Interplay of the transcription factor MRTF-A and matrix stiffness controls mammary acinar structure and protrusion formation

Abstract

Background: Ongoing differentiation processes characterize the mammary gland during sexual development and reproduction. In contrast, defective remodelling is assumed to be causal for breast tumorigenesis. We have shown recently that the myocardin-related transcription factor A (MRTF-A) is essential for forming regular hollow acinar structures. Moreover, MRTF-A activity is known to depend on the biochemical and physical properties of the surrounding extracellular matrix. In this study we analysed the mutual interaction of different matrix stiffnesses and MRTF-A activities on formation and maintenance of mammary acini.

Methods: Human MCF10A acini and primary mature organoids isolated from murine mammary glands were cultivated in 3D on soft and stiff matrices (200-4000 Pa) in conjunction with the Rho/MRTF/SRF pathway inhibitor CCG-203971 and genetic activation of MRTF-A.

Results: Three-dimensional growth on stiff collagen matrices (> 3000 Pa) was accompanied by increased MRTF-A activity and formation of invasive protrusions in acini cultures of human mammary MCF10A cells. Differential coating and synthetic hydrogels indicated that protrusion formation was attributable to stiffness but not the biochemical constitution of the matrix. Stiffness-induced protrusion formation was also observed in preformed acini isolated from murine mammary glands. Acinar outgrowth in both the MCF10A acini and the primary organoids was partially reverted by treatment with the Rho/MRTF/SRF pathway inhibitor CCG-203971. However, genetic activation of MRTF-A in the mature primary acini also reduced protrusion formation on stiff matrices, whilst it strongly promoted luminal filling matrix-independently.

Conclusion: Our results suggest an intricate crosstalk between matrix stiffness and MRTF-A, whose activity is required for protrusion formation and sufficient for luminal filling of mammary acini. Video Abstract.

Keywords: Mammary acini; Mechanotransduction; Mrtf; Protrusion formation; Stiffness.

Fig. 1

Correlation of increasing matrix stiffness with protrusion formation of MCF10A acini. 3D cultures of MCF10A cells on the indicated Matrigel/Collagen I mixtures (100:0, 75:25, 50:50, 25:75, 0:100) were analysed by microscopy. A Phalloidin (green), Laminin V (red) and nuclei (DAPI, blue) staining of acinar structures at day 4 after seeding on matrices with increasing Collagen I percentages. Arrowheads indicate protrusions. The corresponding increase of Young's elastic modulus of the matrix is depicted below. B Diameters of individual acini (diamonds) measured at day 4, day 8 and day 14. Black horizontal lines indicate mean diameter and SEM (whiskers) of three biologically independent replicates. C Quantification of protrusion formation at day 4, day 8 and day 14. At least 11 acini were assessed each day, the percentage of protrusion-positive acini calculated, and the entire experiment repeated 3 times. D Calculated roundness index of individual acini (diamonds). Statistical significance compared to acini formed on pure Matrigel was determined using a one-way ANOVA and the means for each time point. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Error bars, SEM (n = 3). Scale bars, 50 μm

Fig. 2

Acinar protrusions are independent of composition but depend on stiffness of matrix. A, B, C Matrigel (MG) or collagen I (COL1) gels were thinly coated with collagen IV (COL4) or left uncoated (wo COL4) as indicated. Subsequently MCF10A cells were seeded on top and acinar diameter A and protrusion formation B was analysed 4 days post seeding. C Representative micrographs of acini stained with laminin V (red), actin filaments with phalloidin (green) and cell nuclei with DAPI (blue) on day 4 of acini morphogenesis. Arrowheads indicate protrusions. D, E, F MCF10A cells were seeded on pre-cast acrylamide gels with defined stiffness of 0.2 kPa and 4 kPa coated with Matrigel, respectively. Acini diameter D and average percentage of protruding acini (E) were determined 4, 8 and 14 days post seeding as before. F Representative micrographs of acini on day 4 after seeding on the matrix indicated. Statistical significance was determined by ANOVA using the means of 3 independent experiments, each analysing ≥ 22 acini per condition. G, H Relative mRNA expression of ACTA2 G and VIM H normalised to ALAS and GAPDH. Statistical significance according to an unpaired Student’s t-test (n = 4). *p ≤ 0.05, **p ≤ 0.01. Error bars, SEM. Scale bars, 50 μm

Fig. 3

Implication of the MRTF-SRF pathway in stiffness-dependent acini morphogenesis. A Relative MRTF/SRF luciferase reporter activity in transiently transfected MCF10A seeded on Matrigel (MG) or collagen I (COLI) and analysed at day 3 of acini morphogenesis (n = 4). B, C Effects of the Rho/MRTF/SRF pathway inhibitor CCG203971 (20 μM, day 4–8) on acini diameter and average percentage of protrusion-positive acini at day 8. Eleven or more acini were analysed per condition in 3 biologically independent replicates. B Individually measured acini diameter (diamond) and mean ± SEM (horizontal black line ± whiskers). C Percentage of protrusion formation. Significance was tested using an unpaired Student’s t-test. *p ≤ 0.05. Error bars, SEM

Fig. 4

Matrix stiffness and MRTF/SRF activity cooperate on protrusion formation in mature murine acini. Acinar organoids were extracted from the mammary gland of 8–12 week old female NMRI mice. Organoids were seeded on matrices with increasing stiffness, and morphologies were analysed 5 days after seeding by microscopy. A Representative images of primary murine acini stained for nuclei (DAPI, blue) and actin (phalloidin, green) on matrigel or collagen I matrices 5 days after seeding. Arrowheads indicate protrusions. Scale bars, 50 μm. Individual acini diameter B, percentage of protrusion-positive acini C and percentage of filled acini D were quantified (≥ 42 individual acini per condition, one-way ANOVA). (E) Representative images of acini after 4 days of treatment with DMSO or 20 μM CCG203971. Scale bars, 25 μm. Individual acini diameter F, percentage of protrusion-positive acini G and percentage of filled acini H (≥ 24 individual acini per condition, two-way ANOVA). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Error bars or whiskers, SEM

Fig. 5

High MRTF-A activity causes luminal filling in primary mammary acini. Acinar organoids were extracted from the mammary gland of 8–12 week old female wildtype (wt) and transgenic (tg) mice carrying a LSL-MRTF-A cassette and CreERT2. Organoids were seeded on matrices with increasing stiffness and treated for 4 days with 0.25 μM Hydroxy-Tamoxifen (4OHT) or vehicle control. A Representative images of primary murine acini stained for nuclei (DAPI, blue) and actin (phalloidin, green) on matrigel/collagen I matrices 5 days after seeding. Arrowheads indicate protrusions, and asterisks the acinar lumen. B, C Distribution of individual organoid roundness and diameters. D, E Percentage of acini with filled lumen and protrusions. More than 17 acini were analysed per condition. Significance was determined by two-way ANOVA. F Genotyping of 4OHT-treated ( +) and vehicle treated ( − ) tg acini using primer flanking the loxP STOP loxP cassette (LSL, 1500 bp) in comparison to an untreated positive control sample (Ctrl). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Error bars or whiskers, SEM. Scale bars, 25 μm

Fig. 6

Model of the functional interplay between matrix stiffness and MRTF-A activity. Increasing matrix stiffness correlates with increased MRTF-A activity, enhanced protrusion formation and luminal filling. Alteration of the Rho/MRTF/SRF pathway reduces protrusions, indicating that MRTF-A is required but not sufficient for protrusion formation. In addition, increased MRTF-A activity promotes luminal filling independent of matrix stiffness in mammary organoids