Ultrasound-Induced Mechanical Compaction in Acoustically Responsive Scaffolds Promotes Spatiotemporally Modulated Signaling in Triple Negative Breast Cancer

Abstract

Cancer cells continually sense and respond to mechanical cues from the extracellular matrix (ECM). Interaction with the ECM can alter intracellular signaling cascades, leading to changes in processes that promote cancer cell growth, migration, and survival. The present study used a recently developed composite hydrogel composed of a fibrin matrix and phase-shift emulsion, termed an acoustically responsive scaffold (ARS), to investigate effects of local mechanical properties on breast cancer cell signaling. Treatment of ARSs with focused ultrasound drives acoustic droplet vaporization (ADV) in a spatiotemporally controlled manner, inducing local compaction and stiffening of the fibrin matrix adjacent to the matrix-bubble interface. Combining ARSs and live single cell imaging of triple-negative breast cancer cells, it is discovered that both basal and growth-factor stimulated activities of protein kinase B (also known as Akt) and extracellular signal-regulated kinase (ERK), two major kinases driving cancer progression, negatively correlate with increasing distance from the ADV-induced bubble both in vitro and in a mouse model. Together, these data demonstrate that local changes in ECM compaction regulate Akt and ERK signaling in breast cancer and support further applications of the novel ARS technology to analyze spatial and temporal effects of ECM mechanics on cell signaling and cancer biology.

Keywords: Akt; ERK; fibrin; mechanobiology; phase-shift emulsions; triple-negative breast cancer; ultrasound.

Figure 1.. Acoustic droplet vaporization (ADV) resulted in local compaction and stiffening of fibrin surrounding the bubbles.

A) Confocal microscopy images of acoustically-responsive scaffolds (ARSs) before and after ADV are shown. For matrix visualization, ARSs contained Alexa Fluor 647-labeled fibrinogen (fibrinogen₆₄₇). B) Longitudinal, 1D intensity profiles of fibrinogen₆₄₇, were derived from confocal images containing a single ADV-generated bubble (like Day 0 and Day 2 images in panel A). 0 μm corresponds to the bubble-fibrin interface. C) Gaussian fitting was performed on the intensity profiles to calculate the full width at half maximum (FWHM) thickness of the consolidated fibrin region (n=8 (Day 0), n=13 (Day 2), n=18 (Day 3)). Statistically significant differences (p < 0.05) are denoted as follows: α vs. Day 0. D) A 1D profile of the Young’s modulus was generated one hour post-ADV using atomic force microscopy. 0 μm corresponds to the bubble-fibrin interface. Scale bar: 20μm.

Figure 2.. Imaging acoustically-responsive scaffolds containing breast cancer cells to measure cell signaling.

A. Schematic of the acoustically-responsive scaffold (ARS). ARSs containing breast cancer cells and a phase-shift emulsion (left) were exposed to focused ultrasound to generate acoustic droplet vaporization (ADV), which induces bubble formation and drives micromechanical and microstructural changes to the fibrin matrix (middle). These changes increase matrix stiffness closer to a bubble (right). B. Schematic (left) and representative images (right) of ERK and Akt kinase translocation reporters (KTRs). Phosphorylation (+P) and dephosphorylation (−P) of the kinase substrate drives the reporter into the cytoplasm (kinase “on”) or nucleus (kinase “off”), respectively. Scale bars are 20μm. C. Representative images of MDA-MB-231 breast cancer cells containing kinase translocation reporters (KTRs) for both ERK and Akt kinases near a bubble (white arrow) generated by ADV. Scale bar is 50μm.

Figure 3.. Enhanced signaling in MDA-MB-231 breast cancer cells closer to the ADV-induced bubble.

A. Graphs show mean ± SEM for Akt and ERK KTRs in MDA-MB-231 breast cancer cells treated with or without focused ultrasound cultured in ARSs without (left) or with (right) the phase-shift emulsion (n ≥ 117 cells per group). We imaged cells immediately after focused ultrasound (Day 0) and for 3 subsequent days. B. We quantified activation of Akt (left) and ERK (right) (n ≥ 259 cells per group) signaling relative to distance of the cell from the bubble surface, demonstrating that cells closest to the bubble surface signal more. All graphs show a significant non-zero linear regression (p < 0.001). C. Graph shows mean ± SEM log₂ cytoplasmic-to-nuclear ratios (CNR) for Akt and ERK KTRs of cells close (< 100μm) or far (> 100μm) from the bubble at each time point (n ≥ 85 cells per group). The CNR is the log₂ measurement of the fluorescence intensity of the KTR in the cytoplasm relative to the fluorescence intensity of the KTR in the nucleus.

Figure 4.. Cancer cells nearest the bubble surface show increased EGF signaling.

After 3 days in an ARS, we performed time-lapse imaging of Akt and ERK KTRs before (0 min) and 30 min after addition of EGF (50 ng/mL). A. Graphs show summaries (mean ± SD) of Akt (left) and ERK (right) KTR activation at the initial time point before addition of EGF (n = 20 cells per group). * = p < 0.05. Dashed line represents the mean log₂ CNR in cells proximal (< 100μm) to the bubble. B. Single-cell time tracks show EGF-dependent activation of Akt and ERK in individual MDA-MB-231 breast cancer cells with KTR values displayed on a pseudocolor scale. We normalized data to Akt and ERK values at the initial time point for each cell (0 min). The graph sorts cells by distance from the bubble surface with cells nearest the bubble on top. The purple line separates cells nearest the bubble (< 100μm, n = 20 cells) from those furthest (> 100μm, n = 20 cells) from the bubble. C. Graphs show normalized area under the curve (AUC) for Akt (left) and ERK (right) signaling from (B) relative to distance of a cell from the bubble surface, demonstrating that cells most proximal to the bubble surface signal the most. Both graphs show a significant non-zero linear regression (p < 0.01).

Figure 5.. Acoustically-responsive scaffolds (ARSs) increase MDA-MB-231 signaling in vivo.

Graphs show mean ± SEM for Akt and ERK KTRs in MDA-MB-231 breast cancer cells treated with or without targeted ultrasound cultured in ARSs with the phase-shift double emulsion (n ≥ 60 cells per group) in individual mice (n = 2 at Day 0 and n = 3 mice at Day 3) (A) or with the mice combined (B). We imaged cells immediately after targeted ultrasound (Day 0) and 3 days later (Day 3). C. We quantified and combined the activation of Akt (left) and ERK (right) (n = 215 cells per group) signaling relative to distance of a cell from the bubble surface for each mouse, demonstrating that cells closest to the bubble surface signal more. Both graphs show a significant non-zero linear regression (p < 0.001).