IL-33 stimulates the anticancer activities of eosinophils through extracellular vesicle-driven reprogramming of tumor cells.

Immune cell-derived extracellular vesicles (EV) affect tumor progression and hold promise for therapeutic applications. Eosinophils are major effectors in Th2-related pathologies recently implied in cancer. Here, we evaluated the anti-tumor activities of eosinophil-derived EV following activation with the alarmin IL-33. We demonstrate that IL-33-activated mouse and human eosinophils produce higher quantities of EV with respect to eosinophils stimulated with IL-5. Following incorporation of EV from IL-33-activated eosinophils (Eo33-EV), but not EV from IL-5-treated eosinophils (Eo5-EV), mouse and human tumor cells increased the expression of cyclin-dependent kinase inhibitor (CDKI)-related genes resulting in cell cycle arrest in G0/G1, reduced proliferation and inhibited tumor spheroid formation. Moreover, tumor cells incorporating Eo33-EV acquired an epithelial-like phenotype characterized by E-Cadherin up-regulation, N-Cadherin downregulation, reduced cell elongation and migratory extent in vitro, and impaired capacity to metastasize to lungs when injected in syngeneic mice. RNA sequencing revealed distinct mRNA signatures in Eo33-EV and Eo5-EV with increased presence of tumor suppressor genes and enrichment in pathways related to epithelial phenotypes and negative regulation of cellular processes in Eo33-EV compared to Eo5-EV. Our studies underscore novel IL-33-stimulated anticancer activities of eosinophils through EV-mediated reprogramming of tumor cells opening perspectives on the use of eosinophil-derived EV in cancer therapy.

Germinal Center Dark Zone harbors ATR-dependent determinants of T-cell exclusion that are also identified in aggressive lymphoma.

The germinal center (GC) dark zone (DZ) and light zone (LZ) regions spatially separate expansion and diversification from selection of antigen-specific B-cells to ensure antibody affinity maturation and B cell memory. The DZ and LZ differ significantly in their immune composition despite the lack of a physical barrier, yet the determinants of this polarization are poorly understood. This study provides novel insights into signals controlling asymmetric T-cell distribution between DZ and LZ regions. We identify spatially-resolved DNA damage response and chromatin compaction molecular features that underlie DZ T-cell exclusion. The DZ spatial transcriptional signature linked to T-cell immune evasion clustered aggressive Diffuse Large B-cell Lymphomas (DLBCL) for differential T cell infiltration. We reveal the dependence of the DZ transcriptional core signature on the ATR kinase and dissect its role in restraining inflammatory responses contributing to establishing an immune-repulsive imprint in DLBCL. These insights may guide ATR-focused treatment strategies bolstering immunotherapy in tumors marked by DZ transcriptional and chromatin-associated features.

Mutant p53 sustains serine-glycine synthesis and essential amino acids intake promoting breast cancer growth.

Reprogramming of amino acid metabolism, sustained by oncogenic signaling, is crucial for cancer cell survival under nutrient limitation. Here we discovered that missense mutant p53 oncoproteins stimulate de novo serine/glycine synthesis and essential amino acids intake, promoting breast cancer growth. Mechanistically, mutant p53, unlike the wild-type counterpart, induces the expression of serine-synthesis-pathway enzymes and L-type amino acid transporter 1 (LAT1)/CD98 heavy chain heterodimer. This effect is exacerbated by amino acid shortage, representing a mutant p53-dependent metabolic adaptive response. When cells suffer amino acids scarcity, mutant p53 protein is stabilized and induces metabolic alterations and an amino acid transcriptional program that sustain cancer cell proliferation. In patient-derived tumor organoids, pharmacological targeting of either serine-synthesis-pathway and LAT1-mediated transport synergizes with amino acid shortage in blunting mutant p53-dependent growth. These findings reveal vulnerabilities potentially exploitable for tackling breast tumors bearing missense TP53 mutations.

No Clear Clustering Dysbiosis from Salivary Microbiota Analysis by Long Sequencing Reads in Patients Affected by Oral Squamous Cell Carcinoma: A Single Center Study.

BACKGROUND: Advancements in DNA sequencing technology have facilitated the assessment of the connection between the oral microbiome and various diseases. The aim of the present study was to investigate the salivary microbiota composition employing for the first time in the literature the Oxford Nanopore Technology in patients affected by oral squamous cell carcinoma (OSCC). METHODS: Unstimulated saliva samples of 31 patients were collected (24 OSCC patients and 7 controls). DNA was extracted using the QIAamp DNA Blood Kit and metagenomic long sequencing reads were performed using the MinION device. RESULTS: In the OSCC group, 13 were males and 11 were females, with a mean age of 65.5 ± 13.9 years; in the control group, 5 were males and 2 were females, with a mean age of 51.4 ± 19.2 years. The border of the tongue was the most affected OSCC site. The microorganisms predominantly detected in OSCC patients were Prevotella, Chlamydia, Tissierellia, Calothrix, Leotiomycetes, Firmicutes and Zetaproteobacteria. CONCLUSIONS: This study confirmed the predominance of periodontopathic bacteria in the salivary microbiome in the OSCC group. If a direct correlation between oral dysbiosis and OSCC onset was proven, it could lead to new prevention strategies and early diagnostic tools.

A longitudinal study of C1q and anti-C1q autoantibodies in homologous and heterologous pregnancies for predicting pre-eclampsia.

C1q, the recognition molecule of the classical pathway of the complement system, plays a central role in pregnancy. Lack of C1q is characterized by poor trophoblast invasion and pregnancy failure. C1q can be the target of an antibody response: anti-C1q autoantibodies (anti-C1q) are present in several infectious and autoimmune diseases. The presence of these autoantibodies has been detected also in 2-8% of the general population. Recent evidence indicates that women who undergo assisted reproductive technology (ART) have an increased risk of developing pre-eclampsia (PE), particularly oocyte donation (OD) pregnancies. The aim of this study was to characterize the levels of C1q and anti-C1q in PE gestations, in healthy spontaneous, homologous and heterologous ART pregnancies. Serum of the following four groups of women, who were followed throughout two or three trimesters, were collected: PE, patients diagnosed with PE; OD, oocyte donation recipients; HOM, homologous ART women; Sp, spontaneous physiological pregnancy. Our results indicate that PE patients have lower levels of anti-C1q. In ART pregnant women, the trend of C1q and anti-C1q levels were similar to PE patients, even though these women did not develop PE-like symptoms during pregnancy. This finding suggests an immunological dysfunction at the foetal-maternal interface in ART pregnancies, a hypothesis confirmed by the observation of C1q deposition in placentae derived from OD, comparable to PE. Since significantly lower levels of anti-C1q were detected in PE compared to healthy control sera, we hypothesize the possible binding on placental syncytiotrophoblast microvesicles (STBM), which are increased in the circulation of PE mothers. Furthermore, the characterization of the binding-epitope of anti-C1q revealed that "physiological" autoantibodies were mainly directed against C1q globular domain. We concluded that anti-C1q could have a physiological role in pregnancy: during the healthy spontaneous pregnancy the raised levels of these autoantibodies can be important for the clearance of STBM. In PE and in pathological pregnancies (but also in OD pregnancies), the increase in syncytiotrophoblast apoptosis and consequent increase of the circulating STMB levels lead to a consumption of C1q and anti-C1q.

Salivary Microbiota Composition in Patients with Oral Squamous Cell Carcinoma: A Systematic Review.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is one of the most prevalent cancers worldwide. Despite recent advances in diagnosis and treatment, in recent years, an increase in the incidence of OSCC has been registered, and the mortality rate is still high. This systematic review aims to identify a potential association between the composition of salivary microbiota and OSCC. MATERIALS AND METHODS: The protocol for this study was designed following the PRISMA guidelines. Records were identified using different search engines (e.g., Medline/PubMed). Observational studies, in human subjects with histological diagnosis of OSCC, concerning the analysis of salivary microbiota, were selected. RESULTS: Eleven papers were included. The salivary microbiomes of 1335 patients were analysed (n.687 OSCC and n.648 controls). Due to the great heterogeneity of the studies, it was not possible to profile a specific microbiota associated with OSCC. However, periodontal pathogens were the most common bacteria detected in patients with OSCC (i.e., Fusobacterium, Prevotella). CONCLUSIONS: Although there are evident alterations in the salivary microbiota composition in OSCC patients, it is still a challenge to identify a specific microbiota pattern in OSCC patients. If the associations between specific salivary microorganisms and OSCC are confirmed, microbiome analysis could be a useful tool for the screening and follow-up of patients affected by OSCC.

The Abundance of Tumor-Infiltrating CD8+ Tissue Resident Memory T Lymphocytes Correlates with Patient Survival in Glioblastoma.

Glial tumors alone account for 40% of all CNS tumors and present a low survival rate. The tumor microenvironment is a critical regulator of tumor progression and therapeutic effectiveness in glioma. Growing evidence from numerous studies of human solid tumor-infiltrating CD8+ T cells indicates that tissue-resident memory T cells (TRM) represent a substantial subpopulation of tumor-infiltrating lymphocytes (TILs). Although it is reported that some types of cancer patients with high immune infiltration tend to have better outcomes than patients with low immune infiltration, it seems this does not happen in gliomas. This study aimed to characterize TRMs cells in the glioma tumor microenvironment to identify their potential predictive and prognostic role and the possible therapeutic applications. Fluorescence activated cell sorting (FACS) analysis and immunofluorescence staining highlighted a statistically significant increase in CD8+ TRM cells (CD103+ and CD69+ CD8+ T cells) in gliomas compared to control samples (meningioma). In-silico analysis of a dataset of n = 153 stage IV glioma patients confirmed our data. Moreover, the gene expression analysis showed an increase in the expression of TRM-related genes in tumor tissues compared to normal tissues. This analysis also highlighted the positive correlation between genes associated with CD8+ TRM and TILs, indicating that CD8+ TRMs cells are present among the infiltrating T cells. Finally, high expression of Integrin subunit alpha E (ITGAE), the gene coding for the integrin CD103, and high CD8+ TILs abundance were associated with more prolonged survival, whereas high ITGAE expression but low CD8+ TILs abundance were associated with lower survival.

Direct RNA Nanopore Sequencing of SARS-CoV-2 Extracted from Critical Material from Swabs.

In consideration of the increasing prevalence of COVID-19 cases in several countries and the resulting demand for unbiased sequencing approaches, we performed a direct RNA sequencing (direct RNA seq.) experiment using critical oropharyngeal swab samples collected from Italian patients infected with SARS-CoV-2 from the Palermo region in Sicily. Here, we identified the sequences SARS-CoV-2 directly in RNA extracted from critical samples using the Oxford Nanopore MinION technology without prior cDNA retrotranscription. Using an appropriate bioinformatics pipeline, we could identify mutations in the nucleocapsid (N) gene, which have been reported previously in studies conducted in other countries. In conclusion, to the best of our knowledge, the technique used in this study has not been used for SARS-CoV-2 detection previously owing to the difficulties in the extraction of RNA of sufficient quantity and quality from routine oropharyngeal swabs. Despite these limitations, this approach provides the advantages of true native RNA sequencing and does not include amplification steps that could introduce systematic errors. This study can provide novel information relevant to the current strategies adopted in SARS-CoV-2 next-generation sequencing.

RNA Sequencing of Primary Cutaneous and Breast-Implant Associated Anaplastic Large Cell Lymphomas Reveals Infrequent Fusion Transcripts and Upregulation of PI3K/AKT Signaling via Neurotrophin Pathway Genes.

Cutaneous and breast implant-associated anaplastic large-cell lymphomas (cALCLs and BI-ALCLs) are two localized forms of peripheral T-cell lymphomas (PTCLs) that are recognized as distinct entities within the family of ALCL. JAK-STAT signaling is a common feature of all ALCL subtypes, whereas DUSP22/IRF4, TP63 and TYK gene rearrangements have been reported in a proportion of ALK-negative sALCLs and cALCLs. Both cALCLs and BI-ALCLs differ in their gene expression profiles compared to PTCLs; however, a direct comparison of the genomic alterations and transcriptomes of these two entities is lacking. By performing RNA sequencing of 1385 genes (TruSight RNA Pan-Cancer, Illumina) in 12 cALCLs, 10 BI-ALCLs and two anaplastic lymphoma kinase (ALK)-positive sALCLs, we identified the previously reported TYK2-NPM1 fusion in 1 cALCL (1/12, 8%), and four new intrachromosomal gene fusions in 2 BI-ALCLs (2/10, 20%) involving genes on chromosome 1 (EPS15-GNG12 and ARNT-GOLPH3L) and on chromosome 17 (MYO18A-GIT1 and NF1-GOSR1). One of the two BI-ALCL samples showed a complex karyotype, raising the possibility that genomic instability may be responsible for intra-chromosomal fusions in BI-ALCL. Moreover, transcriptional analysis revealed similar upregulation of the PI3K/Akt pathway, associated with enrichment in the expression of neurotrophin signaling genes, which was more conspicuous in BI-ALCL, as well as differences, i.e., over-expression of genes involved in the RNA polymerase II transcription program in BI-ALCL and of the RNA splicing/processing program in cALCL.