STAT3 Is the Master Regulator for the Forming of 3D Spheroids of 3T3-L1 Preadipocytes

To elucidate the currently unknown mechanisms responsible for the diverse biological aspects between two-dimensional (2D) and three-dimensional (3D) cultured 3T3-L1 preadipocytes, RNA-sequencing analyses were performed. During a 7-day culture period, 2D- and 3D-cultured 3T3-L1 cells were subjected to lipid staining by BODIPY, qPCR for adipogenesis related genes, including peroxisome proliferator-activated receptor γ (Pparγ), CCAAT/enhancer-binding protein alpha (Cebpa), Ap2 (fatty acid-binding protein 4; Fabp4), leptin, and AdipoQ (adiponectin), and RNA-sequencing analysis. Differentially expressed genes (DEGs) were detected by next-generation RNA sequencing (RNA-seq) and validated by a quantitative reverse transcription–polymerase chain reaction (qRT–PCR). Bioinformatic analyses were performed on DEGs using a Gene Ontology (GO) enrichment analysis and an Ingenuity Pathway Analysis (IPA). Significant spontaneous adipogenesis was observed in 3D 3T3-L1 spheroids, but not in 2D-cultured cells. The mRNA expression of Pparγ, Cebpa, and Ap2 among the five genes tested were significantly higher in 3D spheroids than in 2D-cultured cells, thus providing support for this conclusion. RNA analysis demonstrated that a total of 826 upregulated and 725 downregulated genes were identified as DEGs. GO enrichment analysis and IPA found 50 possible upstream regulators, and among these, 6 regulators—transforming growth factor β1 (TGFβ1), signal transducer and activator of transcription 3 (STAT3), interleukin 6 (IL6), angiotensinogen (AGT), FOS, and MYC—were, in fact, significantly upregulated. Further analyses of these regulators by causal networks of the top 14 predicted diseases and functions networks (IPA network score indicated more than 30), suggesting that STAT3 was the most critical upstream regulator. The findings presented herein suggest that STAT3 has a critical role in regulating the unique biological properties of 3D spheroids that are produced from 3T3-L1 preadipocytes.

Prostaglandin F2 and EP2 Agonists Exert Different Effects on 3D 3T3-L1 Spheroids during Their Culture Phase

To elucidate the effects of switching a PGF2α agonist, bimatoprost acid (BIM-A), to an EP2 agonist (Omidenepag—OMD; butaprost—Buta) or reversing the switching on adipose tissue, two-dimensional (2D) and three-dimensional (3D) cultures of 3T3-L1 cells were analyzed by lipid staining and according to the mRNA expression of adipogenesis-related genes (Pparγ, Ap2, and Leptin), components of the extracellular matrix (ECM; collagen1 (Col1), Col4, Col6, and fibronectin (Fn)), and the sizes and stiffness of the 3D spheroids. Switching from BIM-A to EP2 agonists caused (1) suppression of lipid staining and downregulation of most adipogenesis-related genes, (2) smaller and stiffer 3D spheroids, and (3) upregulation of Col1 and Fn, downregulation of Col4 (2D), or up-regulation of all ECM genes (3D, BIM-A to OMD), as well as downregulation of Col6 (3D, BIM-A to Buta). In contrast, reversing the switching resulted in (1) an enhancement in lipid staining (2D) and a significant upregulation of adipogenesis-related genes (2D, 3D Buta to BIM-A), (2) larger and slightly stiffer 3D spheroids, and (3) upregulation of Col1 and Fn (2D). These collective findings indicate that the switching orders of BIM-A and EP2 agonists have a significant effect on lipid metabolism, ECM expression, and the physical stiffness of 3T3-L1 cells.

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

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.

A Novel 3D Culture Model of Human ASCs Reduces Cell Death in Spheroid Cores and Maintains Inner Cell Proliferation Compared With a Nonadherent 3D Culture

3D cell culture technologies have recently shown very valuable promise for applications in regenerative medicine, but the most common 3D culture methods for mesenchymal stem cells still have limitations for clinical application, mainly due to the slowdown of inner cell proliferation and increase in cell death rate. We previously developed a new 3D culture of adipose-derived mesenchymal stem cells (ASCs) based on its self-feeder layer, which solves the two issues of ASC 3D cell culture on ultra-low attachment (ULA) surface. In this study, we compared the 3D spheroids formed on the self-feeder layer (SLF-3D ASCs) with the spheroids formed by using ULA plates (ULA-3D ASCs). We discovered that the cells of SLF-3D spheroids still have a greater proliferation ability than ULA-3D ASCs, and the volume of these spheroids increases rather than shrinks, with more viable cells in 3D spheroids compared with the ULA-3D ASCs. Furthermore, it was discovered that the SLF-3D ASCs are likely to exhibit the abovementioned unique properties due to change in the expression level of ECM-related genes, like COL3A1, MMP3, HAS1, and FN1. These results indicate that the SLF-3D spheroid is a promising way forward for clinical application.

Photoimmunotherapy retains its anti-tumor efficacy with increasing stromal content in heterotypic pancreatic cancer spheroids

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by increased levels of desmoplasia that contributes to reduced drug delivery and poor treatment outcomes. In PDAC, the stromal content can account for up to 90% of the total tumor volume. The complex interplay between stromal components, including pancreatic cancer associated fibroblasts (PCAFs), and PDAC cells in the tumor microenvironment (TME) have a significant impact on prognoses and thus needs to be recapitulated in vitro when evaluating various treatment strategies. This study is a systematic evaluation of photodynamic therapy (PDT) in 3D heterotypic coculture models of PDAC with varying ratios of patient derived PCAFs that simulate heterogenous PDAC tumors with increasing stromal content. The efficacy of antibody-targeted PDT (photoimmunotherapy; PIT) using cetuximab photoimmunoconjugates (PICs) of benzoporphyrin derivative (BPD) is contrasted with that of liposomal BPD (Visudyne), which is currently in PDT clinical trials for PDAC. We demonstrate that both Visudyne-PDT and PIT were effective in heterotypic PDAC 3D spheroids with a low stromal content. However, as the stromal content increases above 50% in the 3D spheroids, the efficacy of Visudyne-PDT is reduced by up to 10-fold, while PIT retains its efficacy. PIT was found to be 10-fold, 19-fold and 14-fold more phototoxic in spheroids with 50%, 75% and 90% PCAFs, respectively, as compared to Visudyne-PDT. This marked difference in efficacy is attributed to the ability of PICs to penetrate and distribute within spheroids with a higher stromal content, whereas Visudyne is restricted to the spheroid periphery. This study thus demonstrates how the stromal content in PDAC spheroids directly impacts their responsiveness to PDT and proposes PIT to be a highly suited treatment option for desmoplastic tumors with particularly high degrees of stromal content.

The Influence of Antitumor Unsymmetrical Bisacridines on 3D Cancer Spheroids Growth and Viability

The culture of 3D spheroids is a promising tool in drug development and testing. Recently, we synthesized a new group of compounds, unsymmetrical bisacridines (UAs), which exhibit high cytotoxicity against various human cell lines and antitumor potency against several xenografts. Here, we describe the ability of four UAs—C-2028, C-2041, C-2045, and C-2053—to influence the growth of HCT116 and H460 spheres and the viability of HCT116 cells in 3D culture compared with that in 2D standard monolayer culture. Spheroids were generated using ultra-low-attachment plates. The morphology and diameters of the obtained spheroids and those treated with UAs were observed and measured under the microscope. The viability of cells exposed to UAs at different concentrations and for different incubation times in 2D and 3D cultures was assessed using 7-AAD staining. All UAs managed to significantly inhibit the growth of HCT116 and H460 spheroids. C-2045 and C-2053 caused the death of the largest population of HCT116 spheroid cells. Although C-2041 seemed to be the most effective in the 2D monolayer experiments, in 3D conditions, it turned out to be the weakest compound. The 3D spheroid culture seems to be a suitable method to examine the efficiency of new antitumor compounds, such as unsymmetrical bisacridines.

A Substrate-Mimicking Basement Membrane Drives the Organization of Human Mesenchymal Stromal Cells and Endothelial Cells Into Perivascular Niche-Like Structures

Extracellular matrix-derived products (e.g. Matrigel) are widely used for in vitro cell cultures both as two-dimensional (2D) substrates and as three-dimensional (3D) encapsulation gels because of their ability to control cell phenotypes through biospecific cues. However, batch-to-batch variations, poor stability, cumbersome handling, and the relatively high costs strictly limit their use. Recently, a new substrate known as PhenoDrive-Y has been used as 2D coating of tissue culture plastic showing to direct the bone marrow mesenchymal stromal cells (MSCs) toward the formation of 3D spheroids. When organized into 3D spheroids, the MSCs expressed levels of pluripotency markers and of paracrine angiogenic activity higher than those of the MSCs adhering as fibroblast-like colonies on tissue culture plastic. The formation of the spheroids was attributed to the properties of this biomaterial that resemble the main features of the basement membrane by mimicking the mesh structure of collagen IV and by presenting the cells with orderly spaced laminin bioligands. In this study, PhenoDrive-Y was compared to Matrigel for its ability to drive the formation of perivascular stem cell niche-like structures in 2D co-culture conditions of human endothelial cells and adult bone marrow MSCs. Morphological analyses demonstrated that, when compared to Matrigel, PhenoDrive-Y led endothelial cells to sprout into a more consolidated tubular network and that the MSCs nestled as compact spheroids above the anastomotic areas of this network resemble more closely the histological features of the perivascular stem cell niche. A study of the expressions of relevant markers led to the identification of the pathways linking the PhenoDrive-Y biomimicking properties to the acquired histological features, demonstrating the enhanced levels of stemness, renewal potential, predisposition to migration, and paracrine activities of the MSCs.