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.
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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.
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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
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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
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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).
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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
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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
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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-
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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
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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
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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
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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
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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.
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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
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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.
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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.
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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.
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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
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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.
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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
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