mcf10a  (ATCC)

Journal: bioRxiv

Article Title: Oncogenic Ras-Src-cortactin signaling rewires actin-generated forces to drive basement membrane rupture and initiate breast cancer invasion

doi: 10.64898/2026.04.15.717430

Figure Lengend Snippet: (A) Schematic of the spheroid morphogenesis assay. Single MCF10A/HRAS or MCF10A wild-type cells were cultured in a collagen IV/laminin-rich EHS(Engelbreth-Holm-Swarm) hydrogel to generate basoapically polarized spheroids after 10 days in culture (DiC). (B) Representative immunofluorescence micrographs show differences in basoapical polarization of MCF10A spheroids at 10 DiC depending on HRas activation status. BM (collagen IV, yellow), F-actin cytoskeleton (magenta), nuclei (DAPI, blue) and Golgi protein (GM130, green). (C) HRas activation confirmed by pERK immunofluorescence after 1 hour OHT or EtOH treatment. Representative immunofluorescence intensities of intracellular pERK protein (inverted grey scale) in MCF10A/HRAS spheroids treated with OHT or EtOH for 16 hours. SAC: secondary antibody control. Right, quantification of mean pERK intensity per spheroid (n ≥ 44; 3 independent experiments). Box: interquartile range; whiskers: 5th–95th percentiles; red dots: median. (D) Phase-contrast images show the invasive transition of spheroids (10 DiC) with cell transmigration into the EHS matrix after 65 hours of HRas activation with OHT. EtOH-treated HRas off controls remained non-invasive. Kolmogorov-Smirnov test was performed for the data in C; n.s.: p > 0.05; ****: p ≤ 0.0001. Scale bars: 20 µm (B); 50 μm (C). Position of focal plane used for imaging and analyses is indicated by red bar.

Article Snippet: MCF10A wildtype cells (purchased from ATCC), MCF10A_ER:HRas G12V and MCF10A_HRas G12V cells described in and kindly provided by Buzz Baum were maintained in culture dishes under standard culture conditions (37 °C, 5% CO 2 ) in DMEM/F12 growth medium (ThermoFisher Scientific) containing 5% horse serum (ThermoFisher Scientific) or steroid hormone free horse serum (c.c.pro) (in all experiments from onwards), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 20 ng/mL EGF, 10 μg/mL insulin (all Sigma Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin (both ThermoFisher Scientific).

Techniques: Cell Culture, Immunofluorescence, Activation Assay, Control, Transmigration Assay, Imaging

Journal: bioRxiv

Article Title: Oncogenic Ras-Src-cortactin signaling rewires actin-generated forces to drive basement membrane rupture and initiate breast cancer invasion

doi: 10.64898/2026.04.15.717430

Figure Lengend Snippet: (A) Scheme of BM disruption and cell invasion assay. MCF10A/HRAS spheroids (10 DiC) were isolated from EHS matrix and placed on elastomeric substrates (16 kPa, functionalized with EHS proteins) to count events of local BM rupture and cell transmigration. Mechanical BM stress exertion by breast spheroids at time point of invasion onset was measured by traction force microscopy (TFM): Surface-coupled fluorescent fiducial microbeads were used to track tangential surface deformations from which strain energies were calculated as measure for cell force-generated BM stress. (B) In spheroids, the outer basal cell layer is covered by a BM which itself is in contact to the underlying substrate. Images show the BM integrity of a representative HRas on sample, fixed and stained after adhering (1 hour) to the elastomeric substrate. Collagen IV (yellow), laminin-332 (cyan) and F-actin cytoskeleton (magenta). Zoom in highlights in vivo -like layering of the endogenous BM. (C) Representative sequence of phase-contrast images illustrates the first appearance of protrusive cell bodies (also shown as zoom in), marking onset of BM disruption and cell transmigration. This was counted as a positive event of invasion. (D) Cumulative distribution of BM disruption time, depending on HRas induction on 16 kPa substrates (n ≥ 79 spheroids of ≥ 3 independent experiments). (E) Cumulative distribution of BM disruption time in spheroids treated with blebbistatin for myosin II inhibition and additionally with marimastat for MMP inhibition after HRas induction on stiff 16 kPa substrates (n ≥ 69 spheroids of ≥ 3 independent experiments). (F) Scatter plot shows individual invasion onset time points for the sample conditions analyzed in (D and E) (median and 95% confidence interval (CI)). (G) Calculated strain energies (SE) exerted by individual spheroids at onsets of BM disruption, depending on HRas activation and actomyosin inhibition, (cf. D and E). Representative maps of cell-induced traction stresses per condition from which SE were calculated. Scatter plot: median with 95% CI (n ≥ 48 from 3 independent experiments). Kruskal-Wallis test with Dunn’s multiple comparison test was performed for the data in D and E; n.s.: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001. Scale bars: 20 µm (B, C and G).

Article Snippet: MCF10A wildtype cells (purchased from ATCC), MCF10A_ER:HRas G12V and MCF10A_HRas G12V cells described in and kindly provided by Buzz Baum were maintained in culture dishes under standard culture conditions (37 °C, 5% CO 2 ) in DMEM/F12 growth medium (ThermoFisher Scientific) containing 5% horse serum (ThermoFisher Scientific) or steroid hormone free horse serum (c.c.pro) (in all experiments from onwards), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 20 ng/mL EGF, 10 μg/mL insulin (all Sigma Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin (both ThermoFisher Scientific).

Techniques: Disruption, Invasion Assay, Isolation, Transmigration Assay, Microscopy, Generated, Staining, In Vivo, Sequencing, Inhibition, Activation Assay, Comparison

Journal: bioRxiv

Article Title: Oncogenic Ras-Src-cortactin signaling rewires actin-generated forces to drive basement membrane rupture and initiate breast cancer invasion

doi: 10.64898/2026.04.15.717430

Figure Lengend Snippet: (A) Representative images of MT1-MMP (inversed grey scale) staining of MCF10A/HRas spheroids incubated with 1 µM OHT or EtOH (HRas off control) for 16 hours, and the secondary antibody control (SAC) to measure unspecific background signals. (B) Quantification of fluorescence intensities of MT1-MMP staining (n = 60 and n = 30 for SAC from three and two individual staining experiments, respectively). Scatter plot includes median and 95% CI. Mann-Whitney-U-test was performed for the data (n.s.: p > 0.05). Scale bars: 20 µm. Position of focal plane used for imaging and analyses is indicated by red bar.

Article Snippet: MCF10A wildtype cells (purchased from ATCC), MCF10A_ER:HRas G12V and MCF10A_HRas G12V cells described in and kindly provided by Buzz Baum were maintained in culture dishes under standard culture conditions (37 °C, 5% CO 2 ) in DMEM/F12 growth medium (ThermoFisher Scientific) containing 5% horse serum (ThermoFisher Scientific) or steroid hormone free horse serum (c.c.pro) (in all experiments from onwards), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 20 ng/mL EGF, 10 μg/mL insulin (all Sigma Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin (both ThermoFisher Scientific).

Techniques: Staining, Incubation, Control, Fluorescence, MANN-WHITNEY, Imaging

Journal: bioRxiv

Article Title: Oncogenic Ras-Src-cortactin signaling rewires actin-generated forces to drive basement membrane rupture and initiate breast cancer invasion

doi: 10.64898/2026.04.15.717430

Figure Lengend Snippet: (A) Schematic of the spheroid morphogenesis assay. Single MCF10A/HRAS or MCF10A wild-type cells were cultured in a collagen IV/laminin-rich EHS(Engelbreth-Holm-Swarm) hydrogel to generate basoapically polarized spheroids after 10 days in culture (DiC). (B) Representative immunofluorescence micrographs show differences in basoapical polarization of MCF10A spheroids at 10 DiC depending on HRas activation status. BM (collagen IV, yellow), F-actin cytoskeleton (magenta), nuclei (DAPI, blue) and Golgi protein (GM130, green). (C) HRas activation confirmed by pERK immunofluorescence after 1 hour OHT or EtOH treatment. Representative immunofluorescence intensities of intracellular pERK protein (inverted grey scale) in MCF10A/HRAS spheroids treated with OHT or EtOH for 16 hours. SAC: secondary antibody control. Right, quantification of mean pERK intensity per spheroid (n ≥ 44; 3 independent experiments). Box: interquartile range; whiskers: 5th–95th percentiles; red dots: median. (D) Phase-contrast images show the invasive transition of spheroids (10 DiC) with cell transmigration into the EHS matrix after 65 hours of HRas activation with OHT. EtOH-treated HRas off controls remained non-invasive. Kolmogorov-Smirnov test was performed for the data in C; n.s.: p > 0.05; ****: p ≤ 0.0001. Scale bars: 20 µm (B); 50 μm (C). Position of focal plane used for imaging and analyses is indicated by red bar.

Article Snippet: MCF10A wildtype cells (purchased from ATCC), MCF10A_ER:HRas G12V and MCF10A_HRas G12V cells described in and kindly provided by Buzz Baum were maintained in culture dishes under standard culture conditions (37 °C, 5% CO 2 ) in DMEM/F12 growth medium (ThermoFisher Scientific) containing 5% horse serum (ThermoFisher Scientific) or steroid hormone free horse serum (c.c.pro) (in all experiments from onwards), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 20 ng/mL EGF, 10 μg/mL insulin (all Sigma Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin (both ThermoFisher Scientific).

Techniques: Cell Culture, Immunofluorescence, Activation Assay, Control, Transmigration Assay, Imaging

Journal: bioRxiv

Article Title: Oncogenic Ras-Src-cortactin signaling rewires actin-generated forces to drive basement membrane rupture and initiate breast cancer invasion

doi: 10.64898/2026.04.15.717430

Figure Lengend Snippet: (A) Scheme of BM disruption and cell invasion assay. MCF10A/HRAS spheroids (10 DiC) were isolated from EHS matrix and placed on elastomeric substrates (16 kPa, functionalized with EHS proteins) to count events of local BM rupture and cell transmigration. Mechanical BM stress exertion by breast spheroids at time point of invasion onset was measured by traction force microscopy (TFM): Surface-coupled fluorescent fiducial microbeads were used to track tangential surface deformations from which strain energies were calculated as measure for cell force-generated BM stress. (B) In spheroids, the outer basal cell layer is covered by a BM which itself is in contact to the underlying substrate. Images show the BM integrity of a representative HRas on sample, fixed and stained after adhering (1 hour) to the elastomeric substrate. Collagen IV (yellow), laminin-332 (cyan) and F-actin cytoskeleton (magenta). Zoom in highlights in vivo -like layering of the endogenous BM. (C) Representative sequence of phase-contrast images illustrates the first appearance of protrusive cell bodies (also shown as zoom in), marking onset of BM disruption and cell transmigration. This was counted as a positive event of invasion. (D) Cumulative distribution of BM disruption time, depending on HRas induction on 16 kPa substrates (n ≥ 79 spheroids of ≥ 3 independent experiments). (E) Cumulative distribution of BM disruption time in spheroids treated with blebbistatin for myosin II inhibition and additionally with marimastat for MMP inhibition after HRas induction on stiff 16 kPa substrates (n ≥ 69 spheroids of ≥ 3 independent experiments). (F) Scatter plot shows individual invasion onset time points for the sample conditions analyzed in (D and E) (median and 95% confidence interval (CI)). (G) Calculated strain energies (SE) exerted by individual spheroids at onsets of BM disruption, depending on HRas activation and actomyosin inhibition, (cf. D and E). Representative maps of cell-induced traction stresses per condition from which SE were calculated. Scatter plot: median with 95% CI (n ≥ 48 from 3 independent experiments). Kruskal-Wallis test with Dunn’s multiple comparison test was performed for the data in D and E; n.s.: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001. Scale bars: 20 µm (B, C and G).

Article Snippet: MCF10A wildtype cells (purchased from ATCC), MCF10A_ER:HRas G12V and MCF10A_HRas G12V cells described in and kindly provided by Buzz Baum were maintained in culture dishes under standard culture conditions (37 °C, 5% CO 2 ) in DMEM/F12 growth medium (ThermoFisher Scientific) containing 5% horse serum (ThermoFisher Scientific) or steroid hormone free horse serum (c.c.pro) (in all experiments from onwards), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 20 ng/mL EGF, 10 μg/mL insulin (all Sigma Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin (both ThermoFisher Scientific).

Techniques: Disruption, Invasion Assay, Isolation, Transmigration Assay, Microscopy, Generated, Staining, In Vivo, Sequencing, Inhibition, Activation Assay, Comparison

Journal: bioRxiv

Article Title: Oncogenic Ras-Src-cortactin signaling rewires actin-generated forces to drive basement membrane rupture and initiate breast cancer invasion

doi: 10.64898/2026.04.15.717430

Figure Lengend Snippet: (A) Representative images of MT1-MMP (inversed grey scale) staining of MCF10A/HRas spheroids incubated with 1 µM OHT or EtOH (HRas off control) for 16 hours, and the secondary antibody control (SAC) to measure unspecific background signals. (B) Quantification of fluorescence intensities of MT1-MMP staining (n = 60 and n = 30 for SAC from three and two individual staining experiments, respectively). Scatter plot includes median and 95% CI. Mann-Whitney-U-test was performed for the data (n.s.: p > 0.05). Scale bars: 20 µm. Position of focal plane used for imaging and analyses is indicated by red bar.

Article Snippet: MCF10A wildtype cells (purchased from ATCC), MCF10A_ER:HRas G12V and MCF10A_HRas G12V cells described in and kindly provided by Buzz Baum were maintained in culture dishes under standard culture conditions (37 °C, 5% CO 2 ) in DMEM/F12 growth medium (ThermoFisher Scientific) containing 5% horse serum (ThermoFisher Scientific) or steroid hormone free horse serum (c.c.pro) (in all experiments from onwards), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 20 ng/mL EGF, 10 μg/mL insulin (all Sigma Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin (both ThermoFisher Scientific).

Techniques: Staining, Incubation, Control, Fluorescence, MANN-WHITNEY, Imaging