Three-dimensional cell cultures, such as spheroids and organoids, serve as increasingly important models in fundamental and applied research and start to be used for drug screening purposes. Optical tissue clearing procedures are employed to enhance visualization of fluorescence-stained organs, tissues, and three-dimensional cell cultures. To get a more systematic overview about the effects and applicability of optical tissue clearing on three-dimensional cell cultures, we compared six different clearing/embedding protocols on seven types of spheroid-and chip-based threedimensional cell cultures of approximately 300 µm in size that were stained with nuclear dyes, immunofluorescence, cell trackers, and cyan fluorescent protein. Subsequent whole mount confocal microscopy and semi-automated image analysis were performed to quantify the effects. Quantitative analysis included fluorescence signal intensity and signal-to-noise ratio as a function of z-depth as well as segmentation and counting of nuclei and immunopositive cells. In general, these analyses revealed five key points, which largely confirmed current knowledge and were quantified in this study. First, there was a massive variability of effects of different clearing protocols on sample transparency and shrinkage as well as on dye quenching. Second, all tested clearing protocols worked more efficiently on samples prepared with one cell type than on co-cultures. Third, z-compensation was imperative to minimize variations in signal-tonoise ratio. Fourth, a combination of sample-inherent cell density, sample shrinkage, uniformity of signal-to-noise ratio, and image resolution had a strong impact on data segmentation, cell counts, and relative numbers of immunofluorescence-positive cells. Finally, considering all mentioned aspects and including a wish for simplicity and speed of protocols-in particular, for screening purposes-clearing with 88% Glycerol appeared to be the most promising option amongst the ones tested.

Motoneurons, skeletal muscle fibers, and Schwann cells form synapses, termed neuromuscular junctions (NMJs). These control voluntary body movement and are affected in numerous neuromuscular diseases. Therefore, a variety of NMJ in vitro models have been explored to enable mechanistic and pharmacological studies. So far, selective integration of Schwann cells in these models has been hampered, due to technical limitations. Here we present robust protocols for derivation of Schwann cells from human induced pluripotent stem cells (hiPSC) and their coculture with hiPSC-derived motoneurons and C2C12 muscle cells. Upon differentiation with tuned BMP signaling, Schwann cells expressed marker proteins, S100b, Gap43, vimentin, and myelin protein zero. Furthermore, they displayed typical spindle-shaped morphologies with long processes, which often aligned with motoneuron axons. Inclusion of Schwann cells in coculture experiments with hiPSC-derived motoneurons and C2C12 myoblasts enhanced myotube growth and affected size and number of acetylcholine receptor plaques on myotubes. Altogether, these data argue for the availability of a consistent differentiation protocol for Schwann cells and their amenability for functional integration into neuromuscular in vitro models, fostering future studies of neuromuscular mechanisms and disease.

Most tumors consume large amounts of glucose. Concepts to explain the mechanisms that mediate the achievement of this metabolic need have proposed a switch of the tumor mass to aerobic glycolysis. Depending on whether primarily tumor or stroma cells undergo such a commutation, the terms ‘Warburg effect’ or ‘reverse Warburg effect’ were coined to describe the underlying biological phenomena. However, current in vitro systems relying on 2-D culture, single cell-type spheroids, or basal-membrane extract (BME/Matrigel)-containing 3-D structures do not thoroughly reflect these processes. Here, we aimed to establish a BME/Matrigel-free 3-D microarray cancer model to recapitulate the metabolic interplay between cancer and stromal cells that allows mechanistic analyses and drug testing. Human HT-29 colon cancer and CCD-1137Sk fibroblast cells were used in mono- and co-cultures as 2-D monolayers, spheroids, and in a cell-chip format. Metabolic patterns were studied with immunofluorescence and confocal microscopy. In chip-based co-cultures, HT-29 cells showed facilitated 3-D growth and increased levels of hexokinase-2, TP53-induced glycolysis and apoptosis regulator (TIGAR), lactate dehydrogenase, and: translocase of outer mitochondrial membrane 20 (TOMM20), when compared with HT-29 mono-cultures. Fibroblasts co-cultured with HT-29 cells expressed higher levels of mono-carboxylate transporter 4, hexokinase-2, microtubule-associated proteins 1A/1B light chain 3, and ubiquitin-binding protein p62 than in fibroblast mono-cultures, in both 2-D cultures and chips. Tetramethylrhodamin-methylester (TMRM) live-cell imaging of chip co-cultures revealed a higher mitochondrial potential in cancer cells than in fibroblasts. The findings demonstrate a crosstalk between cancer cells and fibroblasts that affects cellular growth and metabolism. Chip-based 3-D co-cultures of cancer cells and fibroblasts mimicked features of the reverse Warburg effect.

Bone sialoprotein (BSP) has become a target in breast cancer research as it is associated with tumor progression and metastasis. The mechanisms underlying the regulation of BSP expression have been largely elusive. Given that BSP is involved in the homing of cancer cells in bone metastatic niches, we addressed regulatory effects of proteolytic cleavage and extracellular matrix components on BSP expression and distribution in cell culture models. Therefore, MDA-MB-231 human breast cancer cells were kept in 2D and 3D spheroid cultures and exposed to basement membrane extract in the presence or absence of matrix metalloproteinase 9 or the non-polar protease, dispase. Confocal imaging of immunofluorescence samples stained with different antibodies against human BSP demonstrated a strong inducing effect of basement membrane extract on anti-BSP immunofluorescence. Similarly, protease incubation led to acute upregulation of anti-BSP immunofluorescence signals, which was blocked by cycloheximide, suggesting de novo formation of BSP. In summary, our data show that extracellular matrix components play an important function in regulating BSP expression and hint at mechanisms for the formation of bone-associated metastasis in breast cancer that might involve local control of BSP levels by extracellular matrix degradation and release of growth factors.

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