Adoptive cell therapy of triple negative breast cancer with redirected cytokine-induced killer cells

Cytokine-Induced Killer (CIK) cells share several functional and phenotypical properties of both T and natural killer (NK) cells. They represent an attractive approach for cell-based immunotherapy, as they do not require antigen-specific priming for tumor cell recognition, and can be rapidly expanded in vitro. Their relevant expression of Fc\u3b3RIIIa (CD16a) can be exploited in combination with clinical-grade monoclonal antibodies (mAbs) to redirect their lytic activity in an antigen-specific manner. Here, we report the efficacy of this combined approach against triple negative breast cancer (TNBC), an aggressive tumor that still requires therapeutic options. Different primitive and metastatic TNBC cancer mouse models were established in NSG mice, either by implanting patient-derived TNBC samples or injecting MDA-MB-231 cells orthotopically or intravenously. The combined treatment consisted in the repeated intratumoral or intravenous injection of CIK cells and cetuximab. Tumor growth and metastasis were monitored by bioluminescence or immunohistochemistry, and survival was recorded. CIK cells plus cetuximab significantly restrained primitive tumor growth in mice, either in patient-derived tumor xenografts or MDA-MB-231 cell line models. Moreover, this approach almost completely abolished metastasis spreading and dramatically improved survival. The antigen-specific mAb favored tumor and metastasis tissue infiltration by CIK cells, and led to an enrichment of the CD16a+ subset. Data highlight the potentiality of this novel immunotherapy strategy where a nonspecific cytotoxic cell population can be converted into tumor-specific effectors with clinical-grade antibodies, thus providing not only a therapeutic option for TNBC but also a valid alternative to more complex approaches based on chimeric antigen receptor-engineered cells. List of abbreviations: ACT, Adoptive Cell Transfer; ADCC, Antibody-Dependent Cell-mediated Cytotoxicity; ADP, Adenosine diphosphate; BLI, Bioluminescence Imaging; CAR, Chimeric Antigen Receptor; CIK, Cytokine Induced Killer cells; CTX, Cetuximab; DMEM, Dulbecco\u2019s Modified Eagle Medium; EGFR, Human Epidermal Growth Factor 1; ER, Estrogen; FBS, Fetal Bovine Serum; FFPE, Formalin-Fixed Paraffin-Embedded; GMP, Good Manufacturing Practices; GVHD, Graft Versus Host Disease; HER2, Human Epidermal Growth Factor 2; HRP, Horseradish Peroxidase; IFN-\u3b3, Interferon-\u3b3; IHC, Immunohistochemistry; IL-2, Interleukin-2; ISO, Irrelevant antibody; i.t., intratumoral; i.v., intravenous, mAbs, Monoclonal Antibodies; mIHC, Multiplex Fluorescence Immunohistochemistry; MHC, Major Histocompatibility Complex; NK, Natural Killer; NKG2D, Natural-Killer group 2 member D; NSG, NOD/SCID common \u3b3 chain knockout; PARP, Poly ADP-ribose polymerase; PBMCs, Peripheral Blood Mononuclear Cells; PBS, Phosphate-buffered saline; PDX, Patient-derived xenograft; PR, Progesterone; rhIFN-\u3b3, Recombinant Human Interferon-\u3b3; RPMI, Roswell Park Memorial Institute; STR, Short tandem Repeat; TCR, T Cell Receptor; TNBC, Triple Negative Breast Cancer; TSA, Tyramide Signal Amplification

Influence of cover crop termination on ground dwelling arthropods in organic vegetable systems

open7noA key aspect in cover crop management is termination before the cash crop is planted. The aim of this study was to assess the effects of termination methods on ground-dwelling arthropods. The conventional mechanical termination method—i.e., green manuring by means of a disc harrow—was compared to flattening using a roller crimper. Two different crop systems were investigated for two growing seasons; cauliflower was grown in autumn after the termination of a mixture of cowpea, pearl millet, and radish, and tomato was cropped in spring and summer after the termination of a mixture of barley and vetch. Ground beetles (Coleoptera: Carabidae), rove beetles (Coleoptera: Staphylinidae), and spiders (Araneae) were sampled by means of standard pitfall traps throughout the growing season of both cash crops. The roller crimper increased the overall abundance of ground beetles in the first growing season of both cash crops, whereas in the second year, no significant effect could be detected. Rove beetles were more abundant in plots where the cover crops were terminated by the roller crimper. Finally, green manuring increased the abundance of spiders, especially on the first sampling date after cover crop termination. Albeit different taxa showed different responses, the termination of cover crops by a roller crimper generally increased the abundance of ground dwelling arthropods. Given that most of the sampled species were generalist predators, their increased abundance could possibly improve biological control.openDepalo L.; Burgio G.; Magagnoli S.; Sommaggio D.; Montemurro F.; Canali S.; Masetti A.Depalo L.; Burgio G.; Magagnoli S.; Sommaggio D.; Montemurro F.; Canali S.; Masetti A

Ground-dwelling arthropods as biodiversity indicators in maize agroecosystems of Northern Italy

Reliable monitoring of arthropod diversity in a given agroecosystem is essential for the conservation of the related ecosystem services, such as biological control. The often daunting complexity of arthropod collection and identification, however, highlights the need for surrogate taxa that can be easily sampled and be representative of a number of other taxa in term of diversity, general community features and specific composition. In this study, we used pitfall traps to sample three ground-dwelling arthropod taxa important as biocontrol agents (ground beetles, rove beetles and spiders) in 9 conventionally managed maize agroecosystems of Northern Italy over the course of two years, with the goal of characterizing their assemblages and evaluating their reciprocal potential as indicators of activity density, species richness, community turnover and species co-occurrence. Although dominated by few generalist species, sampled arthropod communities were relatively species-rich, and included the first Italian record of the spider Zelotes metellus (Roewer) (Araneae: Gnaphosidae). Ground beetles as a group were confirmed as promising indicators for the species richness and community composition turnover of rove beetles and spiders. Additionally, several abundant arthropod species acted as indicators of the species richness of their respective groups, and the ground beetle Pterostichus macer (Marsham) also worked as an indicator of overall rove beetle activity density. While the co-occurrence of individual arthropod species was limited for the studied taxa, a few species such as the ground beetle Parophonus maculicornis (Duftschmid) did show promise as species-specific bioindicators. Our results could be useful in improving the monitoring and management of these important natural enemies in maize-growing regions

A BARF1-specific mAb as a new immunotherapeutic tool for the management of EBV-related tumors.

The use of monoclonal antibodies (mAb) for the diagnosis and treatment of malignancies is acquiring an increasing clinical importance, thanks to their specificity, efficacy and relative easiness of use. However, in the context of Epstein-Barr virus (EBV)-related malignancies, only cancers of B-cell origin can benefit from therapeutic mAb targeting specific B-cell lineage antigens. To overcome this limitation, we generated a new mAb specific for BARF1, an EBV-encoded protein with transforming and immune-modulating properties. BARF1 is expressed as a latent protein in nasopharyngeal (NPC) and gastric carcinoma (GC), and also in neoplastic B cells mainly upon lytic cycle induction, thus representing a potential target for all EBV-related malignancies. Considering that BARF1 is largely but not exclusively secreted, the BARF1 mAb was selected on the basis of its ability to bind a domain of the protein retained at the cell surface of tumor cells. In vitro, the newly generated mAb recognized the target molecule in its native conformation, and was highly effective in mediating both ADCC and CDC against BARF1-positive tumor cells. In vivo, biodistribution analysis in mice engrafted with BARF1-positive and -negative tumor cells confirmed its high specificity for the target. More importantly, the mAb disclosed a relevant antitumor potential in preclinical models of NPC and lymphoma, as evaluated in terms of both reduction of tumor masses and long-term survival. Taken together, these data not only confirm BARF1 as a promising target for immunotherapeutic interventions, but also pave the way for a successful translation of this new mAb to the clinical use

The mitochondrial calcium uniporter regulates breast cancer progression via HIF-1α

Triple-negative breast cancer (TNBC) represents the most aggressive breast tumor subtype. However, the molecular determinants responsible for the metastatic TNBC phenotype are only partially understood. We here show that expression of the mitochondrial calcium uniporter (MCU), the selective channel responsible for mitochondrial Ca(2+) uptake, correlates with tumor size and lymph node infiltration, suggesting that mitochondrial Ca(2+) uptake might be instrumental for tumor growth and metastatic formation. Accordingly, MCU downregulation hampered cell motility and invasiveness and reduced tumor growth, lymph node infiltration, and lung metastasis in TNBC xenografts. In MCU-silenced cells, production of mitochondrial reactive oxygen species (mROS) is blunted and expression of the hypoxia-inducible factor-1α (HIF-1α) is reduced, suggesting a signaling role for mROS and HIF-1α, downstream of mitochondrial Ca(2+) Finally, in breast cancer mRNA samples, a positive correlation of MCU expression with HIF-1α signaling route is present. Our results indicate that MCU plays a central role in TNBC growth and metastasis formation and suggest that mitochondrial Ca(2+) uptake is a potential novel therapeutic target for clinical intervention

Come to the dark side! The role of functional traits in shaping dark diversity patterns of south-eastern European hoverflies

1. Dark diversity represents the set of species that can potentially inhabit a given area under particular ecological conditions, but are currently 'missing' from a site. This concept allows characterisation of the mechanisms determining why species are sometimes absent from an area that seems ecologically suitable for them. 2. The aim of this study was to determine the dark diversity of hoverflies in south-eastern Europe and to discuss the role of different functional traits that might increase the likelihood of species contributing to dark diversity. Based on expert opinion, the Syrph the Net database and known occurrences of species, the study estimated species pools, and observed and dark diversities within each of 11 defined vegetation types for 564 hoverfly species registered in south-eastern Europe. To detect the most important functional traits contributing to species being in dark diversity across different vegetation types, a random forest algorithm and respective statistics for variable importance were used. 3. The highest dark diversity was found for southwest Balkan sub-Mediterranean mixed oak forest type, whereas the lowest was in Mediterranean mixed forest type. Three larval feeding modes (saproxylic, and phytophagous on bulbs or roots) were found to be most important for determining the probability of a species contributing to hoverfly dark diversity, based on univariate correlations and random forest analysis. 4. This study shows that studying dark diversity might provide important insights into what drives community assembly in south-eastern European hoverflies, especially its missing components, and contributes to more precise conservation prioritisation of both hoverfly species and their habitats.Peer reviewe

Clinical relevance of DNA ploidy and proliferative activity in childhood rhabdomyosarcoma: a retrospective analysis of patients enrolled onto the Italian Cooperative Rhabdomyosarcoma Study RMS88.

Abstract: Purpose: Evaluation of the possible clinical relevance of DNA ploidy and proliferative activity assessed as S-phase fraction (SPF) in childhood rhabdomyosarcoma (RMS). Patients and Methods: We conducted a retrospective study on 59 RMS patients enrolled onto the ICS-RMS88 protocol (seven botryoid, 35 embryonal, and 17 alveolar RMS), for which formalin-fixed paraffin-embedded (FFPE) tissue was available. Nuclear suspensions for cytometric investigation were obtained using a mechanical disaggregation, Tumors were distinguished according to their DNA index (DI) value as follows: diploid (0.9 < DI < 1.1), hyperdiploid (1.1 less than or equal to DI < 1.8 or DI greater than or equal to 2.2), and tetraploid (1.8 less than or equal to DI < 2.2); for analysis of SPF, a cutoff value of 14% was used. Results: DNA histograms were diploid in 19 (33%) cases, hyperdiploid in 29 (49%), and tetraploid in 10 (32%). One patient showed both a hyperdiploid and a tetraploid peek. The 5-year overall survival (OS) rate by ploidy status was 73% in hyperdiploid patients as compared with 33% and 25% in diploid and tetraploid patients, respectively (P = .0012), A striking difference emerged when the 5-year OS for the combined diploid and tetraploid RMS groups was compared with survival of the hyperdiploid RMS group: 30% versus 73%, respectively (P = .0006). In addition, the SPF was prognostically relevant: 5-year OS by SPF less than or greater than 14% was 70% and 36%, respectively (P = .009). Multivariate analysis confirmed the importance of DNA content (P = .0006) and SPF (P = .034) in predicting survival. Conclusion: These findings confirm that ploidy and SPF are important new prognostic factors that are able to identify selected groups of patients at high risk of treatment failure, even if the tumor's presentation is favorable according to standard criteria. (C) 1997 by American Society of Clinical Oncology

Phytophagous hoverflies (Diptera Syrphidae) as indicators of changing landscapes

Spatial and temporal differences in landscape patterns are of considerable interest for understanding ecological processes. In this study, we assessed habitat quality by using the Syrph The Net database and data on decreasing species richness over a 25-year period for the two largest phytophagous hoverfly genera (Merodon and Cheilosia). Furthermore, within this time frame, we explored congruence between ecological responses (species richness and Biodiversity Maintenance Function for these two genera) and landscape structural changes through correlation analysis. Our results indicate that landscapes have experienced changes in aggregation, isolation/connectivity and landscape diversity, with these parameters being significantly correlated with Cheilosia species richness loss and habitat quality. We conclude that the genus Cheilosia is a good bioindicator that can highlight not only the current quality of an area but also temporal changes in landscape patterns.Peer reviewe