Figure Legends
Figure 1. (A) Mechanometabolomics concept was built on the
culture of MDA-MB-231 and MCF-7 breast cancer cell lines in/on the soft
and stiff HTA hydrogels. The effect of matrix stiffness and dimension on
the cancer cell metabolism were tested using GC-MS/LC-qTOF-MS based
metabolomics study. (B) Microstructural images of the soft and stiff HTA
hydrogels.
Figure 2. Microscopic evaluation of the effect of stiffness and
dimension on the behavior of breast cancer cells. (A) Inverted
microscope images of MDA-MB-231 and MCF-7 cells on/in soft and stiff HTA
gels (scale bars 200 µm). (B) SEM images of MDA-MB-231 and MCF-7 cells
on soft and stiff HTA hydrogels with different magnifications. (C) IF
images showing the expression of YAP (green), as well as the morphology
of cells by β-actin (green). DAPI (blue) was used for nuclear
counterstaining (scale bars 200 µm).
Figure 3. Gene expression analyses of mechano-related
genes in MDA-MB-231 and MCF-7 cells on/in hydrogels . (A) The
expressions of YAP , TAZ , Rhoa and FAK in
MDA-MB-231 cells on/in soft and stiff HTA hydrogels. (B) The expressions
of YAP , TAZ , RhoA and FAK in MCF-7 cells
on/in soft and stiff HTA hydrogels. Error bars denote Mean ± SD for
three independent experiments (biological replicate=3, technical
replicate=3) , p-significance as determined by Student’s t-test
(*** p < .0001, ** p < .001, * p < .05).
Figure 4. Metabolic plasticity ofMDA-MB-231 and MCF-7
cells in different matrix conditions (Biological replicate=3, technical
replicate=3). (A and B) Effect of matrix stiffness on breast cancer
cells in 2D and 3D conditions: (a) PLS-DA score plots that represents
metabolomics discrimination between MDA-MB-231 (soft, Green), MCF-7
(soft, Pink), MDA-MB-231 (stiff, Violet), and MCF-7 (stiff, Turquoise).
(b) VIP score plot indicating the top 15 most significant metabolites in
discrimination between the groups, p < 0.05 as determined by
one-way ANOVA. (c) Heat-map with hierarchical clustering analysis
obtained by altered metabolites in MDA-MB-231 and MCF-7 cells in soft
and stiff gels in 2D culture. (C) Pathway impact analysis representing
the affected metabolic pathways by stiffness-mediated differentially
expressed metabolites in 2D and 3D conditions. (D) Venn scheme
summarizing the significantly altered metabolites in 2D and 3D
conditions.
Figure 5. Mechano-metabolomic analyses (biological
replicate=3, technical replicate=3). (A) Effect of matrix stiffness
specifically on individual cell type under 2D and 3D conditions: (a)
Volcano plots demonstrating the significantly up- and down-regulated
metabolites in MDA-MD-231 and MCF-7 cells on soft and stiff matrices.
(b) Pathway impact analyses representing the affected metabolic pathways
by differentially expressed metabolites in MDA-MD-231 and MCF-7 cells on
soft and stiff matrices. (c) Venn scheme summarizing the altered and
common metabolites MDA-MD-231 and MCF-7 cells on soft and stiff
matrices. n= 3 and * p < 0.05 as determined by Student’s
t-test.
Figure 6. DOX anti-cancer drug testing. (A) The
representative scheme of doxorubicin anti-cancer drug treatment
experimental design. (B) Anti-cancer drug (0.2 µg/ml DOX, 72h) response
of MDA-MB-231 and MCF-7 cells in 2D and 3D, soft and stiff gels (n=3).
Percentage cell viability was presented. Error bars denote Mean ± SD for
three independent experiments, p-significance as determined by Student’s
t-test (*** p < .001, ** p < .01, * p <
.05). (C) An illustration explaining the synergistic effects of matrix
stiffness, dimension, and metabolomic plasticity on the anti-cancer drug
sensitivity of breast cancer cells.