Elise Demange

Direct Effect of Bevacizumab on Glioblastoma Cell Lines In Vitro

Abstract Bevacizumab is a humanized monoclonal antibody directed against the pro-angiogenic factor vascular and endothelial growth factor-A (VEGF-A) used in the treatment of glioblastomas. Although most patients respond initially to this treatment, studies have shown that glioblastomas eventually recur. Several non-mutually exclusive theories based on the anti-angiogenic effect of bevacizumab have been proposed to explain these mechanisms of resistance. In this report, we studied whether bevacizumab can act directly on malignant glioblastoma cells. We observe changes in the expression profiles of components of the VEGF/VEGF-R pathway and in the response to a VEGF-A stimulus following bevacizumab treatment. In addition, we show that bevacizumab itself acts on glioblastoma cells by activating the Akt and Erks survival signaling pathways. Bevacizumab also enhances proliferation and invasiveness of glioblastoma cells in hyaluronic acid hydrogel. We propose that the paradoxical effect of bevacizumab on glioblastoma cells could be due to changes in the VEGF-A-dependent autocrine loop as well as in the intracellular survival pathways, leading to the enhancement of tumor aggressiveness. Investigation of how bevacizumab interacts with glioblastoma cells and the resulting downstream signaling pathways will help targeting populations of resistant glioblastoma cells.

Survival of cord blood haematopoietic stem cells in a hyaluronan hydrogel for ex vivo biomimicry

Haematopoietic stem cells (HSCs) and haematopoietic progenitor cells (HPCs) grow in a specified niche in close association with the microenvironment, the so‐called ‘haematopoietic niche’. Scaffolds have been introduced to overcome the liquid culture limitations, mimicking the presence of the extracellular matrix (ECM). In the present study the hyaluronic acid scaffold, already developed in the laboratory, has been used for the first time to maintain long‐term cultures of CD34+ haematopoietic cells obtained from human cord blood. One parameter investigated was the impact on ex vivo survival of CD34+ cord blood cells (CBCs) on the hyaluronic acid surface, immobilized with peptides containing the RGD motif. This peptide was conjugated by coating the hyaluronan hydrogel and cultured in serum‐free liquid phase complemented with stem cell factor (SCF), a commonly indispensable cytokine for haematopoiesis. Our work demonstrated that these hyaluronan hydrogels were superior to traditional liquid cultures by maintaining and expanding the HPCs without the need for additional cytokines, and a colonization of 280‐fold increment in the hydrogel compared with liquid culture after 28 days of ex vivo expansion. Copyright

Biomimetic Three Dimensional Cell Culturing: Colorectal Cancer Micro-Tissue Engineering

Background: Spheroid cultures are known to mimic closely the properties of tumor tissue than monolayer cultures with regard to growth kinetics and metabolic rates. The aim of this paper is to confirm that tumor micro-tissue in a 3D biocompatible microenvironment maintain the cells natural behavior when compared to 2D monolayer culturing. Method: In order to validate our 3D culture system, we compared the 3D culture within a cross-linked hydrogel of hyaluronic acid, one of the major components of the extracellular matrix and the conventional 2D culture system. Results: Interestingly within our culture system, cells could be analyzed either after retrieval from the scaffold or even without being extracted in the 3D form rendering the HA hydrogel an ideal tool for biological applications. We observed the difference in the cell cycle, cell proliferation and behavior in both culture systems. Additionally drug testing was carried out using a chemotherapeutic agent (cis-platinium) that is already in clinical use to unequivocally prove the clinical predictive significance of the test strategy as compared with less complex assay systems and more complex in vivo models. We observed the presence of cell cycle heterogeneity very similar to the situation in vivo human tumors. Moreover, we have confirmed that resistance to chemotherapeutic reagents within this 3D culture system is much higher than those used in 2D cultures, since the tight assembly of cells in 3D culture systems render them more resistant requiring chemotherapeutic doses that recapitulate the drug sensitivity of tumor cells in vivo. Additionally we have observed the difference of apoptotic protein expression between 2D and 3D cell culture.

A biomimetic hydrogel functionalized with adipose ECM components as a microenvironment for the 3D culture of human and murine adipocytes

The lack of relevant in vitro models for adipose tissue makes necessary the development of a more physiological environment providing spatial and chemical cues for the effective maturation of adipocytes. We developed a biofunctionalized hydrogel with components of adipose extracellular matrix: collagen I, collagen VI, and the cell binding domain of fibronectin and we compared it to usual 2D cultures on plastic plates. This scaffold allowed 3D culture of mature adipocytes from the preadipocytes cell lines 3T3‐L1 and 3T3‐F442A, as well as primary Human White Preadipocytes (HWP), acquiring in vivo‐like organization, with spheroid shaped adipocytes forming multicellular aggregates. The size of these aggregates increased with time up to 120 μm in diameter after 4 weeks of maturation, with good viability. Significantly higher lipogenic activity (up to 20‐fold at day 28 for HWP cultures) and differentiation rates were also observed compared to 2D. Gene expression analyses highlighted earlier differentiation and complete maturation of 3D HWP compared to 2D, reinforced by the expression of Perilipin protein after 21 days of nutrition. This increase in adipocytes phenotypic and genotypic markers made this scaffold‐driven culture as a robust adipose 3D model. Retinoic acid inhibition of lipogenesis in HWP or isoprenalin and caffeine induction of lipolysis performed on mouse 3T3‐F442A cells, showed higher doses of molecules than typically used in 2D, underlying the physiologic relevance of this 3D culture system. Biotechnol. Bioeng. 2017;114: 1813–1824.

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 4266: Reproducible spheroid formation using functionalized hyaluronan 3D scaffolds

Extracellular matrix (ECM) characteristics, including stiffness, porosity, composition and spatial interaction with the surrounding cells and soluble factors are key components for cell growth in a tissue microenvironment. However, poor performance of 2D in vitro systems and animal models demands physiologically relevant well controlled 3D platforms for mechanistic assays, drug resistant phenotypes, new drug efficacy, toxicity assessment. Technical limitations in the current use of multicellular spheroids prevent their widespread use in cancer research and drug development. Existing systems for spheroid formation require lengthy processing times, and make simple tasks like media exchange, cell retrieval and microscopy analysis challenging. An ideal 3D cell culture system would form spheroids in an in vivo like microenvironment, while being easy to handle and compatible with all analytical methods. To address this unmet needs, we use a controlled hyaluronic acid-based scaffolds for spheroid formation. We have enriched 2 hyaluronan scaffolds with other components of the ECM such as collagen I, collagen IV, collagen VI, RGDs motif or galactosamine. Functionalised scaffolds overpass the 2D flat culture limitations by recreating cell/cell interactions and cell/matrix interaction to recreate a more physiologically authentic 3D architecture. The 2 functionalized scaffolds recapitulate the microenvironment for Hepatocyte and Adipocyte growth. Once formed, the spheroids can be cultured long-term, the scaffold is transparent allowing reproducible High Content Screening, the spheroids and the cells can be retrieve, avoiding the technical issues of other 3D systems to retain samples. Moreover, the scaffold is compatible with fluorescence/luminescent kit and immunofluorescent microscopy. We demonstrated this technology using cell lines, primary cells (adipocytes, hepatocytes). We assayed the spheroids over time using various endpoint spheroid morphology, growth and viability, resistance to anti-cancer drug, relevant cell organization formation and toxicity endpoint. Thus, this study introduces functionalized HA scaffold for the use of in vitro culture model as that represent native cell environments is ready to ready to use and compatible with HTS and all analytical methods for drug development and compound screening. Citation Format: Pauline Pannetier, Fiona Louis, Zied Souguir, Agathe Devaux, Didier Le Cerf, Jean-Pierre Vannier, Elise Demange, Guillaume Vidal. Reproducible spheroid formation using functionalized hyaluronan 3D scaffolds. [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 4266.