Research article

CD13 is a therapeutic target in human liver

cancer stem cells
Naotsugu Haraguchi,1,2 Hideshi Ishii,1,3 Koshi Mimori,3 Fumiaki Tanaka,3 Masahisa Ohkuma,4
Ho Min Kim,1 Hirofumi Akita,1 Daisuke Takiuchi,1 Hisanori Hatano,1 Hiroaki Nagano,1
Graham F. Barnard,5 Yuichiro Doki,1 and Masaki Mori1
1Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, Osaka, Japan.
2Excellent
Young Researchers Overseas Visit Program, Japan Society for the Promotion of Science (JSPS), Tokyo, Japan. 3Department of Surgery,
Medical Institute of Bioregulation, Kyushu University, Oita, Japan. 4Department of Surgery, Jikei University School of Medicine, Tokyo, Japan.
5Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

Cancer stem cells (CSCs) are generally dormant or slowly cycling tumor cells that have the ability to recon-
stitute tumors. They are thought to be involved in tumor resistance to chemo/radiation therapy and tumor
relapse and progression. However, neither their existence nor their identity within many cancers has been well
defined. Here, we have demonstrated that CD13 is a marker for semiquiescent CSCs in human liver cancer cell
lines and clinical samples and that targeting these cells might provide a way to treat this disease. CD13+ cells
predominated in the G0 phase of the cell cycle and typically formed cellular clusters in cancer foci. Following
treatment, these cells survived and were enriched along the fibrous capsule where liver cancers usually relapse.
Mechanistically, CD13 reduced ROS-induced DNA damage after genotoxic chemo/radiation stress and pro-
tected cells from apoptosis. In mouse xenograft models, combination of a CD13 inhibitor and the genotoxic
chemotherapeutic fluorouracil (5-FU) drastically reduced tumor volume compared with either agent alone.
5-FU inhibited CD90+ proliferating CSCs, some of which produce CD13+ semiquiescent CSCs, while CD13
inhibition suppressed the self-renewing and tumor-initiating ability of dormant CSCs. Therefore, combining
a CD13 inhibitor with a ROS-inducing chemo/radiation therapy may improve the treatment of liver cancer.

Introduction proliferating cancer cells but not reduce the survival of CSCs in
Functional and morphologic heterogeneity exists in a tumor with the dormant or slow-growing phase. Thus, the identification and
a hierarchy in which tumor growth is driven by a small subset of characterization of dormant or slow-growing CSCs are important
cancer stem cells (CSCs) (1). Like normal tissue stem cells, which for developing novel therapeutic approaches.
are capable of self renewal and multidifferentiation, CSCs have the In studies of hepatocellular carcinoma (HCC) (7), the fifth most
ability to reconstitute tumors (2). In the hematologic cell lineage, common cancer in the world, the SP fraction (8), CD133+ (9–11),
stem cells exist in the dormant phase and can be detected as a side CD44+ (11, 12), CD90+ (12, 13), and epithelial cell adhesion mol-
population (SP) (3). Generally, CSCs, like somatic tissue stem cells, ecules (14) were reported as markers of CSCs or cancer-/tumor-ini-
proliferate slowly, i.e., they are in the dormant or slow-growing tiating cells. The majority of CSC studies focus on identification
phase of the cell cycle. This partially accounts for their therapeu- of cell markers to enrich cell populations that have high tumor
tic refractoriness to chemo/radiation therapy, tumor relapse, and initiation ability in immune-deficient mice. In the field of liver
presumably metastasis. The CSCs of acute myeloid leukemia (4) cancer CSCs, there have been few reports describing dormant or
and chronic myeloid leukemia (5) also survive in the dormant G0 slow-growing CSCs that include their cellular characteristics or
phase of the cell cycle in a bone marrow niche after chemotherapy. indicate a way to target these cells based on cytological evidence.
Relapses and metastases of breast cancer often occur after inter- In addition, there have been few reports that clearly indicate the
vals of several decades, suggesting the involvement of a deep dor- interrelationships among these candidate markers.
mant phase for CSCs (6). The majority of liver cancers are superim- In a previous study (similar to hematopoietic and leukemic stud-
posed on a background of chronic hepatitis and hepatic cirrhosis. ies), we reported that the SP fraction enriches the CSC-like frac-
Therefore, it may be difficult to distinguish between intrahepatic tions. Cells of the SP fraction express both hepatocyte and cholan-
metastasis through portal or hepatic venules and metachronous giocyte markers, show high resistance to anti-cancer agents, and
multicentric development of liver cancer in a precancerous back- high tumorigenicity in NOD/SCID mice (8). Based on our previous
ground. However, there are some cases of liver cancer in which can- data (8) and applying the techniques of hematopoietic stem cell
cer recurs in the liver or metastasizes to the lung and bone several studies (3–5), our aims were as follows: first, to clarify the relation-
years after radical hepatectomy or liver transplantation. This sug- ships between reported candidate CSC markers; second, to assess
gests that some slow-growing cancer cells also exist in liver cancer whether dormant CSCs exist in liver cancer and to concentrate on
but these may not be in deep dormancy like breast cancer CSCs. cell-surface markers, which definitively identify potentially dor-
Anticancer reagents in clinical use generally affect division and mant CSCs; third, to clarify the cellular characteristics of poten-
proliferation of cancer cells. This could result in elimination of tially dormant CSCs and to identify the mechanisms that protect
potentially dormant CSCs from chemo/radiation therapy; finally,
Conflict of interest: The authors have declared that no conflict of interest exists. to identify target molecules of liver cancer CSCs to initiate novel
Citation for this article: J Clin Invest. 2010;120(9):3326–3339. doi:10.1172/JCI42550. approaches that could lead to a future radical cure for liver cancer.

3326 The Journal of Clinical Investigation    http://www.jci.org    Volume 120    Number 9    September 2010
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Figure 1
CD13 is a candidate marker of the SP fraction. (A) The strategy used to identify cell-surface markers closely related to the SP fraction. We
determined CD13 and CD31 as candidate markers for identifying SP cells. (B) Both CD13 and CD31 expression in HuH7 cells were compared
in SP and non-SP cells by semiquantitative RT-PCR. Data represent mean ± SD from independent experiments of fractions differentially sorted
by flow cytometry. *P < 0.01; **P = 0.076 versus non-SP fractions. The cut-off lines were determined using isotype controls. (C) Expression of
CD13, CD133, and CD90 in HuH7 (upper panels) and PLC/PRF/5 cells (lower panels). Horizontal and vertical axes denote expression intensity.
(D) The SP fraction is recognized as a “beak” appearing beside the G1 phase fraction. The relationship between CD13+ and CD13− cells and the
SP fraction was studied using multicolor flow cytometry.

Results sion of CD31 was abundant in the G2/M/SP fraction but was
CD13 is a candidate marker closely correlated with SP cells. To identify not universal in the liver cancer cell lines studied by us (HuH7,
specific cell-surface markers that correlate with the SP fraction, PLC/PRF/5, and Hep3B), and the statistical significance was weak
we utilized our previous data sets of SP and non-SP fraction gene (P = 0.076) (Figure 1B and Supplemental Figure 1, A and B).
expression profiles obtained using microarray analyses (8). From a Expression of CD13, CD133, and CD90 was assessed in hepatitis
list of 268 genes upregulated in the SP cells (with a fold change > 2) infection–negative (HuH7) and –positive (PLC/PRF/5) cell lines.
(8), we selected 56 genes that potentially encode cell-surface proteins The expression of CD133 was detected in HuH7 but not in PLC/
via the UniProtKB database (http://www.uniprot.org/). Working PRF/5, and the expression of CD90 was detected in PLC/PRF/5
from the list of 56 upregulated genes (Supplemental Table 1; sup- but not in HuH7. The expression of CD13 was observed in both
plemental material available online with this article; doi:10.1172/ these cell lines as well as in Hep3B (Figure 1C and Supplemental
JCI42550DS1) and an additional 43 markers reported to be closely Figure 1A). In HuH7 in particular, the CD13+ cells typically existed
associated with normal stem cells and CSCs, we tested 47 commer- in a CD133strong fraction (CD13+CD133+).
cially available antibodies (Supplemental Table 2) to identify surface Multicolor analysis with Hoechst staining exhibited clear local-
markers that were enriched in the SP fraction (Figure 1A). ization of CD13+ cells in the SP fraction of HuH7 and PLC/PRF/5,
During this screening, we identified 2 candidate markers, CD13 whereas the CD13−CD133+ and CD90+ fractions were localized to
and CD31. The expression analysis of CD13 was 1.64 ± 0.45 in the the G1-to-G2 fraction and not the SP fraction (Figure 1D). To con-
SP and 0.51 ± 0.03 in the non-SP cell fraction (P < 0.01) (Figure firm the cell-cycle status of the CD13+ cells in PLC/PRF/5, cell-cycle
1B). We focused on CD13 in the current study, since the expres- analysis by combined multicolor analysis and 7-amino-actinomy-

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Figure 2
CD13 is a candidate marker of dormant to slow-growing CSCs. (A) Dormant cells can be identified using the DNA-binding dye Hoechst 33342
and RNA-binding dye PY. Dormant cells contain lower RNA levels than G1 phase cells. Combination analysis of the cell cycle with cell-surface
markers CD13, CD133, and CD90 was performed with reserpine. The cut-off lines were determined using isotype controls. (B) Time-lapse cell-
fate tracing of HuH7 cells. Cells were labeled with PKH26GL, isolated to their CD13+, CD13−CD133+, and CD13−CD133− fractions, and traced
for 238 hours. The dye-retaining cells can be identified as red-labeled cells (white arrow). Original magnification, ×20. (C) Proliferation assay
of the CD13+CD133+, CD13−CD133+, and CD13−CD133− fractions. Data represent mean ± SD from independent experiments of fractions dif-
ferentially sorted by flow cytometry. *P < 0.05. (D) BrdU-retaining cells in serially transplanted control tumor specimens of HuH7 (6 weeks after
BrdU injection) and PLC/PRF/5 (10 weeks after BrdU injection). The sections were stained with anti-CD13 (red), BrdU (green), and DAPI (blue).
Top panels show lower magnification of the sections of HuH7 and PLC/PRF/5 (×10, HuH7 and left panel of PLC; ×20, right panel of PLC). The
lower panels show high magnification (×40) of the place indicated by white arrows in the top panels.

cin D (7-AAD) DNA labeling was performed. The CD13+CD90− relationships with the cell-cycle phase, using the DNA-binding
population was mainly in the G0/G1 phase, and the CD13+CD90+ dye Hoechst 33342 and the RNA-binding dye pyronin Y (PY) (3),
population was clearly in the S to G2/M phase. The CD13−CD90+ indicated that most of the CD13+ fraction exists in the G1/G0
cells were present in all phases of the cell cycle but were more phase and the CD13strong population was clearly localized in G0.
clearly present in the G2/M and S phases when compared with the The CD133+ population in HuH7 and the CD90+ fraction in PLC/
CD13+CD90− population (Supplemental Figure 1C). PRF/5 were distributed in the G1/G0 and G2/M phases, respec-
In these studies, we confirmed CD13 as a universal candidate tively. The relationships between the SP fraction and the G0 cell-
marker that correlates with the liver cancer SP fraction. There were cycle phase were also confirmed, and the SP fraction was clearly
no definitive single markers that showed a stronger correlation to localized in the G0 phase under reserpine-free (ABC transporter
the SP fraction than CD13 and, to a lesser extent, CD31. blocker) conditions (Figure 2A).
CD13 is a marker of tumor-initiating and potentially dormant HCC To study the cell fate and dye-retaining capacity of HuH7 CD13+
cells. Given that hematopoietic and leukemic stem cells are in cells, the cell-surface membrane was labeled with PKH26GL reagent
the G0 phase, identification and characterization of dormant or and cell fate was traced for 238 hours. Equal numbers of cells were
slow-growing cancer cell populations is very important because of seeded for each population. The CD13+CD133+ fraction exhib-
these populations’ relevance to chemo resistance and recurrence. ited very slow growth compared with the CD13−CD133+ fraction,
Studies of CD13 expression in HuH7 and PLC/PRF/5 and their with the doubling time of the CD13+CD133+ fraction estimated

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 research article

Table 1 to single cells and serially trans-

Limiting dilution and serial transplantation assay of HuH7 and PLC/PRF/5 cells planted without isolation of
cell-surface markers. BrdU was
Cell type Assay Marker/cells 1 × 102 5 × 102 1 × 103 5 × 103 1 × 104 injected intraperitoneally. After
HuH7 Limiting dilution CD13+CD133+ 2/4 2/4 3/4 3/4 – 6 weeks for HuH7, and after 10
HuH7 Limiting dilution CD13–CD133+ 0/4 0/4 3/4 4/4 – weeks for PLC/PRF/5, tumors
HuH7 Limiting dilution CD13–CD133– 0/4 0/4 0/4 0/4 – were enucleated and sections
HuH7 Serial transplantation CD13+CD133+ 0/4 1/4 3/4 4/4 3/4 were stained with anti-BrdU
HuH7 Serial transplantation CD13–CD133+ 0/4 0/4 0/4 0/4 1/4 and anti-CD13 antibody. In
HuH7 Serial transplantation Control 0/4 0/6 0/6 0/6 2/6 the tumors derived from HuH7
PLC Limiting dilution CD13+CD90– 2/4 2/2 4/4 3/3 –
cells, very small numbers of
PLC Limiting dilution CD13–CD90+ 0/4 2/2 4/4 3/3 –
BrdU-retaining cells that also
PLC Limiting dilution CD13 CD90
– – 0/4 0/4 0/3 0/4 –
PLC Serial transplantation CD13+CD90– 0/4 2/4 3/4 3/4 3/4 expressed CD13 were observed
PLC Serial transplantation CD13 CD90
– + 0/4 0/4 0/4 0/4 1/4 typically in the edge of tumor
PLC Serial transplantation Control 0/6 0/6 0/6 1/6 2/6 foci. BrdU-retaining cells were
not observed in the tumor
center. Tumors derived from
PLC/PRF/5 cells grew more
slowly than did those derived
at approximately 160 hours. Dye-retaining cells could be observed from HuH7 cells. BrdU-retaining cells could be identified and did
238 hours after cell seeding only in the CD13+CD133+ fraction express CD13. Interestingly, clusters of CD13+BrdU– cells were
(Figure 2B and Supplemental Videos 1–3). The CD13−CD133− observed close to CD13+BrdU+ cells, suggesting that they might
fraction exhibited cell fragmentation and apoptotic changes dur- be derived from the CD13+BrdU+ cells (Figure 2D).
ing cell culture. To confirm CD13 expression in association with HCC-CD13+ cells form spheres and produce the CD90 + phenotype.
cell growth, we performed cell proliferation assays. Data from iso- Sphere formation is a common characteristic of stem cells. To
lated HuH7 populations showed that CD13+CD133+ cells exhib- evaluate CD13 as a candidate CSC marker, the expression of CD13
ited slow cell growth compared with CD13−CD133+ cells 72 hours in spheres derived from HuH7, PLC/PRF/5, and clinical HCC was
after seeding (Figure 2C). The CD13−CD133− population also studied. The expression of CD13 was increased in both HuH7
grew slowly but maintained viability for a week, with difficulty, (2.0% in control vs. 67.0% in spheres; 33.5-fold increase) and PLC/
because of apoptosis. PRF/5 (15.2% in control vs. 83.8% in spheres; 5.51-fold increase)
Next, tumor-formation ability of each fraction was studied in (Figure 3A). There was no significant change in CD133 expression
HuH7 and PLC/PRF/5 cells. Limiting dilution analysis of HuH7 in HuH7. In PLC/PRF/5, expression of CD90 was decreased in the
cells revealed that the CD13 +CD133+ fraction formed tumors spheres (35.7% in control vs. 2.5% in spheres; 14.28-fold decrease).
from 100 cells (2/4), the CD13−CD133+ fraction formed tumors The expression of CD13 compared with that of CD133 and CD90
from 1,000 cells (3/4), and the CD13−CD133− fraction formed no appeared to be associated with a more immature stem-like and
tumors from 5,000 cells (0/4) in NOD/SCID mice after 4 weeks dormant population. Spheres established from clinical HCC sam-
of observation. In PLC/PRF/5 cells, the CD13+CD90− fraction ples localized in the CD13+CD90−CD133− fraction in a manner
formed tumors from 100 cells (2/4), and the CD13−CD90+ fraction similar to that observed in PLC/PRF/5 (Figure 3B).
formed tumors from 5,000 cells (2/2), whereas the CD13−CD90− The time-course changes in the expression of CD13 and CD90 in
cells formed no tumors from 5,000 cells (0/4) in NOD/SCID mice PLC/PRF/5 were studied. Isolation and culture of the CD13+CD90−
after 6 weeks of observation (Table 1). To assess the tumor forma- fraction from the PLC/PRF/5 spheres in serum-containing media
tion ability definitively, formed tumors were digested, and isolated resulted in the production of CD13+CD90+ fraction after 96 hours
CD13+CD133+ and CD13−CD133+ fractions of HuH7 and isolated (Figure 3C). The isolated CD13−CD90− fraction elicited cell death
CD13+CD90− and CD13−CD90+ fractions of PLC/PRF/5 were seri- within a few days and could not be maintained. Interestingly, the
ally transplanted to secondary NOD/SCID mice. As controls, non- isolated CD13−CD90+ fraction rapidly produced the CD13+CD90−
isolated cell fractions of HuH7 and PLC/PRF/5 were also serially fraction within 24 hours (Figure 3C). These findings suggest that
transplanted. Tumor formation ability of CD13+ cells compared potentially dormant CD13+ cells produce proliferating CD90+ cells
with that of CD13− cells in serial transplantation assay was demon- and that some parts of the proliferating CD90+ cells also produce
strated more clearly than that of limiting dilution assay. In HuH7, CD13+ cells. It is important to determine how this CD13+ popula-
after 6 weeks of observation, the CD13+CD133+ fraction formed tion (slow-growing potentially dormant) could be maintained in a
tumors from 500 cells (1/4), whereas the CD13−CD133+ fraction cancer cell line in vitro. Dormant or slow-growing cell populations
and control formed very small tumors only in 10,000 cells (1/4 in may disappear during continuous subculturing. These findings
the CD13−CD133+ fraction, and 2/6 in control). In PLC/PRF/5, may replicate the rapid change from dormant to active status in
after 6 weeks of observation, the CD13+CD90− formed tumors cancer stem-like cells, as revealed by their cell-surface markers, or
from 500 cells (2/4), and the control formed tumors from 5,000 the dormant cells might mimic a certain multipotent condition in
cells (1/6), whereas the CD13−CD90+ fraction formed 1 very small cellular differentiation.
tumor only in 10,000 cells (1/4) (Table 1). CD13+ cells resist chemotherapy, and CD13 inhibition drives cells to apop-
To assess semiquiescent status of CD13+ cells in vivo, BrdU- tosis. The change of cell-surface marker expression before and after
retaining status was studied. Tumors obtained from NOD/SCID doxorubicin (DXR) hydrochloride treatment or 5-fluorouracil
mice xenografted with HuH7 and PLC/PRF/5 cells were digested (5-FU) was studied in HuH7 and PLC/PRF/5. In HuH7, CD13

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Figure 3
CD13+ cells exist as a core in HCC spheres and
produce CD90+ cells. (A) Spheres established
from HuH7 and PLC/PRF/5 cells were dissoci-
ated to single cells and the marker expressions
were compared with control cells. Scale bars: 200
μm. (B) Expression analysis of primary human
HCC cells (control) and spheres established from
original human HCC cells (sphere). Scale bar:
200 μm. (C) The time-course expression analy-
ses of sorted CD13+CD90− cells (upper panels)
and CD13−CD90+ cells (lower panels) from PLC/
PRF/5. The cut-off lines were determined using
isotype controls.

assay in HuH7. The CD13+CD133+ fraction

was highly resistant to DXR compared with the
CD13−CD133+ and CD13−CD133− fractions,
indicating consistent changes in the markers
following DXR treatment (Supplemental Fig-
ure 2A). Although the CD13−CD133− fraction
exhibited slow cell growth in the proliferation
and cell fate study (Figure 2, B and C), this frac-
tion showed high chemosensitivity.
Next, the effect of CD13 inhibition on cell
proliferation in HuH7 was assessed. Cell pro-
liferation was suppressed in a concentration-
dependent manner after 72 hours exposure to
the CD13-neutralizing antibody. At 10 and 20
μg/ml concentrations of the CD13-neutralizing
antibody, cell proliferation was suppressed by
approximately 80% at 24 hours and 95% at 72
hours (Figure 4B). The apoptosis assay showed
that both the CD13-neutralizing antibody and
CD13 inhibitor ubenimex induced apoptosis in
both HuH7 and PLC/PRF/5 after 24 hours (Fig-
ure 4C). The CD13 antibody (clone WM15) has
been shown to be specific to humans and to func-
tion as a neutralizing antibody (15). Reportedly,
ubenimex (bestatin) specifically blocks CD13,
which antagonizes the zinc-binding site of the
aminopeptidase N domain (16–19). Ubenimex
is used as a therapeutic agent for adult acute
nonlymphatic leukemia (20).
We then hypothesized that not only the ABC
transporter (21, 22) but also CD13 is involved
in cell protection against exposure to antican-
cer agents. DXR is a well-known ABC-trans-
expression was increased over 20-fold by DXR or 5-FU treat- porter–dependent anticancer drug. We have established a DXR-
ment compared with control (CD13+CD133+ population in con- resistant HuH7 clone in which 90% of cells survive in 0.5 μg/ml
trol, 2.0%; by DXR treatment, 40.3%; by 5-FU treatment, 44.3%), of DXR, whereas about 99% of parent HuH7 cells die at that con-
although expression of CD133 remained unchanged (87.1% in centration (Supplemental Figure 2, B and C). Inhibition of CD13
control vs. 88.0% after DXR treatment, 88.7% after 5-FU treat- indicated approximately 50% suppression of cell proliferation
ment). In PLC/PRF/5, after treatment with DXR, the CD13+CD90− in this clone (Figure 4D), and this finding suggests that CD13
fraction was also increased and the CD13−CD90+ fraction was inhibition can potentially suppress cells that may have multidrug-
shifted to the CD13 positive (the CD13+CD90− fraction of con- resistance capacities and remain viable after conventional anti-
trol was 15.4% and of DXR treatment was 58.2%). After treatment cancer drug treatments.
with 5-FU, the remaining cells were more clearly localized in the CD13 is expressed preferentially in therapy-resistant HCC cells. To
CD13+CD90− fraction (77.8%) (Figure 4A). The chemo-resistance identify the expression of CD13 in clinical HCC, HCC samples
ability of the CD13+ cells was also confirmed by cell proliferation were digested and hematopoietic Lin–CD45– fractions were further

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Figure 4
CD13+ cells resist chemotherapy, and inhibition of CD13 elicits cellular apoptosis. (A) The HuH7 and PLC/PRF/5 cells were treated with
0.1 μg/ml of DXR or 1 μg/ml of 5-FU for 72 hours. The changes in cell-surface markers were compared with controls. The percentages of
CD13+CD133+ in HuH7 and CD13+CD90– populations in PLC/PRF/5 are shown in figure. (B) Effect of CD13 inhibition on cell proliferation. HuH7
cells were treated with various concentrations of anti-human mouse IgG1 CD13-neutralizing antibody. As a negative control, 10 μg/ml of anti-
human mouse IgG1 antibody was used. (C) Inhibition of CD13 induces cell apoptosis. Cells were treated with 1–20 μg/ml of CD13-neutralizing
antibody or 50–500 μg/ml of ubenimex for 24 hours. Data show each case of 5 μg/ml of CD13-neutralizing antibody and 100 μg/ml of ubenimex
treatment. (D) The effect of CD13-neutralizing antibody on DXR-R HuH7. The DXR-R clone was established with continuous treatment in
1 μg/ml of DXR and a selection of viable colonies. In 0.5 μg/ml of DXR, most control HuH7 cells die after 72 hours, whereas over 90% of DXR-R
cells survive. The DXR-R HuH7 cells were cultured with 1–20 μg/ml of CD13-neutralizing antibody for 72 hours. Control, treated with 10 μg/ml
of anti-human mouse IgG1 antibody.

analyzed by multicolor flow cytometry. In all 12 clinical HCC bigger than other kinds of cancer cells and may be more easily
samples, including 3 cases of non–hepatitis-derived HCC (1 case damaged by mechanical and enzymatic digestion.
recurred after transcatheter arterial embolization [TAE]) and 9 The expression of CD13 was confirmed in fresh frozen surgi-
cases of hepatitis-derived HCC (4 cases recurred after TAE), no cal specimens. The CD13+ HCC cells typically existed along the
CD133 expression was observed. In all cases, CD13 and CD90 fibrous capsule forming cellular clusters after TAE. In non-TAE
expression was observed in the following 4 subpopulations: cases, the CD13+ HCC cells usually formed small cellular clusters
CD13+CD90+, CD13+CD90−, CD13−CD90+, and CD13−CD90−. inside the cancer foci (Figure 5B). CD13 was expressed on the cell
In cases that recurred after TAE, the CD13+CD90− fraction was surface in HCC cases. In normal liver samples, CD13 was expressed
more abundant than that in non-TAE cases (48% ± 12% in TAE in the sinusoid with a linear staining pattern and in bile ducts with
cases vs. 8% ± 4% in non-TAE cases; 6-fold increase), whereas the an intraductal pattern; this was different in the HCC samples. The
CD13−CD90+ fraction was more abundant in non-TAE cases immunohistochemical findings for the post-TAE cases support
than in TAE cases (40% ± 18% in non-TAE cases vs. 12% ± 5% in clinical experience because HCC recurrence after TAE usually
TAE cases; 3.3-fold increase) (Figure 5A). In all 12 clinical HCC occurs at the fibrous capsule and chemoresistant viable HCC cells
samples, the expression patterns were very similar to that of PLC/ exist mainly around the fibrous capsule.
RLF/5, indicating its usefulness as an HCC model. Of course, Interestingly, some small canalicular structures near the bile
the percentages of cells just indicate the percentage that survived ducts expressed CD13 on the cell surface, and these are suggested
after mechanical and enzymatic digestion. The majorities of to be liver stem/progenitor cells, since it has been reported that
HCC cells retain the cellular functions of liver cells, accumulate normal liver stem/progenitor cells express CD13 (23). In our stud-
fat and glycogen, and produce bilirubin. Also, they are relatively ies, spheres established from the normal liver were predominantly

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Figure 5
CD13 expression in clinical HCC samples with or without TAE. (A) Expression analysis of clinical HCC samples. The nonhematopoietic Lin/
CD45− fraction was analyzed. The data show 2 typical TAE and non-TAE cases, nonhepatitis virus infection (NBNC; first row), nonhepatitis B
but hepatitis C infection (NBC; second row), both hepatitis B and C infection (BC; third row), and another nonhepatitis B but hepatitis C infec-
tion (fourth row). The cut-off lines were determined using isotype controls. (B) Immunohistochemical analysis of HCC and normal liver samples
stained with anti-human CD13 (red) and DAPI (blue) for the nucleus. Each middle panel shows a high magnification (×40) of the white dotted
square in each left-hand column (×10). The right columns exhibit H&E stains (×10) of each adjacent frozen section. 3 typical samples from 6
HCC samples are represented. The white arrow indicates the fibrous capsule (Fc) in HCC samples, and tumor cells exist inside Fc. The upper
2 samples show HCC after TAE, and the middle samples are from no-TAE cases. In HCC samples, CD13 is expressed on the cell surface. The
lower panels show immunohistochemical stains of normal liver obtained from surgical sections of colon cancer metastasis. Expression of CD13
in hepatic lobules is linear along the sinusoid. In the bile ducts, CD13 is expressed in an intraductal manner (lower left; bile duct, ×40). In some
small canalicula present near the bile duct, CD13 is expressed on the cell surface (lower right; Glisson, ×40).

CD13+CD90+CD133+, with a multidifferentiation potential in both by treatment with a DNA synthesis inhibitor or not. We used 5-FU,
hepatocyte and cholangiocyte lineages (Supplemental Figure 3). the most common anticancer drug in HCC treatment, to simulate
To assess whether the area of the fibrous capsule contributed to the clinical setting. After 3 days of intraperitoneal administra-
the maintenance of semiquiescent CD13+ cells, we stained fresh tion of 5-FU (30 mg/kg), most Ki67+ active cells were disrupted
frozen tissues obtained from HCC parents with a hypoxia marker, and remained only at small foci, and tumors were replaced by a
carbonic anhydrase 9 (CA9) (24). In hematopoietic stem cells, majority of CD13+Ki67− cells. In the controls, CD13 expression
hypoxia is well known as a hypoxic niche that plays important was limited to a small fraction with cellular clustering, and most
roles in maintaining stem cells in a dormant phase (25, 26). In the cells expressing CD13 were Ki67−. Conversely, in ubenimex-treated
TAE samples, expression of CA9 was localized along the fibrous mice (20 mg/kg, 3 days), most of the CD13+ cells were disrupted
capsule and coexpressed CD13. In the non-TAE samples, CA9 + and replaced with Ki67+ active cells (Figure 6A).
cells formed cellular clusters in cancer foci and coexpressed CD13. PLC/PRF/5 was then used for further analyses. The expression of
In normal livers, CA9 expression was limited to the cell surface of markers in this cell line is similar to that in clinical HCC, and thus,
bile ducts (Supplemental Figure 4). the PLC/PRF/5 cell line is potentially useful as an HCC model. In
CD13 inhibition elicits tumor regression. For preliminary studies, control mice, CD13 expression was limited to a small fraction and
HuH7 cells were transplanted into NOD/SCID mice and treated most of the cells expressed CD90. After treatment with 5-FU (30
with 5-FU to determine whether the CD13+ fraction was enriched mg/kg, 5 days of injection and 2 days of withdrawal, 2 courses),

3332 The Journal of Clinical Investigation    http://www.jci.org    Volume 120    Number 9    September 2010
 research article

Figure 6
CD13 inhibition elicits cancer regression in vivo. (A) HuH7-xenografted mice were treated with 5-FU or ubenimex for 3 days. The sections were
stained with anti-human CD13 (red), Ki-67 (green), and DAPI (blue). Each right-hand panel shows a high magnification (×20, control and 5-FU;
×40, Ube) of the white dot square on the left (×10, control and 5-FU; ×20, Ube). White arrows: cellular clusters express CD13 but not Ki-67 (upper
panels), residual Ki-67+ cancer cells (middle panels), and a residual CD13+ cell (lower panels). (B) PLC/PRF/5-xenografted mice were treated with
5-FU, ubenimex, and ubenimex plus 5-FU for 14 days. The black arrows indicate a small amount of residual cancer. The sections were stained with
H&E (×10), anti-human CD13 (red), anti-human CD90 (green), and DAPI (blue) (×20, control, 5-FU, and Ube; ×40, Ube + 5-FU). Nonspecific and
fragmented expression of CD13 (white arrow). In situ hybridization for DNA fragmentation (low and high magnification). Black dot-like structures
indicate labeled DNA. (C) Tumors of control and ubenimex–plus–5-FU–treated mice. Black arrowheads indicate the tumor margin. (D) The relative
tumor volumes (after treatment [mm3]/before treatment [mm3] × 100%) of the control, 5-FU, ubenimex, and ubenimex–plus–5-FU–treated mice.
Data represent mean ± SD from independent experiments. *NS; **P < 0.01. (E) The CD13+ cell–enriched fractions obtained from 5-FU–treated
mice were serially transplanted into secondary NOD/SCID mice. The mice were treated with ubenimex (Ube; n = 6) or received no treatment
(control; n = 10) from the day after transplantation for 7 days. Tumor growth was observed for 3 weeks.

most of the CD90+ cells were disrupted and tumors were replaced ment groups may have been newly produced from residual CD90+
by a majority of CD13+ cells. After ubenimex treatment (20 mg/kg cells. Costaining of Ki67 and CD13 revealed that CD13+ cells were
every day for 14 days), not only were many CD90+ cells present but negative for the expression of Ki67 (Supplemental Figure 5).
CD13+ cells were also identified. Interestingly, in cases in which The highly deformed nuclei observed in the ubenimex–plus–
both ubenimex and 5-FU were administered, the majority of tumor 5-FU treatment specimens suggested that DNA fragments were
cells were disrupted. We identified atypical, nonspecific CD13 present. The DNA fragmentation status was thus assessed by in
expression in these cases (Figure 6B). Taken together with the find- situ hybridization with terminal deoxynucleotidyl transferase
ings that CD90+ cells produce CD13+ cells within 24 hours and that (TdT). There were a few DNA fragments in both the control and
almost all of the CD13+ cells were disrupted by ubenimex plus 5-FU 5-FU–treated specimens, whereas there were many more in the
treatment, the CD13+ cells that appeared in the ubenimex treat- ubenimex-treated specimens. Especially in the specimens treated

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research article

Figure 7
CD13 + cells contain lower levels of ROS
than CD13 − cells. (A) The expression of
prooxidant DCF-DA in CD13+CD133 + and
CD13−CD133+ HuH7 cells and CD13+CD90−
and CD13 −CD90 + PLC/PRF/5 cells. Con-
trols, treated with 10 μg/ml of mouse anti-
human IgG. As positive controls, cells were
treated with 100 μM of oxidant H 2O 2 for
2 hours. Cells were treated with 5 μg/ml of
CD13-neutralizing antibody and 25 μg/ml of
ubenimex for 4 hours. (B) The expression
of ROS in the CD13+CD90−, CD13−CD90+,
and CD13+CD90+ fractions of 2 clinical HCC
samples. (C) The expression of the ROS
scavenger pathway gene GCLM in isolated
CD13+CD90−, CD13+CD90+, CD13−CD90+,
and CD13 − CD90 − cells from PLC/PRF/5
and clinical HCC samples estimated by
semiquantitative RT-PCR. (D) The time-
course change of ROS expression in DXR or
5-FU treatment. Cells were treated with 1 μg/
ml of DXR and 1 μg/ml of 5-FU continuously.
After 3 hours and 48 hours of treatment, ROS
levels in each population were measured.

with ubenimex plus 5-FU, there were numerous DNA fragments tor, MitoSOX (a highly selective marker for mitochondrial super-
in residual tumor cells (Figure 6B). oxide), which was markedly lower in the PLC and clinical HCC
After 14 days of treatment, the tumor volume was significantly samples and less in HuH7 (Supplemental Figure 6).
decreased in the ubenimex–plus–5-FU groups compared with the To study the correlation between CD13 and the ROS scavenger
control and 5-FU or ubenimex groups (Figure 6, C and D). pathway, the expression of Gclm was assessed by RT-PCR. Gclm
Next, we studied the effects of CD13 inhibition as it pertains to encodes the glutamate-cysteine ligase that catalyses the rate-limit-
the self-renewing ability of cells and repopulation of tumors. The ing synthesis step of glutathione (GSH), which works as a critical
CD13+-enriched fraction obtained from 5-FU–treated mice was cellular reducing agent and anti-oxidant. Gclm was overexpressed
serially transplanted into secondary NOD/SCID mice. Starting in the CD13+CD90− fraction (P < 0.001) compared with the
the day after transplantation, the mice were treated with ubeni- CD13+CD90+, CD13−CD90+, and CD13−CD90− fractions in PLC/
mex (20 mg/kg) for 7 days. After 3 weeks, no tumor formation was PRF/5 and primary HCC cells (Figure 7C).
observed in the ubenimex-treated mice (n = 0/6), whereas 60% of It is well known that cell destruction after exposure to cytotoxic
the untreated mice grew tumors (n = 6/10) (Figure 6E). chemotherapy and ionizing radiation is partially due to free radi-
The CD13+ HCC cells contain lower levels of ROS. We focused on cals (28, 29). Given that the present study indicates a low ROS
the ROS scavenger pathway to determine why DNA fragmenta- concentration in the CD13+ population, we were interested to
tion and apoptosis were induced by CD13 inhibition. It has been see whether chemotherapy agents actually increase ROS level of
reported that self-renewing dormant stem cells normally possess CD13+ population. To study this, ROS levels of CD13+CD133+
low levels of intracellular ROS and that deregulation of ROS levels and CD13–CD133+ populations in HuH7, and CD13+CD90– and
impairs stem cell functions (27). Intracellular ROS levels were mea- CD13–CD90+ populations in PLC/PRF/5 were measured 3 hours
sured by prooxidants using the 2′,7′-dichlorofluorescein diacetate and 48 hours after of DXR or 5-FU treatment. After 3 hours treat-
(DCF-DA) stain. Both in HuH7 and PLC/PRF/5, the CD13+ frac- ment with DXR, ROS levels were increased in both CD13+ and
tion contained lower concentrations of ROS than the CD133strong CD13– populations in HuH7 and PLC/PRF/5. Interestingly, after
and CD90+ fractions. After stimulation of oxidative stress by H2O2, 48-hour treatment with DXR, ROS levels of CD13+ populations
a lower concentration of ROS was clearly observed in the CD13+ were decreased and reached those of control levels. Especially in
fraction compared with the CD13− fraction. Following treatment PLC/PRF/5, CD13+CD90– populations showed 2 peaks of ROS
with the CD13-neutralizing antibody or ubenimex, the ROS con- levels, one of which contained further lowered ROS levels than
centration was significantly increased in CD13+ cells and reached control. With 5-FU treatment, though the power of upregulation
the level of ROS observed in the CD13− fraction (Figure 7A). In of ROS levels was weaker than those of DXR, ROS levels of CD13+
clinical HCC samples, the results were similar to those in PLC/ fractions were actually increased to those of CD13– fractions. As
PRF/5, as the CD13+CD90− fraction exhibited lower ROS levels with the data regarding DXR treatment, after 48 hours of 5-FU
than those in the CD13−CD90+ and CD13+CD90+ fractions (Fig- treatment, CD13+ populations showed lower levels of ROS com-
ure 7B). The CD13+ fraction also contained another ROS indica- pared with those of the CD13– population (Figure 7D). These data

3334 The Journal of Clinical Investigation    http://www.jci.org    Volume 120    Number 9    September 2010
 research article

Figure 8
High levels of ROS scavenger expression parallel DNA damage in CD13+ HCC cells. (A) Isolated cell fractions of CD13+CD90−, CD13+CD90+,
CD13−CD90+, and CD13−CD90− in PLC/PRF/5 and CD13+CD133+, CD13−CD133+, and CD13−CD133− in HuH7 were irradiated with 4 Gy with or
without antioxidant tempol. Data show the tail lengths in the alkaline comet assay of control (blue), 4 Gy irradiation (brown), and antioxidant tem-
pol pretreated (green) cells. *P < 0.01. **NS. (B) HuH7 and PLC/PRF/5 cells were irradiated with 4 Gy, and time course change of gamma-H2AX
expression in each population was assessed. Numbers indicate the percentage of gamma-H2AX in CD13+CD90− PLC/PRF/5 and CD13+CD133+
HuH7 cells (red) and CD13−CD90+ PLC/PRF/5 and CD13−CD133+ HuH7 cells (blue) with ± SD. (C) HuH7 and PLC/PRF/5 cells were irradiated
with 4 Gy, seeded in culture medium, and their expressions analyzed after 24 and 48 hours. Damaged and dead cells were eliminated with
7-AAD. The cut-off lines were determined using isotype controls.

together with the observation that CD13+ cells remained after gamma-H2AX, a marker of double-strand breaks (30), was studied.
treatment with chemotherapy agents (Figure 4A), suggest that ROS In PLC/PRF/5, after 4 Gy of irradiation, gamma-H2AX expression
levels of all of the cells are temporally upregulated when cells are in CD13–CD90+ population increased after 2 hours of irradiation
treated with chemotherapy agents and that this leads to disruption (45.4% ± 5.3%) and then decreased within 6 hours (15.6% ± 4.5%),
of the CD13– population, whereas in the CD13+ cells, ROS levels are whereas gamma-H2AX expression in CD13+CD90– population did
downregulated by the ROS scavenger pathway and the cells survive. not. In HuH7, gamma-H2AX expression increased after 2 hours in
In addition, proliferative CD13– cells are easily affected by the DNA both CD13+CD133+ and CD13–CD133+ populations and decreased
synthesis inhibition effect of chemotherapy agents. rapidly in the CD13+CD133+ population (4.4% ± 2.8%) compared
To assess radiation-induced DNA damage with ROS, purified with the CD13–CD133+ population (38.4% ± 4.6%) (Figure 8B).
CD13+CD90−, CD13+CD90+, CD13−CD90+, and CD13−CD90− After 24 hours of irradiation, the residual cells were localized in the
PLC/PRF/5 cells were irradiated and subjected to an alkaline CD13+ fraction in HuH7 and in the CD13+CD90− fraction in PLC/
comet assay. Although untreated cells did not show significantly PRF/5 (Figure 8C). Although there were some different manners
different levels of DNA damage, there were fewer DNA strand in time-course change of gamma-H2AX in PLC/PRF/5 and HuH7,
breaks in CD13+CD90− cells than in the other 3 fractions (P < 0.01) surviving cells after 24 hours of irradiation were localized in the
after ionizing irradiation. The DNA damage in these 3 fractions CD13+ population, suggesting the radio-resistant characteristics
(but not in the CD13+CD90− fraction) was significantly inhibited of the CD13+ population, due to rapid recovery of DNA damage.
(P < 0.001) by treatment with an antioxidant reagent, tempol (Fig- After 48 hours of irradiation, the residual cells began to prolifer-
ure 8A). In HuH7 cells, the CD13+ fraction also exhibited lower lev- ate and produced CD13−CD133+ cells in HuH7 and CD13+CD90+
els of DNA damage compared with the CD13− fraction. There was cells in PLC/PRF/5 (Figure 8C). These studies support the time-
no significant difference between the irradiated and tempol-treated course studies (Figure 3C) and indicate that CD13+ cells exist as a
groups for the CD13−CD133− fraction (Figure 8A). These findings core fraction in the cellular hierarchy.
reveal that the enhanced ROS defenses in the CD13+ fraction con-
tribute to the reduction in DNA damage after genotoxic cancer Discussion
therapy. To confirm radiation-induced DNA double-strand break To achieve the goal of a radical cure for cancer, recurrence and
status in CD13+ and CD13– populations, time-course change of metastasis caused by residual cancer cells are barriers that need to

The Journal of Clinical Investigation    http://www.jci.org    Volume 120    Number 9    September 2010 3335
research article

Figure 9
The CD13+ CSCs of the liver generate genotoxic resis-
tance through reduced levels of ROS (proposed schema).
(A) Results indicate that CD13+CD90− CSCs of the liver
are dormant and exhibit reduced intracellular ROS lev-
els and, because of increased antioxidants, may result in
resistance to genotoxic chemo/radiation therapy. On the
other hand, CD13−CD90+ CSCs actively proliferate and
are sensitive to therapy. (B) Neutralization or inhibition
of CD13 may result in an increase in intracellular ROS in
CD13+CD90− CSCs and induction of apoptosis.

be overcome. Recently, the presence of CSCs has attracted atten- tive stresses inhibit cellular dormancy and self renewal of hemato-
tion, and it is thought that these CSCs are intimately involved in poietic stem cells (37, 38). In cancer, low ROS levels and radiation
cancer recurrence and resistance. In addition, as with leukemia resistance in CD44+CD24− breast CSCs has been reported (39).
(4, 5), the presence of dormant or slow-growing CSCs is beginning However, an association between ROS and self renewal in CSCs
to be recognized in breast cancer (6). However, dormant or slow- is unknown. In the present study, we demonstrated that CD13+
growing CSCs have yet to be identified in most solid cancers. In cells contain low levels of ROS. The CD13 −CD133+ and CD90+
the present study, we identified CD13 as a functional marker that cells expressed higher levels of the ROS indicators DCF-DA and
can be used to identify potentially dormant liver CSCs resistant to MitoSOX. RT-PCR of the ROS scavenger pathway gene GCLM and
treatment. Our exploration of SP cells has indicated that CD13 + a comet assay also indicated that CD13+ cells protect themselves
cancer cells are closely associated with SP cells. Cell-cycle studies from oxidant stress via the ROS pathway. Continuous treatment
indicated that CD13+ cells exist in lower PY lesions. Cell-fate trac- with anticancer agents predominantly elicits high levels of ROS in
ing assay with PKH26GL and immunohistochemical analysis of the CD13– population. However, in the CD13+ population, it elic-
BrdU-retaining cells demonstrated that CD13+ but not CD13− its low levels of ROS, and these cells survive and are enriched for
cells exhibited long dye retention and relatively slow proliferation after chemotherapy. Mice treated with ubenimex exhibited high
in vitro and in vivo. This population possessed high tumorigenic DNA fragmentation in xenografted tumors. These findings sug-
potential in NOD/SCID mice and also induced chemo resistance. gest that the ROS scavenger pathway and CD13 are essential to
The results of this study are compatible with those of dormancy CSC protection and maintenance in the liver (Figure 9, A and B).
studies on hematopoietic stem/progenitor (3) and malignant cells Importantly, tumorigenicity was completely inhibited by treat-
(4, 5). CD13, also known as amino peptidase N, is a super fam- ment with ubenimex in secondary mice xenografted with a CD13+
ily of zinc-binding metalloproteinases that play roles in cellular cell–enriched tumor fraction obtained from 5-FU–treated mice.
processes such as mitosis, invasion, cell adhesion, angiogenesis, The suppression of CD13 inhibited self renewal and the tumor-
radiation resistance, and antiapoptosis (31–34). To the best of our initiation ability of CD13+ cells. It is thought that deregulation of
knowledge, there have been no reports describing the exclusive ROS pathway may contribute to disruption of CSCs.
expression of CD13 in CSCs of the liver. The hierarchy analysis of PLC/PRF/5 cells revealed that a small
The immunohistochemical findings also support the view that fraction of CD90+ cells produce a small number of CD13+ cells
CD13+ cells play a role in relapse of liver cancer. The apparent in vitro. This finding indicates that activated CD90+ cells should
increase in the number of CD13+ cells near the fibrous capsule also be involved in targeted cancer therapy. The CD90+ cells were
after TAE is consistent with the fact that clinical HCC relapse after resistant and remained in spite of treatment with ubenimex in
TAE is frequent at the capsule site (7). These findings are compat- vivo. The residual CD90+ cells cause cancer regrowth and cancer
ible with the results of studies in mouse models that revealed that recurrence by producing tumor-initiating CD13+ cells. CD13+
CD13+ cells survived and were amplified after 5-FU treatment. In cells have high tumorigenicity and self-renewal ability in vivo. But
addition, the preferential accumulation of CD13+ HCC cells at the unfortunately, in the case of liver cancer, it is difficult to target
capsule but not in the central region after TAE therapy suggests the proliferative CD90+ cells by using conventional anticancer
the attractive hypothesis that cellular components in the fibrous drugs because some parts of CD90+ cells also express CD13. The
capsule may function as a protective niche (3). expression of CD13 is closely related to the multidrug-resistant
The suppression of CD13 by the CD13-neutralizing antibody SP fraction, and CD13 protects cells from apoptosis via the ROS
or ubenimex showed an effect even if the cancer cells were resis- scavenger pathway. Of course, based on CSC concepts, tumors will
tant to the ABC transporter–dependent agent DXR. This finding disappear when CSCs are disrupted completely. This is because the
suggests that CD13+ cells have some mechanism of resistance to loss of CSCs leads to the destruction of the hierarchical structure
anticancer agents in addition to their slow growth and ABC trans- within the tumor. However, it may be difficult to obtain complete
porter (21, 35, 36) expressions. It is known that the control of ROS pharmacokinetic control, especially in vivo. Actually, in this study,
is indispensable for hematopoietic stem cell maintenance. Oxida- we could not achieve complete disappearance of CD13+ cells and

3336 The Journal of Clinical Investigation    http://www.jci.org    Volume 120    Number 9    September 2010
 research article

could not elicit tumor regression by single agent administration of copies was normalized against GAPDH mRNA expression. The PCR primers
ubenimex. To overcome these problems, we established combina- used for amplification were as follows: GCLM, 5′-TGTGTGATGCCACCA-
tion therapy with ubenimex plus 5-FU to efficiently elicit tumor GATTT-3′ and 5′-TTCACAATGACCGAATACCG-3′; GAPDH, 5′-TTGGTATC-
regression. Ubenimex works to disrupt CD13+ cells by its potential GTGGAAGGACTCA-3′ and 5′-TGTCATCATATTTG-GCAGGTTT-3′.
effect of upregulating ROS levels and its inhibition of self-renewal Cell proliferation and chemo-resistance assay. Isolated cells were seeded into
of CD13+ cells. 5-FU inhibits proliferative cancer cells, decreases 96-well culture plates at 5 × 103 cells/well for cell proliferation assays.
tumor size, and improves survival. It is known that cell destruc- After 72 hours, cell viability was determined by an ATP bioluminescence
tion after exposure to cytotoxic chemotherapy and ionizing radia- assay (CellTiter-Glo Luminescent Cell Viability Assay; Promega) and the
tion is partially due to free radicals (29, 39), and it is reported that luminescence signal was detected using a luminometer (ARVO MX; Perkin­
5-FU induces ROS in hematopoietic stem cells and suppresses the Elmer) according to the manufacturer’s protocol. The cells were seeded
hematopoietic stem cell niche (40). We have also confirmed that onto 96-well culture plates at 5 × 103 cells/well. After 24 hours, DXR was
5-FU works to increase the ROS levels of CD13+ populations. By added to the culture medium (0.01, 0.05, and 0.1 μg/ml). After 72 hours
this combination therapy, tumors were drastically regressed com- of exposure to the chemotherapeutic agent, cell viability was determined
pared with single-agent therapy. It is suggest that 5-FU and ubeni- using a method similar to that used in the cell proliferation assay.
mex work in a complementary or additive fashion. Cell-fate tracing. Cells were labeled with 20 μM PKH26GL (Sigma-Aldrich)
Although the majority of the experiments in this study are based according to the manufacturer’s protocol. Purified populations of cells were
on cell lines, the expression, sphere, and ROS analyses support the isolated and seeded onto 4-chamber polystyrene vessel tissue culture-treated
contention that PLC/PRF/5 cells reflect clinical HCC and may glass slides (Falcon; BD Biosciences) at 5 × 103 cells/well. Cells were cultured
hold promise for preclinical studies. This study also suggests that in RPMI 1640 (Invitrogen) medium with 20% FBS (Equitech-Bio). Cell fate
the future development of liver cancer therapy based on CSC con- was studied at each 30-minute time point for 238 hours using a time-lapse
cepts appears promising. We are attempting to establish human fluorescence microscope (BZ-9000 Biorevo; KEYENCE). Data were analyzed
HCC-xenografted preclinical mouse models from clinical HCC using a BZ-II analyzer (KEYENCE). BrdU-retaining cells were identified with
samples to provide necessary confirmation of our contention fresh frozen samples with the modification of using 5-bromo-2′-deoxyuri-
using in vivo assays. dine Labeling & Detection Kit 1 (Roche Applied Science) and CD13 rabbit
polyclonal antibody (Santa Cruz Biotechnology Inc.). As secondary antibody,
Methods anti-rabbit IgG Alexa Fluor 555 (Molecular Probes) was used.
Cell culture. Human liver cancer cells, HuH7 and PLC/PRF/5, obtained from Sphere assay. Cells were seeded on ultra-low attachment culture dishes
the Cell Resource Center for Biomedical Research, Institute of Develop- (Corning) in serum-free medium. DMEM/F-12 serum-free medium
ment, Aging, and Cancer (Tohoku University, Sendai, Japan) were cultured (Invitrogen) contained 2 mM l-glutamine, 1% sodium pyruvate (Invitrogen),
in RPMI 1640 (Invitrogen) medium with 10% FBS (Equitech-Bio). Cells 1% MEM nonessential amino acids (Invitrogen), 1% insulin-transferrin-sele-
were cultured at 37°C in a humidified atmosphere containing 5% CO2. nium-X supplement (Invitrogen), 1 μM dexamethasone (Wako), 200 μM
Flow cytometric analysis and cell sorting. The antibodies used in this study L-ascorbic acid 2-phosphate (Sigma-Aldrich), 10 mM nicotinamide (Wako),
are listed in Supplemental Table 1. Briefly, cells were harvested with trypsin 100 μg/ml penicillin G, and 100 U/ml streptomycin supplemented with
and EDTA. Doublet cells were eliminated using FSC-A/FSC-H and SSC-A/ 20 ng/ml epithelial growth factor and 10 ng/ml fibroblast growth factor-2
SSC-H. Dead and damaged cells were eliminated with 7-AAD (BD Biosci- (PeproTech). Digestion and cell passage were performed every 3 days.
ences — Pharmingen). Isotype controls (BD Biosciences) were used. FcR Differentiation assays from spheres. Each single sphere established from
blocking was performed using an FcR-blocking reagent (Miltenyi-Biotec). normal liver cells was seeded into a culture chamber (BD Biosciences).
FITC-conjugated anti-human CD45 (BD Biosciences — Pharmingen) and Spheres were cultured in sphere medium containing 10% FBS to induce
FITC-conjugated Lineage Cocktail (Lin1; BD Biosciences — Pharmingen), the differentiation process. Three days after the spheres became attached
which contains antibodies against CD3, CD14, CD16, CD19, CD20, and to the bottom of the chamber and spreading cells appeared, cells were fixed
CD56 and is used to detect lymphocytes, monocytes, eosinophils, and and stained with anti-human CD13 mouse monoclonal antibody (clone
neutrophils, were used for eliminating hematopoietic cells in the clinical WM15, dilution 1:50; Santa Cruz Biotechnology Inc.), FITC–anti-human
sample analysis. For sorting, cells were incubated with 1 μg of each anti- albumin goat polyclonal antibody (dilution 1:500; Bethyl Laboratories),
body for 30 minutes. Control experiments involved incubation with each anti-human Cytokeratin 19 mouse monoclonal antibody (clone RCK108,
antibody for 30 minutes and no apparent increase in the number of dead dilution 1:50; Dako), and anti-human α-fetoprotein mouse monoclonal
cells detected by propidium iodide (PI) staining. antibody (clone 189502, concentration 5 μg/ml; R&D Systems).
Cell-cycle assay. To characterize the SP fractions, 1 × 106 cells in 2% FCS/ Immunohistochemistry. The 4-μm–thick sections were obtained using
1 mM HEPES buffer/DMEM were preincubated at 37°C for 30 minutes. cryostat and fixed with 4% paraformaldehyde for 15 minutes. After 1 hour
Cells were then labeled with 10 μg/ml Hoechst 33342 (Molecular Probes) of blocking, the sections were incubated overnight at 4°C in a humidified
in staining medium at 37°C for 70 minutes. A total of 15 μg/ml reserpine chamber with primary antibodies. For primary antibodies, anti-human
(Sigma-Aldrich) was used for the Hoechst staining procedure. For cell- CD13 mouse monoclonal antibodies (clone WM15, dilution 1:50; Santa
cycle analysis by PY staining, cells were first stained with Hoechst 33342 at Cruz Biotechnology Inc.), anti-human CA9 rabbit polyclonal antibodies
37°C. After 50 minutes, 1 μg/ml PY was added and the cells were incubated (dilution 1:1000; Novus Biologicals), anti-human CD90 rabbit monoclo-
at 37°C for 20 minutes. FACSVantage SE DiVa (BD) and FACS SORP Aria nal antibodies (dilution 1:1000; Epitomics), and anti-human Ki-67 rabbit
(BD) were used for analysis and cell sorting. The cell cycle was also studied polyclonal antibodies (dilution 1:100; Santa Cruz Biotechnology Inc.) were
with 10 μg/ml 7-AAD (BD Biosciences — Pharmingen). used. For secondary antibodies, goat anti-mouse IgG1, Alexa Fluor 546–
Gene expression study. Total RNA was prepared using TRIzol reagent conjugated, and highly cross-adsorbed (Molecular Probes) as well as goat
(Invitrogen). Reverse transcription was performed with SuperScript­III anti-rabbit IgG, Alexa Fluor 488–conjugated and highly cross-adsorbed
(Invitrogen). Quantitative real-time RT-PCR was performed using a Light­ (Molecular Probes) antibodies were used. The coverslips were mounted
Cycler TaqMan Master kit (Roche Diagnostics). The expression of mRNA using ProLong Gold and SlowFade Gold Antifade Reagent (Molecular

The Journal of Clinical Investigation    http://www.jci.org    Volume 120    Number 9    September 2010 3337
research article

Probes), and the slides were viewed with a fluorescence microscope (BZ- mice were treated with ubenimex (20 mg/kg) from the day after transplan-
9000 Biorevo). Data were analyzed using BZ-II (Keyence). The continuous tation for 7 days. Tumor growth was observed for 3 weeks. We used 4 or
cryostat sections were also stained with modified H&E. more mice for each model to enable statistical assessment of the results.
Tumor cell preparation. Primary liver cancer samples were obtained from All animal studies were approved by the Animal Experiments Committee
Osaka University with the patients’ informed consent and the approval of at Osaka University.
the Research Ethics Board of Osaka University. Tumor tissues were cut into ROS assay. To study intracellular ROS levels, cells were loaded with 10 μM
approximately 2-mm fragments, further minced with a sterile scalpel, and of DCF-DA at 37°C for 30 minutes. ROS was activated by treatment with
washed twice with DMEM/10% FBS. They were then placed in DMEM/10% 100 μM H2O2 at 37°C for 120 minutes. To study the effect of CD13 inhibi-
FBS with 2 mg/ml collagenase A (Roche Diagnostics) solution. The mix- tion on ROS levels, cells were pretreated with 5 μg/ml of the CD13-neutral-
ture was incubated at 37°C with shaking until digestion was complete. izing antibody or 25 μg/ml of ubenimex at 37°C for 4 hours and stained
Cells were filtered through a 40-μm nylon mesh and washed twice and the with DCF-DA. For mitochondria ROS detection, cells were loaded with
cell fragments and debris were then eliminated by Ficoll (GE Healthcare) 5 μM MitoSOX (Molecular Probes) at 37°C for 20 minutes.
density gradient centrifugation and stained for flow cytometry. DNA fragmentation assay. For the alkaline comet assay, 5,000 isolated cells
Inhibition of CD13. A total of 5 × 103 cells were seeded into 96-well plates were irradiated (4 Gy) on ice and suspended in 0.6% of low melting point
in 200 μl of culture medium. After 24 hours, the medium was replaced with agarose, spread over the wells of slides, and immersed in alkaline solution
fresh culture medium containing 1, 5, 10, and 20 μg/ml mouse monoclo- for 30 minutes using a kit (Trevigen). Alkaline electrophoresis was then
nal anti-human CD13 antibodies (clone WM15; GeneTex) or 25, 50, 100, performed. Slides were stained with silver for visualization. For the tem-
250, and 500 μg/ml ubenimex (Nippon Kayaku). Cell viability was assayed pol experiments, cells were treated with 10 mM of tempol (Sigma-Aldrich)
at 24, 48, and 72 hours using Cell Counting Kit-8 (Dojindo) according for 15 minutes before irradiation. For in situ hybridization detection of
to the manufacturer’s instructions. Absorbance was measured at 450 nm fragmented DNA, 10-μm–thick serial sections obtained from fresh frozen
using a 680 XR microplate reader (Bio-Rad). A total of 10 ng IgG1 mouse samples were hybridized with TdT using tumor TACS in situ apoptosis
monoclonal antibody (GeneTex) was used as the negative control. DXR- detection kit (Trevigen) according to the manufacturer’s protocols.
resistant (DXR-R) HuH7 cells were established by continuous treatment To identify DNA double-strand breaks, Alexa Fluor 488 Mouse Anti-
with 1 μg/ml DXR and selection of resistant colonies. Cellular apopto- H2AX (BD Pharmingen) was used according to the manufacturer’s proto-
sis was measured using PI and APC–annexin V (BD Pharmingen) with an cols. Briefly, cells were irradiated at 4 Gy. Cells were incubated in culture
Apoptosis Detection Kit (BioVision). medium at 37°C in a humidified atmosphere containing 5% CO2 after
In vivo assay. The xenografted mouse model was created by injection of irradiation for 0, 2, 4, and 6 hours. After incubation, cells were stained
1 × 105 HuH7 and PLC/PRF/5 cells into NOD/SCID mice under anesthe- with cell-surface antibodies. Then cells were fixed and permeabilized using
sia. For injection, the cells were resuspended in a 1:1 mixture of medium Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD), and
and Matrigel (BD Biosciences). The HuH7 cell–xenografted mice were stained with Alexa Fluor 488 Mouse Anti-H2AX.
treated with 5-FU (30 mg/kg; intraperitoneal administration) or ubeni- Statistics. We determined statistical significance by 1-tailed Student’s
mex (20 mg/kg; oral administration) for 3 days. On the following day, mice t test. P < 0.05 was defined as significant.
were sacrificed and tumors were enucleated for the immunohistochemical
assay. In the studies of PLC/PRF/5 cell–xenografted mice, mice were treat- Acknowledgments
ed with 5-FU (30 mg/kg, 5 days of intraperitoneal injection and 2 days of We thank T. Shimooka for technical assistance in this study. This
withdrawal, 2 courses; 14 days), ubenimex (20 mg/kg, 14 days of forced work was supported in part by a grant from the Core Research for
oral administration), or ubenimex and 5-FU (combination of 2 courses of Evolutional Science and Technology (CREST), a grant-in-aid for
30 mg/kg of 5-FU and 14 days of 20 mg/kg of ubenimex). The tumor size Scientific Research on Priority Areas (20012039), a grant-in-aid
was calculated as follows: tumor volume (mm3) = a × b/2, where a = long for Scientific Research (category S) (21229015), and a grant-in-aid
axis and b = short axis. The relative tumor volume was calculated as fol- for Young Scientists (category B) (21790274) from the Ministry of
lows: relative tumor volume (%) = a/b × 100, where a = tumor volume before Education, Culture, Sports, Science, and Technology, Japan.
treatment (mm3) and b = tumor volume after 14 days of treatment. The day
after 14 days of treatment, mice were sacrificed and tumors were enucle- Received for publication February 3, 2010, and accepted in revised
ated for immunohistochemical assay. The relative tumor volume was esti- form June 30, 2010.
mated as follows: relative tumor volume (mm3) = tumor volume on the
day after 14 days of treatment (mm3)/tumor volume just before the start Address correspondence to: Masaki Mori, Department of Gastro-
of treatment (mm3) × 100 (%). The residual tumors after 14 days of 5-FU enterological Surgery, Graduate School of Medicine, Osaka Univer-
treatment were enucleated and minced into 2-mm squares and subcutane- sity, 2-2 Yamadaoka, Suita 565-0871, Japan. Phone: 81.6.6879.3251;
ously transplanted into secondary NOD/SCID mice with Matrigel. The Fax: 81.6.6879.3259; E-mail: mmori@gesurg.med.osaka-u.ac.jp.

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