**2. Patients and methods**

**1. Introduction**

120 Stromal Cells - Structure, Function, and Therapeutic Implications

malignant cells [13].

Multiple myeloma (MM) is a B-cell malignancy characterized by an accumulation of malignant plasma cells within the bone marrow (BM) [1]. Contact between MM cells and their microenvironment plays a crucial role in MM survival and proliferation and is able to promote tumor progression and drug resistance. Bone marrow mesenchymal stromal cells (BMMSCs) represent a central component of MM microenvironment supporting its progression and proliferation [2–4]. Alterations in BMSC from MM patients (MM-BMMSC) have become an important research focus. Several studies and our previous data have suggested the genesis of constitutive abnormalities within the BMMSC population through direct and indirect interactions with MM cells [5–8]. The development of a senescence-like state in BMMSC and thereby a modulated secretory profile, worsened osteogenic differentiation potential and inhibition of the T-cell proliferation, was reported [6, 8, 9]. Senescent BMMSCs display an increased senescence-associated β-galactosidase activity (SAβGalA) and irregular cell morphology. Usually, the cell cycle of senescent cells is arrested at the G1

S-transition point in combination with the overexpression of different cell cycle inhibitors as p21 and p16. In spite of the aberrant growth characteristics, senescent cells remain metabolically active, and therefore, the secretion of pro-inflammatory mediators could promote tumorigenesis in neighboring premalignant cells [10–12]. The secretion of pro-inflammatory mediators by senescent BMMSC could therefore promote tumorigenesis in neighboring pre-

Two imprinted clusters in the human genome might contribute to the generation of senescence and the induction of cellular changes in MM-BMMSC [14–17]. The DLK1-DIO3 imprinted domain is located on chromosome 14q32.2, and cluster C19MC is located on chromosome 19q13. The DLK1-DIO3 expresses the non-coding transcripts MEG3, anti-RTL1, 53 microR-NAs (miRNA), and 2 snoRNA clusters on the maternal chromosome. The paternal chromosome is responsible for the transcription of the protein-coding genes DLK1, RTL1, and DIO3 [18, 19]. Allelic expression of these genes is controlled through methylation of a regulatory region (IG-DMR) located upstream of the cluster [20]. The C19MC codes for 59 miRNAs are processed into one primary transcript from the paternal chromosome. Its expression strongly

In addition, there are evidence that the presence of cancer-associated fibroblasts (CAFs), characterized by high α-SMA, FAP, and FSP-1 expression, in the BM samples of MM patients, contributes to altered, tumor favorable, cell-cell interactions and cytokine secretions [22–24]. BMMSC represents an essential part for assistance of MM partly by the secretion of tumor supportive cytokines as interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) [2]. Both of them play a major role in the aberration of multiple signaling pathways such as PI3K, JAK/STAT3, Raf, and NF-κB [25]. In addition, MM cells produce inflammatory molecules, such as TGFβ and TNFα, which lead to NF-κB activation tumor-promoting effects. The activation of the NF-κB pathway in both MM cells and BMMSC results in the downstream upregulation of adhesion molecules and a consequent increase in cell-cell interactions [26, 27].

correlates with the epigenetic modulation of a CpG site located upstream [21].

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