Higher percentage of CD133+ cells is associated with poor prognosis in colon carcinoma patients with stage IIIB.

Abstract

Cancer stem cell model suggested that tumor progression is driven by the overpopulation of cancer stem cells and eradicating or inhibiting the symmetric division of cancer stem cells would become the most important therapeutic strategy. However, clinical evidence for this hypothesis is still scarce. To evaluate the overpopulation hypothesis of cancer stem cells the association of percentage of CD133+ tumor cells with clinicopathological parameters in colon cancer was investigated since CD133 is a putative cancer stem cell marker shared by multiple solid tumors.
Tumor tissues matched with adjacent normal tissues were collected from 104 stage IIIB colon cancer patients who were subject to radical resection between January, 1999 to July, 2003 in this center. The CD133 expression was examined with immunohistochemical staining. The correlation of the percentage of CD133+ cell with clinicopathological parameters and patients' 5-year survival was analyzed.
The CD133+ cells were infrequent and heterogeneous distribution in the cancer tissue. Staining of CD133 was localized not only on the glandular-luminal surface of cancer cells but also on the invasive budding and the poorly differentiated tumors with ductal structures. Both univariate and multivariate survival analysis revealed that the percentage of CD133+ cancer cells and the invasive depth of tumor were independently prognostic. The patients with a lower percentage of CD133+ cancer cells (less than 5%) were strongly associated with a higher 5-year survival rate than those with a higher percentage of CD133+ cancer cells (greater than or equal to 55%). Additionally, no correlation was obtained between the percentage of CD133+ cancer cells and the other clinicopathological parameters including gender, age, site of primary mass, pathologic types, grades, and invasive depth.
The fact that a higher percentage CD133+ cells were strongly associated with a poorer prognosis in patients with locally advanced colon cancer implicated that CD133+ cancer cells contribute to the tumor progression, and the overpopulation hypothesis of cancer stem cell seems reasonable.

Full text

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BioMed Central
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Journal of Translational Medicine
Open Access
Research
Higher percentage of CD133+ cells is associated with poor prognosis
in colon carcinoma patients with stage IIIB
Chun-Yan Li1,2,5, Bao-Xiu Li1,2,6, Yi Liang1,4, Rui-Qing Peng1,2, Ya Ding1,2, Da-
Zhi Xu1,3, Xin Zhang1,2, Zhi-Zhong Pan1,3, De-Sen Wan1,3, Yi-Xin Zeng1,2,4,
Xiao-Feng Zhu1,4 and Xiao-Shi Zhang*1,2
Address: 1State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, Guangzhou, PR China, 2Biotherapy Center,
Cancer Center, Sun Yat-sen University, Guangzhou, PR China, 3Department of Abdominal Oncology, Cancer Center, Sun Yat-sen University,
Guangzhou, PR China, 4Department of Experimental Research, Cancer Center, Sun Yat-sen University, Guangzhou, PR China, 5Biotherapy Center,
The First Affiliated Hospital, Chongqing Medical University, Chongqing, China and 6GuangZhou First Municipal People's Hospital, Guangzhou,
China
Email: Chun-Yan Li - lichyanun@hotmail.com; Bao-Xiu Li - libaoxiu@csco.org.cn; Yi Liang - liangyi_best@163.com; Rui-
Qing Peng - gz13724083175@126.com; Ya Ding - dingya@mail.sysu.edu.cn; Da-Zhi Xu - dazhi@163.com;
Xin Zhang - xingzhang@hotmail.com; Zhi-Zhong Pan - panzhzh@mail.sysu.edu.cn; De-Sen Wan - wds-fk@yahoo.com.cn; Yi-
Xin Zeng - zengyix@mail.sysu.edu.cn; Xiao-Feng Zhu - zuxfeng@mail.sysu.edu.cn; Xiao-Shi Zhang* - zxs617@hotmail.com
* Corresponding author
Abstract
Background: Cancer stem cell model suggested that tumor progression is driven by the overpopulation of cancer stem
cells and eradicating or inhibiting the symmetric division of cancer stem cells would become the most important
therapeutic strategy. However, clinical evidence for this hypothesis is still scarce. To evaluate the overpopulation
hypothesis of cancer stem cells the association of percentage of CD133+ tumor cells with clinicopathological parameters
in colon cancer was investigated since CD133 is a putative cancer stem cell marker shared by multiple solid tumors.
Patients and methods: Tumor tissues matched with adjacent normal tissues were collected from 104 stage IIIB colon
cancer patients who were subject to radical resection between January, 1999 to July, 2003 in this center. The CD133
expression was examined with immunohistochemical staining. The correlation of the percentage of CD133+ cell with
clinicopathological parameters and patients' 5-year survival was analyzed.
Results: The CD133+ cells were infrequent and heterogeneous distribution in the cancer tissue. Staining of CD133 was
localized not only on the glandular-luminal surface of cancer cells but also on the invasive budding and the poorly
differentiated tumors with ductal structures. Both univariate and multivariate survival analysis revealed that the
percentage of CD133+ cancer cells and the invasive depth of tumor were independently prognostic. The patients with a
lower percentage of CD133+ cancer cells (less than 5%) were strongly associated with a higher 5-year survival rate than
those with a higher percentage of CD133+ cancer cells (greater than or equal to 55%). Additionally, no correlation was
obtained between the percentage of CD133+ cancer cells and the other clinicopathological parameters including gender,
age, site of primary mass, pathologic types, grades, and invasive depth.
Conclusion: The fact that a higher percentage CD133+ cells were strongly associated with a poorer prognosis in
patients with locally advanced colon cancer implicated that CD133+ cancer cells contribute to the tumor progression,
and the overpopulation hypothesis of cancer stem cell seems reasonable.
Published: 7 July 2009
Journal of Translational Medicine 2009, 7:56doi:10.1186/1479-5876-7-56
Received: 29 November 2008
Accepted: 7 July 2009
This article is available from: http://www.translational-medicine.com/content/7/1/56
© 2009 Li et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Background
Colorectal cancer is one of the most common causes of
cancer death worldwide. Although the median overall sur-
vival of patients with metastatic colorectal cancer has
increased from 12 months to approximately 24 months
over the past decade as a result of an improvement in sys-
temic therapies including new chemotherapeutic agents
such as CPT-11 and oxaliplatin and monoclonal antibod-
ies against EGFR (cetuximab and panitumumab) and
VEGF (bevacizumab), the 5-year survival is still pessimis-
tic [1-4]. Therefore, one of the main challenges in colorec-
tal carcinoma remains to develop new strategies beyond
chemotherapy to inhibit the disease progression.
A growing body of evidence supports the notion that only
a small subset of cells within a solid tumor have 'stem-
like' characteristics. These tumor-initiating cells, or cancer
stem cells, distinct from non-malignant stem cells, show
low proliferative rates, high self-renewal capacity, propen-
sity to differentiate into active proliferating tumor cells,
and resistance to chemotherapy or radiation [5,6]. Until
now, cancer stem cells have been identified in a great deal
of solid tumors [5-8].
Multiple cancer stem cell-associated markers have been
identified, among which CD133 has received considera-
ble attention. CD133 or prominin-1 gene is located on
chromosome 4p15.32 and encodes a cell surface glyco-
protein compromising five transmembrane domain and
two large glycosylated extracellular loops [9,10]. The tran-
scription of CD133 can be initiated at five tissue specific
promoters, yielding eight alternatively spliced transcripts
[11-13]. Epigenetic mechanism is involved in the regula-
tion of CD133 expression [14-16]. Although the function
of CD133 is unknown, preliminary evidence proposed
that expression of CD133 is associated with the activation
of stemness-related signal pathway, resistance to apopto-
sis and bioenergetic stress [17-22]. Initially identified in
hematopoietic stem cells, CD133 is now shared as cancer
stem cell marker across multiple kinds of solid tumors,
such as those in the brain, breast, lung, liver, colon, pros-
tate, pancreatic carcinomas, medulloblastoma, and
melanoma [5-7,23-29].
As for colorectal cancer, initially, Ricci-vitiani and O'Brien
observed that colon cancer stem cells are located in the
CD133+ subpopulation, which accounts for approxi-
mately 2.5% of the tumor cells [30,31]. Subsequently,
Dalerba and Haraguchi reported that markers for colon
cancer stem cells are EpCAM hi/CD44+/CD166+ [32,33].
In addition, Barker proposed that Lgr5 is another marker
[34]. CD133+ colon cancer cells include EpCAM hi/CD44+
cells, whereas the relationship between CD133+ subset
and Lgr5+ subset is unclear. Therefore, which protein
would be an ideal marker for colorectal cancer stem cells
remains an open question.
Based on the mathematic model, the hypothesis that
development of colorectal carcinoma is driven by over-
population of stem cells has been suggested. It is believed
that the abundance of cancer stem cells is derived from
their symmetric division, whereas their normal partners
are subject to asymmetric division, therefore, eradicating
or inhibiting the symmetric division of cancer stem cells
would become the most important strategy for cancer
treatment [35-39]. If the percentage of cancer stem cells is
associated with the prognosis of cancer patients, the over-
population hypothesis would be substantially supported.
By now, the relationship between the percentage of
CD133 and prognosis of colorectal carcinomas was con-
troversial. Horst reported that CD133 expression is an
independently prognostic marker whereas this kind of
correlation was not confirmed by Kojima[40,41]. Accord-
ingly, more evidence was need to elucidate the relation-
ship between the percentage of CD133+ tumor cells and
the prognosis of colorectal cancer patients. This study
showed that the percentage of CD133+ tumor cells was
associated with the prognosis among patients with locally
advanced colon cancers, implicating that CD133+ cells are
involved in the progression of colon cancer.
Patients and methods
Patients and Follow-up
104 cases of pathologically confirmed specimens were
obtained from colon carcinoma patients with TNM stage
IIIB (the depth of primary invasive spread defined as T3
and T4 with one to three regional node involvement but
no distant metastasis) who were subject to radical resec-
tion between January, 1999 and July, 2003 in the Cancer
Center of Sun Yat-Sen University, Guangzhou, China.
None of the patients had undergone either chemotherapy
or radiotherapy before the collection of the samples. All of
them were subject to 5-Fu based postoperatively adjuvant
chemotherapy for six months. Patients were observed on
an every-three-month basis during the first year, once
every 6 months in the second year, and by telephone or
mail communication once every year thereafter for a total
of 5 years. If recurrence or metastasis occurred, 5-Fu based
chemotherapy was given according to the NCCN guide-
line. Overall survival was defined as the time from opera-
tion to death or was censored at the last known alive data.
Histopaothologic characteristics were confirmed by
blinded review of the original pathology slides. The TNM
classification was used for pathologic staging, and the
World Health Organization classification was used for
pathologic grading.
Immunohistochemical assay
The expression of CD133 in primary tumors matched
with adjacent noncancerous tissue was examined with
immunohistochemical assay. Briefly, formalin-fixed, par-
affin-embedded archived tissues were subject to 4-m sec-
tion. Then, sections were subject to dewax, rehydration,

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blocking with hydrogen peroxide, and antigen retrieval
with microwave in a 10 mM citrate buffer (pH 6.0) for 10
min and cooled to room temperature. After being blocked
with 1% goat serum albumin sections were incubated
with the mouse monoclonal antibodies against human
CD133 at a dilution of 1:150 (Abcam, Cambridge, UK)
overnight at 4°C, followed with horseradish peroxidase-
labeled secondary antibodies for 30 minutes at room tem-
perature. The sections were developed with diaminoben-
zidine tetrahydrochloride (DAB) and counterstained with
hematoxylin. Immunohistochemical assay was per-
formed within 7 days of section preparation. To prevent
antigen degradation sections were stored at 4°C before
immunohistochemical analysis. Tissue derived from gli-
oma was used as positive control and negative controls
were made with primary antibody replaced by PBS.
Referring to Maeda's method, slides were examined under
low power (×40 ~ ×200) microscope to identify the
regions containing the highest percentage of CD133+ cells
(hot spot) in the cancer nest [42]. Ten fields of hot spot
inside the tumor tissue were selected, and expression of
CD133 was evaluated in 1000 tumor cells (100 cells per
field) with high power (×400) microscopy. Specimens
were defined as positive for CD133 expression if there
were tumor cells distinctly stained by the anti-CD133
antibodies. The percentage of CD133+ cells was classified
into two levels: < 5% CD133-positive cells and  5%
CD133-positive cells[42].
Statistical analysis
The following factors were assessed with both univariate and
multivariate analyses for their influence on overall survival:
gender, age (<60 years old vs  60 years old), sites of primary
mass (left hemicolon vs right hemicolon), the T stages (the
depth of primary invasive spread, T3 vs T4), pathological
classifications (papillary carcinoma and tubular adenocarci-
noma vs mucoid adenocarcinoma and signet ring cell carci-
noma), tumor grades (the degree of cellular differentiation,
well differentiated, G1 vs moderate differentiated, G2 vs
poorly differentiated, G3), and the percentage of the CD133+
cells (<5% positive vs 5% positive). The nonparametric cor-
relation Kendall's tau-b test was used to assess associations
between categorical variables. Kaplan-Meier curves were
used to estimate the distributions of clinicopathological
characteristics to survival and compared with the log-rank
test. The Cox regression model was used to correlate assigned
variables with overall survival. All statistical analyses were
carried out using SPSS 15.0 software (SPSS Inc, Chicago, IL,
USA). Statistical significance was assumed for a two-tailed P
< 0.05.
Results
Expression of CD133 in tumor tissue
CD133 brownish signals were observed on the cell mem-
brane, especially on its luminal and basal surface. In gen-
eral, the cases with intensive staining of CD133 had
higher percentage of CD133+ tumor cells. Several nests
with intensive CD133 staining, so-called "hotspot" could
always be seen within the field of cancer nests microscop-
ically (Fig 1A to 1D). The cancer cells within an adenocar-
cinoma nest could actively proliferate and form a group of
cells, which invaded into the surrounding tissue, so-called
"budding", and showed negative or weak staining against
CD133 (Fig 1E). Besides staining on the well differenti-
ated tumors CD133 staining was documented on the
poorly differentiated tumors with ductal structures rather
than those without ductal structures (Fig 1F). The paratu-
morous normal intestinal epithelium could be found in
72 out of 104 specimens used for this study. The CD133
expression of normal intestinal epithelium was only
found in 7 out of the 72 specimens.
Referring to Maeda's method the percentage of CD133+
cells was classified into two levels: < 5% CD133+ cells and
 5% CD133+ cells [39]. In this group of patients, 62 cases
The expression of CD133 in colon cancer patients with stage IIIB (10 × 20~10 × 100)
Figure 1
The expression of CD133 in colon cancer patients
with stage IIIB (10 × 20~10 × 100). The expression of
CD133 was examined with immunohistochemical assay. (A):
<5% CD133+ cells in the cancer nest; (B): 5% CD133+ cells
in the cancer nest; (C) and (D): the staining of CD133 on the
luminal surface and the basal surface of cancer cells; (E): the
staining of CD133 on budding cancer nest; (F): the staining of
CD133 on poor-differentiated cancer nests with ductal
structures.

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of 104 (59.6%) specimens contained less than 5%
CD133-positive tumor cells and 42 cases (40.4%) con-
tained more than 5% CD133-positive tumor cells, among
which the percentage of CD133+ cells varying from 5% to
25% existed in 23 cases, from 26% to 50% in 12 cases,
and more than 50% in 7 cases.
Relationship between the percentage of CD133+ cells and
clinicopathological characteristics
No correlation was observed between the expression of
CD133 and clinicopathological parameters such as age,
gender, sites of primary mass, pathological classifications,
invasive depth, and tumor grades. Otherwise, the analysis
revealed that mucoid adenocarcinomas and signet ring
cell carcinomas had the potential with poorer differentia-
tion (r = 0.459, P < 0.001) and higher frequency occurred
in the right hemicolon (r = 0.215, P = 0.022) (Tab 1).
Relationship between survival and clinicopathological
characteristcs assessed with univariate survival analysis
By the end of the 5-year follow-up, 67 cases were still
alive. So, the 5-year survival rate was 64.4%. Kaplan-Meier
analysis revealed that the percentage of CD133+ cells in
cancer nests and the invasive depth of primary mass were
prognostic. The 5-year survival rate among patients with a
higher percentage of CD133+ cells (5%) in the cancer
nests was 45.2%, whereas those with a lower percentage
of CD133+ cells (<5%) was 77.4% (P = 0.001). In addi-
tion, the 5-year survival rate among patients with T3
tumors (tumors which invade through the muscular pro-
pria into the subserosa, or into nonperitonealizd pericolic
tissue) was 69.6%, whereas the 5-year survival rate among
patients with T4 tumors (tumors which perforate the vis-
ceral peritoneum or directly invade other organs or struc-
ture) was 25.0% (P = 0.001)(Tab 2).
Relationship between survival and clinicopathological
characteristics assessed with multivariate survival analysis
The Cox regression model revealed that the patients with
a lower percentage CD133+ cells (<5%) in the cancer nests
were significantly associated with a higher 5-year survival
rate with -0.987 in partial regression coefficient and 0.373
(95% CI 0.190 ~ 0.732) in relative risk (P = 0.004). Addi-
tionally, a higher T stage (invasive depth) was signifi-
cantly associated with a lower survival rate with 1.209 in
partial regression coefficient and 3.351 (95% CI 1.558
~7.208) in relative risk (P = 0.002). Therefore, the percent-
age of CD133+ cells in cancer nests and T stage were inde-
pendently prognostic factors. No relationship was
observed between the survival and the other clinicopatho-
logical parameters such as age, gender, site of primary
mass, pathological classifications, and grades (Tab 2,
Fig 2).
Discussion
This study showed that a higher percentage of CD133+
cells in cancer nests was strongly associated with the lower
5-year survival rate in colon cancer patients with stage
IIIB, a locally advanced disease among which most of
patients would die from metastasis in spite of adjuvant
chemotherapy, implying that the overpopulation hypoth-
esis of cancer stem cell seems reasonable as CD133 is a
putative marker of colon cancer stem cells.
The evidence concerning the correlation of the percentage
of CD133+ tumor cells with the prognosis of patients was
scarce as a few of observations were reported [43-46].
Recently the relationship between CD133 expression and
prognosis in colorectal carcinomas was examined. Horst
reported that CD133 expression is an independently
prognostic marker whereas this kind of correlation was
not observed by Kojima. [40,41] The discrepancy might
derived from inadequate patient quantity and the mixed
tumor stage. For example, in Kojima's study, a total of 189
patients consisted of 106 cases of colon cancers and 83
cases of rectal cancers with TNM stages varying from I to
VI, that is, one group of patients with a definite stage con-
tained only 20 or 30 cases of colon or rectal cancer
patients, respectively[41]. Similar situation existed in
Horst's study [40]. To narrow the heterogeneity of patients
Table 1: Correlations of CD133 expression with clinicopathological parameters in the Stage IIIB colon carcinomas
Variables genderageInvasive depth Sites of primary mass GradesPathological
classifications
The percentage of
CD133+ cells
gender
age
Invasive depth
Sites of primary mass
Grades
Pathological
classifications
The percentage of
CD133+ cells
P
P
P
P
P
P
.
.242
.541
.792
.129
.129
.242
.
.312
.075
.455
.869
.541
.312
.
.895
.272
.426
.792
.075
.895
.
.936
.022*
.129
.455
.272
.936
.
.000**
.129
.869
.426
.022*
.000**
.
.785
.249
.499
.786
.536
.333
P .785.249 .499.786 .536.333.
*: P < 0.05; **: P < 0.001

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and make the results more reproducible this study
included 104 cases of colon carcinoma patients with stage
IIIB. The results showed that CD133+ cancer cells contrib-
uted to the progression of colon cancer, arguing the
Hosrt's observation.
The discrepancy concerning the pattern and the frequency
of CD133 expression in colon cancer also existed between
the studies mentioned above and this study. Horst and
Kojima reported that CD133 antigen, stained with anti-
bodies from Miltenyi Biotech, Sata Cruz Biotechnology,
or Cell signaling, was localized exclusively on the glandu-
lar-luminal surface of colorectal cancer. Staining of the
CD133 was observed neither on the budding cancer nest
nor on poorly differentiated cancer cells [40,41]. How-
ever, in this study, being stained with antibodies from
Abcam CD133 expression existed not only on the apical
membrane but also on basal surface of tumor cells, both
on the budding cancer nest (the invasive front) and on the
poorly differentiated cancer cells, although the intensity
of staining was weaker. This pattern of CD133 expression
might be more likely consistent with the hypothesis that
CD133+ cancer cells would reveal a more aggressive phe-
notype. Since the intensity of CD133 is cell cycle-depend-
ent, among which the least CD133 immunoreactive cells
are in the G0/G1 portion, and the increased CD133+ cells
is correlated with increased DNA content, and cancer cells
is relatively arrested in the invasive front, so, attenuated
expression of CD133 occurred in the invasive front (bud-
ding)[47,48]. As for the frequency of CD133+ cells in
colorectal cancers the discrepancy also existed. In
Kojima's study CD133 expression was detected in only 29
of the 189 tumors (15.3%). Of these, 21 tumors (11.1%)
showed CD133 over-expression among which CD133
positive area occupied more than 10% of the entire tumor
tissue[41]. Otherwise, in Horst's study tumors with more
than 50% of CD133+ tumor cells exist in 20 out of 79
colorectal cancers (25.3%) [40]. In this study, the percent-
age of CD133+ cells varying from 5% to 25% existed in 23
cases (22.1%), from 26% to 50% in 12 cases (11.5%), and
more than 50% in 7 cases (6.7%). Therefore, it is reason-
able to infer that the heterogeneous patterns and frequen-
cies of CD133 expression in colon cancer derived from the
specificity of antibody clones used. In the future, more
attention should be paid to the specificity of CD133-tar-
geting antibodies, the standardization of the CD133 pos-
itive cells classification system, and homogeneity of
tissues.
Table 2: Assessment of overall survival for stage IIIB colon carcinoma patients by clinicopathological parameters with univariate and
multivariate analysis
Clinicopaothological characteristicsN
(n = 104)
5-year survivalKaplan-Meier analysis
P value
Cox regression model analysis
P value
64.4%
Gender
male
female
0.461 0.479
65
39
61.5%
69.2%
64.4%
Age 0.1480.211
 60 year old
<60 year old
57
47
57.9%
72.3%
64.4%
Sites of primary mass
Left hemicolon
Right hemicolon
0.291 0.381
60
44
68.3%
59.1%
64.4%
Pathological classifications
papillary + tubular
mucoid + signet ring
0.4230.139
82
22
65.9%
59.1%
64.4%
Grades 0.1540.114
G1
G2
G3
560%
68.8%
47.4%
64. 4%
80
19
Invasive depth
T3
T4
0.0010.002
92
12
69.6%
25.0%
64.4%
The percentage of CD133+ cells
5% CD133 positive
<5%CD133 positive
0.001 0.004
42
62
45.2%
77.4%