**4. Discussion**

MM-BMMSCs play a critical role in MM tumor growth and survival. Several studies suggest the existence of constitutive abnormalities in MM-BMMSC, and these lead to abnormal cell characteristics and increased tumor support [5, 6, 9, 23, 53, 54]. In this study, we explored the cellular and genetic aberrations of MM-BMMSCs in order to further identify the molecular mechanisms for these changes.

The enhanced and early senescence of BMMSC has been previously reported for different hematological disorders, including MM [9, 55]. Here, a significant higher senescence level of MM-BMMSC compared to HD-BMMSC was identified. When combined with our qPCR results that revealed an enrichment of cyclin D1 mRNA and the reduced expression of cyclin E1, an arrest of the cell cycle in G1 phase can be assumed. In contrast, André et al. related senescence to an accumulation of MM-BMMSCs in S phase [9]. These contrasting results could be due to diverse patient samples as well as different cell isolation and culture treatment methods. However, early senescence indicates the impairment of MM-BMMSCs. With regard to the relapsed analysis group, therapy might lead to increased cellular stress for MM-BMMSCs resulting in higher senescence levels.

Distinct changes to gene expression profiles were also reported [24, 56–59]. In addition to the abovementioned changes in cyclin expression, an upregulation of the cell adhesion molecules VCAM-1 and ICAM-1, as well as the NF-κB member IKK-α, was found, consistent with previous studies [6, 9, 53]. Overexpression of the cell adhesion molecules and the NF-κB pathway without MM-BMMSC in contact with MM cells suggests the generation of a constitutive myeloma favorable microenvironment.

In contrast to the above studies, data relating to microRNA expression in MM-BMMSCs are limited. Here, overexpression of miR-16, miR-223, miR-485-5p, and miR-519d was identified. These microRNAs possibly influence cell cycle regulation, cell differentiation, and cell migration. Alterations to MM-BMMSCs could therefore result from the specific deregulation of microRNA expression and their corresponding downstream targets [15, 52, 60–66]. The relapsed analysis group displayed a higher senescence level and a strongly increased microRNA expression (mean fold change > 100), supporting their possible function as cell cycle modifiers. Therapy seems to enforce senescence in MM-BMMSCs due to higher cellular stress and could lead to an even more altered cellular phenotype at relapse.

Subsequently, the ROS amount, mitochondrial membrane potential, cell cycle, and SAβGalA of the cells were investigated. Two different HD-BMMSCs were used for these analyses, and from each study, 2–3 replicates were performed. The donors were 73 and 74 years old. Furthermore, four different siRNAs against SIRT3 were used. SIRT3 knockdown in HD-BMMSC induced 1.4 to 1.9-fold increase in ROS levels (*p* < 0.05; **Figure 6A**). This was associated with dissipation of ΔΨM between 1.4- and 1.8-fold depending on the siRNA that was used for transient knockdown of SIRT3 (*p* < 0.04; **Figure 6B**). Furthermore, the inhibition of SIRT3 mimicked cell cycle arrest in S phase previously reported in BMMSC of myeloma patients. The percentage of BMMSC in S phase increased upon SIRT3 knockdown between 6.7 and 9.6% (*p* < 0.039; **Figure 6C**). In addition, it was investigated whether the depletion of SIRT3 increases senescence-associated β-galactosidase activity. It was found that transfection of HD-BMMSC with SIRT3 siRNAs 4 and 5 resulted in an approximately 1.5-fold increase in SAβGalA (*p* < 0.03). In contrast, HD-BMMSCs transfected with siRNA 2 did not show any changes. Similarly, transfections with siRNA 3

MM-BMMSCs play a critical role in MM tumor growth and survival. Several studies suggest the existence of constitutive abnormalities in MM-BMMSC, and these lead to abnormal cell characteristics and increased tumor support [5, 6, 9, 23, 53, 54]. In this study, we explored the cellular and genetic aberrations of MM-BMMSCs in order to further identify the molecular

The enhanced and early senescence of BMMSC has been previously reported for different hematological disorders, including MM [9, 55]. Here, a significant higher senescence level of MM-BMMSC compared to HD-BMMSC was identified. When combined with our qPCR results that revealed an enrichment of cyclin D1 mRNA and the reduced expression of cyclin

cence to an accumulation of MM-BMMSCs in S phase [9]. These contrasting results could be due to diverse patient samples as well as different cell isolation and culture treatment methods. However, early senescence indicates the impairment of MM-BMMSCs. With regard to the relapsed analysis group, therapy might lead to increased cellular stress for MM-BMMSCs

Distinct changes to gene expression profiles were also reported [24, 56–59]. In addition to the abovementioned changes in cyclin expression, an upregulation of the cell adhesion molecules VCAM-1 and ICAM-1, as well as the NF-κB member IKK-α, was found, consistent with previous studies [6, 9, 53]. Overexpression of the cell adhesion molecules and the NF-κB pathway without MM-BMMSC in contact with MM cells suggests the generation of a constitutive

In contrast to the above studies, data relating to microRNA expression in MM-BMMSCs are limited. Here, overexpression of miR-16, miR-223, miR-485-5p, and miR-519d was identified. These microRNAs possibly influence cell cycle regulation, cell differentiation, and cell migration.

phase can be assumed. In contrast, André et al. related senes-

caused only minimal changes in HD-BMMSCs (**Figure 6D**).

132 Stromal Cells - Structure, Function, and Therapeutic Implications

**4. Discussion**

mechanisms for these changes.

E1, an arrest of the cell cycle in G1

resulting in higher senescence levels.

myeloma favorable microenvironment.

Overexpressed miR-485-5p and miR-519d are associated with two imprinted clusters on chromosomes 14 (DLK1-DIO3) and 19 (C19MC), respectively. Since both clusters exhibit a complex composition, including tumor-suppressive as well as tumor-promoting microR-NAs, changes to their epigenetic regulation could account for important changes to the cellular characteristics of MM-BMMSCs [21, 66]. Here, analysis revealed hypomethylation and amplification of both clusters, possibly resulting in a higher transcriptional rate of cluster-associated genes. Several studies have reported the accumulation of genomic and global methylation changes due to in vitro cultivation of BMMSCs [67–72]. Indeed, minimal changes in the HD-BMMSC population, for example, hypo- and hypermethylation, as well as CN values between 2.2 and 2.8, were found. However, these alterations were less than those found in MM-BMMSCs, with distinct clustering of MM-BMMSC values below 20% methylation level and a mean value of more than 3.5 copies of the DLK1-DIO3 and C19MC genomic regions. The detected aberrations could be due to the existence of a CAF population in the MM-BMMSCs because some data highlight the presence of DNA hypomethylation and genetic instability in CAFs [24, 56, 73]. However, genetic instability in CAFs is controversial [74]. Hence, it cannot be excluded that CN variations of DLK1-DIO3 and C19MC result from hypomethylation or vice versa.

Moreover, the effect of MM cells on previously identified gene expression variations was investigated. In this context, a proliferation stimulating influence of KMS12-PE myeloma cells on MM-BMMSCs was apparent. Thus, KMS12-PE cells appear to repress MM-BMMSC senescence entry and increase the cell vitality. This modification could be associated with an increase in cyclin E1 mRNA levels.

Lastly, we investigated whether metabolic changes in MM-BMMSC could be responsible for the early aging status of the cells. For this purpose, we analyzed the expression of the gene and protein of the metabolic molecules SIRT3 and UCP2 and the lactate transporter MCT1 and MCT4. There were no significant differences in the gene expression of MCT1, MCT4, and UCP2 in MM-BMMSC compared to HD-BMMSC. In contrast, a significant lower expression of SIRT3 and a significant increase in mitochondrial mass compared were detected in MM-BMMSC. Interesting, no changes were detected in MGUS-BMMSC, suggesting an association with disease progression.

Our results suggested that MM cells influence the mitochondrial function of MM-BMMSC. This interaction leads to decrease the ROS levels in both cell types and could support their survival and growth. Moreover, the sustained induction of mitochondrial stress response could be the reason for premature senescence in MM-BMMSC. Therefore, the result of MM therapy could be improved through the disabling of metabolic interactions between MM cells and MM-BMMSC.
