Characterization of tissue biomechanics and mechanical signaling in uterine leiomyoma

Abstract

Leiomyoma are common tumors arising within the uterus that feature excessive deposition of a stiff, disordered extracellular matrix (ECM). Mechanical stress is a critical determinant of excessive ECM deposition and increased mechanical stress has been shown to be involved in tumorigenesis. Here we tested the viscoelastic properties of leiomyoma and characterized dynamic and static mechanical signaling in leiomyoma cells using three approaches, including measurement of active RhoA. We found that the peak strain and pseudo-dynamic modulus of leiomyoma tissue was significantly increased relative to matched myometrium. In addition, leiomyoma cells demonstrated an attenuated response to applied cyclic uniaxial strain and to variation in substrate stiffness, relative to myometrial cells. However, on a flexible pronectin-coated silicone substrate, basal levels and lysophosphatidic acid-stimulated levels of activated RhoA were similar between leiomyoma and myometrial cells. In contrast, leiomyoma cells plated on a rigid polystyrene substrate had elevated levels of active RhoA, compared to myometrial cells. The results indicate that viscoelastic properties of the ECM of leiomyoma contribute significantly to the tumor's inherent stiffness and that leiomyoma cells have an attenuated sensitivity to mechanical cues. The findings suggest there may be a fundamental alteration in the communication between the external mechanical environment (extracellular forces) and reorganization of the actin cytoskeleton mediated by RhoA in leiomyoma cells. Additional research will be needed to elucidate the mechanism(s) responsible for the attenuated mechanical signaling in leiomyoma cells.

Fig. 1

Apparatus and method used to quantify pseudo-dynamic modulus in myometrial and leiomyoma surgically obtained tissue samples. a: Schematic of the experimental confined compression apparatus with a porous membrane (40 micron pore size). A 5% constant displacement uniaxial load was applied to the myometrial and leiomyoma tissue. The confined compression chamber was smooth, rigid, and impermeable. b: Representative Force versus Displacement graph for a single leiomyoma specimen.

Fig. 2

Leiomyoma tissue specimens have an increased pseudo-dynamic modulus compared to myometrial tissue samples. a: Summary of mechanical testing in matched surgical specimens. The pseudo-dynamic modulus (megapascals (MPa) per millimeter (mm) over mm, black diamonds) was increased in leiomyomata (L) surgical samples (n=10) relative to myometrium (M; n=7). Mean pseudo-dynamic moduli (open black squares) for myometrium and leiomyomata were 48.1±25.6 and 202.7±27.8 respectively (p<0.001). b: Leiomyoma surgical samples (n=10) held an increased peak stress (black diamonds) compared to myometrium (n=7). Mean peak stress (open black squares) for myometrium and leiomyomata were 1.35±0.70 and 6.96±0.91 respectively (p<0.001). c: Leiomyoma surgical samples (n=10) contained more sulfated glycosaminoglycan (sGAG) (DMMB assay) relative to matched myometrial samples, n=7: Leiomyoma=0.62±0.080 μg of sGAG per μg of DNA; Myometrium=0.19±0.012 μg per μg, p<0.0001. d: Leiomyoma surgical samples contained more collagen (Hydroxyproline assay) relative to matched myometrium: Leiomyoma=246.7±26.2 μg of collagen per μg of DNA; Myometrium=97.5±18.7 μg per μg, p<0.001. Values are reported as means±SEM. All statistical tests used a 2-tailed unpaired t-Test for unequal variance.

Fig. 3

Response of myometrial and leiomyoma cells to cyclic uniaxial strain. a: Cytoimmunofluorescent images of leiomyoma and myometrial cells exposed to either no strain (control) or to 8.9% uniaxial cyclic strain (Strain) for 18 h at 1 Hz. Cells were cultured with or without pre-treatment of the ROCK inhibitor, Y-27632 (Y-27) (10 μM) for 30 min prior to strain or no strain (control). Actin stress fibers and nuclei were visualized by staining for Alexa Fluor-546 Phalloidin and DAPI, respectively. b: Quantitative computerized morphometric measurements of cellular reorientation in response to uniaxial strain with, or without, pre-treatment of Y-27632 (Y-27) for leiomyoma (black bars) or myometrial cells (grey bars). Results are shown as the percentage of cells aligned at 90°+/−30° relative to the direction of the applied strain. Data represent a mean of three independent experiments with a minimum of 45 cells measured per condition. Angular differences between unstrained and strained leiomyoma and myometrial cells differed significantly (p<0.05).

Fig. 4

RhoA levels in leiomyoma and myometrial cells at baseline and in response to applied chemical or mechanical strain. a: Assessment of active RhoA in leiomyoma or myometrial cells cultured on flexible pronectin-coated substrate, or uncoated polystyrene. Y axis=relative level of active RhoA. Leiomyoma cells (black bars) demonstrated increased levels of activated RhoA relative to myometrial cells when cultured on polystyrene (p<0.05). b: Levels of active RhoA in myometrial (gray bars) or leiomyoma cells (black bars) cultured on flexible, pronectin-coated substrate untreated (control) or treated with a chemical activator or RhoA, lysophosphatidic acid (LPA), for minutes as indicated. Y axis= relative level of active RhoA. On the flexible, pronectin-coated substrate levels of activated RhoA in myometrial cells peaked at 3 min. c: Culture of leiomyoma (black bars) or myometrial cells (gray bars) on polystyrene either untreated (control) or treated with LPA for minutes as indicated. Y axis=relative level of active RhoA. Levels of active RhoA were significantly elevated in leiomyoma cells at baseline, and were less affected by LPA treatment. Data in a–c represent the average relative RhoA activation compared to myometrial control from three independent experiments. d: Quantification of active RhoA in myometrial (gray bars) or leiomyoma cells (black bars) to 2 h of applied uniaxial strain. Myometrial cells demonstrated a 2-fold increased active RhoA levels in response to uniaxial strain on pronectin-coated flexible silicone substrate (M Control versus M Strain). Leiomyoma cell active RhoA levels were attenuated and had a muted response (1.3 fold) to mechanical strain (L Control versus L Strain). The cell response was normalized to myometrial control activation of RhoA and reported as the mean±standard deviation from two independent experiments with 6 wells for each condition.

Fig. 5

Response of myometrial or leiomyoma cells to substrates of varied stiffness. a: Leiomyoma and myometrial cells were cultured on collagen-coated polyacrylamide gels of varying stiffness, then treated with calcein AM and fluorescent images were obtained 22 h after plating for assessment of cell spreading. Stiffness as indicated. b: Mean surface area per cell was determined using ImageJ software as indicated for myometrial cells (gray line) or leiomyoma cells (black line). Myometrial cell spreading responded to the increased substrate stiffness more than leiomyoma cells. Values represent a mean of four independent experiments with a minimum of 45 cells measured per condition (** trend comparison: p<0.05).