Whether and under what conditions astrocytes can mount a collective network response has recently become one of the central questions in neurobiology. Here, we address this problem, investigating astrocytic reactions to different biochemical stimuli and ischemic-like conditions in vitro. Identifying an emergent astrocytic network is based on a novel mathematical approach that extracts calcium activity from time-lapse fluorescence imaging and estimates the connectivity of astrocytes. The developed algorithm represents the astrocytic network as an oriented graph in which the nodes correspond to separate astrocytes, and the edges indicate high dynamical correlations between astrocytic events. We demonstrate that ischemic-like conditions decrease network connectivity in primary cultures in vitro, although calcium events persist. Importantly, we found that stimulation under normal conditions with 10 µM ATP increases the number of long-range connections and the degree of corresponding correlations in calcium activity, apart from the frequency of calcium events. This result indicates that astrocytes can form a large functional network in response to certain stimuli. In the post-ischemic interval, the response to ATP stimulation is not manifested, which suggests a deep lesion in functional astrocytic networks. The blockade of Connexin 43 during ischemic modeling preserves the connectivity of astrocytes in the post-hypoxic period.

A novel potent analog of the branched tail oxyquinoline group of hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors, neuradapt, has been studied in two treatment regimes in an in vitro hypoxia model on murine primary hippocampal cultures. Neuradapt activates the expression of HIF1 and HIF2 target genes and shows no toxicity up to 20 μM, which is more than an order of magnitude higher than its biologically active concentration. Cell viability, functional activity, and network connectivity between the elements of neuronal networks have been studied using a pairwise correlation analysis of the intracellular calcium fluctuations in the individual cells. An immediate treatment with 1 μM and 15 μM neuradapt right at the onset of hypoxia not only protects from the death, but also maintains the spontaneous calcium activity in nervous cells at the level of the intact cultures. A similar neuroprotective effect in the post-treatment scenario is observed for 15 μM, but not for 1 μM neuradapt. Network connectivity is better preserved with immediate treatment using 1 μM neuradapt than with 15 μM, which is still beneficial. Post-treatment with neuradapt did not restore the network connectivity despite the observation that neuradapt significantly increased cell viability at 1 μM and functional activity at 15 μM. The preservation of cell viability and functional activity makes neuradapt promising for further studies in a post-treatment scenario, since it can be combined with other drugs and treatments restoring the network connectivity of functionally competent cells.

Research in the past decade on immunogenic cell death (ICD) has shown that the immunogenicity of dying tumor cells is crucial for effective anticancer therapy. ICD induction leads to the emission of specific damage-associated molecular patterns (DAMPs), which act as danger signals and as adjuvants to activate specific anti-tumor immune responses, leading to the elimination of tumor cells and the formation of long-term immunological memory. ICD can be triggered by many anticancer treatment modalities, including photodynamic therapy (PDT). However, due to the variety of photosensitizers used and the lack of a universally adopted PDT protocol, there is a need to develop novel PDT with a proven ICD capability. In the present study, we characterized the abilities of two photoactive dyes to induce ICD in experimental glioma in vitro and in vivo. One dye was from the tetracyanotetra(aryl)porphyrazine group with 9-phenanthrenyl (pz I), and the other was from the 4-(4-fluorobenzyoxy)phenyl (pz III) group in the aryl frame of the macrocycle. We showed that after the photosensitizers penetrated into murine glioma GL261 cells, they localized predominantly in the Golgi apparatus and partially in the endoplasmic reticulum, providing efficient phototoxic activity against glioma GL261 cells upon light irradiation at a dose of 20 J/cm² (λex 630 nm; 20 mW/cm²). We demonstrated that pz I-PDT and pz III-PDT can act as efficient ICD inducers when applied to glioma GL261 cells, facilitating the release of two crucial DAMPs (ATP and HMGB1). Moreover, glioma GL261 cells stimulated with pz I-PDT or pz III-PDT provided strong protection against tumor growth in a prophylactic subcutaneous glioma vaccination model. Finally, we showed that dendritic cell (DC) vaccines pulsed with the lysates of glioma GL261 cells pre-treated with pz-I-PDT or pz-III-PDT could act as effective inducers of adaptive anti-tumor immunity in an intracranial orthotopic glioma mouse model.

The type of cell death has proven to play a crucial role in cancer immunotherapy efficacy. Immunogenic cell death (ICD) enhances tumor adjuvanticity and antigenicity by releasing danger signals and altering the immune peptidome. The immunogenicity of ferroptosis, an iron-dependent form of cell death, remains uncertain. Here, we show that dendritic cell (DC) vaccines loaded with ferroptotic lysates protect mice against glioma growth, inducing IFN-γ production, and promoting robust CD8⁺ T cell infiltration, activation, and effector memory formation in the tumor microenvironment. The intrinsic immunogenicity of ferroptosis was independent of the glioma type and the ferroptosis inducer. Instead, it critically required the presence of the damage-associated molecular patterns calreticulin and ATP, rather than involving HMGB1–TLR4 signaling. However, supplementing these DAMPs into DC vaccines loaded with non-ICD lysates did not restore efficacy to the level of the ferroptosis-based DC vaccine, suggesting a more complex mechanism beyond a purely DAMP-mediated effect. These findings demonstrate that ferroptosis-loaded DC vaccines elicit a potent, tumor-specific immune response, capable of eradicating intracranial gliomas in mice, which highlights their potential in cancer immunotherapy.

Approximately 30% of glioma patients are able to survive beyond one year postdiagnosis. And this short time is often overshadowed by glioma-associated epilepsy. This condition severely impairs the patient’s quality of life and causes great suffering. The genetic, molecular and cellular mechanisms underlying tumour development and epileptogenesis remain incompletely understood, leading to numerous unanswered questions. The various types of gliomas, namely glioblastoma, astrocytoma and oligodendroglioma, demonstrate distinct seizure susceptibility and disease progression patterns. Patterns have been identified in the presence of IDH mutations and epilepsy, with tumour location in cortical regions, particularly the frontal lobe, showing a more frequent association with seizures. Altered expression of TP53, MGMT and VIM is frequently detected in tumour cells from individuals with epilepsy associated with glioma. However, understanding the pathogenesis of these modifications poses a challenge. Moreover, hypoxic effects induced by glioma and associated with the HIF-1a factor may have a significant impact on epileptogenesis, potentially resulting in epileptiform activity within neuronal networks. We additionally hypothesise about how the tumour may affect the functioning of neuronal ion channels and contribute to disruptions in the blood–brain barrier resulting in spontaneous depolarisations.

The current efforts in photodynamic therapy (PDT) of brain cancer are focused on the development of novel photosensitizers with improved photodynamic properties, targeted specific localization, and sensitivity to the irradiation dose, ensuring the effectiveness of PDT with fewer side effects for normal nerve tissue. Here, we characterize the effects of four photosensitizers of the tetracyanotetra(aryl)porphyrazine group (pz I–IV) on the functional activity of neuron-glial networks in primary hippocampal cultures in their application in normal conditions and under PDT. The data revealed that the application of pz I–IV leads to a significant decrease in the main parameters of the functional calcium activity of neuron-glial networks and pronounced changes in the network characteristics. The observed negative effects of pz I–IV were aggravated under PDT. Considering the significant restructuring of the functional architectonics of neuron-glial networks that can lead to severe impairments in synaptic transmission and loss of brain functions, and the feasibility of direct application of PDT based on pz I–IV in the therapy of brain tumors is highly controversial. Nevertheless, the unique properties of pz I–IV retain a great prospect of their use in the therapy of tumors of another origin and cellular metabolism.