**3.4. Co-culturing of MM-BMSC with the KMS12-PE cell line induces the changes of microRNA expression in both cell types**

The expression of four miRNA (miR-16, miR-223, miR-485-5p, and miR-519d) after co-culturing and transwell cultured MM-BMMSC was measured using qPCR (**Figure 4A**). MiR-223 was downregulated in co-cultured MM-BMMSC (*p* < 0.007), whereas no effect was detected in transwell

**Figure 4.** KMS12-PE myeloma cells downregulate miR-223 and miR-485-5p in MM-BMMSC. *P* values: \* <0.05; \*\* <0.01; \*\*\* <0.001; and \*\*\*\* <0.0001. All data were analyzed using the Wilcoxon signed-rank test. (A) Co-cultured MM-BMMSC (*n* = 25) displayed reduced expression of miR-223 and miR-485-5p. Transwell-cultured (*n* = 10) MM-BMMSC showed no changes in miR-223 expression but also decreased miR-485-5p levels. Intensity of changes in miR-485-5p decreased when cell-cell contact was prevented by transwell cultivation. (B) Cell interaction with MM-BMMSC induced changes in the microRNA expression of KMS12-PE myeloma cells (*n* = 10). MiR-221 was upregulated, whereas miR-223 and miR-519d decreased in co-cultured KMS12-PE myeloma cells.

cultured MM-BMMSC. In contrast, downregulation of miR-485-5p was detected in both cell culture systems (*p* < 0.03). Interestingly, cell-cell interaction also altered miRNA expression of KMS12-PE myeloma cells. We found upregulation of miR-221 and significantly downregulation of miR-223 and miR-519d (*p* < 0.02; **Figure 4B**). Expression of miR-485-5p was not detectable in KMS12-PE myeloma cells.

#### **3.5. KMS12-PE cells modulate the gene expression of MM-BMMSC**

**Figure 3.** Overexpressed microRNAs in MM-BMMSC are associated with hypomethylation and CN accumulation of DLK1-DIO3 and C19MC. *P* values: \* <0.05; \*\* <0.01; \*\*\* <0.001; and \*\*\*\* <0.0001. All data were analyzed using the Mann-Whitney *U* test. (A) ND-MM-BMMSC and R-MM-BMMSC showed high overexpression of miR-16, miR-485-5p, miR-519d, and miR-223 compared to HD-BMMSCs. (B) The regulatory regions of DLK1-DIO3 and C19MC were hypomethylated in ND-MM-BMMSC and R-MM-BMMSC compared to HD-BMMSC. (C) CN analysis of C19MC displayed CN accumulation in all three regions in MM-BMMSC compared to HD-BMMSC. (D) CN analysis of DLK1- DIO3 displayed CN accumulation in all three measured positions in MM-BMMSCs compared to HD-BMMSC.

Given that the expression of both clusters is controlled by methylation of their regulatory regions, we analyzed their methylation status using qMSP (**Figure 3B**). Hypomethylation of both clusters in MM-BMMSCs compared to HD-BMMSCs was observed. For DLK1-DIO3, MM-BMMSC exhibited an approximate fivefold lower methylation level of the IG-DMR. The C19MC exhibited a 2.5-fold lower methylation level in MM-BMSC compared to HD-BMMSC (*p =* 0.0062). CN analysis of both clusters displayed CN accumulation in all three regions in

MM-BMMSC (*n* = 38) compared to HD-BMMSC (*n* = 8; **Figure 3C** and **D**).

**microRNA expression in both cell types**

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**3.4. Co-culturing of MM-BMSC with the KMS12-PE cell line induces the changes of** 

The expression of four miRNA (miR-16, miR-223, miR-485-5p, and miR-519d) after co-culturing and transwell cultured MM-BMMSC was measured using qPCR (**Figure 4A**). MiR-223 was downregulated in co-cultured MM-BMMSC (*p* < 0.007), whereas no effect was detected in transwell To explore the influence of KMS12-PE cells on gene expression of adhesion molecules, qPCR analysis of MM-BMMSC, co-cultured for 72 h with KMS12-PE cells in passage 4, was performed (*n* = 25). In mono-cultured BMSC, an upregulation of VCAM-1 (*p* = 0.33), ICAM-1 (*p* = 0.33), and IKK-α (*p* = 0.05) was demonstrated. Furthermore, the expression profile of miRNAs, targeting the analyzed genes or correlating with senescence, was studied (miR-16, miR-221, miR-126, miR-223, miR-485-5p, and miR-519d). MiR-16, miR-223, miR-485-5p, and miR-519d were significantly upregulated (*p* = 0.02; *p* = 0.004; *p* = 0.02; and *p* = 0.002, respectively), whereas miR-221 and miR-126 showed no considerable differences to BMSC obtained from healthy donors. After co-culturing of MM-BMSC with KMS12-PE cells, an enhanced expression of adhesion molecules was apparent. This includes the upregulation of VCAM-1 (*p =* 0.0078), ICAM-1 (*p =* 0.2425), and NF-κB activator IKK-α (*p =* 0.0573), though the values for ICAM-1 and IKK-α were not significant. Hence, MM cells seem to further boost the aberrant expression of adhesion molecules in MM-BMMSCs. Regarding microRNAs, a significant downregulation of miR-223 and miR-485-5p (*p <* 0.009) was detected. In addition, miR-16 and miR-519d showed a trend toward downregulation, though the changes were not significant. No expression alterations to miR-221 or miR-126 were detected (data not shown).

#### **3.6. Expression of metabolic regulators in MM-BMSC**

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.

To explore the influence of MM cells on SIRT3 expression in BMSC, co-culturing for 72 h with KMS12-PE cells (*n* = 20) and transwell experiments (*n* = 10) was performed. Interestingly, we found a fourfold upregulation of SIRT3 expression in MM-BMMSC when co-cultured with

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Moreover, co-cultivation induced depolarization of ΔΨm leading to an approximately twofold JC1 monomers increasing in MM-BMSC and MM cells (**Figure 5D**). Co-cultivation of KMS12-PE and MM-BMSC reduced the amount of ROS in both cell systems (**Figure 5E**).

To further elucidate the involvement of SIRT3 in metabolic and senescence-like alterations of MM-BMMSCs, siRNA was used to transiently "knockdown" this gene in HD-BMMSC.

**Figure 6.** Influence of SIRT3 on ROS in HD-BMMSC. *P* values: \* <0.05; \*\* <0.01; \*\*\* <0.001; and \*\*\*\* <0.0001. (A) The knockdown of SIRT3 in HD-BMMSC caused an increase in the ROS content of all four siRNAs tested compared to the negative and transfection control. (B) Influence of SIRT3 on ΔΨm in HD-BMMSC. The "knockdown" of SIRT3 in HD-BMMSC caused a reduction in the FL-2/FL-1 ratio. For siRNAs 2 and 3, only the proportion of FL-1 negative cells was reduced (R-4), whereas siRNAs 4 and 5 also caused an increase in FL-2 negative cells (R-3). (C) Influence of SIRT3 on cell cycle in HD-BMMSC. The "knockdown" of SIRT3 in HD-BMMSCs led to an accumulation of cells in S phase of the cell cycle (siRNAs 4 and 5). siRNAs 2 and 3 produced effects of the same tendency, but these were very low (<5%). (D) Influence of SIRT3 on senescence-associated β-galactosidase activity HD-BMMSC. Transfection of HD-BMMSCs with siRNAs 4 and

5 produced an increase in SAβGalA. In contrast, no significant effects were observed for siRNA 2 and siRNA.

KMS12-PE myeloma cells (**Figure 5C**). No changes were seen in transwell cultures.

There were no significant differences in the gene expression of MCT1, MCT4, and UCP2 in MM-BMMSC compared to HD-BMMSC (data not shown). In contrast, a significant lower expression of SIRT3 was detected in MM-BMMSC (*p* < 0.001; **Figure 5A**). All data were reproduced at the protein level. In addition, it was investigated whether MM-BMMSCs have an increased mitochondrial mass in comparison with HD-BMMSC. For this purpose, mtDNA was quantified and was normalized to the content of nuDNA. It was shown that MM-BMMSCs show a significant increase in mitochondrial mass compared to HD-BMMSC (*p* = 0.0149; **Figure 5B**). These changes were not detected in MGUS-BMMSC (*n* = 4), suggesting an association with disease progression.

**Figure 5.** SIRT3 expression and mtDNA mass in MM-BMMSC. *P* values: \* <0.05; \*\* <0.01; \*\*\* <0.001; and \*\*\*\* <0.0001. All data were analyzed using the Wilcoxon signed-rank test. (A) MM-BMSC displayed a twofold decrease in the expression of SIRT3 compared to HD-BMSC. MGUS-BMSC showed no changes. (B) MM-BMSC showed a twofold increase in mtDNA mass compared to HD-BMSC. (C) Co-cultured MM-BMSC displayed a fourfold increase in SRT3 mRNA level. No changes were seen in transwell cultures. (D) Co-cultivation KMS12-PE and MM-BMSC induced depolarization of ΔΨm. (E) Co-cultivation KMS12-PE and MM-BMSC reduced the amount of ROS in both cell systems.

To explore the influence of MM cells on SIRT3 expression in BMSC, co-culturing for 72 h with KMS12-PE cells (*n* = 20) and transwell experiments (*n* = 10) was performed. Interestingly, we found a fourfold upregulation of SIRT3 expression in MM-BMMSC when co-cultured with KMS12-PE myeloma cells (**Figure 5C**). No changes were seen in transwell cultures.

**3.6. Expression of metabolic regulators in MM-BMSC**

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an association with disease progression.

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 (data not shown). In contrast, a significant lower expression of SIRT3 was detected in MM-BMMSC (*p* < 0.001; **Figure 5A**). All data were reproduced at the protein level. In addition, it was investigated whether MM-BMMSCs have an increased mitochondrial mass in comparison with HD-BMMSC. For this purpose, mtDNA was quantified and was normalized to the content of nuDNA. It was shown that MM-BMMSCs show a significant increase in mitochondrial mass compared to HD-BMMSC (*p* = 0.0149; **Figure 5B**). These changes were not detected in MGUS-BMMSC (*n* = 4), suggesting

**Figure 5.** SIRT3 expression and mtDNA mass in MM-BMMSC. *P* values: \* <0.05; \*\* <0.01; \*\*\* <0.001; and \*\*\*\* <0.0001. All data were analyzed using the Wilcoxon signed-rank test. (A) MM-BMSC displayed a twofold decrease in the expression of SIRT3 compared to HD-BMSC. MGUS-BMSC showed no changes. (B) MM-BMSC showed a twofold increase in mtDNA mass compared to HD-BMSC. (C) Co-cultured MM-BMSC displayed a fourfold increase in SRT3 mRNA level. No changes were seen in transwell cultures. (D) Co-cultivation KMS12-PE and MM-BMSC induced depolarization of

ΔΨm. (E) Co-cultivation KMS12-PE and MM-BMSC reduced the amount of ROS in both cell systems.

Moreover, co-cultivation induced depolarization of ΔΨm leading to an approximately twofold JC1 monomers increasing in MM-BMSC and MM cells (**Figure 5D**). Co-cultivation of KMS12-PE and MM-BMSC reduced the amount of ROS in both cell systems (**Figure 5E**).

To further elucidate the involvement of SIRT3 in metabolic and senescence-like alterations of MM-BMMSCs, siRNA was used to transiently "knockdown" this gene in HD-BMMSC.

**Figure 6.** Influence of SIRT3 on ROS in HD-BMMSC. *P* values: \* <0.05; \*\* <0.01; \*\*\* <0.001; and \*\*\*\* <0.0001. (A) The knockdown of SIRT3 in HD-BMMSC caused an increase in the ROS content of all four siRNAs tested compared to the negative and transfection control. (B) Influence of SIRT3 on ΔΨm in HD-BMMSC. The "knockdown" of SIRT3 in HD-BMMSC caused a reduction in the FL-2/FL-1 ratio. For siRNAs 2 and 3, only the proportion of FL-1 negative cells was reduced (R-4), whereas siRNAs 4 and 5 also caused an increase in FL-2 negative cells (R-3). (C) Influence of SIRT3 on cell cycle in HD-BMMSC. The "knockdown" of SIRT3 in HD-BMMSCs led to an accumulation of cells in S phase of the cell cycle (siRNAs 4 and 5). siRNAs 2 and 3 produced effects of the same tendency, but these were very low (<5%). (D) Influence of SIRT3 on senescence-associated β-galactosidase activity HD-BMMSC. Transfection of HD-BMMSCs with siRNAs 4 and 5 produced an increase in SAβGalA. In contrast, no significant effects were observed for siRNA 2 and siRNA.

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

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

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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

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

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, sug-

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

even more altered cellular phenotype at relapse.

C19MC result from hypomethylation or vice versa.

gesting an association with disease progression.

increase in cyclin E1 mRNA levels.

MM-BMMSC.
