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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.

Bone sarcomas are tumours belonging to the family of mesenchymal tumours and constitute a highly heterogeneous tumour group. The three main bone sarcomas are osteosarcoma, Ewing sarcoma and chondrosarcoma each subdivided in diverse histological entities. They are clinically characterised by a relatively high morbidity and mortality, especially in children and adolescents. Although these tumours are histologically, molecularly and genetically heterogeneous, they share a common involvement of the local microenvironment in their pathogenesis. This review gives a brief overview of their specificities and summarises the main therapeutic advances in the field of bone sarcoma.

Circulating tumor cells (CTCs) are biomarkers for noninvasively measuring the evolution of tumor genotypes during treatment and disease progression. Recent technical progress has made it possible to detect and characterize CTCs at the single-cell level in blood. Most current methods are based on epithelial cell adhesion molecule (EpCAM) detection, but numerous studies have demonstrated that EpCAM is not a universal marker for CTC detection because it fails to detect both carcinoma cells that undergo epithelial-mesenchymal transition (EMT) and CTCs of mesenchymal origin. Moreover, EpCAM expression has been found in patients with benign diseases. A large proportion of the current studies and reviews about CTCs describe EpCAM-based methods, but there is evidence that not all tumor cells can be detected using this marker. Here we describe the most recent EpCAM-independent methods for enriching, isolating, and characterizing CTCs on the basis of physical and biological characteristics and point out the main advantages and disadvantages of these methods. CTCs offer an opportunity to obtain key biological information required for the development of personalized medicine. However, there is no universal marker of these cells. To strengthen the clinical utility of CTCs, it is important to improve existing technologies and develop new, non-EpCAM-based systems to enrich and isolate CTCs.

Macrophages are specialized cells that control tissue homeostasis. They include non-resident and tissue-resident macrophage populations which are characterized by the expression of particular cell surface markers and the secretion of molecules with a wide range of biological functions. The differentiation and polarization of macrophages relies on specific growth factors and their receptors. Macrophage-colony stimulating factor (CSF-1) and interleukine-34 (IL-34), also known as \"twin\" cytokines, are part of this regluatory landscape. CSF-1 and IL-34 share a common receptor, the macrophage-colony stimulating factor receptor (CSF-1R), which is activated in a similar way by both factors and turns on identical signaling pathways. However, there is some discrete differential activation leading to specific activities. In this review, we disscuss recent progress in understanding of the role of the twin cytokines in macrophage differentiation, from their interaction with CSF-1R and the activation of signaling pathways, to their implication in macrophage polarization of non-resident and tissue-resident macrophages. A special focus on IL-34, its involvement in pathophsyiological contexts, and its potential as a theranostic target for macrophage therapy will be proposed.

Tumor heterogeneity is the major cause of failure in cancer prognosis and prediction. Accurately detecting heterogeneity for the development of biomarkers and the detection of the clones resistant to therapy is one of the main goals of contemporary medicine. Metastases belong to the natural history of cancer. The present review gives an overview on the origin of tumor heterogeneity. Recent progress has made it possible to isolate and characterize circulating tumor cells (CTCs), which are the drivers of the disease between the primary sites and metastatic foci. The most recent methods for characterizing CTCs are summarized and we discuss the power of CTC profiling for analyzing tumor heterogeneity in early and advanced diseases.

The ability to analyze intratumoral heterogeneity is of great interest for both diagnostic and basic research purposes. However, currently available dissection techniques are unsuitable for routine use and hard to access financially. Recently, a novel microcore-based dissection technique has been developed by the company Excilone for studying tissue heterogeneity in formalin-fixed paraffin-embedded (FFPE) microcore samples. The use of FFPE biological samples for transcriptomic studies, coupled with their small size, remains a real barrier to dissection applications. The efficacy of five commercially available RNA extraction kits were analyzed on microcores collected from human and mice FFPE tissues. Thirty microcore samples of healthy tissue (human uterus and stomach, and murine liver and spleen) were collected and distributed equally and randomly to the five kits assessed. Microcores were collected directly from paraffin blocks using 200 µm inner diameter needles with a sample depth, variable regarding to tissue type, ranging from 450 to 600 µm. Overall RNA yield and RNA fragmentation were evaluated, and RT-qPCR analyses were carried out after deparaffinization and compared to non-deparaffinization protocols. RNA yields and RNA fragmentation varied considerably between kits and FFPE tissues analyzed. Although the main limitation of this technique is the small initial sample size, differences in qPCR efficiency were also observed. Interestingly, no significant differences were observed between deparaffinized and non-deparaffinized microcore samples. Ultimately, we demonstrate the feasibility of using FFPE microcore samples for sensitive molecular biology applications, both with and without deparaffinization. The importance of setting up an optimized workflow was emphasized by significant differences observed in outcomes of the different protocols.

Introduction The immune system plays a major role in cancer progression. In solid tumors, 5-40 % of the tumor mass consists of tumor-associated macrophages (TAMs) and there is usually a correlation between the number of TAMs and poor prognosis, depending on the tumor type. TAMs usually resemble M2 macrophages. Unlike M1-macrophages which have pro-inflammatory and anti-cancer functions, M2-macrophages are immunosuppressive, contribute to the matrix-remodeling, and hence favor tumor growth. The role of TAMs is not fully understood in breast cancer progression. Methods Macrophage infiltration (CD68) and activation status (HLA-DRIIα, CD163) were evaluated in a large cohort of human primary breast tumors (562 tissue microarray samples), by immunohistochemistry and scored by automated image analysis algorithms. Survival between groups was compared using the Kaplan-Meier life-table method and a Cox multivariate proportional hazards model. Macrophage education by breast cancer cells was assessed by ex vivo differentiation of peripheral blood mononuclear cells (PBMCs) in the presence or absence of breast cancer cell conditioned media (MDA-MB231, MCF-7 or T47D cell lines) and M1 or M2 inducing cytokines (respectively IFN-γ, IL-4 and IL-10). Obtained macrophages were analyzed by flow cytometry (CD14, CD16, CD64, CD86, CD200R and CD163), ELISA (IL-6, IL-8, IL-10, monocyte colony stimulating factor M-CSF) and zymography (matrix metalloproteinase 9, MMP-9). Results Clinically, we found that high numbers of CD163 + M2-macrophages were strongly associated with fast proliferation, poor differentiation, estrogen receptor negativity and histological ductal type ( p <0.001) in the studied cohort of human primary breast tumors. We demonstrated ex vivo that breast cancer cell-secreted factors modulate macrophage differentiation toward the M2 phenotype. Furthermore, the more aggressive mesenchymal-like cell line MDA-MB231, which secretes high levels of M-CSF, skews macrophages toward the more immunosuppressive M2c subtype. Conclusions This study demonstrates that human breast cancer cells influence macrophage differentiation and that TAM differentiation status correlates with recurrence free survival, thus further emphasizing that TAMs can similarly affect therapy efficacy and patient outcome.

Classically, particle-induced periprosthetic osteolysis at the implant–bone interface has explained the aseptic loosening of joint replacement. This response is preceded by triggering both the innate and acquired immune response with subsequent activation of osteoclasts, the bone-resorbing cells. Although particle-induced periprosthetic osteolysis has been considered a foreign body chronic inflammation mediated by myelomonocytic-derived cells, current reports describe wide heterogeneous inflammatory cells infiltrating the periprosthetic tissues. This review aims to discuss the role of those non-myelomonocytic cells in periprosthetic tissues exposed to wear particles by showing original data. Specifically, we discuss the role of T cells (CD3 + , CD4 + , and CD8 + ) and B cells (CD20 + ) coexisting with CD68 + /TRAP − multinucleated giant cells associated with both polyethylene and metallic particles infiltrating retrieved periprosthetic membranes. This review contributes valuable insight to support the complex cell and molecular mechanisms behind the aseptic loosening theories of orthopedic implants.

The 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.

Background Literature reports that mature microRNA (miRNA) can be methylated at adenosine, guanosine and cytosine. However, the molecular mechanisms involved in cytosine methylation of miRNAs have not yet been fully elucidated. Here we investigated the biological role and underlying mechanism of cytosine methylation in miRNAs in glioblastoma multiforme (GBM). Methods RNA immunoprecipitation with the anti-5methylcytosine (5mC) antibody followed by Array, ELISA, dot blot, incorporation of a radio-labelled methyl group in miRNA, and miRNA bisulfite sequencing were perfomred to detect the cytosine methylation in mature miRNA. Cross-Linking immunoprecipiation qPCR, transfection with methylation/unmethylated mimic miRNA, luciferase promoter reporter plasmid, Biotin-tagged 3’UTR/mRNA or miRNA experiments and in vivo assays were used to investigate the role of methylated miRNAs. Finally, the prognostic value of methylated miRNAs was analyzed in a cohorte of GBM pateints. Results Our study reveals that a significant fraction of miRNAs contains 5mC. Cellular experiments show that DNMT3A/AGO4 methylated miRNAs at cytosine residues inhibit the formation of miRNA/mRNA duplex and leading to the loss of their repressive function towards gene expression. In vivo experiments show that cytosine-methylation of miRNA abolishes the tumor suppressor function of miRNA-181a-5p miRNA for example. Our study also reveals that cytosine-methylation of miRNA-181a-5p results is associated a poor prognosis in GBM patients. Conclusion Together, our results indicate that the DNMT3A/AGO4-mediated cytosine methylation of miRNA negatively. Graphical abstract