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

Dormancy 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.

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.

Drug-tolerant persister cells in cancer: the cutting edges and future directions.

Drug-tolerant persister (DTP) cell populations were originally discovered in antibiotic-resistant bacterial biofilms. Similar populations with comparable features have since been identified among cancer cells and have been linked with treatment resistance that lacks an underlying genomic alteration. Research over the past decade has improved our understanding of the biological roles of DTP cells in cancer, although clinical knowledge of the role of these cells in treatment resistance remains limited. Nonetheless, targeting this population is anticipated to provide new treatment opportunities. In this Perspective, we aim to provide a clear definition of the DTP phenotype, discuss the underlying characteristics of these cells, their biomarkers and vulnerabilities, and encourage further research on DTP cells that might improve our understanding and enable the development of more effective anticancer therapies.

Spatial Distribution of Immune Cells in Primary and Recurrent Glioblastoma: A Small Case Study.

Only a minority of patients with glioblastoma (GBM) respond to immunotherapy, and always only partially. There is a lack of knowledge on immune distribution in GBM and in its tumor microenvironment (TME). To address the question, we used paired primary and recurrent tumors from GBM patients to study the composition and the evolution of the immune landscape upon treatment. We studied the expression of a handful of immune markers (CD3, CD8, CD68, PD-L1 and PD-1) in GBM tissues in 15 paired primary and recurrent GBM. In five selected patients, we used Nanostring Digital Spatial Profiling (DSP) to obtain simultaneous assessments of multiple biomarkers both within the tumor and the microenvironment in paired primary and recurrent GBM. Our results suggest that the evolution of the immune landscape between paired primary and recurrent GBM tumors is highly heterogeneous. However, our study identifies B3-H7 and HLA-DR as potential targets in primary and recurrent GBM. Spatial profiling of immune markers from matched primary and recurrent GBM shows a nonlinear complex evolution during the progression of cancer. Nonetheless, our study demonstrated a global increase in macrophages, and revealed differential localization of some markers, such as B7-H3 and HLA-DR, between GBM and its TME.

A Simple 3D Cell Culture Method for Studying the Interactions between Human Mesenchymal Stromal/Stem Cells and Patients Derived Glioblastoma.

We have developed a 3D biosphere model using patient-derived cells (PDCs) from glioblastoma (GBM), the major form of primary brain tumors in adult, plus cancer-activated fibroblasts (CAFs), obtained by culturing mesenchymal stem cells with GBM conditioned media. The effect of MSC/CAFs on the proliferation, cell-cell interactions, and response to treatment of PDCs was evaluated. Proliferation in the presence of CAFs was statistically lower but the spheroids formed within the 3D-biosphere were larger. A treatment for 5 days with Temozolomide (TMZ) and irradiation, the standard therapy for GBM, had a marked effect on cell number in monocultures compared to co-cultures and influenced cancer stem cells composition, similar to that observed in GBM patients. Mathematical analyses of spheroids growth and morphology confirm the similarity with GBM patients. We, thus, provide a simple and reproducible method to obtain 3D cultures from patient-derived biopsies and co-cultures with MSC with a near 100% success. This method provides the basis for relevant in vitro functional models for a better comprehension of the role of tumor microenvironment and, for precision and/or personalized medicine, potentially to predict the response to treatments for each GBM patient.

Challenges in glioblastoma research: focus on the tumor microenvironment.

Glioblastoma (GBM) is the most deadly type of malignant brain tumor, despite extensive molecular analyses of GBM cells. In recent years, the tumor microenvironment (TME) has been recognized as an important player and therapeutic target in GBM. However, there is a need for a full and integrated understanding of the different cellular and molecular components involved in the GBM TME and their interactions for the development of more efficient therapies. In this review, we provide a comprehensive report of the GBM TME, which assembles the contributions of physicians and translational researchers working on brain tumor pathology and therapy in France. We propose a holistic view of the subject by delineating the specific features of the GBM TME at the cellular, molecular, and therapeutic levels.

Three-dimensional in vitro culture models in oncology research.

Cancer is a multifactorial disease that is responsible for 10 million deaths per year. The intra- and inter-heterogeneity of malignant tumors make it difficult to develop single targeted approaches. Similarly, their diversity requires various models to investigate the mechanisms involved in cancer initiation, progression, drug resistance and recurrence. Of the in vitro cell-based models, monolayer adherent (also known as 2D culture) cell cultures have been used for the longest time. However, it appears that they are often less appropriate than the three-dimensional (3D) cell culture approach for mimicking the biological behavior of tumor cells, in particular the mechanisms leading to therapeutic escape and drug resistance. Multicellular tumor spheroids are widely used to study cancers in 3D, and can be generated by a multiplicity of techniques, such as liquid-based and scaffold-based 3D cultures, microfluidics and bioprinting. Organoids are more complex 3D models than multicellular tumor spheroids because they are generated from stem cells isolated from patients and are considered as powerful tools to reproduce the disease development in vitro. The present review provides an overview of the various 3D culture models that have been set up to study cancer development and drug response. The advantages of 3D models compared to 2D cell cultures, the limitations, and the fields of application of these models and their techniques of production are also discussed.

Cellular Heterogeneity and Cooperativity in Glioma Persister Cells Under Temozolomide Treatment.

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.