Exosome-Based Liquid Biopsy Approaches in Bone and Soft Tissue Sarcomas: Review of the Literature, Prospectives, and Hopes for Clinical Application.

The knowledge of exosome impact on sarcoma development and progression has been implemented in preclinical studies thanks to technological advances in exosome isolation. Moreover, the clinical relevance of liquid biopsy is well established in early diagnosis, prognosis prediction, tumor burden assessment, therapeutic responsiveness, and recurrence monitoring of tumors. In this review, we aimed to comprehensively summarize the existing literature pointing out the clinical relevance of detecting exosomes in liquid biopsy from sarcoma patients. Presently, the clinical utility of liquid biopsy based on exosomes in patients affected by sarcoma is under debate. The present manuscript collects evidence on the clinical impact of exosome detection in circulation of sarcoma patients. The majority of these data are not conclusive and the relevance of liquid biopsy-based approaches in some types of sarcoma is still insufficient. Nevertheless, the utility of circulating exosomes in precision medicine clearly emerged and further validation in larger and homogeneous cohorts of sarcoma patients is clearly needed, requiring collaborative projects between clinicians and translational researchers for these rare cancers.

Bridging therapeutic opportunities: a survey by the Italian molecular tumor board workgroup of Alliance Against Cancer.

BACKGROUND: Molecular tumor boards (MTBs) match molecular alterations with targeted anticancer drugs upon failure of the available therapeutic options. Special and local needs are most likely to emerge through the comparative analysis of MTB networks, but these are rarely reported. This manuscript summarizes the state-of-art of 16 active Italian MTBs, as it emerges from an online survey curated by Alliance Against Cancer (ACC). MAIN TEXT: Most MTBs (13/16) are exclusively supported through local Institutional grants and meet regularly. All but one adopts a fully virtual or a mixed face-to-face/virtual calling/attendance meeting model. It appears that the ACC MTB initiative is shaping a hub-and-spoke virtual MTB network reminiscent of non-redundant, cost-effective healthcare organization models. Unfortunately, public awareness of MTB opportunities presently remains insufficient. Only one center has a website. Dedicated e-mail addresses are for the exclusive use of the MTB staff. More than half of ACC members consider a miscellanea of most or all solid and hematological malignancies, and more than one-third consider neoplasms arising at any anatomical location. The average number of Staff Members in MTBs is 9. More than 10 staff members simultaneously attend MTB meetings in 13 MTBs. A medical oncologist is invariably present and is in charge of introducing the clinical case either with (45%) or without previous discussion in organ-specific multidisciplinary Boards. All but two MTBs take charge of not only patients with no standard-of-care (SoC) therapy option, but also cases receiving NGS profiling in SoC settings, implying a larger number of yearly cases. All MTBs run targeted NGS panels. Three run whole-exome and/or RNAseq approaches. ESCAT-ESMO and/or Onco-KB levels of evidence are similarly used for diagnostic reporting. Most MTBs (11) provide a written diagnostic report within 15 days. Conclusions are invariably communicated to the patient by the medical oncologist. CONCLUSIONS: MTB networking is crucial not only for molecular diagnosis and therapy assignment, but also for healthcare governance. Survey results show that MTBs review therapeutic opportunities at the crossover between standard-of-care with off-label, the former task being much beyond their scope. Societal and scientific implications of this beyond-the-scope MTB function may be relevant for healthcare in Italy and abroad.

Mitochondria dysfunction in circulating tumor cells.

Circulating tumor cells (CTCs) represent a subset of heterogeneous cells, which, once released from a tumor site, have the potential to give rise to metastasis in secondary sites. Recent research focused on the attempt to detect and characterize these rare cells in the circulation, and advancements in defining their molecular profile have been reported in diverse tumor species, with potential implications for clinical applications. Of note, metabolic alterations, involving mitochondria, have been implicated in the metastatic process, as key determinants in the transition of tumor cells to a mesenchymal or stemness-like phenotype, in drug resistance, and in induction of apoptosis. This review aimed to briefly analyse the most recent knowledge relative to mitochondria dysfunction in CTCs, and to envision implications of altered mitochondria in CTCs for a potential utility in clinics.

UC.183, UC.110, and UC.84 Ultra-Conserved RNAs Are Mutually Exclusive with miR-221 and Are Engaged in the Cell Cycle Circuitry in Breast Cancer Cell Lines.

In the human genome, there are about 600 ultra-conserved regions (UCRs), long DNA sequences extremely conserved in vertebrates. We performed a large-scale study to quantify transcribed UCR (T-UCR) and miRNA levels in over 6000 cancer and normal tissue samples to find possible correlation between these kinds of regulatory molecules. Our analysis evidenced several non-coding RNAs showing negative co-regulation with miRNAs; among them, we focused on miR-221 to investigate any relationship with its pivotal role in the cell cycle. We have chosen breast cancer as model, using two cell lines with different phenotypes to carry out in vitro treatments with siRNAs against T-UCRs. Our results demonstrate that the expression of uc.183, uc.110, and uc.84 T-UCRs is mutually exclusive with miR-221 and is engaged in the regulation of CDKN1B expression. In addition, tests with a set of anticancer drugs, including BYL719, AZD5363, AZD8055, AZD7762, and XL765, revealed the modulation of specific T-UCRs without alteration of miR-221 levels.

Heterogeneous Circulating Tumor Cells in Sarcoma: Implication for Clinical Practice.

Bone and soft tissue sarcomas (STSs) represent a group of heterogeneous rare malignant tumors of mesenchymal origin, with a poor prognosis. Due to their low incidence, only a few studies have been reported addressing circulating tumor cells (CTCs) in sarcoma, despite the well-documented relevance for applications of liquid biopsy in precision medicine. In the present review, the most recent data relative to the detection and isolation of viable and intact CTCs in these tumors will be reviewed, and the heterogeneity in CTCs will be discussed. The relevance of epithelial-mesenchymal plasticity and stemness in defining the phenotypic and functional properties of these rare cells in sarcoma will be highlighted. Of note, the existence of dynamic epithelial-mesenchymal transition (EMT)-related processes in sarcoma tumors has only recently been related to their clinical aggressiveness. Also, the presence of epithelial cell adhesion molecule (EpCAM)-positive CTC in sarcoma has been weakly correlated with poor outcome and disease progression, thus proving the existence of both epithelial and mesenchymal CTC in sarcoma. The advancement in technologies for capturing and enumerating all diverse CTCs phenotype originating from these mesenchymal tumors are presented, and results provide a promising basis for clinical application of CTC detection in sarcoma.

The network of non-coding RNAs and their molecular targets in breast cancer.

BACKGROUND: Non-coding RNAs are now recognized as fundamental components of the cellular processes. Non-coding RNAs are composed of different classes, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Their detailed roles in breast cancer are still under scrutiny. MAIN BODY: We systematically reviewed from recent literature the many functional and physical interactions of non-coding RNAs in breast cancer. We used a data driven approach to establish the network of direct, and indirect, interactions. Human curation was essential to de-convolute and critically assess the experimental approaches in the reviewed articles. To enrol the scientific papers in our article cohort, due to the short time span (shorter than 5 years) we considered the journal impact factor rather than the citation number. The outcome of our work is the formal establishment of different sub-networks composed by non-coding RNAs and coding genes with validated relations in human breast cancer. This review describes in a concise and unbiased fashion the core of our current knowledge on the role of lncRNAs, miRNAs and other non-coding RNAs in breast cancer. CONCLUSIONS: A number of coding/non-coding gene interactions have been investigated in breast cancer during recent years and their full extent is still being established. Here, we have unveiled some of the most important networks embracing those interactions, and described their involvement in cancer development and in its malignant progression.

Heterogeneity in Circulating Tumor Cells: The Relevance of the Stem-Cell Subset.

The release of circulating tumor cells (CTCs) into vasculature is an early event in the metastatic process. The analysis of CTCs in patients has recently received widespread attention because of its clinical implications, particularly for precision medicine. Accumulated evidence documents a large heterogeneity in CTCs across patients. Currently, the most accepted view is that tumor cells with an intermediate phenotype between epithelial and mesenchymal have the highest plasticity. Indeed, the existence of a meta-stable or partial epithelial⁻mesenchymal transition (EMT) cell state, with both epithelial and mesenchymal features, can be easily reconciled with the concept of a highly plastic stem-like state. A close connection between EMT and cancer stem cells (CSC) traits, with enhanced metastatic competence and drug resistance, has also been described. Accordingly, a subset of CTCs consisting of CSC, present a stemness profile, are able to survive chemotherapy, and generate metastases after xenotransplantation in immunodeficient mice. In the present review, we discuss the current evidence connecting CTCs, EMT, and stemness. An improved understanding of the CTC/EMT/CSC connections may uncover novel therapeutic targets, irrespective of the tumor type, since most cancers seem to harbor a pool of CSCs, and disclose important mechanisms underlying tumorigenicity.

Heterogeneous expression of EPCAM in human circulating tumour cells from patient-derived xenografts.

BACKGROUND: We aim to characterize the heterogeneous circulating tumour cells (CTCs) in peripheral blood, independently of physical or immunological purification, by using patient-derived xenografts (PDXs) models. CTC studies from blood generally rely on enrichment or purification. Conversely, we devised a method for the inclusive study of human cells from blood of PDX models, without pre-selection or enrichment. METHODS: A qRT-PCR assay was developed to detect human and cancer-related transcripts from CTCs in PDXs. We quantified the EPCAM and keratins CTC markers, in a PDX cohort of breast cancer. The murine beta actin gene was used for normalization. Spearman's rho coefficients were calculated for correlation. RESULTS: We demonstrated, for the first time, that we can quantify the content of CTCs and the expression of human CTC markers in PDX blood using human-specific qRT-PCR. Our method holds strong potential for the study of CTC heterogeneity and for the identification of novel CTC markers. CONCLUSIONS: The identification and the relative quantification of the diverse spectrum of CTCs in patients, irrespective of EPCAM or other currently used markers, will have a great impact on personalized medicine: unrestricted CTCs characterization will allow the early detection of metastases in cancer patients and the assessment of personalized therapies.

SNPs and Somatic Mutation on Long Non-Coding RNA: New Frontier in the Cancer Studies?

In the last decade, it has been demonstrated that long non-coding RNAs (lncRNAs) are involved in cancer development. The great majority of studies on lncRNAs report alterations, principally on their expression profiles, in several tumor types with respect to the normal tissues of origin. Conversely, since lncRNAs constitute a relatively novel class of RNAs compared to protein-coding transcripts (mRNAs), the landscape of their mutations and variations has not yet been extensively studied. However, in recent years an ever-increasing number of articles have described mutations of lncRNAs. Single-nucleotide polymorphisms (SNPs) that occur within the lncRNA transcripts can affect the structure and function of these RNA molecules, while the presence of a SNP in the promoter region of a lncRNA could alter its expression level. Also, somatic mutations that occur within lncRNAs have been shown to exert important effects in cancer and preliminary data are promising. Overall, the evidence suggests that SNPs and somatic mutation on lncRNAs may play a role in the pathogenesis of cancer, and indicates strong potential for further development of lncRNAs as biomarkers.

The Network of Non-coding RNAs in Cancer Drug Resistance.

Non-coding RNAs (ncRNAs) have been implicated in most cellular functions. The disruption of their function through somatic mutations, genomic imprinting, transcriptional and post-transcriptional regulation, plays an ever-increasing role in cancer development. ncRNAs, including notorious microRNAs, have been thus proposed to function as tumor suppressors or oncogenes, often in a context-dependent fashion. In parallel, ncRNAs with altered expression in cancer have been reported to exert a key role in determining drug sensitivity or restoring drug responsiveness in resistant cells. Acquisition of resistance to anti-cancer drugs is a major hindrance to effective chemotherapy and is one of the most important causes of relapse and mortality in cancer patients. For these reasons, non-coding RNAs have become recent focuses as prognostic agents and modifiers of chemo-sensitivity. This review starts with a brief outline of the role of most studied non-coding RNAs in cancer and then highlights the modulation of cancer drug resistance via known ncRNAs based mechanisms. We identified from literature 388 ncRNA-drugs interactions and analyzed them using an unsupervised approach. Essentially, we performed a network analysis of the non-coding RNAs with direct relations with cancer drugs. Within such a machine-learning framework we detected the most representative ncRNAs-drug associations and groups. We finally discussed the higher integration of the drug-ncRNA clusters with the goal of disentangling effectors from downstream effects and further clarify the involvement of ncRNAs in the cellular mechanisms underlying resistance to cancer treatments.

Screen for MicroRNA and Drug Interactions in Breast Cancer Cell Lines Points to miR-126 as a Modulator of CDK4/6 and PIK3CA Inhibitors.

Background: Breast cancer (BC) represents the most common cancer in women worldwide. Due to its heterogeneous nature, breast cancer management might benefit from differential treatments toward personalized medicine. Additionally, drug resistance is a common phenomenon. We systematically investigated the effect of 14 different drugs administered on BC cell lines in combination with microRNAs (miRNA, miR). Methods: Thirty-eight miRNAs, all associated with BC by clinical and molecular parameters including progression, prognosis and subtypes, were tested for their effects on the viability of 12 different BC cell lines. Four miRNAs with the strongest impact on viability were further assayed in combination with 14 BC drugs. Mann-Whitney U-test with Bonferroni correction was used for statistical analysis. Results: In a miRNA only pre-screen we observed effects on BC cell lines' viability for 34 out of 38 candidate miRNAs. We then identified 14 miRNA/drug combinations for which the combination IC50 was lower than that of both miRNA and drug as single agents. miR-181a, paired with GSK1070916, Doxorubicin, XL765 and AMG511, was the only miRNA active on the triple negative (TNBC) MDA-MB-468 cell line. miR-126 was the only miRNA (in combination with CDK4/6 or PIK3CA inhibitors) with significant effects on cell lines from different subtypes: MCF7 (Luminal) and MDA-MB-453 (HER2+). Because of its activity on different BC subtypes, we investigated the genome wide effects of miR-126 using transcriptomics and confirmed that expression of miR-126 in BC cell lines affected cell cycle and mitosis. Conclusion: Our results show that a combination treatment with miRNAs, in particular miR-181a, miR-326, miR-9 and miR-126, enhance the activity of specific BC drugs in vitro, even on the most aggressive BC subtypes, HER2+ and TNBC. Finally, as expected from its drug interactions, based on a whole transcriptome study we could confirm a role for miR-126 in cell cycle regulation.

Loss of miR-204 expression is a key event in melanoma.

Cutaneous melanoma (CM) is a malignancy with increasing occurrence. Its microRNA repertoire has been defined in a number studies, leading to candidates for biological and clinical relevance: miR-200a/b/c, miR-203, miR-205, miR-204, miR-211, miR-23b and miR-26a/b. Our work was aimed to validate the role of these candidate miRNAs in melanoma, using additional patients cohorts and in vitro cultures. miR-26a, miR-204 and miR-211 were more expressed in normal melanocytes, while miR-23b, miR-200b/c, miR-203 and miR-205 in epidermis and keratinocytes. None of the keratinocyte-related miRNAs was associated with any known mutation or with clinical covariates in melanoma. On the other hand, the loss of miR-204 was enriched in melanomas with NRAS sole mutation (Fisher exact test, P = 0.001, Log Odds = 1.67), and less frequent than expected in those harbouring CDKN2A mutations (Fisher exact test, P = 0.001, Log Odds - 1.09). Additionally, miR-204 was associated with better prognosis in two independent melanoma cohorts and its exogenous expression led to growth impairment in melanoma cell lines. Thus, miR-204 represents a relevant mechanism in melanoma, with potential prognostic value and its loss seems to act in the CDKN2A pathway, in cooperation with NRAS.

Profiling of the Predicted Circular RNAs in Ductal In Situ and Invasive Breast Cancer: A Pilot Study.

The recent advantage obtained by next generation sequencing allows a depth investigation of a new "old" kind of noncoding transcript, the circular RNAs. Circular RNAs are nontranslated RNAs, typically nonpolyadenylated, with a resistance to exonucleases that gives them the ability to be more stable than the common linear RNA isoforms. We used a bioinformatic detection tool (CIRCexplorer) to research predictive circRNAs from the next generation sequenced data of five samples of ductal in situ carcinoma (DCIS) and matched adjacent invasive ductal carcinoma (IDC). Furthermore, we also investigated the circular RNAs expressed in MCF7, an invasive breast ductal carcinoma cell line. We described the genomic context of the predicted circular RNAs and we address the hypothetical possible functional roles. This study showed a perspective of a panel of predictive circRNAs identified and the function that circRNAs could exert.

The anti-leukemic activity of sodium dichloroacetate in p53mutated/null cells is mediated by a p53-independent ILF3/p21 pathway.

B-chronic lymphocytic leukemia (B-CLL) patients harboring p53 mutations are invariably refractory to therapies based on purine analogues and have limited treatment options and poor survival. Having recently demonstrated that the mitochondria-targeting small molecule sodium dichloroacetate (DCA) exhibits anti-leukemic activity in p53wild-type B-CLL cells, the aim of this study was to evaluate the effect of DCA in p53mutated B-CLL cells and in p53mutated/null leukemic cell lines. DCA exhibited comparable cytotoxicity in p53wild-type and p53mutated B-CLL patient cell cultures, as well as in p53mutated B leukemic cell lines (MAVER, MEC-1, MEC-2). At the molecular level, DCA promoted the transcriptional induction of p21 in all leukemic cell types investigated, including p53null HL-60. By using a proteomic approach, we demonstrated that DCA up-regulated the ILF3 transcription factor, which is a known regulator of p21 expression. The role of the ILF3/p21 axis in mediating the DCA anti-leukemic activity was underscored by knocking-down experiments. Indeed, transfection with ILF3 and p21 siRNAs significantly decreased both the DCA-induced p21 expression and the DCA-mediated cytotoxicity. Taken together, our results emphasize that DCA is a small molecule that merits further evaluation as a therapeutic agent also for p53mutated leukemic cells, by acting through the induction of a p53-independent pathway.

Intranasal administration of recombinant TRAIL down-regulates CXCL-1/KC in an ovalbumin-induced airway inflammation murine model.

Ovalbumin (OVA)-sensitized BALB/c mice were i.n. instilled with recombinant TNF-related apoptosis inducing ligand (TRAIL) 24 hours before OVA challenge. The total number of leukocytes and the levels of the chemokine CXCL-1/KC significantly increased in the bronchoalveolar lavage (BAL) fluids of allergic animals with respect to control littermates, but not in the BAL of mice i.n. pretreated with recombinant TRAIL before OVA challenge. In particular, TRAIL pretreatment significantly reduced the BAL percentage of both eosinophils and neutrophils. On the other hand, when TRAIL was administrated simultaneously to OVA challenge its effect on BAL infiltration was attenuated. Overall, the results show that the i.n. pretreatment with TRAIL down-modulated allergic airway inflammation.

Sodium dichloroacetate exhibits anti-leukemic activity in B-chronic lymphocytic leukemia (B-CLL) and synergizes with the p53 activator Nutlin-3.

The anti-leukemic activity of the mitochondria-targeting small molecule sodium dichloroacetate (DCA), used alone and in association with the small molecule inhibitor of the p53/MDM2 interaction Nutlin-3, was analyzed in primary B-chronic lymphocytic leukemia (B-CLL) samples (n=22), normal peripheral blood cells (n=10) and in p53wild-type EHEB, JVM-2, JVM-3 B lymphoblastoid cell lines. DCA exhibited a dose-dependent anti-leukemic activity in both primary B-CLL and B leukemic cell lines with a functional p53 status and showed a synergistic cytotoxic activity when used in combination with Nutlin-3. At the molecular level, DCA positively regulated p53 activity, as documented by post-transcriptional modifications of p53 protein and synergized with Nutlin-3 in increasing the expression of the p53-target genes MDM2, PUMA, TIGAR and in particular p21. The potential role of p21 in mediating the DCA+Nutlin-3 anti-leukemic activity was underscored in knocking-down experiments. Indeed, transfection of leukemic cells with p21 siRNAs significantly decreased the DCA+Nutlin-3-induced cytotoxicity. Taken together, our data emphasize that DCA is a molecule that merits to be further evaluated as a chemotherapeutic agent for B-CLL, likely in combination with other therapeutic compounds.

The levels of circulating TRAIL at the onset of type 1 diabetes are markedly decreased in patients with ketoacidosis and with the highest insulin requirement.

Experimental evidence in animal models suggests that TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, might play an important role in type 1 diabetes (T1D). We have performed a retrospective study by analyzing the sera of a cohort of pediatric subjects (age ≤18 years; n = 507) consisting of (1) patients diagnosed with T1D (n = 387), (2) healthy individuals (n = 98, considered as controls), and (3) healthy autoantibody-positive subjects (n = 22). Patients with T1D exhibited significantly decreased levels of circulating TRAIL with respect to the control healthy subjects, as well as to the healthy autoantibody-positive subjects. Within the T1D group, no differences in the levels of circulating TRAIL were observed between patients with or without other concomitant autoimmune pathologies. Of note, the levels of TRAIL were significantly lower in the T1D patients analyzed at onset, although reduction in TRAIL levels persisted also in patients analyzed after disease onset (>1 year from diagnosis). In particular, T1D patients who exhibited ketoacidosis at onset showed significantly lower levels of circulating TRAIL with respect to patients without ketoacidosis at onset. Moreover, the levels of TRAIL at diagnosis correlated inversely with the insulin requirement up to 21 months of follow-up. This is the first study demonstrating that the levels of circulating TRAIL are significantly decreased in T1D, with the lowest levels of TRAIL being observed in patients with ketoacidosis at the onset and with the highest insulin requirement.

The p53 transcriptional pathway is preserved in ATMmutated and NOTCH1mutated chronic lymphocytic leukemias.

By using next generation sequencing, we have analyzed 108 B chronic lymphocytic leukemia (B-CLL) patients. Among genes involved in the TP53 pathway, we found frequent mutations in ATM (n=18), TP53 (n=10) and NOTCH1 (n=10) genes, rare mutations of NOTCH2 (n=2) and CDKN1A/p21 (n=1) and no mutations in BAX, MDM2, TNFRSF10A and TNFRSF10B genes. The in vitro treatment of primary B-CLL cells with the activator of p53 Nutlin-3 induced the transcription of p53 target genes, without significant differences between the B-CLL without mutations and those harboring either ATM or NOTCH1mutations. On the other hand, the subgroup of TP53mutated B-CLL exhibited a significantly lower induction of the p53 target genes in response to Nutlin-3 as compared to the other B-CLL samples. However, among the TP53mutated B-CLL, those showing mutations in the high hot spot region of the DNA binding domain [273-280 aa] maintained a significantly higher p53-dependent transcriptional activity as compared to the other TP53mutated B-CLL samples. Since the ability to elicit a p53-dependent transcriptional activity in vitro has a positive prognostic significance, our data suggest that ATMmutated, NOTCH1mutated and surprisingly, also a subset of TP53mutated B-CLL patients might benefit from therapeutic combinations including small molecule activator of the p53 pathway.

The circulating levels of TRAIL are extremely low after delivery but rapidly recover in both mothers and newborns.

TNF-related apoptosis inducing ligand (TRAIL) plasma levels was measured in plasma samples obtained 1h (time 1) and 2-3 days (time 2) after delivery in a group of healthy women (n=17) who underwent cesarean delivery, and showed a significantly increase from time 1 (39.3 pg/ml median; 41.2 ± 15.9 mean ± SD) to time 2 (71.6 pg/ml median; 73.8 ± 27.8 mean ± SD). Similarly, circulating TRAIL levels were extremely low in the plasma of newborns (n=41) within the first 24h after partum (time 1; 27.5 pg/ml, median; 31.5 ± 15.8 means ± SD), showing a significant increase 2-3 days after partum (time 2; 68.4 pg/ml, median; 75.1 ± 36.7 mean ± SD). It is also noteworthy that the highest levels of plasma TRAIL were observed in newborns with the following characteristics: (i) born at later gestational age, (ii) Apgar score >9, (iii) higher birth weight, (iv) born through vaginal partum. In conclusion, we have demonstrated for the first time that the levels of circulating TRAIL are markedly low in both mothers and children after delivery, rapidly rising thereafter. Moreover, the highest levels of TRAIL are observed in newborns with the best clinical parameters.

The MDM2 inhibitor Nutlin-3 attenuates streptozotocin-induced diabetes mellitus and increases serum level of IL-12p40.

Besides its well-established oncosuppressor activity, a key function of p53 in regulating metabolic pathways has been recently identified. Nevertheless, the role of p53 with respect to diabetes mellitus (DM) appears highly controversial. To address this issue, we have used the cis-imidazoline compound Nutlin-3, an inhibitor of MDM2/p53 interaction, which represents a potent and selective non-genotoxic activator of the p53 pathway both in in vivo and in vitro experimental settings. Experimental DM was induced by intraperitoneal injections of low concentrations of streptozotocin (STZ) in C57BL/6N mice (n = 20). A group of control vehicle-injected mice (n = 10) and of STZ-treated mice (n = 10) was co-injected with Nutlin-3. Mice co-injected with STZ + Nutlin-3 exhibited attenuated features of DM with respect to animals treated with STZ alone. Indeed, STZ + Nutlin-3-treated mice were characterized by significantly (p < 0.05) lower levels of hyperglycemia, reduced weight loss, and increased spleen weight. In addition, STZ alone promoted a marked decrease in the levels of several circulating cytokines, including interleukin-12 (IL-12)p40. On the other hand, co-injection of STZ + Nutlin-3 significantly (p < 0.01) counteracted IL-12p40 down-modulation. In vitro experiments performed on the RAW264.7 macrophagic cell line model, used as cellular source of IL-12p40, demonstrated that Nutlin-3 treatment increased IL-12p40 release, strongly suggesting a direct effect of Nutlin-3 on the immune system. Overall, these data demonstrate that systemic administration of Nutlin-3 ameliorates the severity of STZ-induced DM and increases the levels of circulating IL-12p40.