Acquired chemoresistance drives spatial heterogeneity, chemoprotection and collective migration in pancreatic tumor spheroids.

Tumors display rich cellular heterogeneity and typically consist of multiple co-existing clones with distinct genotypic and phenotypic characteristics. The acquisition of resistance to chemotherapy has been shown to contribute to the development of aggressive cancer traits, such as increased migration, invasion and stemness. It has been hypothesized that collective cellular behavior and cooperation of cancer cell populations may directly contribute to disease progression and lack of response to treatment. Here we show that the spontaneous emergence of chemoresistance in a cancer cell population exposed to the selective pressure of a chemotherapeutic agent can result in the emergence of collective cell behavior, including cell-sorting, chemoprotection and collective migration. We derived several gemcitabine resistant subclones from the human pancreatic cancer cell line BxPC3 and determined that the observed chemoresistance was driven of a focal amplification of the chr11p15.4 genomic region, resulting in over-expression of the ribonucleotide reductase (RNR) subunit RRM1. Interestingly, these subclones display a rich cell-sorting behavior when cultured as mixed tumor spheroids. Furthermore, we show that chemoresistant cells are able to exert a chemoprotective effect on non-resistant cells in spheroid co-culture, whereas no protective effect is seen in conventional 2D culture. We also demonstrate that the co-culture of resistant and non-resistant cells leads to collective migration where resistant cells enable migration of otherwise non-migratory cells.

Cell Adhesion Assessment Reveals a Higher Force per Contact Area on Fibrous Structures Compared to Flat Substrates.

The distribution and density of ligands have a determinant role in cell adhesion on planar substrates. At the same time, planar surfaces are nonphysiological for most cells, and cell behavior on planar and topographical surfaces is significantly different, with fibrous structures being the most natural environment for cells. Despite phenomenological examinations, the role of adhesion ligand density in the fibrous scaffold for cell adhesion strength has so far not been assessed. Here, we established a method to measure the amount of cell ligands on biofunctionalized electrospun meshes and planar substrate coatings with the same chemical composition. With this as a basis for systematic comparison and pure polyester as benchmark substrates, we have cultured L929 mouse fibroblasts and measured the adhesion force to surfaces of different chemistry and topography. In every case, having fibrous structures have led to an increased adhesion force per area also at a lower ligand density, which remarks the importance of such structures in a natural extracellular environment. Conversely, cells migrate more on planar surfaces than on the tested fibrous substrates. We thus established a platform to study cell-matrix interactions on different surfaces in a precise and reproducible manner as a new tool to assess and quantify cell-matrix interactions toward 3D scaffolds.

Human Adipose-Derived Mesenchymal Stromal/Stem Cell Spheroids Possess High Adipogenic Capacity and Acquire an Adipose Tissue-like Extracellular Matrix Pattern.

Adipose-derived mesenchymal stromal/stem cells (ASCs) represent a commonly used cell source for adipose tissue engineering. In this context, ASCs have routinely been cultured in conventional 2D culture and applied as single cell suspension for seeding onto scaffold materials or direct injection. However, this approach is associated with the loss of their intrinsic 3D microenvironment and leads to impaired regenerative capacity of the cells. Thus, the application of ASCs as self-assembled 3D spheroids with cells residing in their own matrix is an attractive alternative. However, characterization of the structural features and differentiation capacity of the spheroids is necessary to effectively apply them as building blocks in adipose tissue engineering. In this study, we focus on extracellular matrix (ECM) development in ASC spheroids, as well as adipogenic differentiation in comparison to conventional 2D culture using different induction protocols. Reproducible assembly of ASCs into spheroids was achieved within 24 h using the liquid overlay technique. Undifferentiated spheroids displayed a stromal ECM pattern, with fibronectin, collagen V, and VI as the main components. In the course of adipogenesis, a dynamic shift in the ECM composition toward an adipogenic phenotype was observed, associated with enhanced expression of laminin, collagen I, IV, V, and VI, similar to native fat. Furthermore, adipogenic differentiation was enhanced in spheroids as compared with 2D cultured cells, with the spheroids needing a distinctly shorter adipogenic stimulus to sustain adipogenesis, which was demonstrated based on analysis of triglyceride content and adipogenic marker gene expression. In summary, culturing ASCs as spheroids can enhance their adipogenic capacity and generate adipose-like microtissues, which may be a promising cell delivery strategy for adipose tissue engineering approaches. Impact statement Adipose-derived mesenchymal stromal/stem cells (ASCs) as a widely used cell source for adipose tissue engineering have been shown to be limited in their regenerative capacity when applied as single cells. As an alternative approach, the delivery as spheroids, consisting of cells in a 3D context, may be favorable. However, insights into extracellular matrix (ECM) development and efficient adipogenic differentiation are required for their effective application. In this study, we show that differentiated ASC spheroids develop an ECM, resembling native adipose tissue. Furthermore, the ASC spheroids exhibited a superior differentiation capacity as compared with conventional 2D culture, and required only a short adipogenic induction stimulus. Our results identify ASC-derived spheroids as an attractive cell delivery method for adipose tissue engineering approaches.

Tailored Melt Electrowritten Scaffolds for the Generation of Sheet-Like Tissue Constructs from Multicellular Spheroids.

Melt electrowriting (MEW) is an additive manufacturing technology that is recently used to fabricate voluminous scaffolds for biomedical applications. In this study, MEW is adapted for the seeding of multicellular spheroids, which permits the easy handling as a single sheet-like tissue-scaffold construct. Spheroids are made from adipose-derived stromal cells (ASCs). Poly(ε-caprolactone) is processed via MEW into scaffolds with box-structured pores, readily tailorable to spheroid size, using 13-15 µm diameter fibers. Two 7-8 µm diameter "catching fibers" near the bottom of the scaffold are threaded through each pore (360 and 380 µm) to prevent loss of spheroids during seeding. Cell viability remains high during the two week culture period, while the differentiation of ASCs into the adipogenic lineage is induced. Subsequent sectioning and staining of the spheroid-scaffold construct can be readily performed and accumulated lipid droplets are observed, while upregulation of molecular markers associated with successful differentiation is demonstrated. Tailoring MEW scaffolds with pores allows the simultaneous seeding of high numbers of spheroids at a time into a construct that can be handled in culture and may be readily transferred to other sites for use as implants or tissue models.

Contextual Control of Adipose-Derived Stem Cell Function: Implications for Engineered Tumor Models.

Adipose-derived stem cells (ASCs) are multipotent and, thus, are an attractive cell source for tissue engineering and regenerative medicine. However, ASCs can also differentiate into myofibroblasts, a cell type known to support tumorigenesis, yet the mechanisms underlying the myofibroblastic differentiation of ASCs and the resulting consequences on tumor pathogenesis are not well understood. This knowledge deficit is due, in part, to a lack of 3D culture models that capture the biological and physical heterogeneity of the microenvironment that influences ASC fate under both physiological and pathological conditions. Advanced biomanufacturing strategies offer new opportunities toward the generation of in vivo-like 3D culture environments that can help study how some of these conditions regulate ASC fate. This review discusses the pro-tumorigenic plasticity of ASCs in the tumor microenvironment as well as other disease contexts (e.g., obesity and aging) and highlights advanced biomanufacturing technologies that could be used to integrate stromal parameters into the next generation of engineered 3D tumor models. Such models will enable new insights into ASC-dependent microenvironmental aspects contributing to tumor initiation and progression that may ultimately be targeted for improved anticancer therapies.

Impact of whole body electromyostimulation on cardiometabolic risk factors in older women with sarcopenic obesity: the randomized controlled FORMOsA-sarcopenic obesity study.

BACKGROUND: Sarcopenic obesity (SO) is characterized by a combination of low muscle and high fat mass with an additive negative effect of both conditions on cardiometabolic risk. The aim of the study was to determine the effect of whole-body electromyostimulation (WB-EMS) on the metabolic syndrome (MetS) in community-dwelling women aged ≥70 years with SO. METHODS: The study was conducted in an ambulatory university setting. Seventy-five community-dwelling women aged ≥70 years with SO living in Northern Bavaria, Germany, were randomly allocated to either 6 months of WB-EMS application with (WB-EMS&P) or without (WB-EMS) dietary supplementation (150 kcal/day, 56% protein) or a non-training control group (CG). WB-EMS included one session of 20 min (85 Hz, 350 µs, 4 s of strain-4 s of rest) per week with moderate-to-high intensity. The primary study endpoint was the MetS Z-score with the components waist circumference (WC), mean arterial pressure (MAP), triglycerides, fasting plasma glucose, and high-density lipoprotein cholesterol (HDL-C); secondary study endpoints were changes in these determining variables. RESULTS: MetS Z-score decreased in both groups; however, changes compared with the CG were significant (P=0.001) in the WB-EMS&P group only. On analyzing the components of the MetS, significant positive effects for both WB-EMS groups (P≤0.038) were identified for MAP, while the WB-EMS group significantly differed for WC (P=0.036), and the WB-EMS&P group significantly differed for HDL-C (P=0.006) from the CG. No significant differences were observed between the WB-EMS groups. CONCLUSION: The study clearly confirms the favorable effect of WB-EMS application on the MetS in community-dwelling women aged ≥70 years with SO. However, protein-enriched supplements did not increase effects of WB-EMS alone. In summary, we considered this novel technology an effective and safe method to prevent cardiometabolic risk factors and diseases in older women unable or unwilling to exercise conventionally.

Development of volume-stable adipose tissue constructs using polycaprolactone-based polyurethane scaffolds and fibrin hydrogels.

Adipose tissue engineering aims at the restoration of soft tissue defects and the correction of contour deformities. It is therefore crucial to provide functional adipose tissue implants with appropriate volume stability. Here, we investigate two different fibrin formulations, alone or in combination with biodegradable polyurethane (PU) scaffolds as additional support structures, with regard to their suitability to generate volume-stable adipose tissue constructs. Human adipose-derived stem cells (ASCs) were incorporated in a commercially available fibrin sealant as well as a stable fibrin hydrogel previously developed by our group. The composite constructs made from the commercially available fibrin and porous poly(ε-caprolactone)-based polyurethane scaffolds exhibited increased volume stability as compared to fibrin gels alone; however, only constructs using the stable fibrin gels completely maintained their size and weight for 21 days. Adipogenesis of ASCs was not impaired by the additional PU scaffold. After induction with a common hormonal cocktail, for constructs with either fibrin formulation, strong adipogenic differentiation of ASCs was observed after 21 days in vitro. Furthermore, upregulation of adipogenic marker genes was demonstrated at mRNA (PPARγ, C/EBPα, GLUT4 and aP2; qRT-PCR) and protein (leptin; ELISA) levels. Stable fibrin/PU constructs were further evaluated in a pilot in vivo study, resulting in areas of well-vascularized adipose tissue within the implants after only 5 weeks. Copyright © 2013 John Wiley & Sons, Ltd.

Engineering vascularized adipose tissue using the stromal-vascular fraction and fibrin hydrogels.

The development of vascularized and functional adipose tissue substitutes is required to improve soft tissue augmentation. In this study, vascularized adipose tissue constructs were generated using uncultured cells from the stromal-vascular fraction (SVF) of adipose tissue as an alternative cell source to adipose-derived stem cells. SVF cell behavior and tissue formation were compared in a stable fibrin formulation developed by our group and a commercial fibrin sealant (TissuCol; Baxter) upon direct subcutaneous implantation in a nude mouse model. Further, the effect of in vitro adipogenic induction on SVF cell development was investigated by implanting stable fibrin constructs after 1 week of precultivation (adipogenic vs. noninduced control). Constructs were thoroughly analyzed before implantation regarding adipogenic differentiation status, cell viability, and distribution as well as the presence of endothelial cells. Before implantation, in vitro precultivation strongly promoted adipogenesis (under adipogenic conditions) and the formation of CD31(+) prevascular structures by SVF cells (under nonadipogenic conditions). Tissue development in vivo was determined after 4 weeks by histology (hematoxylin and eosin, human vimentin) and quantified histomorphometrically. In stable fibrin gels, adipogenic precultivation was superior to noninduced conditions, resulting in mature adipocytes and the formation of distinct vascular structures of human origin in vivo. Strong neovascularization by the implanted cells predominated in noninduced constructs. Without pretreatment, the SVF in stable fibrin gels displayed only a weak differentiation capability. In contrast, TissuCol gels strongly supported the formation of coherent and well-vascularized adipose tissue of human origin, displaying large unilocular adipocytes. The developed native-like tissue architecture was highlighted by a whole mount staining technique. Taken together, SVF cells from human adipose tissue were shown to successfully lead to adipose tissue formation in fibrin hydrogels in vivo. The results render the SVF a promising cell source for subsequent studies both in vitro and in vivo with the aim of engineering clinically applicable soft tissue substitutes.