Multiple myeloma (MM) is a glutamine (Gln)-auxotroph and Gln-addicted cancer, with Gln synthetase (GS)-deficient MM cells avidly taking up extracellular Gln to sustain their metabolism. Thus, MM cells create a peculiar metabolic niche in the patients' bone marrow (BM), where low levels of Gln contribute to the osteolytic bone lesions by inhibiting the osteoblastic differentiation of mesenchymal stromal cells (MSCs). The effects of the altered MM metabolic niche on other BM cell populations remain to be clarified. We demonstrate here that MM cells secrete high amounts of glutamate through the exchange transporter SLC7A11/xCT. In turn, BM MSCs, but neither MM cells nor osteoblasts (OBs), actively take up extracellular glutamate through the transporter EAAT3 (SLC1A1), whose expression decreases during osteogenesis. GS-positive MSCs secrete Gln, a process boosted by extracellular glutamate in undifferentiated MSCs, but not in differentiated OBs. Coculture of MSCs with MM cells promotes the expression of the bidirectional transporter SNAT5 (SLC38A5), suggesting its involvement in Gln efflux. Consistently, MSCs, derived from either patients with MM or healthy donors, sustain MM growth in a low-Gln environment, an effect suppressed by the inhibition or silencing of glutamate uptake or Gln synthesis. In conclusion, a metabolic cycle occurs in MM BM microenvironment, where Gln-auxotroph MM cells extrude glutamate that is converted into Gln by MSC, sustaining in turn MM anabolism through Gln secretion. The inhibition of this metabolic trade-off impairs MM cell growth, thus highlighting novel potential, niche-oriented therapeutic targets.

The role of extracellular vesicles has been extensively studied in physiological and pathological conditions, and growing evidence has pinpointed them as key players in tumor progression, regulation of the metastatic niche, and modulation of anti-tumor immune responses. Indeed, a dynamic transfer of extracellular vesicles between cancer cells and immunological or non-immunological cells homing in the tumor microenvironment exists, and the balance between their release by cancer cells and by normal cells determines cancer progression. Here, we focused on the role of extracellular vesicles in the dysregulation of the bone marrow environment in pediatric tumors such as acute leukemias and neuroblastomata, whose poor prognosis is strictly related to the involvement of such anatomical site. Acute leukemias arise from bone marrow progenitors, whereas approximately 50% of neuroblastoma patients have bone marrow metastases at diagnosis. Thus, here, we discuss the mechanisms underlying the bone marrow dysregulation in pediatric acute leukemias and neuroblastomata with particular emphasis on the involvement of extracellular vesicles.

B-cell type acute lymphoblastic leukemia (B-ALL) is the most common type of childhood malignancy. Although the survival rate nowadays exceeds 90%, central nervous system (CNS) involvement is associated with a poor outcome. Experimental models are needed to study the interaction between leukemia cells and the brain microenvironment to unravel new targets for drug intervention. We developed a novel three-dimensional (3D) ex vivo model utilizing murine organotypic cortical brain slices microinjected with human B-ALL cells, serving as a platform for investigating the influence of Activin A, a pro-leukemic factor, on leukemia invasion into the CNS. After injection, B-ALL cells exponentially increased in the cortical slices, promoting tissue mortality and an anti-inflammatory microenvironment phenotype, as demonstrated by morphological and gene expression alterations in microglia and astrocytes. Of note, Activin A pretreatment increased leukemia proliferation and exacerbated the effects on the microenvironment. Overall, our model presents an ideal platform for investigating the cross-talk between tumors and the brain microenvironment and the influence of disease-modifying factors. Moreover, it could facilitate drug screening across a spectrum of CNS cancers, meanwhile reducing animal usage.

B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) blasts strictly depend on the transport of extra-cellular asparagine (Asn), yielding a rationale for L-asparaginase (ASNase) therapy. However, the carriers used by ALL blasts for Asn transport have not been identified yet. Exploiting RS4;11 cells as BCP-ALL model, we have found that cell Asn is lowered by either silencing or inhibition of the transporters ASCT2 or SNAT5. The inhibitors V-9302 (for ASCT2) and GluγHA (for SNAT5) markedly lower cell proliferation and, when used together, suppress mTOR activity, induce autophagy and cause a severe nutritional stress, leading to a proliferative arrest and a massive cell death in both the ASNase-sensitive RS4;11 cells and the relatively ASNase-insensitive NALM-6 cells. The cytotoxic effect is not prevented by coculturing leukaemic cells with primary mesenchymal stromal cells. Leukaemic blasts of paediatric ALL patients express ASCT2 and SNAT5 at diagnosis and undergo marked cytotoxicity when exposed to the inhibitors. ASCT2 expression is positively correlated with the minimal residual disease at the end of the induction therapy. In conclusion, ASCT2 and SNAT5 are the carriers exploited by ALL cells to transport Asn, and ASCT2 expression is associated with a lower therapeutic response. ASCT2 may thus represent a novel therapeutic target in BCP-ALL.

Extracellular vesicles (EVs) are a new mechanism of cellular communication, by delivering their cargo into target cells to modulate molecular pathways. EV-mediated crosstalk contributes to tumor survival and resistance to cellular stress. However, the role of EVs in B-cell Acute Lymphoblastic Leukaemia (B-ALL) awaits to be thoroughly investigated. We recently published that ActivinA increases intracellular calcium levels and promotes actin polymerization in B-ALL cells. These biological processes guide cytoskeleton reorganization, which is a crucial event for EV secretion and internalization. Hence, we investigated the role of EVs in the context of B-ALL and the impact of ActivinA on this phenomenon. We demonstrated that leukemic cells release a higher number of EVs in response to ActivinA treatment, and they can actively uptake EVs released by other B-ALL cells. Under culture-induced stress conditions, EVs coculture promoted cell survival in B-ALL cells in a dose-dependent manner. Direct stimulation of B-ALL cells with ActivinA or with EVs isolated from ActivinA-stimulated cells was even more effective in preventing cell death. This effect can be possibly ascribed to the increase of vesiculation and modifications of EV-associated microRNAs induced by ActivinA. These data demonstrate that ActivinA boosts EV-mediated B-ALL crosstalk, improving leukemia survival in stress conditions.

Gastrointestinal graft-versus-host disease (GvHD) is a major cause of mortality and morbidity following allogeneic bone marrow transplantation (allo-BMT). Chemerin is a chemotactic protein that recruits leukocytes to inflamed tissues by interacting with ChemR23/CMKLR1, a chemotactic receptor expressed by leukocytes, including macrophages. During acute GvHD, chemerin plasma levels were strongly increased in allo-BM-transplanted mice. The role of the chemerin/CMKLR1 axis in GvHD was investigated using Cmklr1-KO mice. WT mice transplanted with an allogeneic graft from Cmklr1-KO donors (t-KO) had worse survival and more severe GvHD. Histological analysis demonstrated that the gastrointestinal tract was the organ mostly affected by GvHD in t-KO mice. The severe colitis of t-KO mice was characterized by massive neutrophil infiltration and tissue damage associated with bacterial translocation and exacerbated inflammation. Similarly, Cmklr1-KO recipient mice showed increased intestinal pathology in both allogeneic transplant and dextran sulfate sodium–induced colitis. Notably, the adoptive transfer of WT monocytes into t-KO mice mitigated GvHD manifestations by decreasing gut inflammation and T cell activation. In patients, higher chemerin serum levels were predictive of GvHD development. Overall, these results suggest that CMKLR1/chemerin may be a protective pathway for the control of intestinal inflammation and tissue damage in GvHD.

We recently demonstrated that ActivinA (ActA) is abundant in the leukemic bone marrow microenvironment where it exerts a pro-leukemic action by increasing the migratory and invasive properties of B-ALL cells. Extracellular vesicles (EV) are a new way of intercellular communication. They can be categorized in small-EV (sEV) and large-EV (lEV) based on size and origin. Similarly to migration, EV release is dependent upon cytoskeleton activation and membrane remodeling. EV play a prominent role in carcinogenesis, depending on the molecules they enclose, including miRNA that can downmodulate the expression of target mRNAs in recipient cells. The aim of the project was to study the impact of ActA on B-ALL vesiculation: quantity and miRNA cargo. Moreover, we investigated whether deregulated miRNAs could contribute to ActA-mediated leukemic progression. To investigate the role of ActA on B-ALL vesiculation, we used 697 and Nalm-6 cells lines. By means of nanoparticle tracking analysis, we demonstrated that after 24h, ActA significantly increased of 2.5 and 1.4 folds the number of sEV (p<0.0001) and lEV (p<0.0026, n=9) produced by 697 cells, compared to not stimulated cells (NS). A significant increase of both EV was also observed after 48h of ActA stimulation. The ability of ActA to promote B-ALL cell vesiculation was also confirmed on Nalm-6. Furthermore, we investigated the miRNA cargo of EV derived from 697 cells stimulated or not with ActA by OpenArray. After 24h, ActA significantly upregulated miR491-5p, let-7i-3p, and downregulated miR-639, miR135b-3p and miR139-5p. On the other hand, after 48h, ActA significantly upregulated miR15b-3p, let-7-e-5p, miR23b-3p and downregulated miR1236-3p, miR-18a-3p. We focused on miR491-5p, the most upregulated in ActA EV (3 folds at 24h), compared to NS (p<0.0001). ActA-mediated increase of miR491-5p was observed also in 697 cells (FC=1.5, p<0.0001, n=14). To investigate miR491-5p modulation also in Nalm-6 cells, they were stimulated or not for 24h and miRNA levels were evaluated in cells and their EV, isolated by ultracentrifugation. We preliminary observed that ActA increased miRNA expression of 479 folds in Nalm-6 EV (p=0.09, n=3), but only slightly in cells. Interestingly, absolute miRNA levels were higher of 4 folds in NS EV compared to NS cells (p=0.05), and showed an increasing trend in stimulated condition (FC=1552, p=0.09, n=3), suggesting that upon ActA stimulation, miR491-5p is upregulated and mostly encapsulated in EV. It has been described that miR491-5p promotes cell survival and proliferation in solid tumors. Indeed, we investigated whether ActA could impact on B-ALL cell viability through miR491-5p modulation. Indeed, 697 cells were stimulated every 3 days with ActA. Cell counts showed no differences between cells stimulated or not in the exponential growth phase. Interestingly, at plateau (day 7), ActA-stimulated cells were more viable (76%, range: 69%-80%) compared to NS cells (56%, range: 50%-66%, p=0.05, n=3). Alongside, we preliminary observed higher miR491-5p in ActA-stimulated cells (FC=3, n=1). It has also been reported that miR491-5p regulates chemoresistance in several cancers. Interestingly, by using Annexin-V/7-AAD assay, we demonstrated that ActA-stimulated 697 cells had a viability advantage of 11.3% (p=0.006, n=3), compared to NS cells, after 72h of ASNase-based chemotherapy, suggesting a chemoprotective role of ActA in B-ALL. Moreover, we observed that the combination of ActA and ASNase treatments induced the overexpression of miR491-5p compared to NS (FC=2.5, p=0.001) and to cells stimulated with ActA alone (FC=2.3, p=0.04) or ASNase alone (FC=1.4, p=0.004, n=8). To investigate the possible role of miR491-5p, we downregulated it into 697 cells by transfecting a miR491-5p inhibitor. Interestingly, under ASNase treatment, ActA still induced a viability advantage in cell transfected with a random inhibitor (negative control, 9.8%, p=0.006), comparable to untransfected cells. On the contrary, in miR491-5p inhibitor transfected cells, the viability advantage mediated by ActA decreased to 5.9% (p=0.03). Therefore, miR491-5p inhibition reduced of 30% the chemoprotective ability of ActA (p=0.04, n=3). In conclusion, these results suggest that ActA not only increases EV production, but also modifies their miRNA cargo and that miR491-5p could be one of the key players involved in ActA-mediated pro-leukemic action.

Introduction Shwachman-Diamond Syndrome (SDS) is a rare bone marrow (BM) failure disorder. SDS BM biopsies show tortuous and collapsed vessels, highlighting angiogenic abnormalities. Our group previously demonstrated that SDS BM mesenchymal stromal cells (MSCs) have in vitro and in vivo impaired angiogenic potential compared to healthy donors (HD)-MSCs. Interestingly, recent evidence underlines that pathological angiogenesis could be accompanied by altered metabolism and that antioxidants could represent a novel strategy to target angiogenic defects. Aims We aimed to characterize the metabolic status of SDS-MSCs and to investigate if the treatment with antioxidants may restore their impaired angiogenic potential. Methods BM derived HD- and SDS-MSCs were stimulated with DMEM ± N-Acetylcysteine (NAC, 1mM) or dimethylsulfoxide (DMSO, 0.05% v/v) for 48h. Oxidative phosphorylation (OxPhos) was assayed by oximetry and bioluminescent ATP synthesis assay. Spectrophotometric analyses were performed to evaluate ATP/AMP ratio, malondialdehyde (MDA) level, and Complex IV and lactate dehydrogenase (LDH) activity. The amount of reactive oxygen species (ROS) was quantified by flow cytometry. The angiogenic capability was evaluated by performing in vitro Matrigel angiogenesis assay. Results Firstly, we evaluated OxPhos oxygen consumption and ATP production (n=6). Concerning the I-III-IV mitochondrial complexes pathway, SDS-MSCs consumed 57% less oxygen (p=0.004) and produced 64% less ATP compared to HD-MSCs (p=0.002). Accordingly, the analysis of the II-III-IV complexes pathway demonstrated that the oxygen consumption was reduced by 62% (p=0.002) and the ATP synthesis was 67% lower than HD-MSCs (p=0.002). Furthermore, the P/O ratio, index of OxPhos efficiency, was significantly reduced in SDS-MSCs in both the electron transport chain pathways (p=0.002 for both). Therefore, we demonstrated that Complex IV activity was 61% lower in SDS- vs HD-MSCs (p=0.002), highlighting its role in the SDS OxPhos defect. As for the energetic status, SDS-MSCs showed a low intracellular ATP/AMP ratio (mean=1.1, range=0.8-1.6 vs mean=3.6, range=3.1-4.0, in HDs; p=0.002) that was accompanied by 30% increase of LDH activity in SDS-MSCs (p=0.002), an expression of increased anaerobic glycolysis to compensate the mitochondrial defect. The amount of ROS in SDS-MSCs was increased by 27% compared to HD-MSCs (n=5) and also the SDS-MSCs lipid peroxidation level was significantly higher over HDs (mean=12.7 µM of MDA/mg, range=10.5-14.1µM/mg vs mean=6.6µM/mg, range=5.9-7.4µM/mg; p=0.002). Then we treated SDS-MSCs for 48h with NAC, a broad-range antioxidant (n=5), or with DMSO, which acts at very low concentrations as scavenger, specifically on lipid peroxidation products (n=6). Interestingly, NAC and DMSO stimulated SDS-MSCs increased by 75% the oxygen consumption and by 70% the ATP synthesis in both the electron transport chain pathways, thus resulting in levels comparable to HD-MSCs. The SDS OxPhos restoration was respectively associated to a 53% and 60% increase of Complex IV activity after NAC and DMSO stimulation (p<0.0001 for both). Moreover, antioxidants corrected the SDS energetic defect by restoring ATP/AMP ratio and LDH activity to the levels of HDs. Importantly, SDS-MSCs lipid peroxidation level was drastically reduced after NAC and DMSO treatments (mean=12.7µM/mg, range=10.5-14.1µM/mg vs NAC mean=5.1µM/mg, range=4.3-5.4µM/mg and DMSO mean=6.2µM/mg, range=5.8-6.5µM/mg; p<0.0001 for both). Finally, we approached SDS-MSCs angiogenic defect and showed that SDS-MSCs defective capability to recreate a defined capillary-like network under angiogenic stimuli was completely restored after antioxidant stimulations (n≥5). As shown by ImageJ Angiogenesis Analyzer several angiogenic elements, including branches and segments, were significantly increased in NAC and DMSO stimulated SDS-MSCs that become comparable to HD-MSCs. Conclusions We demonstrated that the altered OxPhos metabolism of SDS-MSCs significantly contributes to their angiogenic defect and, importantly, that antioxidants restored the metabolic alterations and the angiogenic potential in SDS-MSCs, paving the way for new therapeutic strategies.

Topic: 1. Acute lymphoblastic leukemia - Biology & Translational Research Background: ActivinA (ActA) is an important regulator of carcinogenesis and it is involved in chemoresistance in several solid tumors. We recently demonstrated that ActA is able to exert a pro-leukemic action by increasing the migratory and invasive properties of leukemic cells. Similarly to cell migration, Extracellular Vesicles (EV) release is dependent upon cytoskeleton activation and membrane remodelling. EV play a prominent role in cancer pathogenesis and therapy resistance, depending on the functional molecules they enclose. Among them, microRNA (miRNA) can modulate the expression of target mRNAs in recipient cells Aims: We aimed to study the effect of ActA on BCP-ALL cell vesiculation and its impact on microRNA cargo Methods: EV were isolated by ultracentrifugation from the 697 BCP-ALL cell line and characterized bynanoparticles tracking analysis. EV-miRNA cargo was screened by OpenArray technology and miR-491-5p levels were investigated by qRT-PCR. 697 cell viability was evaluated by Annexin-V/7-AAD assay. Specific miR-491-5p inhibitor/mimic were transfected in 697 cells, by means of lipofectamine, to downregulate/upregulate miR-491-5p expression. miR-491-5p biological function and targets were predicted by miR-System and miR-Walk databases Results: ActA significantly increased, after 24h of stimulation, the quantity of both small-EV and large-EV released by 697 cells, compared to unstimulated cells (NS) (Fold Change (FC)=2, p<0.0001 and 1.3,p=0.003, respectively,n=16). Surprisingly, we discovered that ActA strongly impacted on EV miRNA cargo. In particular, we focused on miR-491-5p that was upregulated in EV of 3 folds (p<0.0001) and of 1.5 folds at the intracellular level after 24h(p=0.0004). Interestingly, literature data suggest that miR-491-5p could play a key role in modulating chemoresistance in several cancers. To investigate the potential involvement of the ActA/ miR-491-5p axis in chemoprotection in the context of BCP-ALL, we performed experiments in which 697 cells were stimulated or not with ActA and then treated with Asparaginase (ActA+ASNase versus NS+ASNase), a drug commonly used as treatment of paediatric BCP-ALL. Surprisingly, ActA significantly increased the viability of ASNase-treated 697 cells of 11,3% compared to NS (p=0.006). Moreover, the combination of ActA and ASNase induced the most significant increase of miR-491-5p in the cytoplasm of 697 cells (FC=2.5, p=0.0078 compared to control, n=8). To confirm the involvement of miR-491-5p in chemoresistance we modulated its expression by transfecting a specific inhibitor, obtaining a reduction of 40% of ActA-mediated anti-apoptotic action (p=0.0423,n=3). Through miR-System database, we predicted the miR-491-5p involvement in TP53-mediated apoptosis. At this purpose, gene expression profile data obtained on 697 cells demonstrated that ActA stimulation reduced of about 30% the levels of tp53aip1, a pro-apoptotic molecule with a key role in TP53 pathway. Interestingly, by using miR-Walk database, tp53aip1 was predicted as a direct target gene of miR‑491-5p. In accordance, the inhibition of miR491-5p by transfection of its specific inhibitor increased of 5,8 folds the expression levels of tp53aip1 mRNA (p=0.0313,n=6), while the transfection in 697 cells of a miR-491-5p synthetic mimic molecule decreased tp53aip1 mRNA of 20% (p=0,0283,n=4) Summary/Conclusion: Overall, we demonstrated that ActA is able to increase the production of miR-491-5p enriched EV. Furthermore, we identified ActA/ miR-491-5p axis as a pathway in the modulation of ASNase chemoresistance in 697 BCP-ALL cells. Future studies are needed to understand whether the delivery of miR-491-5p by EV could transfer chemoresistance to other BCP-ALL cells Keywords: Chemoresistance, B cell acute lymphoblastic leukemia, Microvesicles

Bone marrow mesenchymal stromal cells (MSCs) have immunomodulatory and regenerative potential. However, culture conditions govern their metabolic processes and therapeutic efficacy. Here we show that culturing donor-derived MSCs in Plasmax™, a physiological medium with the concentrations of nutrients found in human plasma, supports their proliferation and stemness, and prevents the nutritional stress induced by the conventional medium DMEM. The quantification of the exchange rates of metabolites between cells and medium, untargeted metabolomics, stable isotope tracing and transcriptomic analysis, performed at physiologically relevant oxygen concentrations (1%O2), reveal that MSCs rely on a high rate of glucose to lactate conversion, coupled with parallel anaplerotic fluxes from glutamine and glutamate to support citrate synthesis and secretion. These distinctive traits of MSCs shape the metabolic microenvironment of the bone marrow niche and can influence nutrient cross-talks under physiological and pathological conditions.

B-Cell Acute Lymphoblastic Leukemia (B-ALL) is the most common cancer in children. Multidrug high-dose chemotherapy has increased the 5-year event free survival rate to more than 80%. However, the prognosis of refractory/relapsed (r/r) ALL remains dismal. Increasing evidence indicates that treatment failure could be linked to the protective role of leukemic bone marrow (BM) microenvironment. The identification of the molecules involved in the crosstalk between leukemic cells and the BM niche would be crucial to establish niche-targeted therapies to be combined with conventional drugs. Recently, we identified ActivinA (ActA) as a new potentially targetable leukemia-promoting factor, which is significantly overexpressed in the BM plasma of B-ALL patients. We demonstrated that ActA is able to potentiate the migratory and invasive ability of B-ALL cells in vitro and enhance both BM engraftment and metastatic potential of leukemic cells in a xenograft mouse model. Literature data indicate that ActA exerts a pro-tumoral role in several solid tumors, by promoting cancer cell migration, proliferation and chemoresistance. The aim of this work was to ascertain whether ActivinA could mediate resistance to standard chemotherapy agents routinely used in pediatric B-ALL, such as Vincristine (VCR), Dexamethasone (Dex) and Asparaginase (ASNase). To investigate the role of ActA on B-ALL cell chemoresistance, we stimulated or not 697 cells with ActA for 24h. Cells were then treated with increasing concentrations of Dex, ASNase and VCR for 72h to obtain a dose-response curve. Cell viability was evaluated by flow cytometry and used to calculate the inhibitory dose 50 (IC50). Interestingly, the IC50 of ActA stimulated 697 cells for Dex and ASNase was in both cases about ten-fold higher than NS controls. In the case of VCR-treated cells, we did not detect any difference in IC50. These data suggest that ActA is able to convey resistance to Dex and ASNase-induced cell death, while sparing VCR-based leukemic cell killing, possibly due to different mechanisms of action. Validation experiments, with selected doses of Dex and ASNase (7 µg/mL and 1 U/mL, respectively), confirmed the ability of ActA to induce chemoresistance. In detail, the median viability of ActA stimulated 697 cells after 3 days of treatment with Dex was 64.15% (range=51.95-73.75%). This percentage was significantly higher compared to NS cells (median viability=41.95%, range=31.95-60.93%, Wilcoxon test: p<0.0001). Alongside, the viability of ActA stimulated cells (median=53.40%, range=38.70-75.00%) was also significantly increased (median=31.40%, range=22.45-54.25%, p=0.001) after ASNase treatment. To understand the molecular mechanisms underlying Acta-mediated chemoprotection, we firstly evaluated in our experimental setting the percentage of 697 cells expressing the active form of Caspase-3, by flow cytometry. Our results highlight that the activation of Caspase-3 was significantly reduced in ActA pre-stimulated cells treated with Dex (mean fold change (FC) ActA/NS=0.48±0.22, p=0.0003, One-sample t test) and ASNase (mean FC=0.49±0.062, p<0.0001), compared to NS cells. Then, we focused on the impact of ActA on anti- and pro-apoptotic factors playing a key role in drug-mediated apoptosis. By western blot, we demonstrated that Bcl-2 was significantly increased upon Dex treatment in ActA pre-stimulated cells (median FC ActA+Dex/NS untreated cells=0.63, range=0.49-1.12, Wilcoxon test: p<0.05) compared to NS cells (median FC NS+Dex/NS untreated cells=0.43, range=0.26-0.74). In contrast, we did not observe any difference with ASNase. Furthermore, we demonstrated that ActA was able to decrease the drug-induced cleavage activation of pro-apoptotic factors BAX and BAK. Finally, we investigated by confocal microscopy the levels of intracellular Reactive Oxygen Species (ROS), well-known key elements of the drug-induced oxidative stress, resulting in the activation of apoptosis. Interestingly, upon Dex treatment we observed a reduction of ROS levels of 59.7%±4.6% (mean±sd) in ActA-stimulated cells compared to NS. In the case of ASNase treatment the mean reduction was 46.9%±8.8%. Overall, our data suggest that ActA plays a pivotal role in B-ALL cell chemoprotection and that its targeting may be a promising strategy to be used in combination with standard chemotherapy in r/r ALL patients.

Mesenchymal stromal cells (MSCs) are structural components of the bone marrow (BM) niche, where they functionally interact with hematopoietic stem cells and more differentiated progenitors, contributing to hematopoiesis regulation. A growing body of evidence is nowadays pointing to a further crucial contribution of MSCs to malignant hematopoiesis. In the context of B-cell acute lymphoblastic leukemia (B-ALL), MSCs can play a pivotal role in the definition of a leukemia-supportive microenvironment, impacting on disease pathogenesis at different steps including onset, maintenance and progression. B-ALL cells hijack the BM microenvironment, including MSCs residing in the BM niche, which in turn shelter leukemic cells and protect them from chemotherapeutic agents through different mechanisms. Evidence is now arising that altered MSCs can become precious allies to leukemic cells by providing nutrients, cytokines, pro-survivals signals and exchanging organelles, as hereafter reviewed. The study of the mechanisms exploited by MSCs to nurture and protect B-ALL blasts can be instrumental in finding new druggable candidates to target the leukemic BM microenvironment. Some of these microenvironment-targeting strategies are already in preclinical or clinical experimentation, and if coupled with leukemia-directed therapies, could represent a valuable option to improve the prognosis of relapsed/refractory patients, whose management represents an unmet medical need.

Introduction: Acute lymphoblastic leukemia (ALL) is the most common form of childhood malignancy, accounting for 25% of all childhood cancers. Although great strides have been made in the treatment of childhood leukemia, close to 20% of patients will have resistant disease eventually leading to death. In approximately 40% of relapses, the central nervous system (CNS) is involved, alone or in combination with other sites, thus representing a major clinical concern. We recently demonstrated that ActivinA, a pleiotropic cytokine that belongs to the TGF-β superfamily, plays a crucial role within the leukemic niche. In addition, we showed by using a xenograft mouse model of human B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL), the ability of ActivinA to enhance both bone marrow (BM) engraftment and metastatic potential into extra-medullary sites of leukemic cells. Interestingly, an increased leukemic burden was observed in the CNS of mice receiving ActivinA-treated cells (Portale et al., 2019). In this study, we attempted to elucidate how leukemic NALM-6 cell line pre-treated or not with ActivinA modifies the CNS microenvironment by using an in vitro model of organotypic cortical brain slices. Methods:200-µm-thick brain slices from C57BL/6J or CX3CR1+/GFP (that express GFP in microglial cells) postnatal (P1-3) mice were obtained using a vibratome and placed onto a cell culture insert. After 7 days of recovery from cut, slices were microinjected with NALM-6 cells expressing iRFP670 pre-treated or not with ActivinA (50ng/mL for 24h). Proliferation of NALM-6 was quantified as the area of iRFP670 fluorescent signal in the brain slices up to 72h after injection. Brain slice cell death was measured by propidium iodide incorporation assay. Microglia activation was evaluated in CX3CR1+/GFP slices by longitudinally quantifying GFP signal up to 7 days post injection. In addition, microglia morphology was evaluated by shape descriptor parameters at 3 and 7days post-injection. Lastly, qRT-PCR assay to evaluate the modulation of microglial pro-and anti-inflammatory genes, was conducted. Results: We observed an exponential increase of iRFP670+ NALM-6 cells in the brain tissue up to 72h post-injection. Of note, pre-treatment with ActivinA significantly increase NALM-6 proliferation at 48 hours (p<0,05; n=7) and 72 hours (p<0,05; n=7) compared to not treated cells. ActivinA pre-treatment of NALM-6 cells was also associated with an higher mortality of brain slices compared to not stimulated NALM-6 cells at 24 hours (p<0,001; n=6 independent experiments) and 72 hours (p<0,05; n=6 independent experiments) after microinjection. By using CX3CR1+/GFP slices, we observed an increased microglia activation in ActivinA-pretreated NALM-6 microinjected slices compared to not treated at 5 (p<0,05; n=7) and 7 days (p<0.01; n=7) post-injection. Microglia morphological analysis showed that at 3 days post-injection, ActivinA pre-treated NALM-6 cells induced a slight increase of round-shaped microglia compared to CTRL (p<0,05; n=3) and to untreated NALM-6 injected slices (p<0,05; n=3), indicative of microglia reactivity to leukemic cells. However, at 7 days post-injection microglia from ActivinA pre-treated NALM-6 injected slices, showed an increase of cell area and perimeters (p<0,05; n=3), a reduced circularity and solidity (p<0,01; n=3) and a slight increase of aspect ratio (p<0,05; n=3) compared to CTRL slices, indicative of a reduced phagocytic activity of microglial cells. These modifications were associated with significant changes in gene expression, characterized by a down-regulation of microglial pro-inflammatory genes (iNOS, PAI-1; p<0.001; n=7) and an up-regulation of the anti-inflammatory ones (Arginase-1, CD206; p<0.01; n=7). Conclusions: We established an in vitro model to study leukemia cell interactions with brain tissue. Our data indicate that ActivinA pre-treatment promote higher NALM-6 proliferation in organotypic cortical brain slices compared to unstimulated leukemic cells. ActivinA pre-treated NALM-6 induce microglial modifications, characterized by a decrease reactivity and an induction of anti-inflammatory phenotype, favoring leukemic cell survival and proliferation. Our model can aid in answering important questions about brain changes after leukemic cell infiltration and could speed up the process of drug screening for leukemic patients.

Introduction: Shwachman-Diamond Syndrome (SDS) is a rare autosomal recessive bone marrow (BM) failure disorder. SDS BM biopsies showed tortuous vessels with collapsed lumens, which might promote progression of malignant clones. Mesenchymal stromal cells (MSCs) are crucial elements in the BM niche homeostasis. Our group previously demonstrated that the in vivo ability of semi-cartilaginous pellets derived from SDS-MSCs to generate complete heterotopic ossicles was severely impaired in comparison with healthy donors (HDs). In addition, after specific angiogenic stimuli, SDS-MSCs showed an in vitro defective capability to form a well-defined tubular network. The aim of this study was to deeply investigate the cellular and molecular mechanisms underlying the SDS-MSCs impaired angiogenic potential. Methods: The angiogenic capability of BM derived SDS- and HD-MSCs was evaluated by performing in vitro Matrigel angiogenesis assay, by analysing cell viability and the expression of key angiogenic molecules before and after the angiogenic assay (RT-PCRs and/or ELISA). Concerning energy metabolism, oxidative phosphorylation (OxPhos) has been assayed by oximetry and bioluminescent ATP synthesis assay, and ATP/AMP ratio, malondialdehyde (MDA) level, and complex IV and lactate dehydrogenase (LDH) activity have been evaluated by spectrophotometric analyses. Results: We confirmed the impaired angiogenic potential of SDS-MSCs, previously observed in a restricted cohort of patients (n=6), in 13 SDS- compared to 14 HD-MSCs. In detail, after 3h of angiogenic stimuli, SDS-MSCs showed a defective capability to recreate a defined capillary-like network compared to HD-MSCs in a Matrigel-based assay. Accordingly, ImageJ Angiogenesis Analyzer showed that several angiogenic elements, including branches and meshes, were significantly reduced in SDS-MSCs. Furthermore, we demonstrated that the angiogenic defect of SDS-MSCs was correlated neither to a lower cellular viability than HD-MSCs, nor to a different expression of the main angiogenic molecules. Different reports showed a close relationship between aberrant angiogenesis and oxidative stress. Based on previous studies that demonstrated a defective energy metabolism and an increased oxidative stress in SDS patient-derived lymphoblastoid cells, we performed metabolic analyses of 6 SDS- and 6 HD-MSCs from the cohort used for angiogenic assay. In detail, we assayed OxPhos metabolism, evaluating oxygen consumption and ATP production. Concerning the I, III, and IV complexes pathway, we demonstrated that SDS-MSCs consumed 54% less oxygen (p=0.009) and they produced 60% less ATP compared to HD-MSCs (p=0.004). Accordingly, the analysis of the II, III, and IV complexes pathway demonstrated that the oxygen consumption was reduced by 61% (p=0.002) and the ATP synthesis was 67% lower than HD-MSCs (p=0.002). In addition, the P/O ratio, an index of OxPhos efficiency, was significantly reduced in SDS-MSCs in both electron transport chain pathways (p=0.004 for both). Moreover, OxPhos defect of SDS-MSCs was associated to the decreased enzymatic activity of complex IV compared to HD-MSCs (mean=9.5mU of oxidated cytochrome c/mg protein, range=7.8-11.5mU/mg vs mean=25.6mU/mg, range=21.6-31.9mU/mg; p=0.002). Consequently, SDS-MSCs showed a low intracellular ATP/AMP ratio (mean=1.1, range=0.8-1.6 vs mean=3.6, range=3.1-4.0, in HDs; p=0.002). This decrease was accompanied by an increased LDH activity (mean=0.4mU/mg protein, range=0.4-0.6mU/mg vs mean=0.3mU/mg, range=0.27-0.32mU/mg, in HDs; p=0.0022) in SDS-MSCs, showing an attempt to compensate the mitochondrial defect by the anaerobic glycolysis enhancement. Finally, we demonstrated that the level of lipid peroxidation of SDS-MSCs was significantly increased compared to HDs (mean=11.8µM of MDA/mg protein, range=10.4-15.2µM/mg vs mean=6.5µM/mg, range=5.0-7.7µM/mg; p=0.0022), suggesting an oxidative stress production increment. Conclusions: We demonstrated that the angiogenic defect in SDS-MSCs coexists with metabolic alterations. In addition, we demonstrated for the first time to our knowledge, that SDS-MSCs showed a defective oxidative metabolism, characterized by a decreased energy production and a high level of oxidative damage. These results underline the key role of MSCs in SDS BM niche and provide new insights into the pathogenesis of this rare disease.

B-cell acute lymphoblastic leukaemia (B-ALL) reprograms the surrounding bone marrow (BM) stroma to create a leukaemia-supportive niche. To elucidate the contribution of immune cells to the leukaemic microenvironment, we investigated the involvement of monocyte/macrophage compartments, as well as several recruitment pathways in B-ALL development. Immunohistochemistry analyses showed that CD68-expressing macrophages were increased in leukaemic BM biopsies, compared to controls and predominantly expressed the M2-like markers CD163 and CD206. Furthermore, the "non-classical" CD14+ CD16++ monocyte subset, expressing high CX3CR1 levels, was significantly increased in B-ALL patients' peripheral blood. CX3CL1 was shown to be significantly upregulated in leukaemic BM plasma, thus providing an altered migratory pathway possibly guiding NC monocyte recruitment into the BM. Additionally, the monocyte/macrophage chemoattractant chemokine ligand 2 (CCL2) strongly increased in leukaemic BM plasma, possibly because of the interaction of leukaemic cells with mesenchymal stromal cells and vascular cells and due to a stimulatory effect of leukaemia-related inflammatory mediators. C5a, a macrophage chemoattractant and M2-polarizing factor, further appeared to be upregulated in the leukaemic BM, possibly as an effect of PTX3 decrease, that could unleash complement cascade activation. Overall, deregulated monocyte/macrophage compartments are part of the extensive BM microenvironment remodelling at B-ALL diagnosis and could represent valuable targets for novel treatments to be coupled with classical chemotherapy.

Mechanisms underlying the resistance of acute lymphoblastic leukemia (ALL) blasts to L-asparaginase are still incompletely known. Here we demonstrate that human primary bone marrow mesenchymal stromal cells (MSCs) successfully adapt to L-asparaginase and markedly protect leukemic blasts from the enzyme-dependent cytotoxicity through an amino acid tradeoff. ALL blasts synthesize and secrete glutamine, thus increasing extracellular glutamine availability for stromal cells. In turn, MSCs use glutamine, either synthesized through glutamine synthetase (GS) or imported, to produce asparagine, which is then extruded to sustain asparagine-auxotroph leukemic cells. GS inhibition prevents mesenchymal cells adaptation to t-asparaginase, lowers glutamine secretion by ALL blasts, and markedly hinders the protection exerted by MSCs on leukemic cells. The pro-survival amino acid exchange is hindered by the inhibition or silencing of the asparagine efflux transporter SNAT5, which is induced in mesenchymal cells by ALL blasts. Consistently, primary MSCs from ALL patients express higher levels of SNAT5 (P < .05), secrete more asparagine (P < .05), and protect leukemic blasts (P < .05) better than MSCs isolated from healthy donors. In conclusion, ALL blasts arrange a pro-leukemic amino acid trade-off with bone marrow mesenchymal cells, which depends on GS and SNAT5 and promotes leukemic cell survival during L-asparaginase treatment.

Genetic lesions predisposing to pediatric B-cell acute lymphoblastic leukemia (B-ALL) arise in utero, generating a clinically silent pre-leukemic phase. We here reviewed the role of the surrounding bone marrow (BM) microenvironment in the persistence and transformation of pre-leukemic clones into fully leukemic cells. In this context, inflammation has been highlighted as a crucial microenvironmental stimulus able to promote genetic instability, leading to the disease manifestation. Moreover, we focused on the cross-talk between the bulk of leukemic cells with the surrounding microenvironment, which creates a "corrupted" BM malignant niche, unfavorable for healthy hematopoietic precursors. In detail, several cell subsets, including stromal, endothelial cells, osteoblasts and immune cells, composing the peculiar leukemic niche, can actively interact with B-ALL blasts. Through deregulated molecular pathways they are able to influence leukemia development, survival, chemoresistance, migratory and invasive properties. The concept that the pre-leukemic and leukemic cell survival and evolution are strictly dependent both on genetic lesions and on the external signals coming from the microenvironment paves the way to a new idea of dual targeting therapeutic strategy.

In cultured human fibroblasts, SNAT transporters (System A) account for the accumulation of non-essential neutral amino acids, are adaptively up-regulated upon amino acid deprivation and play a major role in cell volume recovery upon hypertonic stress. No information is instead available on the expression and activity of SNAT transporters in human bone marrow mesenchymal stromal cells (MSC), although they are increasingly investigated for their staminal and immunomodulatory properties and used for several therapeutic applications. The uptake of glutamine and proline, two substrates of SNAT1 and SNAT2 transporters, was measured in primary human MSC and an MSC line. The amino acid analogue MeAIB, a specific substrate of these carriers, has been used to selectively inhibit SNAT-dependent transport of glutamine and, through its sodium-dependent transport, as an indicator of SNAT1/2 activity. SNAT1/2 expression and localization were assessed with RT-PCR and confocal microscopy, respectively. Cell volume was assessed from urea distribution space. In all these experiments, primary human fibroblasts were used as the positive control for SNAT expression and activity. Compared with fibroblasts, MSC have a lower SNAT1 expression and hardly detectable membrane localization of both SNAT1 and SNAT2. Moreover, they exhibit no sodium-dependent MeAIB uptake or MeAIB-inhibitable glutamine transport, and exhibit a lower ability to accumulate glutamine and proline than fibroblasts. MSC exhibited an only marginal increase in MeAIB transport upon amino acid starvation and did not recover cell volume after hypertonic stress. In conclusion, the activity of SNAT transporters is low in human MSC. MSC adaptation to amino acid shortage is expected to rely on intracellular synthesis, given the absence of an effective up-regulation of the SNAT transporters.

Mesenchymal stromal cells (MSCs) represent an essential component of the bone marrow (BM) niche and display disease-specific alterations in several myeloid malignancies. The aim of this work was to study possible MSC abnormalities in Philadelphia-negative myeloproliferative neoplasms (MPNs) in relationship to the degree of BM fibrosis. MSCs were isolated from BM of 6 healthy donors (HD) and of 23 MPN patients, classified in 3 groups according to the diagnosis and the grade of BM fibrosis: polycythemia vera and essential thrombocythemia (PV/ET), low fibrosis myelofibrosis (LF-MF), and high fibrosis MF (HF-MF). MSC cultures were established from 21 of 23 MPN patients. MPN-derived MSCs did not exhibit any functional impairment in their adipogenic/osteogenic/chondrogenic differentiation potential and displayed a phenotype similar to HD-derived MSCs but with a decreased expression of CD146. All MPN-MSC lines were negative for the patient-specific hematopoietic clone mutations (JAK2, MPL, CALR). MSCs derived from HF-MF patients displayed a reduced clonogenic potential and a lower growth kinetic compared to MSCs from HD, LF-MF, and PV/ET patients. mRNA levels of hematopoiesis regulatory molecules were unaffected in MSCs from HF-MF compared to HD. Finally, in vitro ActivinA secretion by MSCs was increased in HF-MF compared to LF-MF patients, in association with a lower hemoglobin value. Increased ActivinA immunolabeling on stromal cells and erythroid precursors was also observed in HF-MF BM biopsies. In conclusion, higher grade of BM fibrosis is associated with functional impairment of MSCs and the increased secretion of ActivinA may represent a suitable target for anemia treatment in MF patients.

The critical role of neuroinflammation in favoring and accelerating the pathogenic process in Alzheimer's disease (AD) increased the need to target the cerebral innate immune cells as a potential therapeutic strategy to slow down the disease progression. In this scenario, mesenchymal stem cells (MSCs) have risen considerable interest thanks to their immunomodulatory properties, which have been largely ascribed to the release of extracellular vesicles (EVs), namely exosomes and microvesicles. Indeed, the beneficial effects of MSC-EVs in regulating the inflammatory response have been reported in different AD mouse models, upon chronic intravenous or intracerebroventricular administration. In this study, we use the triple-transgenic 3xTg mice showing for the first time that the intranasal route of administration of EVs, derived from cytokine-preconditioned MSCs, was able to induce immunomodulatory and neuroprotective effects in AD. MSC-EVs reached the brain, where they dampened the activation of microglia cells and increased dendritic spine density. MSC-EVs polarized in vitro murine primary microglia toward an anti-inflammatory phenotype suggesting that the neuroprotective effects observed in transgenic mice could result from a positive modulation of the inflammatory status. The possibility to administer MSC-EVs through a noninvasive route and the demonstration of their anti-inflammatory efficacy might accelerate the chance of a translational exploitation of MSC-EVs in AD.