Valérie Lobjois

Forcing neural progenitor cells to cycle is insufficient to alter cell-fate decision and timing of neuronal differentiation in the spinal cord

BackgroundDuring the development of the nervous system, neural progenitor cells can either stay in the pool of proliferating undifferentiated cells or exit the cell cycle and differentiate. Two main factors will determine the fate of a neural progenitor cell: its position within the neuroepithelium and the time at which the cell initiates differentiation. In this paper we investigated the importance of the timing of cell cycle exit on cell-fate decision by forcing neural progenitors to cycle and studying the consequences on specification and differentiation programs.ResultsAs a model, we chose the spinal progenitors of motor neurons (pMNs), which switch cell-fate from motor neurons to oligodendrocytes with time. To keep pMNs in the cell cycle, we forced the expression of G1-phase regulators, the D-type cyclins. We observed that keeping neural progenitor cells cycling is not sufficient to retain them in the progenitor domain (ventricular zone); transgenic cells instead migrate to the differentiating field (mantle zone) regardless of cell cycle exit. Cycling cells located in the mantle zone do not retain markers of neural progenitor cells such as Sox2 or Olig2 but upregulate transcription factors involved in motor neuron specification, including MNR2 and Islet1/2. These cycling cells also progress through neuronal differentiation to axonal extension. We also observed mitotic cells displaying all the features of differentiating motor neurons, including axonal projection via the ventral root. However, the rapid decrease observed in the proliferation rate of the transgenic motor neuron population suggests that they undergo only a limited number of divisions. Finally, quantification of the incidence of the phenotype in young and more mature neuroepithelium has allowed us to propose that once the transcriptional program assigning neural progenitor cells to a subtype of neurons is set up, transgenic cells progress in their program of differentiation regardless of cell cycle exit.ConclusionOur findings indicate that maintaining neural progenitor cells in proliferation is insufficient to prevent differentiation or alter cell-fate choice. Furthermore, our results indicate that the programs of neuronal specification and differentiation are controlled independently of cell cycle exit.

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.

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.

Abstract 4262: Combined 3D quantitative imaging and 3D cell culture for cancer drug discovery

Monitoring drug activity in multicellular tumor spheroid in vitro models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological screening and anticancer drugs evaluation (1). Optical microscopy coupled to the selective plane illumination microscopy (SPIM)3D microscopy, and, and automatic automated images processing and quantitative analysis are promising approaches to process optimizes the evaluation of drug response in a 3D cell culture models with, in a high/mediumhigh throughput screening waycapabilities. BIOMIMESYS® used as a 3D cell model is based on hyaluronic to provide an in vivo like environment to the cells. The In this study the effects of the topoisomerase I inhibitor Topotecan were investigated on colon adenocarcinoma HCT-116 3D cell culture. Spheroids were prepared and grown in BIOMIMESYS® used as a 3D cell model isa matrix based on hyaluronic acid tothat provides an in vivo like environment to the cells. was monitored and coupled with mMorphometric parameters and with nuclei fluorescence markers were used to monitor analysis for drug activity. Data provideand to characterization characterize of both cytostatic and cytotoxic effects of this drug withon the basis of both quantitative 3D and imaging of the mechanisms. These data highlight the relevance of combining a biomimetic microenvironment for 3D cell culture recreating the the in vivo features of a tumor cell population biology, together with and high resolution content microscopy-based quantification for drug screening. This biomimetic model provides an innovative platform for the in-depth analysis of tumor development and for the discovery or the characterization of novel therapeutic targets. Ref: 1) Breslin S, O’Driscoll L. Drug Discov Today.18: 240-249 (2013) Citation Format: Guillaume Vidal, Valerie Lobjois, Zied Souguir, Pauline Pannetier, Elise Demange, Jean-Michel Lagarde. Combined 3D quantitative imaging and 3D cell culture for cancer drug discovery. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4262.

Abstract 560: Mechanical stress activates a mitotic checkpoint in multicellular tumor spheroids.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnExpanding solid tumors are subjected to mechanical stress that impact on their growth rate and development. However little is known on the mechanisms by which mechanical cues are acting on tumor cell biology.nnTo address that issue, we used MultiCellular Tumor Spheroid (MCTS), a 3D model recapitulating the microenvironment, the proliferative gradient and cell-cell interactions found in a tumor. To test the impact of mechanical stress on tumor cell proliferation, we first designed, produced and used dedicated polymer microdevices in which MCTS engineered to express fluorescent biomarkers were confined to apply mechanical constraints. We observe that under constraints, MCTS display a high proportion of mitotic cells in low proliferative regions of confined spheroids. We show that these cells are being arrested in mitosis for at least 24 hours (EdU incorporation neg.) and that mitotic arrest is not caused by impairment of rounding.nnWe next used live SPIM (Selective Plane Illumination Microscopy) 3D imaging to monitor mitosis progression in isotropically constrained MCTS. We show that constraint impairs bipolar spindle assembly and delays progression toward metaphase-anaphase transition.nnOur data indicate that in a multicellular structure mechanical constraints are responsible for a defect in cell cycle progression associated with a mitotic arrest.nnCitation Format: Annaick Desmaison, Katia Grenier, Celine Frongia, Corinne Lorenzo, Bernard Ducommun, Valerie Lobjois. Mechanical stress activates a mitotic checkpoint in multicellular tumor spheroids. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 560. doi:10.1158/1538-7445.AM2013-560

Checkpoint oriented cell-cycle simulation: critical role for age distribution initialization

In this paper we start to validate a computational cell cycle model, developed in a collaborative work between computer scientists and biologists, evaluating the convergence between in-vitro and in-silico results. Whereas most models are phase-orientated our model deals with a checkpoint orientated paradigm and uses phase orientation as an output to provide the biologists with a relevant view of the simulation result. Throughout this paper, we will show that the initialization state of a multi-cellular culture is a high constraint in the relevance of the results. This initialization state lets emerge computational artifacts as phasing.