Targeted sequencing of BRAF by MinION in archival Formalin-Fixed Paraffin-Embedded specimens allows to discriminate between Hairy Cell Leukemia and Hair Cell Leukemia Variant

Targeted sequencing of BRAF by MinION in archival Formalin-Fixed Paraffin-Embedded specimens allows to discriminate between Hairy Cell Leukemia and Hair Cell Leukemia Varian

Rewiring innate and adaptive immunity with TLR9 agonist to treat osteosarcoma

Background Osteosarcoma (OS) is the most common primary bone tumor in children and adolescent. Surgery and multidrug chemotherapy are the standard of treatment achieving 60-70% of event-free survival for localized disease at diagnosis. However, for metastatic disease, the prognosis is dismal. Exploiting immune system activation in the setting of such unfavorable mesenchymal tumors represents a new therapeutic challenge. Methods In immune competent OS mouse models bearing two contralateral lesions, we tested the efficacy of intralesional administration of a TLR9 agonist against the treated and not treated contralateral lesion evaluating abscopal effect. Multiparametric flow cytometry was used to evaluate changes of the tumor immune microenviroment. Experiments in immune- deficient mice allowed the investigation of the role of adaptive T cells in TLR9 agonist effects, while T cell receptor sequencing was used to assess the expansion of specific T cell clones. Results TLR9 agonist strongly impaired the growth of locally-treated tumors and its therapeutic effect also extended to the contralateral, untreated lesion. Multiparametric flow cytometry showed conspicuous changes in the immune landscape of the OS immune microenvironment upon TLR9 engagement, involving a reduction in M2-like macrophages, paralleled by increased infiltration of dendritic cells and activated CD8 T cells in both lesions. Remarkably, CD8 T cells were needed for the induction of the abscopal effect, whereas they were not strictly necessary for halting the growth of the treated lesion. T cell receptor (TCR) sequencing of tumor infiltrating CD8 T cells showed the expansion of specific TCR clones in the treated tumors and, remarkably, their selected representation in the contralateral untreated lesions, providing the first evidence of the rewiring of tumor-associated T cell clonal architectures. Conclusions Overall these data indicate that the TLR9 agonist acts as an in situ anti-tumor vaccine, activating an innate immune response sufficient to suppress local tumor growth while inducing a systemic adaptive immunity with selective expansion of CD8 T cell clones, which are needed for the abscopal effect

Pseudotemporal ordering of spatial lymphoid tissue microenvironment profiles trails Unclassified DLBCL at the periphery of the follicle

We have established a pseudotemporal ordering for the transcriptional signatures of distinct microregions within reactive lymphoid tissues, namely germinal center dark zones (DZ), germinal center light zones (LZ), and peri-follicular areas (Peri). By utilizing this pseudotime trajectory derived from the functional microenvironments of DZ, LZ, and Peri, we have ordered the transcriptomes of Diffuse Large B-cell Lymphoma cases. The apex of the resulting pseudotemporal trajectory, which is characterized by enrichment of molecular programs fronted by TNFR signaling and inhibitory immune checkpoint overexpression, intercepts a discrete peri-follicular biology. This observation is associated with DLBCL cases that are enriched in the Unclassified/type-3 COO category, raising questions about the potential extra-GC microenvironment imprint of this peculiar group of cases. This report offers a thought-provoking perspective on the relationship between transcriptional profiling of functional lymphoid tissue microenvironments and the evolving concept of the cell of origin in Diffuse Large B-cell Lymphomas

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.Mutant p53 induces serine/glycine synthesis and essential amino acids intake. Under amino acid restriction, mutant p53 is stabilized and activates a transcriptional program that sustains a metabolic adaptive response promoting breast cancer cells growt

Author Correction: 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 mutation

Impaired activation of plasmacytoid dendritic cells via toll-like receptor 7/9 and STING is mediated by melanoma-derived immunosuppressive cytokines and metabolic drift

IntroductionPlasmacytoid dendritic cells (pDCs) infiltrate a large set of human cancers. Interferon alpha (IFN-α) produced by pDCs induces growth arrest and apoptosis in tumor cells and modulates innate and adaptive immune cells involved in anti-cancer immunity. Moreover, effector molecules exert tumor cell killing. However, the activation state and clinical relevance of pDCs infiltration in cancer is still largely controversial. In Primary Cutaneous Melanoma (PCM), pDCs density decreases over disease progression and collapses in metastatic melanoma (MM). Moreover, the residual circulating pDC compartment is defective in IFN-α production.MethodsThe activation of tumor-associated pDCs was evaluated by in silico and microscopic analysis. The expression of human myxovirus resistant protein 1 (MxA), as surrogate of IFN-α production, and proximity ligation assay (PLA) to test dsDNA-cGAS activation were performed on human melanoma biopsies. Moreover, IFN-α and CXCL10 production by in vitro stimulated (i.e. with R848, CpG-A, ADU-S100) pDCs exposed to melanoma cell lines supernatants (SN-mel) was tested by intracellular flow cytometry and ELISA. We also performed a bulk RNA-sequencing on SN-mel-exposed pDCs, resting or stimulated with R848. Glycolytic rate assay was performed on SN-mel-exposed pDCs using the Seahorse XFe24 Extracellular Flux Analyzer.ResultsBased on a set of microscopic, functional and in silico analyses, we demonstrated that the melanoma milieu directly impairs IFN-α and CXCL10 production by pDCs via TLR-7/9 and cGAS-STING signaling pathways. Melanoma-derived immunosuppressive cytokines and a metabolic drift represent relevant mechanisms enforcing pDC-mediated melanoma escape.DiscussionThese findings propose a new window of intervention for novel immunotherapy approaches to amplify the antitumor innate immune response in cutaneous melanoma (CM)

NUPR1, a new target in liver cancer: implication in controlling cell growth, migration, invasion and sorafenib resistance

Sorafenib, an oral multikinase inhibitor, is the only approved agent for the treatment of advanced hepatocellular carcinoma (HCC). However, its benefits are modest, and as its mechanisms of action remain elusive, a better understanding of its anticancer effects is needed. Based on our previous study results, we investigated here the implication of the nuclear protein 1 (NUPR1) in HCC and its role in sorafenib treatment. NUPR1 is a stress-inducible protein that is overexpressed in various malignancies, but its role in HCC is not yet fully understood. We found that NUPR1 expression was significantly higher in primary human HCC samples than in the normal liver. Knockdown of NUPR1 significantly increased cell sensitivity to sorafenib and inhibited the cell growth, migration and invasion of HCC cells, both in vitro and in vivo. Moreover, NUPR1 silencing influenced the expression of RELB and IER3 genes. Unsurprisingly, RELB and IER3 knockdown also inhibited HCC cell viability, growth and migration. Using gene expression profiling of HCC cells following stable NUPR1 knockdown, we found that genes functionally involved in cell death and survival, cellular response to therapies, lipid metabolism, cell growth and proliferation, molecular transport and cellular movement were mostly suppressed. Network analysis of dynamic gene expression identified NF-κB and ERK as downregulated gene nodes, and several HCC-related oncogenes were also suppressed. We identified Runt-related transcription factor 2 (RUNX2) gene as a NUPR1-regulated gene and demonstrated that RUNX2 gene silencing inhibits HCC cell viability, growth, migration and increased cell sensitivity to sorafenib. We propose that the NUPR1/RELB/IER3/RUNX2 pathway has a pivotal role in hepatocarcinogenesis. The identification of the NUPR1/RELB/IER3/RUNX2 pathway as a potential therapeutic target may contribute to the development of new treatment strategies for HCC management

Experiences and reflections on three years of CPS Summer schools within EIT Digital

This article provides an overview of current European Commission effort in term of educational innovation to reduce the gap between research and industry which still is a barrier to the economic development. Entrepreneurial innovation & education driving Europe's digital transformation (EIT Digital for short) is an European-based initiative fostering I&E (innovation and entrepreneurship) by integrating education, research and business at different educational levels. For instance in EIT master programmes, students work together with industries and academics to have a faster go-to-market of research results. Summer schools are part of the master programs; three of them have been organised related to CPS (cyber-physical systems), critical infrastructure and, more recently, Industry 4.0. Past and present events are discussed and the experience from these events is reported. It is further analysed how the general setup of the summer school program is affecting the educational aspects and achievement of the intended learning outcomes

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