Introduction As translational methods improve, liquid biopsies are becoming increasingly relevant in early detection, therapy monitoring, and tumor surveillance. CfDNA has been proposed as a potential analyte, however clinical translation has been limited by cost and technological constraints. Here, we evaluate a novel technology of reduced representation bisulfite sequencing for investigation of cfDNA methylation in HNSCC.
Mesenchymal stromal cells (MSC) are an integral cellular component of the tumor microenvironment. Nevertheless, very little is known about MSC originating from human malignant tissue and modulation of these cells by tumor-derived factors. The aim of this study was to isolate and characterize MSC from head and neck squamous cell carcinoma (HNSCC) and to investigate their interaction with tumor cells.MSC were isolated from tumor tissues of HNSCC patients during routine oncological surgery. Immunophenotyping, immunofluorescence and in vitro differentiation were performed to determine whether the isolated cells met the consensus criteria for MSC. The cytokine profile of tumor-derived MSC was determined by enzyme-linked immunosorbent assay (ELISA). Activation of MSC by tumor-conditioned media was assessed by measuring cytokine release and expression of CD54. The impact of MSC on tumor growth in vivo was analyzed in a HNSCC xenograft model.Cells isolated from HNSCC tissue met the consensus criteria for MSC. Tumor-derived MSC constitutively produced high amounts of interleukin (IL)-6, IL-8 and stromal cell-derived factor (SDF)-1α. HNSCC-derived factors activated MSC and enhanced secretion of IL-8 and expression of CD54. Furthermore, MSC provided stromal support for human HNSCC cell lines in vivo and enhanced their growth in a murine xenograft model.This is the first study to isolate and characterize MSC from malignant tissues of patients with HNSCC. We observed cross-talk of stromal cells and tumor cells resulting in enhanced growth of HNSCC in vivo.
Abstract During inflammatory processes, tissue environmental cues are influencing the immunoregulatory properties of tissue‐resident mesenchymal stem/stromal cells (MSC). In this study, we elucidated one of the molecular and cellular responses of human MSC exposed to combinations of inflammatory cytokines. We showed that during multi‐cytokine priming by TNF‐α, IL‐1β, and IFN‐γ, IL‐1β further augmented the well‐established immunoregulatory activity induced by TNF‐α/IFN‐γ. On the molecular level, TNF‐α and IL‐1β enhanced the expression of IFN‐γ receptor (IFN‐γR) via NF 'kappa‐light‐chain‐enhancer' of activated B‐cells (NF‐κΒ) signaling. In turn, enhanced responsiveness to IFN‐γ stimulation activated STAT5 and p38‐MAPK signaling. This molecular feedback resulted in an increased IL‐8 release and augmented recruitment of polymorphonuclear granulocytes (PMN). Our study suggests the possibility that responses of MSC to multi‐cytokine priming regimens may be exploited therapeutically to fine‐tune inflammatory activity in tissues. This study elucidates molecular mechanisms underlying the immunological priming of mesenchymal stromal cells (MSC) and their interaction with neutrophils.
Abstract Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that has been reported to enhance the aggressiveness and metastatic potential of tumor cells. However, the mechanisms through which MIF influences tumor development and progression are not understood. The objectives of our study were to assess the effects of tumor‐derived MIF on neutrophils in head and neck cancer (HNC) and to identify possible feedback effects on tumor cells. To this end, we used an in vitro system to model the interaction between human HNC cells and neutrophils. In addition, we analyzed expression of MIF in tissues from HNC patients in relation to neutrophilic infiltration and clinical parameters. Our results show that human HNC is infiltrated by neutrophils proportional to the levels of tumoral MIF. Strong MIF expression by the tumor is associated with higher lymph node metastasis and reduced survival in HNC patients. In vitro , MIF modulated functions of human neutrophils by inducing chemokine CXC motif receptor 2(CXCR2)‐dependent chemotaxis, enhancing neutrophil survival and promoting release of chemokine C‐C Motif Ligand 4 (CCL4) and matrix metalloprotease 9(MMP9). Further, neutrophils activated with tumor‐derived MIF enhanced migratory properties of HNC cells. In conclusion, our data indicate that the effects of tumor‐derived MIF on neutrophils represent an additional mechanism by which MIF might contribute to tumor progression.
Mesenchymal stem cells (MSCs) are adult fibroblastoid progenitor cells. Because of their immunoregulatory properties and their so-called trophic effects, MSCs play an important role in tissue regeneration, inflammation and trauma. Tissue trauma and challenge, for example during radiotherapy or infection, result in the release of so-called "danger molecules", which may be derived from dying cells or incoming pathogens. The molecular response of MSCs to this tissue stress remains largely elusive.In this study we examined the cell biological response of MSCs derived from human parotid glands (pgMSCs) and used bacterial endotoxin as a model of tissue stress and inflammation. PgMSCs from 3 donors were isolated, expanded and tested for classical tri-lineage plus myogenic differentiation. The cell biological response to the model "stressor" endotoxin was examined by low density gene expression arrays.Through immunofluorescence and immunohistochemistry we were able to proof osteogenic, adipogenic, chondrogenic, and myogenic differentiation potential characteristic for stem cells. In vitro, gene expression analysis showed a characteristic modulation of MSCs after stimulation with endotoxin Lipopolysaccharide (LPS). Specifically, receptors and ligands typically involved in immune regulation, such as interleukins, TGF-β, tumor necrosis factors (TNF), and toll-like receptors (TLR), were regulated.Our study elucidates some key functions and molecules, which are regulated in MSCs during tissue stress and inflammation. A thorough understanding of their cell biological function will aid future rationale therapeutic application of MSCs.
