Rapid disorganization of mechanically interacting systems of mammary acini

Abstract

Cells and multicellular structures can mechanically align and concentrate fibers in their ECM environment and can sense and respond to mechanical cues by differentiating, branching, or disorganizing. Here we show that mammary acini with compromised structural integrity can interconnect by forming long collagen lines. These collagen lines then coordinate and accelerate transition to an invasive phenotype. Interacting acini begin to disorganize within 12.5 ± 4.7 h in a spatially coordinated manner, whereas acini that do not interact mechanically with other acini disorganize more slowly (in 21.8 ± 4.1 h) and to a lesser extent (P < 0.0001). When the directed mechanical connections between acini were cut with a laser, the acini reverted to a slowly disorganizing phenotype. When acini were fully mechanically isolated from other acini and also from the bulk gel by box-cuts with a side length <900 μm, transition to an invasive phenotype was blocked in 20 of 20 experiments, regardless of waiting time. Thus, pairs or groups of mammary acini can interact mechanically over long distances through the collagen matrix, and these directed mechanical interactions facilitate transition to an invasive phenotype.

Keywords: cancer; mechanobiology.

Fig. 1.

Mechanical remodeling of collagen substrates by mammary acini. (A) Schematic of experiment. Acini are placed on collagen 1 gels and subsequent disorganization is quantified. (B) Acini allowed to settle on collagen gels gradually disorganize as shown by changes in acinar morphology (nuclei stained blue) and actin cytoskeleton (red). (C) Low-magnification overview of a system of ∼200 acini (red) on collagen 1 (green). Initially, the collagen is uniformly distributed and the acini are intact. (D) System of ∼200 acini after 20 h. Many acini (red) have disorganized into single cells (tiny red dots), which are scattering. The collagen has been extensively reorganized; the average green signal has dropped and bright green collagen lines run along geodesics between acini.

Fig. 2.

Acceleration of disorganization in interacting acini. (A) Acini pull collagen radially inward, as shown by gradual bead enrichment near and under the acini. (Scale bar, 100 μm.) (Inset) Initial bead distribution. (B) Two acini interacting via a collagen line (green). Zoom 1, detail of collagen alignment in line. Zoom 2, detail of acinus (nuclei, red) and radially aligned collagen (green). (C) SEM image of collagen ultrastructure between interacting acini. Zoom 1–2, higher magnifications of aligned fibers comprising collagen line. Zoom 3, aligned collagen fibers can coalesce into higher order collagen bundle. (D) Survival analysis using Kaplan–Meier curves and the log-rank test show that interacting acini (blue) disorganize more rapidly than noninteracting acini (red). (E) Change of area of interacting (blue) and noninteracting (red) contractile acini. Interacting acini spread more rapidly than noninteracting acini (significantly different with P < 0.001 for t > 4 h) but note two exceptions (black stars), consisting of rapidly disorganizing noninteracting acini. Red line, mean; Gray shaded region, 95% CI. (F) Local collagen velocity near interacting (blue) and noninteracting (red) contractile acini. Velocities are significantly different with P < 0.001 for 1 < t < 13 h.

Fig. 3.

Mechanical control of transition to invasive phenotype. (A) Example of cutting experiment. Acini are allowed to interact and then the collagen line between them is cut (gap = dashed white line). (B) Some interacting acini are able to bypass the gap by reconnecting via lines that travel around the gap (white arrows, bypass). The image is a detail of a much larger automated multitile acquisition, similar to Fig. 1 C and D. (C) Example of the box-cut geometry (dashed white line), which isolates an acinus from its neighbors and also the bulk gel. Inset 1, before cutting. Inset 2, immediately after cutting. (D) Mechanically isolated acini do not disorganize despite extensive collagen pulling (arrow). (E) Mean collagen intensity vs. time for isolated (i.e., box cut as in C and D), noninteracting, or interacting acini. Observations start at t = 10 h due to time needed to move acini from laser cutting microscope to confocal incubator microscope. (F) Acinar phenotypes vs. box radius. Acini on the smallest boxes are contractile and can change their circularity slightly over 24 h, but they do not spread or scatter (Movie S13).

Fig. 4.

Mechanical reprogramming of protrusion site and direction. Initially, acinus 3 rapidly pulls collagen, and cells begin to leave acinus 2 toward acinus 3 at t = 7 h. Then at t = 15 h, acinus 1 begins to pull more rapidly than acinus 3. Ultimately, the middle acinus disorganizes toward acinus 1. Raw velocity maps, image segmentation, and complete movie are shown in Fig. S7 and Movie S12.

Fig. 5.

Expression changes during acinar disorganization. (A) Cells toward the top of the acinus have robust β-catenin expression (Upper), unlike cells toward the bottom of the acinus and in direct contact with collagen (Lower). (B) The opposite pattern is seen with the mesenchymal marker Vimentin. Individual cells in contact with and spreading on the collagen have high Vimentin (Lower) unlike the majority of cells at the top of the acinus (Upper). (C) Leader cells leaving the acini along collagen lines have prominent perinuclear Vimentin and mesenchymal cell architecture. (D) Cells at the acinus/collagen interface have lost E-cadherin. (Inset) Zoom of a pioneer migratory cell on collagen line with no detectable E-cadherin. (E) Cells leaving acini along lines sense a stiffened environment as judged by nuclear relocation of the YAP mechanosensory protein (white arrows).