Galyna Bila

The air pouch model has emerged as a valuable in vivo platform for investigating the complex interplay between the immune system and tumor development, making it particularly relevant for evaluating novel anticancer immunotherapies. This study highlights the utility of the air pouch model for preclinical assessment of reactive oxygen species (ROS)-enhancing compounds aimed at inducing immunogenic cancer cell death (ICD) and eliciting antitumor immune responses. The air pouch, an experimentally induced subcutaneous cavity, rapidly becomes infiltrated with diverse immune cells, including neutrophils, macrophages, eosinophils, and mast cells, creating a localized environment conducive to studying cellular interactions with administered agents and tumor cells. Injection of monosodium urate crystals (MSU) into the air pouch cavity stimulate abundant neutrophil infilatration and formation of aggregated neutrophil extracellular traps (NETs). Introduction of cancer cells, such as the Nemeth-Kellner (NK/Ly-RB) myeloblastoma cell line, into the air pouch allows for the observation of tumor growth within an immunocompetent host. Here we for the first time demonstrate the dynamics of tumor solification inside the formed airpouch, tumor growth dynamics and morphological changes. Animal survival was also evaluated. Furthermore, the early interaction of immune cells, notably neutrophils, with the inoculated tumor cells and the formation of NETs, cytokine production can be readily assessed in this model. We leveraged the air pouch model to evaluate the efficacy of ROS-modulating anticancer agents with immunotherapeutic potential. For instance, the compound NCure50, a reversible thiol binder that increases lysosomal ROS, demonstrated significant antitumor effects in the NK/Ly-RB air pouch model, extending the lifespan of treated mice and even leading to cures in some cases. Activation of lysosomal ROS resulted in formation of oxidized neoadjuvants. Importantly, surviving mice developed anti-tumor antibodies and exhibited neutrophil-mediated tumor targeting, including NET formation, indicating the induction of systemic and local antitumor immunity [1]. These findings underscore the air pouch model's capacity to provide critical insights into the in vivo efficacy of ROS-enhancing anticancer compounds and their ability to stimulate antitumor immunity. The model allows for the evaluation of local immune responses, such as NET formation, and the subsequent development of systemic immunity, paving the way for the identification and optimization of novel immunotherapeutic strategies against various cancers. Acknowledgement: Current study was supported by EU Horizon2020 FET Open project 861878 NeutroCure.

The unique characteristics of lysosomes in cancer cells, including their number and stability, make them an attractive target for cancer-specific therapies. However, no lysosome-targeting drugs have been approved for clinical use. Here, we describe a novel therapeutic approach utilizing reversible thiol binder 11, which selectively accumulates in the lysosomes of cancer cells. Thiol binder 11 increases lysosomal reactive oxygen species (ROS), leading to lysosomal destabilization, disruption, and subsequent immunogenic cancer cell death—effects that are not observed in healthy cells. In a murine sarcoma Nemeth-Kellner (NK)/Ly-RB model, treatment with thiol binder 11 significantly extended the lifespan of mice from 21 to 85 days and resulted in complete cures in 40% of the cohort. Remarkably, the cured mice developed tumor-specific antibodies, and a subsequent challenge with NK/Ly-RB cells led to 100% survival, compared to 0% in the control group of naïve mice. Ex vivo analyses revealed that neutrophils from the spleens of cured animals contributed to the antitumor response by producing neutrophil extracellular traps (NETs), further supporting the immune-mediated effects. In conclusion, thiol binder 11 exerts a direct antitumor effect, complemented by robust humoral immune responses and primed neutrophil activity, making it a promising candidate for cancer therapy.

The excessive and uncontrolled release of neutrophil extracellular traps (NETs) is increasingly linked to the pathogenesis of various inflammatory diseases, cardiovascular disorders, and cancers. Real-time, non-invasive detection of NETs is crucial for understanding their role in disease progression and developing targeted therapies. Current NETs detection methods often lack the necessary specificity and resolution, particularly in vivo and ex vivo settings. To address this, we have developed novel near-infrared squaraine-peptide conjugates by rational molecular design as reporters of NETosis by targeting the protease activity of neutrophil elastase (NE). These self-quenching, cell-impermeable probes enable the precise real-time detection and imaging of NETs. The Förster resonance energy transfer (FRET)-based probe, Hetero-APA, demonstrated high specificity in detecting NETs in vitro and in vivo, generating strong fluorescence in NETs-rich environments. To overcome the limitations of FRET-based probes for ex vivo imaging, we designed SQ-215-NETP, a non-FRET-based probe that covalently binds to the NE. SQ-215-NETP achieved an unprecedented imaging resolution of 90 nm/pixel in human coronary thrombi, marking the first report of such high resolution with a low molecular weight probe. Additionally, SQ-215-NETP effectively detected NETs by flow cytometry. These results highlight the potential of these probes in NETosis detection, offering promising tools for enhanced diagnostics and therapeutic strategies in managing NET-mediated inflammatory diseases and cancers.

Sepsis is a life-threatening condition with a rising disease burden worldwide. It is a multifactorial disease and is defined as a dysregulated host response to infection. Neutrophils have been shown to be involved in the pathogenesis of sepsis by exacerbating inflammation. However, the exact effector mechanism of action still remains a mystery. Changes in the glycosylation pattern of the immunoglobulin G (IgG) Fc region are described for several diseases including meningococcal sepsis. In this study, we investigated the possible contribution of neutrophils and neutrophil implication, potentially related to degranulation or neutrophil extracellular trap (NET) formation in changing the IgG Fc N-glycosylation pattern in a murine sepsis model. We have measured the serum level of cytokines/chemokines and immunoglobulins, the serum activity of neutrophil elastase (NE), and analyzed the IgG Fc glycosylation pattern by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) and Lectin enzyme-linked immunosorbent assay (ELISA). We observed an increased activity of NE- and neutrophil-associated cytokines such as keratinocyte chemoattractant (KC) with the development of sepsis. Regarding the IgG Fc N-glycosylation, we observed an increase in fucosylation and α1,3-galactosylation and a decrease for sialyation. Interestingly, these changes were not uniform for all IgG subclasses. After depletion of neutrophils, we saw a change in the exposure of fucose and α2,6-linked sialic acid during the time course of our experimental sepsis model. In conclusion, neutrophils can influence changes in the IgG glycosylation pattern in experimental sepsis.

Neutrophil extracellular traps (NETs) have been identified as triggers for a self‐limited inflammatory reaction upon contact with nanoparticles within our bodies. This typically results in entrapping potentially harmful nano‐ or micro‐objects following an immune burst. The demand for potent adjuvants has led to research on particulate‐based adjuvants, particularly those that act via NET formation. Various particles, including hydrophobic nanoparticles, needle‐like microparticles, and other natural and artificial crystals, have been shown to induce NET formation, eliciting a robust humoral and cellular immune response toward co‐injected antigens. The NET formation was found to be the basis of the efficient use of alum as a vaccine adjuvant. Thus, nanoparticles with specific surface properties serve as NET‐stimulating adjuvants. In this mini‐review, we aim to summarize the current knowledge about the surface properties of particulate objects and the molecular pathways involved in inducing NET formation by neutrophils. Additionally, we discuss the potential use of nanoparticles for activating neutrophils in the tissues and the exploitation of such activation for enhancing vaccine adjuvants.

The increasing demand for reliable near-infrared (NIR) probes exhibiting enduring fluorescence in living systems and facile compatibility with biomolecules such as peptides, antibodies or proteins is driven by the increasing use of NIR imaging in clinical diagnostics. To address this demand, a series of carboxy-functionalized unsymmetrical squaraine dyes (SQ-27, SQ-212, and SQ-215) along with non-carboxy-functionalized SQ-218 absorbing and emitting in the NIR wavelength range were designed and synthesized followed by photophysical characterization. This study focused on the impact of structural variations in the alkyl chain length, carboxy functionality positioning, and spacer chain length on dye aggregation and interaction with bovine serum albumin (BSA) as a model protein. In phosphate buffer (PB), the absorption intensity of the dyes markedly decreased accompanied by pronounced shoulders indicative of dye aggregation, and complete fluorescence quenching was seen in contrast to organic solvents. However, in the presence of BSA in PB, there was a enhancement in absorption intensity while regaining the fluorescence coupled with a remarkable increase in the intensity with increasing BSA concentrations, signifying the impact of dye-BSA interactions on preventing aggregation. Further analysis of Job's plot unveiled a 2:1 interaction ratio between BSA and all dyes, while the binding studies revealed a robust binding affinity (K_a) in the order of 10⁷/mol. SQ-212 and SQ-215 were further tested for their in vitro and in vivo imaging capabilities. Notably, SQ-212 demonstrated nonpermeability to cells, while SQ-215 exhibited easy penetration and prominent cytoplasmic localization in in vitro studies. Injection of the dyes into laboratory mice showcased their efficacy in visualization, displaying stable and intense fluorescence in tissues without toxicity, organ damage, or behavioral changes. Thus, SQ-212 and SQ-215 are promising candidates for imaging applications, holding potential for noninvasive cellular and diagnostic imaging as well as biomarker detection when coupled with specific vectors in living systems.

Many known chemotherapeutic anticancer agents exhibit neutropenia as a dose-limiting side effect. In this paper we suggest a prodrug concept solving this problem for camptothecin (HO-cpt). The prodrug is programmed according to Boolean “AND” logic. It requires for its activation the simultaneous presence of triggers T1 (H2O2) and T2 (pH 8 in mitochondria: Mit). The level of H2O2 in cancer cells is higher than that in normal cells. Thus, T1 discriminates cancer cells from the majority of normal cells excluding neutrophils, known to produce elevated levels of H2O2. T2 discriminates cancer cells from neutrophils, since the former cells have a higher number of Mit. We demonstrated that our prodrug exhibits antitumor activity both in vitro and in vivo, but is not toxic to normal cell and neutrophils in contrast to known single trigger prodrugs and the parent drug HO-cpt.

Coronaviruses are sharing several protein regions notable the spike protein (S) on their enveloped membrane surface, with the S1 subunit recognizing and binding to the cellular receptor, while the S2 subunit mediates viral and cellular membrane fusion. This similarity opens the question whether infection with one coronavirus will confer resistance to other coronaviruses? Investigating patient serum samples after SARS-CoV-2 infection in cross-reactivity studies of immunogenic peptides from Middle East respiratory syndrome coronavirus (MERS-CoV), we were able to detect the production of antibodies also recognizing MERS virus antigens. The cross-reactive peptide comes from the heptad repeat 2 (HR2) domain of the MERS virus spike protein. Indeed, the peptide of the HR2 domain of the MERS spike protein, previously proven to induce antibodies against MERS-CoV is sharing 74% homology with the corresponding sequence of SARS-CoV-19 virus. Sera samples of 47 convalescent SARS-CoV-2 patients, validated by RT-PCR-negative testes 30 days post-infection, and samples of 40 sera of control patients (not infected with SARS-CoV-2 previously) were used to establish eventual cross-bind reactivity with the MERS peptide antigen. Significantly stronger binding (p< 0.0001) was observed for IgG antibodies in convalescent SARS-CoV-2 patients compared to the control group. If used as an antigen, the peptide of the HR2 domain of the MERS spike protein allows discrimination between post-Covid populations from non-infected ones by the presence of antibodies in blood samples. This suggests that polyclonal antibodies established during SARS-CoV-2 infection has the ability to recognize and probably decrease infectiveness of MERS-CoV infections as well as other coronaviruses. The high homology of the spike protein domain suggests in addition that the opposite effect can also be true: coronaviral infections producing cross-reactive antibodies affective against SARS-CoV-19. The collected data prove in addition that despite the core HR2 region being hidden in the native viral conformation, its exposure during cell entry makes it highly immunogenic. Since inhibitory peptides to this region were previously described, this opens new possibilities in fighting coronaviral infections.