**3.2. Co-culturing of KMS12-PE cell line represses the senescence entry of MM-BMMSCs**

Co-cultures of the KMS12-PE cell line with MM-BMMSC and HD-BMMSC were carried out to analyze whether MM cells can exert an influence on the senescence characteristics of BMMSC. Experiments were performed with MM-BMMSC (*n* = 20) and HD-BMMSC (*n* = 3). After co-culturing BMSC with MM cells, an inhibition of senescence entry in MM-BMMSC was observed. SAβGalA activity was significantly reduced (**Figure 2A**). A similar effect was detected using transwell cultures to prevent cell-cell contact between MM-BMMSC and KMS12-PE cells (*p <* 0.0313; **Figure 2A**). No effect on the activity of SA-βGal was observed in HD-BMMSC and the HS-5 cell line co-cultured with KMS12-PE myeloma cells. Interestingly, CD138<sup>+</sup> plasma cells from healthy donors induced a downregulation of SAβGalA activity in MM-BMMSC. However, this influence was three- to sixfold lower than that of observed in co-cultures with KMS12-PE cells. These results indicate that MM cells have a higher and more specificity proliferation stimulation effect on BMMSC compared to CD138+ plasma cells.

Also, mRNA expression of co-cultured and transwell cultured MM-BMMSCs was measured (**Figure 2B**). No changes were found for cyclin D1 and p16, whereas cyclin E1 was upregulated in both co-cultured and transwell cultured MM-BMMSC (*p* < 0.05). BMSC interaction with MM cells has induced an upregulation of p21. This effect was lower in transwell cultured MM-BMMSC compared to co-cultured MM-BMMSC (*p* < 0.008).

when compared with mono-cultured MM-BMMSC (*p* = 0.008). Transwell cultured MM-BMMSCs

phase and reduced the amount of cells in S phase in co-cultured and transwell

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**Figure 2.** KMS12-PE myeloma cells reduce SAβGalA and modify cell cycle characteristics of MM-BMMSC. *P* values: \* <0.05; \*\* <0.01; \*\*\* <0.001; and \*\*\*\* <0.0001. All data were analyzed using the Wilcoxon signed-rank test and paired *t*-test (ELISA analysis). HS-5 (CRL-11882)—BMSC line. (A) KMS12-PE myeloma cells reduce SAβGalA in MM-BMMSC upon co-cultivation and cultivation in transwell. The MFI in MM-BMMSC was significantly reduced in both culture systems. No changes were observed for co-cultured HD-BMMSC and HS-5 cells indicating specificity of the measured effect for MM-BMMSC. (B) Cell interaction with KMS12-PE myeloma cells induced increased cyclin E1 and p21 expression in MM-BMMSC compared to MM-BMMSC cultured alone. (C) Protein expression analysis of co-cultured MM-BMMSC (*n* = 3) compared to mono-cultured MM-BMMSC. Cyclin E1 was increased, whereas cyclin D1 and p21 were reduced in co-cultured cells compared to mono-cultures. No change was seen for p16. (D) Cell interaction with KMS12-PE myeloma

We chose six microRNAs, which were previously reported to be deregulated in MM cells and to play a possible role in the generation of senescence or cell cycle arrest (miR-16, miR-485-5p, miR-519d, miR-221, miR-126, and miR-223). Analysis revealed an overexpression of miR-16, miR-223, miR-485-5p, and miR-519d (all with *p <* 0.025) in MM-BMMSCs compared to HD-BMMSCs. No expression differences were detected for miR-221 and miR-126 (**Figure 3A**). We revealed the overexpression of miR-485-5p and miR-519d in MM-BMMSCs. These microRNAs are located on two imprinted clusters on chromosomes 14 (DLK1-DIO3) and 19 (C19MC), respectively, and are reported to play a role in senescence generation [21, 31, 32].

showed the same tendency, but significant changes were not detectable.

**3.3. Deregulation of microRNA expression in MM-BMSC**

/G0

cultured MM-BMMSCs (*n* = 8) compared to the same MM-BMMSC cultured alone.

cells induced an increase in cells in G1

However, some contrary results were detected at the protein level. We have found a reduction in p21 in co-cultured MM-BMMSC (**Figure 2C**). In addition, cyclin D1 protein expression was 1.8-fold reduced upon co-cultivation with KMS12-PE myeloma cells, whereas no change was seen on mRNA level (*p* = 0.0033). The mRNA and protein analysis of cyclin E1 and p16 were concordant.

Next, we analyzed cell cycle distribution of co-cultured and transwell cultured MM-BMMSC (**Figure 2D**). Both cell culture systems led to a slight reduction in cells in S phase compared to MM-BMMSC cultured alone (*p* = 0.008) and an increase in the percentage of cells in G1/G0 phase

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**Figure 2.** KMS12-PE myeloma cells reduce SAβGalA and modify cell cycle characteristics of MM-BMMSC. *P* values: \* <0.05; \*\* <0.01; \*\*\* <0.001; and \*\*\*\* <0.0001. All data were analyzed using the Wilcoxon signed-rank test and paired *t*-test (ELISA analysis). HS-5 (CRL-11882)—BMSC line. (A) KMS12-PE myeloma cells reduce SAβGalA in MM-BMMSC upon co-cultivation and cultivation in transwell. The MFI in MM-BMMSC was significantly reduced in both culture systems. No changes were observed for co-cultured HD-BMMSC and HS-5 cells indicating specificity of the measured effect for MM-BMMSC. (B) Cell interaction with KMS12-PE myeloma cells induced increased cyclin E1 and p21 expression in MM-BMMSC compared to MM-BMMSC cultured alone. (C) Protein expression analysis of co-cultured MM-BMMSC (*n* = 3) compared to mono-cultured MM-BMMSC. Cyclin E1 was increased, whereas cyclin D1 and p21 were reduced in co-cultured cells compared to mono-cultures. No change was seen for p16. (D) Cell interaction with KMS12-PE myeloma cells induced an increase in cells in G1 /G0 phase and reduced the amount of cells in S phase in co-cultured and transwell cultured MM-BMMSCs (*n* = 8) compared to the same MM-BMMSC cultured alone.

when compared with mono-cultured MM-BMMSC (*p* = 0.008). Transwell cultured MM-BMMSCs showed the same tendency, but significant changes were not detectable.

#### **3.3. Deregulation of microRNA expression in MM-BMSC**

The colony-forming unit fibroblast (CFU-F) assay was used to study the self-renewal capacity of BMMSC. MM-BMMSC showed a lower self-renewal capacity compared to HD-BMMSC (**Figure 1B**). Similar to the senescence study, MM-BMMSC obtained from relapsed patients showed a significantly lower self-renewal capacity than MM-BMMSC, which forms newly

MM-BMMSCs are characterized by a lower expression of cyclin E1 and an overexpression of cyclin D1 when compared with HD-BMMSC (**Figure 1D**). In addition, the cell cycle inhibitor p21 was upregulated in MM-BMMSC compared to HD-BMMSC (*p <* 0.05). No changes were observed in the mRNA level of p16. Changes in the mRNA levels were also confirmed using protein analysis (*p* < 0.03; **Figure 1E**). Cyclin E1 was decreased in MM-BMMSC compared to HD-BMMSC (*p* = 0.0416). Cyclin D1 and p21 protein levels were 1.5- to 1.8-fold increased. Protein measurement also showed a slightly reduced level of p16 in MM-BMMSCs, but this change was below 1.5-fold. These results correlated with a higher number of cells in S phase

Co-cultures of the KMS12-PE cell line with MM-BMMSC and HD-BMMSC were carried out to analyze whether MM cells can exert an influence on the senescence characteristics of BMMSC. Experiments were performed with MM-BMMSC (*n* = 20) and HD-BMMSC (*n* = 3). After co-culturing BMSC with MM cells, an inhibition of senescence entry in MM-BMMSC was observed. SAβGalA activity was significantly reduced (**Figure 2A**). A similar effect was detected using transwell cultures to prevent cell-cell contact between MM-BMMSC and KMS12-PE cells (*p <* 0.0313; **Figure 2A**). No effect on the activity of SA-βGal was observed in HD-BMMSC and the HS-5 cell line co-cultured with KMS12-PE myeloma cells. Interestingly,

plasma cells from healthy donors induced a downregulation of SAβGalA activity in

MM-BMMSC. However, this influence was three- to sixfold lower than that of observed in co-cultures with KMS12-PE cells. These results indicate that MM cells have a higher and more specificity proliferation stimulation effect on BMMSC compared to CD138+ plasma cells.

Also, mRNA expression of co-cultured and transwell cultured MM-BMMSCs was measured (**Figure 2B**). No changes were found for cyclin D1 and p16, whereas cyclin E1 was upregulated in both co-cultured and transwell cultured MM-BMMSC (*p* < 0.05). BMSC interaction with MM cells has induced an upregulation of p21. This effect was lower in transwell cultured

However, some contrary results were detected at the protein level. We have found a reduction in p21 in co-cultured MM-BMMSC (**Figure 2C**). In addition, cyclin D1 protein expression was 1.8-fold reduced upon co-cultivation with KMS12-PE myeloma cells, whereas no change was seen on mRNA level (*p* = 0.0033). The mRNA and protein analysis of cyclin E1 and p16 were

Next, we analyzed cell cycle distribution of co-cultured and transwell cultured MM-BMMSC (**Figure 2D**). Both cell culture systems led to a slight reduction in cells in S phase compared to MM-BMMSC cultured alone (*p* = 0.008) and an increase in the percentage of cells in G1/G0 phase

MM-BMMSC compared to co-cultured MM-BMMSC (*p* < 0.008).

phase compared to HD-BMMSCs (*p <* 0.008; **Figure 1C**).

/G0

**3.2. Co-culturing of KMS12-PE cell line represses the senescence entry of** 

diagnosed patients.

**MM-BMMSCs**

CD138<sup>+</sup>

concordant.

and a reduced number of cells in G1

126 Stromal Cells - Structure, Function, and Therapeutic Implications

We chose six microRNAs, which were previously reported to be deregulated in MM cells and to play a possible role in the generation of senescence or cell cycle arrest (miR-16, miR-485-5p, miR-519d, miR-221, miR-126, and miR-223). Analysis revealed an overexpression of miR-16, miR-223, miR-485-5p, and miR-519d (all with *p <* 0.025) in MM-BMMSCs compared to HD-BMMSCs. No expression differences were detected for miR-221 and miR-126 (**Figure 3A**).

We revealed the overexpression of miR-485-5p and miR-519d in MM-BMMSCs. These microRNAs are located on two imprinted clusters on chromosomes 14 (DLK1-DIO3) and 19 (C19MC), respectively, and are reported to play a role in senescence generation [21, 31, 32].

**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.

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

**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

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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

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

KMS12-PE myeloma cells.

decreased in co-cultured KMS12-PE myeloma cells.

were detected (data not shown).

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**).
