Reactivities of a Prostanoid EP2 Agonist, Omidenepag, Are Useful for Distinguishing between 3D Spheroids of Human Orbital Fibroblasts without or with Graves’ Orbitopathy

Abstract

Background. To obtain new insights into the activation of the thyroid-stimulating hormone (TSH) and insulin-like growth factor 1 (IGF-1) receptors in human orbital fibroblasts (n-HOFs), the effects of the prostanoid EP2 agonist, omidenepag (OMD), and a rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) were evaluated using three-dimension (3D) n-HOFs spheroids in the absence and presence of the recombinant human TSH receptor antibodies, M22 and IGF-1. Methods. The effects of 100 nM OMD or 10 μM Rip on the physical properties, size, stiffness, and mRNA expression of several extracellular matrix (ECM) molecules, their regulator, inflammatory cytokines, and endoplasmic reticulum (ER) stress-related factors were examined and compared among 3D spheroids of n-HOFs, M22-/IGF-1-activated n-HOFs and GO-related human orbital fibroblasts (GHOFs). Results. The physical properties and mRNA expressions of several genes of the 3D n-HOFs spheroids were significantly and diversely modulated by the presence of OMD or Rip. The OMD-induced effects on M22-/IGF-1-activated n-HOFs were similar to the effects caused by GHOHs, but quite different from those of n-HOFs. Conclusions. The findings presented herein indicate that the changes induced by OMD may be useful in distinguishing between n-HOFs and GHOFs.

Unexpected Crosslinking Effects of a Human Thyroid Stimulating Monoclonal Autoantibody, M22, with IGF1 on Adipogenesis in 3T3L-1 Cells

“…To support this hypothesis, a previous study demonstrated that flightless-I (FLII), a transcriptional modulator of PPARγ, binds directly to PPARγ through the LRR domain and suppresses the transcriptional activity of cells [ 69 ]. Furthermore, in addition to the current observations related to the Seahorse real-time cellular metabolic analyses of n-HOFs and GHOFs ( Supplemental Figure S2 ), we previously reported that a significant difference existed between GHOFs and n-HOFs that had been treated with M22 and IGF1 in terms of mRNA expression levels of several molecules including ECM molecules (COL1, 4 and 6 and FN), ECM regulatory factors (LOX and CTGF), inflammatory cytokines (IL1B and IL6) and ER stress-related factors, suggesting the presence of some additional, unrelated TSHR effects by M22 and IGF1 [ 30 ]. However, this TSHR-independent cross-reactivity by M22 and IGF1 is speculative at this time, and additional studies such as a TSHR knockdown system and others will be required to reveal the magnitude of this issue.…”

“…In the presence or absence of 100 ng/mL IGF1 and/or 10 ng/mL M22, two-dimensional (2D) planar and three-dimensional (3D) spheroid cultures of 3T3-L1 preadipocytes (#EC86052701-G0, KAK) were maintained for 7 days [ 24 , 25 ], or n-HOFs [ 30 , 70 , 71 ] or GHOFs [ 29 , 72 ] were maintained for 10 days, as described previously. The induction of adipogenic differentiation of the 3T3-L1 cells was also carried out as described previously [ 24 , 25 ].…”

“…In fact, it has been proposed that such a 3D cell culture technique would allow for more representative physiological cell-cell and cell-extracellular matrix (ECM) interactions than conventional 2D culture models [ 22 ]. Quite recently, our group independently succeeded in producing 3D spheroids using 3T3-L1 cells [ 23 , 24 , 25 , 26 , 27 , 28 ] as well as cells from other sources, including human orbital fibroblasts (HOFs) [ 29 , 30 , 31 ], human corneal stromal fibroblasts [ 32 ], human trabecular meshwork (HTM) [ 33 , 34 , 35 , 36 ] and human conjunctival fibroblasts (HconF) [ 37 , 38 , 39 , 40 ].…”

Physical Properties and Cellular Metabolic Characteristics of 3D Spheroids Are Possible Definitive Indices for the Biological Nature of Cancer-Associated Fibroblasts

“…Among the various in vitro 3D cell culture methods, a simpler model, a 3D spheroid culture model, has been the most widely used [28,29]. In our recent investigations, to characterize the microenvironments of various tissues, we independently developed unique 3D drop cell culture methods using several non-cancerous cells including human conjunctival fibroblasts (HconF) [30], human orbital fibroblasts (HOF) [31], human scleral stromal fibroblasts (HSclF) [32] and human scleral fibroblasts (HSSF) [33], and Graves'-diseaserelated HOF (GOF) [34], as well as cancerous cells including an A549 lung adenocarcinoma cell line [35], various malignant melanoma (MM) cell lines [36] and various OSCC. As expected, our findings indicated that the biological natures of the 3D spheroid models were markedly different and distinct from those of 2D cultured models even though both were prepared under exactly identical experimental conditions except that the culture plates that were used were different [37].…”