Murine fetal bone marrow does not support functional hematopoietic stem and progenitor cells until birth.

While adult bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) and their extrinsic regulation is well studied, little is known about the composition, function, and extrinsic regulation of the first HSPCs to enter the BM during development. Here, we functionally interrogate murine BM HSPCs from E15.5 through P0. Our work reveals that fetal BM HSPCs are present by E15.5, but distinct from the HSPC pool seen in fetal liver, both phenotypically and functionally, until near birth. We also generate a transcriptional atlas of perinatal BM HSPCs and the BM niche in mice across ontogeny, revealing that fetal BM lacks HSPCs with robust intrinsic stem cell programs, as well as niche cells supportive of HSPCs. In contrast, stem cell programs are preserved in neonatal BM HSPCs, which reside in a niche expressing HSC supportive factors distinct from those seen in adults. Collectively, our results provide important insights into the factors shaping hematopoiesis during this understudied window of hematopoietic development.

Ribosomal proteins as distinct "passengers" of microvesicles: new semantics in myeloma and mesenchymal stem cells' communication.

Aberrant mesenchymal stem cells (MSCs) in multiple myeloma (MM) bone marrows (BM) promote disease progression and drug resistance. Here, we assayed the protein cargo transported from MM-MSCs to MM cells via microvesicles (MVs) with focus on ribosomal proteins (RPs) and assessment of their influence on translation initiation and design of MM phenotype. Proteomics analysis (mass spectrometry) demonstrated increased levels and repertoire of RPs in MM-MSCs MVs compared to normal donors (ND) counterparts (n = 3-8; P = 9.96E - 08). We limited the RPs load in MM-MSCs MVs (starvation, RSK and XPO1 inhibitions), reapplied the modified MVs to MM cell lines (U266, MM1S), and demonstrated that the RPs are essential to the proliferative effect of MM-MSCs MVs on MM cells (n = 3; P < 0.05). We also observed that inhibition with KPT-185 (XPO1 inhibitor) displayed the most extensive effect on RPs delivery into the MVs (↓80%; P = 3.12E - 05). Using flow cytometry we assessed the expression of select RPs (n = 10) in BM-MSCs cell populations (ND and MM; n ≥ 6 each). This demonstrated a heterogeneous expression of RPs in MM-MSCs with distinct subgroups, a phenomenon absent from ND-MSCs samples. These findings bring to light a new mechanism in which the tumor microenvironment participates in cancer promotion. MVs-mediated horizontal transfer of RPs between niche MSCs and myeloma cells is a systemic way to bestow pro-cancer advantages. This capacity also differentiates normal MSCs from the MM-modified MSCs and may mark their reprogramming. Future studies will be aimed at assessing the clinical and therapeutic potential of the increased RPs levels in MM-MSCs MVs.

Multiple myeloma BM-MSCs increase the tumorigenicity of MM cells via transfer of VLA4-enriched microvesicles.

Multiple myeloma (MM) cells accumulate in the bone marrow (BM) where their interactions impede disease therapy. We have shown that microvesicles (MVs) derived from BM mesenchymal stem cells (MSCs) of MM patients promote the malignant traits via modulation of translation initiation (TI), whereas MVs from normal donors (ND) do not. Here, we observed that this phenomenon is contingent on a MVs' protein constituent, and determined correlations between the MVs from the tumor microenvironment, for example, MM BM-MSCs and patients' clinical characteristics. BM-MSCs' MVs (ND/MM) proteomes were assayed (mass spectrometry) and compared. Elevated integrin CD49d (X80) and CD29 (X2) was determined in MM-MSCs' MVs and correlated with patients' staging and treatment response (free light chain, BM plasma cells count, stage, response to treatment). BM-MSCs' MVs uptake into MM cell lines was assayed (flow cytometry) with/without integrin inhibitors (RGD, natalizumab, and anti-CD29 monoclonal antibody) and recipient cells were analyzed for cell count, migration, MAPKs, TI, and drug response (doxorubicin, Velcade). Their inhibition, particularly together, attenuated the uptake of MM-MSCs MVs (but not ND-MSCs MVs) into MM cells and reduced MM cells' signaling, phenotype, and increased drug response. This study exposed a critical novel role for CD49d/CD29 on MM-MSCs MVs and presented a discriminate method to inhibit cancer promoting action of MM-MSCs MVs while retaining the anticancer function of ND-MSCs-MVs. Moreover, these findings demonstrate yet again the intricacy of the microenvironment involvement in the malignant process and highlight new therapeutic avenues to be explored.

Niche origin of mesenchymal stem cells derived microvesicles determines opposing effects on NSCLC: Primary versus metastatic.

Novel therapeutic approaches that address the malignant cells in their stroma microenvironment are urgently needed in lung cancer. The stroma resident mesenchymal stem cells (MSCs) interact with cancer cells in diverse ways including microvesicles (MVs) that transfer proteins and RNA species thereby modulating recipient cells' phenotype. Previously, we have demonstrated that MSCs' secretome from the primary non-small cell lung cancer (NSCLC) niche (lung) and metastatic niche (bone marrow (BM)) demonstrate opposite effects on NSCLC cells in a translation initiation (TI) dependent manner. Here, we examined the effect of MVs secreted from BM-MSCs' or lung-MSCs (healthy, NSCLC) to NSCLC phenotype. Briefly, NSCLC cell lines treated with Lung or BM-MSCs' MVs were assayed for viability (WST-1), cell count/death (trypan), migration (scratch), TI status and MAPKs activation (immunoblotting). Corresponding to previous published trends, Lung-MSCs' MVs promoted NSCLC cells' assayed traits whereas, BM-MSCs' MVs suppressed them. Activation of MAPKs and autophagy was registered in lung-MSCs MVs treated NSCLC cell lines only. Furthermore, lung-MSCs' MVs' treated NSCLC cells demonstrated an early (5min) activation of MAPKs and TI factors (peIF4E/peIF4GI) not evident in BM-MSCs MVs treated cells. These observations depict a role for MSCs'-MVs in NSCLC phenotype design and display distinct differences between the primary and metastatic niches that correspond to disease progression. In conclusion, the systemic nature of MVs marks them as attractive therapeutic markers/targets and we propose that identification of specific cargoes/signals that differentiate between MSCs MVs of primary and metastatic niches may introduce fresh therapeutic approaches.

BM-MSCs-derived ECM modifies multiple myeloma phenotype and drug response in a source-dependent manner.

Multiple myeloma (MM) malignant plasma cells accumulate in the bone marrow (BM) where their interaction with the microenvironment promotes disease progression and drug resistance. Previously, we have shown that MM cells cocultured with BM-mesenchymal stem cells (MSCs) comodulated cells' phenotype in a MAPKs/translation initiation (TI)-dependent manner. Dissection of the coculture model showed that BM-MSCs secretomes and microvesicles (MVs) participate in this crosstalk. Here, we addressed the role of the BM-MSCs extracellular matrix (ECM). MM cell lines cultured on decellularized ECM of normal donors' (ND) or MM patients' BM-MSCs were assayed for phenotype (viability, cell count, death, proliferation, migration, and invasion), microRNAs (MIR125a-3p, MIR199a-3p) and targets, MAPKs, TI epithelial-to-mesenchymal transition (EMT), CXCR4, and autophagy. Drug (doxorubicin, velcade) response of MM cells cultured on ND/MM-MSCs' ECM with/without adhered MVs was also evaluated. ECM evoked opposite responses according to its origin: MM cells cultured on ND-MSCs' ECM demonstrated a rapid and continued decrease in MAPK/TI activation (↓10%-25%, P < 0.05) (15-24 hours) followed by diminished viability, cell count, proliferation, migration, and invasion (16-72 hours) (↓10%-50%, P < 0.05). In contrast, MM cells cultured on MM-MSCs' ECM displayed activated MAPK/TI, proliferation, EMT, and CXCR4 (↑15%-250%, P < 0.05). Corresponding changes in microRNAs relevant to the MM cells' altered phenotype were also determined. The hierarchy and interdependence of MAPKs/TI/autophagy/phenotype cascade were demonstrated. Finally, we showed that the ECM cooperates with MVs to modulate MM cells drug response. These data demonstrate the contribution of BM-MSCs' ECM to MM niche design and underscore the clinical potential of identifying targetable signals.

Microvesicles derived from normal and multiple myeloma bone marrow mesenchymal stem cells differentially modulate myeloma cells' phenotype and translation initiation.

Multiple myeloma (MM) cells' interaction with the bone marrow (BM) microenvironment critically hinders disease therapy. Previously, we showed that MM co-culture with BM-mesenchymal stem cells (MSCs) caused co-modulation of translation initiation (TI) and cell phenotype and implicated secreted components, specifically microvesicles (MVs). Here, we studied the role of the BM-MSCs [normal donors (ND) and MM] secreted MVs in design of MM cells' phenotype, TI and signaling. BM-MSCs' MVs collected from BM-MSCs (MM/ND) cultures were applied to MM cell lines. After MVs uptake confirmation, the MM cells were assayed for viability, cell count and death, proliferation, migration, invasion, autophagy, TI status (factors, regulators, targets) and MAPKs activation. The interdependence of MAPKs, TI and autophagy was determined (inhibitors). ND-MSCs MVs' treated MM cells demonstrated a rapid (5 min) activation of MAPKs followed by a persistent decrease (1-24 h), while MM-MSCs MVs' treated cells demonstrated a rapid and continued (5 min-24 h) activation of MAPKs and TI (↑25-200%, P < 0.05). Within 24 h, BM-MSCs MVs were internalized by MM cells evoking opposite responses according to MVs origin. ND-MSCs' MVs decreased viability, proliferation, migration and TI (↓15-80%; P < 0.05), whereas MM-MSCs' MVs increased them (↑10-250%, P < 0.05). Inhibition of MAPKs in MM-MSCs MVs treated MM cells decreased TI and inhibition of autophagy elevated cell death. These data demonstrate that BM-MSCs MVs have a fundamental effect on MM cells phenotype in accordance with normal or pathological source implemented via TI modulation. Future studies will aim to elucidate the involvement of MVs-MM receptor ligand interactions and cargo transfer in our model.

Multiple myeloma cells promote migration of bone marrow mesenchymal stem cells by altering their translation initiation.

The role of the bone marrow microenvironment in multiple myeloma pathogenesis and progression is well recognized. Indeed, we have shown that coculture of bone marrow mesenchymal stem cells from normal donors and multiple myeloma cells comodulated translation initiation. Here, we characterized the timeline of mesenchymal stem cells conditioning by multiple myeloma cells, the persistence of this effect, and the consequences on cell phenotype. Normal donor mesenchymal stem cells were cocultured with multiple myeloma cell lines (U266, ARP1) (multiple myeloma-conditioned mesenchymal stem cells) (1.5 h,12 h, 24 h, 48 h, and 3 d) and were assayed for translation initiation status (eukaryotic translation initiation factor 4E; eukaryotic translation initiation factor 4G; regulators: mechanistic target of rapamycin, MNK, 4EBP; targets: SMAD family 5, nuclear factor κB, cyclin D1, hypoxia inducible factor 1, c-Myc) (immunoblotting) and migration (scratch assay, inhibitors). Involvement of mitogen-activated protein kinases in mesenchymal stem cell conditioning and altered migration was also tested (immunoblotting, inhibitors). Multiple myeloma-conditioned mesenchymal stem cells were recultured alone (1-7 d) and were assayed for translation initiation (immunoblotting). Quantitative polymerase chain reaction of extracted ribonucleic acid was tested for microRNAs levels. Mitogen-activated protein kinases were activated within 1.5 h of coculture and were responsible for multiple myeloma-conditioned mesenchymal stem cell translation initiation status (an increase of >200%, P < 0.05) and elevated migration (16 h, an increase of >400%, P < 0.05). The bone marrow mesenchymal stem cells conditioned by multiple myeloma cells were reversible after only 1 d of multiple myeloma-conditioned mesenchymal stem cell culture alone. Decreased expression of microRNA-199b and microRNA-125a (an increase of <140%, P < 0.05) in multiple myeloma-conditioned mesenchymal stem cells supported elevated migration. The time- and proximity-dependent conditioning of normal donor mesenchymal stem cells in our model points to a dynamic interaction between multiple myeloma cells and the bone marrow niche, which causes profound changes in the nonmalignant bone marrow constituents. Future studies are warranted to identify clinically relevant means of blocking this crosstalk and improving multiple myeloma therapy.

Multiple myeloma and bone marrow mesenchymal stem cells' crosstalk: Effect on translation initiation.

Multiple myeloma (MM) malignant plasma cells reside in the bone marrow (BM) and convert it into a specialized pre-neoplastic niche that promotes the proliferation and survival of the cancer cells. BM resident mesenchymal stem cells (BM-MSCs) are altered in MM and in vitro studies indicate their transformation by MM proximity is within hours. The response time frame suggested that protein translation may be implicated. Thus, we assembled a co-culture model of MM cell lines with MSCs from normal donors (ND) and MM patients to test our hypothesis. The cell lines (U266, ARP-1) and BM-MSCs (ND, MM) were harvested separately after 72 h of co-culture and assayed for proliferation, death, levels of major translation initiation factors (eIF4E, eIF4GI), their targets, and regulators. Significant changes were observed: BM-MSCs (ND and MM) co-cultured with MM cell lines displayed elevated proliferation and death as well as increased expression/activity of eIF4E/eIF4GI; MM cell lines co-cultured with MM-MSCs also displayed higher proliferation and death rates coupled with augmented translation initiation factors; in contrast, MM cell lines co-cultured with ND-MSCs did not display elevated proliferation only death and had no changes in eIF4GI levels/activity. eIF4E expression was increased in one of the cell lines. Our study demonstrates that there is direct dialogue between the MM and BM-MSCs populations that includes translation initiation manipulation and critically affects cell fate. Future research should be aimed at identifying therapeutic targets that may be used to minimize the collateral damage to the cancer microenvironment and limit its recruitment into the malignant process. © 2015 Wiley Periodicals, Inc.