Journal: bioRxiv

Article Title: Breast cancer cell migration is potentiated by associated fibroblasts through a laminin-511-Integrin α6β1-transduced Arp2/3 localization

doi: 10.1101/2025.08.10.669320

Figure Lengend Snippet: (A) Representative confocal photomicrographs of NF (left), CAF1 (middle), and CAF2 (right) showing staining using cognate antibodies for laminin γ1 (top, red), laminin β1 (second from top, red), laminin α5 (third from top, green) and laminin α1 (bottom most, green). The DNA is stained with DAPI (white), scale bar: 50 μm (n = 3). (B) Immunoblot showing signals using antibodies against laminin β1 (top) and laminin γ1 (middle) levels in NF (left most), CAF (second from left), NF-CM (third from left) and CAF-CM (right most) normalized to β-Actin (bottom), n = 3. (C) Representative immunohistochemistry images showing signal (in brown) using antibodies against laminin α5 (top), laminin γ1 (middle), and laminin β1 (bottom) in fibroadenoma (left) and invasive ductal breast carcinoma sections (right), n = 4 for breast carcinoma, n = 2 for fibroadenoma, scale bar: 20 μm. (D) Representative confocal photomicrographs of maximum intensity projections of RFP-expressing MDA-MB-231 clusters with NF (top) and CAF (bottom) invading into surrounding collagen I for 24 h and observed with staining for Laminin-511 (green): Only Laminin-511 staining (left), merged with RFP of MDA-MB231 (right), scale bar: 50 μm (n = 3). (Ei) Representative epi-fluorescence micrographs taken at 20 h from time-lapse videography of clusters of RFP-labelled MDA-MB-231 cells invading into surrounding Collagen I, MDA-MB-231 with CAF-shSc (1:1, left most), MDA-MB-231 mixed with CAF-shLAMB1 (1:1, second from left), MDA-MB-231 mixed with CAF-shLAMC1 (1:1, third from left) and MDA-MB-231 mixed with CAF-shLAMA5 (1:1, right most), bright field images on top panel and RFP images on bottom panel, scale bar: 100 μm (n = 3), see also video S5A, S5B, S5C, S5D. (Eii) Scatter plot graph showing number of dispersed single cancer cells in Collagen I normalized to the initial cluster size ( n = 3). Error bars denote mean□±□SEM. One-way ANOVA with Tukey’s multiple comparison test was performed for statistical significance.

Article Snippet: For Arp2/3 inhibition, 100 μM CK-666 (MedChemExpress, HY-16926) or DMSO was added to the wells with cancer cells for 12 h. Integrin-blocking experiments were performed by seeding cells on laminin-511 substrata in presence of 5 μg/ml anti-β1, anti-α3, anti-α6, anti-β4 or 250 μm RGD tripeptide (MedChemExpress, HY-P0278) for 12 h. Time-lapse imaging of migratory cancer cells was then performed on Olympus IX83 Inverted Epi-fluorescence microscope fitted with stage top incubator and 5% carbon dioxide.

Techniques: Staining, Western Blot, Immunohistochemistry, Expressing, Fluorescence, Comparison

Journal: bioRxiv

Article Title: Breast cancer cell migration is potentiated by associated fibroblasts through a laminin-511-Integrin α6β1-transduced Arp2/3 localization

doi: 10.1101/2025.08.10.669320

Figure Lengend Snippet: (Ai) Representative photomicrographs showing adhesion of MDA-MB-231 on BSA (top left), laminin-511 (top right), laminin-111 (bottom left), and laminin-211 (bottom right) substrata, scale bar: 50 μm. Graph depicting cell adhesion on different laminins measured by mean fluorescence intensity of propidium iodide (PI) stained attached cells ( n ≥ 3). (Bi) Epifluorescence photomicrographs of RFP-labelled MDA-MB-231 cells with their migration tracks for 3 hours, on BSA (top left), laminin-511 (top right), laminin-111 (bottom left), and laminin-211 (bottom right) substrata, scale bar: 50 μm. (Bii) Graph depicting mean migration velocity (left) and (Biii) elongation dynamics entropy (right), (n=3, N > 30). See also video S6A, S6B, S6C, S6D (C) Schematic depiction of MDA-MB-231 spheroid migration assay on different laminin matrices. (Di) Representative epi-fluorescence micrographs taken at 24 h from time-lapse videography of clusters of GFP-labelled MDA-MB-231 cells suspended over, and allowed to migrate on, different substrata: BSA (control, left top), laminin-211 (right top), laminin-511 (left bottom) and laminin-111 (right bottom), See also video S7A, S7B, S7C, S7D Scale bar: 50□μm. (Dii) Scatter plot graph showing number of dispersed single cancer cells in normalized to the initial cluster size (top) and (Diii) total distance covered by single cancer cells coming out of the cluster (right) obtained from time lapse videography ( n □= 3). Error bars denote mean□±□SEM. One-way ANOVA with Tukey’s multiple comparison test was performed for statistical significance

Article Snippet: For Arp2/3 inhibition, 100 μM CK-666 (MedChemExpress, HY-16926) or DMSO was added to the wells with cancer cells for 12 h. Integrin-blocking experiments were performed by seeding cells on laminin-511 substrata in presence of 5 μg/ml anti-β1, anti-α3, anti-α6, anti-β4 or 250 μm RGD tripeptide (MedChemExpress, HY-P0278) for 12 h. Time-lapse imaging of migratory cancer cells was then performed on Olympus IX83 Inverted Epi-fluorescence microscope fitted with stage top incubator and 5% carbon dioxide.

Techniques: Fluorescence, Staining, Migration, Control, Comparison

Journal: bioRxiv

Article Title: Breast cancer cell migration is potentiated by associated fibroblasts through a laminin-511-Integrin α6β1-transduced Arp2/3 localization

doi: 10.1101/2025.08.10.669320

Figure Lengend Snippet: (A) Representative photomicrographs showing actin dynamics taken at 0 h, 1.5 h and 3 h (left to right) from time-lapse videography of MDA-MB-231 cells migrating on laminin-511 (top) and laminin-211 (bottom) substrata using SPY555-FastAct fluorescent live cell F-actin probe, black colour showing actin cytoskeleton; red arrowheads representing actin branching (n = 3), see also video S9A, S9B. (B) Representative confocal photomicrographs of MDA-MB-231 cells treated with CAF-CM (top) and with NF-CM (bottom) and observed after 48□h of culture with staining for□DNA (white using DAPI, left panel) and Arp2/3 (cognate antibody; green, right panel (n□= 3). Scale bar: 50□μm. (C) Representative confocal photomicrographs of MDA-MB-231 cells grown on laminin-511 (top), laminin-211 (bottom) and observed after 24□h of culture with staining for Arp2/3 (green, left panel),□DNA (white using DAPI, middle panel), and F-actin (red using phalloidin, right panel (n□= 3). Scale bar: 20□μm. (D) Epifluorescence photomicrographs showing zoomed-in images with signals for F-actin (red; top) and Arp2/3 (green; bottom) on laminin-511 from figure (C). (Ei) Epifluorescence photomicrographs of RFP-labelled MDA-MB-231 cells with their migration tracks for 3 hours, on laminin-511 with vehicle treatment (left), and with Arp2/3 inhibitor on laminin-511 (middle), and laminin-211 (right), scale bar: 50 μm. (Eii) Graph depicting mean migration velocity (left) and (Eiii) elongation dynamics entropy (right), (n=3, N > 14). See video S10A, S10B, S10C. Error bars denote mean□±□SEM. One-way ANOVA with Tukey’s multiple comparison test was performed for statistical significance.

Article Snippet: For Arp2/3 inhibition, 100 μM CK-666 (MedChemExpress, HY-16926) or DMSO was added to the wells with cancer cells for 12 h. Integrin-blocking experiments were performed by seeding cells on laminin-511 substrata in presence of 5 μg/ml anti-β1, anti-α3, anti-α6, anti-β4 or 250 μm RGD tripeptide (MedChemExpress, HY-P0278) for 12 h. Time-lapse imaging of migratory cancer cells was then performed on Olympus IX83 Inverted Epi-fluorescence microscope fitted with stage top incubator and 5% carbon dioxide.

Techniques: Staining, Migration, Comparison

Journal: bioRxiv

Article Title: Breast cancer cell migration is potentiated by associated fibroblasts through a laminin-511-Integrin α6β1-transduced Arp2/3 localization

doi: 10.1101/2025.08.10.669320

Figure Lengend Snippet: (Ai) Epifluorescence photomicrographs of RFP-labelled MDA-MB-231 cells on laminin-511 with their migration tracks for 3 hours, treated with vehicle (left most), function blocking antibodies against integrin-α3 (second from left), Integrin-α6 (third from left), Integrin-β1 (fourth from left), and Integrin-β4 (right most), scale bar: 50 μm, see video S12A, S12B, S12C, S12D, S12E. (Aii) Graph depicting mean migration speed on Laminin-511 under treatment with antibodies shown in Ai (n=3, N =10). (B) Representative confocal photomicrographs of MDA-MB-231 cells on Laminin-511 treated with vehicle (top left), with antibodies against Integrin- β1 (5 µg/ml, top right), Integrin- α6 (5 µg/ml, bottom left) and Integrin- α3 (5 µg/ml, bottom right) and observed after 24□h of culture with staining using cognate antibody for Arp2/3 (green, left panel), □DNA (white using DAPI, middle panel) and F-actin (red using phalloidin, right panel (n□= 3). Scale bar: 50□μm. (Bii) Graph depicting mean percentage of cells with cortical Arp2/3 (n = 3). (C) Representative confocal photomicrographs of MDA-MB-231 cells treated with NF-CM (left) and with CAF-CM (right) and observed after 48□h of culture with staining for DNA (white using DAPI, left panel) and Integrin-α6 (green, right panel (n□= 3). Scale bar: 20□μm. (D) Representative confocal photomicrographs of MDA-MB-231 (left) and HCC1806 (right) cells on laminin-511 (top), and laminin-211 (bottom) and observed after 24□h of culture with staining for Integrin-α6 (green, left panel),□DNA (white using DAPI, middle panel) and F-actin (red using phalloidin, right panel (n□= 3). Scale bar: 20□μm.

Article Snippet: For Arp2/3 inhibition, 100 μM CK-666 (MedChemExpress, HY-16926) or DMSO was added to the wells with cancer cells for 12 h. Integrin-blocking experiments were performed by seeding cells on laminin-511 substrata in presence of 5 μg/ml anti-β1, anti-α3, anti-α6, anti-β4 or 250 μm RGD tripeptide (MedChemExpress, HY-P0278) for 12 h. Time-lapse imaging of migratory cancer cells was then performed on Olympus IX83 Inverted Epi-fluorescence microscope fitted with stage top incubator and 5% carbon dioxide.

Techniques: Migration, Blocking Assay, Staining