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Cancer cells become less deformable and more invasive with activation of β-adrenergic signaling - PubMed

  • ️Fri Jan 01 2016

. 2016 Dec 15;129(24):4563-4575.

doi: 10.1242/jcs.194803. Epub 2016 Nov 14.

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Cancer cells become less deformable and more invasive with activation of β-adrenergic signaling

Tae-Hyung Kim et al. J Cell Sci. 2016.

Abstract

Invasion by cancer cells is a crucial step in metastasis. An oversimplified view in the literature is that cancer cells become more deformable as they become more invasive. β-adrenergic receptor (βAR) signaling drives invasion and metastasis, but the effects on cell deformability are not known. Here, we show that activation of β-adrenergic signaling by βAR agonists reduces the deformability of highly metastatic human breast cancer cells, and that these stiffer cells are more invasive in vitro We find that βAR activation also reduces the deformability of ovarian, prostate, melanoma and leukemia cells. Mechanistically, we show that βAR-mediated cell stiffening depends on the actin cytoskeleton and myosin II activity. These changes in cell deformability can be prevented by pharmacological β-blockade or genetic knockout of the β2-adrenergic receptor. Our results identify a β2-adrenergic-Ca2+-actin axis as a new regulator of cell deformability, and suggest that the relationship between cell mechanical properties and invasion might be dependent on context.

Keywords: Atomic force microscopy; Cancer; Cell mechanical properties; Invasion; Mechanotype; Parallel microfiltration; β2-adrenergic receptor.

© 2016. Published by The Company of Biologists Ltd.

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Conflict of interest statement

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.

Activation of βAR signaling reduces the deformability of cancer cells. (A) Relative retention of MDA-MB-231 cells measured by parallel microfiltration (PMF) and normalized to the vehicle (water) treatment after treating with varying concentrations of isoproterenol (n>5). (B) Density scatterplot showing transit time and cell size from microfluidic deformability cytometry. The crossmark indicates the median transit time and median cell size for each sample. (C) Transit time for cells to pass through the 9 μm×10 μm channel of a microfluidic device (n>59). Unless otherwise stated, the box plot shows the lower and upper quartiles with median (line). The whiskers show the 10–90th percentiles. (D) Representative force curves from AFM measurements. Force curves are fitted to the Hertz–Sneddon model and histograms of Young's moduli are shown in

Fig. S2A,B

. (E) Young's moduli of cells adhered to Matrigel-coated plates (n>15). (F) cAMP levels after treatment with increasing concentrations of isoproterenol (n=3). (G) Relative retention of cells after treatment with agonists (n=4). (H) Relative retention of cells measured by PMF after treatment with vehicle, isoproterenol, propranolol, and co-treatment with isoproterenol and propranolol (n=2). (I) Relative retention of cells after treatment with different concentrations of propranolol (n=2). (J) Relative retention across various cancer cell lines after isoproterenol treatment (n=3). All treatments are for 24 h prior to measurement. Owing to the variability in baseline retention from day to day, we show here data that is normalized to the vehicle control (see

Fig. S1J–L

for non-normalized data). Unless otherwise stated, all error bars represent mean±s.e.m. Iso, isoproterenol; Pro, propranolol; Veh, vehicle. *P<0.05; **P<0.01; ***P<0.001 [one-way ANOVA with Tukey's test (A,F,G,H,I), Mann–Whitney test (C,E) or unpaired t-test (J)].

Fig. 2.
Fig. 2.

Activation of βAR signaling enhances cell invasion and migration. (A) Illustration showing 3D invasion of cells in a transwell migration assay (black arrow, direction of cell invasion; blue, Matrigel; yellow, serum-free medium; red, high-serum medium; green, cells; red triangles, isoproterenol). (B) Representative images used to quantify the number of invaded cells in the transwell migration assay. Scale bar: 100 µm. (C) Quantification of the number of invaded cells normalized to that in the vehicle treatment (n=3). (D) Illustration of the modified 3D scratch wound healing assay (orange, cell culture medium; blue, Matrigel; green, cells; red triangles, isoproterenol). (E) Scratch wound invasion assay of MDA-MB-231 cells through Matrigel (black arrows, direction of cells moving into the scratch wound; yellow, initial scratch wound area; purple, area that is confluent with cells). Scale bar: 300 μm. (F,G) Relative wound density as a function of time (n=3) (F). The dashed blue line indicates the time point (56 h) that is shown in G. (H) Scratch wound migration assay of MDA-MB-231 cells on a tissue-culture treated 2D plastic substrate. Scale bar: 300 μm. (I,J) Relative wound density as a function of time (n=3) (I). The dashed blue line indicates the time point (26 h) that is shown in J. All error bars represent mean±s.e.m.; the box plots show the lower and upper quartiles with median (line), with the whiskers representing the 10–90th percentiles. Iso, isoproterenol; Pro, propranolol; Veh, vehicle. ***P<0.001 (one-way ANOVA with Tukey's test).

Fig. 3.
Fig. 3.

Activation of βAR signaling increases cytoplasmic and cortical F-actin levels in suspended cells. (A) Representative images of live MDA-MB-231 cells in suspension obtained from imaging flow cytometry. BF, brightfield; the nucleus is labeled with Hoechst 33342 and F-actin is visualized using SiR-Actin. Scale bar: 14 µm. (B,C) Quantitative analysis showing the fluorescence intensity of (B) total F-actin and (C) cortical F-actin (n>1168). (D) Confocal images of cells that are adhered to a glass slide and stained with Phalloidin–Alexa-Fluor-546 (Red) and DRAQ5 (Cyan) to visualize F-actin and the nucleus. Scale bar: 20 µm. (E,F) Quantitative analysis of individual cells in confocal images showing (E) the ratio of F-actin intensity in protrusions to total cellular F-actin intensity (n>108) and (F) the perimeter-to-convex-hull ratio (n>283). (G) Representative image showing G- and F-actin expression levels (PC, positive control treatment with phalloidin to stabilize F-actin; G, G-actin; F, F-actin). (H) Representative images showing total actin protein levels. GAPDH is used as an internal loading control (n=2). (I) Quantification of F-actin:G-actin ratios. (J) Quantification of total actin normalized to GAPDH (n=2). Actin levels in each treatment are further normalized to vehicle treatment. All error bars represent mean±s.e.m.; the box plots show the lower and upper quartiles with median (line), with the whiskers representing the 10–90th percentiles. Iso, isoproterenol; Pro, propranolol; Veh, vehicle. ***P<0.001 [Mann–Whitney test (B,C,E,F)]. The results of Iso and Iso + Pro treatment in I and J were not statistically different from Veh control (one-way ANOVA with Tukey's test).

Fig. 4.
Fig. 4.

The βAR-induced reduction in cell deformability depends on Ca2+ and actin remodeling. Relative retention of MDA-MB-231 cells measured by PMF after treatment with vehicle, BAPTA-AM, isoproterenol, and co-treatment with isoproterenol and (A) BAPTA-AM (n=3); (B) cytochalasin D (n=3); and (C) blebbistatin (n=3) (see

Fig. S1M–P

for non-normalized data). All data is normalized to the vehicle. (D) Young's moduli of adhered cells measured by AFM after blebbistatin treatment (n>17). All error bars represent mean±s.e.m.; the box plots show the lower and upper quartiles with median (line), with the whiskers representing the 10–90th percentiles. Iso, isoproterenol; Pro, propranolol; Veh, vehicle; Bleb, blebbistatin. *P<0.05; **P<0.01; ***P<0.001 [unpaired t-test (A,D) or one-way ANOVA with Tukey's test (B,C)].

Fig. 5.
Fig. 5.

Deletion of β2AR abrogates reduced cell deformability and enhanced invasiveness with βAR activation. (A) cAMP levels after non-selective or β2AR selective agonist treatment in parental MDA-MB-231 cells (Ctrl, negative control) and ADRB2 KO cells (n=4). (B) Relative retention of control and KO cells measured by PMF (n=3). (C) Relative retention of KO cells after transfection with GFP or ADRB2 (non-normalized data is displayed in

Fig. S1Q,R

). (D,E) Relative wound density during scratch wound invasion of control and KO-1a cells through Matrigel (n=5) (D). The dashed blue line indicates the time point (52 h) that is shown in E. All error bars represent mean±s.e.m.; the box plots show the lower and upper quartiles with median (line), with the whiskers representing the 10–90th percentiles. Iso, isoproterenol; Veh, vehicle. *P<0.05; **P<0.01; ***P<0.001 [one-way ANOVA with Tukey's test (A,B, and C) or unpaired t-test (E)].

Fig. 6.
Fig. 6.

Schematic illustration of putative mechanisms of how βAR may affect cell deformability and invasive activity. (1) Activation of βAR by endogenous stress neurotransmitters (epinephrine and norepinephrine) or pharmacological agonists such as isoproterenol leads to a cascade of intracellular signaling events including: (2) activation of adenylyl cyclase through G proteins; (3) production of cAMP; (4) activation of PKA by cAMP; (5) release of Ca2+ from the endoplasmic reticulum (ER) and import of Ca2+ from extracellular spaces through Ca2+ channels, which leads to; (6) an increased level of intracellular Ca2+; (7) increased F-actin in suspended cells, and increased F-actin-rich protrusions in adhered cells, which might reflect altered actin structure. In addition, pharmacological perturbation with cytochalasin D or blebbistatin suggest that F-actin remodeling and/or contractility might occur with βAR activation. (8) Activation of βAR signaling results in increased cell invasion and reduced cell deformability. These findings build on previous characterization of signaling pathways that are activated by βAR in MDA-MB-231 cancer cells (Pon et al., 2016). Solid lines, relationships supported by the literature; dotted lines, relationships explored in our study. Images are adopted from Servier Medical Art by Servier (

http://www.servier.com/Powerpoint-image-bank

) and are published under published under a Creative Commons BY license (

https://creativecommons.org/licenses/by-nc/3.0/

). –, no change; ↑, increase.

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