Sarcoma treatment in the era of molecular medicine.

Sarcomas are heterogeneous and clinically challenging soft tissue and bone cancers. Although constituting only 1% of all human malignancies, sarcomas represent the second most common type of solid tumors in children and adolescents and comprise an important group of secondary malignancies. More than 100 histological subtypes have been characterized to date, and many more are being discovered due to molecular profiling. Owing to their mostly aggressive biological behavior, relative rarity, and occurrence at virtually every anatomical site, many sarcoma subtypes are in particular difficult-to-treat categories. Current multimodal treatment concepts combine surgery, polychemotherapy (with/without local hyperthermia), irradiation, immunotherapy, and/or targeted therapeutics. Recent scientific advancements have enabled a more precise molecular characterization of sarcoma subtypes and revealed novel therapeutic targets and prognostic/predictive biomarkers. This review aims at providing a comprehensive overview of the latest advances in the molecular biology of sarcomas and their effects on clinical oncology; it is meant for a broad readership ranging from novices to experts in the field of sarcoma.

Adipose-Derived Mesenchymal Stem Cells do not Affect the Invasion and Migration Potential of Oral Squamous Carcinoma Cells.

Mesenchymal stem cells (MSCs) are commonly isolated from bone marrow and adipose tissue. Depending on the tissue of origin, MSCs have different characteristics and physiological effects. In various cancer studies, MSCs have been found to have either tumor-promoting or tumor-inhibiting action. This study investigated the effect of adipose tissue-MSCs (AT-MSCs) and bone marrow-MSCs (BM-MSCs) on global long interspersed nuclear element-1 (LINE-1) methylation, the expression level of microenvironment remodeling genes and cell proliferation, migration and invasion of oral tongue squamous cell carcinoma (OTSCC). Additionally, we studied the effect of human tongue squamous carcinoma (HSC-3)-conditioned media on LINE-1 methylation and the expression of microenvironment remodeling genes in AT-MSCs and BM-MSCs. Conditioned media from HSC-3 or MSCs did not affect LINE-1 methylation level in either cancer cells or MSCs, respectively. In HSC-3 cells, no effect of MSCs-conditioned media was detected on the expression of ICAM1, ITGA3 or MMP1. On the other hand, HSC-3-conditioned media upregulated ICAM1 and MMP1 expression in both types of MSCs. Co-cultures of AT-MSCs with HSC-3 did not induce proliferation, migration or invasion of the cancer cells. In conclusion, AT-MSCs, unlike BM-MSCs, seem not to participate in oral cancer progression.

LINE-1 Methylation Analysis in Mesenchymal Stem Cells Treated with Osteosarcoma-Derived Extracellular Vesicles.

Methylation-specific probe amplification (MSPA) is a simple and robust technique that can be used to detect relative differences in methylation levels of DNA samples. It is resourceful, requires small amounts of DNA, and takes around 4-5 h of hands-on work. In the presented technique, DNA samples are first denatured then hybridized to probes that target DNA at either methylated or reference sites as a control. Hybridized DNA is separated into parallel reactions, one undergoing only ligation and the other undergoing ligation followed by HhaI-mediated digestion at unmethylated GCGC sequences. The resultant DNA fragments are amplified by PCR and separated by capillary electrophoresis. Methylated GCGC sites are not digested by HhaI and produce peak signals, while unmethylated GCGC sites are digested and no peak signals are generated. Comparing the control-normalized peaks of digested and undigested versions of each sample provides the methylation dosage ratio of a DNA sample. Here, MSPA is used to detect the effects of osteosarcoma-derived extracellular vesicles (EVs) on the methylation status of long interspersed nuclear element-1 (LINE-1) in mesenchymal stem cells. LINE-1s are repetitive DNA elements that typically undergo hypomethylation in cancer and, in this capacity, may serve as a biomarker. Ultracentrifugation is also used as a cost-effective method to separate extracellular vesicles from biological fluids (i.e., when preparing EV-depleted fetal bovine serum [FBS] and isolating EVs from osteosarcoma conditioned media [differential centrifugation]). For methylation analysis, custom LINE-1 probes are designed to target three methylation sites in the LINE-1 promoter sequence and seven control sites. This protocol demonstrates the use of MSPA for LINE-1 methylation analysis and describes the preparation of EV-depleted FBS by ultracentrifugation.

Epigenetic alterations in mesenchymal stem cells by osteosarcoma-derived extracellular vesicles.

Extracellular vesicles (EVs) are central to intercellular communication and play an important role in cancer progression and development. Osteosarcoma (OS) is an aggressive bone tumour, characterized by the presence of malignant mesenchymal cells. The specific tumour-driving genetic alterations that are associated with OS development are currently poorly understood. Mesenchymal stem cells (MSCs) of osteogenic lineage have been postulated as likely candidates as the cells of origin for OS, thus indicating that MSCs and OS stroma cells may be related cell types. Therefore, this study set out to examine the EV-mediated intercellular crosstalk of MSCs and OS. MSCs and pre-osteoblasts were treated with OS-EVs at different time points, and the epigenetic signature of OS-EVs was assessed by methylation analysis of LINE-1 (long interspersed element) and tumour suppressor genes. In addition, surface markers and expression of specific genes were also evaluated. Our data indicated that OS-EVs mediated LINE-1 hypomethylation in MSCs, whereas an opposite effect was seen in pre-osteoblasts, indicating that MSCs but not pre-osteoblasts were susceptible to epigenetic transformation. Thus, OS-EVs modulated the fate of MSCs by modulating the epigenetic status, and also influenced the expression of genes related to bone microenvironment remodelling. Overall, this study provided evidence that epigenetic regulation appears to be an early event in the transformation of MSCs during the development of OS. Elucidating the mechanisms of EV-mediated communication may lead to new avenues for therapeutic exploitation.