XuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Cao, X. Wei, M. Tu, Y. ZhangStudy supervision: Y. Xu
Figure 6.
Therapeutic efficacies of targeted drug delivery liposomes in GA007 PDXmodels. A, Effects on tumor growth of Dox-loaded liposomes. The LoVotumor–bearing mice were intravenously treated with Dox-loaded liposomesat the Dox dose of 0.5 mg/kg every 3 days after tumor volume reachedapproximately 100 mm3. The data are mean �SD, n ¼ 4. B, Representativehematoxylin and eosin (H&E) and Alcian blue staining images of GA007tumor tissues after treatment of Dox-loaded liposomes.
AcknowledgmentsY. Xu is supported by National Natural Science Foundation of China (NSFC)
No. 31571019 and No. 81690262.
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 July 19, 2017; revised December 12, 2017; accepted April 12, 2018;published first April 25, 2018.
References1. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C,
et al. Identification and expansion of human colon-cancer-initiating cells.Nature 2007;445:111–5.
2. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF.Prospective identification of tumorigenic breast cancer cells. PNAS 2003;100:3983–8.
3. Yang ZF, Ho DW, NG MN, Lau CK, Yu WC, Ngai P, et al. Significance ofCD90þ cancer stem cells in human liver cancer. Cancer Cell 2008;13:153–66.
4. Levin TG, Powell AE, Davies PS, Silk AD, Dismuke AD, Anderson EC, et al.Characterization of the intestinal cancer stem cell marker CD166 in thehuman and mouse gastrointestinal tract. Gastroenterology 2010;139:2072–82.e5.
5. Yamashita T, Ji J, Budhu A, Forgues M, Yang W, Wang H, et al. EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells withstem/progenitor cell features. Gastroenterology 2009;136:1012–24.e4.
6. Takebe N, Harris PJ, Warren RQ, Ivy SP. Targeting cancer stem cells byinhibiting Wnt, Notch, and Hedgehog pathways. Nat Rev Clin Oncol2011;8:97–106.
7. Takebe N, Miele L, Harris PJ, Jeong W, Bando H, Kahn M, et al. TargetingNotch, Hedgehog, and Wnt pathways in cancer stem cells: clinical update.Nat Rev Clin Oncol 2015;12:445–64.
8. RuffnerH, Sprunger J, CharlatO, Leighton-Davies J,Grosshans B, SalatheA,et al. R-Spondin potentiates Wnt/b-catenin signaling through orphanreceptors LGR4 and LGR5. PLoS ONE 2012;7:e40976.
9. Glinka A, Dolde C, Kirsch N, Huang Y, Kazanskaya O, Ingelfinger D, et al.LGR4 and LGR5 are R-spondin receptors mediating Wnt/b-catenin andWnt/PCP signalling. EMBO reports 2011;12:1055–61.
10. de Lau W, Barker N, Low TY, Koo BK, Li VSW, Teunissen H, et al. Lgr5homologues associate with Wnt receptors and mediate R-spondin signal-ling. Nature 2011;476:293–7.
11. Carmon KS, Gong X, Lin Q, Thomas A, Liu Q. R-spondins function asligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling. PNAS 2011;108:11452–7.
12. Carmon KS, Lin Q, Gong X, Thomas A, Liu Q. LGR5 interacts andcointernalizes with Wnt receptors to modulate Wnt/-catenin signaling.Mol Cell Biol 2012;32:2054–64.
13. Yamamoto Y, Sakamoto M, Fujii G, Tshiji H, Kenetaka K, Asaka M, et al.Overexpression of orphan G-protein–coupled receptor, Gpr49, in humanhepatocellular carcinomas with b-catenin mutations. Hepatology 2003;37:528–33.
14. Takahashi H, ishii H, Nishida N, Takemasa I, Mizushima T, Ikeda M, et al.Significance of Lgr5þve cancer stem cells in the colon and rectum.Ann Surg Oncol 2011;18:1166–74.
15. UchidaH, Yamazaki K, FukumaM,Hayashida T, HasegawaH, KitajimaM,et al. Overexpression of leucine-rich repeat-containing G protein-coupledreceptor 5 in colorectal cancer. Cancer Science 2010;101:1731–7.
16. Tanese K, Fukuma M, Yamada T, Mori T, Yoshikawa T, Watanabe W, et al.G-Protein-coupled receptor GPR49 is Up-regulated in basal cell carcinomaand promotes cell proliferation and tumor formation. Am J Pathol 2008;173:835–43.
17. Yang L, Tang H, Kong Y, Xie X, Chen J, Song C, et al. LGR5 promotes breastcancer progression and maintains stem-like cells through activation ofWnt/b-catenin signaling. Stem Cells 2015;33:2913–24.
18. Hirsch D, Barker N, McNeil N, Hu Y, Camps J, McKinnon K, et al. LGR5positivity defines stem-like cells in colorectal cancer. Carcinogenesis2014;35:849–58.
19. Schepers AG, Snippert HJ, Stamge DE, van de Born M, van Es JH, van deWetering M, et al. Lineage tracing reveals Lgr5þ stem cell activity in mouseintestinal adenomas. Science 2012;337:730–5.
20. Shimokawa M, Ohta Y, Nishikori S, Matano M, Takano A, Fujii M, et al.Visualization and targeting of LGR5þ human colon cancer stem cells.Nature 2017;545:187–92.
21. Junttila MR, Mao W, Wang X, Wang E, Pham T, Flygare J, et al. TargetingLGR5þ cells with an antibody-drug conjugate for the treatment of coloncancer. Sci Translat Med 2015;7:1–11.
22. de LauWB, Snel B,CleversHC. TheR-spondinprotein family.GenomeBiol2012;13:242.
23. Uster PS, Allen TM,Daniel BE,Mendez CJ, NewmanMS, ZhuGZ. Insertionof poly(ethylene glycol) derivatized phospholipid into pre-formed lipo-somes results in prolonged in vivo circulation time. FEBS Lett 1996;386:243–6.
24. Ishida T, Iden DL, Allen TM. A combinatorial approach to producingsterically stabilized (Stealth) immunoliposomal drugs. FEBS Lett 1999;460:129–33.
25. Mayer LD, BallyMB, Cullis PR.Uptake of adriamycin into large unilamellarvesicles in response to a pH gradient. Biochim Biophys Acta 1986;857:123–6.
26. BallyMB,Mayer LD, Loughrey H, Redelmeier T,Madden TD,Wong K, et al.Dopamine accumulation in large unilamellar vesicle systems inducedby transmembrane ion gradients. Chem Phys Lipids 1988;47:97–107.
27. BarkerN, vanEs JH, Kuipers J, Kujala P, vandenBornM,CozijinsenM, et al.Identification of stem cells in small intestine and colon by marker geneLgr5. Nature 2007;449:1003–7.
28. Becker L, Huang Q, Mashimo H. Immunostaining of Lgr5, an intestinalstem cell marker, in normal and premalignant human gastrointestinaltissue. Sci World J 2008;8:1168–76.
29. McDonald T, Wang R, Bailey W, Xie G, Chen F, Caskey CT, et al. Identi-fication and cloning of an orphan G protein-coupled receptor ofthe glycoprotein hormone receptor subfamily. Biochem Biophys ResCommun 1998;247:266–70.
30. Hsu SY, Liang SG, Hsueh AJ. Characterization of two LGR geneshomologous to gonadotropin and thyrotropin receptors with extracel-lular leucine-rich repeats and a G protein-coupled, seven-transmem-brane region. Mol Endocrinol 1998;12:1830–45.
31. Hsu SY, Kudo M, Chen T, Nakabayashi K, Bhalla A, van der Spek PJ, et al.The three subfamilies of leucine-rich repeat-containing G protein-coupledreceptors (LGR): identification of LGR6 and LGR7 and the signalingmechanism for LGR7. Mol Endocrinol 2000;14:1257–71.
32. Barker N, Tan S, Clevers H. Lgr proteins in epithelial stem cell biology.Development 2013;140:2484–94.
33. Kamata T, Katsube K, MichikawaM, Yamada M, Takada S, Mizusawa H. R-spondin, a novel genewith thrombospondin type 1domain,was expressedin the dorsal neural tube and affected in Wnts mutants. Biochim BiophysActa 2004;1676:51–62.
34. Kazanskaya O, Glinka A, del Barco Barrantes I, Stannek P, Niehrs C,WuW.R-Spondin2 Is a secreted activator of Wnt/b-catenin signaling and isrequired for xenopus myogenesis. Develop Cell 2004;7:525–34.
35. Kim K, Wagle M, Tran K, Zhan X, Dixon MA, Liu S, et al. R-Spondin familymembers regulate the Wnt pathway by a common mechanism. Mol BiolCell 2008;19:2588–96.
36. Allen TM, Brandeis E, Hansen CB, Kao GY, Zalipsky S. A new strategyfor attachment of antibodies to sterically stabilized liposomes resultingin efficient targeting to cancer cells. Biochim Biophys Acta 1995;1237:99–108.
37. Barenholz Y.Doxil�– The first FDA-approved nano-drug: lessons learned.J Controll Rel 2012;160:117–34.
38. Tang H, Chen X, Rui M, Sun W, Chen J, Peng J, et al. Effects of surfacedisplayed targeting ligand GE11 on liposome distribution and extravasa-tion in tumor. Mol Pharmaceutics 2014;11:3242–50.
Cao et al.
Mol Cancer Ther; 17(7) July 2018 Molecular Cancer Therapeutics1484
39. Kirpotin DB, Drummond DC, Shao Y, Shalaby MR, Hong K,Nielsen UB, et al. Antibody targeting of long-circulating lipidicnanoparticles does not increase tumor localization but doesincrease internalization in animal models. Cancer Research 2006;66:6732–40.
40. Working PK, Newman MS, Huang SK, Mayhew E, Vaage J, Lasic DD.Pharmacokinetics, biodistribution and therapeutic efficacy of doxoru-bicin encapsulated in Stealth� liposomes (Doxil�). J Liposome Res1994;4:667–87.
41. Gabizon A, Tzemach D, Mak L, Bronstein M, Horowitz AT. DoseDependency of pharmacokinetics and therapeutic efficacy of pegylatedliposomal doxorubicin (DOXIL) in murine models. J Drug Target 2008;10:539–48.
42. Saunders LR, Bankovich AJ, Anderson WC, Aujay MA, Bheddah S, Black K,et al. A DLL3-targeted antibody-drug conjugate eradicates high-gradepulmonary neuroendocrine tumor-initiating cells in vivo. Sci Transl Med2015;7:1–13.
43. Hoey T, YenW, Axelrod F, Basi J, Donigian L, Dylla S, et al. DLL4 blockadeinhibits tumor growth and reduces tumor-initiating cell frequency.Stem Cell 2009;5:168–77.
44. Wang L, Su W, Liu Z, Zhou M, Chen S, Chen Y, et al. CD44 antibody-targeted liposomal nanoparticles for molecular imaging and therapyof hepatocellular carcinoma. Biomaterials 2012;33:5107–14.
45. Arabi L, Badiee A, Mosaffa F, Jaafari MR. Targeting CD44 expressingcancer cells with anti-CD44 monoclonal antibody improves cellularuptake and antitumor efficacy of liposomal doxorubicin. J ControlledRel 2015;220:275–86.
46. Wang C, Chiou S, Chou C, Chen Y, Huang Y, Peng C. Photothermolysisof glioblastoma stem-like cells targeted by carbon nanotubes conjugat-ed with CD133 monoclonal antibody. Nanomedicine 2011;7:69–79.
47. van Es JH, Clevers H. Notch and Wnt inhibitors as potential new drugsfor intestinal neoplastic disease. Trends Mol Med 2005;11:496–502.
48. Todaro M, Francipane MG, Medema JP, Stassi G. Colon cancer stem cells:promise of targeted therapy. Gastroenterology 2010;138:2151–62.
www.aacrjournals.org Mol Cancer Ther; 17(7) July 2018 1485
2018;17:1475-1485. Published OnlineFirst April 25, 2018.Mol Cancer Ther Jing Cao, Chong Li, Xiaohui Wei, et al. Using RSPO-Conjugated Doxorubicin Liposomes
Cancer Stem Cells+Selective Targeting and Eradication of LGR5
Updated version
10.1158/1535-7163.MCT-17-0694doi:
Access the most recent version of this article at:
