Impaired Seroconversion After Severe Acute Respiratory Syndrome Coronavirus 2 mRNA Vaccine in Patients With Thymic Epithelial Tumors.

INTRODUCTION: Thymic epithelial tumors (TETs) are rare malignancies associated with dysregulation of the immune system and humoral- and cell-mediated immunity abnormalities. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccine is effective in preventing coronavirus disease 2019 morbidity and mortality. The aim of this study was to evaluate the seroconversion in patients with TET after two doses of mRNA vaccine. METHODS: This is a prospective study in which consecutive patients with TET were enrolled before receiving the first dose of SARS-CoV-2 mRNA vaccine (BNT162b2 by Pfizer-BioNTech). SARS-CoV-2 spike-binding immunoglobulin (Ig)G antibody serologic levels were analyzed at different time points, including before first vaccine dose (T0), 1 month after the second dose (T2), and 3 months after the second dose (T3). RESULTS: Overall, 39 patients were included in the analysis. All patients had negative antibody titer results at T0. There were 19 patients (48.7%) in the follow-up with no residual tumor lesion/s (referred as no evidence of disease), and 20 (51.3%) had evidence of disease (ED) and were receiving systemic treatment. Dysregulations of the immune system were diagnosed in 29 patients (74.4%) with Good syndrome (GS) being the most frequent immune disorder (48.7%). At univariate analysis, lack of seroconversion at T2 was significantly associated with ED (p < 0.001) and with GS (p = 0.043). A significant association with impaired seroconversion was confirmed at multivariate analysis for ED (p = 0.00101) but not for GS (p = 0.625). CONCLUSIONS: Our data revealed that patients with TET with ED had substantially higher probability of impaired seroconversion after SARS-CoV-2 mRNA vaccine as compared with patients with no evidence of disease.

Metal-Complexes Bearing Releasable CO Differently Modulate Amyloid Aggregation.

Neurodegenerative diseases are often associated with an uncontrolled amyloid aggregation. Hence, many studies are oriented to discover new compounds that are able to modulate self-recognition mechanisms of proteins involved in the development of these pathologies. Herein, three metal-complexes able to release carbon monoxide (CORMs) were analyzed for their ability to affect the self-aggregation of the amyloidogenic fragment of nucleophosmin 1, corresponding to the second helix of the three-helix bundle located in the C-terminal domain of the protein, i.e., NPM1264-277, peptide. These complexes were two cymantrenes coordinated to the nucleobase adenine (Cym-Ade) and to the antibiotic ciprofloxacin (Cym-Cipro) and a Re(I)-compound containing 1,10-phenanthroline and 3-CCCH2NHCOCH2CH2-6-bromo-chromone as ligands (Re-Flavo). Thioflavin T (ThT) assay, UV-vis absorption and fluorescence spectroscopies, scanning electron microscopy (SEM), and electrospray ionization mass spectrometry (ESI-MS) indicated that the three compounds have different effects on the peptide aggregation. Cym-Ade and Cym-Cipro act as aggregating agents. Cym-Ade induces the formation of NPM1264-277 fibers longer and stiffer than that formed by NPM1264-277 alone; irradiation of complexes speeds the formation of fibers that are more flexible and thicker than those found without irradiation. Cym-Cipro induces the formation of longer fibers, although slightly thinner in diameter. Conversely, Re-Flavo acts as an antiaggregating agent. Overall, these results indicate that metal-based CORMs with diverse structural features can have a different effect on the formation of amyloid fibers. A proper choice of ligands attached to metal can allow the development of metal-based drugs with potential application as antiamyloidogenic agents.

Targeting G-quadruplex motifs interferes with differentiation of adipose-derived mesenchymal stem cells.

BACKGROUND: G-quadruplex (G4) motifs are nucleic acid secondary structures observed in mammalian genomes and transcriptomes able to regulate various cellular processes. Several small molecules have been developed so far to modulate G4 stability, frequently associated with anticancer activity. However, how G4 structures are regulated over homeostatic conditions is mostly unexplored. Here, we used human adipose-derived mesenchymal stem cells (ASCs) to address the role of G4 motifs during adipogenic differentiation. METHODS: Adipocyte differentiation of ASCs was investigated in the presence or absence of a well-known G4 ligand, Braco-19. Cell viability was determined by sulforhodamine B assay. Cell dimension and granularity, DNA G4 motifs and cell cycle were detected by flow cytometry. Lipid droplet accumulation was assessed by Oil Red O staining. Cell senescence was evaluated by ß-galactosidase staining. Gene expression was measured by qPCR. Protein release in the extracellular medium was quantified by ELISA. RESULTS: Braco-19 used at non-cytotoxic concentrations induced morphological changes in mature adipocytes partially restoring an undifferentiated-like status. Braco-19 reduced lipid vacuolization and PPARG, AP2, LEP and TNFA mRNA levels in terminally differentiated cells. No effect was observed in cell senescence, fibrotic markers, IL-6 and IL-8 production, while the secretion of VEGF was dose-dependently reduced. Interestingly, G4 structures were increased in differentiated adipocytes compared to their precursors. Braco-19 treatment reduced G4 content in mature adipocytes. CONCLUSIONS: Our data highlight a new role of G4 motifs as genomic structural elements related to human ASC differentiation into mature adipocytes, with potential implications in physio-pathological processes.

History of how viruses can fight cancer: From the miraculous healings to the approval of oncolytic viruses.

Since the nineteenth century, several reports in the historical medical literature emphasized that, occasionally, cancer patients showed a clinical remission, called "Saint Peregrine tumor" as a result of natural infections. Moreover, additional evidence indicated that viruses show a tropism toward cancer cells, leading to the discovery of oncolytic activity of several viruses, called oncolytic viruses (OVs). With the technological and scientific advancements, the advent of rodent models, the establishment of in vitro cell lines, the introduction of methods for virus propagation, several attempts through the 1950s and 1970s have been made to increase OVs specificity, efficacy and safety; however, inconclusive/negative results have been reached and many researchers abandoned the field. Only in the later 1990s, the genetic engineering and the recombinant DNA techniques that allowed the generation of potent, specific and safe OVs and a better understanding of cancer cells renewed the interest in virotherapy. Currently, virotherapy represents a cancer therapeutic strategy based on the use of OVs that selectively infect and lyse cancer cells, without harming normal cells. Over the past years, several "natural" and "genetic engineered" viruses, have been investigated in clinical studies and some of them revealed encouraging results. Recently, the clinical use of OVs has also been supported by the immune stimulatory property of OVs against tumor cells. Here, we analyze the early oncolytic virotherapy before genetic engineering to highlight the relevant progresses reached, and the mechanism to stimulate host immune response, a significant challenge in current virotherapy field.

Insights into Network of Hot Spots of Aggregation in Nucleophosmin 1.

In a protein, point mutations associated with diseases can alter the native structure and provide loss or alteration of functional levels, and an internal structural network defines the connectivity among domains, as well as aggregate/soluble states' equilibria. Nucleophosmin (NPM)1 is an abundant nucleolar protein, which becomes mutated in acute myeloid leukemia (AML) patients. NPM1-dependent leukemogenesis, which leads to its aggregation in the cytoplasm (NPMc+), is still obscure, but the investigations have outlined a direct link between AML mutations and amyloid aggregation. Protein aggregation can be due to the cooperation among several hot spots located within the aggregation-prone regions (APR), often predictable with bioinformatic tools. In the present study, we investigated potential APRs in the entire NPM1 not yet investigated. On the basis of bioinformatic predictions and experimental structures, we designed several protein fragments and analyzed them through typical aggrsegation experiments, such as Thioflavin T (ThT), fluorescence and scanning electron microscopy (SEM) experiments, carried out at different times; in addition, their biocompatibility in SHSY5 cells was also evaluated. The presented data clearly demonstrate the existence of hot spots of aggregation located in different regions, mostly in the N-terminal domain (NTD) of the entire NPM1 protein, and provide a more comprehensive view of the molecular details potentially at the basis of NPMc+-dependent AML.

Immunological signature of patients with thymic epithelial tumors and Good syndrome.

Background: Thymic epithelial tumors (TETs) are frequently accompanied by Good Syndrome (GS), a rare immunodeficiency, characterized by hypogammaglobulinemia and peripheral B cell lymphopenia. TETs can be also associated to other immunological disorders, both immunodeficiency and autoimmunity. Methods: In this study, we enrolled TET patients with GS to address differences between patients with or without associated autoimmune diseases (AD). We analyzed the immunophenotype from peripheral blood of these patients focusing on selected immune cell subsets (CD4+T cells, CD8+T cells, T regulatory cells, NK cells, B-cells, monocytes, eosinophils, basophils, neutrophils) and serum levels of cytokines, chemokines and growth factors. Results: We observed higher number of leucocytes, in particular lymphocytes, B lymphopenia and lower number of T regulatory cells in TET patients with associated AD compared to TET patients without AD. In the group of TET patients with AD, we also observed increased serum levels of IL-15, VEGF, IP-10, GM-CSF, IL-6, and MIP-1α. Thus, we identified considerable differences in the lymphocyte profiles of TET patients with and without ADs, in particular a reduction in the numbers of B lymphocytes and T-regulatory cells in the former, as well as differences in the serum levels of various immune modulators. Conclusions: Although the pathogenic mechanisms are still unclear, our results add new knowledge to better understand the disease, suggesting the need of surveilling the immunophenotype of TET patients to ameliorate their clinical management.

Combination of dl922-947 Oncolytic Adenovirus and G-Quadruplex Binders Uncovers Improved Antitumor Activity in Breast Cancer.

G-quadruplexes (G4s) are nucleic secondary structures characterized by G-tetrads. G4 motif stabilization induces DNA damage and cancer cell death; therefore, G4-targeting small molecules are the focus of clinical investigation. DNA destabilization induced by G4 ligands might potentiate the anticancer activity of agents targeting DNA or inhibiting its repair such as oncolytic viruses. This study represents the first approach combining G4 ligands, BRACO-19 (B19), pyridostatin (PDS), and the adenovirus dl922-947 in breast cancer cells. We demonstrated that G4 binders and dl922-947 induce cytotoxicity in breast cancer cells (MDA-MB-231 and MCF-7) and at higher doses in other neoplastic cell lines of thyroid (BHT-101 cells) and prostate (PC3 cells). G4 binders induce G4 motifs distributed in the S and G2/M phases in MCF-7 cells. G4 binder/dl922-947 combination increases cell cytotoxicity and the accumulation in subG0/G1. Indeed, G4 binders favor viral entry and replication with no effect on coxsackie and adenovirus receptor. Notably, dl922-947 induces G4 motifs and its combination with PDS potentiates this effect in MCF-7 cells. The agents alone or in combination similarly enhanced cell senescence. Additionally, PDS/dl922-947 combination inactivates STING signaling in MDA-MB-231 cells. Our results suggest that G4 binder/virotherapy combination may represent a novel therapeutic anticancer approach.

Synthesis and In Vitro Characterization of Selective Cannabinoid CB2 Receptor Agonists: Biological Evaluation against Neuroblastoma Cancer Cells.

1,8-naphthyridine-3-carboxamide structures were previously identified as a promising scaffold from which to obtain CB2R agonists with anticancer and anti-inflammatory activity. This work describes the synthesis and functional characterization of new 1,8-naphthyridin-2(1H)-one-3-carboxamides with high affinity and selectivity for CB2R. The new compounds were able to pharmacologically modulate the cAMP response without modulating CB2R-dependent ß-arrestin2 recruitment. These structures were also evaluated for their anti-cancer activity against SH-SY5Y and SK-N-BE cells. They were able to reduce the cell viability of both neuroblastoma cancer cell lines with micromolar potency (IC50 of FG158a = 11.8 µM and FG160a = 13.2 µM in SH-SY5Y cells) by a CB2R-mediated mechanism. Finally, in SH-SY5Y cells one of the newly synthesized compounds, FG158a, was able to modulate ERK1/2 expression by a CB2R-mediated effect, thus suggesting that this signaling pathway might be involved in its potential anti-cancer effect.

Glucosyl Platinum(II) Complexes Inhibit Aggregation of the C-Terminal Region of the Aß Peptide.

Neurodegenerative diseases are often caused by uncontrolled amyloid aggregation. Hence, many drug discovery processes are oriented to evaluate new compounds that are able to modulate self-recognition mechanisms. Herein, two related glycoconjugate pentacoordinate Pt(II) complexes were analyzed in their capacity to affect the self-aggregation processes of two amyloidogenic fragments, Aß21-40 and Aß25-35, of the C-terminal region of the ß-amyloid (Aß) peptide, the major component of Alzheimer's disease (AD) neuronal plaques. The most water-soluble complex, 1Ptdep, is able to bind both fragments and to deeply influence the morphology of peptide aggregates. Thioflavin T (ThT) binding assays, electrospray ionization mass spectrometry (ESI-MS), and ultraviolet-visible (UV-vis) absorption spectroscopy indicated that 1Ptdep shows different kinetics and mechanisms of inhibition toward the two sequences and demonstrated that the peptide aggregation inhibition is associated with a direct coordinative bond of the compound metal center to the peptides. These data support the in vitro ability of pentacoordinate Pt(II) complexes to inhibit the formation of amyloid aggregates and pave the way for the application of this class of compounds as potential neurotherapeutics.

The Oncolytic Caprine Herpesvirus 1 (CpHV-1) Induces Apoptosis and Synergizes with Cisplatin in Mesothelioma Cell Lines: A New Potential Virotherapy Approach.

Malignant mesothelioma (MM) is an aggressive asbestos-related cancer, against which no curative modalities exist. Oncolytic virotherapy is a promising therapeutic approach, for which MM is an ideal candidate; indeed, the pleural location provides direct access for the intra-tumoral injection of oncolytic viruses (OVs). Some non-human OVs offer advantages over human OVs, including the non-pathogenicity in humans and the absence of pre-existing immunity. We previously showed that caprine herpesvirus 1 (CpHV-1), a non-pathogenic virus for humans, can kill different human cancer cell lines. Here, we assessed CpHV-1 effects on MM (NCI-H28, MSTO, NCI-H2052) and non-tumor mesothelial (MET-5A) cells. We found that CpHV-1 reduced cell viability and clonogenic potential in all MM cell lines without affecting non-tumor cells, in which, indeed, we did not detect intracellular viral DNA after treatment. In particular, CpHV-1 induced MM cell apoptosis and accumulation in G0/G1 or S cell cycle phases. Moreover, CpHV-1 strongly synergized with cisplatin, the drug currently used in MM chemotherapy, and this agent combination did not affect normal mesothelial cells. Although further studies are required to elucidate the mechanisms underlying the selective CpHV-1 action on MM cells, our data suggest that the CpHV-1-cisplatin combination could be a feasible strategy against MM.

Cross Talk of Macrophages with Tumor Microenvironment Cells and Modulation of Macrophages in Cancer by Virotherapy.

Cellular compartments constituting the tumor microenvironment including immune cells, fibroblasts, endothelial cells, and mesenchymal stromal/stem cells communicate with malignant cells to orchestrate a series of signals that contribute to the evolution of the tumor microenvironment. In this study, we will focus on the interplay in tumor microenvironment between macrophages and mesenchymal stem cells and macrophages and fibroblasts. In particular, cell-cell interaction and mediators secreted by these cells will be examined to explain pro/anti-tumor phenotypes induced in macrophages. Nonetheless, in the context of virotherapy, the response of macrophages as a consequence of treatment with oncolytic viruses will be analyzed regarding their polarization status and their pro/anti-tumor response.

Self-assembly of bio-inspired heterochiral peptides.

Peptide hydrogels, deriving from natural protein fragments, present unique advantages as compatibility and low cost of production that allow their wide application in different fields as wound healing, cell delivery and tissue regeneration. To engineer new biomaterials, the change of the chirality of single amino acids demonstrated a powerful approach to modulate the self-assembly mechanism. Recently we unveiled that a small stretch spanning residues 268-273 in the C-terminal domain (CTD) of Nucleophosmin 1 (NPM1) is an amyloid sequence. Herein, we performed a systematic D-scan of this sequence and analyzed the structural properties of obtained peptides. The conformational and kinetic features of self-aggregates and the morphologies of derived microstructures were investigated by means of different biophysical techniques, as well as the compatibility of hydrogels was evaluated in HeLa cells. All the investigated hexapeptides formed hydrogels even if they exhibited different conformational intermediates during aggregation, and they structural featured are finely tuned by introduced chiralities.

Conformational consequences of NPM1 rare mutations: An aggregation perspective in Acute Myeloid Leukemia.

Often proteins association is a physiological process used by cells to regulate their growth and to adapt to different stress conditions, including mutations. In the case of a subtype of Acute Myeloid Leukemia (AML), mutations of nucleophosmin 1 (NPM1) protein cause its aberrant cytoplasmatic mislocalization (NPMc+). We recently pointed out an amyloidogenic propensity of protein regions including the most common mutations of NPMc+ located in the C-terminal domain (CTD): they were able to form, in vitro, amyloid cytotoxic aggregates with fibrillar morphology. Herein, we analyzed the conformational characteristics of several peptides including rare AML mutations of NPMc+. By means of different spectroscopic, microscopic and cellular assays we evaluated the importance of amino acid composition, among rare AML mutations, to determine amyloidogenic propensity. This study could add a piece of knowledge to the structural consequences of mutations in cytoplasmatic NPM1c+.

A Comparative Study of the Effects of Platinum (II) Complexes on ß-Amyloid Aggregation: Potential Neurodrug Applications.

Herein the effects of three platinum complexes, namely (SP-4-2)-(2,2'-bipyridine)dichloridoplatinum(II), Pt-bpy, (SP-4-2)-dichlorido(1,10-phenanthroline) platinum(II), Pt-phen, and (SP-4-2)-chlorido(2,2':6',2''-terpyridine)platinum(II) chloride, Pt-terpy, on the aggregation of an amyloid model system derived from the C-terminal domain of Aß peptide (Aß21-40) were investigated. Thioflavin T (ThT) binding assays revealed the ability of Pt(II) compounds to repress amyloid aggregation in a dose-dependent way, whereas the ability of Aß21-40 peptide to interfere with ligand field of metal complexes was analyzed through UV-Vis absorption spectroscopy and electrospray ionization mass spectrometry. Spectroscopic data provided micromolar EC50 values and allowed to assess that the observed inhibition of amyloid aggregation is due to the formation of adducts between Aß21-40 peptide and complexes upon the release of labile ligands as chloride and that they can explore different modes of coordination toward Aß21-40 with respect to the entire Aß1-40 polypeptide. In addition, conformational studies through circular dichroism (CD) spectroscopy suggested that Pt-terpy induces soluble ß-structures of monomeric Aß21-40, thus limiting self-recognition. Noticeably, Pt-terpy demonstrated the ability to reduce the cytotoxicity of amyloid peptide in human SH-SY5Y neuroblastoma cells. Presented data corroborate the hypothesis to enlarge the application field of already known metal-based agents to neurodegenerative diseases, as potential neurodrugs.

Pharmacological Inhibition of WEE1 Potentiates the Antitumoral Effect of the dl922-947 Oncolytic Virus in Malignant Mesothelioma Cell Lines.

Malignant mesothelioma (MM) is a very aggressive asbestos-related cancer, for which no therapy proves to be effective. We have recently shown that the oncolytic adenovirus dl922-947 had antitumor effects in MM cell lines and murine xenografts. Previous studies demonstrated that dl922-947-induced host cell cycle checkpoint deregulation and consequent DNA lesions associated with the virus efficacy. However, the cellular DNA damage response (DDR) can counteract this virus action. Therefore, we assessed whether AZD1775, an inhibitor of the G2/M DNA damage checkpoint kinase WEE1, could enhance MM cell sensitivity to dl922-947. Through cell viability assays, we found that AZD1775 synergized with dl922-947 selectively in MM cell lines and increased dl922-947-induced cell death, which showed hallmarks of apoptosis (annexinV-positivity, caspase-dependency, BCL-XL decrease, chromatin condensation). Predictably, dl922-947 and/or AZD1775 activated the DDR, as indicated by increased levels of three main DDR players: phosphorylated histone H2AX (γ-H2AX), phospho-replication protein A (RPA)32, phospho-checkpoint kinase 1 (CHK1). Dl922-947 also increased inactive Tyr-15-phosphorylated cyclin-dependent kinase 1 (CDK1), a key WEE1 substrate, which is indicative of G2/M checkpoint activation. This increase in phospho-CDK1 was effectively suppressed by AZD1775, thus suggesting that this compound could, indeed, abrogate the dl922-947-induced DNA damage checkpoint in MM cells. Overall, our data suggest that the dl922-947-AZD1775 combination could be a feasible strategy against MM.

Proteostasis unbalance of nucleophosmin 1 in Acute Myeloid Leukemia: An aggregomic perspective.

The role exerted by the nucleus in the regulation of proteostasis in both health and disease is recognized of outmost importance, even though not fully understood. Many recent investigations are focused on its ability to modulate and coordinate protein quality control machineries in mammalian cells. Nucleophosmin 1 (NPM1) is one of the most abundant nucleolar proteins and its gene is mutated in ~30% of Acute Myeloid Leukemia (AML) patients. Mutations are localized in the C-terminal domain of the protein and cause cytoplasmatically delocalized and possibly aggregated forms of NPM1 (NPM1c+). Therapeutic interventions targeted on NPM1c+ are in demand and, to this end, deeper knowledge of NPM1c+ behavior in the blasts' cytosol is required. Here by means of complementary biophysical techniques we compared the conformational and aggregative behavior of the entire C-terminal domains of NPM1wt and type A NPM1c+ (bearing the most common mutation). Overall data show that only Cterm_mutA is able to form amyloid-like assemblies with fibrillar morphology and that the oligomers are toxic in human neuroblastoma SHSY cells. This study adds a novel piece of knowledge to the comprehension of the molecular roles exerted by cytoplasmatic NPM1c+ and suggests the exploitation of the amyloidogenic propensity of NPM1c+ as a new strategy for targeting AML with NPM1 mutations.

Tumor-Associated Macrophage Status in Cancer Treatment.

Tumor-associated macrophages (TAMs) represent the most abundant innate immune cells in tumors. TAMs, exhibiting anti-inflammatory phenotype, are key players in cancer progression, metastasis and resistance to therapy. A high TAM infiltration is generally associated with poor prognosis, but macrophages are highly plastic cells that can adopt either proinflammatory/antitumor or anti-inflammatory/protumor features in response to tumor microenvironment stimuli. In the context of cancer therapy, many anticancer therapeutics, apart from their direct effect on tumor cells, display different effects on TAM activation status and density. In this review, we aim to evaluate the indirect effects of anticancer therapies in the modulation of TAM phenotypes and pro/antitumor activity.

Virotherapy: From single agents to combinatorial treatments.

Virotherpay is emerging as a promising strategy against cancer, and three oncolytic viruses (OVs) have gained approval in different countries for the treatment of several cancer types. Beyond the capability to selectively infect, replicate and lyse cancer cells, OVs act through a multitude of events, including modification of the tumour micro/macro-environment as well as a complex modulation of the anti-tumour immune response by activation of danger signals and immunogenic cell death pathways. Most OVs show limited effects, depending on the viral platform and the interactions with the host. OVs used as monotherapy only in a minority of patients elicited a full response. Better outcomes were obtained using OVs in combination with other treatments, such as immune therapy or chemotherapy, suggesting that the full potential of OVs can be unleashed in combination with other treatment modalities. Here, we report the main described combination of OVs with conventional chemotherapeutic agents: platinum salts, mitotic inhibitors, anthracyclines and other antibiotics, anti-metabolites, alkylating agents and topoisomerase inhibitors. Additionally, our work provides an overview of OV combination with targeted therapies: histone deacetylase inhibitors, kinase inhibitors, monoclonal antibodies, inhibitors of DNA repair, inhibitors of the proteasome complex and statins that demonstrated enhanced OV anti-neoplastic activity. Although further studies are required to assess the best combinations to translate the results in the clinic, it is clear that combined therapies, acting with complementary mechanisms of action might be useful to target cancer lesions resistant to currently available treatments.

Reply to Comment on "Malfitano, A.M. et al. Virotherapy as a Potential Therapeutic Approach for the Treatment of Aggressive Thyroid Cancer" Cancers 2019, 11, 1532.

Our work is focused on the future clinical use of oncolytic viruses (OVs) for the treatment of aggressive thyroid carcinomas. Therefore, we provide a brief description of the overall use of OVs in the clinic. Rigvir is among the few OVs that have already been used for the treatment of patients, and studies describing its effects have been briefly commented and cited in our text [1]. [...].

G-Quadruplex Binders Induce Immunogenic Cell Death Markers in Aggressive Breast Cancer Cells.

BACKGROUND: DNA G-quadruplex (G4) structures represent potential anti-cancer targets. In this study, we compared the effect of two G4-targeting compounds, C066-3108 and the gold standard BRACO-19. METHODS: In breast and prostate cancer cells, cytotoxicity induced by both molecules was measured by a sulforhodamine B assay. In breast cancer cells, cycle, apoptosis, the formation of G4 structures, calreticulin and high mobility group box 1 (HMGB1), as well as T cell activation, were analyzed by flow cytometry and adenosine triphosphate (ATP) by luminescence. RESULTS: Both ligands inhibited cell survival and induced DNA damage. In MCF-7 cells, G4 ligands increased the subG0/G1 phase of the cell cycle inducing apoptosis and reduced intracellular ATP. In untreated MCF-7 cells, we observed a slight presence of G4 structures associated with the G2/M phase. In MDA-MB231 cells, G4 ligands decreased the G1 and enhanced the G2/M phase. We observed a decrease of intracellular ATP, calreticulin cell surface exposure and an increase of HMGB1, accompanied by T cell activation. Both compounds induced G4 structure formation in the subG0/G1 phase. CONCLUSIONS: Our data report similar effects for both compounds and the first evidence that G4 ligands induce the release of danger signals associated with immunogenic cell death and induction of T cell activation.