Giovanna D'AMico

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.

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.

Background: Since one of the suggested mechanisms of action of VNS on epilepsy is the reduction of central inflammation, we carried out a comprehensive analysis of blood inflammatory markers in children considered for VNS surgery. Materials and methods: Five pediatric patients were studied. An extensive analysis of blood inflammatory markers was performed before surgery (T0) and six weeks after VNS implantation (T1). An epileptological outcome was obtained according to the McHugh score. Results: The variations of IgA, IgE, IgG, CD19, and PTX3 displayed a tendency toward a positive statistical correlation between T0 and T1. According to McHugh score, the patients were divided into Group 1 (i.e., Class I) and Group 2 (i.e., Classes II and III). IL-1β and PTX-3 tended to decrease more in Group 1, while TNF-α decreased in Group 2 (−56.65%) and slightly increased (+3.61%) in Group 1 at T1 without statistical correlation. Conclusions: The variation of IL-1β and PTX-3 seem to be related to a better outcome; thus, they do not reach statistical significance. A larger series of patients is needed to determine whether biochemical changes could relay with the clinical improvement of epilepsy.

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.

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 trade-off. 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 l-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.

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.

Multiple myeloma (MM) cells consume huge amounts of glutamine and, as a consequence, the amino acid concentration is lower-than-normal in the bone marrow (BM) of MM patients. Here we show that MM-dependent glutamine depletion induces glutamine synthetase in stromal cells, as demonstrated in BM biopsies of MM patients, and reproduced in vitro by co-culturing human mesenchymal stromal cells (MSCs) with MM cells. Moreover, glutamine depletion hinders osteoblast differentiation of MSCs, which is also severely blunted by the spent, low-glutamine medium of MM cells, and rescued by glutamine restitution. Glutaminase and the concentrative glutamine transporter SNAT2 are induced during osteoblastogenesis in vivo and in vitro, and both needed for MSCs differentiation, pointing to enhanced the requirement for the amino acid. Osteoblastogenesis also triggers the induction of glutamine-dependent asparagine synthetase (ASNS), and, among non-essential amino acids, asparagine rescues differentiation of glutamine-starved MSCs, by restoring the transcriptional profiles of differentiating MSCs altered by glutamine starvation. Thus, reduced asparagine availability provides a mechanistic link between MM-dependent Gln depletion in BM and impairment of osteoblast differentiation. Inhibition of Gln metabolism in MM cells and supplementation of asparagine to stromal cells may, therefore, constitute novel approaches to prevent osteolytic lesions in MM.