Cell Biochemistry and Biophysics

https://doi.org/10.1007/s12013-021-00982-1

ORIGINAL PAPER

Biological properties and development of hypoxia in a breast cancer

3D model generated by hanging drop technique
Madalina Andreea Badea1 Mihaela Balas
●
1 ●
Anca Dinischiotu1

Received: 23 September 2020 / Accepted: 31 March 2021

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract
Hanging drop represents a simple approach designed for the generation of 3D models that have potential to be used for the
study of solid tumors characteristics. The aim of the study was to develop and characterize the breast cancer 3D cellular
models obtained through hanging drop technique using MDA-MB-231 cells. The biological characteristics such as:
morphology, cellular viability, proliferation capacity and hypoxia, were monitored for a six-day time period. The
morphological evaluation indicated that the 3D models presented the aspect of compact (seeding density of 2500 and 5000
cells/drop) and loose (seeding density of 8000 cells/drop) aggregates, with a decrease in diameter and an increase of their
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circularity. The cellular viability and proliferation capacity decreased in time and the level of lactate dehydrogenase (LDH)
increased in a time-dependent manner, suggesting the presence of necrotic cells that were dispersed in the cellular
aggregates. The occurrence of hypoxia process was suggested by the up-regulation of Hsp70 protein expression and
increased level of nitric oxide (NO). Moreover, the up-regulation of HIF-1α and poli-ubiquitinated Nrf2 protein expressions
and decreased level of reduced glutathione (GSH) indicated the presence of an acute hypoxic environment in MDA-MB-231
3D aggregates. In conclusion, the MDA-MB-231 3D models generated through hanging drop are compact and loose
aggregates characterized by an acute hypoxic condition.
Keywords Hanging drop MDA-MB-231 Hypoxia HIF-1α Hsp70
● ● ● ●

Introduction flasks or micro-patterned plates) and scaffold-free methods

Nowadays, the 3D cultures are intensively used for research
applications including the invasive potential of cancer cells
[1], hypoxia [2], cancer stem cell niche [3], cell-cell and
cell-extracellular matrix (ECM) interactions [4], drug
screening and drug delivery [5–8].
The 3D cell culture methods have become indispensable
for in vitro modeling of fundamental developmental pro-
cesses as well as human diseases. Over the years, various
3D culture methods were developed and optimized and at
this moment, these can be classified in scaffold-based
methods (matrix-on-top and matrix embedded methods,
matrix encapsulation methods, methods using spinner
* Mihaela Balas
mihaela.balas@bio.unibuc.ro
1 trolled [5, 9]. At the same time, this technique provides the
Department of Biochemistry and Molecular Biology, Faculty of
Biology, University of Bucharest, 91-95 Splaiul Independentei,
Bucharest R-050095, Romania
(methods using ultra-low attachment plates, hanging drop
technique, methods using magnetic levitation and bio-
printing). Each of these methods presents its limitations
and advantages regarding reproducibility, costs, reagent
consumption, the number of generated spheroids and
applications [6].
From the methods mentioned above, hanging drop is
one of the most well-established and studied methods for
the formation of 3D models. This technique is based on the
cultivation of cells in a suspended drop of medium,
allowing them to aggregate and form 3D models at the
bottom of the droplet [6, 7]. Hanging drop is a simple and
traditional method and its frequent use is correlated with the
advantages provided in spheroids generation. The main
advantages of this technique are represented by the reduced
reagent consumption and no requirement of using specia-
lized equipment. Moreover, the 3D models generated are
uniform in size and this characteristic can be easily con-
possibility to generate co-cultures from established cell
lines [10].

However, hanging drop method faces some limitations
regarding the manipulation and time cultivation of spher-
oids: is tedious handling, time-consuming and for long
term culturing the 3D models should be transferred in
another culture plate that can hold larger culture media
[6, 9]. Therefore, in order to minimize these limitations,
hanging drop array platforms, which allow the more effi-
cient formation of 3D models and their longer culture, were
developed and tested [11].
The 3D experimental models generated by hanging drop
method are cellular models that reproduce with high accu-
racy the microenvironment, the phenotype, the complex
molecular networks and the cellular heterogeneity of
tumors, being described as intermediaries between in vitro
cancer cell line cultures and in vivo tumors [12]. The 3D
models with the highest relevance in cancer research are
cellular multilayers, matrix-embedding cultures, hollow
fiber bioreactor, ex vivo cultures and multicellular tumor
spheroids (MCTSs). However, MCTSs are the most used
experimental models for the study of in vivo solid tumors
characteristics [10].
MCTSs are cellular aggregates described by gradients of
nutrients, gases, growth and signal factors [13]. Due to the
3D structure, the penetration of oxygen in the spheroids
core is limited and leads to the development of hypoxia
process, a phenomenon that occurs also in the in vivo
tumors. Consequently, in spheroids with a diameter gen-
erally greater than 400 μm, a necrotic center surrounded by
quiescent and proliferative cells at their border develops
[14, 15].
Hypoxia and necrotic center progress with the increase
of proliferation rate and spheroids size. Spheroids with a
diameter smaller than 200 µm are characterized only by
proliferating and normoxic cells. Further, the increase of
spheroids diameter to 300 µm lead to the organization of
proliferative zones at spheroid’s border, normoxic quies-
cent zones in the middle and hypoxic zones in the core.
The formation of necrotic zones is highlighted in spher-
oids with a diameter of 500 µm [16]. More specifically, the
organization of spheroids in specific layers is mediated by
hypoxia inducible factors (HIF) [2] and transcription fac-
tors involved in cell adaptation to low oxygen concentra-
tions [17].
In tumor cells, HIF-1 activates the transcription of
downstream genes involved in angiogenesis, cell survival
and proliferation, such as nitric oxide synthase (NOS),
vascular endothelial growth factor (VEGF), glucose trans-
porters 1 and 3 (GLUT 1, GLUT 3). For example, in
hypoxic conditions, the cells require a high glucose uptake
and HIF-1 induces the expression of the enzymes involved
in the glycolysis pathway and overexpression of glucose
transporters. HIF-1 signaling pathway is also regulated by
Mdm2 pathway, p53 genes or Hsp90 protein. Thus, loss of
Cell Biochemistry and Biophysics
p53 gene and high HIF-1α levels were concomitantly
observed in some tumor types, while inhibition of Hsp90
was associated with the nullify of HIF-1α [18].
Other factors involved in the development of hypoxia in
spheroid culture are represented by the oxygen perme-
ability and availability [15] as well as the media compo-
sition that dictates the growth kinetics of spheroids and
the occurrence of nutrients and metabolites gradients [2].
Moreover, biologically, hypoxia is associated with cell
invasion and metastasis [19], tumor progression and che-
moresistance [20, 21], some of the main markers of ther-
apeutic failure that can indicate this process as a potential
therapeutic target.
In this paper, we describe the technical steps for the
generation of an MDA-MB-231 multicellular 3D breast
tumor model by hanging drop and the characteristics of
the 3D culture concerning morphology aspects, viability,
proliferation capacity, and the variation of the level of
some hypoxic markers such as HIF-1α, Nrf2, Hsp70, GSH
and NO.
Materials and methods
Cell culture and generation of 3D cellular models
Triple negative breast cancer cell line MDA-MB-231
(ATCC HTB-26, Manassas, VA) was cultured in Dulbec-
co’s Modified Eagle Medium (DMEM; cat. no. 31600-083,
Gibco, UK) supplemented with 3.5 g/L glucose, 1.5 g/L
NaHCO3, 1% antibiotics-antimycotics solution (cat. no.
A5955, Sigma-Aldrich, St. Louis, MO), and 10% fetal
bovine serum (cat. no. 10270-106, origin South America,
Gibco, by Life Technologies, Carlsbad, CA). Cells were
cultured in 75 cm2 culture flasks until 80–90% confluency
at 37 °C, in a humid atmosphere with 5% CO2. The cells
were trypsinized using a 0.25% trypsin/0.53 mM EDTA
solution and the culture media was refreshed every two
days. The cell number from cellular suspensions was
determined using a Bürker-Türk counting chamber before
cell seeding.
Harvested cells were used to generate MDA-MB-231 3D
cellular models through hanging drop technique. In this
purpose, MDA-MB-231 cells were cultured on the inside lid
of Petri dishes in a drop of culture media at different cellular
densities, respectively, 2500, 5000 and 8000 cells/drop. The
drop volume was 20 µL. To ensure humidity conditions, a
sufficient volume of phosphate-buffered saline (PBS)
solution was pipetted inside the Petri dishes. The culture
media of 3D cellular models was refreshed once every two
days by replacing half of the culture media with a fresh one.
The 3D cellular models were maintained at 37 °C, in a
humid atmosphere with 5% CO2.
Cell Biochemistry and Biophysics
All the biological experiments were performed using 3D
cellular models generated from 5000 cells.
Morphology of 3D cellular models
The morphology of 3D cellular models was analyzed
until the sixth day of culture through optical microscopy
using an Olympus IX73 (Olympus, Tokyo, Japan)
microscope equipped with a Hamamatsu ORCA-03G
camera (A3472-06, Hamamatsu, Japan). The images
were acquired using the CellSens Dimension software
(v1.11, Olympus).
The variation of diameter and circularity was measured
using the ImageJ software (v 1.51k, NIH, Bethesda, MD,
USA) [22]. The diameter was estimated for 3D models
generated from 2500, 5000 and 8000 cells, using the
“freehand selections” function, “Feret” parameter indicating
the value of the 3D model diameter. The circularity of 3D
models generated from 2500, 5000 and 8000 cells was
determined with the “freehand selection” tool and “circu-
larity” parameter, a value of 1 being associated with a
perfect circle [23]. Five 3D models were analyzed for each
time interval.
Cellular viability
The cellular viability of MDA-MB-231 3D models was
evaluated from the third until the sixth day of culture
using the LIVE/DEAD Viability/cytotoxicity kit (L3224,
Invitrogen, Carlsbad, CA). According to the manu-
facturer’s instructions, live cells were labeled in green
with calcein AM, while the dead ones were stained in red
with ethidium homodimer (EthD-1). To perform the
assay, half of the culture media of the 3D models was
replaced with a free serum medium solution containing
2 µM calcein AM and 4 µM EthD-1. After 20 min of
incubation at 37 °C, the labeled 3D models were ana-
lyzed under an Olympus IX73 fluorescence microscope
(Olympus, Tokyo, Japan). The images were acquired
using fluorescein isothiocyanate (FITC) and tri-
rhodamine-isothiocyanate (TRITC) filters and CellSens
Dimension software (v1.11, Olympus). The fluorescence
intensity was quantified using ImageJ software (v1.51k,
NIH, Bethesda, MD, USA). For each time interval were
analyzed three images. The corrected total cell fluores-
cence (CTCF) for live cells (green fluorescence) and dead
cells (red fluorescence) was calculated, using the for-
mula: CTCF = Integrated Density of selected spheroid -
(Area of selected spheroid X Mean fluorescence of
background readings). Using CTCF values, the results
were expressed as a percentage of live cells and respec-
tively dead cells relating to total CTCF value (live and
dead cells) which was considered 100%.
Lactate dehydrogenase (LDH) assessment
The level of lactate dehydrogenase (LDH) activity released
in the culture media of the 3D models was determined up to
the sixth day of culture, using Cytotoxicity Detection Kit
(LDH) (ver. 10, cat. no. 14115700, Roche, Basel, Swit-
zerland). This assay uses a catalyst represented by a mixture
of diaphorase and NAD+ and a dye solution consisting of
iodotetrazolium chloride (INT) and sodium lactate. Thus, in
the first step, LDH catalyzes the oxidation of lactate to
pyruvate and the reduction of NAD+ to NADH. In the
second enzymatic reaction, NADH participates in the con-
version of the yellow tetrazolium salt INT (2-[4-iodophe-
nyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride) to the
formazan salt that is detected spectrophotometrically.
At each tested culture time interval, in a 96-well flat
bottom microplate, 50 µL culture media collected from
five 3D models wells (from the drop volume of 20 µL
culture media were collected 10 µL from each 3D model)
were incubated with 50 µL reaction mixture (catalyst and
dye solution) for 15 min at room temperature, in the dark.
The absorbance of the samples was read at 490 nm using a
Flex Station 3 microplate reader (Molecular Devices, San
Jose, CA, USA). The values of LDH activity were first
divided by cell number and the LDH level in the sample
media was calculated in relation to the one of control
(seeding moment - Day 0). For calculation of LDH level,
the dilution resulted from replacing half of the culture media
was also taken into consideration.
Preparation of cellular lysate
At each time interval, the generated 3D models were col-
lected from Petri culture dishes, centrifuged for 5 min at
1500 rpm, washed and resuspended in PBS. The cellular
lysates were obtained after the sonication of 3D models
on ice (three times, 30 s) using a UP50H ultrasonicator
(Hielscher Ultrasound Technology, Teltow, Germany) at
80% amplitude and their centrifugation for 10 min, at
3000 rpm, 4 °C. The supernatant obtained after the cen-
trifugation was used for further determinations. The protein
concentration was measured by the Bradford method, using
bovine serum albumin (BSA) as standard protein [24].
Immunoblotting of PCNA, HIF-1α, Nrf2 and Hsp70
proteins
Western blot technique was used to analyze the changes in
the expression of the following proteins: proliferating cell
nuclear antigen (PCNA), hypoxia-inducible factor-1-alpha
(HIF-1α), nuclear factor erythroid 2-related factor 2 (Nrf2)
and 70 heat shock protein (Hsp70), during six days of
cultivation of 3D cellular models. The preparation of the

samples included the dilution of the cellular lysates with
PBS and their chemical (using a loading buffer with 2-
mercaptoethanol) and thermic denaturation (5 min, at
95 °C). Further, the samples (30 µg of protein) were loaded
on 10% and 12% polyacrylamide gels and ran in a TRIS-
glycine-SDS buffer, at 90 V, for 2 h. The proteins separated
in SDS-PAGE gels were then transferred on PVDF mem-
branes (cat. no. IPVH00010, Merck, Darmstadt, Germany)
in a wet transfer system, using a TRIS-glycine-methanol
buffer (90 min).
After the transfer step, the membranes were revealed
using Western Breeze Chromogenic Anti-Mouse and
Anti-Rabbit kits (WB7103, WB7105, Invitrogen, Carls-
bad, CA, USA), following the manufacturer’s instruc-
tions. Thus, the membranes were incubated with
blocking solution for 30 min and then overnight with
monoclonal anti-PCNA (sc-56, Santa Cruz, Biotechnol-
ogy, Dallas, TX, USA), anti-HIF-1α (Ab179483, Abcam,
Cambridge, UK), anti-Hsp70 (sc-24, Santa Cruz, Bio-
technology, Dallas, TX, USA), anti-β-actin (A1978,
Sigma-Aldrich, St. Louis, MO, USA) specific antibodies
and polyclonal anti-Nrf2 (sc-722, Santa Cruz, Bio-
technology, Dallas, TX, USA) antibody. The next day,
alkaline phosphatase-conjugated anti-mouse and anti-
rabbit secondary antibodies were added for 30 min. In
the end, the protein bands were detected with BCIP/NBT
substrate, visualized with the ChemiDoc Imaging System
(BioRad, Hercules, CA, USA) and quantified using the
Image Lab (ver. 5.1, Bio-Rad, Hercules, CA, USA)
software. The β-actin was used as a reference protein to
normalize the protein levels.
Determination of reduced glutathione (GSH)
content
The concentration of GSH was assessed up to the sixth
day of culture using the Glutathione Assay Kit (CS0260,
Sigma-Aldrich, St. Louis, MO, USA). In the first step,
the cellular lysates were deproteinized with an equal
volume of 5% sulfosalicylic acid (SSA) through the
centrifugation of the samples at 10,000 rpm, for 10 min,
4 °C. In the second step, 10 µL of the supernatants were
incubated with 150 µL reaction mixture containing 5,5’-
dithio-bis(2-nitrobenzoic) acid (DTNB; D8130, Sigma-
Aldrich, St. Louis, MO, USA) solution, for 10 min, at
room temperature. The absorbance of 5’-thio-2-nitro-
benzoic acid (TNB) generated during the reaction was
measured at 405 nm, using a Flex Station 3 microplate
reader (Molecular Devices, San Jose, CA, USA).
A calibration curve (200-3.125 µM) was similarly
prepared using a solution of 200 µM GSH (G6529,
Sigma-Aldrich, St. Louis, MO, USA) as standard. The
GSH concentration of samples was determined by
Cell Biochemistry and Biophysics
extrapolation on the calibration curve and was calculated
in relation to the GSH level of control (Day 0).
Quantification of nitric oxide (NO) production
The level of NO released in the culture media of the 3D
models was determined up to the sixth day of culture, using
colorimetric Griess reaction [25]. Griess reagent, used in
this reaction, is made of equal volumes of 10% N-(1-
Naphthyl) ethylenediamine dihydrochloride (cat. no.
222488, Sigma-Aldrich, St. Louis, MO, USA) and sulfa-
nilamide (S9251, Sigma-Aldrich, St. Louis, MO, USA)
prepared in 85% phosphoric acid (H3PO4). To perform the
assay, 80 µL culture media were collected from eight 3D
models (from the drop volume of 20 µL culture media were
collected 10 µL from each 3D model) and mixed with 80 µL
Griess reagent. In the end, the absorbance of the samples
was determined at 540 nm at a Flex Station 3 microplate
reader (Molecular Devices, San Jose, CA, USA). The
values of NO concentration were first divided by cell
number and all samples were reported to the control values.
For calculation of NO level, the dilution resulted from
replacing half of the culture media was also taken into
consideration.
Statistical analysis
All experiment data were represented as an average of three
biological replicates. Statistical significance was determined
by Student’s t test, a P value < 0.05 being considered sig-
nificant. All sample values were reported to the control ones
(Day 0 samples).
Results
Morphology of hanging drop generated breast
cancer 3D cellular aggregates
The morphology of breast cancer 3D cellular aggregates
was visualized by optical microscopy and the diameter and
circularity of the models were monitored within six days.
The 3D models were generated starting from a density of
2500, 5000, and 8000 cells per drop.
The bright-field images showed that breast cancer 3D
models were characterized by spherical morphology, with
an irregular border. Depending on the seeding cellular
density, the generated 3D models can form compact (2500
and 5000 cells/drop) or loose (8000 cells/drop) cellular
aggregates. As can be observed in Fig. 1a, 3D models
obtained from 2500 and 5000 cells are characterized by
high compaction of cells starting with the third day of
culture, particularity that was not observed for a cellular
Cell Biochemistry and Biophysics

Fig. 1 Morphology of breast

cancer 3D cellular aggregates
generated by hanging drop
technique. a Bright-field images
of cellular aggregates generated
from 2500, 5000 and 8000 cells
after 1, 3, 4 and 6 days of culture
and the variation in time of their
(b) diameter and (c) circularity.
Columns represent mean value
± S.D. relative to Day 0 sample
(n = 5). The asterisks
correspond to statistical
significance of aggregates
generated in Day 3, 4 and 6
relative to those generated in
Day 2. *P < 0.05, **P < 0.01,
***P < 0.001 vs. Day 2. Scale
bar: 500 µm

density of 8000 cells. Thus, the 3D models generated from
2500 and 5000 cells can be considered compact aggregates,
whereas those obtained from 8000 cells were described as
loose aggregates.
The diameter of cellular aggregates decreased in a time-
dependent manner until the sixth day in a relationship with
the initial cellular density (Fig. 1b). The high compaction of
cells observed in the third day of culture on bright-field
images was associated with a high difference of diameter
between the second and the third day (for 3D aggregates
generated from 5000 cells, the diameter varied from
571.31 ± 87.26 µm in the second day to 446 ± 30.29 µm in
the third day). Also, the diameter of 3D aggregates seeded
at 2500 cells/drop decreased from 430.71 ± 24.1 µm in the
second day to 250.54 ± 17.29 µm in the last day. However,
the most significant variation of diameter was recorded for
3D aggregates generated from 8000 cells (a decrease from
996.19 ± 99.06 µm in the second day to 474 ± 5.91 µm in
the sixth day).
The circularity of 3D cellular aggregates generated from
2500 and 5000 cells varied similar in the six-day interval.
Thus, the circularity of aggregates generated from 2500 and
5000 cells increased from day 2 (0.73 ± 0.06 and respec-
tively 0.73 ± 0.072) to day 3 of culture (Fig. 1c), when it
reached a value of 0.85 ± 0.05 and 0.86 ± 0.02 respectively,
closely related to a value of 1 corresponding to a perfect
circle. After this period, the circularity remained almost
constant. Instead, the circularity of 3D cellular aggregates
generated from 8000 cells reached a value closely to that of
3D aggregates obtained from lower cellular densities only in
the sixth day of culture (0.83 ± 0.01).
Viability and proliferative capacity of hanging drop
generated breast cancer 3D cellular aggregates
The cellular viability of compact aggregates was evaluated
by fluorescence labeling of live cells in green and dead cells
in red, and by quantification of LDH level released in the
culture media, as a marker of membrane integrity and
necrosis. The proliferation rate of hanging drop generated
3D models was assessed by measuring the PCNA protein
expression during six days of culture.
The percentages of the live and dead cells that form
the structure of the 3D cellular aggregates generated by
hanging drop in this study are presented in Fig. 2a, b.
The dead cells were found dispersed in the entire cellular
mass and not organized in a necrotic center as is
expected from a 3D tumoral model [26]. The quantifi-
cation of fluorescence images revealed an increase in the
percentage of dead cells, in a time-dependent manner.

Fig. 2 Cell viability and proliferation of hanging drop generated
MDA-MB-231 breast cancer 3D cellular aggregates on a period of
6 days. a Fluorescence microscopy images: green fluorescence (cal-
cein AM) indicates live cells and red labeling (ethidium homodimer -
EthD-1) corresponds to dead cells. Scale bar: 500 µm. b Quantification
of fluorescence images (n = 3). The asterisks correspond to statistical
significance of 3D cellular aggregates generated in Day 4, 5 and 6
relative to those generated in Day 3. *P < 0.05 vs. Day 3. c The
From the third until the sixth day of culture, the per-
centage of dead cells increased from 17.61% ± 1.8 to
33.71% ± 2.65 thus decreasing the viability of 3D cel-
lular aggregates.
In accordance with these results, an increase of LDH
level was registered starting with the second day of culture
comparing with the seeding moment (Fig. 2c). Moreover,
the highest level of LDH was recorded in the last day of
culture (7.42 ± 0.036-fold).
The PCNA protein expression presented in Fig. 2d was
analyzed as a proliferation marker [27]. Thus, in the second
day of culture a decrease by 5.18% ± 2.06 was recorded
compared with the control and by 29.36% ± 0.45 in the
fourth day, compared with the second day. The last day
analyzed was characterized by a constant expression of
PCNA (Fig. 2e).
Hypoxia analysis
The evolution of hypoxia process in the hanging drop
generated 3D cellular aggregates was monitored by ana-
lyzing the markers specific to this process: HIF-1α, Nrf2
and Hsp70 proteins and the concentration of GSH and NO.
The expression of HIF-1α increased significantly from
the second until the sixth day of culture compared to Day 0
(Fig. 3b). Thus, in the second and fourth day, the expression
increased by 45.18% ± 0.67, respectively 45.93% ± 5.06
over Day 0. In the sixth day, the expression of HIF-1α
reached the highest level (54.9% ± 1.95 increase compared
to control). Similarly, the expression of Nrf2 protein
Cell Biochemistry and Biophysics
relative level of LDH released in the culture medium of cellular
aggregates. Columns represent mean values ± S.D. (n = 3) relative to
Day 0 sample (seeding moment). *P < 0.05, ***P < 0.001 vs. control.
d Blot images presenting PCNA and β-actin protein expression.
e Quantification of PCNA protein expression normalized to β-actin.
Columns represent mean values ± S.D. (n = 3) relative to Day 0 sam-
ple. *P < 0.05, ***P < 0.001 vs. Day 0
increased significantly starting from the second day of
culture reaching the highest level in the fourth day by
132.5% ± 6.4 over control. Although, a slight decrease of
Nrf2 expression compared with this level (by 15.57 % ±
12.63) was recorded in the last day (Fig. 3c), this level still
remained high when relating to control. On the other hand,
the expression of Hsp70 was similar in the second and the
fourth day, when the expression increased by 57.62% ±
3.25 and respectively 57.02% ± 5.92 compared to control,
and reached a maximum in the sixth day, exceeding by
123.28% ± 10.22 the control level (Fig. 3d).
The amount of intracellular GSH content was sig-
nificantly modified only in the last day of culture when a
decrease by 29.5% ± 13.11 compared with the control was
recorded (Fig. 3e). The level of NO released in the culture
media in which were grown the MDA-MB-231 3D cellular
aggregates increased in a time-dependent manner. Thus, in
the second day of culture, the level of NO raised by 0.81 ±
0.08-fold compared to control, in the fourth day by 3.03 ±
0.42-fold, and on the last day, this was maximum, being
raised by 7.11 ± 0.02-fold versus control level (Fig. 3f).
Discussion
By hanging drop method, the cells settle down to the bottom
of a drop of culture medium, under the gravity [28, 29] and
form via self-aggregation a 3D cellular structure. The 3D
cultures obtained by us were represented by multicellular
aggregates of small dimensions, characterized by a spherical
Cell Biochemistry and Biophysics
Fig. 3 Expression of hypoxia markers in breast cancer 3D cellular
aggregates generated through hanging drop technique within 6 days of
culture. a Blot images of HIF-1α, Nrf2 and Hsp70 protein expressions.
Quantification of protein expressions of (b) HIF-1α, (c) Nrf2 and (d)
Hsp70 from blots images. Columns represent mean values ± S.D.
(n = 3) relative to Day 0 sample. **P < 0.01, ***P < 0.001 vs. Day
shape and compactness which were dependent on the den-
sity of cells at the seeding moment. Previous studies
showed that, the MDA-MB-231 cells lack the E-cadherin
protein expression [30], a key adhesion molecule that
mediate the interactions involved in breast cancer spheroid
formation [4] thus making difficult to form highly compact
3D models from these cells. A higher degree of compac-
tation could be acquired by supplementing the culture
medium with reconstituted basement membrane or Matrigel
[31–33]. In human breast carcinomas, β1 integrin played a
critical role in mediation of cell-ECM interactions which are
required for the transition of MDA-MB-231 aggregates into
spheroids [4].
0 sample. e Intracellular GSH content. Columns represent mean
values ± S.D. (n = 3) relative to Day 0 sample. *P < 0.05 vs. Day 0.
f Relative level of NO released in the culture medium, within six days
of culture. Columns represent mean values ± S.D. (n = 3) relative to
Day 0 sample. **P < 0.01 vs. Day 0
Considering the degree of cellular compaction and nature
of adhesion molecules on the cells surface [4, 31, 32], 3D
models obtained in this study by hanging drop technique
can be classified in compact (from cellular densities of 2500
and 5000 cells/drop) and loose (from cellular density of
8000 cells/drop) aggregates. Utilization of the 3D models
generated without a scaffold by hanging drop technique
presents the benefit of a reduced cost when screening the
effects of a high number of chemotherapeutic drugs com-
pared with those with Matrigel. Moreover, the Matrigel
might contain endogenous growth factors that are not found
in the human tumor environment and can influence
experimental results [34].

The circularity and size of the diameter of 3D cellular
aggregates varied dependently on cellular densities and
the time of culture incubation. We observed an increase
of circularity and a decrease of cellular aggregates dia-
meter in time and we associated these with the com-
paction process of cellular aggregates. According to
Leung et al. [35], circularity refers to the symmetry of a
spheroid with an ideal equidistant symmetry from the
center of the spheroid to any point on the surface. The
3D cellular aggregates generated in this study from 5000
cells/drop had a maximum circularity of 0.86 from 1.
The circularity of spheroids is cell line dependent and
can increase in the presence of additives (collagen,
MethoCel) [35]. Raghavan et al. [36] compared the cir-
cularity of breast cancer spheroids generated through
hanging drop and other techniques, but no significant
differences were observed.
Previous works showed that when the size of the
spheroid increases, the oxygen concentration in the center
of the sphere decreases leading to the formation of a
necrotic core surrounded by a layer of nonproliferating
quiescent cells and only the external rim of the spheroid
remains in a proliferating state. Spheroids below 500 µm
present a small necrotic center or not at all [9, 37]. In our
study, the 3D aggregates generated from 5000 cells/drop
reached in the sixth day around 300 µm in diameter. The
fluorescence images indicated the presence of necrotic
cells, but not their organization in a necrotic center in the
middle of the aggregates. Generally, 3D cellular aggre-
gates with 200-300 µm in diameter present a typical
zonation with proliferative zones at the surface co-existing
with normoxic quiescent zones in the middle and hypoxic
zones in the core [16]. When diameter increases and
hypoxic areas develop in time, more necrotic cells appear.
One possible explanation of necrotic cells distribution
throughout the cellular mass of aggregates could be the
high degree of compaction without diameter increase that
can induce the organization of a necrotic center, but also
the absence of a reconstituted basement membrane that can
sustain the nutrition and implicitly the high viability of
cells from the border of the aggregates.
An inhibition of cell proliferation was observed in our
3D aggregates indicated by the decrease of PCNA protein
expression during the cultivation time which might be a
result of the action of cytostatic factors diffusing out of the
necrotic cells [38]. The presence of necrotic cells in the
cellular mass of aggregates was confirmed also by level of
LDH activity released in the culture media, which increased
significantly in time when the cellular membrane was
damaged [39]. Previous studies associated the increased
production of LDH activity in culture media with the
response of cancer cells to hypoxia [40]. Hypoxia is one
of the main causes of resistance to chemotherapy and
Cell Biochemistry and Biophysics
radiotherapy, and also of increased metastases [41]. Due to
this, this is of critical importance in cancer research studies.
In the cellular environment, hypoxia is mediated by the
family of hypoxia-inducible factors (HIF) transcription
factors that regulate the adaptation of cells to low oxygen
concentrations [42]. The highly conserved transcriptional
complex HIF-1 is a dimeric transcription factor with an
integral role in hypoxia and other associated processes that
consist of two subunits: HIF-1α, an oxygen-regulated sub-
unit and HIF-1β, a constitutively expressed subunit [17].
Previous studies demonstrated that in hypoxic conditions,
HIF-1α induces the up-regulation of other proteins expres-
sion, such as Hsp70 (70-kDa heat shock proteins), central
components of folding processes, with housekeeping func-
tions [43]. Thus, primary chondrocytes responded to
hypoxic conditions through the enhancement of HIF-1α and
Hsp70 mRNA and protein expressions, with the protection
of cells from apoptosis [44]. A significant increase of Hsp70
in hypoxia, as a general mechanism of cancer cells, was
observed also in MCF-7 breast cancer cells [45]. Consistent
with previous studies, we noticed an up-regulation of HIF-
1α and Hsp70 proteins that indicates the presence of
hypoxic cells in MDA-MB-231 3D aggregates during the
cultivation time. According to previous data, a high stability
and activation of HIF-1α is maintained during acute
hypoxic conditions, while chronic hypoxia is associated
with a down-regulation of HIF-1α [46, 47], which can
suggest the development of an acute hypoxic medium in
MDA-MB-231 cellular aggregates generated in our study.
It was observed that in hypoxic conditions, Nrf2 protein,
a member of the basic leucine zipper transcription factor
subfamily with a vital role in maintaining cellular home-
ostasis, especially under oxidative stress, is activated,
leading to the transcription of the specific genes [48, 49]. A
significant increase in the Nrf2-ARE binding activity, Nrf2
protein and mRNA expression of Nrf2-mediated antioxidant
genes was observed in mouse skeletal muscle after 48 h of
hypoxia, all these modifications being associated with the
involvement of HIF-1α protein [50]. The increased level of
HIF-1α concomitant with the activation of Nrf2 was
observed also in breast cancer cells under hypoxic condi-
tions [51, 52]. In the present study, we quantified the
expression of poli-ubiquitinated Nrf2 that migrated at
~100 kDa [53] and we observed an increase of its expres-
sion starting with the second day that could indicate its
accumulation in the cytoplasm of cells and a hypoxic con-
dition developed in MDA-MB-231 3D cellular aggregates
that does not reach the threshold required for the activation
of Nrf2. This could also suggest that the acute hypoxic
condition occurs, indicated also by an increased expression
of HIF-1α, due to the dispersion of necrotic cells in the
cellular mass of aggregates and not their organization in a
defined necrotic structure.
Cell Biochemistry and Biophysics
Nrf2 activation is determined by the presence of reac-
tive oxygen species (ROS) in oxidative stress conditions
which could be counteracted by enzymatic and non-
enzymatic antioxidants including reduced glutathione
(GSH), the most important non-enzymatic cellular anti-
oxidant. In this study, the level of GSH in the cellular 3D
aggregates decreased in a time-dependent manner, most
probably as a result of the hypoxia environment. These
results were in accordance with a study conducted by
Mansfield et al. [54] that showed the exposure of HEK293
and Hep3B cells to hypoxia (1.5% O2) caused a time-
dependent decrease of total cellular GSH mediated by
hypoxia-induced mitochondrial ROS. The results indicated
a mechanism that is not dependent on HIF transcriptional
activity but can be based on inhibition of GSH synthesis
due to changes in the levels or activity of the rate-limiting
synthetic enzyme γ-glutamylcysteine synthase or due to
availability of the substrate cysteine, which is transported
into cells as cystine. The critical role of mitochondrial
GSH during hypoxia was underlined also by Lluis et al.
[55] who observed its involvement in the survival of
hepatocytes through the regulation of oxidative stress.
However, the variation of GSH levels most likely depends
on the intensity of the hypoxia process. Previous studies
demonstrated that intracellular GSH levels could be also
influenced by NO through a mechanism based on the
induction of cystine uptake.
NO is a short-lived gas that acts as a molecule signaling
in the body where it modulates several physiological and
pathological mechanisms but also contributes to the
hypoxia process [56]. In 2013, Liu and Xu [57] reported
the increase of NO accompanied by an up-regulation of the
HIF1-α gene expression and a decrease of the respiration
rate. An increase of NO production was observed also
when cardiomyoblasts were exposed to hypoxia [58].
During the cultivation time of MDA-MB-231 3D aggre-
gates, NO level recorded an increase in a time-dependent
manner which might be correlated with the changes of
intracellular GSH content in hypoxic conditions. Li et al.
[59] demonstrated that acute exposure to NO induced
depletion of GSH level, while chronic exposure induced
elevation of GSH one.
In our study, the presence of hypoxia environment in 3D
cellular aggregates generated by hanging drop technique was
proved by the increase of NO level, Hsp70 and HIF-1α
protein expressions during the cultivation time, and decrease
of GSH level in the last day of culture which could indicate
an acute level of hypoxia. These findings are strengthened by
the increased expression of Nrf2, its accumulation in the
cytoplasm of MDA-MB-231 cells and the dispersion of
necrotic cells in the cellular mass of the aggregates.
Through this study, we characterized the 3D cellular
aggregates generated by hanging drop technique following
some of the key features of spheroids and in vivo solid
tumors (3D cellular growth, necrosis and hypoxia).
The elaborated characterization of hypoxia process
development specifically contributes to the novelty of this
study. Also, the biological characterization presented in our
research can contribute to the understanding of the
mechanisms involved in hypoxia which is associated with
the induction and development of chemoresistance in drug
screening research.
In conclusion, the MDA-MB-231 3D models generated
through hanging drop technique can be characterized as
compact or loose aggregates, depending on initial cellular
density, with a decreased proliferation rate in time. Also,
the occurrence and development in time of hypoxia fol-
lowed by the appearance of necrotic zones in MDA-MB-
231 3D cellular aggregates was proved by the high levels
of Hsp70 protein expression as well as NO and LDH levels
from culture media. Furthermore, the up-regulation of
HIF-1α protein expression, decreased level of GSH and
accumulation of Nrf2 protein in cytoplasm indicated the
acute hypoxic medium in MDA-MB-231 3D models
generated through hanging drop technique. Taking into
account these biochemical and molecular biomarkers used
to characterize the MDA-MB-231 3D cellular aggregates,
the rational design of new drug against breast cancer could
be facilitated.
Acknowledgements This work was supported by a grant of the
Romanian National Authority for Scientific Research and Innovation,
UEFISCDI, project number PN-III-P2-2.1-PED-2016-0904, within the
PNCDI III program.
Compliance with ethical standards
Conflict of interest The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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