Asparagine transport through SLC1A5/ASCT2 and SLC38A5/SNAT5 is essential for BCP-ALL cell survival and a potential therapeutic target.

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.

Ecology- and genome-based identification of the Bifidobacterium adolescentis prototype of the healthy human gut microbiota.

Bifidobacteria are among the first microbial colonizers of the human gut, being frequently associated with human health-promoting activities. In the current study, an in silico methodology based on an ecological and phylogenomic-driven approach allowed the selection of a Bifidobacterium adolescentis prototype strain, i.e., B. adolescentis PRL2023, which best represents the overall genetic content and functional features of the B. adolescentis taxon. Such features were confirmed by in vitro experiments aimed at evaluating the ability of this strain to survive in the gastrointestinal tract of the host and its ability to interact with human intestinal cells and other microbial gut commensals. In this context, co-cultivation of B. adolescentis PRL2023 and several gut commensals revealed various microbe-microbe interactions and indicated co-metabolism of particular plant-derived glycans, such as xylan.IMPORTANCEThe use of appropriate bacterial strains in experimental research becomes imperative in order to investigate bacterial behavior while mimicking the natural environment. In the current study, through in silico and in vitro methodologies, we were able to identify the most representative strain of the Bifidobacterium adolescentis species. The ability of this strain, B. adolescentis PRL2023, to cope with the environmental challenges imposed by the gastrointestinal tract, together with its ability to switch its carbohydrate metabolism to compete with other gut microorganisms, makes it an ideal choice as a B. adolescentis prototype and a member of the healthy microbiota of adults. This strain possesses a genetic blueprint appropriate for its exploitation as a candidate for next-generation probiotics.

Amorphous silica nanoparticles and the human gut microbiota: a relationship with multiple implications.

Amorphous silica nanoparticles (ASNP) are among the nanomaterials that are produced in large quantities. ASNP have been present for a long time in several fast-moving consumer products, several of which imply exposure of the gastrointestinal tract, such as toothpastes, food additives, drug excipients, and carriers. Consolidated use and experimental evidence have consistently pointed to the very low acute toxicity and limited absorption of ASNP. However, slow absorption implies prolonged exposure of the intestinal epithelium to ASNP, with documented effects on intestinal permeability and immune gut homeostasis. These effects could explain the hepatic toxicity observed after oral administration of ASNP in animals. More recently, the role of microbiota in these and other ASNP effects has attracted increasing interest in parallel with the recognition of the role of microbiota in a variety of conditions. Although evidence for nanomaterial effects on microbiota is particularly abundant for materials endowed with bactericidal activities, a growing body of recent experimental data indicates that ASNPs also modify microbiota. The implications of these effects are recounted in this contribution, along with a discussion of the more important open issues and recommendations for future research.

The SLC38A5/SNAT5 amino acid transporter: from pathophysiology to pro-cancer roles in the tumor microenvironment.

SLC38A5/SNAT5 is a system N transporter that can mediate net inward or outward transmembrane fluxes of neutral amino acids coupled with Na+ (symport) and H+ (antiport). Its preferential substrates are not only amino acids with side chains containing amide (glutamine and asparagine) or imidazole (histidine) groups, but also serine, glycine, and alanine are transported by the carrier. Expressed in the pancreas, intestinal tract, brain, liver, bone marrow, and placenta, it is regulated at mRNA and protein levels by mTORC1 and WNT/ß-catenin pathways, and it is sensitive to pH, nutritional stress, inflammation, and hypoxia. SNAT5 expression has been found to be altered in pathological conditions such as chronic inflammatory diseases, gestational complications, chronic metabolic acidosis, and malnutrition. Growing experimental evidence shows that SNAT5 is overexpressed in several types of cancer cells. Moreover, recently published results indicate that SNAT5 expression in stromal cells can support the metabolic exchanges occurring in the tumor microenvironment of asparagine-auxotroph tumors. We review the functional role of the SNAT5 transporter in pathophysiology and propose that, due to its peculiar operational and regulatory features, SNAT5 may play important pro-cancer roles when expressed either in neoplastic or in stromal cells of glutamine-auxotroph tumors.NEW & NOTEWORTHY The transporter SLC38A5/SNAT5 provides net influx or efflux of glutamine, asparagine, and serine. These amino acids are of particular metabolic relevance in several conditions. Changes in transporter expression or activity have been described in selected types of human cancers, where SNAT5 can mediate amino acid exchanges between tumor and stromal cells, thus providing a potential therapeutic target. This is the first review that recapitulates the characteristics and roles of the transporter in physiology and pathology.

Nanoemulsions Embedded in Alginate Beads as Bioadhesive Nanocomposites for Intestinal Delivery of the Anti-Inflammatory Drug Tofacitinib.

Oral administration of nanoparticles (NPs) is a promising strategy to overcome solubility and stability issues of many active compounds. However, this route faces major obstacles related to the hostile gastrointestinal (GI) environment, which impairs the efficacy of orally administered nanomedicines. Here, we propose nanocomposites as a promising approach to increase the retention time of NPs in the intestinal tract by using bio- and mucoadhesive matrixes able to protect the cargo until it reaches the targeted area. A microfluidic-based approach has been applied for the production of tailored nanoemulsions (NEs) of about 110 nm, used for the encapsulation of small hydrophobic drugs such as the anti-inflammatory JAK-inhibitor tofacitinib. These NEs proved to be efficiently internalized into a mucus-secreting human intestinal monolayer of Caco-2/HT29-MTX cells and to deliver tofacitinib to subepithelial human THP-1 macrophage-like cells, reducing their inflammatory response. NEs were then successfully encapsulated into alginate hydrogel microbeads of around 300 µm, which were characterized by rheological experiments and dried to create a long-term stable system for pharmaceutical applications. Finally, ex vivo experiments on excised segments of rats' intestine proved the bioadhesive ability of NEs embedded in alginate hydrogels compared to free NEs, showing the advantage that this hybrid system can offer for the treatment of intestinal pathologies.

Designation of optimal reference strains representing the infant gut bifidobacterial species through a comprehensive multi-omics approach.

The genomic era has resulted in the generation of a massive amount of genetic data concerning the genomic diversity of bacterial taxa. As a result, the microbiological community is increasingly looking for ways to define reference bacterial strains to perform experiments that are representative of the entire bacterial species. Despite this, there is currently no established approach allowing a reliable identification of reference strains based on a comprehensive genomic, ecological, and functional context. In the current study, we developed a comprehensive multi-omics approach that will allow the identification of the optimal reference strains using the Bifidobacterium genus as test case. Strain tracking analysis based on 1664 shotgun metagenomics datasets of healthy infant faecal samples were employed to identify bifidobacterial strains suitable for in silico and in vitro analyses. Subsequently, an ad hoc bioinformatic tool was developed to screen local strain collections for the most suitable species-representative strain alternative. The here presented approach was validated using in vitro trials followed by metagenomics and metatranscriptomics analyses. Altogether, these results demonstrated the validity of the proposed model for reference strain selection, thus allowing improved in silico and in vitro investigations both in terms of cross-laboratory reproducibility and relevance of research findings.

Functional Consequences of Low Activity of Transport System A for Neutral Amino Acids in Human Bone Marrow Mesenchymal Stem Cells.

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.

Oligodendroglioma Cells Lack Glutamine Synthetase and Are Auxotrophic for Glutamine, but Do not Depend on Glutamine Anaplerosis for Growth.

In cells derived from several types of cancer, a transcriptional program drives high consumption of glutamine (Gln), which is used for anaplerosis, leading to a metabolic addiction for the amino acid. Low or absent expression of Glutamine Synthetase (GS), the only enzyme that catalyzes de novo Gln synthesis, has been considered a marker of Gln-addicted cancers. In this study, two human cell lines derived from brain tumors with oligodendroglioma features, HOG and Hs683, have been shown to be GS-negative. Viability of both lines depends from extracellular Gln with EC50 of 0.175 ± 0.056 mM (Hs683) and 0.086 ± 0.043 mM (HOG), thus suggesting that small amounts of extracellular Gln are sufficient for OD cell growth. Gln starvation does not significantly affect the cell content of anaplerotic substrates, which, consistently, are not able to rescue cell growth, but causes hindrance of the Wnt/ß-catenin pathway and protein synthesis attenuation, which is mitigated by transient GS expression. Gln transport inhibitors cause partial depletion of intracellular Gln and cell growth inhibition, but do not lower cell viability. Therefore, GS-negative human oligodendroglioma cells are Gln-auxotrophic but do not use the amino acid for anaplerosis and, hence, are not Gln addicted, exhibiting only limited Gln requirements for survival and growth.

GPNA inhibits the sodium-independent transport system L for neutral amino acids.

L-γ-Glutamyl-p-nitroanilide (GPNA) is widely used to inhibit the glutamine transporter ASCT2, although it is known that it also inhibits other sodium-dependent amino acid transporters. In a panel of human cancer cell lines, which express the system L transporters LAT1 and LAT2, GPNA inhibits the sodium-independent influx of leucine and glutamine. The kinetics of the effect suggests that GPNA is a low affinity, competitive inhibitor of system L transporters. In Hs683 human oligodendroglioma cells, the incubation in the presence of GPNA, but not ASCT2 silencing, lowers the cell content of leucine. Under the same conditions the activity of mTORC1 is inhibited. Decreased cell content of branched chain amino acids and mTORC1 inhibition are observed in most of the other cell lines upon incubation with GPNA. It is concluded that GPNA hinders the uptake of essential amino acids through system L transporters and lowers their cell content.

Genomic and ecological approaches to identify the Bifidobacterium breve prototype of the healthy human gut microbiota.

Members of the genus Bifidobacterium are among the first microorganisms colonizing the human gut. Among these species, strains of Bifidobacterium breve are known to be commonly transmitted from mother to her newborn, while this species has also been linked with activities supporting human wellbeing. In the current study, an in silico approach, guided by ecology- and phylogenome-based analyses, was employed to identify a representative strain of B. breve to be exploited as a novel health-promoting candidate. The selected strain, i.e., B. breve PRL2012, was found to well represent the genetic content and functional genomic features of the B. breve taxon. We evaluated the ability of PRL2012 to survive in the gastrointestinal tract and to interact with other human gut commensal microbes. When co-cultivated with various human gut commensals, B. breve PRL2012 revealed an enhancement of its metabolic activity coupled with the activation of cellular defense mechanisms to apparently improve its survivability in a simulated ecosystem resembling the human microbiome.

Colon-available mango (poly)phenols exhibit mitigating effects on the intestinal barrier function in human intestinal cell monolayers under inflammatory conditions.

This study investigated the impact of in vivo available colon-mango (poly)phenols on stress-induced impairment of intestinal barrier function. Caco-2/HT29-MTX cells were incubated with six extracts of ileal fluid collected pre- and 4-8 h post-mango consumption before being subjected to inflammatory stress. (Poly)phenols in ileal fluids were analysed by UHPLC-HR-MS. Epithelial barrier function was monitored by measurement of trans-epithelial electrical resistance (TEER) and the production of selected inflammatory markers (interleukin-8 (IL-8) and nitric oxide (NO)) and the major mucin of the mucosal layer (MUC2). Post-mango intake ileal fluids contained principally benzoic acids, hydroxybenzenes and galloyl derivatives. There was a high interindividual variability in the levels of these compounds, which was reflected by the degree of variability in the protective effects of individual ileal extracts on inflammatory changes in the treated cell cultures. The 24 h treatment with non-cytotoxic doses of extracts of 4-8 h post-mango intake ileal fluid significantly reduced the TEER decrease in monolayers treated with the inflammatory cytomix. This effect was not associated with changes in IL-8 expression and secretion or claudine-7 expression. The mango derived-ileal fluid extract (IFE) also mitigated cytomix-dependent nitrite secretion, as a proxy of NO production, and the MUC2 reduction observed upon the inflammatory challenge. These insights shed light on the potential protective effect of mango (poly)phenols on the intestinal barrier exposed to inflammatory conditions.

GH136-encoding gene (perB) is involved in gut colonization and persistence by Bifidobacterium bifidum PRL2010.

Bifidobacteria are commensal microorganisms that typically inhabit the mammalian gut, including that of humans. As they may be vertically transmitted, they commonly colonize the human intestine from the very first day following birth and may persist until adulthood and old age, although generally at a reduced relative abundance and prevalence compared to infancy. The ability of bifidobacteria to persist in the human intestinal environment has been attributed to genes involved in adhesion to epithelial cells and the encoding of complex carbohydrate-degrading enzymes. Recently, a putative mucin-degrading glycosyl hydrolase belonging to the GH136 family and encoded by the perB gene has been implicated in gut persistence of certain bifidobacterial strains. In the current study, to better characterize the function of this gene, a comparative genomic analysis was performed, revealing the presence of perB homologues in just eight bifidobacterial species known to colonize the human gut, including Bifidobacterium bifidum and Bifidobacterium longum subsp. longum strains, or in non-human primates. Mucin-mediated growth and adhesion to human intestinal cells, in addition to a rodent model colonization assay, were performed using B. bifidum PRL2010 as a perB prototype and its isogenic perB-insertion mutant. These results demonstrate that perB inactivation reduces the ability of B. bifidum PRL2010 to grow on and adhere to mucin, as well as to persist in the rodent gut niche. These results corroborate the notion that the perB gene is one of the genetic determinants involved in the persistence of B. bifidum PRL2010 in the human gut.

Epidemiology, Patients' Journey and Healthcare Costs in Early-Stage Non-Small-Cell Lung Carcinoma: A Real-World Evidence Analysis in Italy.

This real-world analysis aims to estimate the epidemiology and economic burden related to early-stage non-small-cell lung carcinoma (eNSCLC) in the clinical practice Italian setting. An observational analysis was performed using administrative databases linked to pathological anatomy data, covering around 2.5 mln health-assisted individuals. From 2015 to mid-2021, eNSCLC patients staged II-IIIA treated with chemotherapy after surgery were included. Patients were stratified into those presenting loco-regional or metastatic recurrence during follow-up and annualized healthcare direct costs covered by the Italian National Health System (INHS) were estimated. In 2019-2020, the prevalence of eNSCLC was 104.3-117.1/million health-assisted subjects, and the annual incidence was 38.6-30.3/million. Data projected to the Italian population estimated 6206 (2019) and 6967 (2020) prevalent and 2297 (2019) and 1803 (2020) incident cases. Overall, 458 eNSCLC patients were included. Of them, 52.4% of patients had a recurrence (5% loco-regional-recurrence, 47.4% metastatic-recurrence). Healthcare total direct costs/patient averaged EUR 23,607, in particular, in the first year after recurrence, costs averaged EUR 22,493 and EUR 29,337 in loco-regional and metastatic-recurrence patients, respectively. This analysis showed that about one-half of eNSCLC patients stage II-IIIA experience a recurrence, and in recurrence patients, total direct costs were almost two-fold those of no-recurrence patients. These data highlighted an unmet clinical need, as the therapeutic optimization of patients at early stages.

Identification of a prototype human gut Bifidobacterium longum subsp. longum strain based on comparative and functional genomic approaches.

Bifidobacteria are extensively exploited for the formulation of probiotic food supplements due to their claimed ability to exert health-beneficial effects upon their host. However, most commercialized probiotics are tested and selected for their safety features rather than for their effective abilities to interact with the host and/or other intestinal microbial players. In this study, we applied an ecological and phylogenomic-driven selection to identify novel B. longum subsp. longum strains with a presumed high fitness in the human gut. Such analyses allowed the identification of a prototype microorganism to investigate the genetic traits encompassed by the autochthonous bifidobacterial human gut communities. B. longum subsp. longum PRL2022 was selected due to its close genomic relationship with the calculated model representative of the adult human-gut associated B. longum subsp. longum taxon. The interactomic features of PRL2022 with the human host as well as with key representative intestinal microbial members were assayed using in vitro models, revealing how this bifidobacterial gut strain is able to establish extensive cross-talk with both the host and other microbial residents of the human intestine.

Genetic strategies for sex-biased persistence of gut microbes across human life.

Although compositional variation in the gut microbiome during human development has been extensively investigated, strain-resolved dynamic changes remain to be fully uncovered. In the current study, shotgun metagenomic sequencing data of 12,415 fecal microbiomes from healthy individuals are employed for strain-level tracking of gut microbiota members to elucidate its evolving biodiversity across the human life span. This detailed longitudinal meta-analysis reveals host sex-related persistence of strains belonging to common, maternally-inherited species, such as Bifidobacterium bifidum and Bifidobacterium longum subsp. longum. Comparative genome analyses, coupled with experiments including intimate interaction between microbes and human intestinal cells, show that specific bacterial glycosyl hydrolases related to host-glycan metabolism may contribute to more efficient colonization in females compared to males. These findings point to an intriguing ancient sex-specific host-microbe coevolution driving the selective persistence in women of key microbial taxa that may be vertically passed on to the next generation.

Disentangling the interactions between nasopharyngeal and gut microbiome and their involvement in the modulation of COVID-19 infection.

The human organism is inhabited by trillions of microorganisms, known as microbiota, which are considered to exploit a pivotal role in the regulation of host health and immunity. Recent investigations have suggested a relationship between the composition of the human microbiota and COVID-19 infection, highlighting a possible role of bacterial communities in the modulation of the disease severity. In this study, we performed a shotgun metagenomics analysis to explore and compare the nasopharyngeal microbiota of 38 hospitalized Italian patients with and without COVID-19 infection during the third and fourth pandemic waves. In detail, the metagenomic analysis combined with specific correlation analyses suggested a positive association of several microbial species, such as S. parasanguinis and P. melaninogenica, with the severity of COVID-19 infection. Furthermore, the comparison of the microbiota composition between the nasopharyngeal and their respective fecal samples highlighted an association between these different compartments represented by a sharing of several bacterial species. Additionally, lipidomic and deep-shotgun functional analyses of the fecal samples suggested a metabolic impact of the microbiome on the host's immune response, indicating the presence of key metabolic compounds in COVID-19 patients, such as lipid oxidation end products, potentially related to the inflammatory state. Conversely, the patients without COVID-19 displayed enzymatic patterns associated with the biosynthesis and degradation of specific compounds like lysine (synthesis) and phenylalanine (degradation) that could positively impact disease severity and contribute to modulating COVID-19 infection. IMPORTANCE The human microbiota is reported to play a major role in the regulation of host health and immunity, suggesting a possible impact on the severity of COVID-19 disease. This preliminary study investigated the possible correlation between nasopharyngeal microbiota and COVID-19 infection. In detail, the analysis of the nasopharyngeal microbiota of hospitalized Italian patients with and without COVID-19 infection suggested a positive association of several microbial species with the severity of the disease and highlighted a sharing of several bacteria species with the respective fecal samples. Moreover, the metabolic analyses suggested a possible impact of the microbiome on the host's immune response and the disease severity.

The TLR4/NFκB-Dependent Inflammatory Response Activated by LPS Is Inhibited in Human Macrophages Pre-Exposed to Amorphous Silica Nanoparticles.

Amorphous silica nanoparticles (ASNP) are present in a variety of products and their biological effects are actively investigated. Although several studies have documented pro-inflammatory effects of ASNP, the possibility that they also modify the response of innate immunity cells to natural activators has not been thoroughly investigated. Here, we study the effects of pyrogenic ASNP on the LPS-dependent activation of human macrophages differentiated from peripheral blood monocytes. In macrophages, 24 h of pre-exposure to non-cytotoxic doses of ASNP markedly inhibited the LPS-dependent induction of pro-inflammatory (TNFα, IL-6) and anti-inflammatory cytokines (IL-10). The inhibitory effect was associated with the suppression of NFκB activation and the increased intracellular sequestration of the TLR4 receptor. The late induction of glutamine synthetase (GS) by LPS was also prevented by pre-exposure to ASNP, while GS silencing did not interfere with cytokine secretion. It is concluded that (i) macrophages exposed to ASNP are less sensitive to LPS-dependent activation and (ii) GS induction by LPS is likely secondary to the stimulation of cytokine secretion. The observed interference with LPS effects may point to a dampening of the acute inflammatory response after exposure to ASNP in humans.

Mesenchymal stromal cells cultured in physiological conditions sustain citrate secretion with glutamate anaplerosis.

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.

The Role of Amino Acids in the Crosstalk Between Mesenchymal Stromal Cells and Neoplastic Cells in the Hematopoietic Niche.

Within the bone marrow hematopoietic cells are in close connection with mesenchymal stromal cells (MSCs), which influence the behavior and differentiation of normal or malignant lymphoid and myeloid cells. Altered cell metabolism is a hallmark of cancer, and changes in nutrient pools and fluxes are important components of the bidirectional communication between MSCs and hematological cancer cells. Among nutrients, amino acids play a significant role in cancer progression and chemo-resistance. Moreover, selected types of cancer cells are extremely greedy for glutamine, and significantly deplete the extracellular pool of the amino acid. As a consequence, this influences the behavior of MSCs in terms of either cytokine/chemokine secretion or differentiation potential. Additionally, a direct nutritional interaction exists between MSCs and immune cells. In particular, selected subpopulations of lymphocytes are dependent upon selected amino acids, such as arginine and tryptophan, for full differentiation and competence. This review describes and discusses the nutritional interactions existing in the neoplastic bone marrow niche between MSCs and other cell types, with a particular emphasis on cancer cells and immune cells. These relationships are discussed in the perspective of potential novel therapeutic strategies based on the interference on amino acid metabolism or intercellular fluxes.

[18F](2S,4R)-4-Fluoroglutamine as a New Positron Emission Tomography Tracer in Myeloma.

The high glycolytic activity of multiple myeloma (MM) cells is the rationale for use of Positron Emission Tomography (PET) with 18F-fluorodeoxyglucose ([18F]FDG) to detect both bone marrow (BM) and extramedullary disease. However, new tracers are actively searched because [18F]FDG-PET has some limitations and there is a portion of MM patients who are negative. Glutamine (Gln) addiction has been recently described as a typical metabolic feature of MM cells. Yet, the possible exploitation of Gln as a PET tracer in MM has never been assessed so far and is investigated in this study in preclinical models. Firstly, we have synthesized enantiopure (2S,4R)-4-fluoroglutamine (4-FGln) and validated it as a Gln transport analogue in human MM cell lines, comparing its uptake with that of 3H-labelled Gln. We then radiosynthesized [18F]4-FGln, tested its uptake in two different in vivo murine MM models, and checked the effect of Bortezomib, a proteasome inhibitor currently used in the treatment of MM. Both [18F]4-FGln and [18F]FDG clearly identified the spleen as site of MM cell colonization in C57BL/6 mice, challenged with syngeneic Vk12598 cells and assessed by PET. NOD.SCID mice, subcutaneously injected with human MM JJN3 cells, showed high values of both [18F]4-FGln and [18F]FDG uptake. Bortezomib significantly reduced the uptake of both radiopharmaceuticals in comparison with vehicle at post treatment PET. However, a reduction of glutaminolytic, but not of glycolytic, tumor volume was evident in mice showing the highest response to Bortezomib. Our data indicate that [18F](2S,4R)-4-FGln is a new PET tracer in preclinical MM models, yielding a rationale to design studies in MM patients.