*3.5. Stiff Simulated ECM Enhances the Adhesion and Invasion of Multicellular JAR Spheroids*

During the complex biophysical process of embryo implantation, trophoblasts contribute to successful implantation via attachment and invasion. Numerous studies demonstrated many similarities between embryo implantation and tumor progression [57–60]. Components that are crucial to tumor cell migration and invasion are shared by the human trophoblast, including the involvement of the extracellular matrix (ECM), proteases (including serine proteases, cathepsins, and matrix-metalloproteinases), and cell-surface receptors (integrins) [59]. F-actin remodeling regulated by fascin plays a critical role in both cancer metastasis and trophoblast migration and invasion [60,61].

As such, we inferred that if the migration of individual JAR cells is affected by the mechanical forces of their microenvironment, the invasion behaviors of multicellular JAR spheroids could also be regulated by ECM stiffness. During the first step of the embryo implantation process, the blastocyst, a spheroid, establishes adhesion to the endometrium. Wong et al. demonstrated that ECM stiffness regulates the self-assembling of 3D placental trophoblast spheroids [16], but few studies directly demonstrated how ECM stiffness affects adhesion or spreading of 3D human trophoblast spheroids.

Based on this, we referred to tumor research methods to study the effect of ECM stiffness on invasion of multicellular JAR spheroid [62]. Multicellular JAR spheroids were formed in ultra-low attachment 96-well plates, as described in Methods, and were then seeded onto PA hydrogels with different stiffness and allowed to attach for 24 h (Figure 5A). JAR spheroids on stiff and intermediate-stiff simulated ECM had larger adhesion areas and showed a higher degree of invasion compared to spheroids on soft simulated ECM (Figure 5B–D). No significant difference was found in the degree of spheroid invasion on the stiff and intermediate-stiff simulated ECM. This demonstrated that the invasion of multicellular JAR cell spheroids is regulated by simulated ECM stiffness.

**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.

#### **4. Discussion**

Although ECM stiffness was demonstrated to be a key regulator of several developmental processes, the importance of extracellular mechanics for embryo implantation, especially for embryo attachment, was not established. This work identified trophoblasts as mechano-responding malignant tumor-like cells. Using PA hydrogels that mimic Young's modulus values of the human endometrium, we demonstrated that stiffer substrate enhances various cellular processes closely related to trophoblast adhesion and invasion, including JAR cell morphology, migration, contractility, and multicellular spheroid disaggregation. Our results demonstrate that adhesion and invasion of trophoblasts could be regulated by the mechanical properties (e.g., stiffness) of the endometrium. Altogether, these results suggest that mechanobiological properties may regulate the adhesion and invasion of human embryo during the process of implantation, and that the stiffness of the endometrium may affect the selection of embryo implantation sites and the subsequent invasion process.

Scar pregnancy (CSP) occurs when an embryo implants on a scar in the uterus, and the incidence of CSP increases with the number of previous cesarean deliveries [63]. The exact pathogenesis of CSP is not known, but the mechanical properties of the uterine scar significantly differ from other sites. The stiffness of the uterine scar appears to be significantly increased compared to the intact myometrium, as measured by ultrasound elastography [12], which could also affect embryo implantation. In addition, the stiffness of the endometrium in the pathological state is significantly higher than that of the normal state [6,13]. Our experiments demonstrated that in the context of normal and diseased human endometrium tissue characteristics (difference in stiffness), the mechanobiological regulation of trophoblast migration and attachment likely plays a critical role in implantation.

More broadly, this work demonstrated that trophoblast migration and adhesion are mechanically sensitive, which highlights the importance of strategies using extracellular tissue engineering to better understand and develop treatments for diseases related to implantation. This knowledge of mechanically mediated mechanisms for migration and adhesion can be further leveraged to create better technologies to increase the success rate of in vitro fertilization (IVF). In addition, the identification of this process will help to identify new regulatory mechanisms of embryonic adhesion and provide new ideas for the development of therapeutic strategies related to pregnancy.

Considering the difficulty in obtaining pure, primary, first-trimester human trophoblast cells, we used human choriocarcinoma (JAR) cell lineage instead of primary trophoblast cell line in this study. However, compared with primary trophoblast cells, choriocarcinoma cell lines have different transcriptomic profiles, are malignant and contain an abnormal number of chromosomes, which is unfavorable for studying the uniquely invasive extravillous trophoblast (EVT) cell behavior [64]. Therefore, in the subsequent study of embryo invasion, we will choose human embryonic stem cell (hESCs) or adult progenitor cells to derive trophoblast organoids. Endometrial epithelial cells are also involved in the embryo implantation process [65,66]. Under normal conditions, the trophoblast cells interact with endometrial epithelial cells to achieve maternal–fetal adhesion. The stiffness of the ECM could also affect the function of endometrial epithelial cells and the expression of related proteins. Therefore, to better simulate the in vivo environment, it is necessary to consider the mechanism of interaction between these two cells under the regulation of mechanical properties. Furthermore, since mechanical stiffness affects the invasion behaviors of trophoblast cells, the mechanobiological regulation of trophoblast migration and adhesion is most likely to be related to integrin-related signaling pathways. The integrin protein mediates the adhesion between cells and ECM. After being affected by mechanical forces, integrin binds to its ligands and mediates FAK, PI3K, AKT/PKB, and other signaling pathways that regulate cell proliferation, migration, and epithelial-mesenchymal transition [67,68]. By upregulating integrin-β1, the invasion of human trophoblasts can be promoted [69]. We also examined the expression of integrin-β1 of JAR cells on different regions and found no significant differences either between soft and inter or between inter and stiff, with stiff being slightly higher than soft (Figure S4), and the expression of other subunits of integrin did not change significantly with substrate stiffness [16]. In addition, several studies demonstrated the significant role of Rho protein in the process of implantation [26,70,71]. For example, Rho GTPase, most likely RhoA, regulates the adhesion of human trophoblasts to uterine epithelial RL95-2 cells [26].RhoA can also regulate trophoblast migration through cytoskeleton reorganization [70]. By interfering with related proteins such as integrin and Rho, we can further study the precise molecular mechanism of this phenomenon, which is also the content of our follow-up research.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/bioengineering10030384/s1, Figure S1: Fabricated sample of polyacrylamide hydrogel with stiffness gradient. Scale bar = 10 mm. Figure S2: SEM analysis of PA gel surface. Scale bar = 30 µm. Figure S3: Elastic modulus measured by AFM. Data reported as mean ± standard deviation for *N* = 3 independent experiments. Figure S4: Left: Immunofluorescence staining of JAR cells cultured on different regions (green: integrin-β1; blue: DAPI, scale bar: 20 µm). Right: mean fluorescence intensity of integrin-β1 in JAR cells cultured on different regions. *n* = 12, 13, 13 for Soft, Inter and Stiff, respectively. Data reported as mean ± standard deviation.

**Author Contributions:** J.C., Y.W., X.G., D.G., J.D. and Y.F. designed the study; J.C., H.L. and H.T. performed experiments and data analysis; Y.W., J.D. and Y.F. conceived and supervised this project and prepared the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Natural Science Foundation of China (12222201, 82273500, U20A20390, 11827803 and 11972351), Fundamental Research Funds for the Central Universities (ZG140S1971), National Key R&D Program of China (2021YFA0719302), Peking University Health Science Center-X Cross Seed Fund (BMU2020MX014), and Incubation fund for reserve candidates of Peking University Third Hospital (BYSYFY2021007).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.

**Conflicts of Interest:** The authors declare no conflict of interest.
