A Proteomics Approach Identifies RREB1 as a Crucial Molecular Target of Imidazo-Pyrazole Treatment in SKMEL-28 Melanoma Cells.

Cutaneous melanoma is the most dangerous and deadly form of human skin malignancy. Despite its rarity, it accounts for a staggering 80% of deaths attributed to cutaneous cancers overall. Moreover, its final stages often exhibit resistance to drug treatments, resulting in unfavorable outcomes. Hence, ensuring access to novel and improved chemotherapeutic agents is imperative for patients grappling with this severe ailment. Pyrazole and its fused systems derived thereof are heteroaromatic moieties widely employed in medicinal chemistry to develop effective drugs for various therapeutic areas, including inflammation, pain, oxidation, pathogens, depression, and fever. In a previous study, we described the biochemical properties of a newly synthesized group of imidazo-pyrazole compounds. In this paper, to improve our knowledge of the pharmacological properties of these molecules, we conduct a differential proteomic analysis on a human melanoma cell line treated with one of these imidazo-pyrazole derivatives. Our results detail the changes to the SKMEL-28 cell line proteome induced by 24, 48, and 72 h of 3e imidazo-pyrazole treatment. Notably, we highlight the down-regulation of the Ras-responsive element binding protein 1 (RREB1), a member of the zinc finger transcription factors family involved in the tumorigenesis of melanoma. RREB1 is a downstream element of the MAPK pathway, and its activation is mediated by ERK1/2 through phosphorylation.

Lipid Metabolism Reprogramming and Trastuzumab Resistance in Breast Cancer Cell Lines Overexpressing the ERBB2 Membrane Receptor.

Trastuzumab (Tz), an antibody targeting ERBB2, has significantly improved the prognosis for breast cancer (BCa) patients with overexpression of the ERBB2 receptor. However, Tz resistance poses a challenge to patient outcomes. Numerous mechanisms have been suggested to contribute to Tz resistance, and this study aimed to uncover shared mechanisms in in vitro models of acquired BCa Tz resistance. Three widely used ERBB2+ BCa cell lines, adapted to grow in Tz, were examined. Despite investigating potential changes in phenotype, proliferation, and ERBB2 membrane expression in these Tz-resistant (Tz-R) cell lines compared to wild-type (wt) cells, no common alterations were discovered. Instead, high-resolution mass spectrometry analysis revealed a shared set of differentially expressed proteins (DEPs) in Tz-R versus wt cells. Bioinformatic analysis demonstrated that all three Tz-R cell models exhibited modulation of proteins associated with lipid metabolism, organophosphate biosynthesis, and macromolecule methylation. Ultrastructural examination corroborated the presence of altered lipid droplets in resistant cells. These findings strongly support the notion that intricate metabolic adaptations, including lipid metabolism, protein phosphorylation, and potentially chromatin remodeling, may contribute to Tz resistance. The detection of 10 common DEPs across all three Tz-resistant cell lines offers promising avenues for future therapeutic interventions, providing potential targets to overcome Tz resistance and potentially improve patient outcomes in ERBB2+ breast cancer.

Fraisinib: a calixpyrrole derivative reducing A549 cell-derived NSCLC tumor in vivo acts as a ligand of the glycine-tRNA synthase, a new molecular target in oncology.

Background and purpose: Lung cancer is the leading cause of death in both men and women, constituting a major public health problem worldwide. Non-small-cell lung cancer accounts for 85%-90% of all lung cancers. We propose a compound that successfully fights tumor growth in vivo by targeting the enzyme GARS1. Experimental approach: We present an in-depth investigation of the mechanism through which Fraisinib [meso-(p-acetamidophenyl)-calix(4)pyrrole] affects the human lung adenocarcinoma A549 cell line. In a xenografted model of non-small-cell lung cancer, Fraisinib was found to reduce tumor mass volume without affecting the vital parameters or body weight of mice. Through a computational approach, we uncovered that glycyl-tRNA synthetase is its molecular target. Differential proteomics analysis further confirmed that pathways regulated by Fraisinib are consistent with glycyl-tRNA synthetase inhibition. Key results: Fraisinib displays a strong anti-tumoral potential coupled with limited toxicity in mice. Glycyl-tRNA synthetase has been identified and validated as a protein target of this compound. By inhibiting GARS1, Fraisinib modulates different key biological processes involved in tumoral growth, aggressiveness, and invasiveness. Conclusion and implications: The overall results indicate that Fraisinib is a powerful inhibitor of non-small-cell lung cancer growth by exerting its action on the enzyme GARS1 while displaying marginal toxicity in animal models. Together with the proven ability of this compound to cross the blood-brain barrier, we can assess that Fraisinib can kill two birds with one stone: targeting the primary tumor and its metastases "in one shot." Taken together, we suggest that inhibiting GARS1 expression and/or GARS1 enzymatic activity may be innovative molecular targets for cancer treatment.

Regioselective Synthesis, Structural Characterization, and Antiproliferative Activity of Novel Tetra-Substituted Phenylaminopyrazole Derivatives.

A small library of highly functionalized phenylaminopyrazoles, bearing different substituents at position 1, 3, and 4 of the pyrazole ring, was prepared by the one-pot condensation of active methylene reagents, phenylisothiocyanate, and substituted hydrazine (namely, methyl- and benzyl-hydrazine). The identified reaction conditions proved to be versatile and efficient. Furthermore, the evaluation of alternative stepwise protocols affected the chemo- and regio-selectivity outcome of the one-pot procedure. The chemical identities of two N-methyl pyrazole isomers, selected as prototypes of the whole series, were unambiguously identified by means of NMR and mass spectrometry studies. Additionally, semiempirical calculations provided a structural rationale for the different chromatographic behavior of the two isomers. The prepared tetra-substituted phenylaminopyrazoles were tested in cell-based assays on a panel of cancer and normal cell lines. The tested compounds did not show any cytotoxic effect on the selected cell lines, thus supporting their pharmaceutical potentials.

A Circulating Risk Score, Based on Combined Expression of Exo-miR-130a-3p and Fibrinopeptide A, as Predictive Biomarker of Relapse in Resectable Non-Small Cell Lung Cancer Patients.

To date, the 5-year overall survival rate of 60% for early-stage non-small cell lung cancer (NSCLC) is still unsatisfactory. Therefore, reliable prognostic factors are needed. Growing evidence shows that cancer progression may depend on an interconnection between cancer cells and the surrounding tumor microenvironment; hence, circulating molecules may represent promising markers of cancer recurrence. In order to identify a prognostic score, we performed in-depth high-throughput analyses of plasma circulating markers, including exosomal microRNAs (Exo-miR) and peptides, in 67 radically resected NSCLCs. The miRnome profile selected the Exo-miR-130a-3p as the most overexpressed in relapsed patients. Peptidome analysis identified four progressively more degraded forms of fibrinopeptide A (FpA), which were depleted in progressing patients. Notably, stepwise Cox regression analysis selected Exo-miR-130a-3p and the greatest FpA (2-16) to build a score predictive of recurrence, where high-risk patients had 18 months of median disease-free survival. Moreover, in vitro transfections showed that higher levels of miR-130a-3p lead to a deregulation of pathways involved in metastasis and angiogenesis, including the coagulation process and metalloprotease increase which might be linked to FpA reduction. In conclusion, by integrating circulating markers, the identified risk score may help clinicians predict early-stage NSCLC patients who are more likely to relapse after primary surgery.

Blocking glutamate mGlu5 receptors with the negative allosteric modulator CTEP improves disease course in SOD1G93A mouse model of amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: The pathogenesis of amyotrophic lateral sclerosis (ALS) is not fully clarified, although excessive glutamate (Glu) transmission and the downstream cytotoxic cascades are major mechanisms for motor neuron death. Two metabotropic glutamate receptors (mGlu1 and mGlu5 ) are overexpressed in ALS and regulate cellular disease processes. Expression and function of mGlu5 receptors are altered at early symptomatic stages in the SOD1G93A mouse model of ALS and knockdown of mGlu5 receptors in SOD1G93A mice improved disease progression. EXPERIMENTAL APPROACH: We treated male and female SOD1G93A mice with 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP), an orally available mGlu5 receptor negative allosteric modulator (NAM), using doses of 2 mg·kg-1 per 48 h or 4 mg·kg-1 per 24 h from Day 90, an early symptomatic disease stage. Disease progression was studied by behavioural and histological approaches. KEY RESULTS: CTEP dose-dependently ameliorated clinical features in SOD1G93A mice. The lower dose increased survival and improved motor skills in female mice, with barely positive effects in male mice. Higher doses significantly ameliorated disease symptoms and survival in both males and females, females being more responsive. CTEP also reduced motor neuron death, astrocyte and microglia activation, and abnormal glutamate release in the spinal cord, with equal effects in male and female mice. No differences were also observed in CTEP access to the brain. CONCLUSION AND IMPLICATIONS: Our results suggest that mGlu5 receptors are promising targets for the treatment of ALS and highlight mGlu5 receptor NAMs as effective pharmacological tools with translational potential.

GeenaR: A Web Tool for Reproducible MALDI-TOF Analysis.

Mass spectrometry is a widely applied technology with a strong impact in the proteomics field. MALDI-TOF is a combined technology in mass spectrometry with many applications in characterizing biological samples from different sources, such as the identification of cancer biomarkers, the detection of food frauds, the identification of doping substances in athletes' fluids, and so on. The massive quantity of data, in the form of mass spectra, are often biased and altered by different sources of noise. Therefore, extracting the most relevant features that characterize the samples is often challenging and requires combining several computational methods. Here, we present GeenaR, a novel web tool that provides a complete workflow for pre-processing, analyzing, visualizing, and comparing MALDI-TOF mass spectra. GeenaR is user-friendly, provides many different functionalities for the analysis of the mass spectra, and supports reproducible research since it produces a human-readable report that contains function parameters, results, and the code used for processing the mass spectra. First, we illustrate the features available in GeenaR. Then, we describe its internal structure. Finally, we prove its capabilities in analyzing oncological datasets by presenting two case studies related to ovarian cancer and colorectal cancer. GeenaR is available at http://proteomics.hsanmartino.it/geenar/.

Bicyclic Basic Merbarone Analogues as Antiproliferative Agents.

Pyrimido-pyrimidine derivatives have been developed as rigid merbarone analogues. In a previous study, these compounds showed potent antiproliferative activity and efficiently inhibited topoisomerase IIα. To further extend the structure-activity relationships on pyrimido-pyrimidines, a novel series of analogues was synthesized by a two-step procedure. Analogues 3-6 bear small alky groups at positions 1 and 3 of the pyrimido-pyrimidine scaffold whereas at position 6a (4-chloro)phenyl substituent was inserted. The basic side chains introduced at position 7 were selected on the basis of the previously developed structure-activity relationships. The antiproliferative activity of the novel compounds proved to be affected by both the nature of the basic side chain and the substituents on the pyrimido-pyrimidine moiety. Derivatives 5d and 5e were identified as the most promising molecules still showing reduced antiproliferative activity in comparison with the previously prepared pyrimido-pyrimidine analogues. In topoisomerase IIα-5d docking complex, the ligand would poorly interact with the enzyme and assume a different orientation in comparison with 1d bioactive conformation.

The Activation of Prothrombin Seems to Play an Earlier Role than the Complement System in the Progression of Colorectal Cancer: A Mass Spectrometry Evaluation.

Colorectal cancer (CRC) is the second cause of death in men and the third in women. This work deals with the study of the low molecular weight protein fraction of sera from patients who underwent surgery for CRC and who were followed for several years thereafter. MALDI-TOF MS was used to identify serum peptidome profiles of healthy controls, non-metastatic CRC patients and metastatic CRC patients. A multiple regression model was applied to signals preliminarily selected by SAM analysis to take into account the age and gender differences between the groups. We found that, while a signal m/z 2021.08, corresponding to the C3f fragment of the complement system, appears significantly increased only in serum from metastatic CRC patients, a m/z 1561.72 signal, identified as a prothrombin fragment, has a significantly increased abundance in serum from non-metastatic patients as well. The findings were also validated by a bootstrap resampling procedure. The present results provide the basis for further studies on large cohorts of patients in order to confirm C3f and prothrombin as potential serum biomarkers. Thus, new and non-invasive tests might be developed to improve the classification of colorectal cancer.

Fibrinogen αC-regions are not directly involved in fibrin polymerization as evidenced by a "Double-Detroit" recombinant fibrinogen mutant and knobs-mimic peptides.

BACKGROUND: Fibrin polymerization, following fibrinopeptides A and B (FpA, FpB) cleavage, relies on newly exposed α- and ß-chains N-termini (GPR, GHR; A-, B-knobs, respectively) engaging preexistent a and b pockets in other fibrin(ogen) molecules' γ- and (B)ß-chains C-terminal regions. A role for mostly disordered (A)α-chains C-terminal regions "bridging" between fibrin molecules/fibrils has been proposed. OBJECTIVES: Fibrinogen Detroit is a clinically observed mutation (AαR19 â†’ S) with nonengaging GPS A-knobs. By analogy, a similar Bß-chain mutation, BßR17 â†’ S, should produce nonengaging GHS B-knobs. A homozygous "Double-Detroit" mutant (AαR19 â†’ S, BßR17 â†’ S; DD-FG) was developed: with A-a and B-b engagements endogenously blocked, other interactions would become apparent. METHODS: DD-FG, wild-type recombinant (WT-FG), and human plasma (hp-FG) fibrinogen self-association was studied by turbidimetry coupled with fibrinopeptides release high-performance liquid chromatography (HPLC)/mass spectrometry analyses, and by light-scattering following size-exclusion chromatography (SE-HPLC). RESULTS: In contrast to WT-FG and hp-FG, DD-FG produced no turbidity increase, irrespective of thrombin concentration. The SE-HPLC profile of concentrated DD-FG was unaffected by thrombin treatment, and light-scattering, at lower concentration, showed no intensity and hydrodynamic radius changes. Compared with hp-FG, both WT-FG and DD-FG showed no FpA cleavage difference, while ~50% FpB was not recovered. Correspondingly, SDS-PAGE/Western-blots revealed partial Bß-chain N-terminal and Aα-chain C-terminal degradation. Nevertheless, ~70% DD-FG molecules bearing (A)αC-regions potentially able to associate were available. Higher-concentration, nearly intact hp-FG with 500-fold molar excess GPRP-NH2 /GHRP-NH2 knobs-mimics experiments confirmed these no-association findings. CONCLUSIONS: (A)αC-regions interactions appear too weak to assist native fibrin polymerization, at least without knobs engagement. Their role in all stages should be carefully reconsidered.

Fibrin Gels Entrapment of a Poly-Cyclodextrin Nanocarrier as a Doxorubicin Delivery System in an Orthotopic Model of Neuroblastoma: Evaluation of In Vitro Activity and In Vivo Toxicity.

PURPOSE: Fibrin gels (FBGs) are potential delivery vehicles for many drugs, and can be easily prepared from purified components. We previously demonstrated their applicability for the release of different doxorubicin (Dox) nanoparticles used clinically or in an experimental stage, such as its inclusion complex with the amino ß-cyclodextrin polymer (oCD-NH2/Dox). Here we extend these studies by in vitro and in vivo evaluations. METHODS: An in vitro cytotoxicity model consisting of an overlay of a neuroblastoma (NB) cell-containing agar layer above a drug-loaded FBG layer was used. Local toxicity in vivo (histology and blood analysis) was studied in a mouse orthotopic NB model (SHSY5YLuc+ cells implanted into the left adrenal gland). RESULTS: In vitro data show that FBGs loaded with oCD-NH2/Dox have a slightly lower cytotoxicity against NB cell lines than those loaded with Dox. Fibrinogen (FG), and Ca2+ concentrations may modify this activity. In vivo data support a lower general and local toxicity for FBGs loaded with oCD-NH2/Dox than those loaded with Dox. CONCLUSION: Our results suggest a possible increase of the therapeutic index of Dox when locally administered through FBGs loaded with oCD-NH2/Dox, opening the possibility of using these releasing systems for the treatment of neuroblastoma.

Author Correction: Faithful animal modelling of human glioma by using primary initiating cells and its implications for radiosensitization therapy.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Faithful animal modelling of human glioma by using primary initiating cells and its implications for radiosensitization therapy [ARRIVE 1].

It has been reported that the ATM kinase inhibitor KU60019 preferentially radiosensitizes orthotopic high grade gliomas (HGG) driven by established U87 and U1242 cell lines bearing specific TP53 mutations. We wished to determine whether those results could be extended to tumors driven by primary glioma initiating cells (GIC) that closely mimic clinical tumors. Orthotopic HGG were developed in immunodeficient non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice by intracranial injection of primary GIC isolated from the adult glioblastoma COMI (acronym of patient's name) and the pediatric anaplastic astrocytoma 239/12. Similar to the clinical tumors of origin, the orthotopic tumors COMI and 239/12 displayed different growth properties with a voluminous expansive lesion that exerted considerable mass effect on the adjacent structures and an infiltrating, gliomatosis-like growth pattern with limited compressive attitude, respectively. Significant elongations of median animal survival bearing the adult COMI tumor was observed after one KU60019 convection enhanced delivery followed by total 7.5 Gy of ionizing radiation delivered in fifteen 0.5 Gy fractions, as compared to animals treated with vehicle + ionizing radiation (105 vs 89 days; ratio: 0.847; 95% CI of ratio 0.4969 to 1.198; P:0.0417) [ARRIVE 16]. Similarly, a trend to increased median survival was observed with the radiosensitized pediatric tumor 239/12 (186 vs 167 days; ratio: 0.8978; 95% CI of ratio: 0.5352 to 1.260; P: 0.0891) [ARRIVE 16]. Our results indicate that radiosensitization by KU60019 is effective towards different orthotopic gliomas that faithfully mimic the clinical tumors and that multiple GIC-based animal models may be essential to develop novel therapeutic protocols for HGG transferable to the clinics.

A novel calix[4]pyrrole derivative as a potential anticancer agent that forms genotoxic adducts with DNA.

meso-(p-acetamidophenyl)-calix[4]pyrrole 3 was found to exhibit remarkable cytotoxicity towards A549 cancer cells. A comparative study including the isomer of 3 meso-(m-acetamidophenyl)-calix[4]pyrrole 5, as well as molecules containing 'fragments' of these structures, demonstrated that both the calix[4]pyrrole and the acetamidophenyl units are essential for high cytotoxicity. Although calix[4]pyrroles and other anion-complexing ionophores have recently been reported to induce apoptosis by perturbing cellular chloride concentrations, in our study an alternative mechanism has emerged, as proven by the isolation of covalent DNA adducts revealed by the 32P postlabelling technique. Preliminary pharmacokinetic studies indicate that 3 is able to cross the Blood-Brain-Barrier, therefore being a potential drug that could kill primary and brain metastatic cancer cells simultaneously.

Characterization of drug release from fibrin gels loaded with different pharmaceutical and experimental doxorubicin formulations.

BACKGROUND: Local delivery of anticancer drugs represents a desirable type of treatment. Nevertheless, characteristics such as availability, biocompatibility, ease of operation, and efficacy sometimes represent difficult to overcome hurdles. Fibrin gels (FBGs) may be attractive biomaterials for local treatment when loaded with different chemotherapeutics or with polymer-anticancer-drug conjugates and nanoparticles. These components, linked together, might represent candidates to counteract local recurrences or reduce the volume of inoperable tumors. In the present study we analyzed the features of in vitro release of different formulations of doxorubicin (DOXO) from FBGs, and in vivo FBGs degradation. METHODS: In vitro DOXO release from FBGs was studied as a function of thrombin and Ca2+ ion concentrations. DOXO was loaded in FBGs either alone or pre-incorporated in nanoparticles characterized by different physical features. The FBGs in vivo degradation was analyzed after sc or ip positioning. RESULTS: Our results suggest that each of the factors involved in the FBGs preparation may have different effects on drug release. In particular, the fibrinogen (FG) concentration and, above all, the DOXO formulation were found to have the greatest impact. Not surprisingly, we have also found a longer permanence in vivo of FBGs prepared at the highest thrombin, Ca2+ ion, and FG concentrations. CONCLUSIONS: The aim of this work was to study the effect of several conditions for preparing drug delivery systems based on FBGs loaded with different clinical or experimental formulations of DOXO. Our data identify some of these modalities that will be tested in vivo to evaluate their antitumor activity.

ATR kinase inhibitors NVP-BEZ235 and AZD6738 effectively penetrate the brain after systemic administration.

Ataxia Telangiectasia and Rad3 related protein (ATR) is a central mediator of the response to DNA damage that may cause the quiescent resistance of cancer initiating cells to genotoxic radiotherapy. NVP-BEZ235 is a dual PI3K/mTOR inhibitor that also effectively targets ATR with IC50 = 21 × 10- 9 M in cells. AZD6738 does not target significantly PI3K/mTOR-related kinases but specifically inhibits ATR with IC50 = 74 × 10- 9 M in cells. Both drugs have been proposed as radiosensitizers of different tumors including glioblastoma (GB), the most malignant brain tumor. In order to study the radiosensitizing properties of ATR inhibitors NVP-BEZ235 and AZD6738 towards GB, we have preliminarily investigated their capacity to penetrate the brain after systemic administration. Tumor-free CD-1 mice were inoculated i.p. with 25 mg/Kg body weight of NVP-BEZ235 or AZD6738. 1, 2, 6 and 8 h later, blood was collected by retro-orbital bleeding after which the mice were euthanized and the brains explanted. Blood and brain samples were then extracted and NVP-BEZ235 and AZD6738 concentrations determined by High Performance Liquid Chromatography/Mass Spectrometry. We found for NVP-BEZ235 and especially for AZD6738, elevated bioavailability and effective brain penetration after intraperitoneal administration. Albeit low drug and radiation dosages were used, a trend to toxicity of NVP-BEZ235 followed by ionizing radiation (IR) towards mice bearing primary glioma initiating cells (GIC)-driven orthotopic tumors was yet observed, as compared to AZD6738 + IR and vehicle+IR. Survival was never improved with median values of 99, 86 and 101 days for vehicle+IR, NVP-BEZ235 + IR and AZD6738 + IR-treated mice, respectively. Although the present results indicate favorable pharmacokinetics properties of ATR inhibitors NVP-BEZ235 and AZD6738, they do not lend support to their use as radiosensitizers of GB.

Small molecules targeting histone demethylase genes (KDMs) inhibit growth of temozolomide-resistant glioblastoma cells.

In glioblastoma several histone demethylase genes (KDM) are overexpressed compared to normal brain tissue and the development of Temozolomide (TMZ) resistance is accompanied by the transient further increased expression of KDM5A and other KDMs following a mechanism that we defined as "epigenetic resilience". We hypothesized that targeting KDMs may kill the cells that survive the cytotoxic therapy.We determined the effect of JIB 04 and CPI-455, two KDM inhibitors, on glioblastoma cells and found that both molecules are more effective against TMZ-resistant rather than native cells.Because of its lower IC50, we focused on JIB 04 that targets KDM5A and other KDMs as well. We have shown that this molecule activates autophagic and apoptotic pathways, interferes with cell cycle progression, inhibits cell clonogenicity and dephosphorylates Akt thus inactivating a potent pro-survival pathway. We performed combination temozolomide/JIB 04 in vitro treatments showing that these two molecules, under certain conditions, have a strong synergic effect and we hypothesize that JIB 04 intercepts the cells that escape the G2 block exerted by TMZ. Finally we studied the permeability of JIB 04 across the blood-brain barrier and found that this molecule reaches bioactive concentration in the brain; furthermore a pilot in vivo experiment in an orthotopic GB xenograft model showed a trend toward longer survival in treated mice with an Hazard Ratio of 0.5.In conclusion we propose that the combination between cytotoxic drugs and molecules acting on the epigenetic landscape may offer the opportunity to develop new therapies for this invariably lethal disease.

Zoledronate can induce colorectal cancer microenvironment expressing BTN3A1 to stimulate effector γδ T cells with antitumor activity.

Amino-bis-phosphonates (N-BPs) such as zoledronate (Zol) have been used in anticancer clinical trials due to their ability to upregulate pyrophosphate accumulation promoting antitumor Vγ9Vδ2 T cells. The butyrophilin 3A (BTN3A, CD277) family, mainly the BTN3A1 isoform, has emerged as an important structure contributing to Vγ9Vδ2 T cells stimulation. It has been demonstrated that the B30.2 domain of BTN3A1 can bind phosphoantigens (PAg) and drive the activation of Vγ9Vδ2 T cells through conformational changes of the extracellular domains. Moreover, BTN3A1 binding to the cytoskeleton, and its consequent membrane stabilization, is crucial to stimulate the PAg-induced tumor cell reactivity by human Vγ9Vδ2 T cells. Aim of this study was to investigate the relevance of BTN3A1 in N-BPs-induced antitumor response in colorectal cancer (CRC) and the cell types involved in the tumor microenvironment. In this paper, we show that (i) CRC, exposed to Zol, stimulates the expansion of Vδ2 T lymphocytes with effector memory phenotype and antitumor cytotoxic activity, besides sensitizing cancer cells to γδ T cell-mediated cytotoxicity; (ii) this effect is partially related to BTN3A1 expression and in particular with its cellular re-distribution in the membrane and cytoskeleton-associated fraction; (iii) BTN3A1 is detected in CRC at the tumor site, both on epithelial cells and on tumor-associated fibroblasts (TAF), close to areas infiltrated by Vδ2 T lymphocytes; (iv) Zol is effective in stimulating antitumor effector Vδ2 T cells from ex-vivo CRC cell suspensions; and (v) both CRC cells and TAF can be primed by Zol to trigger Vδ2 T cells.

Geena 2, improved automated analysis of MALDI/TOF mass spectra.

BACKGROUND: Mass spectrometry (MS) is producing high volumes of data supporting oncological sciences, especially for translational research. Most of related elaborations can be carried out by combining existing tools at different levels, but little is currently available for the automation of the fundamental steps. For the analysis of MALDI/TOF spectra, a number of pre-processing steps are required, including joining of isotopic abundances for a given molecular species, normalization of signals against an internal standard, background noise removal, averaging multiple spectra from the same sample, and aligning spectra from different samples. In this paper, we present Geena 2, a public software tool for the automated execution of these pre-processing steps for MALDI/TOF spectra. RESULTS: Geena 2 has been developed in a Linux-Apache-MySQL-PHP web development environment, with scripts in PHP and Perl. Input and output are managed as simple formats that can be consumed by any database system and spreadsheet software. Input data may also be stored in a MySQL database. Processing methods are based on original heuristic algorithms which are introduced in the paper. Three simple and intuitive web interfaces are available: the Standard Search Interface, which allows a complete control over all parameters, the Bright Search Interface, which leaves to the user the possibility to tune parameters for alignment of spectra, and the Quick Search Interface, which limits the number of parameters to a minimum by using default values for the majority of parameters. Geena 2 has been utilized, in conjunction with a statistical analysis tool, in three published experimental works: a proteomic study on the effects of long-term cryopreservation on the low molecular weight fraction of serum proteome, and two retrospective serum proteomic studies, one on the risk of developing breat cancer in patients affected by gross cystic disease of the breast (GCDB) and the other for the identification of a predictor of breast cancer mortality following breast cancer surgery, whose results were validated by ELISA, a completely alternative method. CONCLUSIONS: Geena 2 is a public tool for the automated pre-processing of MS data originated by MALDI/TOF instruments, with a simple and intuitive web interface. It is now under active development for the inclusion of further filtering options and for the adoption of standard formats for MS spectra.