Mesenchymal stem cells secretomes' affect multiple ...

Cellular Signalling 28 (2016) 620–630

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Mesenchymal stem cells secretomes' affect multiple myeloma translation initiation H. Marcus a,c, O. Attar-Schneider a,c, M. Dabbah a,c, V. Zismanov a,c, S. Tartakover-Matalon a,c, M. Lishner a,b,c,1, L. Drucker a,c,⁎,1 a b c

Oncogenetic Laboratory, Tel Aviv University, Tel Aviv, Israel Internal Medicine Department, Meir Medical Center, Kfar Saba, Tel Aviv University, Tel Aviv, Israel Sackler faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

a r t i c l e

i n f o

Article history: Received 16 December 2015 Received in revised form 1 March 2016 Accepted 8 March 2016 Available online 11 March 2016 Keywords: bone marrow mesenchymal stem cells (BMMSCs) Multiple myeloma Translation initiation eIF4E eIF4GI Microvesicles

a b s t r a c t Bone marrow mesenchymal stem cells' (BM-MSCs) role in multiple myeloma (MM) pathogenesis is recognized. Recently, we have published that co-culture of MM cell lines with BM-MSCs results in mutual modulation of phenotype and proteome (via translation initiation (TI) factors eIF4E/eIF4GI) and that there are differences between normal donor BM-MSCs (ND-MSCs) and MM BM-MSCs (MM-MSCs) in this crosstalk. Here, we aimed to assess the involvement of soluble BM-MSCs' (ND, MM) components, more easily targeted, in manipulation of MM cell lines phenotype and TI with specific focus on microvesicles (MVs) capable of transferring critical biological material. We applied ND and MM-MSCs 72 h secretomes to MM cell lines (U266 and ARP-1) for 12-72 h and then assayed the cells' (viability, cell count, cell death, proliferation, cell cycle, autophagy) and TI (factors: eIF4E, teIF4GI; regulators: mTOR, MNK1/2, 4EBP; targets: cyclin D1, NFκB, SMAD5, cMyc, HIF1α). Furthermore, we dissected the secretome into N100 kDa and b 100 kDa fractions and repeated the experiments. Finally, MVs were isolated from the ND and MM-MSCs secretomes and applied to MM cell lines. Phenotype and TI were assessed. Secretomes of BM-MSCs (ND, MM) significantly stimulated MM cell lines' TI, autophagy and proliferation. The dissected secretome yielded different effects on MM cell lines phenotype and TI according to fraction (N 100 kDa- repressed; b100 kDa- stimulated) but with no association to source (ND, MM). Finally, in analyses of MVs extracted from BM-MSCs (ND, MM) we witnessed differences in accordance with source: ND-MSCs MVs inhibited proliferation, autophagy and TI whereas MM-MSCs MVs stimulated them. These observations highlight the very complex communication between MM and BM-MSCs and underscore its significance to major processes in the malignant cells. Studies into the influential MVs cargo are underway and expected to uncover targetable signals in the regulation of the TI/proliferation/autophagy cascade. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Cells communicate with their microenvironment through a complex network of signals generated by cell-extracellular matrix (ECM), cell– cell adhesion and secreted soluble components (secretome) [1,2]. The importance of the microenvironment in cancer is well recognized and it is established that the BM microenvironment is significant to the pathophysiology of multiple myeloma (MM) [3,4]. In fact the localization of the malignant MM plasma cells to the bone marrow

⁎ Corresponding author at: Oncogenetic Laboratory, Meir Medical Center, Kfar-Saba, 44281, Israel. E-mail address: [email protected] (L. Drucker). 1 Equal contribution.

http://dx.doi.org/10.1016/j.cellsig.2016.03.003 0898-6568/© 2016 Elsevier Inc. All rights reserved.

(BM) is a major hallmark and diagnostic criterion in this disease [5–7]. In-vitro studies have shown that cell–cell contact between MM cells and BM stromal cells plays a critical role in the growth and survival of the plasma cells [8]. More recent studies extend the knowledge on stroma-MM cell communication to include the involvement of various membrane vesicles [9,10]. Importantly, it is proposed that the variability in the genomic changes in MM may be attributed to epigenetic forces including microRNAs that may originate from resident cells and components of the BM such as mesenchymal stem cells (BM-MSCs) [11,12]. BM-MSCs, an important component of the BM niche, have been reported to differ in cases of MM [13–15]. The changes that occur in BM-MSCs exposed to MM cells were reported to persist in in-vitro cultures in the absence of MM cells [13] and appeared irreversible even after an exposure of only several hours [16]. Transformation included

H. Marcus et al. / Cellular Signalling 28 (2016) 620–630

altered expression of growth factors, extracellular matrix components, and adhesion molecules [13,14,16]. Thus, MM cells have the ability to alter the BM, rather than just reside in it, by converting it into a specialized neoplastic niche that promotes growth and spreading of tumor cells [17]. The nature and time frame of these described changes indicate a rapid yet broad cellular response, compatible with protein translation. In a published study, we have shown that co-culture of MM cell lines with BM-MSCs results in mutual modulation of phenotype and proteome [12]. Moreover, we have demonstrated that normal donor BMMSCs (ND-MSCs) differ in their affect on and response to MM cells from MM BM-MSCs (MM-MSCs) [12]. MM-MSCs caused elevated proliferation and death of co-cultured MM cell lines as well as increased expression and activity of eIF4E and eIF4GI translation initiation factors. In contrast, ND-MSCs caused increased MM cell death only and did not elevate translation initiation factors [12]. The significance of translation initiation factors eIF4E and eIF4GI to MM cell lines phenotype and proteome was recently demonstrated by us in detail in a high throughput system [18]. Our major findings show that each factor has a specific imprint on the malignant cells' protein repertoire and that their expression is positively correlated with cell proliferation and traits compatible with disease progression [18]. Therefore, it stands to reason that regulating translation initiation is of the utmost importance to disease development, progression and drug response. In the current study we aimed to assess and compare the involvement of soluble BM-MSCs (ND, MM) components, more easily targeted, in manipulation of MM cell lines phenotype and translation initiation.

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a Countess Automated cell counter and a phase-contrast microscope [19]. 2.5. Cell viability assay Assessment of viability was performed on MM cell lines using cell proliferation reagent WST-1 (Roche, Basel, Switzerland) as described before [20]. 2.6. Cell cycle and death Cell survival was determined by flow cytometry (FACS) (Navios, Beckman Coulter, USA). Cells were harvested and stained with annexin V-PE (250 μg/ml) (BioVision, CA, USA) and 7AAD (0.05 μg/μl) (eBioscience, CA, USA). Annexin V+/7AAD− cells were considered apoptotic and annexin V+/7AAD+ cells were considered necrotic or late apoptotic cells. For cell cycle harvested cells were exposed to 40 μg/ml propidium iodide (PI) and 100 μg/ml Ribonuclease A (Sigma, St. Louis, MO, USA) in PBS for 30 min at room temperature in the dark and analyzed by FACS. 2.7. Inhibitors 4EGI-1 inhibitor was purchased from EMD Millipore (Darmstadt, Germany) and was dissolved in DMSO to a final concentration of 100 μM as determined by us previously [21]. Autophagy was inhibited by using the autophagy inhibitor 3 methyladenine (3MA) (7.5 mM) (Sigma) dissolved in ddH2O.

2. Materials and methods

2.8. Immunoblotting

2.1. Cell lines

Cells were lysed, proteins were extracted and western blot was preformed as described elsewhere [22]. The following proteins were detected with rabbit/mouse anti-human: peIF4E(Ser209)/ total eIF4E, peIF4GI(Ser1108)/total eIF4GI, p4EBP(Ser65)/total 4EBP, pmTOR(Ser2448)/total mTOR, Beclin-1, HistonH3, Caspase3 (Cell Signaling Technology, Danvers, MA, USA); pMNK(Thr197/Thr202)/total MNK, SMAD5 (Epitomics, Burlingame, CA, USA); c-Myc, HIF1α, NFkB, PCNA (Santa-Cruz, CA, USA); tubulin, LC3-II (Sigma). Bound antibodies were visualized using peroxidase-conjugated secondary goat anti rabbit or mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), followed by enhanced chemiluminescence (ECL) detection (Millipore). Products were visualized with LAS3000 Imager (Fujifilm, Greenwood, SC, USA). Integrated optical densities of the immunoreactive protein bands were measured as arbitrary units employing Multi Gauge software v3 (Fujifilm).

Multiple myeloma cell lines U266 and ARP-1 were cultured in RPMI 1640 supplemented with 20% heat-inactivated fetal bovine serum (FBS), antibiotics and glutamine (Biological Industries, Kibbutz Beit Haemek, Israel). 2.2. BM-MSCs isolation and propagation BM samples were obtained from femur head BM samples of normal donors (ND), generally healthy, undergoing elective full hip replacement surgery due to osteoarthritis (n = 15) and MM patients' BM aspirates taken for medical purposes (n = 16) at Meir Medical Center, Israel. All participants signed informed consent forms approved by Meir Medical Center Helsinki Committee. MSCs' cells were isolated, propagated and differentiated as described by us before [12].

2.9. Immunocytochemistry 2.3. MM cell lines treatment with BM-MSCs secretomes Conditioned media (secretomes) of BM-MSCs (ND, MM) and MM cell lines (U266, ARP-1) were collected after 72 h culture and mixed with fresh media (350 μl:50 μl ratio, respectively). BM-MSCs secretomes (treatment), MM cell lines secretomes (control), and fresh media (control) were used for the culturing of MM cell lines, respectively (i.e.U266 with U266 secretome; ARP-1 with ARP-1 secretome). The treatments were applied to 100,000 MM cells/24 well for various time periods (12 h, 24 h, 36 h, 48 h, 72 h) after which MM cells were harvested and assayed. This experiment was conducted with at least 3 different ND BM-MSCs and with no less than 3 different MM BM-MSCs, using two different cell lines (U266, ARP-1).

Cells were cytospinned, fixated (4% paraformealdehyde, 100% methanol), blocked (5.5% goat serum) and incubated with primary antibody KI67 for MM cells proliferation (Zymed, San Francisco, CA,USA)) over night at 4°C. Slides were washed and incubated with secondary antibody horseradish peroxidase conjugated polymer, washed, and developed with AEC system (Covance Research Products, MA, USA) and hematoxylin stain was used for nuclei staining (Sigma). Cells were visualized with a BX41 microscope (40 ×); images were taken with DP70 digital camera and DP Controller software (OLYMPUS, Center Valley, PA, USA). Blind manual counting intensity grading of KI-67 staining as described previously [23]. 2.10. MSCs secretome fractionation

2.4. Trypan blue Total cell counts as well as the respective proportion of viable and dead cells were enumerated by Trypan blue dye exclusion using

MSCs secretome was separated for two fractions with 100 kDa cutoff by Amicon Ultra-15 centrifugal filter devices (Merck Millipore) according to manufactures' instructions. Briefly, MSCs secretome was applied

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into Amicon devices, and centrifuged (15-30 min, 4000 × g) until approximately 30 μl remain in the upper chambers. The N100 kDa (upper) fraction was suspended in RPMI 3% FBS. 2.11. Microvesicles (MVs) separation MVs were separated as described by Shefler et al. [24]. Briefly, conditioned BM-MSCs media were centrifuged 4 times: first time (5 min, 800×g) to remove cells, second time (5 min, 4500×g) to discard large debris, third time (60 min, 20,000 × g, 4°C) to embed MVs, and forth time (60 min, 20,000×g, 4°C) after resuspension of the MVs with PBS. MVs protein concentration was measured at 280 nm using NanoDrop spectrophotometers (Thermo Fisher Scientific, Wilmington, DE, USA). MVs were kept at −80°C. 2.11.1. MVs validation MVs were resuspended in PBS, and adsorbed to Formvar coated copper grids (EMS, Hatfield, PA, USA). Grids were stained with 1% (w/v) uranyl acetate and air-dried. Samples were viewed with Tecnai 12 transmission electron microscope (TEM) 100 kV (Phillips, Eindhoven, the Netherlands) equipped with MegaView II CCD camera and Analysis version 3.0 software (SoftImaging System GmbH, Münstar, Germany). TEM work was done in the Bio-Imaging Unit, the Alexander Silberman Institute of Life Science, Edmond Safra Campus, the Hebrew University, Jerusalem, Israel. 2.12. Statistical analysis The data distribution was assayed with Shapiro–Wilk test in order to determine normality. Student's paired and unpaired t tests, Wilcoxon non parametric, Mann Whitney or one way ANOVA tests were employed in analysis of differences between cohorts, each when appropriate. An effect was considered significant when P-value was equal to or less than 0.05. All experiments were conducted at least three separate times. 3. Results 3.1. Research model BM-MSCs cultures were prepared from mononuclear cells isolated from BM samples of NDs undergoing hip replacement surgery for orthopedic purposes and from MM patients' BM aspirates taken for medical purposes at Meir Medical Center (complying with Helsinki regulations) detailed in material and methods and described previously [12]. Once a BM-MSCs culture reached 80% confluence the conditioned 72 h media (secretome) was collected and applied to MM cell lines (U266, ARP1). As controls, MM cell lines' 72 h secretomes were collected and also applied to MM cell lines (U266, ARP-1), respectively. MM cells were harvested after being cultured with the respective secretome for 12 h72 h and analyzed for phenotype and translation initiation factors. An additional technical control of fresh media was used throughout the study. Unsurprisingly, MM cell lines treated with corresponding 72 h MM secretome displayed reduced viability and increased death due to nutrient depletion of the “used” media. Interestingly, the BM-MSCs' secretomes did not cause the same “starved” response in the MM cell lines (supplemental Fig. 1). Based on these data and similar studies by others [25–28], all experimental results of MM cell lines treated with BM-MSCs secretomes were compared to the observations made in MM cell lines treated with the corresponding MM cell line's 72 h secretomes. 3.2. BM-MSCs' secretomes (ND, MM) promote MM cell lines proliferation Viability of MM cell lines treated with secretomes of BM-MSCs or MM cell lines (72 h) was assayed with WST1. Significantly elevated

viability was observed in both cell lines, cultured with both types of BM-MSCs (MM and ND) secretomes (↑ 120–660%; p b 0.05) (Fig. 1A). Of note, a significant difference was registered between viability of U266 and ARP-1 treated with MM BM-MSCs and between U266 treated with ND BM-MSCs' secretome compared to MM BM-MSCs (p b 0.05) (Fig. 1A). This was not pursued further in the current study so as not to stray away from the central objectives of this study. Significant difference also existed between U266 treated with ND BM-MSCs' secretome and MM BM-MSCs (p b 0.05) (Fig. 1A), yet this was not observed in ARP1 and therefore was not considered noteworthy. Next, total and dead cells were enumerated using the trypan blue exclusion dye assay and the objective Countess. Significantly elevated total and live cell counts were observed in both cell lines, cultured with both types of BM-MSCs' (MM and ND) secretomes (↑ 50–110%; p b 0.05) (Fig. 1B). However, no significant changes in dead cell counts were observed. Cell death was also assayed with Annexin V-PE/7AAD, yet consistent with trypan blue findings, no increase in death was witnessed in both cell lines (data not shown). Cell cycle was also analyzed in MM cell lines treated with BM-MSCs or respective MM cell line's secretome. No biologically significant changes were recorded in U266 and ARP-1 (G1 31–51%, S 20–36%, G2/M 17– 20%). In order to corroborate that the major effect of the BM-MSCs' secretomes was elevated proliferation we tested the expression of an established proliferation marker. U266 and ARP-1 cells were harvested after 72 h exposure to BM-MSCs secretomes or 72 h MM Cell lines secretome (control), centrifuged and fixated on slides and assayed by immunocytochemistry for Ki-67. Indeed, elevated levels of Ki-67 protein (↑ 180–240%; p b 0.05) were observed in MM cells treated with BM-MSCs secretome (regardless of source)(60–75% Ki67 positive cells) in comparison to MM cells treated with MM 72 h secretome in both cell lines (27–37% Ki67 positive cells). Enclosed are representative images of these results (Fig. 1C). Previous reports showed there is a distinct correlation between cell size and proliferation and protein synthesis [29]. Therefore, we examined cell size indicated in flow cytometry forward scatter of MM cell lines treated with BM-MSCs' secretomes or respective MM cell line's 72 h secretomes (control). In concordance with our results so far, we determined significant increases in cell size in both cell lines (U266, ARP1) treated with both types of BM-MSCs (ND, MM) (↑ ~ 10%; p b 0.01) (Fig. 1D). These observations support elevation in protein translation and coincide with elevated cell proliferation upon exposure to BMMSCs secretomes. Preceding studies have demonstrated that autophagy has a central role in MM, and can support proliferation [22,30,31]. Therefore, we examined the expression levels of autophagy markers in MM cells treated with BM-MSCs and MM 72 h secretomes for 12 h-72 h. Significantly elevated levels of LC3-II and Beclin-1 were observed in both cell lines (U266, ARP-1) treated with both types of BM-MSCs (ND, MM) (↑30–200%; p b 0.05) (Fig. 2A). Interestingly, there seems to be a difference in the autophagy between MM cells treated with MM BM-MSCs and MM cells treated with ND BM-MSCs reflected in the reaction time; cells treated with MM BM-MSCs reacted faster (12 h-24 h) compared to cells treated with ND-BM-MSCs that displayed a slower reaction time (72 h). Moreover, there was a significant difference in the autophagy levels of U266 treated with MM BM-MSCs secretome (milder) and ND BM-MSCs secretome. Next, we inhibited the autophagy process using 3MA, in order to examine if indeed autophagy contributed to the MM cells proliferation. Specifically, U266 and ARP-1 cells were co-treated with 3MA along with secretome (U266 or ARP-1, ND BM-MSCs, and MM BM-MSCs). After 36 h 3MA was removed, and appropriate secretome was reintroduced. After 72 h (36 h post 3MA removal) cells were harvested, dyed with trypan blue and counted. Decreased levels of total cells were witnessed in all 3MA treated secretome conditioned MM cells (U266 and ARP-1, ND BM-MSCs, MM BM-MSCs) compared to MM cells treated with respective secretomes


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Fig. 1. BM-MSCs' secretome elevates MM cells' viability and proliferation but does not affect cell death. MM cell lines (ARP-1 and U266) were cultured with BM-MSCs' secretome (ND or MM) for 72 h and assayed for (A) viability (B) total and live cell count compared to MM cell lines 72 h secretome (control). Results are presented as percent (Mean ± SE, n N 3) of control cells (dotted line). (C) Following 72 h the MM cell lines were also cytospinned onto slides, stained with Ki-67 antibody (brown) and hematoxylin (blue). Representative images of control and BM-MSCs secretome treated cells are presented. (D) The effect of BM-MSCs secretomes on MM cell lines cell size was determined by forward scatter measured in flow cytometer and expressed relative untreated matching controls (dotted line). Statistically significant differences between cohorts (*; p b 0.05, **; p b 0.01) and between MM cell lines' responses to MM-MSCs and ND-MSCs' secretomes ($) are indicated.

only (↓ ~ 30–40% p b 0.05) (Fig. 2B). The differences in total cells was attributed to decreased proliferation as evidenced in slighter fractions of live cells (↓ ~ 30–40% p b 0.05) (Fig. 2C) and no increase on cell death.

3.3. BM-MSCs' secretomes elevate MM cell lines translation initiation Previous findings by us and others have shown the connection between cell proliferation and protein synthesis [29]. Moreover, the effect of BM-MSCs on MM translation initiation was demonstrated by us in a co-culture model [12]. In line with these published observations and our current finding that BM-MSCs' secretomes elevated MM proliferation we wondered whether the secretomes also promoted MM cell lines' translation initiation. MM cell lines treated with BM-MSCs secretomes (ND, MM) were harvested after 72 h, lysed and immunoblotted for p-eIF4E (the active form of eIF4E), t-eIF4E, p-eIF4GI and t-eIF4GI. Significantly elevated levels of p-eIF4E (↑ ~ 150%; p b 0.05), p-eIF4GI (↑ ~ 50%; p b 0.05) and t-eIF4GI (↑ ~ 100%; p b 0.05) were observed in both cell lines (U266, ARP-1) with both secretomes (72 h) (Fig. 3A). Elevated t-eIF4E was observed in both cell lines with ND-MSCs secretome only (↑ ~ 40%; p b 0.05)(Fig. 3A right). These results demonstrate a strong effect of BM-MSCs secretome on the central translation initiation factors with no significance to the BM-MSCs source (MM or ND).

Next, we wanted to establish the involvement of known translation initiation factors' regulatory signals. Taking into account the time line of the signaling cascade responsible for translation initiation factors' reaction we obviously expected eIF4E/eIF4GI regulators' activation to precede the increase in translation initiation factors levels. Therefore, cells treated with BM-MSCs' secretomes or respective control MM 72 h secretomes were harvested after 12-24 h (p-mTOR and t-mTOR) or after 48-72 h (p-MNK1, t-MNK1, p-4EBP1 and t4EBP1), lysed and immunoblotted for stated eIF4E and eIF4GI regulators. Significantly elevated levels of p-mTOR (↑50–100%; p b 0.05), pMNK1 (↑ 50–100%; p b 0.05), t-MNK1 (↑ 50–250%; p b 0.05), p-4EBP1 (↑70–150%; p b 0.05) and t-4EBP1 (↑50–100%; p b 0.05) were observed (Fig. 3B). Finally, we tested the expression of eIF4E and eIF4GI established targets as indication for the translation initiation factors' activity. Cells were harvested after 48 h-72 h, lysed and immunoblotted for eIF4E targets: Cyclin D1 and NFκB; eIF4GI target: Smad5; and for common targets: c-Myc and Hif1α. Significantly elevated levels of Cyclin D1 (↑ ~ 60%; p b 0.05), NFκB (↑ ~ 30%; p b 0.05), Smad5 (↑ ~ 50%; p b 0.05), c-Myc (↑ ~ 70%; p b 0.05) and Hif1α (↑ ~ 100%; p b 0.05 in all but U266 treated with ND BM-MSCs) were observed (Fig 3C). These results confirm that the BM-MSCs' secretomes increase eIF4E and eIF4GI dependent translation in MM cell lines. Throughout the study up to this point there were some statistically significant differences between the MM cell lines' responses to MM-

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Fig. 2. BM-MSCs secretomes increase MM cells' autophagy instrumental to their proliferation. MM cell lines (ARP-1, U266) were cultured with BM-MSCs 72 h secretome (ND, MM) or with MM cell lines 72 h secretome (control, CTRL). (A) Cells were harvested after 12 h, 24 h, 48 h, and 72 h, lysed and immunoblotted for Beclin-1 and LC3-II. Tubulin served as loading control. Significant results are presented in bar graphs (left) and representative immunoblots (right). Of note, exposure of MM cell lines to MM BM-MSCs secretome yielded significant changes in Beclin (12 h) and LC3-II (24 h) earlier than with ND BM-MSCs secretome (72 h). Next, cells were cultured with different secretomes (cell line, ND BM-MSCs, and MM BM-MSCs) and in combined with 3MA autophagy inhibitor. Following 36 h 3MA was removed and appropriate secretome was reintroduced. After additional 36 h cells were assayed for (B) total cell counts and (C) live cell counts. Results are expressed as percent (Mean ± SE, n N 3) of control cells (dotted line). Statistically significant differences between cohorts (*; p b 0.05, **; p b 0.01) and between MM cell lines' responses to MM-MSCs and ND-MSCs' secretomes ($) are indicated.

MSCs and ND-MSCs secretomes, yet all trends were similar and there was no consistency. Therefore, we did not pursue these findings.

3.5. BM-MSCs' secretomes' fractions (b100 kDa and N 100 kDa) have diverse effects on translation initiation in MM cell lines independent of secretomes' source (ND, MM)

3.4. Function of eIF4E/eIF4GI complex in MM cell line is essential for BMMSCs influence on MM cell proliferation and autophagy

Next, we tried to unravel the BM-MSCs' secretomes' action on the translation initiation factors in the MM cells. Using centrifugal filtration with a semi-permeable membrane of a 100 kDa molecular weight cutoff (Centricon) we separated 72 h secretomes (ND and MM; n = 4) into a b 100 kDa fraction that contained soluble proteins, cytokines, and exosomes (b100 kDa) and a N 100 kDa fraction that generally contained large extracellular microvesicles (MVs) (N100 kDa). The separation procedure yielded a high molecular weight fraction of N100 kDa and all media with remaining solubles as the lower molecular weight fraction b100 kDa. Therefore, we used whole undivided BM-MSCs secretomes as control for the b100kDA fraction. For the N 100kDA fraction that was suspended in low serum fresh media of identical volume as the original sample, we used low serum fresh media as control. MM cell lines treated with BM-MSCs secretomes N 100 kDa fraction (ND, MM) were harvested after 72 h, lysed and immunoblotted for peIF4E, t-eIF4E, p-eIF4GI and t-eIF4GI. Surprisingly, significantly decreased levels of p-eIF4E (↓ 20–70%; p b 0.05), t-eIF4E (↓ ~ 20%; p b 0.05) p-eIF4GI (↓ 30–70%; p b 0.05) and t-eIF4GI (↓ 30–70%; p b 0.05) were observed in both cell lines (U266, ARP-1) treated with both secretomes (ND, MM)(Fig 5A). These unexpected results are

MM cell lines (U266, ARP-1) were co-treated with BM-MSCs secretomes (ND, MM) and eIF4F complex inhibitor, 4EGI (100 μg/ml). Cells were harvested after 72 h, lysed and assayed for proliferation and autophagy (Fig 4). Significantly decreased total cell counts were observed in MM cell lines simultaneously exposed to ND or MM BM-MSCs secretomes and 4EGI (↓50–75%; p b 0.05) (Fig 4A). Moreover, a considerable decrease in live cell counts was also witnessed in MM cells treated with MSCs secretomes (ND, MM) and 4EGI (↓60–90%; p b 0.05) (Fig 4B), yet no difference in death rates was evident (NS). Taken together, these results support diminished MSCs secretome induced proliferation upon inhibition of eIF4E-eIF4GI association. Next, we tested the levels of autophagy markers LC3-II and Beclin-1 by immunoblotting protein extracts of MM cell lines (U266, ARP-1) co-treated with BM-MSCs (ND, MM) and 4EGI. In keeping with our findings so far, we witnessed a profound decrease in both LC3-II and Beclin-1 indicating that eIF4EeIF4GI activity is in control of autophagy in this experimental setting (Fig 4C, representative images).


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Fig. 3. BM-MSCs' secretome elevate expression and activity of eIF4E/eIF4GI and their regulators and targets in MM cell lines. MM cell lines (ARP-1, U266) were cultured with BMMSCs 72 h secretome (ND- right, MM- left) or with MM cell lines 72 h secretome (control, CTRL). Cells were harvested after 12 h, 24 h, 48 h, and 72 h, lysed and immunoblotted for (A) p-eIF4E, t-eIF4E and p-eIF4GI, t-eIF4GI (earliest significant elevation at 72 h) (B) Regulators: mTOR (12 h/24 h with ND/MM BM-MSCs' secretome, respectively), MNK1, 4EBP1 (both 72 h) (C) Targets: Cyclin D1, NFkB, Smad5, c-Myc and Hif1α (all 72 h). Tubulin served as loading control. Results are presented in bar graphs (left) and representative immunoblots (right) and expressed as percent (Mean ± SE, n ≥ 7) of control cells (dotted line). Statistically significant differences between cohorts (*; p b 0.05, **; p b 0.01) and between MM cell lines' responses to MM-MSCs and ND-MSCs' secretomes ($) are indicated.

inconsistent with the effect of whole/un-fractionated BM-MSCs secretome on MM cell lines. Our observations do demonstrate unequivocally a strong attenuating effect of BM-MSCs secretome N 100 kDa fraction on the central translation initiation factors in treated MM cell lines. In contrast, MM cell lines treated with BM-MSCs secretomes' b100 kDa fraction (i.e. lacking the N100 kDa fraction) (ND, MM) were harvested after 72 h, lysed and immunoblotted for p-eIF4E, t-eIF4E, peIF4GI and t-eIF4GI. Significantly elevated levels of p-eIF4E (↑ 20– 100%; p b 0.05) and p-eIF4GI (↑ 20–200%; p b 0.05) were observed in both cell lines (U266, ARP-1) with both b100 kDa secretomes. Elevated t-eIF4E was observed only in U266 cell line treated with ND-MSCs b100 kDa secretome (↑20%; p b 0.05). Elevated levels of t-eIF4GI were observed in both cell lines (U266 and ARP-1) treated with MM BMMSCs (↑ 120–220%; p b 0.05) (Fig 5B). Of note, MM BM-MSCs b100 kDa secretomes displayed a greater effect than ND BM-MSCs secretomes on translation initiation factors (p-eIF4E and t-eIF4GI in U266, and p-eIF4E and p-eIF4GI in ARP-1) (↑ 120–220%; p b 0.05). These findings are in concordance with the results of the un-separated conditioned media. 3.6. BM-MSCs' MVs have a differential effect on MM cells' eIF4E/eIF4GI according to source (ND, MM) Our observations with the secretome fractions supported the existence of components in both fractions that actively regulate translation

initiation machinery. Accumulating evidence indicates the involvement of MVs in cancer progression and specifically in MM [32–34]. Moreover, it was reported that B-CLL MVs promote stromal cells' protein synthesis [35]. Thus, we wanted to explore the possible involvement of BMMVs (ND, MM) in manipulation of translation initiation. Employing ultracentrifugation we isolated MVs from ND and MM BM-MSCs, validated their identity by transmission electron microscope (TEM) that demonstrated double membrane vesicles at the appropriate size (100–1000 nm)(Fig 6A). MVs (25 μg/ml) were applied to MM cell line U266 for 72 h. Interestingly, for the first time in the analyses of BM-MSCs secretomes we registered a distinct difference in activity in accordance with the secretomes' source (ND, MM). Generally, ND BM-MSCs derived MVs decreased U266 translation initiation, autophagy and proliferation; whereas MM BM-MSCs derived MVs augmented the expression of the translation initiation factors, autophagy and proliferation. In detail, MM cell line U266 was treated with MVs secreted from BM-MSCs (ND, MM), after 72 h cells were harvested, lysed and immunoblotted for p-eIF4E, t-eIF4E, p-eIF4GI and t-eIF4GI. Significantly decreased levels of p-eIF4E (↓ 30%; p b 0.01), p-eIF4GI (↓ 40%; p b 0.01), t-eIF4E (↓ 40%; p b 0.05) and t-eIF4GI (↓40; p b 0.05) were observed in MM cells treated with MVs secreted from ND BM-MSCs (Fig 6B). Furthermore, reduced levels of Beclin-1 and LC3-II autophagy markers were also observed (↓ 28% and ↓ 20%, respectively; p b 0.05) (Fig 6C). Finally, proliferation marker PCNA expression was diminished as well (↓35%; p b 0.05) and

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Fig. 4. Disassociation of eIF4E/eIF4GI complex results in decrease of total and live cell number. U266 and ARP-1 cells were co-treated with 72 h BM-MSCs' secretome (ND, MM) and 4EGI (1OOμg/ml) for 72 h and assayed for (A) total cell counts and (B) live cell. Results were compared to U266 and ARP-1 treated with matching secretome only and expressed as percent (Mean ± SE, n = 4) of control cells (dotted line). Next, cells were lysed and immunoblotted for the autophagy markers Beclin-1 and LC3-II. HistonH3 served as loading control. Representative images are presented. Statistically significant differences between cohorts (*; p b 0.05, **; p b 0.01) and between MM cell lines' responses to MM-MSCs and ND-MSCs' secretomes ($) are indicated.

caspase 3 cleavage was increased supporting elevated death (Fig 6C). On the contrary, MM cells treated with MVs secreted from MM BMMSCs displayed significantly elevated levels of p-eIF4E (↑ 40%; p b 0.05), p-eIF4GI (↑ 70%; p b 0.05), t-eIF4E (↑ 50%; p b 0.05) and teIF4GI (↑ 70%; p b 0.05), Beclin-1 (↑ 38%; p b 0.05), LC3-II (↑ 44%; p b 0.05) and PCNA (↑35%; p b 0.05) yet no significant change in caspase 3 cleavage (Fig 6D,E). Taken together, our results indicate MVs from MSCs modulate translation initiation and phenotype of MM cells. Moreover, the source of the MVs determines the trend of the effect. MVs from ND-MSCs down regulate MM cells' translation initiation, autophagy and proliferation whereas MVs from MM-MSCs stimulate these characteristics in the MM cells. 4. Discussion We as others have demonstrated [1,36–38] that cardinal communication exists between MM cells and their microenvironment BM-MSCs [3,4]. This study inspected the particular contribution of BM-MSCs soluble factors to the design of MM cells phenotype and translation initiation, particularly eIF4E/eIF4GI. Interestingly, we demonstrated heterogeneous effects of BM-MSCs' secretome components on MM cells' translation initiation and an active role of microvesicles in this dialogue. A major finding of this study is the unequivocal promotion of MM cells proliferation by BM-MSCs secretomes regardless of the MSCs source (ND or MM). This corresponds to reports of continued contribution of the microenvironment to the progression of the malignant disease [39]. Furthermore, this underscores the non-toxic function of a healthy BM-MSCs secretome in regards with MM disease progression. Interestingly; these results are dissimilar to the previous observations made by us and recently published that depict distinct differences in the effect of co-cultured BM-MSCs on MM cell lines with respect to the MSCs source (ND or MM) [12]. Indeed, in this study we detected elevated proliferation of MM cells co-cultured with MM BM-MSCs yet no increase in proliferation of MM cells co-cultured with ND BM-MSCs [12]. Taken together, we suggest that both cell–cell contact and secreted

components contribute to the final outcome of the MM cells interaction with the BM-MSCs. For instance, cell–cell contact of the ND BM-MSCs may attenuate the soluble compartment's effect on proliferation. Furthermore, the current study specifies that the healthy BM-MSCs' environment secretes tumor promoting elements even without receiving any signals from the MM cells. The discrepancy between the observations made in the two research models (co-culture, secretome only) may also be attributed to the co-culture dynamic communication and reciprocal feedback between the populations. Further studies are required for the resolution of these possibilities. Intrigued by our findings we tried to locate specific soluble participants in the BM-MSCs secretomes capable of modulating eIF4E/eIF4GI. Interestingly, once we fractionated the secretomes (b100 kDa, N100 kDa) we observed opposing activities in terms of translation initiation factors yet still did not observe any significance to the source of secretome (ND, MM). Of note, it is not possible to deduce from the fractions onto the whole because different solvents and controls were used (fresh low serum media in the suspension of the N100 kDa fraction and whole BM-MSCs secretome with the b100 kDa fraction). Yet, these data do underscore the complexity and multi-factorial system involved in the intercellular communication and balance of translation initiation. The N100 kDa fraction, that contains MVs caused a decrease in translation initiation process. We were particularly interested in this response because of its therapeutic potential in decreasing translation initiation in MM. Thus, we examined the effect of MVs from ND BMMSCs and MM BM-MSCs on eIF4E/eIF4GI in MM. We also assayed effects on proliferation, autophagy and death. Our initial expectation was to witness the inhibitory effect of the N100 kDa fraction common to ND and MM BM-MSCs secretomes with the application of the MVs only. Surprisingly, for the first time in this study we observed a differential response of the MM cells' to MVs from MM BM-MSCs and ND BMMSCs. The ND BM-MSCs indeed secreted vesicles that decreased the translation initiation, proliferation, and autophagy, as observed with the N100 kDa. However, the MM BM-MSCs secreted vesicles that increased the translation initiation process, proliferation, and autophagy.


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Fig. 5. BM-MSCs' secretome fractions (N100 kDa, b100 kDa) differentially affect eIF4E/eIF4GI in MM cell. MM cell lines (ARP-1, U266) were cultured for 72 h with fresh low serum media containing (A) N100 kDa fraction or (B) lacking the N100 kDa of BM-MSCs 72 h secretome (ND, MM). After 72 h the cells were harvested, lysed and immunoblotted for eIF4E/eIF4GI. Tubulin served as loading control. Results are presented in bar graphs (left) and representative immunoblots (right) and expressed as percent (Mean ± SE, n ≥ 3) of respective protein expression in control cells (dotted line). Statistically significant differences between cohorts (*; p b 0.05, **; p b 0.01) and between MM cell lines' responses to MM-MSCs and ND-MSCs' secretomes ($) are indicated.

The discrepancy between the N 100 kDa fraction results and the MVs results may indicate a collaboration between the MVs and other secretome components. Another explanation may lie in a persistant contamination of the MVs in the N100 kDa fraction with cell debris or protein aggregates that were not cleared by the low speed centrifugation dictated by the protocol [40,41]. In contrast, the MVs isolation process included multiple centrifugation steps in escalating speeds, with washes between each step, leading to a purer product [42]. Another interesting alteration in MM cells' phenotype upon exposure to BM-MSCs secretomes was elevated autophagy, which is activated in MM cells to begin with [22,31]. We also established that the increased autophagy supported the MM cells' survival and proliferation. The differences in response time and extent of MM cells to ND BMMSCs' (higher and later) and MM BM-MSCs secretomes (moderate and earlier) may indicate the utilization of different autophagy regulatory pathways (PI3K–Akt–mTOR; AMPK; NFκB; MAPK; and p53) [43]. In our study we have shown simultaneous increases in both autophagy and translation initiation, which contradicts established understanding of the processes. General knowledge has it that both autophagy and translation initiation are regulated by mTOR [43–45]. Yet, classic autophagy mechanism involves inhibition of mTOR followed by instigation of autophagy promoting cascades [46], while the classic translation initiation mechanism requires activation of mTOR in order to phosphorylate 4EBP1, disengage from eIF4E and allow assembly of the translation initiation machinery [47]. Thus, protein synthesis and autophagic degradation are regulated in an opposite manner by mTOR, which is consistent with cellular response to starvation and stress [48]. Interestingly and directly pertaining to our findings, it has recently been shown that under certain conditions there is

compartmentalization of mTOR that is suggested to allow for the coexistence of the opposing autophagy and synthesis [48]. This is suggested to afford cellular benefits in rapid protein turnover [48]. Our study analyzed the steady state of mTOR at a given time point with disregard to its cellular location. Additional experiments that examine the dynamic and temporal expression of mTOR are needed to clarify its simultaneous role in both autophagy and translation. The importance of translation initiation to cancer progression is increasingly acknowledged [49–51]. Indeed, we have previously shown that manipulation of translation initiation affects MM cells' phenotype and protein repertoire [18,20,21,52]. Here, we showed that BM-MSCs secretomes promote the translation of specific eIF4E/eIF4GI targets of recognized importance to cancer and specifically MM progression (NFκB, c-Myc, Cyclin D1, Hif1α, and Smad5) [10,20,21,52–54]. Since not all changes in protein expressions in MM can be attributed to genomic occurrences it is tempting to speculate that the underlying mechanism may be the exposure of the malignant cells to BM-MSCs secretomes' and modulation of MM translation initiation. Once again ND BM-MSCs and MM BM-MSCs secretomes may have comparable impact on disease progression in regard to these particular parameters. Another interesting point worth mentioning is that since the BMMSCs secretome components are soluble, they can impact distant cells and locations, and may have far reaching and long term effects. It could also intimate as to why the BM niche is the natural environment for MM cells, and their preferred site of residence. Furthermore, several lines of evidence from other studies by us and others demonstrate that eIF4E/eIF4GI control cancer cells proliferation and autophagy thus situating them at the top of the cascade initiated by the secreted BM-MSCs components [25,55–57](Fig. 7).

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Fig. 6. BM-MSCs' MVs have a differential effect on MM cells' eIF4E/eIF4GI according to source (ND, MM): Using ultracentrifugation we isolated MVs from ND and MM BM-MSCs. (A) First, we validated their identity by transmission electron microscope (TEM) and staining with uranyl acetate that demonstrated double membrane vesicles at the appropriate size (100–1000 nm)(designated by arrow). Next, U266 MM cell line was cultured for 72 h with MVs (25 μg/ml) derived from (B + C) ND-BM-MSCs or (D + E) MM-BM-MSCs added to a low serum media, or with fresh low serum media (control). Cells were harvested after 72 h, lysed and immunoblotted for (B + D) eIF4E and eIF4GI and (C + E) LC3-II, Beclin-1, PCNA, Caspase3. Tubulin served as loading control. Results are presented in bar graphs (left) and representative immunoblots (right) and expressed as percent (Mean ± SE, n ≥ 4) of respective protein expression in control cells (dotted line). Statistically significant differences between cohorts (*; p b 0.05, **; p b 0.01) and between MM cell lines' responses to MM-MSCs and ND-MSCs' secretomes ($) are indicated.

Fig. 7. Summary of the secretome fractions effects on MM cells: The figure presents phenotypic and TI alterations following exposure of MM cells to the different secretomes we presented in the paper (cell lines, ND/MM BM-MSCs, ND/MM MSCs MV). The arrows indicate the trend of change.


From a therapeutic point of view, it is important to consider that the overall soluble factors secreted from ND BM-MSCs as well as from MM BM-MSCs may contribute to MM drug resistance. Of note, many of the modulated signals regulated by eIF4E/eIF4GI are significant to MM pathogenesis and progression. Indeed, It was demonstrated that constitutive activation of NFκB confers drug resistance and its inhibition reverses CAM-DR in MM [58,59]; inhibition of c-Myc in drug resistant MM cell lines inhibited cells proliferation [60]; and inhibition of Hif1α restored sensitivity to therapeutic agents previously resistant to [61,62]. All things considered, and in light of increased expressions of NFκB, c-Myc, and Hif1α in MM cells exposed to BM-MSCs, perhaps the secretome and not only cell adhesion contribute to MM drug resistance. We propose that further studies may be worthwhile. 5. Conclusion In total, our results indicate that the vesicles secreted by the ND BMMSCs differ from the vesicles secreted from the MM BM-MSCs. While the whole secretome from both sources cause elevation in translation initiation, disintegrating it into fractions reveals a much more complex system with a multitude of actions that may work together or cancel each other out. This very intriguing finding warrants further studies already underway by our group. A characterization of the MVs cargos will be instigated and hopefully differential expressions will lead to the isolation and identification of eIF4E/eIF4GI modulating agents (proteins, mRNAs, microRNAs, lipids). Importantly, the expected data may promote the understanding of the crosstalk machinery and uncover novel therapeutic targets. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cellsig.2016.03.003. Acknowledgement This work was supported by the Bernard Jacobson fund, Tel Aviv University, research Grant number #0601243672. This work was performed in partial fulfillment of the requirements for a M.Sc. degree of Hila Marcus, Sackler Faculty of Medicine, Tel Aviv University, Israel. We are grateful to the staff of the Hematocytological Laboratory at Meir Medical Center for their dedicated technical support. We also thank Dr. Irit Shefler for her generous support and useful advice. There are no conflicting interests. References [1] M.J. Bissell, W.C. Hines, Why don't we get more cancer? A proposed role of the microenvironment in restraining cancer progression, Nat. Med. 17 (2011) 320–329. [2] M. Gronborg, T.Z. Kristiansen, A. Iwahori, R. Chang, R. Reddy, N. Sato, H. Molina, O.N. Jensen, R.H. Hruban, M.G. Goggins, A. Maitra, A. Pandey, Biomarker discovery from pancreatic cancer secretome using a differential proteomic approach, Mol. Cell. Proteomics 5 (2006) 157–171. [3] S. Manier, A. Sacco, X. Leleu, I.M. Ghobrial, A.M. Roccaro, Bone marrow microenvironment in multiple myeloma progression, J. Biomed. Biotech. 2012 (2012) 157496. [4] Y. Kawano, M. Moschetta, S. Manier, S. Glavey, G.T. Gorgun, A.M. Roccaro, K.C. Anderson, I.M. Ghobrial, Targeting the bone marrow microenvironment in multiple myeloma, Immunol. Rev. 263 (2015) 160–172. [5] R.A. Kyle, S.V. Rajkumar, Criteria for diagnosis, staging, risk stratification and response assessment of multiple myeloma, Leukemia 23 (2009) 3–9. [6] T. Hideshima, C. Mitsiades, G. Tonon, P.G. Richardson, K.C. Anderson, Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets, Nat. Rev. Cancer 7 (2007) 585–598. [7] S.V. Rajkumar, Myeloma Today: Disease Definitions and Treatment Advances, Am. J. Hematol. (2015), http://dx.doi.org/10.1002/ajh.24236. [8] S.R. Wallace, M.M. Oken, K.L. Lunetta, A. Panoskaltsis-Mortari, A.M. Masellis, Abnormalities of bone marrow mesenchymal cells in multiple myeloma patients, Cancer 91 (2001) 1219–1230. [9] H.G. Wendel, R.L. Silva, A. Malina, J.R. Mills, H. Zhu, T. Ueda, R. Watanabe-Fukunaga, R. Fukunaga, J. Teruya-Feldstein, J. Pelletier, S.W. Lowe, Dissecting eIF4E action in tumorigenesis, Genes Dev. 21 (2007) 3232–3237. [10] D. Silvera, R. Arju, F. Darvishian, P.H. Levine, L. Zolfaghari, J. Goldberg, T. Hochman, S.C. Formenti, R.J. Schneider, Essential role for eIF4GI overexpression in the pathogenesis of inflammatory breast cancer, Nat. Cell Biol. 11 (2009) 903–908.

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