*Article* **Stiff Extracellular Matrix Promotes Invasive Behaviors of Trophoblast Cells**

**Jialing Cao 1,2, Hangyu Li 3,4 , Hongyan Tang 1 , Xuenan Gu 1 , Yan Wang 5, \*, Dongshi Guan 3,4, \* , Jing Du 1, \* and Yubo Fan 1, \***


**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:** embryo implantation; human choriocarcinoma cell; extracellular matrix; stiffness; durotaxis

### **1. Introduction**

Embryo implantation is a critical feature of mammalian pregnancy and requires a series of interactions between the embryo and the uterus, which can be divided into three different steps: apposition, attachment, and invasion [1]. In brief, after hatching from the zona pellucida, the spheroid of cells floats freely and finds the proper implantation site (apposition), and the trophoblasts firmly attach to the uterine wall (adhesion). The trophoblasts then differentiate into cytotrophoblasts and syncytiotrophoblasts under tight regulation, thereby invading the endometrium (invasion) and leading to compromised placental development and pregnancy complications [2].

Recent research on embryo implantation primarily focused on biochemical aspects, including some molecular mechanisms and related signaling pathways. During the early stages of implantation, there are many molecular mediators coordinated by ovarian steroid hormones involved in the initial maternal-fetal communication and interaction. These mediators include adhesion molecules, cytokines, growth factors, and lipids [3,4]. Meanwhile, using human endometrium Ishikawa and PL95-2 cells, Ming Yu et al. found that N-glycosylation of the endometrium is necessary to maintain the receptive functions of the uterus [5].

**Citation:** Cao, J.; Li, H.; Tang, H.; Gu, X.; Wang, Y.; Guan, D.; Du, J.; Fan, Y. Stiff Extracellular Matrix Promotes Invasive Behaviors of Trophoblast Cells. *Bioengineering* **2023**, *10*, 384. https://doi.org/10.3390/ bioengineering10030384

Academic Editors: Gary Chinga Carrasco and Christina Zong-Hao Ma

Received: 12 February 2023 Revised: 12 March 2023 Accepted: 17 March 2023 Published: 21 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Several studies recently uncovered the role of mechanical forces before and during human embryo implantation [6–8]. The mechanical properties of the human endometrium are complex, and spatiotemporal changes exist during embryo implantation [8,9]. Mechanical indentation was used to demonstrate that mechanical properties significantly differ between anatomical locations in both nonpregnant and pregnant uterine tissue [10]. For instance, in pregnant tissue, Young's modulus of fundus tissue is higher than that of the posterior and anterior tissue. The stiffness of decidual basalis (implantation site) is much higher than the decidua parietalis, nonpregnant endometrium, and placenta (~10<sup>3</sup> Pa vs. ~10<sup>2</sup> Pa) [11]. When the uterus is in a pathological state or there is a scar in the endometrium, the stiffness will increase [12,13]. For example, the shear modulus in a pathological state is about 7 kPa (calculated elastic modulus: 18.2 kPa), and the shear modulus in an extreme pathological state is about 17.4 kPa (calculated elastic modulus: 45.2 kPa) [6,13]. Endometrium extracellular matrix (ECM) stiffness is lower during the secretory phase than during the proliferative phase (shear modulus: 3.34 kPa vs. 1.97 kPa; calculate elastic modulus: 8.68 kPa vs. 5.12 kPa) [14].

Recent work demonstrated that the implantation process may be regulated by mechanical factors. Firm adhesion between trophoblast-type cells and endometrium epithelial cells was only observed when the trophoblast-functionalized tip indented the apical surface of the epithelial cell [15], indicating the role of mechanical forces during the maternal-fetal interaction [7]. Meanwhile, recent work studied the mechanobiological regulation of placental trophoblast fusion through ECM stiffness [6]. Using a stiffness-tunable hydrogel culture system, Ma et al. suggested that stiffer ECM promotes the spreading and fusion of trophoblast cells [6]. Wong et al. demonstrated that thicker ECM promotes the selfassembly of 3D trophoblast cells spheroids [16]. These might suggest an effect of ECM stiffness on the invasion of trophoblast cells, but they do not directly demonstrate how substrate stiffness regulates the invasion behaviors of trophoblast cells. Therefore, it is important to reveal the effect of ECM stiffness on trophoblast cell adhesion, migration, and invasion of multicellular spheroids.

Polyacrylamide hydrogels are widely used as a biocompatible material with easy fabrication and adjustable stiffness to study the effect of substrate stiffness on cell behaviors [17–19]. In this study, we generated collagen-coated polyacrylamide (PA) hydrogels with different stiffness gradients to mimic the microenvironment of the uterus and investigate the role of ECM stiffness in trophoblast cell morphology, migration, and invasion. We chose the human choriocarcinoma (JAR) cell lineage, which is derived from human choriocarcinoma and is the most widely-used cytotrophoblast-like cell model [20–27]. Our results showed that as the simulated ECM stiffness increased, the spreading area of JAR cells gradually promoted, and the number of focal adhesions increased, while the cytoskeleton became more robust and filamentous. We also found that individual trophoblast cells exhibited durotaxis behavior, and simulated ECM stiffness enhanced the invasion capacity of trophoblast spheroids. This study complements the effect of mechanical forces on the invasion behaviors of trophoblast cells, revealing the important role of ECM stiffness in embryonic implantation, while being able to provide some references for the study of diseases related to implantation.
