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iNKT cells account for a relevant fraction of effector T-cells in the intestine and are considered an attractive platform for cancer immunotherapy. Although iNKT cells are cytotoxic lymphocytes, their functional role in colorectal cancer (CRC) is still controversial, limiting their therapeutic use. Thus, we examined the immune cell composition and iNKT cell phenotype of CRC lesions in patients (n = 118) and different murine models. High-dimensional single-cell flow-cytometry, metagenomics, and RNA sequencing experiments revealed that iNKT cells are enriched in tumor lesions. The tumor-associated pathobiont Fusobacterium nucleatum induces IL-17 and Granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in iNKT cells without affecting their cytotoxic capability but promoting iNKT-mediated recruitment of neutrophils with polymorphonuclear myeloid-derived suppressor cells-like phenotype and functions. The lack of iNKT cells reduced the tumor burden and recruitment of immune suppressive neutrophils. iNKT cells in-vivo activation with α-galactosylceramide restored their anti-tumor function, suggesting that iNKT cells can be modulated to overcome CRC-associated immune evasion. Tumor co-infiltration by iNKT cells and neutrophils correlates with negative clinical outcomes, highlighting the importance of iNKT cells in the pathophysiology of CRC. Our results reveal a functional plasticity of iNKT cells in CRC, suggesting a pivotal role of iNKT cells in shaping the tumor microenvironment, with relevant implications for treatment.

Lineage dedifferentiation towards a mesenchymal-like state is a common mechanism of adaptive response and resistance to targeted therapy in melanoma. Yet, the transcriptional network driving this phenotypic plasticity remains elusive. Remarkably, this cellular state displays myofibroblast and fibrotic features and escapes MAPK inhibitors (MAPKi) through extracellular matrix (ECM) remodeling activities. Here we show that the anti-fibrotic drug Nintedanib/BIBF1120 is active to normalize the fibrous ECM network, enhance the efficacy of MAPK-targeted therapy and delay tumor relapse in a pre-clinical model of melanoma. We also uncovered the molecular networks that regulate the acquisition of this resistant phenotype and its reversion by Nintedanib, pointing the miR-143/-145 pro-fibrotic cluster as a driver of the therapy-resistant mesenchymal-like phenotype. Upregulation of the miR-143/-145 cluster under BRAFi/MAPKi therapy was observed in melanoma cells in vitro and in vivo and was associated with an invasive/undifferentiated profile of resistant cells. The 2 mature miRNAs generated from this cluster, miR-143-3p and miR-145-5p collaborated to mediate phenotypic transition towards a drug resistant undifferentiated mesenchymal-like state by targeting Fascin actin-bundling protein 1 (FSCN1), modulating the dynamic crosstalk between the actin cytoskeleton and the ECM through the regulation of focal adhesion dynamics as well as contributing to a fine-tuning of mechanotransduction pathways. Our study brings insights into a novel miRNA-mediated regulatory network that contributes to non-genetic adaptive drug resistance and provides proof-of-principle that preventing MAPKi-induced pro-fibrotic stromal response is a viable therapeutic opportunity for patients on targeted therapy.

Mammary carcinoma, including triple-negative breast carcinomas (TNBC) are tumor-types for which human and canine pathologies are closely related at the molecular level. The efficacy of an oncolytic vaccinia virus (VV) was compared in low-passage primary carcinoma cells from TNBC versus non-TNBC. Non-TNBC cells were 28 fold more sensitive to VV than TNBC cells in which VV replication is impaired. Single-cell RNA-seq performed on two different TNBC cell samples, infected or not with VV, highlighted three distinct populations: naïve cells, bystander cells, defined as cells exposed to the virus but not infected and infected cells. The transcriptomes of these three populations showed striking variations in the modulation of pathways regulated by cytokines and growth factors. We hypothesized that the pool of genes expressed in the bystander populations was enriched in antiviral genes. Bioinformatic analysis suggested that the reduced activity of the virus was associated with a higher mesenchymal status of the cells. In addition, we demonstrated experimentally that high expression of one gene, DDIT4, is detrimental to VV production. Considering that DDIT4 is associated with a poor prognosis in various cancers including TNBC, our data highlight DDIT4 as a candidate resistance marker for oncolytic poxvirus therapy. This information could be used to design new generations of oncolytic poxviruses. Beyond the field of gene therapy, this study demonstrates that single-cell transcriptomics can be used to identify cellular factors influencing viral replication., Author summary The identification of cellular genes influencing viral replication/propagation has been studied using hypothesis-driven approaches and/or high-throughput RNA interference screens. In the present report, we propose a methodology based on single-cell transcriptomics. We have studied, in the context of oncolytic virotherapy, the susceptibility of different grades of primary low-passage mammary carcinoma cells of canine origin to an oncolytic vaccinia virus (VV). We highlight a fault in replication of VV in cells that originated from high-grade triple-negative breast carcinomas (TNBC). Single-cell RNA-seq performed on TNBC cell samples infected with VV suggested that the reduced activity of the virus was associated with a higher mesenchymal status of the cells. We also demonstrate that high expression of one gene, DDIT4, is detrimental to VV production. Considering that DDIT4 is associated with a poor prognosis in various cancers including TNBC, our data highlight DDIT4 as a candidate resistance marker for oncolytic poxvirus therapy. Beyond the field of cancer gene therapy, we demonstrate here that single-cell transcriptomics increases the arsenal of tools available to identify cellular factors influencing viral replication.

Airborne ultrafine particles (UFP) mainly derive from combustion sources (e.g., diesel exhaust particles&mdash, DEP), abrasion sources (non-exhaust particles) or from the unintentional release of engineered nanoparticles (e.g., metal oxide nanoparticles&mdash, NPs), determining human exposure to UFP mixtures. The aim of the present study was to analyse the combined in vitro effects of DEP and metal oxide NPs (ZnO, CuO) on human lung A549 cells. The mixtures and the relative single NPs (DEP, ZnO, CuO) were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and inductively coupled plasma-optic emission spectroscopy (ICP-OES). Cells were exposed for different times (3&ndash, 72 h) to mixtures of standard DEP at a subcytotoxic concentration and ZnO and CuO at increasing concentrations. At the end of the exposure, the cytotoxicity was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and clonogenic tests, the pro-inflammatory potential was evaluated by interleukin-8 (IL-8) release and the cell morphology was investigated by fluorescence and transmission electron microscopy. The obtained results suggest that the presence of DEP may introduce new physico-chemical interactions able to increase the cytotoxicity of ZnO and to reduce that of CuO NPs.

Resume Objectifs Examiner les representations de la population vis-a-vis du craquement associe aux manipulations vertebrales (MV) de haute velocite et basse amplitude (« thrust ») et comparer les croyances des sujets avec ou sans antecedents de MV. Methodes Cent participants ont ete inclus dans cette etude : 60 (groupe « no-MV ») sans antecedent de MV (40 sujets asymptomatiques avec ou sans antecedents de douleurs rachidiennes et 20 patients souffrant de douleurs rachidiennes non specifiques) et 40 (groupe « MV ») avec antecedent de MV (20 sujets asymptomatiques et 20 patients). Tous les participants ont ete convies a une seance individuelle au cours de laquelle ils ont ete soumis a un questionnaire au sujet de leurs antecedents de MV et de leurs croyances concernant le bruit associe aux MV. Resultats La population etait âgee en moyenne de 43,5 ± 15,4 ans. La question relative a l’origine du bruit a indique que 49 % des participants pensent que le bruit resulte de la « remise en place d’une vertebre » ou du « frottement entre deux vertebres » (23 %) ; seuls 9 % des participants ont evoque, a juste titre, la formation d’une bulle de gaz. Quarante pour cent des sujets ont explique a tort que le bruit signifiait qu’une manipulation etait reussie. Aucune difference en termes de croyances n’a ete mise en evidence entre les groupes « MV » et « no-MV ». Conclusions Compte tenu des effets deleteres demontres de certaines croyances, celles-ci, tres presentes en ce qui concerne les MV comme en temoigne la presente etude, devraient etre davantage prises en consideration par les therapeutes et plus particulierement par ceux effectuant des MV.

Objectives To examine beliefs about cracking sounds heard during high-velocity low-amplitude (HVLA) thrust spinal manipulation in individuals with and without personal experience of this technique. Methods We included 100 individuals. Among them, 60 had no history of spinal manipulation, including 40 who were asymptomatic with or without a past history of spinal pain and 20 who had nonspecific spinal pain. The remaining 40 patients had a history of spinal manipulation; among them, 20 were asymptomatic and 20 had spinal pain. Participants attended a one-on-one interview during which they completed a questionnaire about their history of spinal manipulation and their beliefs regarding sounds heard during spinal manipulation. Results Mean age was 43.5 ± 15.4 years. The sounds were ascribed to vertebral repositioning by 49% of participants and to friction between two vertebras by 23% of participants; only 9% of participants correctly ascribed the sound to the formation of a gas bubble in the joint. The sound was mistakenly considered to indicate successful spinal manipulation by 40% of participants. No differences in beliefs were found between the groups with and without a history of spinal manipulation. Conclusions Certain beliefs have documented adverse effects. This study showed a high prevalence of unfounded beliefs regarding spinal manipulation. These beliefs deserve greater attention from healthcare providers, particularly those who practice spinal manipulation.

The crested porcupine Hystrix cristata is the largest rodent in Italy; it is strictly protected according to national and international laws. Despite this, porcupines in Italy are still poached for meat and complained for crop damages. The species is almost exclusively nocturnal and quite elusive. Its presence may be yet recorded through the detection of quills lost on the ground. In the last 40 years, the crested porcupine showed a remarkable range expansion in Italy. In 2013, in the framework of the www.naturaesocialmapping.it recording site, a web page devoted to H. cristata was set up. The aim of the project was to give people the opportunity to contribute to a continuous updating of the distributional map of this rodent. Samples (quills) were also collected as being easily available sources of DNA. In 2016, some field samplings provided for the first time evidence of the presence of another species of porcupine: the Indian porcupine H. indica. Species identification was carried out through DNA barcoding analysis, since it cannot be performed only through quill morphology. We increased the international genetic database with sequences of both species, so to allow a rapid identification of samples through molecular analyses. Both species were introduced to Italy by man (H. cristata in historical times). Our findings also support the relevance of citizen science contribution in the process of updating species’ distribution range and the potential of spotting new invasive species.

Capitalismo, globalizzazione, donne, genere, lavoro, Sicilia