Stiff Extracellular Matrix Promotes Invasive Behaviors of Trophoblast Cells

Abstract

The effect of extracellular matrix (ECM) stiffness on embryonic trophoblast cells invasion during mammalian embryo implantation remains largely unknown. In this study, we investigated the effects of ECM stiffness on various aspects of human trophoblast cell behaviors during cell-ECM interactions. The mechanical microenvironment of the uterus was simulated by fabricating polyacrylamide (PA) hydrogels with different levels of stiffness. The human choriocarcinoma (JAR) cell lineage was used as the trophoblast model. We found that the spreading area of JAR cells, the formation of focal adhesions, and the polymerization of the F-actin cytoskeleton were all facilitated with increased ECM stiffness. Significantly, JAR cells also exhibited durotactic behavior on ECM with a gradient stiffness. Meanwhile, stiffness of the ECM affects the invasion of multicellular JAR spheroids. These results demonstrated that human trophoblast cells are mechanically sensitive, while the mechanical properties of the uterine microenvironment could play an important role in the implantation process.

Keywords: durotaxis; embryo implantation; extracellular matrix; human choriocarcinoma cell; stiffness.

Figure 1

Simulated ECM stiffness regulates trophoblast cell morphology and spreading area. (A) (Top): schematic diagram of PA gel with stiffness gradient. The Young’s modulus of the PA gel is 1 kPa, and its apparent Young’s modulus gradually increases with the decrease in the gel thickness. According to the change of the apparent Young’s modulus, the surface of the PA gel is evenly divided into three regions, namely Stiff, Intermediate, and Soft. (Bottom): schematic representation of JAR cells cultured on different regions. E indicates the apparent Young’s modulus. (B) Phase contrast images of JAR cells cultures on different regions scale bar: 20 μm. The yellow dashed line indicates the boundary of cells. (C) Measured average cell spreading area of JAR cells cultured on different regions. **** p < 1 × 10⁻⁶, n = 31, 27, 23 for Soft, Inter and Stiff, respectively. Data reported as mean ± standard deviation for N = 3 independent experiments. Each scatter indicates each cell being measured, and each color indicates an independent experiment.

Figure 2

F-actin organization and focal adhesion formation are affected by simulated ECM stiffness in JAR cells. (A) Immunofluorescence staining of JAR cells cultured on different regions (red: F-actin; green: vinculin, scale bar: 20 μm). (B) Measured focal adhesion area of JAR cells cultured on different regions. **** 1 × 10⁻⁶, n = 18, 27, 24 for Soft, Inter, and Stiff, respectively. Data reported as mean ± standard deviation for N = 3 independent experiments. Each scatter indicates each focal adhesion being measured. (C) Skeletonization of F-actin in JAR cells cultured on different regions (scale bar: 20 μm). (D) Measured cytoskeleton length of JAR cells cultured on different regions. **** 1 × 10⁻⁶, n = 35, 27, 24 for Soft, Inter, and Stiff, respectively. Data reported as mean ± standard deviation for N = 3 independent experiments. Each scatter indicated each F-actin filament being measured.

Figure 3

Stiff simulated ECM enhances JAR cell motility (A) (Top): Vectors of JAR cell migration. (Bottom): heatmap of velocity magnitude on different regions of simulated ECM (scale bar: 50 μm, color bar: 0~0.2 μm/min). (B) Tracking of JAR cells cultured on different regions (scale bar: 100 μm, time bar: 5 h). Each scatter indicated each cell being analyzed. (C,D) Velocity and track length of JAR cell migration. **** p < 1 × 10⁻⁶, *** p < 0.001, n = 11, 11, 9 for Soft, Inter, and Stiff, respectively. Data reported as mean ± standard deviation for N = 3 independent experiments. Each scatter indicated each cell being analyzed. (E) Track displacement of JAR cells cultured on different regions. *** p < 0.001, ** p < 0.01, n = 11, 11, 9 for Soft, Inter, and Stiff, respectively. Data reported as mean ± standard deviation for N = 3 independent experiments.

Figure 4

JAR cells exhibit durotaxis. (A) Tracking of JAR cells cultured on stiff region (scale bar: 50 μm, time bar: 9.5 h). E indicates the apparent Young’s modulus. Arrows indicated the displacement of each cell, (B) Representative JAR cell migration plots on stiff region over 9.5 h. The total cell number n = 56, number of independent experiments N = 3. (C) Vector map of JAR cell migration on stiff region (scale bar: 150 μm). Arrows indicated the vector of velocity. (D) Rose diagram of cell migration direction, which displays the angular between migration and stiffness gradient and the frequency of each class. The total cell number n = 56, number of independent experiments N = 3.

Figure 5

Stiff simulated ECM enhances the adhesion and invasion of multicellular JAR spheroids. (A) (Left): schematic diagram of JAR spheroid invasion assay. (Right): schematic diagram of the calculation of the invasion ratio. (B) Image of JAR spheroid invasion taken by an inverted microscope (scale bar: 200 μm). (C) Calculated adhesion area of JAR spheroids on different regions. ** p < 0.01, n = 24, 17, 12 for Soft, Inter, and Stiff, respectively. Data reported as mean ± standard deviation for N = 3 independent experiments. Each scatter indicated each spheroid being measured. (D) Calculated invasion ratio of JAR spheroids on different regions. **** p < 1 × 10⁻⁶, n = 43, 35, 25 for Soft, Inter, and Stiff, respectively. Data reported as mean ± standard deviation for N = 3 independent experiments. Each scatter indicated each spheroid being analyzed.