Alginate-embedded HuH-7 cells increase MMP-9 and reduce OCLN expression in vitro
Angiolini et al. Cancer Cell Int
Alginate-embedded HuH-7 cells increase MMP-9 and reduce OCLN expression in vitro
Virginia Andrea Angiolini 0 2 3
Carolina Uribe Cruz 0 3
Mónica Luján López 0 1 3
Laura Simon 0 3
Ursula Matte 0 1 2 3
0 Gene Therapy Center, Hospital de Clínicas de Porto Alegre , Rua Ramiro Barcelos 2350, Porto Alegre, RS 90035-903 , Brazil
1 Post-Graduation Program in Genetics and Molecular Biology, Federal University of Rio Grande do Sul , Porto Alegre , Brazil
2 Post-Graduation Program in Child and Adolescent Health, Federal University of Rio Grande do Sul , Porto Alegre , Brazil
3 Gene Therapy Center, Hospital de Clínicas de Porto Alegre , Rua Ramiro Barcelos 2350, Porto Alegre, RS 90035-903 , Brazil
Background: Hepatocellular carcinoma is a common cancer, ranking third in cancer-associated deaths. An important cause of cancer patients' mortality is metastasis. At the start of metastasis progression, there is an epithelialmesenchymal transition, characterized by matrix degradation, junction reductions and vessels formation. HuH-7 is a cell line used in research as an in vitro model for hepatocellular carcinoma. It is known that two-dimensional growth reflects tumor characteristics poorly. In contrast, three-dimensional cultures provide a better approach to the study of tumorigenic potential. The purpose of this work was to mimic a three-dimensional environment in order to assess gene expression of some epithelial-mesenchymal transition and metastasis progression markers in HuH-7 cells and compare them with traditional two-dimensional culture model. Methods: HuH-7 cells were encapsulated in sodium alginate (three-dimensional model) to be compared with cells grown in two-dimensional flasks. After 4 days in culture, gene expression of Matrix metallopeptidase 9, Occludin, p65, Intercellular adhesion molecule 1 and Vascular endothelial growth factor A was analyzed by qPCR and cytoskeleton assessment was performed by rhodamine-phalloidin staining. Results: Differences were found in gene expression, with a high increment of Matrix metallopeptidase 9 and Occludin reduction. The cytoskeleton morphology also showed differences, with a cytoplasm restricted only near the nuclei in the three-dimensional model. Conclusions: This work shows the effects of using sodium alginate capsules as a three-dimensional model to the study of HuH-7. Cells in this 3D system show key markers of epithelial-mesenchymal transition, such as Matrix metallopeptidase 9 overexpression and Occludin down-regulation.
HuH-7; Epithelial-mesenchymal transition; Encapsulation; 3D culture
Hepatocellular carcinoma (HCC) is a common cancer,
ranking sixth in the prevalence cancer list and third in
cancer associated deaths . The numbers of cases vary
considerably worldwide and incidence is increasing over
the years . HCC is the fifth most common cancer in
men worldwide (523,000 cases per year, 7.9% of all
cancers) and the seventh most common type in women
(226,000 cases per year, 6.5% of all cancers) .
In vitro studies using patient-derived cancer cell lines
are useful as a first approach to evaluate new therapeutic
targets. HuH-7 is a cell linage cells obtained from a
welldifferentiated hepatocellular carcinoma , and it is
actually used in research as an in vitro model [5–7]. Although
useful, there are several limitations of models using
two-dimensional growth (2D), e.g., poor sensitivity to
chemotherapy [8, 9], with less tumorigenicity and lower
metastatic potential . On the other hand, it is known
that a three dimensional (3D) environment in cell culture
provides a better approach to drug screening, toxicity
studies and metabolic behavior , better mimicking
the physiological contacts between cells and the
mechanical properties of cells . The 3D surroundings can also
represent better tumor bioenergetics by generating
gradients of nutrients and oxygen concentration .
© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
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Epithelial-mesenchymal transition (EMT) is another
process not well-represented in 2D cultures and it is
closely related to metastasis, an important cause of
patients’ mortality caused by the spread of primary
tumor cells to other tissues . EMT is a process
characterized by the reduction of cell to cell contact in which
proteins, such as occludins (OCLN) (responsible for tight
junction) are down-regulated. At the same time, there is
an increase of matrix degradation by metalloproteinases,
mainly by metalloproteinase 9 (MMP-9) . These
process are crucial to start the progression of metastasis [10,
14]. A following step is the formation of new blood
vessels to supply the necessary nutrients for the metastatic
tumor [15, 16]. In this study, different markers of EMT
and metastatic progression were compared between
2D and 3D cultures using a simple and non expensive
sodium alginate matrix.
HuH‑7 cells culture
HuH-7 cells, acquired from the Cell Bank of Río de
Janeiro, were grown in complete medium: DMEM
(Gibco®, USA), 10% fetal bovine serum (FBS; Gibco®,
USA) and 1% Streptomycin and penicillin (S/P; Gibco®,
USA) and maintained in a humidified incubator at
37 °C with 5% CO2. The culture medium was renewed
every 3 days. When confluent, cells were trypsinized
(trypsin + 0.25% EDTA) (Gibco®, USA) and seeded for
expansion. For the 2D model, 105 cells/cm2 were plated
and maintained for 4 days.
HuH-7 cells were re-suspended (3 × 106 cells/mL)
in 1.5% sodium alginate (Sigma Aldrich®, Germany)
previously diluted in sterile complete medium (as
described above). The cell suspension was passed
through a syringe mounted to the encapsulation
equipment (Nisco, Zurich, Switzerland) according to the
following parameters: rate of 40 mL/h and air flow 4.5 L/
min . The suspension was dropped in 125 mM
Calcium Chloride, which was replaced by complete
medium after capsule formation. A total of 400 μL
capsules (approximately 1.2 × 106 cells) per well (n = 3)
were maintained for 4 days under the same conditions
After 4 days, capsules were collected, washed twice with
1× PBS and soaked in 1 mL of Sodium Citrate 125 Mm
(Synth, Brazil) for 3 min. Subsequently, they were
centrifuged at 270 g for 10 min and washed with 1× PBS.
The final pellet was stored at −80 °C for later RNA
Total RNA extraction and cDNA conversion
Total RNA extraction of cells cultured in 2D and 3D was
performed using RNeasy® kit (Qiagen, Germany)
according to manufacturer’s instructions. It was quantified by
NanoDrop 1000 Spectophotometer (Thermo Scientific,
USA) and subsequently converted to cDNA by using
SuperScript™III Reverse Transcriptase kit (Invitrogen,
USA) following the manufacturer’s protocol.
Gene expression analysis
Markers characteristic of EMT and metastatic process
such as, MMP-9, OCLN, p65, intercellular adhesion
molecule 1 (I-CAM-1) and vascular endothelial growth
factor (VEGF) were analyzed by qPCR using SYBR® Green
(Invitrogen, USA). The list of primers, with their
respective fragments sizes and annealing temperatures, are
described in Table 1. Gene expression was calculated by
the 2−ΔΔCt value , using Glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) as a normalizing gene.
A sample of encapsulated cells was fixed for 24 h with 10%
formalin, washed three times with 1× PBS for 5 min and
permeabilized with Triton-X-100 0.25% for 10 min.
Immediately, the cytoskeleton dye rhodamine-phalloidin, diluted
in a concentration of 1:100 in PBS, was applied to the
capsules. It was incubated for 1 h at 4 °C in a wet and dark
chamber. Finally, DAPI was dripped onto the capsules for
nuclei staining and capsules were observed in Leica TCS
SP5 (Leica, USA) confocal microscope at 40×. The same
staining procedure was applied to cells grown in 2D.
Photographs for 2D model were taken with an inverted
Olympus IX71 (Olympus, USA) fluorescence microscope.
Statistical analysis was performed using SPSS for
Windows version 18.0. The values were expressed as mean
(±standard deviation). Student’s t test was used,
considering p values <0.05 as statistically significant.
Cells previously expanded in 2D (Fig. 1a) were
encapsulated in sodium alginate (Fig. 1b) and maintained for
a period of 4 days. The capsules retained their integrity
and morphology after the time of culture and the cells
remained within them, showing that the encapsulation
process is a suitable model of a 3D environment for cells.
The HuH-7 cells encapsulated in sodium alginate
matrix showed to be affected by the presence of the
extracellular environment by changing their gene expression
pattern. There was a marked increase in expression of
the matrix remodeling protein (MMP-9) (545.03 ± 0.75
vs. 139.46 ± 0.44) (Fig. 2a). Furthermore, the expression
f: 5′- ACAAGCGGTTTTATCCAGAGTC-3′
Table 1 Primers used with respective amplicon size and annealing temperatures
Fig. 1 HuH-7 cells cultured in 2D flasks (a) or in 3D alginate capsules at day 4 (b). ×10
of occludin (OCLN) was significantly reduced in the 3D
environment (0.32 ± 0.10 vs. 1.04 ± 0.29 2D) (Fig. 2b).
However, other genes that characterize the process
of EMT and metastasis progression (p65, I-CAM and
VEGF) showed no differences of expression between cells
in the 2D or the 3D environments (Fig. 2c–e).
As expected, changes in culture environment were also
reflected in the cytoskeleton. Confocal microscopy with
rhodamine-phalloidin staining revealed those changes
(Fig. 3). Cells grown in 2D have a flattened shape and a
spread cytoplasm over the plate surface (Fig. 3a). By
contrast, in the 3D model, cells have a round shape with the
cytoplasm being well-distributed to the nuclear
periphery (Fig. 3b). Note that few cells were stained in 3D
environment, probably because the dye penetrates the
capsules with difficulty.
The use of 3D models for cell culture has many
advantages over monolayer cell culture. Many different 3D
models were developed for different approaches  and
one of them is the use of alginate capsules . Sodium
alginate is one of the materials most commonly chosen
because it form hydrogels with desirable characteristics
such as lightweight, flexibility and mechanical stability
. Alginate encapsulation was reported to increment
cell division, aid in extracellular matrix assembly and
exacerbate tumor characteristics [21–23].
Our results show that HuH-7 cells change their
behavior when undergo 3D culture environment, as described
for other tumor cells . Moreover, these changes occur
in important genes for tumor progression, such as
proteases and adhesion molecules.
Fig. 2 Extracellular environment provided by the alginate matrix changes the gene expression profile of HuH-7 cells towards EMT. Gene expression
studies of a MMP-9, b OCLN, c p65, d VEGF and e I-CAM 1, comparing cells in 2D and 3D environment after 4 days. *p < 0.05
Fig. 3 HuH-7 cells stained with rhodamine-phalloidin and DAPI in a 2D culture cells observed under inverted fluorescent microscopy (×20) and b
in 3D cultured cells observed under confocal microscopy (×40)
Proteases are important to trigger cell proliferation,
differentiation, matrix remodeling, vascularization and
migration. All these events take place during normal
development but also during malignant progression.
MMP-9 is a protease widely studied because it is
important to promote the whole process of cell colonization,
hence proliferation, tumor invasion and metastasis
. According to our results, cell culture in capsules
promotes a high increment in MMP-9 expression. It
is reasonable to think that the activation of this kind of
molecule is triggered by the surrounding matrix formed
by sodium alginate.
Although the three-dimensional cell culture aggregates
do not always lead to the formation of a complex 3D
structure, they introduce biomechanical and
biophysical stimuli that promote a controlled environment
promoting the assembly of “tumors tissue type” . As the
cells in 2D are exposed to a uniform environment with
sufficient availability of nutrients and oxygen, they don’t
experience the three-dimensional solid tumor
environment with a gradient of chemical and critical biological
signals that can benefit or harm cell growth and tumor
development . Hypoxia is one of the triggers of EMT,
characterized by decreasing cellular junctions for the
subsequent infiltration of surrounding tissues. This study
observed a decrease in the expression of OCLN, which
participates in the tight junctions between cells. This
result may not due to lack of oxygen, but simply because
during the process of encapsulation cells are isolated
from one another, surrounded by the alginate matrix,
thus unable to establish such cell contacts. However, the
possibility that cells inside the capsule receive less oxygen
cannot be discarded. Hypoxia-related genes and oxygen
measurements must be subsequently analyzed.
However, other analyzed EMT and metastasis
markers (p65, VEGF and I-CAM) did not differ after 4 days
in 3D culture. These molecules are frequently
overexpressed in 3D cultures and in cancer cells in vivo [16,
27–29]. The reasons why we failed to detect a difference
in these molecules in the present study are not clear. It
may be related to the length of time cells were kept in
culture. An interesting analysis would be to compare
results at different times, plotting temporal changes in
gene expression . Studies have already shown the
viability of encapsulated cells for longer culture periods,
up to 45 days . In order to improve the model, an
increment in time culture could be carried out to asses
if a longer period induces different expression of other
markers. It would also be important to asses different
alginate concentrations to investigate the effect of this
variable on EMT and metastatic progression markers
in HuH-7 cells, according to studies performed for viral
On the other hand, some limitations should be
considered. Because of the small size for handling and the
modifications suffering during the fixing process, it is difficult
to obtain slides for histological analysis of good quality
from 3D cultures. Also, as can be seen in Fig. 3, not all the
encapsulated cells could be marked because of the lack of
permeability of the alginate. This could hamper
cytological analysis using antibodies. It would be very interesting
to confirm gene expression data with protein analysis by
western blot. Unfortunately, in our experience, it is very
difficult to obtain good quality protein fraction from
encapsulated cells, as alginate usually contaminates cell
extracts, even after dissolving the beads. Nevertheless,
the use of capsules as a 3D model has the advantage of
decreasing the use of animals for research. Moreover, it is
a cost-effective method, thus providing a useful platform
for the study of cancer cells .
Huh-7 cells change their behavior when cultured in a 3D
environment, showing markers of EMT. A key marker
for cell mobility and remodeling (MMP-9) was
overexpressed in cells cultured in 3D, whereas OCLN
expression was reduced. These results reinforce the use of
alginate as a scaffold for 3D cultures of cancer cells.
VAA carried out experiments, analyzed data and wrote the manuscript. UM
contributed with study design, data analysis, request for funding and critical
revision of the manuscript. CUC, MLL and LS contributed with data analysis
and revision of the manuscript. All authors read and approved the final
We would like to thank Nélson Alexandre Kretzmann Filho for providing us
with the primers used in this study and Ana Helena Paz and Natalia Schneider
Nunes for providing us with rhodamine-phalloidin dye.
Funding institutions have no role in study design, data collection, data analysis
and interpretation, article writing or submitting decision for publication. The
authors declare no competing interests.
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