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A full elucidation of events occurring inside the cancer microenvironment is fundamental for the optimization of more effective therapies. In the present study, the cross-talk between cancer and immune cells was examined by employing mice deficient (KO) in interferon regulatory factor (IRF)-8, a transcription factor essential for induction of competent immune responses. The in vivo results showed that IRF-8 KO mice were highly permissive to B16.F10 melanoma growth and metastasis due to failure of their immune cells to exert proper immunosurveillance. These events were found to be dependent on soluble factors released by cells of the immune system capable of shaping the malignant phenotype of melanoma cells. An on-chip model was then generated to further explore the reciprocal interactions between the B16.F10 and immune cells. B16.F10 and immune cells were co-cultured in a microfluidic device composed of three culturing chambers suitably inter-connected by an array of microchannels; mutual interactions were then followed using time-lapse microscopy. It was observed that WT immune cells migrated through the microchannels towards the B16.F10 cells, establishing tight interactions that in turn limited tumor spread. In contrast, IRF-8 KO immune cells poorly interacted with the melanoma cells, resulting in a more invasive behavior of the B16.F10 cells. These results suggest that IRF-8 expression plays a key role in the cross-talk between melanoma and immune cells, and under-score the value of cell-on-chip approaches as useful in vitro tools to reconstruct complex in vivo microenvironments on a microscale level to explore cell interactions such as those occurring within a cancer immunoenvironment. [ABSTRACT FROM AUTHOR]

SARS-CoV-2-specific CD8+ T cell immunity is expected to counteract viral variants in both efficient and durable ways. We recently described a way to induce a potent SARS-CoV-2 CD8+ T immune response through the generation of engineered extracellular vesicles (EVs) emerging from muscle cells. This method relies on intramuscular injection of DNA vectors expressing different SARS-CoV-2 antigens fused at their N-terminus with the Nefmut protein, i.e., a very efficient EV-anchoring protein. However, quality, tissue distribution, and efficacy of these SARS-CoV-2-specific CD8+ T cells remained uninvestigated. To fill the gaps, antigen-specific CD8+ T lymphocytes induced by the immunization through the Nefmut-based method were characterized in terms of their polyfunctionality and localization at lung airways, i.e., the primary targets of SARS-CoV-2 infection. We found that injection of vectors expressing Nefmut/S1 and Nefmut/N generated polyfunctional CD8+ T lymphocytes in both spleens and bronchoalveolar lavage fluids (BALFs). When immunized mice were infected with 4.4 lethal doses of 50% of SARS-CoV-2, all S1-immunized mice succumbed, whereas those developing the highest percentages of N-specific CD8+ T lymphocytes resisted the lethal challenge. We also provide evidence that the N-specific immunization coupled with the development of antigen-specific CD8+ T-resident memory cells in lungs, supporting the idea that the Nefmut-based immunization can confer a long-lasting, lung-specific immune memory. In view of the limitations of current anti-SARS-CoV-2 vaccines in terms of antibody waning and efficiency against variants, our CD8+ T cell-based platform could be considered for a new combination prophylactic strategy. [ABSTRACT FROM AUTHOR]

The immunological mechanisms that modulate protection during Mycobacterium tuberculosis ( Mtb) infection or vaccination are not fully understood. Secretion of IFN-γ and, to a lesser extent, of IL-17 by CD4+ T cells plays a major role both in protection and immunopathology. Few Mtb Ags interacting with DCs affect priming, activation, and regulation of Ag-unrelated CD4+ T-cell responses. Here we demonstrate that Pst S1, a 38 k Da-lipoprotein of Mtb, promotes Ag-independent activation of memory T lymphocytes specific for Ag85 B or Ag85 A, two immunodominant protective Ags of Mtb. Pst S1 expands CD4+ and CD8+ memory T cells, amplifies secretion of IFN-γ and IL-22 and induces IL-17 production by effector memory cells in an Ag-unrelated manner in vitro and in vivo. These effects were mediated through the stimulation of DCs, particularly of the CD8α− subtype, which respond to Pst S1 by undergoing phenotypic maturation and by secreting IL-6, IL-1β and, to a lower extent, IL-23. IL-6 secretion by Pst S1-stimulated DCs was required for IFN-γ, and to a lesser extent for IL-22 responses by Ag85 B-specific memory T cells. These results may open new perspectives for immunotherapeutic strategies to control Th1/ Th17 immune responses in Mtb infections and in vaccinations against tuberculosis. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) and natural killer (NK) cells are essential components of the innate immunity and play a critical role in the first phase of host defense against infection. Interactions between DCs and NK cells have been demonstrated in a variety of settings, with evidence emerging of complex bidirectional crosstalk between the two cell types. The accessory HIV-1 Nef protein is a crucial determinant for viral replication and pathogenesis. We previously demonstrated that Nef, hijacking DC functional activity, subverts the DC arm of immune response to escape the adaptive immune attack. Here, we monitor the effect of Nef on the outcome of the innate immune response, focusing on the impact of Nef on DC/NK crosstalk. We demonstrate that Nef up-regulates the ability of DCs to stimulate the immunoregulatory NK cells (CD56bright) as assessed by the activated phenotype, up-regulation of their proliferative response and INF-γ release. On the other hand, Nef-pulsed DCs inhibit cytotoxic NK cells (CD56dim), as assessed by the reduced HLA-DR surface expression, reduced proliferation and cytotoxic activity. Moreover, in the presence of Nef-pulsed DCs, we found a significant up-regulation of TNF-γ secretion and a Significant reduction of IL-10, GM-CSF, MIP-1α and RANTES secretion. Our findings suggest that the Nef-induced dysregulation in the DC/NK cell crosstalk may represent a potential mechanism through which HIV escapes innate immune surveillance. [ABSTRACT FROM AUTHOR]

Transmission of the malaria parasite depends on specialized gamete precursors (gametocytes) that develop in the bloodstream of a vertebrate host. Gametocyte/gamete differentiation requires controlled patterns of gene expression and regulation not only of stage and gender-specific genes but also of genes associated with DNA replication and mitosis. Once taken up by mosquito, male gametocytes undergo three mitotic cycles within few minutes to produce eight motile gametes. Here we analysed, in two Plasmodium species, the expression of SET, a conserved nuclear protein involved in chromatin dynamics. SET is expressed in both asexual and sexual blood stages but strongly accumulates in male gametocytes. We demonstrated functionally the presence of two distinct promoters upstream of the set open reading frame, the one active in all blood stage parasites while the other active only in gametocytes and in a fraction of schizonts possibly committed to sexual differentiation. In ookinetes both promoters exhibit a basal activity, while in the oocysts the gametocyte-specific promoter is silent and the reporter gene is only transcribed from the constitutive promoter. This transcriptional control, described for the first time in Plasmodium, provides a mechanism by which single-copy genes can be differently modulated during parasite development. In male gametocytes an overexpression of SET might contribute to a prompt entry and execution of S/M phases within mosquito vector. [ABSTRACT FROM AUTHOR]

Isoginkgetin (Iso) is a natural bioflavonoid isolated from the leaves of Ginkgo biloba, this natural substance exhibits many healing properties, among which the antitumor effect stands out. Here we tested the effect of Iso on the growth of U87MG glioblastoma cells. Growth curves and MTT toxicity assays showed time and dose-dependent growth inhibition of U87MG after treatment with Iso (15/25 µM) for 1, 2, and 3 days. The cell growth block of U87MG was further investigated with the colony formation test, which showed that iso treatment for 24 h reduced colony formation. The present study also aimed to evaluate the effect of Iso on U87MG glioblastoma cell migration. The FACS analysis, on the other hand, showed that treatment with Iso 15 µM determines a blockage of the cell cycle in the S1 phase. Further investigation shows that Iso treatment of U87MG altered the protein pathways of homeostasis including autophagy and apoptosis. The present study demonstrated, for the first time, that Iso could represent an excellent adjuvant drug for the treatment of glioblastoma by simultaneously activating multiple mechanisms that control the growth and migration of neoplastic cells. [ABSTRACT FROM AUTHOR]

Background and objectives: There is an urgent need to discover blood-based biomarkers of multiple sclerosis (MS) to better define the underlying biology of relapses and monitor disease progression. The main goal of this study is to search for candidate biomarkers of MS relapses associated with circulating extracellular vesicles (EVs), an emerging tool for biomarker discovery. Methods: EVs, purified from unpaired plasma and CSF samples of RRMS patients by size-exclusion chromatography (SEC), underwent proteomic analysis to discover novel biomarkers associated with MS relapses. The candidate biomarkers of disease activity were detected by comparison approach between plasma- and CSF-EV proteomes associated with relapses. Among them, a selected potential biomarker was evaluated in a cohort of MS patients, using a novel and highly reproducible flow cytometry-based approach in order to detect low abundant EV subsets in a complex body fluid such as plasma. Results: The proteomic profiles of both SEC-purified plasma EVs (from 6 patients in relapse and 5 patients in remission) and SEC-purified CSF EVs (from 4 patients in relapse and 3 patients in remission) revealed a set of proteins associated with MS relapses significant enriched in the synaptic transmission pathway. Among common proteins, excitatory amino-acid transporter 2, EAAT2, responsible for the majority of the glutamate uptake in CNS, was worthy of further investigation. By screening plasma samples from 110 MS patients, we found a significant association of plasma EV-carried EAAT2 protein (EV-EAAT2) with MS relapses, regardless of disease-modifying therapies. This finding was confirmed by investigating the presence of EV-EAAT2 in plasma samples collected longitudinally from 10 RRMS patients, during relapse and remission. Moreover, plasma EV-EAAT2 levels correlated positively with Expanded Disability Status Scale (EDSS) score in remitting MS patients but showed a negative correlation with age in patients with secondary progressive (SPMS). Conclusion: Our results emphaticize the usefulness of plasma EVs as a source of accessible biomarkers to remotely analyse the CNS status. Plasma EV-EAAT2 showed to be a promising biomarker for MS relapses. Further studies are required to assess the clinical relevance of this biomarker also for disability progression independent of relapse activity and transition from RRMS towards SPMS. [ABSTRACT FROM AUTHOR]

The cell cycle and the transcriptome dynamics of yeast exposed to extracellular self-DNA during an aerobic batch culture on glucose have been investigated using cytofluorimetric and RNA-seq analyses. In parallel, the same study was conducted on yeast cells growing in the presence of (heterologous) nonself-DNA. The self-DNA treatment determined a reduction in the growth rate and a major elongation of the diauxic lag phase, as well as a significant delay in the achievement of the stationary phase. This was associated with significant changes in the cell cycle dynamics, with slower exit from the G0 phase, followed by an increased level of cell percentage in the S phase, during the cultivation. Comparatively, the exposure to heterologous DNA did not affect the growth curve and the cell cycle dynamics. The transcriptomic analysis showed that self-DNA exposure produced a generalized downregulation of transmembrane transport and an upregulation of genes associated with sulfur compounds and the pentose phosphate pathway. Instead, in the case of the nonself treatment, a clear response to nutrient deprivation was detected. Overall, the presented findings represent further insights into the complex functional mechanisms of self-DNA inhibition. [ABSTRACT FROM AUTHOR]

EphB2 and EphA2 control stemness and differentiation in the intestinal mucosa, but the way they cooperate with the complex mechanisms underlying tumor heterogeneity and how they affect the therapeutic outcome in colorectal cancer (CRC) patients, remain unclear. MicroRNA (miRNA) expression profiling along with pathway analysis provide comprehensive information on the dysregulation of multiple crucial pathways in CRC. Through a network-based approach founded on the characterization of progressive miRNAomes centered on EphA2/EphB2 signaling during tumor development in the AOM/DSS murine model, we found a miRNA-dependent orchestration of EphB2-specific stem-like properties in earlier phases of colorectal tumorigenesis and the EphA2-specific control of tumor progression in the latest CRC phases. Furthermore, two transcriptional signatures that are specifically dependent on the EphA2/EphB2 signaling pathways were identified, namely EphA2, miR-423-5p, CREB1, ADAMTS14, and EphB2, miR-31-5p, mir-31-3p, CRK, CXCL12, ARPC5, SRC. EphA2- and EphB2-related signatures were validated for their expression and clinical value in 1663 CRC patients. In multivariate analysis, both signatures were predictive of survival and tumor progression. The early dysregulation of miRs-31, as observed in the murine samples, was also confirmed on 49 human tissue samples including preneoplastic lesions and tumors. In light of these findings, miRs-31 emerged as novel potential drivers of CRC initiation. Our study evidenced a miRNA-dependent orchestration of EphB2 stem-related networks at the onset and EphA2-related cancer-progression networks in advanced stages of CRC evolution, suggesting new predictive biomarkers and potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Glioblastoma, isocitrate dehydrogenase‐wildtype (GB), is the most common and aggressive primary brain malignancy with poor outcome. Immune checkpoint inhibitors (ICIs) have been tested in GB and, despite disappointing results, the identification of a small subgroup of responders underlies the need to improve our understanding of the tumour microenvironment (TME) immunity. This study aimed to determine whether the expression of selected immune checkpoints on tissue‐resident memory T cells (Trm) may predict patient outcome. We conducted a single cohort observational study. Tumour samples were collected from 45 patients with histologically confirmed GB (WHO grade 4) and processed to obtain single‐cell suspensions. Patients were assessed for the correlation of Trm phenotype with overall survival (OS) or progression‐free survival (PFS) using multiparametric flow cytometry and uni/multivariate analyses. Levels of Trm expressing programmed cell death protein 1 (PD1) and T cell immunoglobulin and mucin domain‐containing protein 3 (TIM3) were found to be linked to clinical outcome. Low frequency of Trm expressing PD1 or TIM3 or both markers defined subgroups as independent positive prognostic factors for patient survival. On multivariate analysis, low CD8+CD103+PD1+TIM3+ Trm and Karnofsky performance status (KPS) ≥70 were confirmed to be the most predictive independent factors associated with longer OS (hazard ratios—HR [95%CI]: 0.14 [0.04–0.52] p < 0.001, 0.39 [0.16–0.96] p = 0.04, respectively). The CD8+CD103+ Trm subgroups were also age‐related predictors for survival in GB. [ABSTRACT FROM AUTHOR]

Background: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0–6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions: A crucial step of melanoma progression does occur at melanoma intermediate –stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement. [ABSTRACT FROM AUTHOR]

Background: Clear cell RCC (ccRCC) accounts for approximately 75% of the renal cancer cases. Surgery treatment seems to be the best efficacious approach for the majority of patients. However, a consistent fraction (30%) of cases progress after surgery with curative intent. It is currently largely debated the use of adjuvant therapy for high-risk patients and the clinical and molecular parameters for stratifying beneficiary categories. In addition, the treatment of advanced forms lacks reliable driver biomarkers for the appropriated therapeutic choice. Thus, renal cancer patient management urges predictive molecular indicators and models for therapy-decision making. Methods: Here, we developed and optimized new models and tools for ameliorating renal cancer patient management. We isolated from fresh tumor specimens heterogeneous multi-clonal populations showing epithelial and mesenchymal characteristics coupled to stem cell phenotype. These cells retained long lasting-tumor-propagating capacity provided a therapy monitoring approach in vitro and in vivo while being able to form parental tumors when orthotopically injected and serially transplanted in immunocompromised murine hosts. Results: In line with recent evidence of multiclonal cancer composition, we optimized in vitro cultures enriched of multiple tumor-propagating populations. Orthotopic xenograft masses recapitulated morphology, grading and malignancy of parental cancers. High-grade but not the low-grade neoplasias, resulted in efficient serial transplantation in mice. Engraftment capacity paralleled grading and recurrence frequency advocating for a prognostic value of our developed model system. Therefore, in search of novel molecular indicators for therapy decision-making, we used Reverse-Phase Protein Arrays (RPPA) to analyze a panel of total and phosphorylated proteins in the isolated populations. Tumor-propagating cells showed several deregulated kinase cascades associated with grading, including angiogenesis and m-TOR pathways. Conclusions: In the era of personalized therapy, the analysis of tumor propagating cells may help improve prediction of disease progression and therapy assignment. The possibility to test pharmacological response of ccRCC stem-like cells in vitro and in orthotopic models may help define a pharmacological profiling for future development of more effective therapies. Likewise, RPPA screening on patient-derived populations offers innovative approach for possible prediction of therapy response. [ABSTRACT FROM AUTHOR]