Technical report: liquid overlay technique allows the generation of homogeneous osteosarcoma, glioblastoma, lung and prostate adenocarcinoma spheroids that can be used for drug cytotoxicity measurements

Introduction: The mechanisms involved in cancer initiation, progression, drug resistance, and disease recurrence are traditionally investigated through in vitro adherent monolayer (2D) cell models. However, solid malignant tumor growth is characterized by progression in three dimensions (3D), and an increasing amount of evidence suggests that 3D culture models, such as spheroids, are suitable for mimicking cancer development. The aim of this report was to reaffirm the relevance of simpler 3D culture methods to produce highly reproducible spheroids, especially in the context of drug cytotoxicity measurements.Methods: Human A549 lung adenocarcinoma, LnCaP prostate adenocarcinoma, MNNG/HOS osteosarcoma and U251 glioblastoma cell lines were grown into spheroids for 20 days using either Liquid Overlay Technique (LOT) or Hanging Drop (HD) in various culture plates. Their morphology was examined by microscopy. Sensitivity to doxorubicin was compared between MNNG/HOS cells grown in 2D and 3D.Results: For all cell lines studied, the morphology of spheroids generated in round-bottom multiwell plates was more repeatable than that of those generated in flat-bottom multiwell plates. HD had no significant advantage over LOT when the spheroids were cultured in round-bottom plates. Finally, the IC50 of doxorubicin on MNNG/HOS cultured in 3D was 18.8 times higher than in 2D cultures (3D IC50 = 15.07 ± 0.3 µM; 2D IC50 = 0.8 ± 0.4 µM; *p < 0.05).Discussion: In conclusion, we propose that the LOT method, despite and because of its simplicity, is a relevant 3D model for drug response measurements that could be scaled up for high throughput screening

Cellular Heterogeneity and Cooperativity in Glioma Persister Cells Under Temozolomide Treatment

Factor de impacto: 6,684 Q1.We have observed a drug-tolerant/persister state in a human glioblastoma (GBM) cell line after exposure to temozolomide, the standard-of-care chemotherapeutic agent for GBM. We used a multicolor lentiviral genetic barcode labeling to follow cell population evolution during temozolomide treatment. We observed no change in the distribution of the different colored populations of cells in persister or resistant cells suggesting that pre-existing minor subpopulations, which would be expected to be restricted to a single color, were not amplified/selected during the response to the drug. We have previously identified four genes (CHI3L1, FAT2, KLK5, and HB-EGF) that were over-expressed during the persister stage. Single-cell analysis of these four genes indicated that they were expressed in different individual cells ruling out the existence of a single persister-specific clone but suggesting rather a global answer. Even so, the transitory silencing of CHI3L1, FAT2, or KLK5 influenced the expression of the other three genes and the survival of U251 cells in absence of temozolomide. Since proteins encoded by the four genes are all localized in the extracellular matrix or interact within the extracellular compartment, we propose that cellular interactions and communications are important during the persister stage before the acquisition of chemo-resistance. Thus, persisters might be a new therapeutically relevant target in GBM.This research was founded by a grant from the “Ligue contre le Cancer-Grand Ouest” and a Région Pays de la Loire special fund (ERRATA program).S

Development of three-dimensional cell culture models to study tumor progression and tor drug screening

Bien que les tumeurs malignes solides se développent en trois-dimensions (3D) dans l'organisme, les mécanismes impliqués dans les différentes étapes du cancer sont traditionnellement étudiés par le biais de modèles in vitro en monocouche adhérente (2D). L'objectif des travaux présentés dans ce manuscrit a été de développer des modèles de culture 3D et de mettre en évidence leur avantage par rapport aux culture 2D pour ce qui est du criblage de molécules, de l'étude de mécanismes de la progression tumorale et du développement de résistances aux thérapies. Pour ce faire, des lignées cellulaires de deux cancers communs (adénocarcinomes pulmonaire et prostatique) et de deux cancers rares (glioblastome et ostéosar-come) ont été cultivées par Liquid Overlay Technique ou Hanging Drop dans différents supports de culture afin de générer des sphéroïdes structurellement répétables. La diminution de la sensibilité à la doxorubicine des cellules d'ostéosarcome cultivées en sphéroïde souligne la pertinence de ces modèles dans le contexte du criblage de molécules thérapeutiques par rapport aux culture 2D. Ces modèles 3D ont également fait leur preuve dans le cas de la dormance tumorale, puisqu'un ralentissement de la prolifération cellulaire a pu être observée dans des sphéroïdes d'ostéosarcome par rapport à la culture 2D. Une signature transcriptomique de la dormance a ainsi pu être décrite et les différents gènes impliqués ont été étudiés. Pour conclure, les résultats présentés dans ce manuscrit encouragent ainsi une utilisation plus régulière des modèles 3D dans les projetsde recherche sur le cancer, car ils sont souvent plus représentatifs de la maladie que leurs équivalents 2D.Although solid malignant tumors growin three-dimensions (3D) in the body, the mechanisms involved in the different steps of cancer progression are traditionally investigated using adherent monolayer (2D) in vitro cell models. The aim of the work described here was to develop 3D culture models and to demonstrate their advantages over 2D cultures, especially for drug screening, the study of tumor progression mechanisms and the development of drug resistance. Cell lines from two common cancers (lung and prostate adenocarcinomas) and two rare cancers (glioblastoma and osteosarcoma) were grown in 3D using Liquid Overlay Technique or Hanging Drop in different types of culture vessels to generate spheroids with a repeatable structurellement répétables. La diminution de la sensibilité à la doxorubicine des cellules d'ostéosarcome cultivées en sphéroïde souligne la pertinence de ces modèles dans le contexte du criblage de molécules thérapeutiques par rapport aux culture 2D. Ces modèles 3D ont également fait leur preuve dans le cas de la dormance tumorale, puisqu'un ralentissement de la prolifération cellulaire a pu être observée dans des sphéroïdes d'ostéosarcome par rapport à la culture 2D. Une signature transcriptomique de la dormance a ainsi pu être décrite et les différents gènes impliqués ont été étudiés. Pour conclure, les résultats présentés dans ce manuscrit encouragent ainsi une utilisation plus régulière des modèles 3D dans les projetsde recherche sur le cancer, car ils sont souvent plus représentatifs de la maladie que leurs équivalents 2D. organization. The decreased sensitivity to doxorubicin of osteosarcoma cells grown as spheroids underlines the relevance of this modelin the case of drug screening compared to 2D cultures. These 3D models also allowed to better study cancer dormancy, as a reduction in proliferation rate was observed in osteosarcoma cells grown as spheroids. Thanks to the 3D models, a transcriptomic signature of cancer dormancy was obtained, and the involvement of various genes was i nvestifated. ln conclusion, the results described here support a more frequent use of 3D models in cancer research projects, as they are often more suitable than their 2D counterparts

Circulating tumor cell-derived pre-clinical models for personalized medicine

Source at https://doi.org/10.3390/cancers11010019The main cause of death from cancer is associated with the development of metastases, resulting from the inability of current therapies to cure patients at metastatic stages. Generating preclinical models to better characterize the evolution of the disease is thus of utmost importance, in order to implement effective new cancer biomarkers and therapies. Circulating Tumor Cells (CTCs) are good candidates for generating preclinical models, making it possible to follow up the spatial and temporal heterogeneity of tumor tissues. This method is a non-invasive liquid biopsy that can be obtained at any stage of the disease. It partially summarizes the molecular heterogeneity of the corresponding tumors at a given time. Here, we discuss the CTC-derived models that have been generated so far, from simplified 2D cultures to the most complex CTC-derived explants (CDX models). We highlight the challenges and strengths of these preclinical tools, as well as some of the recent studies published using these models

Circulating tumor cells and ctDNA in sarcomas

International audienceSarcomas are clustered in two oncological entities named bone and soft-tissue sarcomas. Both are rare cancers originating from the mesenchyme, characterized by their propensity to induce the development of lung metastases. Sarcoma cells escaping from the primary tumor site, spread to the pulmonary tissue through the bloodstream where they found a favorable microenvironment to establish metastatic foci. The low number of patients, the high histological, genetic and molecular heterogeneity of sarcomas combined with the absence of specific markers expressed by cancer cells make the detection and follow up of the minimal residual disease challenging. Over the last decade, tremendous technological progress has been made towards the detection of recurrent diseases. The literature is now enriched of information describing the use of liquid biopsies in clinical care of sarcoma patients. The present chapter aims to give a brief overview of the most recent data available on the detection of circulating tumor cells and circulating tumor DNA in sarcomas

Biological evidence of cancer stem-like cells and recurrent disease in osteosarcoma

Sarcomas are a large family of cancers originating in the mesenchyme. Composed of more than 100 histological subtypes, soft tissue and bone sarcomas remain clinically challenging, particularly in children and adolescents in whom sarcomas are the second most common malignant entities. Osteosarcoma is the main primary bone tumor in adolescents and young adults and is characterized by a high propensity to induce distant metastatic foci and become multi-drug resistant. The innate and acquired resistance of osteosarcoma can be explained by high histological heterogeneity and genetic/molecular diversity. In the last decade, the notion of cancer stem-like cells (CSCs) has emerged. This subset of cancer cells has been linked to drug resistance properties, recurrence of the disease, and therapeutic failure. Although CSCs remain controversial, many elements are in favor of them playing a role in the development of the drug resistance profile. The present review gives a brief overview of the most recent biological evidence of the presence of CSCs in osteosarcomas and their role in the drug resistance profile of these rare oncological entities. Their use as promising therapeutic targets is discussed

Identification of MCM4 and PRKDC as new regulators of osteosarcoma cell dormancy based on 3D cell cultures

International audienceDormancy is a potential way for tumors to develop drug resistance and escape treatment. However, the mechanisms involved in cancer dormancy remain poorly understood. This is mainly because there is no in vitro culture model making it possible to spontaneously induce dormancy. In this context, the present work proposes the use of three-dimensional (3D) spheroids developed from osteosarcoma cell lines as a relevant model for studying cancer dormancy. MNNG-HOS, SaOS-2, 143B, MG-63, U2OS and SJSA-1 cell lines were cultured in 3D using the Liquid Overlay Technique (LOT). Dormancy was studied by staining cancer cells with a lipophilic dye (DiD), and long-term DiD+ cells were considered as dormant cancer cells. The role of the extracellular matrix in inducing dormancy was investigated by embedding cells into methylcellulose or Geltrex™. Gene expression of DiD+ cells was assessed with a Nanostring™ approach and the role of the genes detected in dormancy was validated by a transient down-expression model using siRNA treatment. Proliferation was measured using fluorescence microscopy and the xCELLigence technology. We observed that MNNG-HOS, 143B and MG-G3 cell lines had a reduced proliferation rate in 3D compared to 2D. U2OS cells had an increased proliferation rate when they were cultured in Geltrex™ compared to other 3D culture methods. Using 3D cultures, a transcriptomic signature of dormancy was obtained and showed a decreased expression of 18 genes including ETV4, HELLS, ITGA6, MCM4, PRKDC, RAD21 and UBE2T. The treatment with siRNA targeting these genes showed that cancer cell proliferation was reduced when the expression of ETV4 and MCM4 were decreased, whereas proliferation was increased when the expression of RAD21 was decreased. 3D culture facilitates the maintenance of dormant cancer cells characterized by a reduced proliferation and less differential gene expression as compared to proliferative cells. Further studies of the genes involved has enabled us to envisage their role in regulating cell proliferation

In vitro three-dimensional cell cultures for bone sarcomas

International audienceBone sarcomas are rare tumour entities that arise from the mesenchyme most of which are highly heterogeneous at the cellular, genetic and epigenetic levels. The three main types are osteosarcoma, Ewing sarcoma, and chondrosarcoma. These oncological entities are characterized by high morbidity and mortality and an absence of significant therapeutic improvement in the last four decades. In the field of oncology, in vitro cultures of cancer cells have been extensively used for drug screening unfortunately with limited success. Indeed, despite the massive knowledge acquired from conventional 2D culture methods, scientific community has been challenged by the loss of efficacy of drugs when moved to clinical trials. The recent explosion of new 3D culture methods is paving the way to more relevant in vitro models mimicking the in vivo tumour environment (e.g. bone structure) with biological responses close to the in vivo context. The present review gives a brief overview of the latest advances of the 3D culture methods used for studying primary bone sarcomas