Salo et al. BMC Cancer (2015) 15:981
DOI 10.1186/s12885-015-1944-z
RESEARCH ARTICLE
Open Access
A novel human leiomyoma tissue derived
matrix for cell culture studies
Tuula Salo1,2,3*, Meeri Sutinen1,2, Ehsanul Hoque Apu1,2, Elias Sundquist1,2, Nilva K. Cervigne4,5,
Carine Ervolino de Oliveira5, Saad Ullah Akram6,7, Steffen Ohlmeier8,9, Fumi Suomi7,8, Lauri Eklund7,8, Pirjo Juusela3,
Pirjo Åström1,2, Carolina Cavalcante Bitu1,2, Markku Santala10, Kalle Savolainen11, Johanna Korvala1,2,
Adriana Franco Paes Leme12 and Ricardo D. Coletta5
Abstract
Background: The composition of the matrix molecules is important in in vitro cell culture experiments of e.g.
human cancer invasion and vessel formation. Currently, the mouse Engelbreth-Holm-Swarm (EHS) sarcoma -derived
products, such as Matrigel®, are the most commonly used tumor microenvironment (TME) mimicking matrices for
experimental studies. However, since Matrigel® is non-human in origin, its molecular composition does not
accurately simulate human TME. We have previously described a solid 3D organotypic myoma disc invasion assay,
which is derived from human uterus benign leiomyoma tumor. Here, we describe the preparation and analyses of a
processed, gelatinous leiomyoma matrix, named Myogel.
Methods: A total protein extract, Myogel, was formulated from myoma. The protein contents of Myogel were
characterized and its composition and properties compared with a commercial mouse Matrigel®. Myogel was
tested and compared to Matrigel® in human cell adhesion, migration, invasion, colony formation, spheroid culture
and vessel formation experiments, as well as in a 3D hanging drop video image analysis.
Results: We demonstrated that only 34 % of Myogel’s molecular content was similar to Matrigel®. All test results
showed that Myogel was comparable with Matrigel®, and when mixed with low-melting agarose (Myogel-LMA) it
was superior to Matrigel® in in vitro Transwell® invasion and capillary formation assays.
Conclusions: In conclusion, we have developed a novel Myogel TME matrix, which is recommended for in vitro
human cell culture experiments since it closely mimics the human tumor microenvironment of solid cancers.
Keywords: Tumor microenvironment matrix, Invasion, Migration, Hanging drop, Colony formation, Spheroid
formation, Capillary formation
Background
Translational cancer research almost completely lacks
human tissue in vitro models that mimic the natural
tumor microenvironment matrix (TMEM). This also was
partially fulfilled by our organotypic leiomyoma 3D solid
disc model [1], which has been successfully used in numerous cancer invasion studies [2–7]. In this model, the hypoxic tumor matrix provides an authentic environment
* Correspondence: tuula.salo@oulu.fi
1
Cancer and Translational Medicine Research Unit, Faculty of Medicine,
University of Oulu, PO Box 5281FI-90014 Oulu, Finland
2
Medical Research Center Oulu, Oulu University Hospital and University of
Oulu, FI-90014 Oulu, Finland
Full list of author information is available at the end of the article
including e.g. fibroblasts, vessels, collagen fibers, laminins,
glycoproteins, cytokines and proteases [8].
Matrigel® (BD Biosciences), the mouse EngelbrethHolm-Swarm (EHS) tumor-derived commercial product
[9], is widely used for in vitro adhesion, invasion and capillary formation assays [10, 11]. However, the tumor
matrix of rodents clearly differs from the respective human TMEM [12]. These differences most likely affect
human cancer invasion processes and underscore the
need for soluble human TMEM products. In addition to
classic Matrigel® other EHS tumor derived products are
also available, such as ECM gel (Sigma), Cultrex® BME
(Amsbio), Geltrex® (Gibco Life Technologies) and
ECMatrix™ (Millipore). All these products have the same
© 2015 Salo et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Salo et al. BMC Cancer (2015) 15:981
disadvantage for human studies; they are mouse tumor
tissue homogenates that differ in composition from human TMEM.
Since collagens are the most abundant proteins in the
extracellular matrix (ECM), gels from purified rodent
collagens are commonly used to embed cells into 3D
cultures [13, 14]. In organotypic 3D cultures, type I collagen derived from rat tail is probably the most abundant ECM mimicking matrix. Other commercially
available ECM molecules, like fibronectin [15], fibrin
[16] and hyaluronic acid [17], are also used for in vitro
studies. In addition, synthetic ECM or peptide matrices
are available from various manufacturers. However, one
purified molecule, a mixture of them, or totally synthetic
matrices do not adequately simulate the complex effects
of natural ECM due to the obvious lack of hundreds of
cytokines or protease cleavage sites identified in natural
tumor ECM [18, 19]. Moreover, the excessive presence
of one molecule or a mixture of basement membrane
components rich in growth factors does not reflect the
ECM composition synthesized by stromal cells. In vivo the
combinations of multiple TMEM factors are important for
cell-ECM interactions during cancer progression [20].
Three recent reports [21–23] use the term myogel for
an extracellular matrix material that is derived from human, mouse, rat or pig normal skeletal muscles using
procedures similar to those of Kibbey [9] for the preparation of EHS tumor extract. The myogel material was
shown to be adipogenic [21, 23] and to support the ex
vivo amplification of corneal epithelial cells [22]. Vivo
Biosciences Inc. is marketing HuBiogel, an ECM gel derived from normal human amnion tissue containing
laminin, collagen types I and IV, entactin, tenascin and
heparan sulfate proteoglycan, but lacking endogenous
growth factors (EGF, TGF-α, TGF-ß, FGF and PDGF) as
well as MMP-2 and MMP-9 [24]. These commercial
products and other human ECM matrices used in research are derived from normal tissues (skeletal muscle,
amnion membrane, placenta) or are in vitro, cell culture
-derived [MaxGel™ Human ECM (Sigma), AlphaMAX3D
(Neuromics)].
Here we describe a novel product prepared from human uterus benign leiomyoma tumor tissue [1] according to the method described for the preparation of
Matrigel® [9]. We formulated a solution/gel of the total
protein extracts, characterized the protein contents, and
compared with Matrigel® using a set of in vitro experiments. Based on the results we conclude that the tumor
tissue solution/gel derived from human leiomyoma offers an excellent human TMEM tool for analyzing human carcinoma cells in vitro. This novel Myogel
product, combined with low melting agarose (LMA),
provides an accessible method for analyzing cancer cell
invasive, adhesive or migratory properties; potential
Page 2 of 16
chemotherapeutic compounds; as well as to test capillary
formation. Hence Myogel-LMA together with myoma
discs could offer a practical human “TMEM tissue kit”
for translational cancer research purposes.
Methods
Myogel preparation
Human uterus leiomyoma tissue (after taking samples
for histopathological analyses) was received from the
Oulu University Hospital, Department of Gynecology [1]
and the Tampere University Hospital, Department of
Gynecology after obtaining the patients’ written informed consent. The use of myoma tissue was approved
by the Ethics Committee of both the Oulu University
Hospital, and the Tampere University Hospital. Myogel
was prepared according to the method described [9] for
the preparation of EHS sarcoma derived Matrigel® (BD
Biosciences), with minor modifications. Briefly, myoma
tissue, frozen with liquid nitrogen, was ground to a powder with a CryoMill (Retsch, Haan, Germany), and 10 g
of tissue powder was suspended in 20 ml of ice cold
3.4 M, pH 7.4 NaCl buffer. After centrifugation, the pellet was homogenized in another 20 ml of the same NaCl
buffer using a T18 Ultra-Turrax (IKA®-Werke GmbH &
Co. KG, Staufen, Germany). A T18 Ultra-Turrax was
also used in all the following homogenizations. The protein concentration in each preparation was measured
using a DC Protein Assay (Bio-Rad) according to the
manufacturer’s instructions. The absorbance at 590 nm
was measured using a Victor3V 1420 Multilabel Counter
and Wallac 1420 Manager (Perkin Elmer Life and Analytical Sciences, Turku, Finland). The protein concentrations in various Myogel batches were diluted using cell
culture media described in the cell culture -section (see
below) to match those of Matrigel®. The Myogel solution
was then stored in small (≤1 ml) aliquots at −20 °C.
Gradient SDS-PAGE of Myogel and Matrigel® and proteomic
analyses of Myogel
Samples (20 μg) of four different Myogel batches (preparation numbers 12, 15, 16 and 17) and their mixture, as
well as four different Matrigel® (BD Matrigel Matrix, BD
Biosciences, Cat. Number 354234) samples were loaded
on a gradient (4 %, 8 %, 15 %) SDS-PAGE gel and separated using 15 mA for 90 min. A PageRuler Plus Prestained Protein Ladder (Thermo Scientific) was used as
a molecular weight marker. Proteins were stained using
Coomassie Blue and the gel was washed with elution
buffer to remove excess staining. The gel was photographed over a stripping table.
For proteomic analysis, 10 μg and 20 μg of four different Myogel batches (preparation numbers 3, 4, 6, 9)
were separated as above. The gel was viewed over a
stripping table, and individual bands were cut and
Salo et al. BMC Cancer (2015) 15:981
collected from the gel. After digestion (0.3 μg of trypsin
was used/band) each band was resuspended with formic
acid (see μl/band in the Additional file 1: Figure S1B) on
three selected Myogel samples (preparations 3, 6, 9) and
stored in −20 °C until gel digestion for mass spectrometry. The gel digestion of selected samples for mass
spectrometry was done according to Hanna et al. [25]
with some modifications.
Mass spectrometry analysis
An aliquot of 4.5 μl of the resulting peptide mixture was
analyzed on an ETD-enabled LTQ Orbitrap Velos mass
spectrometer (Thermo Fisher Scientific, Waltham, MA)
connected to a nanoflow liquid chromatography (LCMS/MS) instrument by an EASY-nLC system (Proxeon
Biosystems, West Palm Beach, FL) with a Proxeon
nanoelectrospray ion source. Peptides were separated
with a 2–90 % acetonitrile gradient in 0.1 % formic acid
using an analytical column PicoFrit Column (20 cm x
ID75 μm, 5 μm particle size, New Objective, Woburn,
MA) at a flow of 300 nl/min over 27 min. The nanoelectrospray voltage was set to 2.2 kV and the source
temperature was 275 °C. All of the instrument methods
were set up in the data-dependent acquisition mode.
The full scan MS spectra (m/z 300–1600) were acquired
in the Orbitrap analyzer after accumulation to a target
value of 106. The resolution in the Orbitrap was set to r
= 60,000 and the 20 most intense peptide ions with
charge states ≥ 2 were sequentially isolated to a target
value of 5000 and fragmented in the linear ion trap
using low-energy CID (normalized collision energy of
35 %). The signal threshold for triggering an MS/MS
event was set to 1000 counts. Dynamic exclusion was
enabled with an exclusion size list of 500, exclusion duration of 60 s, and a repeat count of 1. An activation q =
0.25 and activation time of 10 ms were used.
Data analysis
Peak lists (msf ) were generated from the raw data files
using the Proteome Discoverer software version
1.3.0.339 (Thermo Fisher Scientific) with the Sequest
search engine and searched against the UniProt Human
Protein Database (release July 11, 2012; 69,711 entries),
with the following parameters: carbamidomethylation as
the fixed modification (+57.021 Da), oxidation of methionine (+15.995 Da) as the variable modification, one
trypsin missed cleavage and a tolerance of 10 ppm for
precursor and 1 Da for fragment ions. All datasets were
processed using the workflow feature in the Proteome
Discoverer software, and the resulting search data were
further analyzed in the software ScaffoldQ + v.3.3.1
(Proteome Software, Inc.). The scoring parameters
(Xcorr and Peptide Probability) in the ScaffoldQ+ software were set to obtain a false discovery rate (FDR) of
Page 3 of 16
less than 1 %, using the number of total spectra output
from the ScaffoldQ+ software, strict parsimony principle
was enabled, and leucine and isoleucine were considered
equal. A normalization criterion, the quantitative value,
was applied to the spectral counts.
Two-Dimensional Gel Electrophoresis (2-DE)
For the proteomic analyses two Myogel and one Matrigel® (BD Matrigel Matrix, BD Biosciences, Cat. Number
354234) samples were further purified by buffer exchange using an Amicon Ultra ultrafiltration unit with a
10 kDa cutoff (Millipore) and urea buffer (7 M urea,
2 M thiourea, 4 % [w/v] CHAPS, 30 mM Tris, pH 8.5).
After that the protein samples were sonicated and centrifuged. The protein concentrations in the supernatants
were determined in duplicate with a Bradford-based
assay according to the manufacturer’s instructions
(Roti®-Nanoquant, Carl Roth) with urea buffer as a control and aliquots were stored at −20 °C. For 2-DE,
100 μg of the protein solution of each sample was adjusted with rehydration urea buffer (7 M urea, 2 M thiourea, 4 % [w/v] CHAPS, 0.15 % [w/v] DTT, 0.5 % [v/v]
carrier ampholytes 3–10, Complete Mini protease inhibitor cocktail (Roche)) to a final volume of 400 μl. In-gel
rehydration with IPG strips (pH 4–7, 18 cm, GE Healthcare) was performed overnight. Isoelectric focusing (IEF)
was carried out with the Multiphor II system (GE
Healthcare) under paraffin oil for 55 kVh. SDS-PAGE
was performed overnight in polyacrylamide gels
(12.5 % T, 2.6 % C) with the Ettan DALT II system (GE
Healthcare) at 1–2 W per gel and 12 °C. The gels were
silver stained as described by Ohlmeier et al. [26] and
analyzed with the 2-D PAGE image analysis software
Melanie 3.0 (GeneBio).
Zymography
Gelatinolytic enzymes in Myogel and Matrigel® (BD
Matrigel Matrix, BD Biosciences, Cat. Number 354234)
were detected by a zymography method using fluorescently labeled gelatin [27]. Prestained low-range SDSPAGE Standards (Bio-Rad) as well as purified control
MMP-2 and MMP −9 samples were loaded in adjacent
wells. After electrophoresis, gelatinases were activated by
incubating the gels with zymography buffer (50 mM
Tris–HCl, 5 mM CaCl2, 1 μM ZnCl2, 0.02 % NaN3,
pH 7.5) overnight at 37 °C. Gelatin degradation was visualized under long wave UV light and photographed
using an AlphaDigiDoc® RT Gel Documentation System
(Alpha Innotech, San Leandro, CA).
Assessing the pH of Myogel and Matrigel®
For pH comparison, Matrigel® (BD Matrigel Matrix, BD
Biosciences, Cat. Number 354234) was diluted with an
equal volume of serum free medium, and the same
Salo et al. BMC Cancer (2015) 15:981
amount of total protein for Myogel was obtained by diluting it 10 + 6 with serum free medium. The pH of both
gels was measured at the beginning, after 17 h, and at
the end of the 48 h experiment. The gels were incubated
at 37 °C in a 5 % CO2 humidified cell culture chamber
with or without HSC-3 cells (see below for the culture
conditions) on top of the gels.
Cell lines
Aggressive human oral tongue squamous cell carcinoma
cell line HSC-3 (Japan Health Sciences Foundation,
Japan) was cultured in a 1:1 DMEM/F-12 medium (Life
Technologies) supplemented with 100 U/ml penicillin,
100 μg/ml streptomycin, 250 ng/ml fungizone, 50 μg/ml
ascorbic acid and 0.4 μg/ml hydrocortisone (all from
Sigma-Aldrich) and 10 % heat inactivated fetal bovine
serum (FBS; Life technologies). HSC-3 cells labeled with
GFP were generated by stable transduction with nonsilencing GIPZ lentiviral shRNAmir control particles
(pGIPZ vector contains GFP in order to track shRNAmir
expression; Thermo Fischer Open Biosystems) with
puromycin (Sigma-Aldrich) selection according to the
manufacturer’s instructions. Nuclear histone-2B (H2B)coupled mCherry expression vector pLenti6.2 V5/DEST
(a gift from Dr. Cindy E. Dieteren, Department of Cell
Biology, Radboud UMC, Netherlands) was introduced
into the HSC-3 cells with cytosolic GFP labeling using
lentivirus mediated infection and selected in culture
media containing 5 μg/ml blasticidin-S (Merck Millipore). HSC-3 cells expressing cytoplasmic GFP and
H2B-coupled mCherry were cultured in DMEM/F12
medium (Gibco/Thermo Fisher Scientific), 10 % heatinactivated FBS (HyClone/Thermo Fisher Scientific), 100
U/ml penicillin, 100 μg/ml streptomycin (Sigma-Aldrich), 50 μg/ml L-ascorbic acid (Sigma-Aldrich) and
2 mM L-glutamine (Sigma-Aldrich). HSC-3 cells labeled
with RFP were generated by stable transduction with
commercial lentiviral particles containing a non-coding
control sequence (Amsbio) and selected with puromycin.
They were cultured as normal HSC-3 cells.
The SCC-9 cell line (American Type Culture Collection, ATCC) was maintained in DMEM/F-12 medium
(Invitrogen) supplemented with 10 % FBS (Cultilab),
400 ng/mL hydrocortisone, and antibiotic/antimycotic
solution (Invitrogen). SCC-9 cells were labeled with
ZsGreen protein and implanted subcutaneously into the
footpads of the left front limb of BALB/c nude mice and
LN-1 and LN-2 cell lines with increased metastatic potential were derived by in vivo selection from axillary
lymph nodes with metastatic cells as described earlier
[6]. Both LN-1 and LN-2 cells were maintained in culture as SCC-9.
Normal oral gingival fibroblasts (GF) were established
from palatal gingiva mucosa biopsies and cultured in
Page 4 of 16
DMEM medium (high glucose, GlutaMAXTM and pyruvate) supplemented with 10 % FBS, 50 U/ml penicillin,
50 μg/ml streptomycin and 2.5 μg/ml amphotericin B
(all from Gibco). After obtaining written informed consent, the palatal tissue biopsies were taken from healthy
volunteers for another study to be used as a starting material for control fibroblast cell line cultures. The volunteer consent encompassed the use of obtained cell lines
for other studies as well. The use of palatal tissue was
approved by the Ethics Committee of the Helsinki University Hospital. The carcinoma associated fibroblast
(CAF) cell lines were generated from fragments of
tongue squamous cell carcinomas by using tissue explants [28]. They were cultured in DMEM medium supplemented with 100 U/ml penicillin, 100 μg/ml
streptomycin, 50 μg/ml ascorbic acid, 250 ng/ml fungizone, 1 mmol/L sodium pyruvate (Sigma-Aldrich) and
10 % heat inactivated FBS.
Melanoma cell lines SK-Mel-25 and A2058 (ATCC)
were maintained in RPMI medium (Invitrogen) supplemented with 10 % FBS (Cultilab) as described earlier
[29]. Human umbilical vein endothelial cells (HUVEC,
ATCC) were cultured in a 1:1 mixture of DMEM/F12
medium (Invitrogen) supplemented with 10 % FBS and
400 ng/ml hydrocortisone (Sigma-Aldrich).
All the cells were cultured in a humidified atmosphere
of 5 % CO2 at 37 °C and passaged routinely using
trypsin-EDTA (Sigma-Aldrich). The media were changed
every 2–3 days. They were regularly tested and confirmed to be negative for mycoplasma infection using a
MycoTrace PCR Detection Kit (PAA Laboratories
GmbH). Cell line identity was not routinely performed.
Adhesion assay
A cell adhesion assay was conducted to determine how
many cells bind to Myogel compared to Matrigel® (BD
Matrigel Matrix, BD Biosciences, Cat. Number 354234).
In this assay, HSC-3 cells were cultured to subconfluence. Wells in a 96-well plate were coated for 24 h either
with 100 μl of PBS, BSA (bovine serum albumin, 10 μg/
ml, Sigma-Aldrich), Matrigel® or Myogel (two different
batches). Matrigel® was diluted to 1:10 in PBS and Myogel was diluted to the same protein concentration. At
the same time, the cell culture medium was changed to
serum-free medium. The next day the excess liquids
were removed and the culture plates were incubated
with 100 μl/well of 0.1 % BSA for 2 h and washed with
PBS. HSC-3 cells (6000) in 100 μl of serum-free medium
were added to each well and the wells were incubated at
37 °C in a 5 % CO2 humidified atmosphere for 2 h. The
non-adherent cells were rinsed off, and the remaining
cells were fixed with 10 % trichloroacetic acid (TCA),
stained with crystal violet and quantified using an ELISA
reader at 540 nm.
Salo et al. BMC Cancer (2015) 15:981
Adhesion of GFs on top of Myogel was studied using
6-well plates coated at 37 °C in a 5 % CO2 humidified atmosphere with 0.62 mg/ml Myogel diluted with DMEM
without supplements. After 2 h the excess liquids were
removed and 150,000 GFs were added in their normal
culture medium. The cultures were photographed with
Olympus CKX41 inverted microscope (Hamburg,
Germany) after 2.5 h and 9.5 h with 20 x magnification
to record the morphology of the cells.
Terminal deoxynucleotidyl transferase dUTP nick end
labeling (TUNEL)
An 8-well Nunc™ Lab-Tek™ chambered slide (Thermo
Scientific) was used for the experiment. Wells were
coated with either rat tail collagen type I (BD Biosciences), one of three different batches of Myogel or an
equal (50–50 %) mixture of collagen type I and one type
of Myogel. A total of 150 μl of coating mixtures were
prepared, with a final protein concentration of each gel
mixture of 0.62 mg/ml, which were adjusted with the
addition of culture medium. Two wells were not coated
and were kept for positive and negative controls for the
subsequent TUNEL assay. The Lab-Tek™ chamber slide
was covered and placed in the incubator at 37 °C in 5 %
CO2 for 2 h. Subsequently, 5000 CAFs were added on
each well and the slide placed in the incubator overnight. On the following day, the wells were washed twice
with PBS, air-dried for few min and fixed with freshly
prepared 4 % (w/v) paraformaldehyde in PBS (pH 7.4)
for 1 hour at RT. After 2 washes with PBS for 5 min
each, the TUNEL assay was performed using a commercially available kit (In Situ Cell Death Detection Kit,
Roche). At the end of the assay, samples were counterstained with DAPI for 10 min at RT. After coverslip
mounting with a water-based medium, 100 cells were
counted under a confocal microscope using the blue
channel (405 nm) and apoptosis was detected using the
green channel (488 nm).
CAFs cultured within Myogel
Myogel for CAF embedding was prepared as described
for collagen organotypic culture in Nurmenniemi et al.
[1]. Briefly, a gel mixture including 8 volumes of Myogel
(4.5 mg/ml), 1 volume of 10 × DMEM (Sigma Aldrich)
and 1 volume of FBS with CAFs (final concentration
70,000 cells/ 400 μl gel mixture) was prepared on ice
and 400 μl aliquots were added into the wells of a 48well plate. After 40 min polymerization, 100 μl of
complete CAF medium was added on the polymerized
gels. The plates were incubated at 37 °C in a 5 %
CO2 humidified atmosphere. Cells were fed twice a
week and pictures were taken with an EVOS microscope (Advanced Microscopy Group, Bothell, WA)
weekly for three weeks.
Page 5 of 16
Myogel as a supplement in soft agar colony formation
-assay
For the soft agar mimicking assay, one percent sterile
base low melting agarose (LMA, Sea Plaque Low Melting Agarose, Lonza) melted in PBS was mixed with 10x
DMEM/F12, 100 % FBS to give a final 0.8 % agarose
with 1x medium, 10 % FBS. One-half ml of the mixture
was added to wells in a 24-well plate and allowed to solidify for at least 30 min in the laminar flow hood. 10
000 HSC-3 (H2B-GFP) cells in 50 μl of FBS were mixed
with 50 μl of 10x DMEM/F12 and 0.4 ml of 0.5 % LMA
(as a control, final agarose concentration 0.4 %) or with
50 μl of 10x DMEM/F12, 0.2 ml of 1.0 % agarose and
0.2 ml Myogel (final agarose concentration 0.4 %, final
Myogel protein concentration 2.2 mg/ml). Myogel was
centrifuged at 4000 rpm for 10 min prior to the procedure. The agarose mixture was gently mixed by swirling,
and 0.5 ml was added on the top of the agarose base.
The plates were incubated at 37 °C in a 5 % CO2 humidified atmosphere for 28 days. The cells were fed
twice a week with 0.25 ml normal HSC-3 medium. Pictures of the colonies were taken with transmitted light
and in the GFP & RFP channels in different objectives
(10x, 20x & 40x) using an EVOS inverted microscope.
Cells in colonies were calculated from the pictures and
ImageJ software (Rasband, W.S., ImageJ, U.S. National
Institutes of Health, Bethesda, Maryland, USA) was used
to measure colony area.
Hanging drop spheroid cultures in Myogel with low-melting
agarose (Myogel-LMA)
The spheroids were formed according to published
protocol [30]. 20 μl drops of the cell suspension (70,000
HSC-3 (H2B-GFP) cells per drop) suspended in DMEM/
F12 medium with 10 % FBS were placed onto the lids of
10 cm dishes, which were inverted over dishes containing 10 ml PBS. Hanging drop cultures were incubated
for 72 h, the resulting cellular aggregates were harvested
by a pipette and embedded in two different conditions
(Myogel-LMA & LMA) into the wells of a 48- well plate
as in the soft agar colony formation –assay above. After
6 h incubation at 37 °C in a 5 % CO2 humidified atmosphere, 150 μl normal HSC-3 cell culture medium was
added per well. Pictures of the spheroids were taken
with 4x objective using an EVOS inverted microscope at
0 h, 24 h, 48 h and 72 h after embedding. ImageJ software was used to measure spheroid area and the results
were calculated as a ratio to the area of the implanted
spheroid right after embedding (without media).
Microarray
For microarray analysis, 90,000 HSC-3 cells transduced
with RFP were seeded into uncoated or Myogel coated
6-well plates (three wells each). The next day the cells
Salo et al. BMC Cancer (2015) 15:981
were harvested for RNA extraction using a Qiagen RNA
kit. Three samples of each group (on top of plastic or
Myogel coating) were pooled; the pools contained an
equal amount of RNA from each sample. Affymetrix
GeneChip Human Genome U133 Plus 2.0 Arrays were
used for microarray analysis and experimental procedures were performed according to the Affymetrix GeneChip Expression Analysis Technical Manual. Briefly,
1 μg of total RNA was used as a template to synthesize
biotinylated cRNA by means of the GeneChip 3’IVT Express kit (Affymetrix) according to the manufacturer’s
instructions. The cRNA was fragmented to 35 to 200 nt
prior to hybridization to Affymetrix Human Genome
U133 Plus 2.0 arrays containing approximately 55,000
human transcripts. The array was washed and stained
with streptavidin-phycoerythrin (Molecular Probes). Finally, biotinylated anti-streptavidin (Vector Laboratories
Inc.) was used to amplify the staining signal and a second
staining was performed with streptavidin-phycoerythrin.
The arrays were scanned on a GeneChip Scanner 3000
(Affymetrix, High Wycombe, United Kingdom). The expression data was analyzed to find genes with fold changes
(FC) of 1.5 or more using dChip software [31]. The genes
with FC 1.2 or more were divided into Gene Ontology
(GO) categories using a dChip enrichment analysis tool.
Scratch assay
To analyze the effects of Myogel and Matrigel® (BD
Matrigel Matrix, BD Biosciences, Cat. Number 354234)
on the migration of HSC-3 cells, 24-well plates were
coated with 0.62 mg/ml Myogel (two different batches)
or 0.62 mg/ml Matrigel®. The coating was left to solidify
for 2 h at 37 °C and then washed twice with PBS. HSC-3
cells (90,000) were allowed to attach overnight, and were
then wounded with a pipette tip, rinsed twice with PBS,
post-coated for 1 h and rinsed before 1 % FBS medium
was added. The wounds were photographed with an
EVOS photomicroscope at 0 h and 24 h after scratching.
The area of the open wound was measured using ImageJ
software and the results were calculated as a percentage
of wound closure.
Transwell invasion through Myogel and Matrigel®
To compare Myogel with Matrigel® (BD Matrigel Matrix,
BD Biosciences, Cat. Number 354234) as an invasion
assay material, experiments were carried out according
to BD Biosciences instructions to coat the filter membranes with Matrigel®. Fifty μl of either 1 + 1 Matrigel® or
Myogel (three different batches at the same protein concentration as Matrigel®) diluted with serum-free medium
was added to the upper chamber of a Transwell® nylon
filter membrane insert (Corning Inc.), incubated at 37 °C
in a 5 % CO2 humidified atmosphere for 30 min after
which 50,000 HSC-3 cells suspended in 100 μl of serum-
Page 6 of 16
free medium were seeded onto the upper compartment
of the Transwell® chambers. The Transwell® inserts were
incubated for 12 h - 48 h at 37 °C in a 5 % CO2 humidified atmosphere, after which the cells were fixed in 10 %
TCA for 15 min, rinsed and air-dried overnight. Once
dry the membranes were stained with crystal violet for
20 min and the excess stain was removed by water rinsing. The non-invasive cells from the upper side of the
membrane were removed by carefully sweeping with a
cotton swab. Next, the membranes were removed from
the inserts and placed on microscope slides and the
number of invaded cells through Myogel and Matrigel®
were counted. We also tested different mixtures of Myogel and Matrigel® (2 + 1, 1 + 1 and 1 + 2) in the invasion
assay.
Transwell invasion with Myogel solidified with low-melting
agarose (Myogel-LMA)
We used a final concentration of 0.2 % LMA in 3.2 mg/
ml final Myogel protein concentration to solidify Myogel
for invasion assays. To compare the results with Matrigel® we diluted Matrigel® (BD Matrigel Matrix, BD Biosciences, Cat. Number 354234) 1 + 4 (final protein
concentration 2.0 mg/ml) with the same concentration
of 0.2 % agarose. Serum-free cell culture medium was
used for gel dilution. Fifty μl of the Myogel-LMA or
Matrigel agarose mixtures was added on the upper
chamber of Transwell® nylon filter membrane insert, incubated ½ h at RT and thereafter at 37 °C in a 5 % CO2
humidified atmosphere until the cells were ready to be
seeded on the top of the gel. HSC-3 cells were trypsinized and counted, trypsin inhibitor instead of serumcontaining medium was used to inactivate trypsin. Five
hundred μl of 10 % serum containing medium was
added into the lower chamber of the Transwell®, and
50,000 HSC-3 cells suspended into 100 μl of medium
containing 0.5 % lactalbumin instead of serum were
seeded into the upper compartment of the Transwell®
chamber. The cells were allowed to invade for one to
three days and the invasion was quantified by staining
the cells with crystal violet followed by counting the cell
number. In this preliminary assay we tested different
agaroses and concentrations with only one Transwell® in
each condition and 0.2 % LMA was chosen for further
experiments. In another set of experiments, the invasion
of SCC-9, LN-1, LN-2, HSC-3, SK-Mel-25 and A2058
cells was studied in Myogel-LMA and growth factorreduced Matrigel® (Matrigel®-GFR, BD Matrigel Matrix,
BD Biosciences, Cat. Number 35430) diluted 1 + 1 with
serum free medium without LMA. The invasion was
quantified with Toluidine Blue -staining. Briefly, after
72 h invasion, the cells were fixed with 4 % formaldehyde for 1 h at RT, washed once with PBS, stained for
5–10 min in Toluidine blue solution (filtered 1 %
Salo et al. BMC Cancer (2015) 15:981
Toluidine Blue + 1 % disodium tetraborate in ddH20) at
RT, excess dye was rinsed out with deionized water, excess gel and the cells from the upper side of the membrane were removed by using cotton swabs and when
necessary, excess dye was further removed from the inside and outside of the Transwell® inserts with cotton
swabs immersed in a solution of 1:1 water/ethanol. The
dye was eluted by dipping the Transwell® inserts in a solution of 1 % SDS (500 μl). The 1 % SDS solution containing the eluted dye was transferred into a 96-well
plate and the absorbance was measured at 650 nm.
Hanging drop method
The gel from rat tail type I collagen (BD Biosciences)
was prepared according to the manufacturer’s protocol,
with a final collagen concentration of 1.7 mg/ml. The
collagen/Matrigel® (BD Matrigel Matrix, BD Biosciences,
Cat. Number 354234) (1.5 mg/ml, 1.5 mg/ml) and collagen/Myogel (1.5 mg/ml, 4.3 mg/ml) mixtures were prepared similarly. The hanging drop technique was used to
observe the cell movement under 3D culture conditions.
HSC-3 (H2B-GFP) cells were washed with PBS, trypsinized and 70,000 cells in 10 μl of DMEM/F12 medium
(2 % FBS) were mixed with 50 μl of the matrix mixture.
Twenty μl of the cell suspension in each matrix was
dropped on the 4 compartment plate. The plate was
inverted after 5 min incubation in culturing conditions
and incubated for 3 h in a humidified chamber in culturing conditions. Mimosine (200 μM) was added to the
medium to synchronize the cell cycle. Images were taken
with a Zeiss Axio Observer.Z1 with a EC Plan-Neofluar
40x/0.75 M27 objective (Göttingen, Germany). Images
consisting of 1024 x 1024 pixels were taken every
15 min with a 19.8 μm Z-stack volume (0.6 μm thickness) with a Hamamatsu Camera#2 controlled by Zeiss
Zen Blue software (Zeiss) for 20 h. This assay was performed twice. The analysis of the cells in hanging drops
is described in the Additional file 2: Supplementary
Method.
In vitro capillary tube formation assay
96-well culture plates were coated with Myogel with 2 %
low melting agarose (Myogel-LMA), Matrigel®-GFR (BD
Matrigel Matrix, BD Biosciences, Cat. Number 35430)
or ECMatrix™ (ECMatrix – In vitro Angiogenesis Assay
Kit, Cat. Number ECM625 – Millipore) previously
thawed overnight on ice, in a total volume of 50 μl/well
and allowed to solidify overnight at 37 °C. HUVEC cells
were trypsinized, neutralized with DMEM/F12 with
10 % FBS, washed once with PBS and resuspended in
DMEM/F12 at a density of 450,000/ml. Hundred μl of
this cell suspension was added into each well and the
plates were incubated at 37 °C for 12 h. Tube formation
was observed under an inverted microscope (4x, Eclipse
Page 7 of 16
Ti-S, Nikon, Tokyo, Japan) and photos were taken and
analyzed using the Motic Images Plus 2.0 software
(Motic). Tubule perimeters were assessed by drawing a
line around each tubule and measuring the length of the
line.
Statistical analysis
SPSS for Windows software version 21.0 (IBM) or
GraphPad Prism 6 (GraphPad Software) were used for
statistical analyses. To establish the statistical significance of differences between the two independent cell
culture groups, a Mann–Whitney U test was used to
compare the groups.
Results
The protein composition in different Myogel batches is
reproducible and differs from that of Matrigel®
The reproducibility of different Myogel batches was first
investigated in Coomassie Blue stained gradient SDSPAGE gels. Different Myogel preparations and the mixture of various batches showed relatively similar protein
band patterns, and only slight variations in the intensities of differently sized bands were visible (Additional
file 1: Figures S1A and S1B). Two Myogel batches were
further investigated by 2-DE (Additional file 1: Figure
S1C) which confirmed with almost similar protein patterns in the silver stained 2D gels likewise the high reproducibility of these Myogel preparations.
After that the protein compositions of Myogel and
Matrigel® were compared. In SDS-PAGE gels more bands
were detected for the Myogel (Additional file 1: Figure
S1A) and also the 2-DE separation showed a significant
difference between Matrigel® and Myogel samples
(Additional file 1: Figure S1C). If similar protein
amounts (100 μg) were separated significantly more
spots were visible for the Myogel. Increasing the
amount of Matrigel® proteins to 300 μg resulted in a significantly higher number of detectable spots (Additional
file 1: Figure S1C). This suggests that a few high abundant
proteins, including these visible in the gel with lower protein amount, comprise a major part of the Matrigel® proteome. However, also with higher protein amounts
Matrigel® and Myogel showed still distinct protein patterns. The here shown differences between Myogel and
Matrigel® proteomes are possibly due to the difference between species (human vs. mouse) and the nature of the
starting material (leiomyoma vs. sarcoma).
We next analyzed the protein content of Myogel and
compared it with the published data of Matrigel® [32–
34]. For further proteomic analyses, different Myogel
batches were separated by gradient SDS-PAGE (Additional file 1: Figure S1B). Since Myogel batch number 4
differed most from the other three batches (3, 6 and 9;
Additional file 1: Figure S1B), it was omitted from the
Salo et al. BMC Cancer (2015) 15:981
Page 8 of 16
mass spectrometry analysis. Two of the Myogel samples
gave successful results, and altogether 765 proteins were
identified (Additional file 3: Table S1). Among the Myogel proteins, 34 % (259 proteins) were the same as in
Matrigel® where 1030 proteins were identified. Based on
the comparison, for instance laminin, type IV collagen,
heparan sulfate proteoglycans, nidogen and epidermal
growth factor were found in both. Based on mass spectrometry analysis, Myogel lacked enactin, which is present
in Matrigel®, but contained for example tenascin-C, collagen types XII and XIV, etc., which were lacking in Matrigel®. Based on zymography, Myogel contained both latent
and active forms of MMP-2, whereas in Matrigel® latent
and active forms of both MMP-2 and MMP-9 were present
(Additional file 1: Figure S1D) as shown earlier [35].
The pH of Myogel is neutral and more stable than the pH
of Matrigel®
The pH of Myogel was initially between 7.0 and 7.5, and
after 48 h incubation the pH remained the same. The
pH of Myogel with HSC-3 cells on top dropped slightly
from 7.0–7.5 to 6.5–7.0 (0 h and 48 h incubations, respectively), whereas in Matrigel® the pH at the same
time dropped from a slightly alkali 8.0–8.5 to as low as
6.0–6.5 (Additional file 4: Table S2). This indicates that
Myogel samples are closer than Matrigel® to neutral pH,
and Myogel keeps the pH more stable than Matrigel®
during cancer cells culture experiments.
Fibroblasts adhere to Myogel, but only HSC-3 cells form
colonies within Myogel- soft agar assay
HSC-3 cells adhered significantly more to plates precoated with Myogel than with BSA, or to the plain plates
kept in PBS. However, they adhered even more readily
to Matrigel® coated plates (Fig. 1). The adhesion experiment with gingival fibroblasts (GFs) showed that after
9.5 h incubation on top of Myogel, fibroblasts were well
spread and vital (not shown). Based on the TUNEL
assay, from 98 to 99 % of the carcinoma associated fibroblasts (CAFs) seeded on top of various batches of Myogel were alive even after 24 h incubation (not shown).
However, when CAFs were embedded within the Myogel
matrix, almost all died within 21 days (not shown). In
contrast, embedded HSC-3 cells stayed alive up to
28 days, divided and formed colonies within Myogel
combined with LMA. The results with HSC-3 cells were
relatively similar using either Myogel-LMA or a conventional soft agarose (LMA) assay (Figs. 2a and b); in
Myogel-LMA the colony number was six percent less
than in LMA (47 vs. 50). However, more colonies with
the lowest cell number were present in LMA, while the
highest cell number/colony was present in Myogel-LMA
(Fig. 2b). According to nuclear RFP expression, 92 % of
the cancer cells were alive in Myogel-LMA colonies,
Fig. 1 Adhesion of HSC-3 cells to Myogel and Matrigel®. HSC-3 cells
were left to adhere to wells for 2 h. Wells coated with BSA and plain
wells kept in PBS served as controls for adhesion. The adherent cells
were fixed with 10 % trichloroacetic acid (TCA), stained with crystal
violet and quantified using an ELISA reader at 540 nm. The number
of wells was altogether 54 in PBS and BSA, 101 in Myogel and 53 in
Matrigel® in three independent experiments. Horizontal lines indicate
mean values, Mann–Whitney U test, *** P < 0.001
whereas 85 % were alive in LMA colonies. The total
average area of cell colonies in Myogel-LMA was three
percent less than in LMA (not shown). In HSC-3 cell
hanging drop spheroid cultures the area of the spheroids
embedded in Myogel-LMA grew more in 24 h incubation than in spheroids embedded in plain LMA (Fig. 2c
and d). The area growing rate remained higher in
Myogel-LMA even during 72 h follow-up and the spheroid area enlarged more in ratio to 0 h area in MyogelLMA cultures than in plain LMA cultures (Fig. 2c and
d). On average the increase at 72 h in ratio to 0 h was
21.5 % in Myogel-LMA and 12.2 % in plain LMA. In the
most enlarged spheroids, the area increase at 72 h in
Myoogel-LMA was 63.5 % while in plain LMA it was
only 43.1 % (Fig. 2d). In less enlarged spheroids, the
average increase at 72 h in Myogel-LMA was 11.0 %
whereas in plain LMA it was 4.5 % compared to 0 h
spheroids (Fig. 2d).
Gene expression is changed when HSC-3 cells are cultured
on top of Myogel compared to the same cells cultured on
plastic
Gene expression assays are usually done with cells
grown on tissue culture plastic. We wanted to see if
Myogel coating has an effect on expressed genes in
Salo et al. BMC Cancer (2015) 15:981
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Fig. 2 Colony formation and hanging drop spheroid culture of HSC-3 cells within Myogel combined with LMA and plain LMA. Colonies were
photographed with 10x and 40x objectives (a). Cell numbers per colony were counted from the photographs (b). Spheroids were photographed
with 4x objective (c). Spheroid area in ratio to 0 h spheroid area was calculated after 24 h, 48 h and 72 h incubations (d). Altogether 47 colonies
were calculated from four Myogel-LMA and 50 colonies from four LMA wells in two independent experiments. Enlargement of altogether five
spheroids in both Myogel-LMA and LMA was followed in two independent experiments
HSC-3 cells compared to cells grown on plain plastic.
Approximately 1.4 % of the genes (totally 751) were differentially expressed (FC 1.5 or more) when the HSC-3
cells cultured on the top of plastic were compared to the
corresponding cells grown on Myogel (raw data presented
in Additional file 5: Table S3 showing 807 probes). When
the genes were divided into groups according to their biological function we found that the Myogel coating affected
those genes that are related to intracellular organelle and
cytoskeleton organization and biogenesis. A significant
change in pathway analysis was found only in the G13 signaling pathway that is related to actin polymerization and
reorganization (www.genmapp.org).
The vertical migration of HSC-3 cells is faster on Myogel
than on Matrigel® coated wells
In the scratch assay, HSC-3 cells migrated significantly
more in Myogel coated wells than in wells coated with
Matrigel® (Fig. 3a and b). However, in uncoated wells their
migration was faster than in coated wells (Fig. 3a and b).
HSC-3 cells invade more efficiently through Myogel than
Matrigel®
In order to test the use of Myogel on cancer invasion
Transwell® -assays, we first compared the invasion of the
most aggressive oral tongue carcinoma cell line (HCS-3)
using Myogel and Matrigel®. The HSC-3 cells invaded
significantly more efficiently through Myogel than
Matrigel® (Fig. 4a). Invasion varied slightly in different
Myogel batches, but HSC-3 cells invaded in all Myogel
samples more than in Matrigel® (Fig. 4b). When Myogel
and Matrigel® were mixed, HSC-3 cells invaded more efficiently when the mixture contained more Myogel, and
less when the portion of Matrigel® increased (Fig. 4c).
The invasion pattern of HSC-3 cells was different in
Myogel and Matrigel®. The cells invaded more evenly
throughout the whole Transwell® membrane in Myogel
Salo et al. BMC Cancer (2015) 15:981
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Fig. 3 Migration of HSC-3 cells on Myogel and Matrigel®. HSC-3 cells migrated for 24 h in the scratch assay on Myogel and Matrigel® coated wells
(a). Dash line in (b) represents the edges of the wounds. Altogether twelve wounds without coating (PBS) and twenty with Myogel or Matrigel®
coating were measured in two independent experiments. Horizontal lines indicate mean values, Mann–Whitney U test, n.s. not significant,
** P < 0.01, *** P < 0.001
Fig. 4 Invasion of HSC-3 cells through Myogel and Matrigel®. HSC-3 cells were allowed to invade for 12 – 48 h through Myogel and Matrigel® (a).
Three different Myogel batches were compared to Matrigel® in HSC-3 cells invasion (b). Invasion of HSC-3 cells in different mixtures of Myogel
and Matrigel® (c). Invasion pattern of HSC-3 cells through Myogel and Matrigel® (d). In a and b the number of Transwell®s was three in every
group, in c Transwell® number was six in the control group and three in the other groups, cells from six areas of each Transwell® were calculated.
Each invasion assay (a, b and c) was performed as an independent experiment. Controls were Transwell®s without coating. Horizontal lines
indicate mean values, Mann–Whitney U test, n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001
Salo et al. BMC Cancer (2015) 15:981
coated filters, whereas in Matrigel® experiments they invaded more in clumps (Fig. 4d).
Myogel solidified with agarose (Myogel-LMA) is a more
feasible matrix for invasion assays
Since Myogel batches vary slightly in their protein content,
as seen in the gradient gel in Additional file 1: Figures S1A
and S1B, we created a more homogenous, reliable and easier to handle matrix by adding low-melting agarose into
the Myogel mixture. We noticed that HSC-3 cells did not
invade through a plain agarose coated membrane, but they
Page 11 of 16
invaded through a Myogel-agarose (Myogel-LMA) mixture (Fig. 5a and b). Interestingly, the passage number of
the HSC-3 cells seemed to affect the invasion through
Myogel- and Matrigel®-mixtures; cells with higher passages invaded slightly less through Matrigel® mixtures than
through Myogel-containing mixtures, whereas cells with a
low passage number invaded slightly less through MyogelLMA than through Matrigel®-LMA (Fig. 5a). The invasion
pattern of HSC-3 cells through agarose-containing mixtures was similar to that observed in plain Myogel or
Matrigel®; in Myogel-LMA mixtures cells invaded more
Fig. 5 Invasion of oral squamous cell carcinoma and melanoma cells through Myogel-LMA and Matrigel® or Matrigel®-GFR. HSC-3 cells were allowed
to invade for three days through Myogel and Matrigel® mixed with agarose, 09-HSC-3 cells have a higher passage number than 13-HSC-3 cells
(a). Invasion pattern of HSC-3 cells through different mixtures of Myogel, Matrigel® and LMA (b). Oral squamous cell carcinoma and melanoma
cells were allowed to invade for 72 h through Myogel-LMA and through Matrigel®-GFR (c). In a one Transwell® was analyzed for each group
except for control at 72 h there were two filters (controls were Transwell®s without coating), six areas per filter were calculated, in c altogether six
Transwell®s were analyzed in each group in two independent experiments. As only one filter was analyzed in preliminary experiment with LMA in
a, statistical significances were not calculated in that experiment. In c differences were not statistically compared to plain LMA. Horizontal lines
indicate mean values, Mann–Whitney U test, ** P < 0.01
Salo et al. BMC Cancer (2015) 15:981
evenly throughout the whole Transwell® membrane than
in Matrigel®-LMA mixtures (Fig. 5b).
In another experiment, Myogel-LMA was compared
with Matrigel®-GFR. In general, all the oral squamous
cell carcinoma cell lines used for these invasion assays
(HSC-3, LN-1, LN-2 and SCC-9) preferred MyogelLMA to Matrigel®-GFR during the invasion (Fig. 5c).
HSC-3 and LN-2 (in this order) seemed to have a higher
potential to invade in both Myogel-LMA and Matrigel®GFR, compared to the LN-1 and SSC-9 cell lines. The
cells invading the Myogel-LMA seemed to keep more of
the morphological characteristics observed in monolayer
cultures. In contrast, the cells grouped together as they invaded the Matrigel®-GFR matrix, and lost the “fusiformstarshape” characteristic of oral carcinoma cell lines (not
shown). In addition to oral carcinoma cell lines, we tested
Myogel-LMA on SK-Mel and A2058 melanoma cell lines
and found that also they invaded Myogel-LMA more efficiently than Matrigel®-GFR (Fig. 5c).
HSC-3 cells move faster in Myogel-collagen matrix than in
Matrigel®-collagen matrix in the 3D hanging-drop
cultures
To observe the cell behavior in 3D culture in different
matrices, we have established an optimized hanging drop
method modified from the previously reported protocol
[36]. HSC-3 cells moved in a relatively similar fashion in
pure collagen, Myogel-collagen and Matrigel®-collagen
matrices (Additional file 6: Movie S1, Additional file 7:
Movie S2 and Additional file 8: Movie S3). However, interestingly the speed of the cells was highest in the Myogelcollagen matrix and lowest in the Matrigel®-collagen matrix
(Additional file 1: Figure S2, Additional file 6: Movie S1,
Additional file 7: Movie S2 and Additional file 8: Movie S3).
Myogel efficiently induces endothelial cell tube formation
In vitro angiogenesis assays are commonly used to assess
pro- or anti-angiogenic drug properties [37]. Here, tube
formation could be quantified in all matrix-assays tested
(Fig. 6a). With Myogel-LMA, tube formation was visible
after 12 h, and even after 72 h the endothelial cells were
alive (Fig. 6a), unlike in Matrigel®-GFR or ECMatrix™
(not shown), where most of the HUVEC cells were
already apoptotic after 24 h. We found that the number
of tubules formed was three times higher in MyogelLMA compared to ECMatrix™ (Fig. 6b). Otherwise,
measuring the diameters of the capillaries, the tubule parameters in Matrigel®-GFR, and especially in ECMatrix™
assays, were significantly higher than in tubes formed in
Myogel-LMA (Fig. 6c).
Discussion
There has been a lack of a gelatinous soluble human
tumor microenvironment matrix (TMEM) for human
Page 12 of 16
cell line experiments in vitro. Today, the most widely
used TMEM gelatinous material is mouse sarcomaderived Matrigel® and other commercial and modified
derivatives of mouse EHS tumors. Here, we described
the preparation and use of a novel TMEM matrix, Myogel, which is derived from human uterus leiomyoma tissue. We have previously reported the use of leiomyoma
tumor solid discs in several invasion studies [1–8]. Now
we demonstrate that both Myogel and its combination
with low melting agarose, Myogel-LMA, provide practical in vitro gelatinous TMEM for testing cancer cells
invasive, adhesive or migratory properties, that is similar
to Matrigel®, Matrigel®-GFR or ECMatrix™. The benefit
of Myogel over various EHS tumor derived products is
that the protein composition of human Myogel differs
significantly from the mouse sarcoma matrix as seen by
gradient SDS-PAGE gels, 2-DE, zymography and proteomics analyses. Moreover, the pH is more stable in Myogel than in Matrigel® during cell culture. In addition,
human uterus myomas, which are by-products of surgical operations, are an ethically superior starting material
compared to EHS tumors in mice, which are grown
purely for preparing TMEM for research purposes. Myogel and in particular Myogel-LMA, is easy to prepare
and handle. Finally, and most importantly, in all assays
we have tested thus far, the results have been reproducible
and similar or even superior to Matrigel®. Namely, the
various cancer cells analyzed invaded through Myogel or
Myogel-LMA generally more efficiently than through
Matrigel® or Matrigel®-GFR. Embedded HSC-3 cells tended
to form larger colonies and spheroids in soft agar colony
formation -assay and in hanging drop spheroid cultures,
respectively, within Myogel-LMA than within plain LMA.
HUVEC cells in Myogel-LMA formed more and smaller
tubules which lasted longer than in Matrigel-GFR® or in
ECMatrix. In addition, we demonstrated in hanging drop
assays that carcinoma cells were able to move more rapidly
in a Myogel-collagen matrix than in collagen, or in Matrigel®-collagen combinations.
Uterine leiomyomas are benign smooth muscle tumors
affecting the health of millions of females. The tumors
have at least four molecular subclasses and contain
chromothripsis, which has previously been associated
with aggressive cancer [38, 39]. As a patient derived material, there is heterogeneity between myomas. The operating surgeon omits peculiar ones. The prepared batch is
always tested in an invasion assay with our reference
HSC-3 cells. To ensure the reproducibility between the
Myogel batches, we combine several myomas for each
batch. As a solid tumor mass, easy to obtain and handle,
myoma tissue is an ideal matrix material for several experimental cancer study applications in vitro. At present,
all the other human ECM matrices to investigate the
properties of cancer cell lines are derived either from
Salo et al. BMC Cancer (2015) 15:981
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Fig. 6 In vitro capillary tube formation assay. Photomicrographs showing the typical appearance of tubules formed by HUVECs in the Myogel-LMA,
Matrigel®-GFR and ECMatrix™ after 12, 24 and 72 h (only for Myogel-LMA) with the original magnifications of 4x and 10x (a). The number of tubules
formed (b), and the diameter of the tubules formed (c) in each of the three matrices is shown. Three wells were coated for each coating and three
visual fields per each well were analyzed, the average value per well was used for statistical analyses. Altogether nine wells were measured in triplicate.
Error bars, mean ± s.d., Mann–Whitney U test, * P < 0.05, *** P < 0.001
normal human tissues or cultured cells. Since the composition of Myogel simulates well the TMEM of human
solid cancers, it clearly offers improved characteristics
also over mouse tumor matrix derivative Matrigel® which
is mouse EHS tumor-derived and has batch-to-batch
variation. Myogel, used for coating cell culture wells, offered a matrix on which normal and transformed cells
attached more efficiently than on BSA or plain plastic
wells with PBS. It was beneficial also in vertical scratch
assays. In addition, it was interesting to note that a Myogel coating affected the expression of 750 genes in HSC3 carcinoma cells compared to plain plastic, the method
commonly used in RNA-microarray analyses. These
genes were related to actin filament reorganization and
contributed to cell motility providing some insight into
the mechanisms behind the more intense migration of
cancer cells on, through or in Myogel containing matrix
in the experiments measuring cell movement. The factors in this G13 signaling pathway, such as WiskottAldrich syndrome-like (WASP), play important roles in
cell response to extracellular stimuli by actin filament
reorganization, and they are also known to contribute crucially to cancer cell motility [40]. Therefore, our results
suggest that Myogel provides a more complex, natural human TMEM substrate for oncogenic assays than cell culture on plain plastic or even wells coated with individual
molecules, such as collagen, fibronectin or vitronectin.
The mesenchymal fibroblasts (GFs and CAFs) remained
viable when seeded on Myogel, but when embedded in a
thick layer of Myogel-LMA they became apoptotic.
Salo et al. BMC Cancer (2015) 15:981
However, carcinoma cells formed vital colonies even for up
to 28 days. This indicates that Myogel, as a homogenous
protein solution, cannot provide the collagenous fiber
structures required for non-transformed cells, like fibroblasts, to survive. Instead, Myogel-LMA is ideal for soft
agar -type assay commonly used to select normal from
transformed cells [41]. Adding Myogel in agarose provides
the cells a more natural TMEM, which most probably affects their growing potential. Here we saw a slight tendency of the carcinoma cells to form more large colonies
in Myogel-LMA than in the plain LMA matrix. The hanging drop spheroids embedded in Myogel-LMA also
seemed to grow more than the ones embedded in plain
LMA.
The Transwell® invasion assay, conducted classically by
applying either Matrigel® or Matrigel®-GFR to Transwell®
inserts [11], worked well with either Myogel or MyogelLMA. Interestingly, we were able to show that carcinoma cells invaded in Myogel even more efficiently than
in Matrigel®. Cancer cells also invaded through MyogelLMA mixtures more evenly throughout the whole membrane, whereas in Matrigel®-mixtures the cells tended to
invade in clumps. Myogel seems to offer an excellent,
natural human TMEM, which helps to simulate the
proper, natural interaction between TMEM and cancer
cells. In our preliminary experiments we have also succeeded in using Myogel-LMA, instead of Matrigel®, in
several chemotherapeutic and irradiation in vitro experiments (data not shown). Based on these experiments, we
believe that Myogel-LMA is able to highly improve the
quality of various preclinical human cancer studies.
For the study of tumor cell behavior, it is important to
observe the cells under biological conditions since the
cellular motility, form and growth in monolayer culture
differ from that observed in 3D culture [42]. The hanging drop is a method of 3D cell culture, which mimics
the environment of the cells within a tissue [43, 44].
This method is preferred compared to the other 3D cell
culture settings since it allows the cells to form spheroids, in which cells are cultured in close contact to each
other, making a small aggregated component. In hanging
drop assay, spheroid is a form of an aggregated cluster
of cells cultured in 3D physiological condition. The
spheroids developed using the hanging drop method are
widely used as a model for the study of cell-cell interactions [45], in vivo tumor tissue organization and microenvironment [46], and the melanoma reconstruction
model [47]. In this study, we have optimized the method
for the observation of single tumor cell motility in different matrices. When the speed of cell movement was
compared between the matrices, the difference was
clear: HSC-3 cells moved most rapidly in Myogelcollagen and their movement was the slowest in Matrigel®-collagen.
Page 14 of 16
We demonstrated that Myogel-LMA could also be applied to angiogenesis studies, where it represented a reliable way to produce and analyze data regarding vascular
tube formation. In Myogel-LMA the number of tubes
formed was higher but they were smaller in circumference compared to the other matrices. The vessel formation assay using Myogel-LMA could thus be suitable for
pro- or anti-angiogenic drug assays where the number
of tubes is the most critical factor to be evaluated. The
differences between Matrigel®-GFR and ECMatrix™ assays could be explained by the fact that the levels of
stimulatory cytokines and growth factors have been
markedly reduced in the growth factor reduced Matrigel®
to avoid problems associated with the over-stimulation
of endothelial cells. However, according to the manufacturer, ECMatrix™ is derived from EHS tumors and is
similar to “normal” Matrigel® and contains e.g. laminin,
collagen type IV, heparan sulfate proteoglycans, entactin
and nidogen. It also includes various growth factors
(TGF-β, EFG) and proteolytic enzymes (plasminogen,
tPA, MMPs) that should optimize it for maximal tubeformation. In all matrices, cellular network structures
were fully developed by 12 h, with the first signs apparent after 5–6 h. However, using Myogel-LMA, tube formation continued for as long as for 72 h, whereas at that
time the endothelial cells in Matrigel®-GFR and ECMatrix™ were already in apoptosis.
Conclusions
In conclusion, Myogel, especially easy-to-use MyogelLMA, is well suited for in vitro cancer and angiogenesis
studies. In some experiments Myogel is even superior to
the Matrigel®, or rat tail type I collagen -based matrices.
Using easily obtained, human uterus leiomyoma tissue
to produce Myogel, production costs are relatively low.
In addition, the use of myoma tissue is ethically superior
to the sacrifice thousands of mice with EHS tumors required for the production of Matrigel®. Myogel together
with the myoma disc organotypic model, could offer a
natural human TMEM based kit to study various cancer
cell lines, chemotherapy or irradiation effects in vitro. In
theory, these materials may well be usable also in the future for personalized medicine. Myogel-LMA could facilitate the culture of cells from a fresh tumor tissue
biopsy or a cell suspension and the effects of drugs or
chemoradiation therapies tested to optimize the treatment for the patient.
Availability of Data
The microarray data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database,
www.ncbi.nlm.nih.gov/geo (accession no. GSE68722).
Salo et al. BMC Cancer (2015) 15:981
Additional files
Additional file 1: Figure S1. Gradient SDS-PAGE, 2-DE and zymography
from Myogel and Matrigel®. Coomassie Blue stained gradient SDS-PAGE
gel from four Myogel batches (12, 15–17) and their mixture and four
commercial Matrigel® samples (20 μg protein, A). Coomassie Blue stained
gradient SDS-PAGE gel from four Myogel samples (3, 4, 6 and 9) for
Myogel proteomics by mass spectrometry (10 μg and 20 μg protein, B).
Silver 2D gels of Myogel (batches 16 and 17, 100 μg protein) and Matrigel®
(100 and 300 μg protein) samples (C). Gelatinolytic activities in Myogel and
Matrigel® detected under long wave UV light by zymography. The empty
lanes between the standard and Myogel-sample, Myogel- and Matrigel®samples and Matrigel®-sample and control MMP-2 were deleted from the
picture and these lanes are divided by white lines, control MMP-2 and −9
were initially in adjacent wells. (D). Gradient gel in A and zymography were
run twice, gradient gel for proteomics and 2-DE gels once. Figure S2.
Average speed of HSC-3 cell movement in hanging drops. HSC-3 cells were
cultured in hanging drops in pure collagen, Myogel-collagen and Matrigel®collagen matrices. Pictures were taken every 15 min for 20 h with a 40x
objective. The hanging drop -assay was performed twice, but the analysis
was performed from one experiment. (PDF 660 kb)
Additional file 2: Supplementary Method. The analysis of the cells in
hanging drops. (PDF 591 kb)
Additional file 3: Table S1. Mass spectrometry results on myogel.
(PDF 640 kb)
Additional file 4: Table S2. The pH-measurements of Myogel and
Matrigel®. (PDF 111 kb)
Additional file 5: Table S3. Microarray results showing genes with FC
1.5 or more when the HSC-3 cells cultured on the top of plastic were
compared to the corresponding cells grown on Myogel. (PDF 1329 kb)
Additional file 6: Movie S1. Nuclear stain, collagen.avi. Video showing
cell movements in the hanging drop culture in pure collagen matrix
tracked by nuclear stain. (AVI 4793 kb)
Additional file 7: Movie S2. Nuclear stain, Myogel-collagen.avi. Video
showing cell movements in the hanging drop culture in Myogel-collagen
matrix tracked by nuclear stain. (AVI 3832 kb)
Additional file 8: Movie S3. Nuclear stain, Matrigel-collagen.avi. Video
showing cell movements in the hanging drop culture in Matrigel®-collagen
matrix tracked by nuclear stain. (AVI 4273 kb)
Abbreviations
2-DE: Two-dimensional gel electrophoresis; ATCC: American type culture
collection; BSA: Bovine serum albumin; CAF: Carcinoma associated fibroblast;
ECM: Extracellular matrix; EGF: Epidermal growth factor; EHS: EngelbrethHolm-Swarm; FBS: Fetal bovine serum; FC: Fold change; FDR: False discovery
rate; FGF: Fibroblast growth factor; GEO: Gene expression omnibus;
GF: Gingival fibroblast; GFP: Green fluorescent protein; GO: Gene ontology;
H2B: Histone-2B; HUVEC: Human umbilical vein endothelial cell;
IEF: Isoelectric focusing; LC: Liquid chromatography; LMA: Low melting
agarose; MMP: Matrix metalloproteinase; MS: Mass spectrometry; MyogelLMA: Myogel mixed with low-melting agarose; PBS: Phosphate-buffered
saline; PDGF: Platelet-derived growth factor; TCA: Trichloroacetic acid;
TGF-α: Transforming growth factor alpha; TGF-ß: Transforming growth factor
beta; TME: Tumor microenvironment; TMEM: Tumor microenvironment
matrix; tPA: Tissue plasminogen activator; TUNEL: Terminal deoxynucleotidyl
transferase dUTP nick end labeling; WASP: Wiskott-Aldrich syndrome-like.
Competing interests
The authors (TS, MeSu, ES and FS) have filed a patent on Myogel.
Authors’ contributions
TS conceived the project, planned the experiments, interpreted data and
wrote the manuscript. MeSu planned and performed experiments, analyzed
and interpreted data and wrote the manuscript. EHA, ES, NKC, CEdO, SUA,
SO, FS, PJ, PÅ, CCB and JK planned and performed experiments, analyzed
and interpreted data. MaSa and KS were responsible for myoma collection
and LE, AFPL and RDC supervised experiments and interpreted data. All
authors revised the manuscript.
Page 15 of 16
Authors’ information
Current affiliation for FS: Research Programs Unit – Molecular Neurology, and
Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki, Helsinki,
Finland.
Acknowledgements
Mrs Maija-Leena Lehtonen and Mrs Eeva-Maija Kiljander are acknowledged
for their excellent technical help in the experiments. We thank Dr Sami
Yokoo, Dr Bianca Alves Pauletti and Dr Romênia R. Domingues, Laboratório
Nacional de Biociências, LNBio, CNPEM, Campinas, Brazil, for their valuable
help and advice with the laboratory work for mass spectrometry and analyzing
the results. We acknowledge Dr. Cindy E. Dieteren, Department of Cell Biology,
Radboud UMC, Netherlands, for providing the H2B-coupled mCherry expression
vector. We thank the Biocenter Oulu Virus Core laboratory for providing help
and the facilities for the work with lentiviral particles and the Biocenter Oulu
DNA sequencing and expression analysis center for performing microarray
analyses and training and help in their data analysis. We acknowledge the
personnel of the Department of Obstetrics and Gynecology from the Oulu
University Hospital for contributing to the research by providing the leiomyoma
tissue. This work was supported by the Science without Borders (CAPES
Program, Project 109/2012, AUXPE-PVES 570/2013) linked to the Special Visiting
Researcher Program and grants from the Sigrid Juselius Foundation, Finnish
Cancer Foundation, and Finnish Cultural Foundation and research funds from
the Medical Faculty of the University of Oulu and Oulu University Hospital
special state support for research and Medical Research Center Oulu.
Author details
1
Cancer and Translational Medicine Research Unit, Faculty of Medicine,
University of Oulu, PO Box 5281FI-90014 Oulu, Finland. 2Medical Research
Center Oulu, Oulu University Hospital and University of Oulu, FI-90014 Oulu,
Finland. 3Department of Oral and Maxillofacial Diseases, University of Helsinki,
FI-00014 Helsinki, Finland. 4Clinical Department, Faculty of Medicine of
Jundiai (FMJ), Jundiai, São Paulo SP-13202-550, Brazil. 5Department of Oral
Diagnosis, Oral Pathology Division, Piracicaba Dental School, University of
Campinas, Piracicaba, São Paulo SP-13414-903, Brazil. 6Center for Machine
Vision Research, University of Oulu, FI-90014 Oulu, Finland. 7Biocenter Oulu,
University of Oulu, FI-90014 Oulu, Finland. 8Faculty of Biochemistry and
Molecular Medicine, University of Oulu, FI-90014 Oulu, Finland. 9Proteomics
Core Facility, Biocenter Oulu, University of Oulu, FI-90014 Oulu, Finland.
10
Department of Obstetrics and Gynecology, Oulu University Hospital and
University of Oulu, FI-90029 Oulu, Finland. 11Department of Obstetrics and
Gynecology, Tampere University Hospital and University of Tampere,
FI-33521 Tampere, Finland. 12Laboratório Nacional de Biociências, LNBio,
CNPEM, Campinas SP-13083-970, Brazil.
Received: 1 July 2015 Accepted: 19 November 2015
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