LGR5 positivity defines stem-like cells in colorectal cancer

Abstract

Like normal colorectal epithelium, colorectal carcinomas (CRC) are organized hierarchically and include populations of cells with stem-like properties. Leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) is associated with these stem cells in normal colorectal epithelium, however the precise function of LGR5 in CRC remains largely unknown. Here, we analyzed the functional and molecular consequences of short hairpin RNA-mediated silencing of LGR5 in CRC cell lines SW480 and HT-29. Additionally, we exposed Lgr5-EGFP-IRES-CreERT2 mice to azoxymethane/dextrane sodium sulfate (AOM/DSS) which induces inflammation-driven colon tumors. Tumors were then flow-sorted into fractions of epithelial cells that expressed high or low levels of Lgr5 and were molecularly characterized using gene expression profiling and array comparative genomic hybridization. Silencing of LGR5 in SW480 CRC cells resulted in a depletion of spheres but did not affect adherently growing cells. Spheres expressed higher levels of several stem cell-associated genes than adherent cells, including LGR5. Silencing of LGR5 reduced proliferation, migration and colony formation in vitro, and tumorigenicity in vivo. In accordance with these results, NOTCH signaling was down-regulated upon LGR5 silencing. In AOM/DSS-induced colon tumors Lgr5 high cells showed higher levels of several stem cell-associated genes and higher Wnt signaling than Lgr5 low tumor cells and Lgr5 high normal colon cells. Array comparative genomic hybridization revealed no genomic imbalances in either tumor cell fraction. Our data elucidate mechanisms that define the role of LGR5 as a marker for stem-like cells in CRC.

Full text

Published by Oxford University Press 2013.
Carcinogenesis vol.35 no.4 pp.849–858, 2014
doi:10.1093/carcin/bgt377
Advance Access publication November 26, 2013
LGR5 positivity denes stem-like cells in colorectalcancer
DanielaHirsch1,2, NickBarker3, NicoleMcNeil1, YueHu1,
JordiCamps1, KatherineMcKinnon4, HansClevers5,
ThomasRied1,* and TimoGaiser6
1Section of Cancer Genomics, Genetics Branch, Center for Cancer Research,
National Cancer Institute, National Institutes of Health, Bethesda, MD
20892, USA, 2Experimental Medicine and Therapy Research, University of
Regensburg, 93053 Regensburg, Germany, 3Institute of Medical Biology,
Singapore 138648, Singapore, 4FACS Core Facility, Vaccine Branch, Center
for Cancer Research, National Cancer Institute, National Institutes of
Health, Bethesda, MD 20892, USA, 5Hubrecht Institute, 3584 CT Utrecht,
The Netherlands and 6Institute of Pathology, University Medical Center
Mannheim, 68167 Mannheim, Germany
*To whom correspondence should be addressed. Tel:+1 301 5943118;
Fax: +1 301 4021204;
Email: riedt@mail.nih.gov
Correspondence may also be addressed to Timo Gaiser. Tel:+49 621
3832556; Fax: +49 621 3832005;
Email: timo.gaiser@umm.de
Like normal colorectal epithelium, colorectal carcinomas (CRCs)
are organized hierarchically and include populations of cells with
stem-like properties. Leucine-rich-repeat-containing G-protein-
coupled receptor 5 (LGR5) is associated with these stem cells
in normal colorectal epithelium; however, the precise function
of LGR5 in CRC remains largely unknown. Here, we analyzed
the functional and molecular consequences of short hairpin
RNA-mediated silencing of LGR5 in CRC cell lines SW480 and
HT-29. Additionally, we exposed Lgr5-EGFP-IRES-CreERT2
mice to azoxymethane/dextrane sodium sulfate (AOM/DSS),
which induces inammation-driven colon tumors. Tumors were
then ow-sorted into fractions of epithelial cells that expressed
high or low levels of Lgr5 and were molecularly characterized
using gene expression proling and array comparative genomic
hybridization. Silencing of LGR5 in SW480 CRC cells resulted
in a depletion of spheres but did not affect adherently growing
cells. Spheres expressed higher levels of several stem cell-asso-
ciated genes than adherent cells, including LGR5. Silencing of
LGR5 reduced proliferation, migration and colony formation in
vitro and tumorigenicity in vivo. In accordance with these results,
NOTCH signaling was downregulated upon LGR5 silencing. In
AOM/DSS-induced colon tumors, Lgr5 high cells showed higher
levels of several stem cell-associated genes and higher Wnt sign-
aling than Lgr5 low tumor cells and Lgr5 high normal colon
cells. Array comparative genomic hybridization revealed no
genomic imbalances in either tumor cell fraction. Our data elu-
cidate mechanisms that dene the role of LGR5 as a marker for
stem-like cells in CRC.
Introduction
Colorectal tumorigenesis is associated with the accumulation of a
number of specic genetic changes, which drive the transition from
normal epithelium through adenomas to invasive carcinomas. These
genetic changes include mutations of specic genes, such as adeno-
matous polyposis coli (APC) and KRAS, and tumor-specic genomic
imbalances (1–3). Similar to normal colorectal epithelium, colorectal
tumors consist of heterogeneous cell populations at various levels
of differentiation. Although a few years ago all cells within a tumor
were considered to be tumorigenic, recent ndings suggested that
only a subpopulation of tumor cells can regenerate the tumor (4,5).
These cells, termed cancer stem cells (CSCs), may also be involved
in therapy resistance, tumor relapse and metastasis. Accordingly, the
identication and characterization of these cells was the subject of
considerable research efforts. With respect to colorectal carcinomas
(CRCs), putative CSCs can be identied by leucine-rich-repeat-con-
taining G-protein-coupled receptor 5 (Lgr5; also known as G-protein-
coupled receptor 49, Gpr49). Lgr5, a Wnt target gene that acts as
receptor for the Wnt agonist R-spondin, is a marker gene for adult
intestinal stem cells as revealed by in vivo lineage tracing (6–8).
Selective deletion of Apc in the mouse in either Lgr5 positive intes-
tinal stem cells or more differentiated cells revealed that mainly the
Lgr5 positive stem cell fraction is capable of forming tumors upon
Wnt pathway activation, suggesting Lgr5 positive stem cells as the
cells-of-origin of intestinal epithelial tumors (9). Although the cell-
of-origin for tumorigenesis and the CSC, which propagates the
tumor, need not necessarily be identical, in vivo lineage tracing pro-
vides direct evidence for a stem cell activity of Lgr5 positive cells
in mouse intestinal adenomas generated by deletion of Apc in Lgr5
positive stem cells (10,11). Resembling the situation in normal intes-
tinal epithelium, adenomas contain a small fraction of Lgr5 positive
cells (5–10%) that are able to generate all cell types present within
the adenomas, including additional Lgr5 positive cells (11). In human
CRC, LGR5 expression is highly enriched in EPHB2 positive cells,
which have similar expression proles to normal intestinal stem cells
and—in contrast to EPHB2 negative cells—display reproducible
tumorigenic capacity in immunodecient mice (12). Cataloging the
genetic idiosyncrasies of LGR5 positive and negative cells might help
to identify the mechanisms that cause these differences in tumorigenic
potential. We have therefore investigated the functional and molecular
consequences of short hairpin RNA (shRNA)-mediated LGR5 silenc-
ing in CRC cell lines SW480 and HT-29. To date, studies on LGR5 in
primary CRC samples have been constrained by the lack of a reliable
antibody against LGR5. We therefore induced inammation-driven
colon tumors in mice that were engineered to contain one enhanced
green uorescent protein (EGFP)-tagged Lgr5 allele (6). This allowed
ow cytometric separation of Lgr5 high and low cells based on GFP
expression and thus enabled a genome and transcriptome characteri-
zation of these two cell fractions. Our loss-of-function experiments
conclusively indicate that LGR5 acts as a marker for stem-like cells
in CRC.
Materials and methods
Cell lines and lentiviral transduction
The six human CRC cell lines (Caco-2, HCT 116, HT-29, SW480, SW620 and
T84) were purchased from the American Type Culture Collection (Manassas,
VA). All cell lines were cultured in media as recommended by the American
Type Culture Collection supplemented with fetal bovine serum (10% v/v),
-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 µg/ml).
Lentiviral shRNA transduction of SW480 and HT-29 cells was done using
high-titer lentivirus (Clone ID: V3LHS_635055, Open Biosystems, Thermo
Fisher Scientic, Lafayette, CO) according to the manufacturer’s instructions.
Mice
Athymic nude mice (strain NCr-nu/nu) were obtained from Frederick National
Laboratory for Cancer Research (Frederick, MD). Heterozygous Lgr5-EGFP-
IRES-CreERT2 mice [strain B6.129P2-Lgr5tm1(cre/ERT2)Cle/J, henceforth
referred to as 'Lgr5-EGFP mice'] were ordered from Jackson Laboratory (Bar
Harbor, ME) (6). All mice were bred and housed in a pathogen-free environ-
ment and used in experiments in accordance with institutional guidelines at
the Center for Cancer Research, National Cancer Institute, National Institutes
of Health. All experimental procedures conducted in this study were approved
by the Animal Care and Use Committee of the National Institutes of Health.
Abbreviations: AOM, azoxymethane; APC, adenomatous polyposis coli; CRC,
colorectal carcinoma; CSC, cancer stem cell; DSS, dextrane sodium sulfate;
EGFP, enhanced green uorescent protein; LGR5, leucine-rich-repeat-containing
G-protein-coupled receptor 5; mRNA, messenger RNA; qRT–PCR, quantitative
reverse transcription–polymerase chain reaction; shRNA, short hairpin RNA.
849

D.Hirsch etal.
Tumorigenicityassay
Tumorigenicity assay was performed as described previously (13).
Microarray gene expression proling of celllines
Total RNA was isolated from SW480 shLGR5 and control cells and from
SW480 spheres and adherent cells using the RNeasy Mini Kit (Qiagen,
Hilden, Germany) including DNase Itreatment (RNase-Free DNase Set,
Qiagen). RNA integrity was assessed by 2100 Bioanalyzer (RNA 6000
Nano LabChip Kit, Agilent Technologies, Santa Clara, CA). Appropriate
LGR5 status was confirmed by real-time quantitative reverse transcrip-
tion–polymerase chain reaction (qRT–PCR). Total RNA (700 ng) was
labeled using the Quick Amp Labeling Kit, one-color (Agilent) and sub-
sequently hybridized on Human GE 4x44K v2 Microarrays (Agilent)
according to the manufacturer’s protocol version 6.5. Slides were scanned
with microarray scanner G2565BA (Agilent). Images were analyzed and
data were quality controlled using Feature Extraction software version
10.7.1.1 (Agilent).
Carcinogen-induced inammation-driven colon tumorigenesismodel
To induce colon tumors, Lgr5-EGFP mice aged 2–4 months were injected
with azoxymethane (AOM, 12.5µg/g body weight; A5486, Sigma, St Louis,
MO) twice and subjected to three cycles of dextrane sodium sulfate (DSS,
2.5%; molecular weight = 36 000–50 000, MP Biomedicals, Solon, OH) in
the drinking water (14,15). Tumor growth was monitored by colonoscopies.
About 100days after the rst AOM injection, mice were killed.
RNA amplication and microarray gene expression proling of ow-sorted
normal mouse colons and mouse colontumors
RNA isolated from ow-sorted normal mouse colons and mouse colon
tumors was amplied together with spike-ins (1µl of a 1:50 000 dilution
per reaction, One-Color RNA Spike-In Kit, Agilent) using the Ovation Pico
WTA System (NuGEN Technologies, San Carlos, CA). The amplied com-
plementary DNA was labeled using the BioPrime® Total Genomic Labeling
Module (Invitrogen, Life Technologies, Carlsbad, CA) and subsequently 4µg
of Alexa Fluor® 3 labeled target was hybridized on Whole Mouse Genome
Microarrays 4x44K (Agilent) according to NuGEN’s Agilent Solution
Application Note #1. Scanning, image analysis and data quality control were
done as for cell lines.
DNA amplication and array comparative genomic hybridization of ow-
sorted mouse colontumors
DNA amplication and array comparative genomic hybridization were per-
formed as described previously and are summarized in Supplementary
Materials and methods, available at Carcinogenesis Online (16). Array-
based comparative genomic hybridization data have been deposited in Gene
Expression Omnibus database (data accession number: GSE46711).
Statistical analysis
Differences between groups were estimated by Student’s t-test, Fisher’s exact
test or two-way repeated measures analysis of variance (one factor repetition).
P<0.05 was considered signicant.
Microarray gene expression analysis
Log2 intensities were normalized to the 75% percentile according to the manu-
facturer’s protocol (Agilent). Only probes with intensities higher than 50 were
used for the analysis. Unsupervised hierarchical clustering with Euclidean
distance and Ward method was performed with Genomics Suite™ software
(Partek Incorporated, St Louis, MO). The corresponding functional annota-
tion of differentially expressed genes and their afliation with specic genetic
pathways were interrogated using Ingenuity Pathway Analysis software
(Ingenuity Systems, Redwood City, CA). Microarray gene expression data
have been deposited in Gene Expression Omnibus database (data accession
number: GSE46200).
Results
LGR5 is overexpressed in human CRC celllines
LGR5 expression is upregulated in primary CRC samples compared
with normal colorectal epithelium (17). Using real-time qRT–PCR,
we could show that LGR5 expression was also upregulated in CRC
cell lines Caco-2, HT-29, SW480, SW620 and T84 (Figure 1A)
(18,19). The only cell line without overexpression of LGR5 was HCT
116. This is consistent with recent ndings, suggesting that HCT 116
may not be organized hierarchically and may therefore not contain a
stem-like cell fraction (13,20,21).
LGR5 silencing leads to depletion of a morphologically distinct
subpopulation of SW480 CRCcells
To investigate the function of LGR5 in CRC, we transduced two
CRC cell lines (SW480 and HT-29), which both showed a simi-
lar moderate overexpression of LGR5, with lentiviral shRNA con-
structs. This signicantly reduced LGR5 expression compared with
empty vector control (henceforth referred to as ‘control’) cells as
assessed by real-time qRT–PCR (Figure1B). In SW480, silencing
of LGR5 led to a remarkable morphologic change, whereas HT-29
did not display an apparent difference (Figure1C). Unlike HT-29,
SW480 typically comprises two morphologically distinct subpopu-
lations, i.e. spheres and adherent cells, when cultured in serum-con-
taining medium. Upon LGR5 silencing, spheres were completely
depleted and only an adherent cell layer remained. Spheres, along
with LGR5 expression, remained undetectable in SW480 shLGR5
cells over more than 12 months of continuous passage, demon-
strating long-lasting effects of the shRNA on LGR5 expression. To
exclude that the disappearance of spheres was induced by cell split-
ting, we next separately analyzed LGR5 messenger RNA (mRNA)
expression in spheres and adherent cells of SW480 control cells.
Although LGR5 was highly expressed in spheres, adherent cells
displayed virtually no LGR5 expression, supporting our conclu-
sion that the morphologic change was caused by silencing of LGR5
(Figure1D).
LGR5 silencing in SW480 and HT-29 CRC cells reduces prolifera-
tion, migration and colony formation in vitro
To evaluate the inuence of LGR5 on cell proliferation, we com-
pared the cleavage of the tetrazolium salt WST-1 between shLGR5
and control cells. Upon silencing of LGR5, cleavage of WST-1 was
signicantly decreased in HT-29 cells (Figure 2A). SW480 cells
showed the same tendency although not reaching statistical signi-
cance (P=0.096). This might be attributable to the loss of spheres
in SW480 upon silencing of LGR5; spheres usually grow slower than
isogenic adherent cells (22). In other words, the proliferating cells of
SW480 reside within the LGR5 low adherent cells and not within the
LGR5 high spheres. This explains the small effect of LGR5 silencing
on proliferation inSW480.
The effect of LGR5 on migration was assessed by wound healing
and transwell migration assays. In the wound healing assay, SW480
shLGR5 cells tended to cover a smaller area of the initial scratch than
respective control cells at all time points, reaching statistical signi-
cance after 72 h (Figure2B). To reduce the impact of proliferation
as a confounding variable, we additionally monitored migration for
each group through transwell membranes and observed a signi-
cantly smaller number of migrated shLGR5 cells compared with con-
trol cells after incubating for 24 h (Figure2C). For HT-29, none of
the assays allowed a proper assessment of migration dependent on
LGR5 status owing to too little migratory activity of HT-29 cells in
our experiments.
In the colony formation assay, both cell lines formed signicantly
fewer colonies when LGR5 was silenced compared with control cells
(Figure2D).
LGR5 silencing reduces tumorigenic capacity of SW480 CRC cells
after xenotransplantation
To corroborate our in vitro colony formation results in vivo, we
injected CRC cells with differential LGR5 expression levels subcu-
taneously into the anks of nude mice and followed the appearance
of tumors. SW480 shLGR5 cells were less tumorigenic than control
cells and the tumors grew slower (Table I and Supplementary Figure
S1A–C, available at Carcinogenesis Online). Reduced tumorigenic-
ity was not observed for HT-29 cells upon LGR5 silencing (Table I).
However, tumors derived from HT-29 shLGR5 cells also grew slower
than those from control cells (Supplementary Figure S1D, available
at Carcinogenesis Online). Sufcient silencing of LGR5 in xenografts
was conrmed by real-time qRT–PCR (Supplementary Figure S1E,
available at Carcinogenesis Online).
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LGR5 in colorectalcancer
Lacking a reliable LGR5 antibody, we utilized the phenotypic
change of SW480 upon LGR5 silencing to separate LGR5 high cells
(spheres) and LGR5 low cells (adherent cells), which we then injected
subcutaneously into the anks of nude mice as a proof-of-principle
experiment. Conrming our hypothesis, only LGR5 high cells gener-
ated tumors; however, the small number of animals prevented reach-
ing statistical signicance (P=0.40) (Table I).
NOTCH signaling is downregulated upon LGR5 silencing
To examine whether differential LGR5 expression levels in CRC
cells would be reected in specic changes to the cellular transcrip-
tome, we performed microarray gene expression proling of SW480
shLGR5 and control cells and of SW480 spheres and adherent cells.
As LGR5 silencing in SW480 resulted in a profound morphologic
change, we reasoned that genes regulated by LGR5 might overlap
with genes differentially expressed between spheres and adherent
cells. Indeed, gene expression proling revealed a signicant overlap
of deregulated genes (false discovery rate < 1.0E-103; Figure3A).
These genes included known oncogenes (e.g. MYB, MYCN) and cer-
tain drug efux genes (e.g. ABCB1, ABCC2), whose expression was
positively correlated with LGR5 expression. The expression differ-
ences between spheres and adherent cells were more pronounced than
between shLGR5 and control cells, yet unsupervised hierarchical clus-
tering showed a clear separation of all four cell fractions (Figure3B).
Ingenuity Pathway Analysis revealed that the NOTCH signaling
pathway was downregulated when LGR5 was silenced. Conversely,
it was upregulated in LGR5 high spheres. Apart from LGR5, spheres
also expressed higher levels of several other stem cell-associated
genes including SOX2, ALDH1A1 and SMOC2 when compared
with adherent cells (12,23–25). Our gene expression results were
Fig.1. Expression levels of LGR5 in CRC cell lines and shRNA-mediated silencing of LGR5 in SW480 and HT-29 CRC cells. (A) LGR5 mRNA levels in CRC
cell lines as determined by real-time qRT–PCR. LGR5 mRNA levels were normalized to YWHAZ and are expressed as fold changes relative to normal colorectal
epithelial cells. Columns and error bars represent means ± SEM of two independent experiments using triplicate measurements in each experiment. (B) LGR5
silencing efciency in CRC cell lines SW480 and HT-29 upon lentiviral shRNA transduction as determined by real-time qRT–PCR. LGR5 mRNA levels were
normalized to YWHAZ and are expressed as fold changes relative to control. Columns and error bars represent means ± SEM of three independent experiments
using triplicate measurements in each experiment. (C) Representative phase-contrast images of SW480 and HT-29 cells upon LGR5 silencing. SW480 is
composed of two morphologically distinct subpopulations (spheres and adherent cells), whereas HT-29 shows one phenotype. After LGR5 silencing, spheres
in SW480 were completely lost with no apparent changes in HT-29. Scale bars, 100µm. (D) Expression of LGR5 in spheres versus adherent cells of SW480.
Spheres express high levels of LGR5, whereas in adherent cells, virtually no LGR5 expression can be detected by real-time qRT–PCR. LGR5 mRNA levels were
normalized to YWHAZ and are expressed as fold changes relative to control. Data represent means ± SEM of three independent experiments using triplicate
measurements in each experiment. *P<0.05, **P<0.005 and ***P<0.0005.
851

D.Hirsch etal.
Fig.2. Functional effects of LGR5 silencing on SW480 and HT-29 CRC cells. (A) LGR5 silencing decreases proliferation of SW480 and HT-29 CRC cells
as quantied based on cleavage of WST-1. Columns and error bars represent means ± SEM of three independent experiments using triplicate measurements
in each experiment. (B) Silencing of LGR5 reduces migration in wound healing assays. Columns and error bars represent means ± SEM from one experiment
representative of three independent experiments (n=6 scratches per cell type and time point in each experiment). Scale bars, 100µm. (C) In line with the results
of the wound healing assay, migration of SW480 shLGR5 cells is also decreased in transwell migration assays. Columns and error bars represent means ± SEM
from one experiment representative of three independent experiments using triplicate measurements in each experiment. Scale bars, 200µm. (D) LGR5 silencing
substantially reduces colony formation in vitro. Each 250 shLGR5 and control cells were cultured for 2 weeks. The number of colonies was then assessed using
crystal violet staining. Columns and error bars represent means ± SEM of three independent experiments using triplicate measurements in each experiment.
*P<0.05 and **P<0.005.
852

LGR5 in colorectalcancer
exemplarily validated by immunohistochemistry against two differ-
entially expressed genes, cleaved NOTCH1 and SOX6 (Figure3C).
Lgr5 is overexpressed in AOM/DSS-induced mouse colon tumors
but expression is, like in normal colon epithelium, restricted to a
small percentage ofcells
We next aimed to determine a detailed molecular characterization
of Lgr5 positive and negative cells in primary colon tumors. As the
lack of a reliable LGR5 antibody prevented these analyses in primary
human CRC, we exposed heterozygous Lgr5-EGFP mice, harbor-
ing one EGFP-tagged Lgr5 allele, to a carcinogen-induced inam-
mation-driven colon tumorigenesis model based on AOM and DSS
(Supplementary Figure S2A, available at Carcinogenesis Online)
(6,14). All AOM/DSS-induced tumors were restricted to the colon,
predominantly located in the rectosigmoid colon, sometimes forming
multiple lesions; no lymph node or hematogenous metastases were
detectable (Supplementary Figure S2B, available at Carcinogenesis
Online). Histologically, AOM/DSS-induced colon tumors in Lgr5-
EGFP mice resembled well-differentiated human tubular adenocarci-
nomas, mainly of the intramucosal type (Supplementary Figure S2C,
available at Carcinogenesis Online).
We rst examined expression, distribution and frequency of Lgr5
in AOM/DSS-induced colon tumors. Real-time qRT–PCR analysis
using RNA isolated from histologically dened tumor and non-tumor
regions revealed a signicant overexpression of Lgr5 in AOM/DSS-
induced tumors compared with normal colon mucosa (Figure4A and
Supplementary Figure S3A, available at Carcinogenesis Online).
Immunohistochemistry against GFP showed that, consistent with
previous reports, Lgr5-EGFP expressing cells in normal colons were
located at the crypt bottoms (Supplementary Figure S3B, available
at Carcinogenesis Online) (6). In AOM/DSS-induced tumors, Lgr5-
EGFP expression was found to be restricted to small populations of
scattered cells (Figure4B). Quantication by ow cytometric analysis
revealed that normal colons contained on average 3.8% of Lgr5-EGFP
high cells. This percentage was similar to that of AOM/DSS-induced
tumors, which harbored on average 3.4% of Lgr5-EGFP high cells
(Figure 4C). Supported by the results from our immunohistochem-
istry-based analysis of Lgr5-EGFP expression, this suggests that a
stem cell hierarchy is preserved in AOM/DSS-induced tumors (9,11).
However, there were 3 out of 14 (21.4%) AOM/DSS-induced tumors
without detectable Lgr5-EGFP expressing cells based on GFP expres-
sion, indicating that occasionally also Lgr5-EGFP low cells could
acquire the capacity for tumor initiation and/or maintenance (10).
This is consistent with observations in human CRC samples, in which
LGR5-expressing cells were not detectable in one-third of the ana-
lyzed samples, arguing for a frequent stem cell and a rare non-stem
cell driven carcinogenesis (12). We conrmed by real-time qRT–PCR
that GFP high ow-sorted cells had signicantly higher Lgr5 expres-
sion levels than GFP low ow-sorted cells (Figure4D).
Transcriptome proles of Lgr5 high and low epithelial cells from
AOM/DSS-induced mouse colon tumors are clearly distinct
To examine whether the differential Lgr5 expression levels in nor-
mal mouse colons and AOM/DSS-induced mouse colon tumors
would be reected in specic gene expression proles, we performed
microarray gene expression proling of normal colons and AOM/
DSS-induced tumors ow-sorted into Lgr5 high and low fractions
based on GFP expression. Reassuringly, the previously described
intestinal stem cell-specic genes Lgr5 and Smoc2 were upregulated
in Lgr5 high normal and tumor cells compared with Lgr5 low normal
and tumor cells (12,25). In addition, Lgr5 high tumor cells showed
increased expression of EphB2, which is known to be coexpressed
with Lgr5 (12). Unsupervised hierarchical clustering showed a clear
separation between Lgr5 high and low tumor cells; however, separa-
tion according to Lgr5 expression levels was not as clear in normal
cells (Figure5A). Ingenuity Pathway Analysis revealed that the Wnt
signaling pathway was upregulated in Lgr5 high tumor cells com-
pared with Lgr5 low tumor cells and also with Lgr5 high normal colon
epithelial cells. Gene expression results were exemplarily validated
by immunohistochemistry against Sox6 (Figure5B).
Lgr5 high and low epithelial cells from AOM/DSS-induced mouse
colon tumors are both chromosomallystable
Genomic imbalances inuence gene expression patterns (26). To
exclude that the transcriptional differences between Lgr5 high and
low tumor cells were imposed by distinct patterns of chromosomal
aberrations in the two cell fractions, we additionally performed array
comparative genomic hybridization from the ow-sorted AOM/DSS-
induced mouse colon tumors. All eight tumors were chromosomally
stable, and thus, no difference between Lgr5 high and low cells could
be detected (Supplementary Figure S4, available at Carcinogenesis
Online) (27). This indicates that other mechanisms, for instance
epigenetics, may be the driving force in AOM/DSS-induced mouse
colon tumors (28). In conclusion, these data conclusively indicate
that LGR5 marks stem-like CRC cells and denes a cell compart-
ment in which proliferating, migrating and tumorigenic CRC cells
are enriched. This is consistent with the observed upregulation of
stem cell-related signaling pathways such as NOTCH or Wnt in
LGR5 high CRC cells.
Discussion
Here, we studied the functional and molecular consequences of
LGR5 silencing in CRC cell lines and identied LGR5 as a marker
for stem-like cells in CRC. Based on Lgr5 expression, we dened a
gene expression signature for stem-like cells in CRC using an inam-
mation-driven mouse colon tumorigenesis model based on AOM and
DSS, which mimics sporadic CRC development.
To understand the role of LGR5 in colorectal tumorigenesis, which
is controversial, we silenced its expression in two CRC cell lines
(SW480 and HT-29) (18,29–34). Silencing of LGR5 in these cell
lines resulted in reduced proliferation, migration and colony forma-
tion in vitro as well as reduced tumorigenicity in vivo. This is consist-
ent with previous studies targeting LGR5 in various cancer entities
including basal cell carcinoma, gastric cancer, glioblastoma and CRC
(18,29,31–34). In these cancers, expression of LGR5 was associated
with increased cell proliferation, migration and invasion and decreased
apoptosis. In turn, silencing of LGR5 in these cancers decreased pro-
liferation, colony formation and tumorigenicity and enhanced apop-
tosis. Upregulation of LGR5 in non-tumorigenic NIH3T3 broblasts
Table I. Quantication of tumor initiation in nude mice after subcutaneous injection of CRC cells with differential levels of LGR5 expression
Cell line shLGR5
(no. of tumors/no. of injections)
Control
(no. of tumors/no. of injections)
No. of
injected cells
P value
SW480 4/10 9/10 2000 0.0052
2/5 5/5 20 000
HT-29 8/10 10/10 2000 0.47
Cell line Adherent cells
(no. of tumors/no. of injections)
Spheres
(no. of tumors/no. of injections)
No. of
injected cells
P value
SW480 0/3 2/3 2000 0.40
853

D.Hirsch etal.
Fig.3. Gene expression proling of shLGR5 versus control cells and adherent cells versus spheres of SW480. (A) Venn diagram showing a signicant overlap
of differentially expressed genes when comparing shLGR5 versus control cells and adherent cells versus spheres of SW480 (false discovery rate < 1.0E-103). (B)
Unsupervised hierarchical clustering based on gene expression proles of shLGR5 and control cells as well as adherent cells and spheres from SW480. Samples
cluster by cell type rst. The different LGR5 fractions are clearly separated. (C) In line with our gene expression data, immunohistochemistry against both
cleaved NOTCH1 and SOX6 also shows a lower expression in SW480 shLGR5 than in SW480 control cell derived xenograft tumors. Scale bars, 20µm.
854

LGR5 in colorectalcancer
and in HaCat keratinocytes induced colony formation and promoted
tumorigenicity (18,29).
For reasons that remain to be understood, all those results are in
contrast to the ndings by Walker and colleagues in CRC cell lines
LIM1215 and LIM1899. Upon silencing of LGR5, they reported
increased migration, colony formation and tumorigenicity, and oppo-
site phenotypes when LGR5 was overexpressed (30).
The reduced tumorigenic capacity of SW480 in vivo upon LGR5
silencing, along with the decreased colony formation in vitro, suggests
that LGR5 expression is associated with stem-like properties in this cell
line. The observation that shLGR5 and control HT-29 cells were equally
tumorigenic upon xenotransplantation is somewhat contradictory but
might be either explained by the lower silencing efciency or because
tumorigenicity of HT-29 does not depend on LGR5 expression. Also,
HT-29 has much lower Wnt activity (35). The number of injected cells
might have been too high to reveal differences since HT-29 is a highly
tumorigenic cell line. Alternatively, the reduction of tumor growth rate
but not tumor incidence upon LGR5 silencing in HT-29 might suggest
that LGR5 silencing affects tumor propagation rather than tumor initia-
tion in this cell line. The slower tumor growth rate upon silencing of
LGR5 for both SW480 and HT-29 cells is consistent with previous data
showing that Lgr5 positive cells are actively proliferating (6).
Fig.4. Expression, distribution and frequency of Lgr5 in AOM/DSS-induced mouse colon tumors. (A) Varying degrees of overexpression of Lgr5 in AOM/DSS-
induced colon tumors as determined by real-time qRT–PCR. Lgr5 mRNA levels were normalized to Gapdh and are expressed as fold changes relative to normal
adjacent colon. Horizontal lines represent means. Triplicate measurements were used for each data point. (B) Lgr5-EGFP expression in AOM/DSS-induced
colon tumors is restricted to small populations of scattered cells. Scale bars, 50µm. (C) Quantication of Lgr5-EGFP high cells by ow cytometric analysis
revealed that both normal colons and AOM/DSS-induced tumors contain similar small percentages of Lgr5-EGFP high cells. N, normal colon; T, tumor. (D) Lgr5
expression is enriched in ow-sorted Lgr5-EGFP high normal colon or colon tumor cells compared with respective Lgr5-EGFP low normal colon or colon tumor
cells as determined by real-time qRT–PCR. Lgr5 mRNA levels were normalized to Gapdh. Horizontal lines represent means. Triplicate measurements were used
for each data point. *P<0.05 and **P<0.005.
855

D.Hirsch etal.
Our functional analyses were complemented by studying global
gene expression levels of cell fractions with differential LGR5 expres-
sion. Spheres (in contrast to adherent cells) of SW480 expressed
several stem cell-associated genes including LGR5 and showed an
upregulation of NOTCH signaling. Consistently, spheres appeared to
be more tumorigenic than adherent cells when xenografted in nude
mice. Silencing of LGR5 resulted in the depletion of spheres. In line
with our ndings, LGR5 is upregulated in spheroid cultures of colo-
rectal CSCs and, conversely, becomes downregulated during in vitro
differentiation of these CSCs (32). Taken together, these ndings sug-
gest that LGR5 identies a stem-like cell compartment in CRC, as it
does in normal colorectal epithelium. This has recently been shown
directly by in vivo lineage tracing in mouse intestinal adenomas (11).
NOTCH signaling was downregulated in SW480 CRC cells upon
LGR5 silencing, whereas genes involved in NOTCH signaling were
overexpressed in LGR5 high SW480 spheres. Consistently, Lgr5
deciency in the mouse during intestine development also seems
to have an inhibitory effect on the Notch signaling pathway (36).
In addition, downregulation of NOTCH signaling in CRC cell lines
and primary CRC samples via reduction of NOTCH1 or RBPJk
decreases proliferation, colony formation and tumorigenicity,
whereas upregulation of NOTCH signaling via NOTCH1 results in
opposite changes (3,37,38). This has also been demonstrated for
other tumor entities including pancreatic, lung or breast cancer
(39–41). Consistent with our gene expression analysis, it appears
most likely that the reduced proliferation and migration after LGR5
Fig.5. Gene expression proling of normal mouse colons and AOM/DSS-induced mouse colon tumors ow-sorted for Lgr5-EGFP. (A) Unsupervised
hierarchical clustering based on gene expression proles of Lgr5 high and low fractions from normal colons and AOM/DSS-induced tumors. Samples cluster
by entity (normal colon or tumor) rst. In tumors, the different Lgr5 fractions are clearly separated; however, in normal colons, they are separated by both Lgr5
and individual mice. (B) In line with our gene expression data, Sox6 expression could also be demonstrated by immunohistochemistry (positivity in a small
population of scattered cells in AOM-/DSS-induced tumors). Scale bar, 20µm.
856

LGR5 in colorectalcancer
silencing in CRC cells are a consequence of reduced NOTCH sign-
aling (3).
Hence, our gene expression results substantiate our ndings from
functional assays upon LGR5 silencing in CRC cell lines at a molec-
ular level, suggesting LGR5 as a potential therapeutic target. For
instance, Honokiol inhibits CRC growth by targeting NOTCH signal-
ing in colorectal CSCs (42).
Pursuing stem cells as therapeutic targets might help to overcome
some of the frustrations associated with current cancer treatment regi-
mens. Cataloging gene expression data from these stem cells serves
as a rst step in understanding the molecular features distinguishing
these cell types from the bulk of tumor cells or from normal adult tis-
sue stem cells (43–46).
Despite the advantages of in vitro cultures to analyze features
of stemness potential, such as ease of propagation, there are con-
cerns that molecular changes might be induced when cells are cul-
tured in the absence of their physiological context (13,47). Thus,
we extended our gene expression proling to ex vivo isolated cells.
Normal mouse colons and AOM/DSS-induced mouse colon tumors
from Lgr5-EGFP mice were ow-sorted into Lgr5 high and low epi-
thelial cell fractions. Global gene expression analyses of ow-sorted
fractions revealed an overexpression of the Wnt signaling pathway
in Lgr5 high tumor cells compared with both Lgr5 low tumor cells
and Lgr5 high normal cells. This supports previous ndings showing
that Wnt activity denes colorectal CSCs (48). Although Horst etal.
(49) found that Wnt activity alone might not be sufcient to convey
stem-like potential, our ndings suggest that the combination of both
Wnt signaling and Lgr5 might help to determine cells with stem-like
properties in CRC.
The association of LGR5 and NOTCH signaling seen in cell lines
could not be recapitulated in vivo when ow sorting AOM/DSS-
induced mouse colon tumors for Lgr5. Also, in contrast to ow-
sorted Lgr5 high and low cells from AOM/DSS-induced tumors,
the Wnt signaling pathway was not signicantly altered in our loss-
of-function experiments in cell lines, though tending to be higher
in LGR5 high spheres compared with LGR5 low adherent cells
(P = 0.16). The biological difference of cell lines being cultured
without their physiological context might explain these ndings.
Increasing evidence indicates that not only intrinsic factors but also
extrinsic factors like the microenvironment can inuence the CSC
phenotype. For instance, Vermeulen etal. (48) showed in CRC that
stromal myobroblasts surrounding CSCs not only can maintain
Wnt signaling activity in CSCs but also can activate Wnt signal-
ing in more differentiated tumor cells and thereby induce the CSC
phenotype. Furthermore, there is a methodological difference that
might contribute to the heterogeneity of our ndings in cell lines
and ex vivo isolated tumor cells. In the ex vivo model, ow-sorted
Lgr5 high and low cells were compared, whereas in the cell line
experiment, LGR5 was silenced actively via shRNA and compared
with the original cell population. Overall, the observed heterogene-
ity across the different lines and models reects one of the major
problems in CSC research.
In summary, our comprehensive functional and molecular analy-
sis of LGR5 in CRC cell lines and AOM/DSS-induced mouse colon
tumors conclusively links LGR5 to stem-like cells in CRC. LGR5
did not only serve as a marker for these stem-like CRC cells but was
also of functional relevance for CRC cells, thus representing a poten-
tial therapeutic target, in particular as conditional deletion of Lgr5
in mouse guts does not seem to negatively affect normal intestinal
epithelium (8). To further specify the role of LGR5 in human CRC,
studies of LGR5 in primary human CRC specimens will be needed in
the future and, as a prerequisite, the development of a reliable LGR5
antibody.
Supplementary material
Supplementary Material and methods, Table S1 and Figures S1–S4
can be found at http://carcin.oxfordjournals.org/
Funding
Intramural Research Program, National Institutes of Health, National
Cancer Institute; German Academic Exchange Service (D.H.).
Acknowledgements
The authors thank B.Chen for help with gures and IT-related support,
X.Lu for help with cell culture-related experiments, M.E.Jorge (Veterinary
Technician, Animal Facility, National Institutes of Health) for help with nude
mouse tumor measurements, D.Despres and B.Klaunberg (Mouse Imaging
Facility, National Institutes of Health) for advice and assistance with mouse
colonoscopies, B.Taylor (FACS Core Laboratory, Center for Cancer Research,
National Cancer Institute, National Institutes of Health) for expert technical
assistance with ow sorting, K.Wolk (Immunohistochemistry Laboratory,
Institute of Pathology, University Medical Center Mannheim) for help with
immunohistochemical staining and C.A.Klein (Experimental Medicine and
Therapy Research, University of Regensburg) for providing expert advice.
Conict of Interest Statement: None declared.
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Received August, 1, 2013; revised October 29, 2013;
accepted November 6, 2013
858