Tumor Biol.
DOI 10.1007/s13277-016-5214-8
ORIGINAL ARTICLE
Primary cultures of human colon cancer as a model to study
cancer stem cells
Sergey Koshkin 1 & Anna Danilova 2 & Grigory Raskin 3 & Nikolai Petrov 1 &
Olga Bajenova 4,5 & Stephen J. O’Brien 4 & Alexey Tomilin 1 & Elena Tolkunova 1
Received: 22 December 2015 / Accepted: 14 July 2016
# International Society of Oncology and BioMarkers (ISOBM) 2016
Abstract The principal cause of death in cancer in-
volves tumor progression and metastasis. Since only a
small proportion of the primary tumor cells, cancer stem
cells (CSCs), which are the most aggressive, have the
capacity to metastasize and display properties of stem
cells, it is imperative to characterize the gene expression
of diagnostic markers and to evaluate the drug sensitiv-
ity in the CSCs themselves. Here, we have examined
the key genes that are involved in the progression of
colorectal cancer and are expressed in cancer stem cells.
Primary cultures of colorectal cancer cells from a pa-
tient’s tumors were studied using the flow cytometry
and cytological methods. We have evaluated the clinical
and stem cell marker expression in these cells, their
resistance to 5-fluorouracil and irinotecan, and the abil-
ity of cells to form tumors in mice. The data shows the
role of stem cell marker Oct4 in the resistance of pri-
mary colorectal cancer tumor cells to 5-fluorouracil.
* Sergey Koshkin
Koshkin31@mail.ru; http://www.cytspb.rssi.ru
1
Institute of Cytology, Russian Academy of Sciences, Tikhoretsky
ave. 4, St. Petersburg, Russia 194064
2
N.N. Petrov Oncology Research Institute, St. Petersburg 197758,
Russia
3
Russian Research Centre for Radiology and Surgical Technologies,
St. Petersburg 197758, Russia
4
Theodosius Dobzhansky Center for Genome Bioinformatics, St.
Petersburg State University, St. Petersburg 199034, Russia
5
Department of Genetics and Biotechnology, St. Petersburg State
University, St. Petersburg 199034, Russia
Keywords Colon cancer . Cancer stem cells . Oct4 . CD133 .
Drug resistance . 5-Fu
Colorectal cancer is one of the leading causes of morbidity
and mortality worldwide [1]. The 5-year survival rate for pa-
tients with local colorectal cancer is 80–90 %, whereas that
with distant metastasis is only 10–20 % [2, 3]. Such unfavor-
able outcome for this pathology is caused by frequent late
diagnosis, treatment failure at advanced stages, and
metastasis.
The current standard treatment for metastatic colorectal
cancer includes FOLFOX (oxaliplatin plus infusional 5-
fluorouracil (5-FU) and leucovorin) or FOLFIRI (5-FU,
leucovorin, and irinotecan). Irinotecan is a camptothecin de-
rivative that exerts cytotoxic effects on cancer cells by
inhibiting topoisomerase I, and FOLFIRI has been proven to
be effective as a second-line treatment of patients with meta-
static colorectal cancer [4, 5].
Based on the concept of cancer stem cells (CSCs) [6], a
tumor represents a mixed-cell population that consists of
healthy and cancerous cells with varying degrees of differen-
tiation. The most differentiated cancer cells in the primary
tumor have the features of epithelial tissue from which the
tumor originated, enabling its identification. The least differ-
entiated cells have the characteristics of stem cells that confer
to the tumor the ability to de- and re-differentiate, metastasize,
and acquire drug resistance. CSCs, according to publications,
comprise about 0.1–3 % of the whole cell number in a tumor
and differ from the remaining mass of differentiated cancer
cells of primary tumors in their abilities of assymetric division,
self-renewal and differentiation, elevated resistance to radio-
therapy and chemotherapy, and expression of some stem cell
markers and telomere length [7–10]. CSCs have the ability to
reproduce the parental tumor in vivo. In fact, as with any other

kind of tissue, a malignant tumor has a structure composed of
genetically heterogenous cells in different state: pre-cancer
cells, primary cancer cells, differentiated cells, migrating cells,
and CSC, which play a role in the development of the disease
[11].
It is well established that cancer stem-like cells are charac-
terized by expression of several stem cell markers [12, 13].
The most common membrane markers used for CSC cell
sorting and analysis are CD133, CD44, Lgr5, and aldehyde
dehydrogenase (ALDH1). CD133+ colorectal cancer cells
were shown to form tumors in the immune-deficient mice.
However, the correlation between CD133 expression and the
clinical-pathological factors in colorectal cancer remains
unclear.
Lgr5 was first identified in human colon cancer as a Wnt
target gene [14, 15]. It was shown to be overexpressed in other
human malignancies such as ovarian, hepatocellular, esopha-
geal, and basal cell carcinomas [16]. Lgr5-expressing cells are
responsible for the complete restoration of small and large
intestine epithelium in vivo [17]. The available evidence sug-
gests that Lgr5 might represent a useful marker to identify and
target CSCs in colon cancer.
Most methods rely on specific surface antigen recognition
and thus are restricted by the availability of highly specific
antibodies. In addition, labeling of cell-surface markers by
antibodies could trigger signaling pathways and induce cell
modification and differentiation. Therefore, the development
of methods that do not rely on marker labeling is vital.
In the recent years, many studies have demonstrated that
aberrant expression of stem cell-associated nuclear transcrip-
tion factors, such as Oct4, Sox2, Nanog, and Klf4, can con-
tribute to tumorigenesis in various cancers [18, 19].
The aim of this study is to investigate the significance of
Oct4 and CD133 in the development of colon cancer and
delineate their CSC properties. We make an attempt to extract
a population of cancer stem cells from primary cell culture of
colorectal cancer. We have also assessed the role of Oct4 tran-
scription factor in drug resistance properties of colorectal can-
cer cells.
Material and methods
Processing of primary samples
The material for this study was obtained on the basis of pa-
tients’ informed consent from the LG Sokolov Clinical
Hospital No. 122. The resulting fragments were placed in a
saline solution for 2 h and brought to the laboratory. This
study included six tumor samples. From six endoscopic tissue
samples, two cell lines were obtained: BSC-1 and BSC-6.
Based on the immuno-histochemical studies, the first tissue
sample was characterized by positive staining of 20 % of cells
Tumor Biol.
with CK20, 90 % of the cells with CDX2, and membrane
cytoplasmic localization of beta-catenin in 90 % of cells; the
index of Ki-67 was 60 %. From the first tissue sample later on,
the BSC-1 cell line was selected. The tissue sample from
which we obtained the BSC-6 cell line was also characterized
by a high index of Ki-67 of 60 % and a high level of expres-
sion of CDX2. It was defined as a solid tumor in the wall of
the colon.
The cell isolation from the primary tissue was carried out
on the basis of recommendations of Yu et al. [17]. Each sam-
ple was cut into pieces of approximately 1 mm−3 in size and
incubated in 10-fold (by volume) excess of trypsin (Gibco,
USA) overnight in a refrigerator at 4 °C, followed by an hour
at 37 °C. The action of trypsin was inhibited by RPMI medi-
um with 10 % FBS. This suspension of disintegrated cells was
filtrated through a Falcon Cell strainer (100 μm) and centri-
fuged to collect the cells (150 g for 5 min).
The remaining Bundigested tissue^ was treated with a so-
lution of collagenase in RPMI medium with serum containing
500 units/ml of enzyme, incubated in a Petri dish at 37 °C for
1 h. The released cells had been collected after filtration by
centrifugation (150 g for 5 min) combined with cells previ-
ously obtained after trypsin treatment and were placed in
DMEM medium (Gibco, USA) with 10 % FBS (Sigma,
USA) for culturing in the incubator. The cells were passaged
using trypsin solution (Gibco, USA), once for 3 days at a ratio
of 1:3.
Cell culturing in order to obtain spheroids
To obtain cell spheroids, we cultured cells in plastic dishes
with a low-adhesive surface and in the serum-free culture
medium of the following composition: DMEM medium with
6 mg/ml glucose, 1 mg/ml sodium bicarbonate, 5 mM
HEPES, 2 mM L-glutamine, 4 mg/ml heparin, 4 mg/ml
BSA, 10 ng/ml bFGF, 20 ng/ml EGF, 100 μg/ml
apotransferrin, 25 μg/ml insulin, 9.6 μg/ml putrescine,
30 nM anhydrous sodium selenite, and 20 nM final concen-
tration of progesterone in a concentration of 300,000 cells per
milliliter. Such culture conditions were chosen to preferential-
ly maintain immature (poorly differentiated) tumor cells,
which proliferate and produce cell aggregates called spher-
oids. The above medium composition was described by
Todaro et al. [18].
Packing of viral particles and infected cells in vitro
with a virus
To produce the viral particles, we used human embryonic
kidney cell line HEK293T, which was cultured in DMEM,
containing 10 % fetal bovine serum (Sigma, USA). Cells were
split every 3 days at a ratio of 1:4.
Tumor Biol.
To assemble the viral particles, 293T cells were transfected
with the lentivirus using the calcium phosphate method. This
method was based on the integration of the lentiviral vector
and helper plasmid for the assembly of viral particles
(http://tcf.epfl.ch/page-6766-en.html). The medium with
transfected cells was collected every 48 h, and the virus
particles were concentrated by ultracentrifugation using a
standard technique (http://tcf.epfl.ch/page-6764.html).
Lentivirus infection of cancer cells and selection
of resistant clones
Cells were plated in 24-well plates 1 day before the infection
at a density of 105 cells per well. The infection was performed
by adding 10 μl of concentrated virus to 100 μl DMEM/10 %
FBS medium per well. After 2 days, cells were trypsinized and
plated on a 10-cm culture dish (Falcon, USA). The selection
was carried out by changing the media next day per medium
with antibiotic puromycin (Sigma, USA) at a concentration of
30 μg/ml. We change the medium every 2 days during
14 days. Grown colonies were picked individually,
trypsinized, and plated on 3-cm plates. After a week in culture,
the cells were frozen in the liquid nitrogen.
Test on the formation of tumors in mice
Cell cultures were trypsinized to obtain single cell suspension,
and cells were washed three times with Hank’s solution. After
counting, the cell concentration was adjusted to 106 cells/ml.
The resulting suspension was injected into immuno-deficient
nude mice subcutaneously into the thigh region, 106 cells per
injection. To prevent any leakage of fluid from the syringe, the
needle was introduced through the top of the thigh muscles
into the gaps between the skin and muscles of the lower side.
Each mouse has received two injections—to the right and left
thighs. Group of animals injected with original cells served as
a control standard to the experimental group, injected with
Oct4+ cells. Grown tumors were measured every 10 days
starting from the 20th day after the injection. Measurements
were made with a caliper by the maximum diameter up to
1 mm. We measured the greatest longitudional diameter, x,
and the greatest transverse diameter, y. The tumor volume
was calculated from these two transcutaneous diameters (1/2
L × W2) [20].
MTT viability test
Assessment of cell viability was performed by colorimetric
MTT assay (Sigma, USA). This method is based on the fact
that in the live cells mitochondrial oxidoreductase reduces
yellow MTT to purple formazan. The amount of the devel-
oped formazan is correlated with the number of viable cells in
the population. The cells were spun down from the RPMI
medium, put into PBS containing 0.5 mg/ml MTT, and incu-
bated for 2 h at 37 °C under 5 % CO2. The resulting precip-
itates (crystals) of formazan were dissolved in DMSO (Sigma,
USA). The absorbance was measured at 570 nm with the
Multiskan EX spectrophotometer (Thermo Electron, USA).
Analysis of drug resistance
To determine the sensitivity of CRC cells to the cytostatic
drugs (5-FU and irinotecan), the cells were seeded in 96-
well plates at 50,000 per well in a volume of 100 μl. Each
experimental point was measured six times, and the standard
error of the mean was calculated. The experiment was repeat-
ed three times with obtaining similar results. The final con-
centration of 5-FU was 100 ng/ml, for IT 20 ng/ml. For each
studied cell lines, we had six wells of control, without the
cytostatic. After 48 h, 10 μl of MTT solution (5 mg/ml in
PBS buffer) was added to each well and the cells were incu-
bated for 2 h. Then, PBS buffer was removed and formazan
crystals were dissolved in 100 μl DMSO. Further measure-
ments were carried out with a Multiskan EX spectrophotom-
eter (Thermo Electron, USA). Results were expressed as per-
centage of cells surviving after the drug treatment vs the con-
trol group (untreated cells).
Histological and enzyme-linked immunosorbent staining
was performed by G.A. Raskin at The Department of
Pathology of the Russian Scientific Center of Radiology and
Surgical Technologies, headed by Dr. K.M. Pozharissky. The
selection of CD133+ by specific antibodies was performed
according to the manufacturer’s CD133 MicroBead human
Kit protocol (MACS Miltenyi Biotec, Germany).
Results
Primary tumors cell culture
Our work was carried out with the biopsies of six patients that
were processed as described in the BMaterials and methods^
section. From six biopsy specimens taken from different pa-
tients, we were able to select two cell lines. The resulting cell
lines, called BSC-1 and BSC-6, were further characterized by
their ability to grow in the form of a monolayer and spheroids.
To obtain the monolayer cultures, CRC cells, obtained
from the endoscopic material, were cultured in DMEM medi-
um with 10 % FBS as a monolayer. Through the passages, the
structure of the cell population changed (Fig. 1). The initial
cell suspension contained a large amount of red blood cells,
sometimes visible as Brouleaux.^ After 5–7 days in a culture,
small colonies of fibroblast-like cells appeared. As illustrated
in Fig. 1, the cells initially had fibroblast-like morphology
(Fig. 1a). After 1 month in culture, the primary cells devel-
oped dense colonies of polygonal cells, similar in morphology
 Tumor Biol.

Fig. 1 Primary colon cancer cell

cultures were grown as a
monolayer or spheroids on the
low-adhesive substrate. a Small
colonies appearing on the fifth
day in culture. b–d Colonies
developed after 1 month in culture
in a monolayer. e Spheroids are
formed when cells are plated on
the low-adhesive plastic in the
serum-free medium. f Spheroid
formed on agarose on the third
day in culture

to the embryonic stem cells surrounded by a small number of
fibroblast-like cells (Fig. 1b–d).
These cells were able to grow in size in a serum-free me-
dium supplemented with growth factors and to form spheroids
on a low-adhesive plastic (Fig. 1e). When agarose was used as
a low-adhesive substrate, the primary cells formed spheroids
(Fig. 1f). Aggregates of five to eight cells could be seen, fol-
lowing the seeding on low-adhesive plastic, suggesting that
these spheres are not the cell clones but cell aggregates. We
noticed that tumor cells have high adhesion to the plastic. If
the serum-free medium was not changed every day, the
spheres will have started attaching to the plastic and spread-
ing, looking similar to the cells in the monolayer culture. On a
highly adhesive plastic in a serum-free medium, the colonies
could detach, and they often started growing as spheres.
Next, we compared the expression of clinical CRC markers
in the BSC-6 cell line and in the original primary tumor tissue
using specific staining with the antibodies to PAN-cytokeratin
and receptor CDX2 (Fig. 2). The data show that after ten
passages in culture, the BSC-6 cell line retains the expression
of PAN-cytokeratin and CDX2 (Fig. 2b, d, f), which corre-
sponds to the staining of initial material—a moderately differ-
entiated adenocarcinoma of the colon (Fig. 2a, c, e). The
figure shows a positive staining by anti-CK-PAN antibodies
in 80 % of cells and by the anti-CDX2 antibody in 100 % of
the cells, which is similar to the staining of original cancer
biopsy.
Enrichment of endogenous Oct4 expression by infection
with the lentiviral construct
We have recently developed a bicistronic DNA construct
2A2B-TKiresPur which features the thymidine kinase (TK)
and puromycin resistance (Puror) genes under the control of
the 2A2B enhancer from the pluripotency-associated gene
Oct4 [21]. When stably integrated into the pluripotent cell
genome, the construct allows highly selective enrichment
(via Puror gene) or elimination (via TK gene) of Oct4+ undif-
ferentiated pluripotent cells from the mixed populations, fol-
lowing the exposure to puromycin or ganciclovir (GCV), re-
spectively [21]. In the current study, we used the 2A2B-
TKiresPuro construct embedded into the lentiviral vector
(Fig. 2b). It was suggested that the same strategy would allow
to positively select CSC cells that, according to our working
hypothesis [22], express Oct4. Transfected colorectal cancer
cells were selected in DMEM medium with 10 % FBS,
Tumor Biol.

Fig. 2 Original colon cancer

tumor (a, c, e) and derived thereof
cell line BSC-6 (b, d, f) were
stained with hematoxylin-eosin
(a, b) or with the antibodies to the
clinical markers CK-20 (c, d) or
CDX2 (e, f), followed by
peroxidase-conjugated secondary
antibodies. The figure shows a
positive staining by anti-CK-PAN
antibodies in 80 % of cells and by
anti-CDX2 antibody in 100 % of
the cells, which is similar to the
staining of original cancer biopsy

containing 2 μg/ml puromycin. Resistant cell lines were called
BSC-1R (Oct4+) and BSC-6R (Oct4+). To keep the cells pu-
romycin resistant, we cultured them in media, containing pu-
romycin. After five passages, cells continue to express Oct4,
which was checked by RT-PCR with Oct4-specific primers
(data not shown). As a matter of controlling lentiviral trans-
duction efficiency, we used lentivirus-expressing green fluo-
rescent protein (EGFP). The data shows the high efficiency of
infection—60–70 % of cells (Fig. 3a, b).
(Oct4+) and BSC-6R (Oct4+) and of parental cell lines are
similar.
To study the nature of the tumors, they were restrained in
polyformaldehyde and stained with anti-CK-20, beta-catenin,
CDX2, and ALDH1 antibodies (Fig. 4). The data shows that
after prolonged passaging, and regardless of culturing
methods, the established CRC cell lines preserve the ability

In vivo mouse experiments

In our next step, we evaluated the ability of CRC cells, cul-

tured in monolayer or as spheroids, to give rise to tumors in
immune-deficient nude mice (Table 1). In this step, after 10
passages, culture BSC-1 cells were injected subcutaneously
into mice; the presence and the size of the tumors were esti-
mated after 30 days. Tumor measurements were made with a
caliper by the maximum diameter. BSC-1 cells cultured in a Fig. 3 Primary colon cancer cells infected with the lentiviral construct.
Upper panel—a scheme of the suicidal lentiviral construct carrying the
monolayer and the same cells cultured as spheroids gave rise 2A2B-TKtkPur cassette. Lower panel—cell lines BSC-1 (a) and BSC-6
to tumors of a similar size (data not shown). Our data show (b) that are transfected with the virus expressing green fluorescent pro-
that tumorigenic potentials of puromycin-selected BSC-1R tein. The figure shows high efficiency of virus transduction
 Tumor Biol.

Table 1 Size of tumors,

developed in vivo after injection Cell line Average volume, Percentage of animals that developed Numbers of injected
of colorectal cancer cells in mice mm3 tumors mice

BSC-1 388 ± 124 75 % 4

BSC-1R 407 ± 343 50 % 4
BSC-6 372 ± 308 57 % 7
BSC-6 150 ± 46 60 % 8
R

BSC-1 (four mice) and BSC-6 (seven mice) original cell lines served as a control standard for experimental
groups, injected with Puror (Oct4+) cells (four mice for BSC-1R and eight mice for BSC-6R)

to form tumors in nude mice (Fig. 4b, d, f, h) and are suitable
to study CSCs. The data revealed that the expression patterns
of these clinical markers are very similar to those in primary
human tumor tissues (compare Fig. 4a, c, e, g).
To find out if there is a genetic heterogeneity in puromycin-
resistant cell cultures, we have characterized individual clones
of the BSC-6R (Oct4+) cell line. The quantification by flow
cytometry of the percentage of cells expressing Oct4, Sox2,
and Lgr5 markers of 16 BSC-6R (Oct4+) clones confirmed
that the levels of Oct4+ cells (98–100 %) and Sox2+ cells (98–
100 %) were similar in all of them. For the Oct4+ cell clones,
which have also shown a high number of cells with enhanced
Sox2 expression, we observed a different expression of colo-
rectal cancer stem cell marker Lgr5. There were substantial
differences in the proportions of cells expressing the Lgr5
marker in different clones (21 to 92 % of cells were Lgr5+).

Fig. 4 Immuno-histochemical
staining of initial colorectal
cancer tumor tissue (a, c, e, g),
and tumors developed in mice (b,
d, f, h) by BSC-1 cells. Samples
were stained with antibodies to
CK-20 (a, b); beta-catenin (c, d);
CDX2 (e, f); ALDH1 (g, h). The
data show the same pattern of
expression of colon cancer
markers in original tumor, and
developed in mice
Tumor Biol.

Not all cells, expressing Oct4, express also Lgr5. When cells
were regrown in culture, they were able to reconstitute the cell
heterogeneity of the starter culture.

Drug resistance of colorectal cancer cells to irinotecan

and 5-FU

5-FU is the Buniversal^ anti-cancer drug. It is included in the

therapeutic regimens received by more than half of all cancer
patients, although the response to the treatment with 5-FU
alone in patients is no more than 10–15 % [23]. Irinotecan
targets DNA topoisomerase I, causing the inhibition of DNA
Fig. 5 The differences in the resistance of colorectal cancer cell lines
replication and subsequent cell death—a standard treatment BSC-1 and BSC-6 to irinotecan (20 mkM) and 5-FU (100 mkM). Y-axis
regimen for metastatic colorectal cancer, especially in combi- shows the percentage of cells that survived treatment vs the control cells
nation with oxaliplatin [24]. It improves the response rate of (no drug treatment) measured by optical density in MTT assay. The data
patients with colorectal cancer in up to 40–50 %. The effects show that both lines are resistant to irinotecan treatment, but have differ-
ent sensitivity to 5-FU. BSC-1 is resistant; BSC-6 is sensitive
of these two anti-cancer drugs were tested on BSC-1 and
BSC-6 cell lines. The results show (Fig. 5) that BSC cells
Discussion
derived from different patients responded differently to 5-
FU. BSC-6 cells were sensitive to 5-FU treatment as more
Gastrointestinal cancers account for approximately 30 % of
than 50 % of cells could not survive, whereas BSC-1 cells
the total cancer patient population worldwide [26, 27]. 5-FU
were resistant to 5-FU treatment (Fig. 5).
with oxaliplatin for colorectal cancer has shown limited clin-
We choose the BSC-6 cell line to further assess how these
ical utility [28, 29], especially due to the high rate of tumor
cells, enriched for CSC marker Oct4 and CD133, will respond
recurrence. Emerging evidence suggests that the poor re-
to the indicated drugs. The stem cell marker enrichment has
sponse to the current treatment modalities for epithelial can-
been done in two ways. First, the cells were transfected with
cers is due to the aberrations in multiple signaling pathways
the lentivirus carrying the suicidal vector that allows to select
together with the presence of a small subpopulation of drug-
cells expressing endogenous Oct4, and second, by sorting
resistant CSCs that have the propensity to promote tumor
cells with the antibodies to the surface antigen CD133.
recurrence, invasion, and metastasis [30]. Although chemo-
The following enriched populations were analyzed (Fig. 6):
therapies target the majority of tumor cells, the CSCs in the
the parental cells BSC-6 cells, (BSC-6), the same cells after
tumor mass are nonresponsive, resulting in tumor recurrence
MACS for CD133 (CD133+) and the flow-through (CD133
[31].
−), BSC-6R cells selected on puromycin (Oct4+), and the
same cells after MACS for CD133 (Oct4+CD133+) and the
flow-through (Oct4+CD133−). The data shows that these cell
populations express similar response to irinotecan treatment
(Fig. 7a). In contrast, sensitivity of selected cell populations to
5-FU is different. As is shown in Fig. 7b, the cell population
enriched in endogenous expression of Oct4 (Oct+) has 33 %
higher resistance to 5-FU than the original cell line. CD133+
cells, otherwise, did not obtain a high resistance compared to
CD133− cells. CD133− cells are more resistant to 5-FU than
CD133+ cells. According to our results, CD133+ cells are
twice more sensitive to 5-FU than CD133− cells. That is cor-
rect for both original cell lines and for Oct4+ enriched cells.
The fact that the cell population enriched by the endoge-
Fig. 6 Scheme of BSC6 cell enrichment by CSC markers Oct4 and
nous expression of nuclear factor Oct4 has significantly great- CD133. Cells were infected with reporter construct and selected on
er resistance to cytostatic drugs is consistent with the obser- puromycin. Starting cell line (BSC-6) and Puror (Oct4+) cells were sorted
vations that CSCs have higher resistance to modern by MACS Technology (Miltenyi Biotec). CD133+ and CD133− cells
chemotherapeutic agents [25]. The lack of resistance to were separated. As a result of the two-step enrichment technique, we
received six cell lines: starting cell line (BSC-6), Oct4+-enriched line
fluorouracil of CD133+ cells makes the possibility of using and CD133+ and CD133− lines from starting line (BSC-6 CD133+,
CD133 as a marker for CSC population in colorectal cancer BSC-6 CD133−) and from Oct4+-enriched line (BSC-6 Oct4+CD133+,
questionable. BSC-6 Oct4+CD133−)

Fig. 7 The resistance of BSC-6 cells, enriched by Oct4 and CD133
markers to irinotecan (a) and 5-FU (b). Y-axis shows the percentage of
cells that survived the treatment vs control cells (no drug treatment)
measured by optical density in MTT assay. (*P < 0.05, Mann-Whitney
U test). The data show that all cell lines are resistant to irinotecan treat-
ment. Oct4+ has higher resistance to 5-FU than the original cell line.
CD133+ cells are twice more sensitive to 5-FU than CD133− cells.
That is correct for both original cell lines and for Oct4+-enriched cells
A number of studies have highlighted the detection of low-
level expression of Oct4 in tumor cells and its enrichment in a
subpopulation of CSCs. Based on such information, Oct4 in
CSCs act via mechanisms different from those in pluripotent
stem cells [32]. To investigate the significance of Oct4 expres-
sion in acquiring cancer stem-like properties, we established
the cell lines from primary tumors in monolayer forms that
have maintained the stem cell markers of the primary tumors.
Low level of Oct4 protein in somatic tumors makes it difficult
to detect it by Western hybridization and to enrich the CSC
subpulation by sorting [33, 34]. We have found that the level
of Oct4 expression in primary cultures of CRC is sufficient to
enrich this subpopulation by integration of the 2A2B-
containing construct. Our study has indicated that primary
colorectal cancer cells retain their tumorigenic potential after
being cultured, regardless of the culturing method (monolayer
or spheroids). The results of this research indicate that obtain-
ed cell lines can be used as in vitro model to study colorectal
cancer stem-like cells.
Populations of immature stem-like cells were successfully
isolated from primary cancer tissues, but the molecular
Tumor Biol.
networks supporting their resistant behavior are poorly under-
stood. Identification of a specific marker of CSCs could have a
significant impact on the design of effective therapies against
drug-resistant cancers [35]. For the first time, the new method
of enrichment of the endogenous Oct4 gene expression by
infection with lentiviral construct was developed. This suicid-
al vector allows not only to enhance the proportion of cells
with endogenous expression of Oct4 protein in the CSC pop-
ulation on puromycin but also to select the population of Oct4-
expressing cells using another antibiotic, ganciclovir.
Cancer stem cells possess a stronger tumorigenetic activity
and resistance to radiotherapy and chemotherapy [36]. CSCs
may be responsible for the treatment failure in multiple tu-
mors, because they are more resistant to chemotherapy and
radiotherapy than other tumor cells [37–39]. However, any
correlation of chemotherapeutic drug resistance and expres-
sion of Oct4 in colorectal cancer remains unclear. The fact that
in our experiments the cell population enriched in endogenous
Oct4 expression had significantly greater resistance to 5-FU is
consistent with the fact that CSCs can survive chemotherapy
[40].
As recently shown, transcription factor Oct4 is involved in
cancer development and causes epithelial dysplasia of somatic
cells [25]. The presence of Sox2- and Oct4-positive cells in
endometrial adenocarcinomas was recently confirmed by
Pitynski et al. [41]. High levels of nuclear expression of both
Sox2 and Oct4 have been observed in cervical cancer, small
cell lung cancer, and squamous cell esophageal cancer
[42–44]. In patients with squamous cell esophageal cancer,
both Sox2 and Oct4 overexpression were related to signifi-
cantly shorter overall survival rate compared to patients with
negative stained tumors. There is a higher risk of distant re-
currence in rectal cancer if Sox2 and Oct4 are overexpressed
in the primary tumor [45]. Also, Oct4 expression has been
implicated in the malignancy and prognosis of glioblastoma
[46]. Osteosarcoma also contained stem-like cells involved in
tumor development that express Oct4 and Nanog stem cell
markers.
As was shown by Kashihara et al., CD133 expression is
correlated with poor prognosis in colorectal cancer. [47].
Vincent et al. [48] claim that CD133 is the only reliable mark-
er for CSC characterization in the Colo205 CRC cell line. It
also has been shown that Wnt pathway activity could be re-
sponsible for the chemoresistance of CD133+ cells in colorec-
tal cancer [49]. Deng et al. [50] demonstrated that 5-FU can
upregulate Wnt activity in CD133+ colon cancer stem-like
cells. Moreover, if Dickkopf-1 is overexpressed in CRC cells,
then the expression of CD133 and Lgr5 markers will be also
decreased in CRC cells. It also reduced the proliferation, mi-
gration, and invasion of CRC cells [51].
In our experiments, to identify colorectal cancer stem cells,
we are using early passages of newly established colorectal
cancer cell lines from clinical specimens. Based on the recent
Tumor Biol.
data, the sorting by surface markers (CD133, CD44) did not
reliably enrich for stemness when the CRC cell lines
established a long time ago were used [52]. The authors sug-
gest to use the newly established cell lines and cells from
clinical specimens to identify the stem cell-enriched popula-
tion. We expect, via CD133 sorting, to obtain chemoresistant
tumor stem cell enrichment. Contrary to expectations, there
was no increase in the resistance to 5-FU in the CD133+
subpopulation of cancer cells, which makes the possibility of
using CD133 as a marker for cancer stem-like cell population
from clinical colon cancer specimens questionable.
Concerning the use of Oct4 as such a marker, our data show
that the expression of the Oct4 transcription factor can be used
as a marker of colorectal cancer and a potential target for
cancer therapy.
In this study, we demonstrated differences in the levels of
Lgr5 expression in cell clones with Oct4 expression. Since
Lgr5 plays an important role not only in the early but also in
the late event of tumorigenesis, we consider that the reason for
this different proportion of positive cells in the final Oct4-
positive clones may reflect the original heterogeneity of tumor
cell culture, which was used for CSC enrichment. Lgr5 is a
marker of normal intestinal stem cells and also could have an
important role in cancer cell stemness [53, 54]. We think that
in vivo experiments will help us to validate our speculation,
which states that the level of Lgr5 can be important for the
tumor development in nude mice and may be used as addi-
tional marker of CSCs.
Altogether, this approach can be used in developing new
therapeutic strategies aimed at eradicating the tumorigenic
subpopulation of CSCs within colorectal cancer tumors. Our
results provide the rationale that new therapeutic methods of
targeting of Oct4-positive cancer cells should be developed to
gain the maximal clinical benefits.
Compliance with ethical standards
Conflicts of interest None
Grant support This work was financially supported by the Russian
Science Foundation (project 14-50-00068) to the Molecular Stem Cell
Biology Laboratory, Institute of Cytology RAS, and by the Government
of the Russian Federation mega grant 11G34.31.0068 to Dr. S. J. O’Brien.
References
1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer
J Clin. 66(1):7–30.
2. Chang GJ, CY H, Eng C, Skibber JM, Rodriguez-Bigas MA.
Practical application of a calculator for conditional survival in colon
cancer. J Clin Oncol. 2009;27:5938–43.
3. Ishida H, Fujita K, Akiyama Y, Sunakawa Y, Yamashita K, Mizuno
K, et al. Regimen selection for first-line FOLFIRI and FOLFOX
based on UGT1A1 genotype and physical background is feasible in
Japanese patients with advanced colorectal cancer. Jpn J Clin
Oncol. 2011;41:617–23.
4. Sanli UA, Karabulut B, Uslu R, Korkut M, Goker E. Single-agent
irinotecan for recurrent/metastatic colorectal cancer: a retrospective
analysis. Med Princ Pract. 2006;15:288–92.
5. Valent P, Bonnet D, De Maria R, Lapidot T, Copland M, Melo JV,
et al. Cancer stem cell definitions and terminology: the devil is in
the details. Nat Rev Cancer. 2012;12:767–75.
6. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-
Cortes J, et al. A cell initiating human acute myeloid leukaemia
after transplantation into SCID mice. Nature. 1994;367:645–8.
7. Ricci-Vitiani L, Fabrizi E, Palio E, De Maria R. Colon cancer stem
cells. J Mol Med (Berl). 2009;87:1097–104.
8. Fabian A, Barok M, Vereb G, Szollosi J. Die hard: are cancer stem
cells the Bruce Willises of tumor biology? Cytometry A. 2009;75:
67–74.
9. Vaiopoulos AG, Kostakis ID, Koutsilieris M, Papavassiliou AG.
Colorectal cancer stem cells. Stem Cells. 2012;30:363–71.
10. Mathonnet M, Perraud A, Christou N, Akil H, Melin C, Denizot Y,
et al. Hallmarks in colorectal cancer: angiogenesis and cancer stem-
like cells. World J Gastroenterol. 2014;20:4189–96.
11. O’Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer
cell capable of initiating tumour growth in immunodeficient mice.
Nature. 2007;445:106–10.
12. Van de Wetering M, Sancho E, Verweij C, de Lau W, Oving I,
Hurlstone A, van der Horn K, Clevers H. The beta-catenin/TCF-4
complex imposes a crypt progenitor phenotype on colorectal cancer
cells. Cell. 2002;111:241–50.
13. XS W, Xi HQ, Chen L. Lgr5 is a potential marker of colorectal
carcinoma stem cells that correlates with patient survival. World J
Surg Oncol. 2012;10:244.14.
14. Ritsma L, Ellenbroek SI, Zomer A, Snippert HJ, de Sauvage FJ, van
Rheenen J, et al. Intestinal crypt homeostasis revealed at single-
stem-cell level by in vivo live imaging. Nature. 2014;507:362–5.
15. Gargett CE, Schwab KE, Zillwood RM, Nguyen HP, Wu D.
Isolation and culture of epithelial progenitors and mesenchymal
stem cells from human endometrium. Biol Reprod. 2009;80:
1136–45.
16. Ebben JD, Treisman DM, Zorniak M, Kutty RG, Clark PA, Kuo JS.
The cancer stem cell paradigm: a new understanding of tumor de-
velopment and treatment. Expert Opin Ther Targets. 2010;14:621–
32.
17. CS Y, Huang AC, Lai KC, Huang YP, Lin MW, Chung JG, et al.
Diallyltrisulfide induces apoptosis in human primary colorectal
cancer cells. Oncol Rep. 2012;28:94954.
18. Todaro M, Orlando V, Cicero G, Caccamo N, Meraviglia S, Dieli F,
et al. Chemotherapy sensitizes colon cancer initiating cells to
Vy9V52 T cell-mediated cytotoxicity. PLoS One. 2013;8.
19. Khan IN, Al-Karim S, Bora RS, Chaudhary AG, Saini KS. Cancer
stem cells: a challenging paradigm for designing targeted drug ther-
apies. Drug Discov Today. 2015;20(10):1205–16.
20. Tomayko MM, Reynolds CP, Euhus DM, Hudd C, LaRegina MC,
Johnson FE. Determination of subcutaneous tumor size in athymic
(nude) mice. Cancer Chemother Pharmacol. 1989;24(3):148–54.
21. Liskovykh MA, Chuikin IA, Ranjan A, Safina DA, Tolkunova EN,
Tomilin AN, et al. Generation of rat induced pluripotent stem cells:
the analysis of reprogramming and culturing media. Tsitologiia.
2011;53:939–45.
22. Davydov-Sinitsyn AP, Bazhenova OV, Liskovykh MA,
Ponomartsev CV, Tomilin AN, Tolkunova EN, et al. In vitro der-
ivation and characterization of a colorectal cancer stem cell sub-
population. Tsitologiia. 2013;55:318–23.

23. Wolmark N, Rockette H, Fisher B, Wickerham DL, Redmond C,
Petrelli NJ, et al. The benefit of leucovorin-modulated fluorouracil
as postoperative adjuvant therapy for primary colon cancer: results
from National Surgical Adjuvant Breast and Bowel Project protocol
C-03. J ClinOncol. 1993;11:187987.
24. Cunningham D, Pyrhonen S, James RD, Punt CJ, Hickish TF,
Herait P, et al. Randomised trial of irinotecan plus supportive care
versus supportive care alone after fluorouracil failure for patients
with metastatic colorectal cancer. Lancet. 1998;352:1413–8.
25. Hochedlinger K, Yamada Y, Beard C, Jaenisch R. Ectopic expres-
sion of Oct-4 blocks progenitor-cell differentiation and causes dys-
plasia in epithelial tissues. Cell. 2005;121:465–77.
26. DeSantis CE, Lin CC, Mariotto AB, Siegel RL, Stein KD, Jemal A.
Cancer treatment and survivorship statistics, 2014. CA Cancer J
Clin. 2014;64:252–71.
27. Siegel R, DeSantis C, Jemal A. Colorectal cancer statistics, 2014.
CA Cancer J Clin. 2014;64:104–17.
28. Gungor C, Hofmann BT, Wolters-Eisfeld G, Bockhorn M.
Pancreatic cancer. Br J Pharmacol. 2014;171:849–58.
29. Kaddis N, Saif MW. Second-line treatment for pancreatic cancer.
JOP. 2014;15:344347.
30. Tanase CP, Neagu AI, Necula LG, Mambet C, Enciu AM,
Albulescu R, et al. Cancer stem cells: involvement in pancreatic
cancer pathogenesis and perspectives on cancer therapeutics. World
J Gastroenterol. 2014;20:10790–801.
31. Muqbil I, Bao GW, El-Kharraj R, Shah M, Mohammad RM, Azmi
AS et al. Systems and network pharmacology approaches to cancer
stem cells research and therapy. J Stem Cell Res Ther. 2012;suppl 7.
32. Wang YJ, Herlyn M. The emerging roles of Oct4 in tumor-initiating
cells. Am J Phys Cell Phys. 2015;309(11):C709–18.
33. Poursani EM, Mohammad Soltani B, Mowla SJ. Differential ex-
pression of OCT4 pseudogenes in pluripotent and tumor cell lines.
Cell J. 2016;18(1):28–36.
34. Kobayashi I, Takahashi F, Nurwidya F, Nara T, Hashimoto M,
Takahashi K. Oct4 plays a crucial role in the maintenance of
gefitinib-resistant lung cancer stem cells. Biochem Biophys Res
Commun. 2016;473(1):125–32.
35. Fazlul H. Sarkar. Novel holistic approaches for overcoming therapy
resistance in pancreatic and colon cancers. Med Princ Pract 2015.
36. Meacham CE, Morrison SJ. Tumour heterogeneity and cancer cell
plasticity. Nature. 2013;501:328–37.
37. Todaro M, Alea MP, Di Stefano AB, Cammareri P, Vermeu-len L,
Stassi G. Colon cancer stem cells dictate tumor growth and resist
cell death by production of interleukin-4. Cell Stem Cell. 2007;1:
389–402.
38. Dallas NA, Xia L, Fan F, Gray MJ, Gaur P, Ellis LM, et al.
Chemoresistant colorectal cancer cells, the cancer stem cell pheno-
type, and increased sensitivity to insulin-like growth factor-I recep-
tor inhibition. Cancer Res. 2009;69:1951–7.
39. Dieter SM, Ball CR, Hoffmann CM, Nowrouzi A, Herbst F, Glimm
H, et al. Distinct types of tumor-initiating cells form human colon
cancer tumors and metastases. Cell Stem Cell. 2011;9:357–65.
Tumor Biol.
40. Pointer KB, Clark PA, Zorniak M, Alrfaei BM, Kuo JS.
Glioblastoma cancer stem cells: biomarker and therapeutic ad-
vances. Neurochem Int. 2014;71:1–7.
41. Pitynski K, Banas T, Pietrus M, Milian-Ciesielska K, Ludwin A,
Okon K. SOX-2, but not Oct4, is highly expressed in early-stage
endometrial adenocarcinoma and is related to tumour grading. Int J
Clin Exp Pathol. 2015;8:8189–98.
42. Ji J, Wei X, Wang Y. Embryonic stem cell markers Sox-2 and OCT4
expression and their correlation with WNT signal pathway in cer-
vical squamous cell carcinoma. Int J Clin Exp Pathol. 2014;7:2470–
86.
43. Yang F, Gao Y, Geng J, Qu D, Han Q, Chen G, et al. Elevated
expression of SOX2 and FGFR1 in correlation with poor prognosis
in patients with small cell lung cancer. Int J Clin Exp Pathol.
2013;6:2846–54.
44. Wang Q, He W, Lu C, Wang Z, Wang J, Suo Z, et al. Oct3/4 and
Sox2 are significantly associated with an unfavorable clinical out-
come in human esophageal squamous cell carcinoma. Anticancer
Res. 2009;29:1233–41.
45. Saigusa S, Tanaka K, Toiyama Y, Yokoe T, Okugawa, Kusunoki M,
et al. Correlation of CD133, OCT4, and SOX2 in rectal cancer and
their association with distant recurrence after chemoradiotherapy.
Ann Surg Oncol. 2009;16:3488–98.
46. Hosokawa Y, Takahashi H, Inoue A, Kawabe Y, Funahashi Y,
Tanaka J, et al. Oct-3/4 modulates the drug- resistant phenotype
of glioblastoma cells through expression of ATP binding cassette
transporter G2. Biochim Biophys Acta. 2015;1850:1197–205.
47. Kashihara H, Shimada M, Kurita N, Iwata T, Sato H, Matsumoto N,
et al. CD133 expression is correlated with poor prognosis in colo-
rectal cancer. Hepato-Gastroenterology. 2014;61:1563–7.
48. Vicente-Duenas C, Gutierrez de Diego J, Rodriguez FD, Jimenez R,
Cobaleda C. The role of cellular plasticity in cancer development.
Curr Med Chem. 2009;16:3676–85.
49. Ren F, Sheng WQ, Du X. CD133: a cancer stem cells marker, is
used in colorectal cancers. World J Gastroenterol. 2013;19:2603–
11.
50. Deng YH, XX P, Huang MJ, Xiao J, Zhou JM, Lin EH, et al. 5-
Fluorouracil upregulates the activity of Wnt signaling pathway in
CD133-positive colon cancer stemlike cells. Chin J Cancer.
2010;29:810–5.
51. Qi L, Sun B, Liu Z, Li H, Gao J, Leng X. Dickkopf-1 inhibits
epithelial-mesenchymal transition of colon cancer cells and contrib-
utes to colon cancer suppression. Cancer Sci. 2012;103:828–35.
52. Fan F, Bellister S, Lu J, Ye X, Boulbes DR, Ellis LM, et al. The
requirement for freshly isolated human colorectal cancer (CRC)
cells in isolating CRC stem cells. Br J Cancer. 2015;112:539–46.
53. Barker N, Clevers H. Leucine-rich repeat-containing G-protein-
coupled receptors as markers of adult stem cells.
Gastroenterology. 2010;138:1681–96.
54. Sangiorgi E, Capecchi MR. Bmi1 is expressed in vivo in intestinal
stem cells. Nat Genet. 2008;40:915–20.