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.