selective targeting and eradication of lgr5+ cscs using rspo … · preparation and...

1

Selective targeting and eradication of LGR5+ cancer stem cells

using RSPO conjugated doxorubicin liposomes

(Running title: Liposome targeting of LGR5+ CSCs)

Jing Cao1; Chong Li1; Xiaohui Wei1; Meiqing Tu1; Yan Zhang1; Fengwei Xu1; Yuhong

Xu1,2# 1 Shanghai Jiaotong University, School of Pharmacy 2 Dali University, College of Pharmacy and Chemistry

# correspondence to: Yuhong Xu, 800 Dong Chuan Road, Shanghai Jiaotong University,

Shanghai 200030, PRChina. Email: [email protected]

Keywords: CSC, RSPO, LGR5, liposome, targeting

Financial supports:

Y. Xu is supported by National Natural Science Foundation of China (NSFC)

No. 31571019 & No. 81690262.

The authors claimed no conflict of interests related to the subject of this

study.

Research. on February 13, 2020. © 2018 American Association for Cancermct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 25, 2018; DOI: 10.1158/1535-7163.MCT-17-0694

mailto:[email protected]

http://mct.aacrjournals.org/

2

ABSTRACT

Cancer stem cells (CSCs) that may account for only a small fraction of tumor

mass were found to play crucial roles during tumor initiating, progression and

metastasis. However, they are usually difficult to be treated and notoriously

resilient to drug eradication. In this study we aimed at the Wnt signaling

characteristic of cancer stem cells and designed a liposomal drug delivery

system to target CSCs. Liposomes decorated with RSPO1 on the surface were

constructed for specific interactions with the Wnt pathway co-receptor LGR5.

Doxorubicin carried by the RSPO1-liposomes was more effective at lower

concentrations than the same drug loaded in PEG-liposomes. More

importantly, we showed using a Patient Derived Xerograft (PDX) tumor model

where LGR5+ CSCs co-existed with LGR5- cells, the RSPO1-liposomes were

able to access more CSC cells and deliver the drug specifically and efficiently.

Such a focused effect in eradicating LGR5+ cells led to massive tumor tissue

necrosis and growth inhibition even when only a fraction of the conventional

drug dose was used. These data clearly demonstrated the advantages of CSC

targeted drug delivery and would support the development of such

approaches as a new cancer treatment strategy.




3

INTRODUCTION

Targeted drug therapies to specific cancer related signal transduction

pathways have been quite successful in clinical treatment. But there are also

growing evidences suggesting the critical roles of CSCs (cancer stem cells)

resulting in drug resistances and escape mutations (1-5). CSCs were initially

defined based on the observation that a small fraction of drug-treated cells

could escape growth arrest and result in re-growth of the entire tumor cell

population. Although serial transplantation experiments in rodent models

represent the gold standard for defining cancer stem cells, various surface

markers including CD133, CD44, CD24 are also commonly used. It was only

recently that more mechanistic studies were reported that identified key

players in various development pathways as potential CSC targets to curb

cancer progression and metastasis (6-7).

The leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) was

found to be an important co-receptor of the Wnt/β-catenin signaling pathway

(8-12) and over-expressed in many types of tumors, including hepatocellular,

colorectal, ovarian, basal cell and breast cancer (13-17). Several studies used

LGR5 expression to characterize CSCs and demonstrated their with tumor-

initiating properties (18-19). Most recently, Shimokawa et al demonstrated

that selective ablation of LGR5+CSCs could lead to tumor regression (20).

Junttila et al reported the development of a LGR5 ADC (antibody-drug-

conjugate) for targeted therapy towards CSCs with potent anti-tumor efficacy

(21).

Compared to the ADC format of target drug delivery, we reasoned that a

nanoparticle system may be able to load more drug and enable better drug

accumulate in the tumor tissue based on the EPR effect. Therefore, in this

study, we prepared nanoliposomes encapsulating doxorubicin and containing

surface conjugation of the LGR5 natural ligand RSPO1. We used both a

LGR5+ cancer cell model and a PDX (Patient Derived Xerograph) model

containing only some LGR5+ CSCs and demonstrated improved drug delivery




4

and efficacy by the RSPO1-liposomes. Most importantly, we showed that

dramatic anti-tumor activities can be achieved by focusing and eradicating

only the CSCs using a very small drug dose. Such CSC targeted treatment

strategy may be highly efficient and should be included in various

combination therapy for cancer.

MATERIALS AND METHODS：

Cell lines and PDX samples：

A549, Caco2, and TCF/LEF reporter-HEK293 cells were grown in Dulbecco’s

Modified Eagle’s medium supplemented with 10% fetal bovine serum.

HCT116, AsPC1 and RAW264.7 cells were grown in RPMI 1640 medium

supplemented with 10% fetal bovine serum. LoVo cells were cultured in

DMEM/F12 medium supplemented with 20% fetal bovine serum. The TCF/LEF

reporter-HEK293 cell line was provided by Curegenix Inc. Other cell lines were

originally from Type Culture Collection of Chinese Academy of Sciences. All

cell lines were authenticated by STR analysis at the time of receipt and

determined to be mycoplasma free (MycoProbe, R&D). Cells were used within

15 passages of their initial freeze down. The PDX model was purchased from

Shanghai LIDE Biotech. The tumor tissues were implanted into the

subcutaneous flank of nude mice to generate heterotopic PDX model.

RSPO1-PEG-DSPE Synthesis

RSPO1 protein (StemRD) was incubated with 50-fold molar excess of tris(2-

carboxyethyl) phosphine (TCEP) for 1 h at room temperature to maintain the

free cysteine residue in reduced form. 1,2-distearoyl-sn-glycero-3-

phosphoethanolamine-N-[maleimide (polyethylene glycol)−2000] (DSPE-

PEG2000-Mal) (Pharmaron) was dissolve in 30mM HEPES buffer (pH6.5)

saturated with nitrogen. The reduced protein was immediately added to

DSPE-PEG2000-Mal micelle solution at 1:40~1:60 molar ratio while

maintaining mixing under nitrogen at 10 °C overnight. Protein conjugation to

the lipid was confirmed by gel electrophoresis. Samples were loaded in a 10%

SDS-PAGE gel under reducing conditions and stained with Coomassie brilliant




5

blue G250. Quantification of lipid conjugated protein molecules was done by

determination of grey level intensity of protein bands on the gels using

ImageJ software. Known concentrations of protein (1 μg) were used for

calibration.

In vitro characterization of RSPO1 binding and activities

Biological activity of RSPO1 protein was detected using the TCF/LEF-

Luciferase reporter gene assay. TCF cells cultured in 96-well plates were

treated with RSPO1/RSPO1-PEG-DSPE and incubated in a humidified

incubator at 37 °C for 18 hours. Luciferase assay was performed using Bright-

Glo Luciferase Assay Kit (Promega) according to the manufacturer’s

instructions.

Preparation and characterization of RSPO1-conjugated liposomes

The lipid composition of empty liposomes was Egg phosphatidylcholine (EPC)

(NOF) and Cholesterol (Avanti Polar Lipids) at the molar ratio of 2:1. The

fluorescence labeled liposomes with the same formulation of empty liposomes

were made by incorporating 1% (molar percentage) of either N-(fluorescein-

5-thiocarbamo-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine

(fluorescein-DHPE) (Invitrogen) or DiI (Sigma). Drug loaded liposomes were

composed of hydrogenated soybean phosphatidylcholine (HSPC) (NOF),

Cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-

[methoxy (polyethylene glycol)-2000] (DSPE-PEG2000) (NOF) at the molar

ratio of 55:40:2. The lipid concentration was about 20 mg/ml. Doxorubicin

was loaded into the liposomes using the ammonium sulfate gradient method.

The loaded drug concentration was 2 mg/ml for Doxorubicin HCl. A post-

insertion method was used to decorate liposomes with RSPO1-PEG2000-

DSPE. The liposomes were added to the RSPO1-PEG-DSPE or PEG-DSPE

micelle solution and stirred at 37 °C for 24 h. The liposomal size distribution

and zeta potential were measured by ZetaSizer 3000HSA (Malven). Liposome

morphology was confirmed by JEM-2010HT transmission electron microscopy

(JEOL) and cryogenic transmission electron microscopy (FEI).




6

Western blot analysis of LGR5 expression

The LGR5 expression levels of various cell lines were determined by western

blotting. Cells (A549, Caco2, HCT116, AsPC1, LoVo) were washed with ice-

cold PBS (pH7.4) and lysed with ice-cold RIPA Lysis Buffer (Thermo Scientific)

supplemented with protease inhibitors at 4 °C for 1 h. Samples were

centrifuged at 12,000g for 10 min at 4 °C. Equal amount of proteins were

subjected to SDS-PAGE and transferred to a nitrocellulose (NC) membrane

(Applygen). The NC membrane was blocked with blocking buffer (5% skim

milk in TBST buffer) and incubated with primary antibody against lgr5

(ab75850, Abcam) followed by IRDye 800CW Donkey Anti-Rabbit IgG (LI-

COR). The blotting signals were detected using Odyssey scanner (LI-COR).

FACS Analysis

LoVo and RAW264.7 cells were seeded in 24-well cell culture plates at 5 X 105

cells per well and allowed to grow for 48 h. They were then incubated with

serum-free medium containing either FITC-labeled RSPO1-PEG-liposomes or

FITC-labeled PEG-liposome at lipid concentration of 200 μM for 1 h at 37°C.

After an ice-cold PBS wash, cells were collected by adding 0.25% trypsin-

EDTA followed by suspension in 500 μl PBS. They were analyzed using a flow

cytometer (BD FACS Caliber). For the analysis, the signals were gated to

exclude cell debris and dead cells and collected for 10,000 cells counts.

Liposomal uptake was calculated by dividing the mean log of FITC

fluorescence from liposome-bound viable cells by the mean log of FITC

fluorescence of control cells.

For the analysis of LGR5+ cells in PDX007 tumor, the explanted tumor was

digested for 2 hours in the mixture of 2 U/ml of dispase (Yisen Bio) and 2000

U/ml of collagenase VI (Yisen Bio) at 37 °C with vigorous shaking. The

resulted cell suspension was filtered (40 μm pore size) and washed in PBS.

2x106 cells were collected in 300μl of PBS containing diluted primary antibody

LGR5 (1:40) and incubated on ice for 30min. The cells were rinsed and




7

labeled with diluted fluorescent dye conjugated secondary antibody (1:100)

(CWBio) for 30 minutes on ice. Flow cytometric analysis was performed using

a Beckman Coulter MoFloTMXDP Sorter.

Confocal analysis

LoVo cells were seeded on 16mm circular cover glasses placed in 12-well cell

culture plate and allowed to grow for 48 h until reaching 60% confluence.

The regular culture medium was removed and replaced with serum-free

medium containing the DiI labeled liposomes at 100 μM lipid concentration.

After 15, 30, 60 min of incubation at 37 °C or 4 °C with or without free

RSPO1 protein, cells were washed with ice-cold PBS and fixed with 4%

paraformaldehyde for 10 min at room temperature. The cover slips were

washed thoroughly with PBS and mounted cell-side down on microscope

slides with Anti-fade Mounting Medium (Vector Laboratories). The slides were

observed using a Leica SP2 confocal microscope (Leica Microsystems).

Cell viability assay

LoVo cells were seeded in 96-well cell culture plates at 2 X 104 cells per well

and allowed to grow for 48 h. The cells were incubated with lipo-Dox, PEG-

lipo-Dox or RSPO1-PEG-lipo-Dox at Dox concentration of 7.5~30 μg/ml for 4

h, the medium were removed and the cells were incubated for additional 24

and 48 h in fresh complete medium. After incubation, cell viability assay was

performed using a Cell Counting Kit-8 (Sigma) according to the

manufacturer’s protocol.

Tumor Tissue staining and Analysis

For Immunohistology analysis of the tumor tissue, explanted tumors were

fixed with 4% paraformaldehyde and embedded in paraffin blocks. 5-μm thick

sections were cut and mounted on slides. Sections were blocked with 1% BSA

(Sangon Biotech) and incubated overnight at 4 °C with primary antibody

against LGR5 (ab75850, Abcam) after dewaxing and rehydration and HRP-

conjugated secondary antibody at 37 °C for 1 h. H&E staining was done by




8

soaking the tissue sections on slides in hematoxylin solution for 10min,

washing in running water for 5min, rinsing in 95% ethanol, and

counterstaining in eosin solution for 1min. The Alcian Blue staining of the

paraffin sections were done by immersing of tissue slides in 3% (vol/vol)

Acetic Acid solution for 3 min and staining with 1% Alcian Blue (Sigma) in 3%

Acetic acid solution at pH 1.0 for 30 min.

For fluorescence labeled liposome distribution studies, tumor samples were

fast frozen after embedding. Tumor sections (7 μm) were mounted on

microscope slides, fixed in 4% paraformaldehyde for 10 min at room

temperature. TUNEL staining was performed following the manufacturer’s

instructions (Roche Laboratories). Immunohistological staining using CD34

antibodies and DAPI were also done. Images were taken using a scanning

confocal microscope (Leica TCS SP2).

Animal studies

Female BALB/c nude mice at the age of 6 weeks were purchased from SLAC

Laboratory Animal Center and housed in groups of 5 with free access to food

and water. All animal procedures were approved by Shanghai Jiao Tong

University Animal Care and Use Committee. For LoVo tumor model, mice were

injected subcutaneously with LoVo cells (1 X 106 per animal, suspended in

serum-free medium) over the left flank. Thirty-five mice were randomly

divided into five groups and treated by intravenous injections of PBS, free

doxorubicin, Doxil, PEG-Lipo-Dox, and RSPO1-PEG-Lipo-Dox (n=7).

Treatment started when the tumor volume reached 50-100 mm3 at Dox doses

of 0.5 mg/kg. The doses were repeated every 3 days for 8 times. For GA007

PDX model, the GA007 tumor tissue was diced into 2X2X3 mm pieces with

scissor, placed in DMEM medium, and implanted subcutaneously into mice

over the left flank. Intravenous injection of PBS, PEG-Lipo-Dox, or RSPO1-

PEG-Lipo-Dox at Dox doses of 0.5 mg/kg started when the tumor volume

reached 50-100 mm3. The doses were repeated every 3 days for 6 times.

Tumor volume and body weight were recorded twice a week for all tumor-




9

bearing mice. Tumor volumes were calculated using the formula: V=(length)2

X width X 0.5. Mice were euthanized when the tumor size of any group

reached 1500 mm3.

Statistical analysis.

Data are expressed as mean ± SD. Statistical analysis were performed using

t-test or 2way ANOVA using GraphPad prism 5 software (GraphPad Software).

RESULTS:

The design and construction of RSPO1 decorated liposomes

containing doxorubicine

The RSPO protein family contained 4 members that were expressed by

different cells but with similar functions (22). We examined their structure

models and found there was a free cysteine residue in the N-terminal

signaling sequence of RSPO1 that may be exposed on the surface (Figure

1A). We used this cysteine residue to react with DSPE-PEG2000 containing a

maleimide functional group (DSPE-PEG2000-Mal) (Figure 1B). The reactant

was analyzed using SDS-PAGE (Figure 1C) to locate the DSPE-PEG2000-

RSPO1 band which was roughly 3 kDa higher in molecular weight. The band

was smeared because of the molecular weight variation of the PEG segment.

We can estimate based on the intensities that roughly more than 95% of the

RSPO proteins were reacted. The biological activities of RSPO1 after

conjugation were evaluated using a luciferase reporter cell line driven by a

promoter containing multiple TCF/LEF binding sites. As shown in Figure 1D,

both RSPO1 and RSPO1-PEG2000-DSPE were able to potentiate Wnt pathway

signaling with similar concentration dependency. The EC50 values were 7.37

ng/ml and 24.47 ng/ml respectively, indicating that RSPO1-PEG2000-DSPE

retained most of the binding capability of RSPO1 after chemical conjugation.

RSPO1 decorated liposomes were prepared by incubation and insertion of

RSPO1-PEG2000-DSPE into pre-formed and drug preloaded liposomes (23-

24). It’s an established method to ensure liposome surface decoration of




10

RSPO1 and unbounded RSPO1 were removed with a membrane dialysis step.

The PEG molecules and RSPO1 proteins were estimated to account for about

5% mol. and 0.1% mol. of the surface molecules respectively. In the binding

experiment that compared different RSPO1 densities (Figure 2B), the

liposomes contained 0.025%, 0.05% or 0.1% mol. of RSPO1 protein.

The cytotoxic drug doxorubicin were loaded using the method developed by

Mayer and Bally with high encapsulation efficiency up to 100% (25-26). Any

un-encapsulated drug was also removed by membrane dialysis. The Cyro-

TEM images of the liposomes (figure 1E) confirmed they are small uni-

lamellar vesicles with average diameters of 90-110 nm.

FACS analysis of RSPO1-PEG-liposome binding to LGR5+ cells

We screening various cell lines for the target molecule LGR5 expression and

found the LoVo cell as a great LGR5+ cell model (Figure 2A). RAW264.7 is a

macrophage cell line and was used as a LGR5- cell model. RSPO1-PEG-

liposome containing different numbers of RSPO1 but same fluorescence

probes on the surface were prepared and compared for binding based on

FACS analysis. The cell-associated fluorescence intensity increased with the

numbers of RSPO1 proteins per liposome surface (Figure 2B). Assuming all

the liposomes were 100 nm in diameter and complete RSPO1-PEG-DSPE

incorporation, we estimated that there were about 25, 50 or 100 RSPO1

molecules per liposome in those samples. In the presence of two-fold or five-

fold of free RSPO1, a dose-dependent decreases of bound fluorescence were

observed (Figure 2C). In contrast, for the LGR5- RAW264.7 cells, the bound

fluorescence intensities were minimal and independent of the presence of ant

RSPO1 on liposome surface ( Figure 2D).

Confocal fluorescence microscopy studies of RSPO1-PEG-liposome

distribution in vitro and in vivo




11

DiI labeled liposomes were first examined for their binding and uptake by

LoVo cells in vitro. The RSPO1-PEG-liposome were shown to be taken up by

the cells quickly upon binding at 37°C, while the PEG-liposomes remained to

be minimum associated (Figure 3A). But the binding and energy dependent

endocytosis processes were inhibited either when the cells were incubated at

4°C or in medium containing 5-fold excess of free RSPO1 (Figure 3B). The in

vivo distribution and binding of fluorescence labeled liposomes were also

examined under confocal microscopy. We took tissue section samples of

LOVO tumor bearing mice at 12hrs after iv injection. Figure 3C showed the

colon and tumor tissue images co-stained with FITC labeled CD34 antibodies

for the vascular endothelium cells and DAPI for the nucleus. Most DiI labeled

liposomes were found still circulating in the micro vessels in colon. On the

other hand, they were more diffused through out the perivascular region in

the tumor tissue.

Drug delivery and activities of RSPO1-PEG-liposomes in the LOVO

colon cancer model

The RSPO1-liposomes were loaded with drugs and examined for drug

activities in vitro and in vivo. For the in vitro cytotoxicity, Doxorubicin loaded

liposomes with or without the RSPO1 surface ligand were incubated with

LOVO cells for 4 hours at different concentrations and cell viabilities were

evaluated after 24 hours or 48 hours. The results shown in Figure 4A

indicated that RSPO1-PEG-Liposome-Dox were more effective in killing cancer

cells than PEG-Liposome-Dox. The cytotoxicity test of empty RSPO1-PEG

liposomes on LoVo cells shows that presence of RSPO1 conjugated liposomes

dose not affect the cell viability up to 500μg/ml lipid.

The cytotoxicity of doxorubicin loaded liposomes in vivo were also examined.

RSPO1-PEG-liposome-Dox and PEG-liposome-Dox were injected intravenously

at 2.5 mg/kg Dox dose. 24 hours or 48 hours later after the injection, tumor

tissues were explanted and stained for TUNEL activities (Figure 4B). The




12

numbers of apoptotic cells were counted and summarized in Figure 4C.

Clearly, one single injection of RSPO1-PEG-liposome-Dox resulted in

significantly more apoptotic cells than that of the PEG-liposome-Dox. In

addition, cell apoptosis resulted from PEG-liposome-Dox was mostly detected

after 48 hrs of injection, indicating a gradual drug release effect, while the

results from RSPO1-PEG-liposome-Dox were more prompt and dramatic. The

comparison of overall anti-tumor activities after repeated injections (every 3

days for 8 times) in the LOVO xenograft model was plotted in Figure 4D. The

entire study was done using a very low doxorubicin dose (0.5mg/kg) in each

injection. At this dose, the drug by itself had almost no anti-tumor activity.

The commercial doxorubicin liposome product Doxil and PEG-lipo-Dox were

also not too much different. But the RSPO1-PEG-liposomes were much more

effective and delayed tumor growth at almost all time points after the first

injection.

Drug delivery and activities of RSPO1-PEG-liposomes in a PDX tumor

model

We then used a clinically more relevant Patient Derived Xenograft (PDX)

model to analyze the targeted drug delivery effect of of RSPO1-PEG-

liposomes. GA007 is a human gastric tumor PDX model obtained from

Shanghai Lide Biotech. The tumor tissue was found to consist of

heterogeneous cell populations with only some LGR5+ cells (Figure 5A). We

also did FACS analysis of single cell suspensions of the tissue and sorted out

the LGR5+ and LGR5- cells (Figure 5B).

All the drug dosing studies were done in vivo. After a single dose (2.5 mg/kg)

of the liposomal doxorubicin, the tumor tissues were collected 24 hours or 48

hours afterwards and stained for TUNEL (Figure 5C, D). Treatment with

RSPO1 conjugated doxorubicin liposomes yielded 3.9- and 4.4-fold greater

number of apoptosis cells than treatment with nontargeted liposomes after

24hr and 48hr incubation. The number of apoptotic cells kept increasing from




13

24hr to 48hr after RSPO1 conjugated doxorubicin liposome injection,

suggesting the continues activity of the targeted liposomes.

Figure 6 summarized the anti-tumor activities in the PDX model after

repeated doses (every 3 days for 6 times). Since only part of the tumor mass

in the PDX tissue contained LGR5+ cells, the partial eradication effect of

RSPO1-PEG-lipo-dox did not immediately affect the tumor growth curve

(Figure 6A). But after 4-5 times injections, the differences in tumor volume

became more clear (Table 1). The real differences were shown concerning

the internal structures inside the tumor tissues (Figure 6B). The tissues

sections of tumors in the RSPO-PEG-Lipo-Dox treated group contained

massive area of necrotic cells, while the PEG-lipo-Dox treated tumors showed

only some toxicity.




14

DISCUSSIONS：

LGR5 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) family and structurally homologous to LGR4 & LGR6

(29-31). LGR5+ cells were identified as stem cells in the stomach, small

intestine, colon, and hair follicles. They were also proposed to be the cells-of-

origin of various gastrointestinal cancers and play important roles in

maintaining and promoting tumor growth and metastasis (19, 32).

Shimokawa et al in a very recent report demonstrated that selective ablation

of LGR5+ 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 nM) and participate in

the regulation of Wnt signaling (11). Structurally, they all contain a N-terminal

signal peptide, independent of the two adjacent cysteine-rich furin-like (CR)

domains that are essential and sufficient for LGR5 binding (33-35). We chose

RSPO1 because there is a free cysteine residue in the signal peptide that’s

easily accessible on the surface and can be used for site-specific conjugation.

Indeed, we were able to achieve higher than 95% conjugation efficacy and

almost complete preservation of its binding affinity (Figure 1). The RSPO1

conjugated DSPE-PEG were incorporated into preformed (drug loaded)

liposomes by incubation at 37oC for 24 hours. The conventional method

requires incubation at 55oC (23, 24, 36). But in order to preserve RSPO1

stability, we used a lower incubation temperature but longer incubation time.

After the RSPO1-PEG-DSPE incorporation, the liposomes were able to bind to

LGR5+ cells efficiently and specifically (Figure 2 and 3A). The more RSPO1 on

the surface, the more liposomes were bound (Figure 2B).

In vivo, after intravenous injection, we showed that the liposomes were able

to extravasate through tumor vasculatures into the tumor tissues but at the

same time stay inside the microvessels in normal tissues (figure 3C). Such a

EPR effect is considered characteristic of nano drug carriers irrespective of the




15

surface targeting ligands. It was initially demonstrated in the development of

FDA approved doxorubicin formulation Doxil®/Caelyx® which contains no

surface ligands (37). Later studies incorporating peptides and antibodies on

these liposomes reported no effect on tissue distribution but there was great

improvement on targeted cell uptake (38-39). In our study, we compared the

targeted drug delivery effects in one tumor model (LOVO) consisted of all

LGR5+ cells and another (GA007) containing both LGR5+ and LGR5- cells. By

comparing the distribution of apoptotic cells resulted from a single dose of

RSPO1-lipo-dox and PEG-lipo-dox, we were able to provide a more in-depth

analysis of the LGR5+ cell targeting effect in vivo inside the tumor tissue. In

Figure 4B and Figure 5C, apoptotic cells were labeled and counted at 24hrs

and 48hrs after a single injection. There were very limited numbers of

apoptotic cells in PEG-lipo-dox treated tumor sections at 24hrs and a little bit

more at 48hrs. Since there were limited interactions between PEG-lipo-dox

and cells, these apoptotic cells were the results of drugs gradually released

from adjacent liposomes. For the RSPO1-lipo-dox, however, the liposomes

could be actively taken up by LGR5+ cells and drugs released inside the cells,

so many more apoptotic cells were observed. More interestingly, the number

of apoptotic cells peaked at 24hrs in the LOVO tumor model while there was a

big increase from 24hrs to 48hrs in the GA007 model. Such a difference

implies the interplay between liposome diffusion and their affinity to the

targeted cells in vivo. In the LOVO model, the liposomes were surrounded by

LGR5+ cells and their diffusion may be limited by an “affinity barrier”. In the

GA007 model, there was less a barrier so the liposomes could travel further.

But diffusion takes time so we saw a gradual increase of apoptotic cells from

24hrs to 48 hrs after injection. Here we have to emphasize that these

differences could only be observed when using a small lipo-dox dose (sub-

therapeutic in terms of PEG-lipo-dox). Most previous studies dosed

doxorubicin liposomes at a higher dose (10mg/kg) (40-41) when the drugs

released may overflow to the neighboring cells to result in a by-stander

effect.




16

For the repeated dose experiments, we also tried to limit the drug dose to

enable specific eradication of LGR5+ cells only. Most CSC targeted

therapeutic effects using ADCs (21, 42), Bite (bispecific T cell engager) (43),

or nanoparticles (44-46) were supported by xenotransplantation of isolated

CSCs. But it has been difficult to take into consideration of the CSC plasticity

and pinpoint the real on-target effect. Shimokawa et al. had to use a genetic

knock in model to recapitulation the stem cell hierarchy. They also employed

a genetic tool to eradicate LGR5+ cells and LGR5+ cells only. In our study,

we used a PDX model that’s considered more relevant to real human tumor.

We showed that there were LGR5+ cells and LGR5- cells co-existing in PDX

tissue but only the LGR5+ cells behaved like CSCs. When a small dose of

RSPO1-lipo-dox was applied, they would mostly interact with LGR5+ cells to

insert the cytotoxic effect, while limited by-stander effects was seen in LGR5-

cells (Figure 5C). The dose was further reduced to 0.5mg/kg in the repeated

dose efficacy study to minimize the amount of drugs leaked before liposome

interacting with LGR5+ cells. As shown in Figure 6, the RSPO1-lipo-dox

treatment resulted in massive tissue necrosis in areas beyond LGR5+ cell

existence. In comparison, the effect of repeated injections of PEG-lipo-Dox

were still patchy and localized. Apparently, the death of LGR5+ CSCs in the

targeted treatment had affected the growth of LGR5- cells as well to result in

extensive tumor tissue damage. This agrees with reports that destruction of

CSCs and their functions might be sufficient for tumor regression (20, 47, 48).

Since the PDX models are more relevant to real tumor scenarios, our data

support the further development of RSPO1-PEG-Liposomes in cancer stem cell

targeted drug delivery and treatment plans.




17

REFERENCES:

1. 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;445:111–115.

2. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF.

Prospective identification of tumorigenic breast cancer cells. PNAS

2003;100:3983–3988.

3. Yang ZF, Ho DW, NG MN, Lau CK, Yu WC, Ngai P, et al. Significance of

CD90+ cancer stem cells in human liver cancer. Cancer Cell 2008;13:153–166.

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 the

Human and Mouse Gastrointestinal Tract. Gastroenterology 2010;139:2072–

2082.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 With

Stem/Progenitor Cell Features. Gastroenterology 2009;136:1012–1024.e4.

6. Takebe N, Harris PJ, Warren RQ, Ivy SP. Targeting cancer stem cells by

inhibiting Wnt, Notch, and Hedgehog pathways. Nat Rev Clin Oncol

2011;8:97–106.

7. Takebe N, Miele L, Harris PJ, Jeong W, Bando H, Kahn M, et al. Targeting

Notch, Hedgehog, and Wnt pathways in cancer stem cells: clinical update. Nat

Rev Clin Oncol 2015;12:445-464.

8. Ruffner H, Sprunger J, Charlat O, Leighton-Davies J, Grosshans B, Salathe A,

et al. R-Spondin Potentiates Wnt/β-Catenin Signaling through Orphan

Receptors 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/β-catenin and

Wnt/PCP signalling. EMBO reports 2011;12:1055–1061.

10. de Lau W, Barker N, Low TY, Koo BK, Li VSW, Teunissen H, et al. Lgr5

homologues associate with Wnt receptors and mediate R-spondin signalling.

Nature 2011;476:293–297.

11. Carmon KS, Gong X, Lin Q, Thomas A, Liu Q. R-spondins function as ligands

of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin

signaling. PNAS 2011;108:11452–11457.

12. Carmon KS, Lin Q, Gong X, Thomas A, Liu Q. LGR5 Interacts and

Cointernalizes with Wnt Receptors To Modulate Wnt/ -Catenin Signaling.

Molecular and Cellular Biology 2012;32:2054–2064.

13. Yamamoto Y, Sakamoto M, Fujii G, Tshiji H, Kenetaka K, Asaka M, et al.

Overexpression of orphan G-protein–coupled receptor, Gpr49, in human

hepatocellular carcinomas with β-catenin mutations. Hepatology 2003;37:528–

533.

14. Takahashi H, ishii H, Nishida N, Takemasa I, Mizushima T, Ikeda M, et al.

Significance of Lgr5+ve Cancer Stem Cells in the Colon and Rectum. Ann

Surg Oncol 2011;18:1166–1174.

15. Uchida H, Yamazaki K, Fukuma M, Hayashida T, Hasegawa H, Kitajima M, et

al. Overexpression of leucine-rich repeat-containing G protein-coupled

receptor 5 in colorectal cancer. Cancer Science 2010;101:1731–1737.

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 Carcinoma




18

and Promotes Cell Proliferation and Tumor Formation. The American Journal

of Pathology 2008;173:835–843.

17. Yang L, Tang H, Kong Y, Xie X, Chen J, Song C, et al. LGR5 Promotes Breast

Cancer Progression and Maintains Stem-Like Cells Through Activation of

Wnt/β-Catenin Signaling. Stem Cells 2015;33:2913–2924.

18. Hirsch D, Barker N, McNeil N, Hu Y, Camps J, McKinnon K, et al. LGR5

positivity defines stem-like cells in colorectal cancer. Carcinogenesis

2014;35:849–858.

19. Schepers AG, Snippert HJ, Stamge DE, van de Born M, van Es JH, van de

Wetering M, et al. Lineage tracing reveals Lgr5+ stem cell activity in mouse

intestinal adenomas. Science 2012;337:730–735.

20. Shimokawa M, Ohta Y, Nishikori S, Matano M, Takano A, Fujii M, et al.

Visualization and targeting of LGR5+ human colon cancer stem cells. Nature

2017;545:187–192.

21. Junttila MR, Mao W, Wang X, Wang E, Pham T, Flygare J, et al. Targeting

LGR5+ cells with an antibody-drug conjugate for the treatment of colon

cancer. Science Translational Medicine 2015;7:1-11.

22. de Lau WB, Snel B, Clevers HC. The R-spondin protein family. Genome Biol

2012;13:242.

23. Uster PS, Allen TM, Daniel BE, Mendez CJ, Newman MS, Zhu GZ. Insertion

of poly(ethylene glycol) derivatized phospholipid into pre-formed liposomes

results in prolonged in vivo circulation time. FEBS Lett 1996;386:243–246.

24. Ishida T, Iden, DL, Allen TM. A combinatorial approach to producing

sterically stabilized (Stealth) immunoliposomal drugs. FEBS Lett

1999;460:129–133.

25. Mayer LD, Bally MB, Cullis PR. Uptake of adriamycin into large unilamellar

vesicles in response to a pH gradient. Biochim Biophys Acta 1986;857:123–

126.

26. Bally MB, Mayer LD, Loughrey H, Redelmeier T, Madden TD, Wong K, et al.

Dopamine accumulation in large unilamellar vesicle systems induced by

transmembrane ion gradients. Chem Phys Lipids 1988;47:97–107.

27. Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijinsen M, et al.

Identification of stem cells in small intestine and colon by marker gene Lgr5.

Nature 2007;449:1003–1007.

28. Becker L, Huang Q, Mashimo H. Immunostaining of Lgr5, an intestinal stem

cell marker, in normal and premalignant human gastrointestinal tissue.

Scientific World Journal 2008;8:1168–1176.

29. McDonald T, Wang R, Bailey W, Xie G, Chen F, Caskey CT, et al.

Identification and cloning of an orphan G protein-coupled receptor of the

glycoprotein hormone receptor subfamily. Biochemical and Biophysical

Research Communications 1998;247:266–270.

30. Hsu SY, Liang SG, Hsueh AJ. Characterization of two LGR genes homologous

to gonadotropin and thyrotropin receptors with extracellular leucine-rich

repeats and a G protein-coupled, seven-transmembrane region. Mol Endocrinol

1998;12:1830–1845.

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-coupled

receptors (LGR): identification of LGR6 and LGR7 and the signaling

mechanism for LGR7. Mol Endocrinol 2000;14:1257–1271.




19

32. Barker N, Tan S, Clevers H. Lgr proteins in epithelial stem cell biology.

Development 2013;140:2484–2494.

33. Kamata T, Katsube K, Michikawa M, Yamada M, Takada S, Mizusawa H. R-

spondin, a novel gene with thrombospondin type 1 domain, was expressed in

the dorsal neural tube and affected in Wnts mutants. Biochim. Biophys. Acta

2004;1676:51–62.

34. Kazanskaya O, Glinka A, del Barco Barrantes I, Stannek P, Niehrs C, Wu W.

R-Spondin2 Is a Secreted Activator of Wnt/β-Catenin Signaling and Is

Required for Xenopus Myogenesis. Developmental Cell 2004;7:525-534.

35. Kim K, Wagle M, Tran K, Zhan X, Dixon MA, Liu S, et al. R-Spondin family

members regulate the Wnt pathway by a common mechanism. Mol Biol Cell

2008;19:2588–2596.

36. Allen TM, Brandeis E, Hansen CB, Kao GY, Zalipsky S. A new strategy for

attachment of antibodies to sterically stabilized liposomes resulting in efficient

targeting to cancer cells. Biochim Biophys Acta 1995;1237:99-108.

37. Barenholz Y. Doxil®-- The first FDA-approved nano-drug: Lessons learned.

Journal of Controlled Release 2012;160:117–134.

38. Tang H, Chen X, Rui M, Sun W, Chen J, Peng J, et al. Effects of Surface

Displayed Targeting Ligand GE11 on Liposome Distribution and Extravasation

in Tumor. Mol Pharmaceutics 2014;11:3242–3250.

39. Kirpotin DB, Drummond DC, Shao Y, Shalaby MR, Hong K, Nielsen UB, et

al. Antibody Targeting of Long-Circulating Lipidic Nanoparticles Does Not

Increase Tumor Localization but Does Increase 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 doxorubicin

encapsulated in Stealth® liposomes (Doxil®). Journal of Liposome Research

1994;4:667-687.

41. Gabizon A, Tzemach D, Mak L, Bronstein M, Horowitz AT. Dose Dependency

of Pharmacokinetics and Therapeutic Efficacy of Pegylated Liposomal

Doxorubicin (DOXIL) in Murine Models. Journal of Drug Targeting

2008;10:539–548.

42. Saunders LR, Bankovich AJ, Anderson WC, Aujay MA, Bheddah S, Black K,

et al. A DLL3-targeted antibody-drug conjugate eradicates high-grade

pulmonary neuroendocrine tumor-initiating cells in vivo. Sci Transl Med

2015;7:1-13.

43. Hoey T, Yen W, Axelrod F, Basi J, Donigian L, Dylla S, et al. DLL4 Blockade

Inhibits Tumor Growth and Reduces Tumor-Initiating Cell Frequency. Stem

Cell 2009;5:168–177.

44. Wang L, Su W, Liu Z, Zhou M, Chen S, Chen Y, et al. CD44 antibody-targeted

liposomal nanoparticles for molecular imaging and therapy of hepatocellular

carcinoma. Biomaterials 2012;33:5107–5114.

45. Arabi L, Badiee A, Mosaffa F, Jaafari MR. Targeting CD44 expressing cancer

cells with anti-CD44 monoclonal antibody improves cellular uptake and

antitumor efficacy of liposomal doxorubicin. Journal of Controlled Release

2015;220:275–286.

46. Wang C, Chiou S, Chou C, Chen Y, Huang Y, Peng C. Photothermolysis of

glioblastoma stem-like cells targeted by carbon nanotubes conjugated with

CD133 monoclonal antibody. Nanomedicine 2011;7:69–79.




20

47. van Es JH, Clevers H. Notch and Wnt inhibitors as potential new drugs for

intestinal neoplastic disease. Trends in Molecular Medicine 2005;11:496–502.

48. Todaro M, Francipane MG, Medema JP, Stassi G. Colon Cancer Stem Cells:

Promise of Targeted Therapy. Gastroenterology 2010;138:2151–2162.




21

TABLES:

Dose and Regimen T/C* (%)

Mean tumor

volume change

(mm3 ± SEM)

Vehicle 5 ml/kg, q3d X6 100 640 ± 51.1

PEG-Lipo-Dox 0.5 mg/kg, q3d X6 53.5 342.2 ± 43.4

RSPO1-PEG-Lipo-Dox 0.5 mg/kg, q3d X6 32.5 208 ± 39.4

Drug

Doxorubicin

Table 1. Tumor growth inhibition results of Doxorubicin loaded liposomes efficacy on GA007 PDX tumor models. *T/C (%) values represent the treated to control ratios of relative median tumor volumes, n=4.




22

FIGURE LEGENDS:

Figure 1. Preparation of RSPO1 conjugated liposomes. (A) The human

RSPO1 protein sequence containing the Cysteine residue labeled in red. (B)

Conjugation reaction of DSPE-PEG2000-maleimide to RSPO1. (C) SDS-PAGE

analysis of RSPO1-PEG2000-DSPE. 1μg 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.

Figure 2. Binding of FITC labeled liposomes to LGR5+ cells and LGR5- cells.

(A) LGR5 protein expression was determined by western blotting in cancer

cell lines, β-actin was used as a loading control. (B) Binding of FITC labeled

liposomes containing different numbers of RSPO1 to LGR5+ LoVo cells. (C)

Liposomes binding in the presence of 2-fold or 5-fold of free RSPO1 protein.

(D) Liposomes binding to LGR5- RAW264.7 cells. Left panel: representative

histogram plot of the FACS analysis. Right panel: mean fluorescence

intensities of all the cells. The data were plotted as mean ±SD, n=3. The

differences between the means were analyzed based on unpaired Student’s t-

test, ***P<0.001, **P<0.01.

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 μm. (C)

DiI labeled liposomes extravasation and distribution in vivo.

Figure 4. Cytotoxicity of Doxorubicin 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 Doxorubicin 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




23

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 were intravenous treated with Dox and Dox loaded liposomes at

the Dox dose of 0.5 mg/kg every 3 days after tumor volume reached ~100

mm3 (total eight injections). The data are mean ± SD, n=7. **P<0.01,

***P<0.001.

Figure 5. Targeted drug delivery and efficacy towards LGR5+ cells in PDX

tumor models. (A) The identification of LGR5+ cells in GA007 PDX tissues. (B)

The coexistence of LGR5+ and LGR5- cells in the GA007 tumor tissues. (C)

TUNEL assays of PDX tumor 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. The data are mean ±SD, n=3. ***P<0.001.

Figure 6. Therapeutic efficacies of targeted drug delivery liposomes in GA007

PDX models. (A) Effects on tumor growth of doxorubicin loaded liposomes.

The LoVo tumor bearing mice were intravenous treated with Dox loaded

liposomes at the Dox dose of 0.5 mg/kg every 3 days after tumor volume

reached ~100 mm3. The data are mean ±SD, n=4. (B) Representative H&E

and Alcian blue staining images of GA007 tumor tissues after treatment of

Dox loaded liposomes.




Published OnlineFirst April 25, 2018.Mol Cancer Ther Jing Cao, Chong Li, Xiaohui Wei, et al. RSPO conjugated doxorubicin liposomesSelective targeting and eradication of LGR5+ CSCs using

Updated version

10.1158/1535-7163.MCT-17-0694doi:

Access the most recent version of this article at:

Manuscript

Authorbeen edited. Author manuscripts have been peer reviewed and accepted for publication but have not yet

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/early/2018/04/25/1535-7163.MCT-17-0694To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-17-0694

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/early/2018/04/25/1535-7163.MCT-17-0694


selective targeting and eradication of lgr5+ cscs using rspo … · preparation and...

Documents