Journal: Advanced Science

Article Title: Aged Breast Extracellular Matrix Drives Mammary Epithelial Cells to an Invasive and Cancer‐Like Phenotype

doi: 10.1002/advs.202100128

Figure Lengend Snippet: Aged breast ECM leads to deformation of KTB21 cell spheroids and delocalization of E‐CAD from cell membrane to cytosol. A) KTB21 spheroids on the matrices monitored for 12 days. B) Microscopy images showing the spheroid presence on matrices at day 15. Top: bright field and bottom: second harmonic generation (SHG) images. Inset: image at a deeper focal plane of the dashed square. Arrowheads point to spheroids. C,D) E‐CAD, MMP2, and COL1 staining (day 15). C) E‐CAD localization in cells. Top: representative confocal microscopy images of matrices. n = 3. Bottom: plot profile of E‐CAD signal intensity across a representative cell. n = 6 cells/matrix for young and 9 for aged. D) MMP2 and COL1 staining. Top: representative confocal microscopy images of matrices. Bottom: quantification of the signal intensities. n = 3. E‐CAD and COL1: Alexa fluor 488 (green), MMP2: Alexa fluor 647 (magenta). Nucleic acid: DAPI (blue). Quantifications are performed using the Fiji software. Data are presented as the mean ± SD. Statistical test: two‐tailed student's t ‐test.

Article Snippet: Briefly, matrices at day 15 of culture were fixed with 4% PFA and permeabilized with 0.3% Triton X‐100, and then incubated for 45 min in 5% goat serum, overnight at 4 °C in mouse anti‐human E‐CAD (Abcam), mouse anti‐human COL1 (Abcam), rabbit anti‐human MMP2 (CusaBio), rabbit anti‐human MMP9 (Abcam), and mouse anti‐human LOX (LSBio) monoclonal antibodies with 1:100 dilutions, and then for 1 h in Alexa fluor 488‐labelled goat anti‐mouse IgG (Abcam), and Alexa fluor 647‐labelled goat anti‐rabbit IgG (Abcam) with 1:400 dilutions.

Techniques: Membrane, Microscopy, Staining, Confocal Microscopy, Software, Two Tailed Test

Journal: Advanced Science

Article Title: Aged Breast Extracellular Matrix Drives Mammary Epithelial Cells to an Invasive and Cancer‐Like Phenotype

doi: 10.1002/advs.202100128

Figure Lengend Snippet: LOX knockdown reverses the phenotypic and biochemical changes in KTB21 cells induced by the aged microenvironment. A) Microscopy images showing MMP2, MMP9, E‐CAD, and LOX expression, and spheroid stability on matrices (day 15). siRNAs applied between days 8–10. Fluorescence microscopy images showing MMP2 and MMP9 (magenta, Column 1), E‐CAD and LOX (green, Column 2), and nucleic acid (DAPI, blue, Column 3) staining, and merged images (Column 4). Bright field images (Column 5) showing the spheroids on matrices. n = 4. Left: young matrices, and right: aged matrices. Upper panel: scramble siRNA, and lower panel: LOX siRNA treated matrices. Arrowheads show spheroids. Results representative of two independent experiments. B) Plot profile of E‐CAD signal intensity along the diameter of representative cells in (A) showing E‐CAD localization in the cells. n = 4 images/matrix, and 8–10 cells/image. C) Cell number‐normalized signal intensities of MMP2, MMP9, E‐CAD, and LOX as quantified from (A). n = 4 matrices. Representative of three independent experiments. D,E) Heat map showing the dot blot‐based cytokine profiling of the cells on matrices (day 15). D) Cytokines and E) cancer‐associated proteins expressed by the cells. n = 3 pooled samples. Also see Tables and , Supporting Information. Quantifications were performed using the Fiji software. Data are presented as the mean ± SD. Statistical tests: one‐way ANOVA followed by Tukey's post hoc.

Article Snippet: Briefly, matrices at day 15 of culture were fixed with 4% PFA and permeabilized with 0.3% Triton X‐100, and then incubated for 45 min in 5% goat serum, overnight at 4 °C in mouse anti‐human E‐CAD (Abcam), mouse anti‐human COL1 (Abcam), rabbit anti‐human MMP2 (CusaBio), rabbit anti‐human MMP9 (Abcam), and mouse anti‐human LOX (LSBio) monoclonal antibodies with 1:100 dilutions, and then for 1 h in Alexa fluor 488‐labelled goat anti‐mouse IgG (Abcam), and Alexa fluor 647‐labelled goat anti‐rabbit IgG (Abcam) with 1:400 dilutions.

Techniques: Knockdown, Microscopy, Expressing, Fluorescence, Staining, Dot Blot, Software