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Introduction: Hypoxia can drive tumor progression, suppress anti-tumor immunity, and reduce the effectiveness of radiotherapy and chemotherapy. This study aimed to assess the impact of remote ischemic conditioning (RIC) on tumor oxygenation (sO2) and the anti-tumor immune response. Material and methods: Fourteen B16-Ova tumor-bearing C57BL/6N mice received six 5-minute RIC cycles, while another fourteen underwent anesthesia only. Pimonidazole was administered 1.5 hours before sacrifice. Blood flow, sO2, and hemoglobin levels were measured in the non-ischemic hind limb and tumor. Tumor hypoxia was assessed using pimonidazole and CA IX immunohistochemistry, and T cell infiltration by CD3 and FoxP3 staining. Serum levels of 23 cytokines were analyzed using a multiplex immunoassay. Results: Isoflurane anesthesia caused a slight intraindividual increase in blood flow (p = 0.07) and sO2 (p = 0.06) of the hind limb and a sole increase in tumor sO2 (p = 0.035), whereas RIC improved sO2 of the tumor in relation to the hind limb (p=0.03). The median of the tumor oxygen saturation reached 51.4% in the control group and 62.7% in the RIC group (p = 0.09), exhibiting a slight tendency towards better oxygenation in the RIC group. Pimonidazole (p=0.24) and CA IX hypoxia score (p=0.48) did not reveal statistically significant differences between the two groups. In RIC-treated tumors, the number of CD3 (p=0.006), but not FoxP3- positive cells (p = 0.84), in the tumor core was significantly higher compared to the control group. In the RIC group, the mean fluorescence intensity (MFI) of IL-17 was significantly higher (p=0.035), and TNF-α was trend-wise higher (p=0.063) compared to the control group. Conclusion: Both isoflurane anesthesia and RIC have an impact on microcirculation. The application of RIC counteracted some of the effects of isoflurane, primarily in healthy tissue, and led to a significant improvement in relative tumor tissue oxygenation compared to the non-ischemic hind limb. RIC selectively enhanced immune infiltration within the tumor center, probably by previously activated tumor infiltrating T cells, while having no significant impact on T-regulatory cells. RIC appears to impact the cytokine profile, as indicated by elevated MFIs of TNF-α and IL-17. [ABSTRACT FROM AUTHOR]

Prognosis of glioblastoma patients is still poor despite multimodal therapy. The highly brain‐infiltrating growth in concert with a pronounced therapy resistance particularly of mesenchymal glioblastoma stem‐like cells (GSCs) has been proposed to contribute to therapy failure. Recently, we have shown that a mesenchymal‐to‐proneural mRNA signature of patient derived GSC‐enriched (pGSC) cultures associates with in vitro radioresistance and gel invasion. Importantly, this pGSC mRNA signature is prognostic for patients' tumor recurrence pattern and overall survival. Two mesenchymal markers of the mRNA signature encode for IKCa and BKCa Ca2+‐activated K+ channels. Therefore, we analyzed here the effect of IKCa‐ and BKCa‐targeting concomitant to (fractionated) irradiation on radioresistance and glioblastoma spreading in pGSC cultures and in pGSC‐derived orthotopic xenograft glioma mouse models. To this end, in vitro gel invasion, clonogenic survival, in vitro and in vivo residual DNA double strand breaks (DSBs), tumor growth, and brain invasion were assessed in the dependence on tumor irradiation and K+ channel targeting. As a result, the IKCa‐ and BKCa‐blocker TRAM‐34 and paxilline, respectively, increased number of residual DSBs and (numerically) decreased clonogenic survival in some but not in all IKCa‐ and BKCa‐expressing pGSC cultures, respectively. In addition, BKCa‐ but not IKCa‐blockade slowed‐down gel invasion in vitro. Moreover, systemic administration of TRAM‐34 or paxilline concomitant to fractionated tumor irradiation increased in the xenograft model(s) residual number of DSBs and attenuated glioblastoma brain invasion and (numerically) tumor growth. We conclude, that KCa‐blockade concomitant to fractionated radiotherapy might be a promising new strategy in glioblastoma therapy. [ABSTRACT FROM AUTHOR]

The intermediate-conductance calcium-activated potassium channel KCa3.1 has been proposed to be a new potential target for glioblastoma treatment. This study analyzed the effect of combined irradiation and KCa3.1-targeting with TRAM-34 in the syngeneic, immune-competent orthotopic SMA-560/VM/Dk glioma mouse model. Whereas neither irradiation nor TRAM-34 treatment alone meaningfully prolonged the survival of the animals, the combination significantly prolonged the survival of the mice. We found an irradiation-induced hyperinvasion of glioma cells into the brain, which was inhibited by concomitant TRAM-34 treatment. Interestingly, TRAM-34 did neither radiosensitize nor impair SMA-560's intrinsic migratory capacities in vitro. Exploratory findings hint at increased TGF-β1 signaling after irradiation. On top, we found a marginal upregulation of MMP9 mRNA, which was inhibited by TRAM-34. Last, infiltration of CD3+, CD8+ or FoxP3+ T cells was not impacted by either irradiation or KCa3.1 targeting and we found no evidence of adverse events of the combined treatment. We conclude that concomitant irradiation and TRAM-34 treatment is efficacious in this preclinical glioma model. [ABSTRACT FROM AUTHOR]

Simple Summary: A potential new treatment for glioma patients is the blockade of KCa3.1 potassium channels. In our study, we performed experiments with the KCa3.1 blocker TRAM-34 in five glioma cell lines. To broaden our findings, effects on cultures enriched in glioma stem cells which are thought to be responsible for treatment failure and relapse were delineated in addition to standard culture conditions. Accordingly, stem-cell enriched cultures were found to be more resistant towards irradiation therapy. Effects of TRAM-34 were dependent on cell line and culture condition and included direct tumoricidal effects, but also temozolomide- and irradiation-sensitizing effects, showing its synergistic potential with current treatment strategies. TRAM-34 effects were mostly found in stem-cell enriched cultures. Overall, our results underline the importance of testing new interventions in several cell lines and different culture conditions to mimic in vivo inter- and intra-tumoral heterogeneity. Reportedly, the intermediate-conductance Ca2+-activated potassium channel KCa3.1 contributes to the invasion of glioma cells into healthy brain tissue and resistance to temozolomide and ionizing radiation. Therefore, KCa3.1 has been proposed as a potential target in glioma therapy. The aim of the present study was to assess the variability of the temozolomide- and radiation-sensitizing effects conferred by the KCa3.1 blocking agent TRAM-34 between five different glioma cell lines grown as differentiated bulk tumor cells or under glioma stem cell-enriching conditions. As a result, cultures grown under stem cell-enriching conditions exhibited indeed higher abundances of mRNAs encoding for stem cell markers compared to differentiated bulk tumor cultures. In addition, stem cell enrichment was paralleled by an increased resistance to ionizing radiation in three out of the five glioma cell lines tested. Finally, TRAM-34 led to inconsistent results regarding its tumoricidal but also temozolomide- and radiation-sensitizing effects, which were dependent on both cell line and culture condition. In conclusion, these findings underscore the importance of testing new drug interventions in multiple cell lines and different culture conditions to partially mimic the in vivo inter- and intra-tumor heterogeneity. [ABSTRACT FROM AUTHOR]

Many tumor cells express highly elevated activities of voltage-gated K+ channels in the plasma membrane which are indispensable for tumor growth. To test for K+ channel function during DNA damage response, we subjected human chronic myeloid leukemia (CML) cells to sub-lethal doses of ionizing radiation (0–8 Gy, 6 MV photons) and determined K+ channel activity, K+ channel-dependent Ca2+ signaling, cell cycle progression, DNA repair, and clonogenic survival by whole-cell patch clamp recording, fura-2 Ca2+ imaging, Western blotting, flow cytometry, immunofluorescence microscopy, and pre-plating colony formation assay, respectively. As a result, the human erythroid CML cell line K562 and primary human CML cells functionally expressed hERG1. Irradiation stimulated in both cell types an increase in the activity of hERG1 K+ channels which became apparent 1–2 h post-irradiation. This increase in K+ channel activity was paralleled by an accumulation in S phase of cell cycle followed by a G2/M cell cycle arrest as analyzed between 8 and 72 h post-irradiation. Attenuating the K+ channel function by applying the hERG1 channel inhibitor E4031 modulated Ca2+ signaling, impaired inhibition of the mitosis promoting subunit cdc2, overrode cell cycle arrest, and decreased clonogenic survival of the irradiated cells but did not affect repair of DNA double strand breaks suggesting a critical role of the hERG1 K+ channels for the Ca2+ signaling and the cell cycle control during DNA damage response. [ABSTRACT FROM AUTHOR]

Prognosis of glioblastoma patients is still poor despite multimodal therapy. The highly brain-infiltrating growth in concert with a pronounced therapy resistance particularly of mesenchymal glioblastoma stem-like cells (GSCs) has been proposed to contribute to therapy failure. Recently, we have shown that a mesenchymal-to-proneural mRNA signature of patient derived GSC-enriched (pGSC) cultures associates with in vitro radioresistance and gel invasion. Importantly, this pGSC mRNA signature is prognostic for patients' tumor recurrence pattern and overall survival. Two mesenchymal markers of the mRNA signature encode for IK Ca and BK Ca Ca 2+ -activated K + channels. Therefore, we analyzed here the effect of IK Ca - and BK Ca -targeting concomitant to (fractionated) irradiation on radioresistance and glioblastoma spreading in pGSC cultures and in pGSC-derived orthotopic xenograft glioma mouse models. To this end, in vitro gel invasion, clonogenic survival, in vitro and in vivo residual DNA double strand breaks (DSBs), tumor growth, and brain invasion were assessed in the dependence on tumor irradiation and K + channel targeting. As a result, the IK Ca - and BK Ca -blocker TRAM-34 and paxilline, respectively, increased number of residual DSBs and (numerically) decreased clonogenic survival in some but not in all IK Ca - and BK Ca -expressing pGSC cultures, respectively. In addition, BK Ca - but not IK Ca -blockade slowed-down gel invasion in vitro. Moreover, systemic administration of TRAM-34 or paxilline concomitant to fractionated tumor irradiation increased in the xenograft model(s) residual number of DSBs and attenuated glioblastoma brain invasion and (numerically) tumor growth. We conclude, that K Ca -blockade concomitant to fractionated radiotherapy might be a promising new strategy in glioblastoma therapy., (© 2024 The Author(s). International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Many tumor cells express highly elevated activities of voltage-gated K + channels in the plasma membrane which are indispensable for tumor growth. To test for K + channel function during DNA damage response, we subjected human chronic myeloid leukemia (CML) cells to sub-lethal doses of ionizing radiation (0-8 Gy, 6 MV photons) and determined K + channel activity, K + channel-dependent Ca 2+ signaling, cell cycle progression, DNA repair, and clonogenic survival by whole-cell patch clamp recording, fura-2 Ca 2+ imaging, Western blotting, flow cytometry, immunofluorescence microscopy, and pre-plating colony formation assay, respectively. As a result, the human erythroid CML cell line K562 and primary human CML cells functionally expressed hERG1. Irradiation stimulated in both cell types an increase in the activity of hERG1 K + channels which became apparent 1-2 h post-irradiation. This increase in K + channel activity was paralleled by an accumulation in S phase of cell cycle followed by a G 2 /M cell cycle arrest as analyzed between 8 and 72 h post-irradiation. Attenuating the K + channel function by applying the hERG1 channel inhibitor E4031 modulated Ca 2+ signaling, impaired inhibition of the mitosis promoting subunit cdc2, overrode cell cycle arrest, and decreased clonogenic survival of the irradiated cells but did not affect repair of DNA double strand breaks suggesting a critical role of the hERG1 K + channels for the Ca 2+ signaling and the cell cycle control during DNA damage response., (Copyright © 2020 Palme, Misovic, Ganser, Klumpp, Salih, Zips and Huber.)

Tumor treating fields (TTFields) represent a novel FDA-approved treatment modality for patients with newly diagnosed or recurrent glioblastoma multiforme. This therapy applies intermediate frequency alternating electric fields with low intensity to the tumor volume by the use of non-invasive transducer electrode arrays. Mechanistically, TTFields have been proposed to impair formation of the mitotic spindle apparatus and cytokinesis. In order to identify further potential molecular targets, here the effects of TTFields on Ca 2+ -signaling, ion channel activity in the plasma membrane, cell cycle, cell death, and clonogenic survival were tested in two human glioblastoma cell lines in vitro by fura-2 Ca 2+ imaging, patch-clamp cell-attached recordings, flow cytometry and pre-plated colony formation assay. In addition, the expression of voltage-gated Ca 2+ (Ca v ) channels was determined by real-time RT-PCR and their significance for the cellular TTFields response defined by knock-down and pharmacological blockade. As a result, TTFields stimulated in a cell line-dependent manner a Ca v 1.2-mediated Ca 2+ entry, G₁ or S phase cell cycle arrest, breakdown of the inner mitochondrial membrane potential and DNA degradation, and/or decline of clonogenic survival suggesting a tumoricidal action of TTFields. Moreover, inhibition of Ca v 1.2 by benidipine aggravated in one glioblastoma line the TTFields effects suggesting that Ca v 1.2-triggered signaling contributes to cellular TTFields stress response. In conclusion, the present study identified Ca v 1.2 channels as TTFields target in the plasma membrane and provides the rationale to combine TTFields therapy with Ca 2+ antagonists that are already in clinical use.