Abstract
The stiffness of the tumor microenvironment (TME) is dynamic and drives
metabolic reprogramming in cancer cells as a consequence of tumor
progression. To demonstrate the possibility to modulate the
mechano-metabolomic profile of breast cancers by tuning the mechanical
property and dimensionality of extracellular matrices (ECMs), we
cultured triple-negative MDA-MB-231 and luminal MCF-7 cells on 2D and in
3D hydrogels based on tyramine functionalized hyaluronic acid (HTA).
Using high-throughput metabolomics analyses, we established that we can
differentially regulate breast cancer mechano-metabolome. The stiff
hydrogels resulted in upregulated lipid and amino acid metabolism along
with increasing malignancy and chemoresistancy. Reprogramming in glucose
metabolism is primarily observed in cells seeded on 2D hydrogels,
whereas modifications in amino acid metabolism is predominant in cells
embedded in 3D stiff hydrogels. These findings suggest that matrix
stiffness and dimensions have decisive roles in reprogramming breast
cancer metabolome, which is the hallmark of breast cancer development
and progression.
Introduction
Cancer microenvironment and mechanical homeostatic processes are altered
from tissue-level modifications to changed cellular signaling pathways
associated with mechanotransduction. In breast cancer, the interactions
between cell microenvironment and mechanical cues are envisaged as
fundamental concepts of breast cancer
mechanobiology1,2 . Breast cancer progression
is generally driven by the biophysical and biochemical
microenvironmental cues including extracellular matrix (ECM) stiffness
and dimensionality (2D and 3D)3 . For example,
the level of crosslinking of collagen in a dynamically stiffening ECM is
a focal adhesion regulator in breast cancer
tumorigenesis4 . However, in vitromodels to better-mimicking mechanotransduction mechanisms are still
required to advance the study of breast cancer mechanobiology.
Biomaterial-based approaches with tunable stiffness and dimension
present a unique opportunity to demonstrate the ECM mimetic modulatory
effects of mechanical cues on selected cancer cell populations and their
behaviors. For example, matrix stiffness of gastric cancer
microenvironment was found as an epigenetic regulator of
mechanotransduction-related YAP in a 3D collagen–alginate
interpenetrating network (IPN)-based matrices, which were adjusted
across a range of elastic moduli of gastric cancer tissues
(~0.5 kPa> G ′>
~6.8 kPa)5 . Varying migration
patterns of breast cancer cells and metastasis responding changes in
matrix stiffness were driven by gelatin-methacrylate hydrogels with
stiffness ranging from soft to stiff (0.8 to 5
kPa)6 . Oncogenic reprogramming of normal
cells was displayed through 2D fibronectin-coated polyacrylamide
hydrogels with varying stiffnesses (0.5 kPa, 1 kPa, 2 kPa, 4 kPa and 40
kPa), which resulted in RTK-Ras oncogenes to support mammary
gland oncogenic reprogramming rely on YAP/TAZ. Additionally, pancreatic
acinar cells in 3D gelatin-hyaluronan-based hydrogels (0.5 kPa and 9
kPa) were observed to support pancreatic tumorigenesis overRas -mediated YAP/TAZ nuclear localization in response to matrix
stiffening7 .
Here, we investigate the plasticity of the metabolome of breast cancer
cells in response to altering microenvironmental conditions. To this
end, we developed 2D and 3D cell culture systems based on tyramine
functionalized hyaluronic acid (HTA) hydrogels. The HTA hydrogels were
cross-linked using horseradish peroxidase (HRP)-mediated oxidative
coupling reaction as we previously
demonstrated8 . Hyaluronic acid (HA) is an
integral molecular component of the breast cancer ECM and generally
presents CD44 receptor, which is critical for cell adhesion, invasion,
and mechanosensing. Therefore, HA-based hydrogels are suitable platform
to investigate breast cancer cell behaviors9 .
Using this material platform, we demonstrated the possibility to
modulate the breast cancer cells metabolome by tuning the mechanical
properties and dimensions of HTA hydrogels (referred the
mechano-metabolomics). A rigorous elucidation of changes in the
metabolite profiles of MCF-7 cells and triple-negative MDA-MB-231 cells
on 2D and in 3D HTA hydrogels having tunable matrix stiffness reveals
that the cancer cells display differential metabolomic plasticity in
response to changes in the matrix stiffness and dimension.
Materials and Methods
MDA-MB-231 and MCF-7 Breast Cancer Cell Cultures
MDA-MB-231 was kindly provided by Prof. Sedat Odabas (Ankara University,
Turkey), and cultured in Dulbecco’s Modified Eagle’s Medium High Glucose
(DMEM), with Fetal Bovine Serum (FBS, 10% v/v) and
antibiotic–antimycotic solution (P/S, 1% v/v), in an incubator under
37 °C and 5% CO2 conditions. The medium was refreshed
every 2–3 days. MCF-7 cells (kindly provided by Prof. Ahmet Acar,
Middle East Technical University, Turkey) were cultured in DMEM with FBS
(20% v/v) and P/S (1% v/v), in an incubator under 37 °C and 5%
CO2 conditions. The medium was refreshed every 2–3
days.
Preparation of HTA Gels with Differential Stiffness
Pure HTA powder (synthesized and characterized
previously)10 was dissolved in Dulbecco’s
Phosphate Buffer Saline (DPBS) at desired concentrations (1% and 5%
wt.). HRP (2 U/mL) was added to the prepared pre-gel solutions to drive
enzyme-mediated oxidative coupling reaction. Hydrogelation of 1% and
5% HTA pre-gels was triggered in well-plates by adding hydrogen
peroxide (H2O2, 2mM) and incubation at
room temperature for 1 min. After a washing step, cells were seeded on
HTA hydrogels at the desired density. The cells were allowed to adhere
to the materials for 30 min before top up the fresh media to ensure an
effective cell adhesion. To prepare 3D cultures, HTA was added to wells,
the cells were inoculated inside the gels, and
H2O2 (2 mM) was injected to trigger
instant gelation. Fresh media was added after the washing step. To
monitor the effect of hydrogels on cell viability and proliferation,
Live/dead staining and
2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide
(XTT) tests were performed with HTA (1% and 5% wt.) hydrogels before
proceeding with the further experiments (details are provided in
Supplementary Information).
Microstructural Characterizations of HTA Gels
Scanning electron microscopy (SEM) imaging was performed to examine the
morphology and structure of 1% and 5% HTA hydrogels. HTA hydrogels
were dehydrated by the critical point drying (CPD) method and then
coated with gold/palladium. Images were obtained at 10 kV voltage and at
different magnifications.
Immunostaining for YAP and β-actin
To visualize the breast cancer cell-matrix interaction and the response
of cells to the matrix stiffness, we performed an immunofluorescent
staining for β-actin (cell cytoskeleton protein) and mechanosensing
protein YAP (see Supporting Information).
Gene Expression Study
The effect of matrix stiffness and context on MDA-MB-231 and MCF-7 cells
were investigated at the molecular level by performing a reverse
transcriptase-quantitative polymerase chain reaction (RT-qPCR) study
(additional notes can be found in the Supplementary Information). The
effect of matrix stiffness and matrix context on the expression of
mechanotransduction markers in MCF-7 and MDA-MB-231 cells was examined
by gene expression analyses of YAP , TAZ , RhoA andFAK .
Metabolomics
Metabolomics analysis was performed for MDA-MB-231 and MCF-7 cells, that
were cultured on/in soft and stiff hydrogels, to specify the metabolites
that alter in response to matrix stiffness and context. To this aim,
MDA-MB-231 and MCF-7 cells (500.000 cells/gel) were seeded on/in
hydrogels in well-plates, and the cell cultures were maintained for 3
days. At the end of the culture period, metabolomic analyses were
performed as previously described (additional notes can be found in the
Supplementary Information)11 .
Drug testing
The cells (~50.000 cells/well, 96-well plate) were
cultured for 48-hours to enable cell adhesion and stabilize the cell
metabolism. Later, Doxorubicin (DOX, 0.2
µg/ml)12 was applied in MDA-MB-231 and MCF-7
cells that were cultured on/in soft and stiff hydrogels. After DOX
treatment for 72 h, an XTT test (Biological Industries, USA) was applied
for MDA-MB-231 and MCF-7 cells to assess cell proliferation and
chemoresistance on/in the gels. Absorbance values were recorded at 490
nm with a Multiskan Sky Microplate Spectrophotometer (Thermo Fisher).
Statistical analysis
Partial least squares-discriminant analysis (PLS-DA) was performed to
reveal the discrimination between groups. Variable Importance in
Projection (VIP) graphs were used to highlight the top 15 differentially
expressed metabolites. Pathway analyzes were performed with the
significantly altered metabolites (Student’s t-tests , p
< 0.05) using MetaboAnalyst
(https://www.metaboanalyst.ca/,ver.5.0). The number of significantly
differentiated metabolites between groups was illustrated on the Venn
scheme and their names, pathways, and cellular locations were summarized
in Table S1 (dimensionality), Table S2(matrix stiffness in 2D), and Table S3 (matrix
stiffness in 3D). Gene expression and drug testing results were analyzed
with One-way ANOVA and pairwise comparisons performed using
Student’s t-tests , bars represent mean ± SEM and symbols
represent each experiment replicate, using GraphPad Prism 9 (for
Windows, GraphPad Software, San Diego, California USA,
www.graphpad.com).
Results and Discussion
The rationale of the study
The mechanometabolome of breast cancer was examined with MCF-7
(non-invasive) and triple–negative MDA-MB-231 (highly invasive) cell
lines. Breast cancer was selected as a model in this work to represent
an aggressive epithelial malignancy mediated by micro-environmental
cues. In addition, it is well-known that breast tumor microenvironment
undergoes a dynamic stiffening compared to healthy breast tissue
(Figure 1A )13 . The tumor-mimetic
mechanical tunability of HTA was harnessed to create 2D and 3D matrices
for breast cancer cells culture. The stiffness of breast tumors varies
between 2 to 10 kPa in vivo . The tunable gelation of HTA enabled
us to prepare hydrogels with controllable mechanical properties –
ranging from soft (1% wt., ~1.95 kPa) to stiff (6%
wt., ~9.6 kPa)8 . SEM images
of the hydrogels revealed a porous microstructure, as expected. Soft HTA
hydrogels are characterized by a high density of interconnected large
pores, while stiff HTA hydrogels are composed of compacted networks with
narrow pores (Figure 1B ). The different morphology of the dried
hydrogels is indicative of the different physico-chemical properties
determined by the range of concentration and crosslinking levels
explored. Taken together, HTA is an ideal material candidate to
investigate cell-material interactions and breast cancer
mechanotransduction.
Assessment of cytotoxicity of HTA hydrogels for breast cancer
cells
HA is one of the main components of breast tumor
ECM6 . To assess the potential applicability
and toxicity of soft and stiff HTA hydrogels as cell culture scaffolds,
MDA-MB-231 and MCF-7 breast cancer cells were cultured on the hydrogels
for 2 and 5 days. We carried out a Calcein-AM/EtBr-1 double staining to
visualize live cells as green stained and dead cells as red stained, as
well as an XTT assay to further assess cell proliferation
(Figure S1 ). Both MDA-MB-231 and MCF-7 cells were observed with
low levels of dead cells. The proliferation of breast cancer cells was
also promoted by HTA hydrogels (Figure S1 ).
Microscopic evaluation of the effect of stiffness and
dimension on cell behavior
To assess the adhesion and morphologies of MDA-MB-231 and MCF-7 cells,
the cells were monitored on the soft and stiff hydrogels in 2D and 3D
culture systems on days 1 and 3 (Figure 2A ). On the soft HTA
hydrogels, MDA-MB-231 cells displayed a clustered morphology from day 1
to day 3, whereas a mixture of aggregated and adherent morphology were
observed on stiff gels. Therefore, MDA-MB-231 cells assumed a more
invasive morphology on the soft matrix, and a less invasive morphology
on the stiffer matrix. On the other hand, MCF-7 cells exhibited a spread
morphology on the soft HTA hydrogels whereas they assumed a clustered
morphology on the stiff hydrogels after 3 days in culture
(Figure 2A ).
To monitor the cell morphology in 3D culture conditions, we encapsulated
MDA-MB-231 cells and MCF-7 cells in both soft and stiff 3D hydrogels
imitating native tumor ECM to characterize their mechanosensitive
responses in the similitude of a native tumor ECM. Optical micrographs
of MDA-MB-231 and MCF-7 cells embedded in the soft 3D hydrogels reveal
an adherent character after day 1 and 3. It is noteworthy that a small
number of tiny aggregates of MCF-7 were also observed in the soft
hydrogels after day 3 in culture. On the other hand, MDA-MB-231 cells
embedded within the stiff hydrogels formed spheroidal aggregates after
day 3 in culture while MCF-7 cells predominantly assumed an adherent
morphology with few clusters (Figure 2A ). The scanning electron
micrographs further showed that both MDA-MB-231 and MCF-7 cells can
adhere to soft and stiff hydrogels possibly owing to their inherent
affinity for HA in the native tumor ECM9(Figure 2B ); however, MDA-MB-231 cells were shown to tend to
form aggregates while MCF-7 cells were more likely to adhere and grow
filopodia.
We investigated the sensitivity of YAP in the breast cancer cells to
increased stiffness. To this end, we performed immunostaining (YAP and
β-actin) for breast cancer cells that were cultured on soft and stiff
HTA hydrogels. Immunofluorescent images of the cells showed that YAP was
spread throughout the cytoplasm of MDA-MB-231 and MCF-7 cells cultured
on soft hydrogels, while it was accumulated around the nuclei of cells
seeded on stiff hydrogels (Figure 2C ). Cell aggregation was
also visualized by β-actin staining on MDA-MB-231 and MCF-7 cells that
were cultured on soft and stiff hydrogels. β-actin staining further
confirmed that MDA-MB-231 and MCF-7 cells respond to matrix stiffness
differently. The MDA-MB-231 cells form aggregates on stiff hydrogels
while MCF-7 cells form a highly adherent morphology (Figure
2C ).
Effect of matrix stiffness and dimension on mechano-related
gene expressions
To determine mechanosensitive response of MDA-MB-231 and MCF-7 cells to
changing matrix stiffness and dimension, we assessed the expressions of
genes associated with mechanotransduction pathways in cancer cells using
RT-qPCR. The expressions of YAP , TAZ, RhoA , andFAK genes were assessed after day 3 in culture. In MDA-MB-231
cells, the expression of YAP was elevated with the increasing
stiffness in both 2D and 3D conditions (Figure 3A ). Unlike in
the 3D culture system where reduced expression of YAP was
observed, YAP was highly expressed by MCF-7 cells in the 2D
culture system as the hydrogel stiffness increases (Figure 3B ).
On the other hand, the expression of YAP in MCF-7 cells in 3D
conditions was shown to decrease possibly due to the non-invasive
character of the cells and the resulting relatively weak cell-ECM
interaction.
The expression of TAZ gene by MDA-MB-231 cells in both our 2D and
3D culture systems was upregulated when the matrix stiffness was
increased (Figure 3A ), which might be the underlying reason for
the enhanced cell proliferation in and on our hydrogel scaffolds
(Figure S1B ). FAK has a role as non-receptor tyrosine
kinase providing signaling functions at the integrin binding sites,
leading to cell migration. Our results showed that FAK expression
was upregulated with increasing matrix stiffness in MDA-MB-231 and MCF-7
cells cultured in both 2D and 3D hydrogels (Figure 3B ). In
order to shed light on the role of RhoA , a member of RhoGTPases, in breast cancer, we investigated the effect of matrix
stiffness on RhoA expression in our hydrogels. While we observed
an upregulated expression of RhoA in the highly invasive
MDA-MB-231 cells cultured on stiff 2D hydrogels, MCF-7 cells displayed a
down-regulated expression of RhoA in the stiff 2D hydrogels. In
contrast, MDA-MB-231 cells showed no significant alteration to the
expression of RhoA in the stiff 3D hydrogels, whereas, the
expression was upregulated in MCF-7 cells cultured under similar
conditions (Figure 3A and 3B ).
Evaluation of matrix
stiffness and dimension on breast cancer cell metabolic plasticity
To examine the putative mechanisms of plasticity in breast cancer
metabolome, we performed a comprehensive untargeted metabolomics
analysis by GC-MS and LC-qTOF-MS. PLS-DA score plots showed clear
discrimination between the metabolic phenotypes of both MDA-MB-231
(invasive) and MCF-7 (non-invasive) cells that were cultured in soft and
stiff gels using 2D and 3D culture systems (Figure 4Aa and
4Ba ). One-way ANOVA was further used to confirm the results(Figure S2A and S2B) . This metabolic discrimination in
MDA-MB-23 and MCF-7 cells was more significant in the stiff 2D matrix
than the 3D counterpart. The most significantly altered 15 metabolites
were reported in the VIP graphs (Figure 4Ab and 4Bb ). For
example, lysophasphotidylcholines (LysoPCs, LPC, a phospholipid),
fucoxanthin, and phosphatidic acid (PA, a glycerophospholipid) were seen
to be differentially expressed in the 2D system, while
phosphatidylcholine (PC, a phospholipid), 4-cholesten-3-one,
palmitoylcarnitine (a carnitine ester), and hexadecane (a hydrocarbon)
were some of the main metabolites that were significantly altered in the
3D culture system with increasing matrix stiffness. The altered
metabolites between MDA-MB-231 and MCF-7 cells cultured in soft and
stiff gels and in 2D and 3D conditions were further depicted in
hierarchical cluster analysis (Figure 4Ac and 4Bc ). A distinct
pattern between the groups with high stiffness was observed. Strikingly,
the levels of lysophosphatidylcholines were up-regulated in MCF-7 cells
on soft gels (2D), while it was down-regulated in MDA-MB-231 in all
conditions (2D and 3D, soft and stiff matrices) (Figure 4Ac ).
Similarly, D-Mannose, D-sphingosine, and cysteine were up-regulated in
MDA-MB-231 cells on soft gels in 2D and down-regulated in 3D; as well as
they were down-regulated in MCF-7 cells in all conditions. Importantly,
the levels of phytosphingosine, dihydrosphingosine, C17-sphingosine, and
5-cholestan-3-beta were up-regulated commonly in both MDA-MB-231 and
MCF-7 cells cultured within the soft hydrogels, whereas these
metabolites were down-regulated in MDA-MB-231 and MCF-7 cells within the
stiff hydrogel (Figure 4Ac ). Pathway analysis showed that the
altered metabolites mainly affected specific pathways including
galactose metabolism, taurine hypotaurine and sphingolipid pathways in
the 2D system whereas taurine hypotaurine metabolism, sphingolipid
metabolism, glycerophospholipid metabolism, and arginine biosynthesis
were affected in the 3D systems (Figure 4C ). The metabolites
that were significantly altered both in 2D and 3D were determined and
illustrated in Venn diagram (Figure 4D ). We established that 49
metabolites were commonly altered in both matrix dimensions (2D and 3D)
and they are mainly associated with a fatty acid, phospholipid and
sphingolipid metabolisms (Table S1 ). Hence, our data indicate
that lipid metabolism-mediated matrix and membrane remodeling pathways
were modulated independent of matrix dimensionality in breast cancer.
Investigation of the cell invasiveness on breast cancer
mechano-metabolome
To specify the effects of matrix stiffness on individual breast cancer
cell lines, we compared the metabolite profiles of each cell type in
specific conditions (soft or stiff, 2D or 3D). The metabolic structure
of MDA-MB-231 cells was affected by matrix stiffness, which is
consistent with the PLS-DA results (Figure S3A) . Similarly,
MCF-7 cells responded to increased matrix stiffness (Figure
S3A ). The top 15 metabolites in discrimination between groups were
provided in VIP plots (Figure S3B ). Additionally, heat-maps of
altered metabolites in MDA-MB-231 and MCF-7 cells on soft and stiff
matrices were depicted with hierarchical cluster analyses
(Figure S3C ). The results were validated by t-tests(Figure S4) .
To elaborate on the differentially regulated metabolites between groups,
the up- and down-regulated metabolites, that created patterns on the
heat-map analyses, were visualized on volcano plots (Figure
5Aa ). We observed that various metabolites affecting the phospholipid
biosynthesis, protein degradation and biosynthesis, and
glycerophospholipid metabolism were up-regulated in MDA-MD-231 cells
with the increased stiffness. In MCF-7 cells, fatty acid degradation
(palmitocarnitine, L-acetyl carnitine), phospholipid biosynthesis (LPC,
LysoPC), ECM amino acid remodeling (isoleucine, 5-methoxytryptophol)
processes related metabolites were up-regulated with increased
stiffness. The pathway impact analysis confirmed that cellular membrane
and ECM remodeling-related lipid metabolism pathways were stimulated
mostly in MDA-MD-231 cells with increasing stiffness in the 2D system
(Figure 5Ab ). Arginine biosynthesis, glycerophospholipid, TCA
cycle and sphingolipid metabolism pathways were profoundly associated
with cellular membrane and ECM-remodelling of the 2D culture of
MDA-MB-231. In MCF-7 cells, phenylalanine-tyrosine-tryptophan,
glycerophospholipid, and valine-isoleucine-leucine metabolism were the
obvious pathways biased (Figure 5Ab ). The Venn diagram revealed
that 39 metabolites were commonly altered in both MDA-MB-231 and MCF-7
cells cultured on the 2D system (Figure 5Ac ), and they were the
active driver for phospholipid, glycerophospholipid and fatty acid
metabolisms (Table S2 ).
Similarly, to reveal the effect of matrix stiffness in 3D conditions on
the metabolomics structure of the various types of breast cancer cells
examined, we compared the metabolite profiles of both MDA-MD-231 and
MCF-7 cells cultured in soft and stiff matrices. The metabolic
structures of MDA-MD-231 and MCF-7 cells were affected by matrix
stiffness in the 3D culture system, which is in agreement with the
PLS-DA results (Figure S5A) . The top 15 metabolites in
discrimination between groups were provided in VIP plots (Figure
S5B ), while the metabolomic discriminations were represented in
heat-maps with hierarchical cluster analyses (Figure S5C ).
Volcano plot obtained for MDA-MB-231 cells that compares the metabolite
profiles of cells cultured in soft and stiff gels showed that
piperidinecarboxaldehyde, phosphatidylcholine, calcipotriol,
glutamylglycine, and monoacylglyceride (18:2(9Z,12Z)/0:0/0:0) were
mostly up-regulated in the stiff matrix. With MCF-7 cells,
4-cholesten-3-one, L-acetyl carnitine, citramalic acid, palmityl
carnitine, D-sphingosine, and phosphatidylcholines were some of the
up-regulated metabolites driven by high matrix stiffness (Figure
5Ba ). These up-regulated metabolites significantly altered selected
pathways including the linoleic acid, alpha-linoleic acid metabolism,
and valine isoleucine biosynthesis pathways (Figure 5Bb ). In
the MCF-7 cells, valine, leucine and isoleucine, aminoacyl-tRNA, and
arginine biosynthesis pathways were affected by the increased matrix
stiffness in 3D culture (Figure 5Bb ). Venn diagram revealed
that 17 metabolites were significantly altered in common for MDA-MB-231
and MCF-7 cells with increasing matrix stiffness and in 3D culture
conditions (Figure 5Bc ), and they mainly affect the arginine
and proline metabolism (Table S3 ).
Differential matrix parameters bring about the metabolic
reprogramming in breast cancer tumor progression and malignancy
To investigate the effects of matrix stiffness and dimension on
metabolomic plasticity and the possible effects of plasticity on tumour
progression, we constructed pathway impact diagrams. Our findings show
breast cancer cells in the 2D conditions undergo a set of modulations in
the glucose, lipid, and energy metabolisms in response to increasing
matrix stiffness (Figure 4C ). On the other hand, modulation in
amino acid metabolism accompanied by altered lipid and energy
metabolisms was observed in the 3D culture system (Figure 4C ).
When we compared MDA-MB-23 and MCF-7 cells in the 2D system,
sphingolipid metabolism, arginine biosynthesis, and citrate cycle were
elucidated as varied in MCF-7 cells that differentiate predominantly
through aromatic amino acid and branched amino acid metabolism pathways.
Lastly, our results showed that MDA-MB-231 and MCF-7 cells within the
hydrogels with the high stiffness displayed an induced amino acid
metabolic reprogramming.
Effect of matrix stiffness and dimension on anti-cancer drug
response
Tumor microenvironment mechanical cues also play a crucial role in not
only promoting tumorigenesis but also chemoresistance. To examine the
effect of matrix stiffness and matrix context on the chemotherapeutic
response of breast cancer cells, MDA-MB-231 and MCF-7 cells were
cultured on/in soft and stiff HTA hydrogels for 2 days, then treated
with a widely used anti-cancer drug Doxorubicin (0.2 µg/ml) for 72 hours
(Figure 6A ). MDA-MB-231 and MCF-7 cells showed a higher
chemoresistance and increased cell viability against doxorubicin after
72-hour of drug treatment in stiff hydrogels compared to soft hydrogels.
Both cell lines respond to increasing stiffness. We observed increased
cellular viability of MDA-MB-231 cells in 2D (soft=47%, stiff=55%) and
in 3D (soft=51.1%, stiff=83%) hydrogels with increasing stiffness.
Similarly, the cellular viability of MCF-7 cells was seen to increase
with the increasing matrix stiffness both in 2D (soft=65%,
stiff=96.8%) and 3D (soft=23.4%, stiff=41.7%) (Figure 6B ).
Evidently, the ECM stiffness could control the chemosensitivity of
breast cancer cells and poor disease
prognosis14 .
Discussion
In this work, we aimed to reveal an unmet question so far, which is how
matrix conditions, i.e., stiffness and dimension, affect cancer
metabolome. Such a correlation provides opportunities to dissect
fundamental information on the potential roles of metabolomics
plasticity in tumour progression, as well as it may help develop drugs
considering tumour matrix conditions to be more efficient.
To this goal, we first assessed the effects of matrix stiffness on the
morphological characters of invasive (MDA-MD-231) and non-invasive
(MCF-7) cells on 2D culture. We observed that MDA-MB-231 cells formed
clusters on soft matrix and a mixture of adherent and clustered cells
were observed on stiff matrix (Figure 2A ). Whereas, MCF-7 cells
depicted a spread morphology on soft matrix and clustered morphology on
stiff matrix (Figure 2A ). This observation was an indicative of
a barely invasive morphology on the soft matrix and a more invasive
morphology on the stiff matrix. This is consistent with previous studies
which reported on how cell spreading regulates proliferation, apoptosis,
invasion, and metastasis13 in a broad range
of cancer cell type including lung carcinoma
cells15 , colorectal cancer
cells16 and breast cancer
cells14 .
Solid tumors display a 3D architecture of cancer cells, non-cancer cells
and ECM. In light of this, 3D hydrogel systems have been extensively
used to recapitulate the architectural and functional framework of
various tumor microenvironments17 . MDA-MB-231
cells were seen to display an adherent and clustered morphology in soft
and stiff matrix, respectively, while MCF-7 cells exhibited an adherent
character both in the soft and stiff matrix (Figure 2A ). Put to
gather, we can conclude that increasing matrix stiffening in 3D
condition promotes malignant phenotype, which resembles in vivotumor progression.
The transcriptional regulator YAP is modulated in several cancer types
as a universal mechanotransducer18 . To
consolidate our inverted phase-contrast microscope observations, we
performed an immunofluorescent study to investigate YAP expressions in
breast cancer cells on soft and stiff 2D hydrogels. As expected, YAP was
seen to be distributed across cytoplasm in the cells seeded on soft
hydrogels, whereas it was seen to be accumulated around nucleus when the
cells seeded on stiff hydrogels (Figure 2C ). Our observation is
consistent with a previously published study that reports nuclear YAP
accumulation in mammary epithelial cells on a dynamically stiffening
matrix19 . Gene expression study
(Figure 3A, B ) further concretized the immunofluorescent
results and are consistent with previous
reports20 . According to established
knowledge, low expression of YAP gene , which is a tumor
suppressor, is generally associated with high
malignancy21 . Thus, suggesting that
MDA-MB-231 cells displayed malignant tumor-related mechanosensitive
response on the stiff 2D hydrogel as observed. Upregulated expression of
TAZ-protein and activity promotes cell proliferation, and EMT in breast
cancer22 . In addition, upregulated expression
of FAK in epithelial cancer has previously been recognized as an
active driver of tumor invasiveness23 .
Clearly, our assessments of the gene expression profile of the examined
breast cancer cell lines showed how the heterogenous cell populations in
a tumor microenvironment can display differential expression of hub
genes related to mechano-transduction including YAP , TAZ ,FAK , and RhoA in matrix stiffness and dimension-dependent
manner.
Metabolic reprogramming is a key process in
tumorigenesis24 . Particularly, the
deregulation of fatty acid and amino acid anabolic/catabolic pathways
have an impact on the metabolic regulation of tumor
growth25,26 . We determined that increasing
matrix stiffness modulate glucose, lipid, and energy metabolisms in 2D.
While, modulation in amino acid metabolism and altered lipid and energy
metabolisms was observed in the 3D culture conditions (Figure
4C) . It is well-known that tumor growth and progression are closely
associated with three main metabolic pathways – glucose metabolism,
lipid metabolism, and amino acid metabolism, that also control tumor
cell proliferation, survival and
malignancy25-28 . The malignant proliferation
of tumor cells presents rapid glycolysis (Warburg effect) in different
environments28 . Glucose metabolism is
required for proliferating cancer cells that have a high demand for
energy29 . The altered metabolites and
metabolic pathways in both 2D and 3D conditions have effects on cell
proliferation (see Supplementary Information), tumor progression, and
increased malignant phenotype mediated high matrix stiffness.
In addition, we compared the metabolomics differences between MDA-MB-23
and MCF-7 cells cultured either in 2D or 3D. In 2D, sphingolipid
metabolism, arginine biosynthesis, and citrate cycle were seen to be
altered in MCF-7 cells (Figure 4C) . Sphingolipids are
responsible for cell adhesion and
migration30 , while the citrate cycle
metabolic pathway is related to the growth and invasion of cancer cells.
In addition, arginine can modulate metastasis and anti-apoptotic
signaling pathways in cancer cells31,32 .
Therefore, it can be concluded that increasing the invasion and
metastasis of MDA-MB-231 cells on hydrogels by increasing the stiffness
of the substrate is expected. In 3D, both in MCF-7 and MDA-MB-231,
increasing stiffness was found to induce amino acid metabolic
reprogramming. This reprogramming is known to modulate cellular
proliferation33 , epigenetic
modifications34 , tumor
growth35 , in either MCF-7 or MDA-MB-231
cells. In addition, an aberrant aminoacyl–tRNA biosynthesis and
arginine biosynthesis in MCF-7 cells can be linked to tumor growth and
metastasis, respectively36 . Moreover,
linoleic acid and alpha-linoleic metabolism reprogramming might be
responsible for an increased malignant phenotype of MDA-MB-231
cells27 .
It is known that TME has a critical role in the efficacy of anti-cancer
drug response37 . We have seen that MDA-MB-231
and MCF-7 cells responded to increasing matrix stiffness in both 2D and
3D conditions, and showed a higher chemoresistance to the introduced
anti-cancer drug, doxorubicin (Figure 6 ). This change in
chemo-sensitivity is in strict correlation with metabolism. Metabolic
reprogramming has a causative effect on signaling and proliferative
inputs that characterize the resistance of cancer
cells38 . Lipids are involved in signal
transduction and regulation of cell growth, proliferation,
differentiation, survival, apoptosis, membrane homeostasis, motility,
and drug resistance in cancer. Tumor metabolic reprogramming,
dysregulation of lipid metabolism and oncogenic signaling enhance lipid
biosynthesis to supply the building blocks for membrane formation and
maintain the high proliferation rate of cancer cells, leading to
increased drug resistance39 . Thus, the
increased drug resistance observed in MCF-7 and MDA-MB-231 breast cancer
cells in our stiff 2D and 3D systems can be linked to the altering lipid
metabolism. In addition to lipid metabolism, reprogramming in the amino
acid metabolism has previously been shown to play crucial roles in tumor
growth and survival and resistance to anti–cancer
drugs40 . Thus, particularly in our stiff and
3D condition, the increased chemoresistance can be associated with
altering amino acid metabolism.
Disclosure statement: The authors declare no competing
interests.
Contributions: B.D. and B.S. developed the concept and designed
the experiments. B.S. performed the experiments and analyzed the data.
B.S., C.C.E., and E.N. accomplished metabolomics analyses. M.D.
supervised the hyaluronic acid-based experiments. B.S and B.D. wrote the
paper. B.D. and B.O.O. edited and commented on the manuscript. All
authors approved the final version of the manuscript.