Molecular Profiling of Lymphatic Endothelial Cell Activation In Vitro.

The lymphatic vascular system plays a key role in cancer progression. Indeed, the activation of lymphatic endothelial cells (LECs) through the lymphangiogenic process allows for the formation of new lymphatic vessels (LVs) that represent the major route for the dissemination of solid tumors. This process is governed by a plethora of cancer-derived and microevironmental mediators that strictly activate and control specific molecular pathways in LECs. In this work we used an in vitro model of LEC activation to trigger lymphangiogenesis using a mix of recombinant pro-lymphangiogenic factors (VFS) and a co-culture system with human melanoma cells. Both systems efficiently activated LECs, and under these experimental conditions, RNA sequencing was exploited to unveil the transcriptional profile of activated LECs. Our data demonstrate that both recombinant and tumor cell-mediated activation trigger significant molecular pathways associated with endothelial activation, morphogenesis, and cytokine-mediated signaling. In addition, this system provides information on new genes to be further investigated in the lymphangiogenesis process and open the possibility for further exploitation in other tumor contexts where lymphatic dissemination plays a relevant role.

GASOLINE: detecting germline and somatic structural variants from long-reads data.

Long-read sequencing allows analyses of single nucleic-acid molecules and produces sequences in the order of tens to hundreds kilobases. Its application to whole-genome analyses allows identification of complex genomic structural-variants (SVs) with unprecedented resolution. SV identification, however, requires complex computational methods, based on either read-depth or intra- and inter-alignment signatures approaches, which are limited by size or type of SVs. Moreover, most currently available tools only detect germline variants, thus requiring separate computation of sample pairs for comparative analyses. To overcome these limits, we developed a novel tool (Germline And SOmatic structuraL varIants detectioN and gEnotyping; GASOLINE) that groups SV signatures using a sophisticated clustering procedure based on a modified reciprocal overlap criterion, and is designed to identify germline SVs, from single samples, and somatic SVs from paired test and control samples. GASOLINE is a collection of Perl, R and Fortran codes, it analyzes aligned data in BAM format and produces VCF files with statistically significant somatic SVs. Germline or somatic analysis of 30[Formula: see text] sequencing coverage experiments requires 4-5 h with 20 threads. GASOLINE outperformed currently available methods in the detection of both germline and somatic SVs in synthetic and real long-reads datasets. Notably, when applied on a pair of metastatic melanoma and matched-normal sample, GASOLINE identified five genuine somatic SVs that were missed using five different sequencing technologies and state-of-the art SV calling approaches. Thus, GASOLINE identifies germline and somatic SVs with unprecedented accuracy and resolution, outperforming currently available state-of-the-art WGS long-reads computational methods.

High-resolution Nanopore methylome-maps reveal random hyper-methylation at CpG-poor regions as driver of chemoresistance in leukemias.

Aberrant DNA methylation at CpG dinucleotides is a cancer hallmark that is associated with the emergence of resistance to anti cancer treatment, though molecular mechanisms and biological significance remain elusive. Genome scale methylation maps by currently used methods are based on chemical modification of DNA and are best suited for analyses of methylation at CpG rich regions (CpG islands). We report the first high coverage whole-genome map in cancer using the long read nanopore technology, which allows simultaneous DNA-sequence and -methylation analyses on native DNA. We analyzed clonal epigenomic/genomic evolution in Acute Myeloid Leukemias (AMLs) at diagnosis and relapse, after chemotherapy. Long read sequencing coupled to a novel computational method allowed definition of differential methylation at unprecedented resolution, and showed that the relapse methylome is characterized by hypermethylation at both CpG islands and sparse CpGs regions. Most differentially methylated genes, however, were not differentially expressed nor enriched for chemoresistance genes. A small fraction of under-expressed and hyper-methylated genes at sparse CpGs, in the gene body, was significantly enriched in transcription factors (TFs). Remarkably, these few TFs supported large gene-regulatory networks including 50% of all differentially expressed genes in the relapsed AMLs and highly-enriched in chemoresistance genes. Notably, hypermethylated regions at sparse CpGs were poorly conserved in the relapsed AMLs, under-represented at their genomic positions and showed higher methylation entropy, as compared to CpG islands. Analyses of available datasets confirmed TF binding to their target genes and conservation of the same gene-regulatory networks in large patient cohorts. Relapsed AMLs carried few patient specific structural variants and DNA mutations, apparently not involved in drug resistance. Thus, drug resistance in AMLs can be mainly ascribed to the selection of random epigenetic alterations at sparse CpGs of a few transcription factors, which then induce reprogramming of the relapsing phenotype, independently of clonal genomic evolution.

ß3-adrenergic receptor on tumor-infiltrating lymphocytes sustains IFN-γ-dependent PD-L1 expression and impairs anti-tumor immunity in neuroblastoma.

Neuroblastoma (NB) is a heterogeneous extracranial tumor occurring in childhood. A distinctive feature of NB tumors is their neuroendocrine ability to secrete catecholamines, which in turn, via ß-adrenergic receptors ligation, may affect different signaling pathways in tumor microenvironment (TME). It was previously demonstrated that specific antagonism of ß3-adrenergic receptor (ß3-AR) on NB tumor cells affected tumor growth and progression. Here, in a murine syngeneic model of NB, we aimed to investigate whether the ß3-AR modulation influenced the host immune system response against tumor. Results demonstrated that ß3-AR antagonism lead to an immune response reactivation, partially dependent on the PD-1/PD-L1 signaling axis involvement. Indeed, ß3-AR blockade on tumor-infiltrating lymphocytes (TILs) dampened their ability to secrete IFN-γ, which in turn reduced the PD-L1 expression, caused by TILs infiltration, on NB tumor cells. Further investigations, through a genomic analysis on NB patients, showed that high ADRB3 gene expression correlates with worse clinical outcome compared to the low expression group, and that ADRB3 gene expression affects different immune-related pathways. Overall, results indicate that ß3-AR in NB TME is able to modulate the interaction between tumor and host immune system, and that its antagonism hits multiple pro-tumoral signaling pathways.

Macrophages and Schwann cell TRPA1 mediate chronic allodynia in a mouse model of complex regional pain syndrome type I.

Complex regional pain syndrome type I (CRPS-I) is characterized by intractable chronic pain. Poor understanding of the underlying mechanisms of CRPS-I accounts for the current unsatisfactory treatment. Antioxidants and antagonists of the oxidative stress-sensitive channel, the transient receptor potential ankyrin 1 (TRPA1), have been found to attenuate acute nociception and delayed allodynia in models of CRPS-I, evoked by ischemia and reperfusion (I/R) of rodent hind limb (chronic post ischemia pain, CPIP). However, it is unknown how I/R may lead to chronic pain mediated by TRPA1. Here, we report that the prolonged (day 1-15) mechanical and cold allodynia in the hind limb of CPIP mice was attenuated permanently in Trpa1-/- mice and transiently after administration of TRPA1 antagonists (A-967079 and HC-030031) or an antioxidant (α-lipoic acid). Indomethacin treatment was, however, ineffective. We also found that I/R increased macrophage (F4/80+ cell) number and oxidative stress markers, including 4-hydroxynonenal (4-HNE), in the injured tibial nerve. Macrophage-deleted MaFIA (Macrophage Fas-Induced Apoptosis) mice did not show I/R-evoked endoneurial cell infiltration, increased 4-HNE and mechanical and cold allodynia. Furthermore, Trpa1-/- mice did not show any increase in macrophage number and 4-HNE in the injured nerve trunk. Notably, in mice with selective deletion of Schwann cell TRPA1 (Plp1-CreERT;Trpa1fl/fl mice), increases in macrophage infiltration, 4-HNE and mechanical and cold allodynia were attenuated. In the present mouse model of CRPS-I, we propose that the initial oxidative stress burst that follows reperfusion activates a feed forward mechanism that entails resident macrophages and Schwann cell TRPA1 of the injured tibial nerve to sustain chronic neuroinflammation and allodynia. Repeated treatment one hour before and for 3 days after I/R with a TRPA1 antagonist permanently protected CPIP mice against neuroinflammation and allodynia, indicating possible novel therapeutic strategies for CRPS-I.