Atypical nuclear localization of CD133 plasma membrane glycoprotein in rhabdomyosarcoma cell lines

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CD133 (also known as prominin-1) is a cell surface glycoprotein that is widely used for the identification of stem cells. Furthermore, its glycosylated epitope, AC133, has recently been discussed as a marker of cancer stem cells in various human malignancies. During our recent experiments on rhabdomyosarcomas (RMS), we unexpectedly identified an atypical nuclear localization of CD133 in a relatively stable subset of cells in five RMS cell lines established in our laboratory. To the best of our knowledge, this atypical localization of CD133 has not yet been proven or analyzed in detail in cancer cells. In the present study, we verified the nuclear localization of CD133 in RMS cells using three independent anti-CD133 antibodies, including both rabbit polyclonal and mouse monoclonal antibodies. Indirect immunofluorescence and confocal microscopy followed by software cross-section analysis, transmission electron microscopy and cell fractionation with immunoblotting were also employed, and all the results undeniably confirmed the presence of CD133 in the nuclei of stable minor subpopulations of all five RMS cell lines. The proportion of cells showing an exclusive nuclear localization of CD133 ranged from 3.4 to 7.5%, with only minor differences observed among the individual anti-CD133 antibodies. Although the role of CD133 in the cell nucleus remains unclear, these results clearly indicate that this atypical nuclear localization of CD133 in a minor subpopulation of cancer cells is a common phenomenon in RMS cell lines.

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# Atypical nuclear localization of CD133 plasma membrane glycoprotein in rhabdomyosarcoma cell lines

ALENA NUNUKOVA 1, JAKUB NERADIL 1,2, JAN SKODA 1,3, JOSEF JAROS 4, ALES HAMPL 4,

JAROSLAV STERBA 2,3 and RENATA VESELSKA 1,3

1Department of Experimental Biology, Faculty of Science, Masaryk University; 2Regional Centre for
Applied Molecular Oncology, Masaryk Memorial Cancer Institute; 3 Department of Pediatric Oncology,

University Hospital Brno; 4 Department of Histology and Embryology, Faculty of Medicine,

Masaryk University, 61137 Brno, Czech Republic

Received February 5, 2015; Accepted April 27, 2015

DOI: 10.3892/ijmm.2015.2210

Abstract. CD133 (also known as prominin-1) is a cell surface
glycoprotein that is widely used for the identification of stem
cells. Furthermore, its glycosylated epitope, AC133, has
recently been discussed as a marker of cancer stem cells in
various human malignancies. During our recent experiments
on rhabdomyosarcomas (RMS), we unexpectedly identified
an atypical nuclear localization of CD133 in a relatively stable
subset of cells in five RMS cell lines established in our laboratory. To the best of our knowledge, this atypical localization of
CD133 has not yet been proven or analyzed in detail in cancer
cells. In the present study, we verified the nuclear localization
of CD133 in RMS cells using three independent anti-CD133
antibodies, including both rabbit polyclonal and mouse monoclonal antibodies. Indirect immunofluorescence and confocal
microscopy followed by software cross-section analysis,
transmission electron microscopy and cell fractionation with
immunoblotting were also employed, and all the results undeniably confirmed the presence of CD133 in the nuclei of stable
minor subpopulations of all five RMS cell lines. The proportion
of cells showing an exclusive nuclear localization of CD133
ranged from 3.4 to 7.5%, with only minor differences observed
among the individual anti-CD133 antibodies. Although the

_Correspondence to:_ Professor Renata Veselska, Department of
Experimental Biology, Faculty of Science, Masaryk University,
Kotlarska 2, 61137 Brno, Czech Republic
E-mail: veselska@sci.muni.cz

_Abbreviations:_ BSA, bovine serum albumin; CSCs, cancer stem
cells; DMEM, Dulbecco's modified Eagle's medium; FISH, fluor
escence _in situ_ hybridization; HRP, horseradish peroxidase; NSCLC,
non-small cell lung cancer; PBS, phosphate-buffered saline; PVDF,
polyvinylidene difluoride; RMS, rhabdomyosarcoma; SDS, sodium
dodecyl sulfate; TBS, Tris-buffered saline; TEM, transmission electron
microscopy

_Key words:_ CD133, prominin-1, rhabdomyosarcoma, cell nuclei,
immunodetection

role of CD133 in the cell nucleus remains unclear, these results
clearly indicate that this atypical nuclear localization of CD133
in a minor subpopulation of cancer cells is a common phenomenon in RMS cell lines.

Introduction

CD133 (also known as prominin-1) is a glycoprotein that is typically localized to the plasma membrane. The molecule consists
of five transmembrane domains, two large extracellular loops,
an extracellular N-terminus and an intracellular C-terminus.
Eight potential glycosylation sites have been identified within
the extracellular domains, with four per loop. Human CD133 is
encoded by the _PROM1_ gene, which is located in chromosomal
region 4p15.32. At least seven CD133 isoforms resulting from
alternative splicing have been described in humans (1,2).

CD133 is widely used to identify stem cells, and its glycosylated epitope, AC133, has recently been discussed as a marker of
cancer stem cells (CSCs) in various human malignancies (2-4).
In our previous studies, we identified CD133-positive cells
that presented typical membrane positivity in two of the most
common types of pediatric sarcomas, osteosarcoma (5) and
rhabdomyosarcoma (RMS) (6). The expression of CD133
in these two solid tumors, as well as the tumorigenicity of
CD133-positive cells, has been confirmed by other research
groups (7-10). Therefore, CD133 is currently accepted as one
of the markers of a CSC phenotype in pediatric sarcomas,
including RMS (11-13).

During our recent study aimed at the analysis of CSC
markers in pediatric sarcomas, we noted a surprising result: a
stable subset of cells in each of five RMS cell lines examined
exhibited an exclusive nuclear localization of CD133 (these
data are published in this article). To date, a similar localization of this antigen has been described only in one case report
of breast cancer (14) and in a large study on lung cancer (15)
using immunohistochemical methods, nevertheless, without
any verification or systematic description. For this reason, in
this study, we sought to analyze this interesting phenomenon
in detail using three independent anti-CD133 commercial
antibodies (Fig. 1).

Materials and methods

_Cell culture._ Five cell lines derived from pediatric patients with
RMS were included in this study: NSTS-8, NSTS-9, NSTS‑11,
NSTS-22 and NSTS-28. The first three cell lines were described
in our previous study (6), and the last two were derived using
the same procedure to generate primary cultures (16). All cell
lines were authenticated by the immunodetection of MyoD, and
the subtype was distinguished using _FKHR_ break detection by
fluorescence _in situ_ hybridization (FISH). Authentication using
MyoD detection was performed in the same passages as the
experiments; FISH analysis of the _FKHR_ break was completed
up to passage 10. The cell lines were maintained in Dulbecco's
modified Eagle's medium (DMEM) supplemented with
20% fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin
and 100 µg/ml streptomycin (all purchased from GE Healthcare
Europe GmbH, Freiburg, Germany). The cells were maintained
under standard conditions at 37˚C in a humified atmosphere
containing 5% CO2 and were subcultured once or twice per
week. The Research Ethics Committee of the School of
Science (Masaryk University, Brno, Czech Republic) approved
the study protocol, and a written statement of informed consent
was obtained from each participant or his/her legal guardian
prior to participation in this study. A brief description of the
cohort of patients included in this study is provided in Table I.

_Indirect immunofluorescence._ The cells were cultivated on
coverslips in Petri dishes for one day and then rinsed with
phosphate-buffered saline (PBS) and fixed with 3% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) at room
temperature for 20 min. After washing again with PBS, nonspecific binding was blocked with 3% bovine serum albumin
(BSA; Sigma-Aldrich) in PBS for 10 min. The cells were
then incubated with primary antibody at 37˚C for 60 min,
washed three times in PBS and then incubated with the
corresponding secondary antibody at 37˚C for 45 min. Rabbit
polyclonal anti-CD133 (Cat. no. ab19898, dilution 1:70; Abcam,
Cambridge, UK), mouse monoclonal anti-CD133 (clone 17A6.1,
Cat. no. MAB4399, dilution 1:100; Millipore, Billerica,
MA, USA), mouse monoclonal anti-AC133 (clone AC133,
Cat. no. 130-090-422, dilution 1:4; Miltenyi Biotec, Bergisch
Gladbach, Germany), and mouse monoclonal anti‑α‑tubulin
antibody (clone: TU-01, Cat. no. 11-250, dilution 1:100; Exbio,
Vestec, Czech Republic), which served as a control, were
used as the primary antibodies. Anti-rabbit Alexa Fluor 488
(Cat. no. A11008, dilution 1:200) and anti-mouse Alexa
Fluor 488 antibody (Cat. no. A11001, dilution 1:200) (both from
Invitrogen, Paisley, UK) were used as the secondary antibodies.
After a final wash with PBS, the cell nuclei were counterstained
with 0.05% Hoechst 33342 (Life Technologies, Carlsbad, CA,
USA) for 10 min, and the coverslips were mounted using Dako
fluorescence mounting medium (Dako, Glostrup, Denmark).
An Olympus BX-51 microscope was used for sample evaluation; micrographs were captured using an Olympus DP72
CCD camera and analyzed using the Cell P imaging system
(Olympus, Tokyo, Japan). At least 200 cells were evaluated
overall within discrete areas of each sample, and the samples
were prepared from at least three independent passages of all
examined cell lines. The mean percentages of cells showing
exclusive nuclear CD133 localization were determined for entire

samples of individual cell lines. For the detailed examination
of CD133 nuclear localization, the same protocol for indirect
immunofluorescence was employed, and the specimens were
then examined using an Olympus FluoView-500 confocal
imaging system combined with an inverted Olympus IX-81
microscope. The images were recorded using an Olympus
DP70 CCD camera and analyzed using analySIS FIVE software (Soft Imaging System GmbH, Muenster, Germany) and
an Olympus FluoView Confocal Laser Scanning Microscope
System 4.3.

_Transmission electron microscopy (TEM)._ To perform the
immunodetection of CD133 in ultrathin sections, the cells
grown on coverslips were rinsed with PBS and fixed in
3% paraformaldehyde (Sigma-Aldrich) and 0.1% glutaraldehyde (AppliChem GmbH, Darmstadt, Germany) in PBS
at room temperature for 60 min. Following a PBS rinse
and dehydration, the cells were embedded in LR White
medium (Polysciences Inc., London, UK). The labeling of
the ultrathin sections was performed on grids. CD133 was
detected using mouse monoclonal anti-CD133 antibody (dilution 1:25; Millipore) and a gold particle-conjugated secondary
antibody (anti-mouse IgG 20 nm gold, Cat. no. ab27242,
dilution 1:40; Abcam). Ultrathin sections incubated without
primary antibody or with the TU-01 primary monoclonal
antibody against α-tubulin (dilution 1:200; Exbio) were used
as controls. Following immunodetection, the specimens
were contrasted with 2.5% uranyl acetate (PLIVA-Lachema,
Brno, Czech Republic) for 10 min and with Reynolds solution
(Sigma‑Aldrich) for 6 min at room temperature. The specimens
were then observed under a Morgagni 268(D) transmission
electron microscope (FEI Co., Hillsboro, OR, USA). The
images were captured using an Olympus Veleta TEM CCD
camera and analyzed using iTEM Olympus Soft Imaging
Solution (Olympus).

_Immunoblot analysis._ To analyze the nuclear and cytoplasmic fractions, a Nuclear Protein Extraction kit (Thermo
Fisher Scientific, Rockford, IL, USA) was used according
to the manufacturer's instructions. A 20 µl sample of
protein extract was loaded onto an 8% sodium dodecyl
sulfate (SDS)‑polyacrylamide gel and separated by electrophoresis. Subsequently, the proteins were transferred onto
polyvinylidene difluoride (PVDF) membranes (Bio-Rad
Laboratories, Hercules, CA, USA), blocked in 5% non-fat milk
at room temperature for 60 min, and incubated with primary
antibodies, rabbit polyclonal anti-CD133 (Abcam), mouse
monoclonal anti-α-tubulin (Exbio), or rabbit monoclonal
anti-lamin B2 antibody (clone: D8P3U, Cat. no. 12255S; Cell
Signaling Technology, Danvers, MA, USA) at a 1:1,000 dilution overnight at 4˚C. Anti-α-tubulin and anti-lamin B2 served
as the controls for the purity of the cytoplasmic and nuclear
cell fractions, respectively. After washing with Tris-buffered
saline (TBS)-Tween-20, the membranes were incubated with
the corresponding horseradish peroxidase (HRP)-conjugated
secondary antibodies anti-mouse IgG-HRP (cat. no. A9917,
dilution 1:5,000; Sigma-Aldrich) and anti-rabbit IgG-HRP
antibodies (cat. no. 7074, dilution 1:5,000; Cell Signaling
Technology) at room temperature for 60 min. Signal detection
was performed using ECL Prime Western Blotting Detection

Table I. Description of patients from whom tumor samples were obtained to establish the rhabdomyosarcoma cell lines and the
results concerning the mean percentage of cells with an exclusive nuclear localization of CD133.

Mean percentage of cells with exclusive nuclear localization of CD133
-----------------------------------------------------------------------------------------------------------------------------------Age RMS No. of passages Anti-CD133 antibody Anti-CD133 antibody Anti-AC133 antibody
Cell line Gender (years) type analyzed (rabbit polyclonal) (mouse monoclonal) (mouse monoclonal)

NSTS-8 F 21 A 15-18 4.6 6.1 3.4
NSTS-9 M 17 A 13-18 4.6 5.2 4.1
NSTS-11 F 16 E 11-19 3.8 4.6 6.6
NSTS-22 F 5 A 11-15 4.0 6.0 6.4
NSTS-28 M 8 A 12-16 4.1 5.2 7.5

M, male; F, female; RMS, rhabdomyosarcoma. Tumor type: A, alveolar; E, embryonal.

Figure 1. Overview of epitopes of the anti-CD133 and anti-AC133 antibodies used in this study. For each antibody, the catalogue number and manufacturer are
indicated. Potential glycosylation sites, as well as length of the N-terminal region, the intracellular and extracellular loops and the C-terminal region of CD133
are depicted.

Reagent (GE Healthcare) according to the manufacturer's
instructions.

Results

For all five RMS cell lines examined in this study, we
performed a detailed analysis of the presence of cells with

nuclear CD133 positivity using indirect immunofluorescence
with three independent anti-CD133 antibodies (Fig. 1). A subset
of cells showed only nuclear CD133 positivity, i.e., no detectable membrane or cytoplasmic positive signal. The results were
markedly similar in all five cell lines analyzed, regardless of
the primary antibody utilized, and the proportion ranged from
3.4 to 7.5%, with only minor differences observed among the

Figure 2. Nuclear localization of CD133 in rhabdomyosarcoma cells. (A) Example of the frequency of cells with CD133 nuclear positivity in the NSTS-11 cell
line, as detected using three independent primary antibodies. Cells with exclusive nuclear positivity for CD133 are indicated by arrows; cells with the typical
membrane positivity are indicated by arrowheads. (B) Details of cells that exhibited exclusive nuclear positivity for CD133 in all five rhabdomyosarcoma cell
lines. CD133 was stained by indirect immunofluorescence using Alexa Fluor 488-labeled secondary antibodies (green), and the nuclei were counterstained with
Hoechst 33342 (blue). Scale bars, (A) 50 µm and (B) 10 µm.

individual anti-CD133 antibodies (Fig. 2A and Table I). We
also performed a detailed morphological analysis of the cells
that exhibited exclusive nuclear positivity for CD133 (Fig. 2B);
as can be seen on these micrographs, the pattern of CD133
nuclear positivity was markedly similar in all of the cell lines.

To confirm the presence of CD133 in the nuclei of the
RMS cells visualized using indirect immunofluorescence, we
employed confocal microscopy and software cross-section
analysis through these CD133-positive nuclei (Fig. 3). As is
apparent from the results, the localization of the fluorescence
signal for CD133 was detected within the cell nuclei both on
the software cross-sections (Fig. 3B) and on the plot diagrams
of the fluorescence intensity (Fig. 3D).

Furthermore, we also used immunogold labeling with
TEM to verify the localization of CD133 in the nuclei of the
RMS cells. To avoid any artifacts associated with this methodological approach, the accumulation of three or more gold
particles together was considered to indicate a positive signal.
The results clearly indicated the presence of CD133 in both the
nuclei and nucleoli (Fig. 4A and B).

These results are all completely consistent with the microscopic observations described above (Fig. 2): the software
cross-sections also showed clear, punctuate signals for CD133
within the nucleus (Fig. 3B and D), i.e., no diffuse positivity
throughout the entire nucleus was observed. Nevertheless,
the TEM micrographs also showed the presence of CD133

Figure 3. Confocal microscopy analysis of CD133-positive cell nuclei. (A) The planes of software cross-sections through the CD133-positive nuclei are highlighted by simple yellow lines. (B) The cross-sections at these yellow lines are shown at the bottom. (C) The yellow arrows indicate lines drawn across individual
CD133-positive nuclei in the confocal image; (D) matching plots reporting the fluorescence intensity according to these arrows are given below. CD133 was
stained by indirect immunofluorescence using Alexa Fluor 488-labeled secondary antibodies (green), and nuclei were counterstained with Hoechst 33342 (blue).
Scale bars, 5 µm.

in the nucleoli (Fig. 4B), and this observation corresponds
with the diffuse positivity for CD133 observed in some of
the nucleoli (Fig. 2B). In addition to cells with the typical
membrane positivity or exclusive nuclear positivity for CD133,
we also sporadically noted clusters of positive signals in the
cytoplasm near the cell nucleus or very close to the nuclear
envelope (Fig. 4B).

Final confirmation of the results achieved through microscopic methods was carried out by immunoblot analysis of the
cytoplasmic and nuclear fractions of all five RMS cell lines.
The presence of CD133-specific bands of various intensities
was detected in all nuclear fractions, in addition to the strong
CD133-specific bands in the cytoplasmic fractions (Fig. 4C).

The purity of both subcellular fractions was confirmed by the
presence/absence of α-tubulin and lamin B2. These results are
completely in accordance with our other findings (reported
above) achieved by indirect immunofluorescence and TEM,
i.e., in all five RMS cell lines, the presence of a small subpopulation of cells with CD133 in the nucleus was revealed.

Discussion

As described above, we unexpectedly identified an atypical
nuclear localization of CD133 in a relatively stable subset of
cells in five RMS cell lines established in our laboratory. To
date, this atypical localization of CD133 was described in one

Figure 4. Detection of CD133 in the nuclei of rhabdomyosarcoma cells using transmission electron microscopy and immunoblot analysis. (A) Presence of CD133
in cell nuclei and nucleoli; more detailed images of the positive signal for CD133 in the highlighted square areas are presented on the bottom row. (B) The
presence of CD133 in the cytoplasm near the nucleus (dashed square) and very close to the nuclear envelope (solid square). More detailed images of the positive
signal for CD133 in the highlighted square areas, as indicated above, are shown on the right side. Representative labeling of CD133 in NSTS-28 cells is shown;
CD133 was detected using 20 nm gold particles. Scale bars: (A) 250 nm and (B) 500 nm. (C) Nuclear/cytoplasmic fractionation followed by immunoblotting was
performed using a rabbit polyclonal anti-CD133 primary antibody; α-tubulin and lamin B2 were used to confirm the purity of the fractions.

case report on breast cancer (14) and in a large study of prognostic markers on lung cancer (15) using immunohistochemical
methods. Nevertheless, published data on CD133 expression
in human cancer cells are partly inconsistent, possibly due to
different analytical tools, as well as methodological limitations
and pitfalls (2). For this reason, results obtained by immunohistochemistry or flow cytometry must be confirmed with
alternative antibodies and should be complemented by the
utilization of different detection methods of either protein or
transcript (2). Furthermore, the glycosylation of CD133 epitopes in relation to the CSC phenotype should be also taken into
account, particularly if the antibodies against AC133 epitope
are commercially available (17).

In this study, we verified the nuclear localization of CD133
in RMS cells using three independent anti-CD133 antibodies,
including both rabbit polyclonal and mouse monoclonal antibodies (Fig. 1). Indirect immunofluorescence and confocal
microscopy followed by software cross-section analysis,
TEM and cell fractionation with immunoblotting were also
employed, and all the results undeniably confirmed the presence of CD133 in the nuclei of stable minor subpopulations of
all five RMS cell lines.

These results strongly support the hypothesis that a stable
subpopulation of cells with nuclear positivity for CD133 is a
common phenomenon in RMS cell lines. Surprisingly, and
to the best of our knowledge, similar results have not been
reported to date for RMS cells. Although certain micrographs
from our previous study showed cells with an accumulation of
fluorescent signal for CD133 in the nucleus (6), we assumed that
this finding was an artifact resulting from the use of a rabbit
polyclonal antibody against CD133, (which was the only antiCD133 antibody available at the time), although we had never
detected similar nuclear positivity for CD133 in osteosarcoma
or glioblastoma cell lines using the same antibody (5,18). Other
authors investigating CD133 expression in RMS and RMS cell
lines have not described the pattern of CD133 positivity in
detail, and no micrographs of the individual cells are available
in their published articles (9,10).

Very recently, one publication has mentioned the nuclear
localization of CD133 in triple-negative breast cancer cells as
revealed by immunohistochemistry; nevertheless, this study is
a case report based on only one simple descriptive method and
therefore does not include any continuing systematic analysis
of this apparently interesting finding. Moreover, two of three
methods listed in this article, quantitative RT-PCR and flow
cytometry, are not suitable for identifying the cell surface,
cytoplasmic or nuclear localization of any protein (14).

To date, another study concerning the possible value of
CD133 as a prognostic indicator of survival in patients with
non-small cell lung cancer (NSCLC) was just published. These
interesting results suggest that CD133 expression in the nucleus
of NSCLC cells was related to tumor diameter, tumor differentiation and the TNM stage. Kaplan-Meier survival and Cox
regression analyses revealed that a high CD133 expression in
the nucleus, as well as in the cytoplasm also predicted the poor
prognosis of NSCLC (15).

As mentioned above, in addition to cells with the typical
membrane positivity or exclusive nuclear positivity for CD133,
clusters of positive signals in the cytoplasm near the cell
nucleus or very close to the nuclear envelope were also sporadi

cally noted. This finding is in accordance with our previously
published observations of sporadic cytoplasmic positivity for
CD133 in RMS cells (6), as well as with the deposition of
CD133 in cytoplasmic vesicles that has been described in osteosarcoma (19) and the recently suggested mechanism of CD133
internalization and trafficking into lysosomes through interactions between CD133 and the histone deacetylase HDAC6 (20).

Taken together, our results undeniably confirmed the presence of CD133 in the cell nuclei of stable minor subpopulations
in RMS cell lines. These results, although surprising and
novel, were achieved through three independent methods using
three independent antibodies purchased from three separate
suppliers.

Nevertheless, the main question of what is the exact role
of CD133 in the nucleus of RMS cells remains unanswered.
In a previous study on breast cancer, the authors suggested
that CD133 in the nucleus may act as transcriptional regulator
and is most likely associated with a poor prognosis; however,
this conclusion is largely speculative in this case report (14).
By contrast, the most recent findings on NSCLC undoubtedly
proved the association of nuclear positivity for CD133 poor
prognosis in these patients (15).

Although a similar function of another type of surface
molecule internalized into the cell nucleus, receptor tyrosine
kinases, has been reported (21-23), CD133 belongs to a distinct
class of cell membrane proteins, and the analogies to this
process are therefore limited. Furthermore, recent studies also
discuss the involvement of internalized CD133 in cell signaling
pathways, such as the canonical Wnt pathway (20), or report an
association between CD133 and the PI3K/Akt pathway (24-26).
Regardless, the elucidation of the possible role of CD133 in the
nucleus of cancer cells should be based on detailed descriptions of the localization and interactions of CD133 with other
molecules in the cell nucleus. These experiments will be the
focus of our upcoming study on this interesting phenomenon.

Acknowledgements

This study was supported by grant IGA NT13443-4 of the
Ministry of Healthcare of the Czech Republic. We thank
Johana Maresova and Dobromila Klemova for their skilful
technical assistance.

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