The distribution of neuromuscular junctions depends on muscle pennation, when botulinum neurotoxin receptors and SNAREs expression are uniform in the rat.

BACKGROUND: Botulinum neurotoxins (BoNTs) are used to treat spastic disorders. Depending on muscle size, one or multiple injections are recommended according to labels to target neuromuscular junctions (NMJ). However, information about NMJ distribution and number in muscles, as well as expression of receptors and molecular targets of toxins is scarce in human and animal models. METHODS: Seven muscles from adult rats were used to identify expression of BoNT receptors and SNAREs using immunohistochemistry (IHC), and fluorescent α-Bungarotoxin combined to light-sheet microscopy used to determine their distribution. RESULTS: The location, number, and density of NMJ were muscle specific and mostly dependent on the type of pennation (myofiber orientation). In the Flexor Digitorum Brevis (a very small muscle) NMJ were as numerous as in the Gastrocnemius lateralis. A strong expression of SV2C, Synaptotagmin 2, SNAP25 and VAMP1 were observed in all muscles, and SV2A, Synaptotagmin 1 and VAMP2 were never detected. CONCLUSION: This work highlights the specific distribution of NMJ in muscles which seems to depend on the type of pennation. Detailed observation of myofibers organization might help clinicians to better evaluate the location of NMJ in humans; the molecular phenotyping of NMJ will contribute to better integrate the rat model into research of BoNT therapeutics.

Screening for anti-adipogenic, pro-lipolytic and thermogenic plant extracts by models associating intestinal epithelial cells with human adipose cells.

PURPOSE: Excessive fat mass accumulation in obesity leads to diverse metabolic disorders, increased risks of cardiovascular diseases and in some cases, mortality. The aim of this study was to screen the actions of botanical extracts intended for oral use on human adipose tissue, using an in vitro screening model combining human intestinal cells with human adipose cells. This was to find the most effective extracts on lipid accumulation, UCP1 expression and ATP production in pre-adipocytes and on adipocyte lipolysis. METHODS: In this study, 25 individual plant extracts were screened for their effects on human adipose cells. Consequently, an original in vitro model was set up using the Caco-2 cell line, to mimic the intestinal passage of the extracts and then exposing human adipose cells to them. The biological actions of extracts were thus characterized, and compared with a coffee extract standard. The most effective extracts, and their combinations, were retained for their actions on lipid accumulation, the expression of the thermogenic effector UCP1 and ATP production in pre-adipocytes as well as on lipolysis activity of mature adipocytes. RESULTS: The biphasic culture system combining human Caco-2 cells with human adipose cells was verified as functional using the green coffee extract standard. Out of the 25 plant extracts studied, only 7 and their combinations were retained due to their potent effects on adipose cells biology. The data showed that compared to the coffee extract standard, Immortelle, Catechu, Carrot and Rose hip extracts were the most effective in reducing lipid accumulation and increased UCP1 expression in human pre-adipocytes. CONCLUSION: This study reveals the potential inhibitory effects on lipid accumulation and thermogenic activity of Immortelle, Catechu, Carrot and Rose hip extracts, and for the first time synergies in their combinations, using an in vitro model mimicking as closely as possible, human intestinal passage linked to adipose cells. These findings need to be confirmed by in vivo trials.

3D Model Characterization by 2D and 3D Imaging in t(14;18)-Positive B-NHL: Perspectives for In Vitro Drug Screens in Follicular Lymphoma.

Follicular lymphoma (FL) is an indolent B cell lymphoproliferative disorder of transformed follicular center B cells, which accounts for 20-30 percent of all non-Hodgkin lymphoma (NHL) cases. Great advances have been made to identify the most relevant targets for precision therapy. However, no relevant models for in vitro studies have been developed or characterized in depth. To this purpose, we generated a 3D cell model from t(14;18)-positive B-NHL cell lines cultured in ultra-low attachment 96-well plates. Morphological features and cell growth behavior were evaluated by classical microscopy (2D imaging) and response to treatment with different drugs was evaluated by a high-content analysis system to determine the robustness of the model. We show that the ultra-low attachment (ULA) method allows the development of regular, spherical and viable ULA-multicellular aggregates of lymphoma cells (MALC). However, discrepancies in the results obtained after 2D imaging analyses on drug-treated ULA-MALC prompted us to develop 3D imaging and specific analyses. We show by using light sheet microscopy and specifically developed 3D imaging algorithms that 3D imaging and dedicated analyses are necessary to characterize morphological properties of 3D models and drug effects. This study proposes a new method, but also imaging tools and informatic solutions, developed for FL necessary for future preclinical studies.

A checkpoint-oriented cell cycle simulation model.

Modeling and in silico simulations are of major conceptual and applicative interest in studying the cell cycle and proliferation in eukaryotic cells. In this paper, we present a cell cycle checkpoint-oriented simulator that uses agent-based simulation modeling to reproduce the dynamics of a cancer cell population in exponential growth. Our in silico simulations were successfully validated by experimental in vitro supporting data obtained with HCT116 colon cancer cells. We demonstrated that this model can simulate cell confluence and the associated elongation of the G1 phase. Using nocodazole to synchronize cancer cells at mitosis, we confirmed the model predictivity and provided evidence of an additional and unexpected effect of nocodazole on the overall cell cycle progression. We anticipate that this cell cycle simulator will be a potential source of new insights and research perspectives.

Reversible growth arrest of 3D tumor spheroids stored in oxygen absorber-induced anoxia.

Multicellular tumor spheroids models are of increasing interest in preclinical studies and pharmacological evaluation. However, their storage and transport is often a limitation because it requires adapted and expensive procedures. Here, we propose a very simple method to store 3D spheroids, using a procedure based on oxygen absorber-induced anoxia. We report that oxygen absorbers allow generating an anoxic environment for spheroid storage in culture plates. Oxygen absorber-induced anoxia fully and reversibly arrests spheroid growth for 4 days at 37°C and up to 18 days at 4°C. We then show that the response to etoposide is comparable in spheroids preserved in conditions of absorber-induced anoxia at 4°C and spheroids kept in normoxia at 37°C. These results represent a major improvement that should simplify the storage, transport and use of 3D spheroids.

Oxygen Partial Pressure Is a Rate-Limiting Parameter for Cell Proliferation in 3D Spheroids Grown in Physioxic Culture Condition.

The in situ oxygen partial pressure in normal and tumor tissues is in the range of a few percent. Therefore, when studying cell growth in 3D culture systems, it is essential to consider how the physiological oxygen concentration, rather than the one in the ambient air, influences the proliferation parameters. Here, we investigated the effect of reducing oxygen partial pressure from 21% to 5% on cell proliferation rate and regionalization in a 3D tumor spheroid model. We found that 5% oxygen concentration strongly inhibited spheroid growth, changed the proliferation gradient and reduced the 50% In Depth Proliferation index (IDP50), compared with culture at 21% oxygen. We then modeled the oxygen partial pressure profiles using the experimental data generated by culturing spheroids in physioxic and normoxic conditions. Although hypoxia occurred at similar depth in spheroids grown in the two conditions, oxygen partial pressure was a major rate-limiting factor with a critical effect on cell proliferation rate and regionalization only in spheroids grown in physioxic condition and not in spheroids grown at atmospheric normoxia. Our findings strengthen the need to consider conducting experiment in physioxic conditions (i.e., tissue normoxia) for proper understanding of cancer cell biology and the evaluation of anticancer drugs in 3D culture systems.

Evaluation by quantitative image analysis of anticancer drug activity on multicellular spheroids grown in 3D matrices.

Pharmacological evaluation of anticancer drugs using 3D in vitro models provides invaluable information for predicting in vivo activity. Artificial matrices are currently available that scale up and increase the power of such 3D models. The aim of the present study was to propose an efficient and robust imaging and analysis pipeline to assess with quantitative parameters the efficacy of a particular cytotoxic drug. HCT116 colorectal adenocarcinoma tumor cell multispheres were grown in a 3D physiological hyaluronic acid matrix. 3D microscopy was performed with structured illumination, whereas image processing and feature extraction were performed with custom analysis tools. This procedure makes it possible to automatically detect spheres in a large volume of matrix in 96-well plates. It was used to evaluate drug efficacy in HCT116 spheres treated with different concentrations of topotecan, a DNA topoisomerase inhibitor. Following automatic detection and quantification, changes in cluster size distribution with a topotecan concentration-dependent increase of small clusters according to drug cytotoxicity were observed. Quantitative image analysis is thus an effective means to evaluate and quantify the cytotoxic and cytostatic activities of anticancer drugs on 3D multicellular models grown in a physiological matrix.

Cell-Cell Adhesion and Cytoskeleton Tension Oppose Each Other in Regulating Tumor Cell Aggregation.

Cell aggregation is frequently impaired during the growth of primary tumors and the formation of metastatic lesions. Cell aggregation depends on cell-cell adhesion; however, no rigorous approach exists to monitor and quantify it accurately in the absence of the confounding factors of cell-substrate adhesion and the resulting cell motility on the substrate. We report here a highly reproducible, automated, microscopy-based quantification of tumor-cell spheroid formation in the absence of cell-substrate adhesion and use it to characterize cell aggregation dynamics in the early steps of this process. This method is based on fluorescence and bright-field microscopy and on a custom MATLAB program to quantify automatically the cells' aggregation kinetics. We demonstrate that the cell-cell adhesion protein E-cadherin and the desmosome proteins DSG2 and DSC2 are important for aggregation. Furthermore, we show that inhibition or silencing of myosin IIa enhances aggregation, suggesting that cytoskeleton tension inhibits tumor cell aggregation. This work opens new avenues to study the principles that govern multicellular aggregation, to characterize the aggregation properties of various tumor cell types, as well as to screen for drugs that inhibit or promote aggregation.