Non-irradiated Cells Research Articles

TMET-06. CLIP3 DOWNREGULATION LEADS TO RADIORESISTANCE IN GLIOBLASTOMA BY PROMOTING GLYCOLYTIC FLUX

Abstract Glioblastoma Multiforme (GBM), a highly aggressive primary brain tumor, often develops resistance to standard treatments such as ionizing radiation (IR), which leads to a median survival of about 15 months. GBM contains glioblastoma stem cells (GSCs), which are crucial in driving therapeutic resistance and recurrence. In our study, we conducted comprehensive analyses to evaluate the molecular changes associated with radioresistance and explored the role of the Spy1-CLIP3 axis as a key regulator of GSC maintenance. Our methods included quantitative PCR and immunoblotting to measure CLIP3 mRNA and protein levels, revealing a significant reduction in expression among radioresistant GBM cells. We found that exposure to IR leads to the upregulation of Spy1 and downregulation of CLIP3, contributing to the development of radioresistance in surviving GBM cells. We used metabolic flux analysis techniques to observe changes in glycolytic activity, which increased in correlation with CLIP3 downregulation. IR induces CLIP3 downregulation, increasing glycolytic activity by facilitating the trafficking of GLUT3, the primary glucose transporter in GSCs. Additionally, colony-forming ability was evaluated using colony-forming assay after treatment of glimepiride with or without IR. Glimepiride didn’t reduce colony-forming ability in non-irradiated cells, but when combined with IR, it significantly reduced colony-forming ability, possibly by disrupting maintenance of GSCs. Importantly, our data reveal that glimepiride, a CLIP3 activator targets GSCs metabolism and enhances radiosensitization while maintaining low toxicity. The radioresistant GBM cells that survive radiotherapy exhibit increased stemness and glycolytic activity mediated by the Spy1-CLIP3 axis. Overall, our findings suggest that clinical trials using glimepiride for GBM patients may potentially improve survival rates, particularly for those experiencing recurrence after radiotherapy. This research highlights the importance of targeting cellular metabolism and stem cell properties in the context of GBM radioresistance and suggests potential avenues for improving the effectiveness of existing treatments.

STEM-16. EXPLORING THE ROLE OF HYPOXIA AND MACROPHAGES ON GLIOBLASTOMA QUIESCENCE AND STEMNESS AS MEDIATORS OF RADIORESISTANCE

Abstract Our ultimate goal is to identify, characterize and therapeutically target the glioma cell population that is responsible for treatment resistance and recurrence following chemoradiation. Two cell types that may have overlapping phenotypes and are known to influence radioresistance are 1) quiescent cells and 2) glioblastoma (GBM) stem cells (GSCs). The extent of overlap between these populations is unknown as are the influence of hypoxia and immune cells on their radioresistant properties. We have shown that hypoxia and co-culturing with THP-1 macrophages dramatically increases the percentage of GSCs. Using the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) system expressed in patient-derived gliomas, we used flow cytometry to distinguish cells based on their cell cycle status (G1/G0 and G2/M) and classified them as quiescent (high p27, low Ki67) or GSC (high CD133/CD44 co-expression). We found that the fraction of cells in G0 expressed high p27 and low Ki67, indicating cell cycle suppression and low proliferation, respectively. G0 glioma cells also contained a high percentage of GSCs, but GSCs were also distributed in G1 and G2/M. Following exposure to hypoxia, flow cytometry of FUCCI GBM cells showed an increase of cells in G0. After 3 Gy radiation under normoxic conditions, GBM cells demonstrated a shift to increased levels of G2/M cells compared to non-irradiated cells. In hypoxia, however, GBM cells sustained their population of cells in G0 and did not exhibit such cell cycle shifts. Our results indicate that both hypoxia and macrophages cause GSC enrichment and that hypoxia increases the proportion of quiescent cells in G0. Additional study is necessary to determine the extent of overlap between the GSCs and quiescent cells and to identify the intrinsic cell properties that drive tumor recurrence.

Structurally similar porphyrins and porphyrazines perform differently under green or red light irradiation against non-melanoma skin tumor cells

A porphyrin (H2P) and a porphyrazine (H2Pz) and their respective zinc complexes (ZnP and ZnPz), with the substituent N-ethylpyridyl-3-yl, were synthesized and their photophysical and phototoxic properties were compared. These substances showed high solubility in water due to the cationic moieties and without notable aggregation in this solvent. Photophysical studies showed that H2P and H2Pz have fluorescence quantum yields (ΦF) of 2.04% and 0.48% in water, respectively, that, upon metalation, decreases to 1.4% for the metalloporphyrin (ZnP) and increases to 6.1% for the metalloporphyrazine (ZnPz). Regarding the singlet oxygen production efficiency (ΦΔ) of the substances in water, a considerable increase from 16.2% to 41% was obtained for the porphyrin upon coordination to zinc, while the porphyrazines practically do not produce singlet oxygen (< 1%) in this solvent. When tested against human squamous cell carcinoma cells (A-431) and immortalized human keratinocytes (HaCaT), the toxicity of all compounds increased using green (520 ± 5 nm) and red (625 ± 5 nm) radiation when compared to the non-irradiated cells, however, phototoxicity was higher for the porphyrins using green light, while the porphyrazines performed better under red radiation. Furthermore, porphyrins had a higher phototoxic index than porphyrazines and greater selectivity for A-431 cancer cells compared to HaCaT cells. To the best of our knowledge, this is the first work that compares the photophysical response and in vitro anticancer activity of porphyrins and porphyrazines with the same substituent.

Nannochloropsis oceanica Lipid Extract Moderates UVB-Irradiated Psoriatic Keratinocytes: Impact on Protein Expression and Protein Adducts.

Psoriasis is characterized by excessive exfoliation of the epidermal layer due to enhanced pro-inflammatory signaling and hyperproliferation of keratinocytes, further modulated by UV-based anti-psoriatic treatments. Consequently, this study aimed to evaluate the impact of a lipid extract derived from the microalgae Nannochloropsis oceanica on the proteomic alterations induced by lipid derivatives in non-irradiated and UVB-irradiated keratinocytes from psoriatic skin lesions compared to keratinocytes from healthy individuals. The findings revealed that the microalgae extract diminished the viability of psoriatic keratinocytes without affecting the viability of these cells following UVB exposure. Notably, the microalgae extract led to an increased level of 4-HNE-protein adducts in non-irradiated cells and a reduction in 4-hydroxynonenal (4-HNE)-protein and 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2)-protein adducts in UVB-exposed keratinocytes from psoriasis patients. In healthy skin cells, the extract decreased the UV-induced elevation of 4-HNE-protein and 15d-PGJ2-protein adducts. The antioxidant/anti-inflammatory attributes of the lipid extract from the Nannochloropsis oceanica suggest its potential as a protective agent for keratinocytes in healthy skin against UVB radiation's detrimental effects. Moreover, it could offer therapeutic benefits to skin cells afflicted with psoriatic lesions by mitigating their proliferation and inflammatory responses during UV radiation treatment.

Protective effects of PDGF-AB/BB against cellular senescence in human intervertebral disc.

Cellular senescence, characterized by a permanent state of cell cycle arrest and a secretory phenotype contributing to inflammation and tissue deterioration, has emerged as a target for age-related interventions. Accumulation of senescent cells is closely linked with intervertebral disc (IVD) degeneration, a prevalent age-dependent chronic disorder causing low back pain. Previous studies have highlighted that platelet-derived growth factor (PDGF) mitigated IVD degeneration through anti-apoptosis, anti-inflammation, and pro-anabolism. However, its impact on IVD cell senescence remains elusive. In this study, human NP and AF cells derived from aged, degenerated IVDs were treated with recombinant human (rh) PDGF-AB/BB for 5 days and changes of transcriptome profiling were examined through mRNA sequencing. NP and AF cells demonstrated similar but distinct responses to the treatment. However, the effects of PDGF-AB and BB on human IVD cells were comparable. Specifically, PDGF-AB/BB treatment resulted in downregulation of gene clusters related to neurogenesis and response to mechanical stimulus in AF cells while the downregulated genes in NP cells were mainly associated with metabolic pathways. In both NP and AF cells, PDGF-AB and BB treatment upregulated the expression of genes involved in cell cycle regulation, mesenchymal cell differentiation, and response to reduced oxygen levels, while downregulating the expression of genes related to senescence associated phenotype, including oxidative stress, reactive oxygen species (ROS), and mitochondria dysfunction. Network analysis revealed that PDGFRA and IL6 were the top hub genes in treated NP cells. Furthermore, in irradiation-induced senescent NP cells, PDGFRA gene expression was significantly reduced compared to non-irradiated cells. However, rhPDGF-AB/BB treatment increased PDGFRA expression and mitigated the senescence progression through increased cell population in the S phase, reduced SA-β-Gal activity, and decreased expression of senescence related regulators including P21, P16, IL6, and NF-κB. Our findings reveal a novel anti-senescence role of PDGF in the IVD, demonstrating its ability to alleviate the senescent phenotype and protect against the progression of senescence. This makes it a promising candidate for preventing or treating IVD degeneration by targeting cellular senescence.

In Vitro Photoselective Gene Transfection of Hepatocellular Carcinoma Cells with Hypericin Lipopolyplexes.

The lipopolyplex, a multicomponent nonviral gene carrier, generally demonstrates superior colloidal stability, reduced cytotoxicity, and high transfection efficiency. In this study, a new concept, photochemical reaction-induced transfection, using photosensitizer (PS)-loaded lipopolyplexes was applied, which led to enhanced transfection and cytotoxic effects by photoexcitation of the photosensitizer. Hypericin, a hydrophobic photosensitizer, was encapsulated in the lipid bilayer of liposomes. The preformed nanosized hypericin liposomes enclosed the linear polyethylenimine (lPEI)/pDNA polyplexes, resulting in the formation of hypericin lipopolyplexes (Hy-LPP). The diameters of Hy-LPP containing 50 nM hypericin and 0.25 μg of pDNA were 185.6 ± 7.74 nm and 230.2 ± 4.60 nm, respectively, measured by dynamic light scattering (DLS) and atomic force microscopy (AFM). Gel electrophoresis confirmed the encapsulation of hypericin and pDNA in lipopolyplexes. Furthermore, in vitro irradiation of intracellular Hy-LPP at radiant exposures of 200, 600, and 1000 mJ/cm2 was evaluated. It demonstrated 60- to 75-fold higher in vitro luciferase expression than that in nonirradiated cells. The lactate dehydrogenase (LDH) assay supported that reduced transfection was a consequence of photocytotoxicity. The developed photosensitizer-loaded lipopolyplexes improved the transfection efficiency of an exogenous gene or induced photocytotoxicity; however, the frontier lies in the applied photochemical dose. The light-triggered photoexcitation of intracellular hypericin resulted in the generation of reactive oxygen species (ROS), leading to photoselective transfection in HepG2 cells. It was concluded that the two codelivered therapeutics resulted in enhanced transfection and a photodynamic effect by tuning the applied photochemical dose.

Characteristics of sphingomyelin metabolism in the MCF7 and BT474 radiotherapy‑resistant HER2‑positive breast cancer cell lines.

Breast cancer is the most common cancer globally in terms of incidence. This cancer is classified into subtypes based on histological or immunological characteristics. HER2-positive cases account for 15-25% of breast cancer cases, and one of the first events in breast carcinogenesis is HER2 upregulation. Furthermore, HER2 expression increases the detection rate of metastatic or recurrent breast cancers by 50-80%. The epidermal growth factor receptor family includes HER2, which is a transmembrane receptor protein. In our previous case report, patients who were resistant to anti-HER2 monoclonal antibody therapy, chemotherapy and radiotherapy had higher concentrations of phospholipid metabolites such as phosphatidylcholine and sphingomyelin (SM), which was associated with cancer recurrence progression. To better understand the relationship between radiotherapy resistance and SM expression, breast cancer cell lines with and without HER2 expression (MCF7 and BT474) after exposure to ionizing radiation (IR) were examined. In the cell culture supernatant, similar levels of SM in MCF7 cells were identified after 1-4 Gy exposure. However, SM levels in BT474 cells were upregulated compared with those of in the control group. Intracellular SM levels were upregulated in BT474 cells exposed to 1 and 4 Gy compared with the non-irradiated control group. Furthermore, significantly increased mRNA expression levels of sphingomyelin synthase 2 (SGMS2) in BT474 cells exposed to IR were observed compared with those in nonirradiated cells; however, the SGMS2 levels in MCF7 cells did not differ significantly among the 0, 2 and 4 Gy groups. These findings suggested that a higher dose of IR induced the secretion of SM and its associated gene expression in HER2-positive breast cancer cells.

Radiotolerant endophytic bacteria and analysis of the effects of 137Cesium on the metabolome of Pantoea sp.

Some bacteria have developed mechanisms to withstand the stress caused by ionizing radiation. The ability of these radioresistant microorganisms to survive high levels of radiation is primarily attributed to their DNA repair mechanisms and the production of protective metabolites. To determine the effect of irradiation on bacterial growth, we propose to compare the metabolites produced by the irradiated isolates to those of the control (non-irradiated isolates) using mass spectrometry, molecular networking, and chemometric analysis. We identified the secondary metabolites produced by these bacteria and observed variations in growth following irradiation. Notably, after 48h of exposure to radiation, Pantoea sp. bacterial cells exhibited a significant 6-log increase compared to non-irradiated cells. Non-irradiated cells produce exclusively Pyridindolol, 1-hydroxy-4-methylcarbostyril, N-alkyl, and N-2-alkoxyethyl diethanolamine, while 5'-methylthioadenosine was detected only in irradiated cells. These findings suggest that the metabolic profile of Pantoea sp. remained relatively stable. The results obtained from this study have the potential to facilitate the development of innovative strategies for harnessing the capabilities of endophytic bacteria in radiological protection and bioremediation of radionuclides.

Enhanced expression of galectin-9 in triple negative breast cancer cells following radiotherapy: Implications for targeted therapy.

Optimizations are expected in the development of immunotherapy for the treatment of Triple-negative breast cancer (TNBC). We studied the expression of galectin-9 (Gal-9) after irradiation and assessed the differential impacts of its targeting with or without radiotherapy. Tumor resections from TNBC patients who received neoadjuvant radiotherapy revealed higher levels of Gal-9 in comparison to their baseline level, only in non-responder patients. Gal-9 expression was also found to be increased in TNBC tumor biopsies and cell lines after irradiation. We investigated the therapeutic advantage of targeting Gal-9 after radiotherapy in mice. Irradiated 4T1 cells or control non-irradiated 4T1 cells were injected into BALB/c mice. Anti-Gal-9 antibody treatment decreased tumor progression only in mice injected with irradiated 4T1 cells. This proof-of-concept study demonstrates that Gal-9 could be considered as a dynamic biomarker after radiotherapy for TNBC and suggests that Gal-9 induced-overexpression could represent an opportunity to develop new therapeutic strategies for TNBC patients.

Study on the anticancer effects of cold atmospheric plasma on deep subcutaneous tumor cells

AbstractCold atmospheric plasma (CAP) therapy has been proven to effectively inhibit the growth of subcutaneous tumors. However, there is no research to explain how plasma‐induced anticancer effects can be transmitted to cancer cells through normal cells. Here, we monitored the effects of plasma‐irradiated HaCaT cells on nonirradiated A375 and HaCaT cells in terms of cell viability and intracellular reactive oxygen species levels in vitro. The experimental results indicated that plasma irradiation may induce plasma‐irradiated HaCaT cells to release soluble factors into the cell culture medium, thereby selectively killing nonirradiated A375 cells. The plasma‐generated short‐lived species play a crucial role in the release of soluble factors. Our research showed that plasma‐induced anticancer effects can be transmitted to deep nonirradiated cancer cells through plasma‐irradiated normal cells.

The effect of different parameters of low-level laser used in the treatment of oral mucositis, on the viability and apoptosis of oral squamous cell carcinoma cells: Invitro study.

Oral mucositis is a complication of chemo/radiotherapy. To assess the impact of various power levels of diode-laser on the survival and expression of apoptosis-related genes in oral cancer cells, it is crucial to consider the potential existence of malignant cells within the treatment region and the reliance of laser effectiveness on its specific characteristics. Cal-27 cells were cultivated and exposed to a 660 nm-diode-laser at power levels of 20, 40, and 80 mW, alongside non-irradiated control cells. Viability and expression of Bax and Bcl-2 mRNA were assessed with Methyl Thiazolyl Tetrazolium (MTT) and Real-time Polymerase Chain Reaction (RT-PCR), respectively. The results were analyzed using one-way ANOVA and Tukey post-hoc test (p < 0.05). A significant reduction in viability was found only in the 20 mW group compared to controls (p = 0.001). Cell survival was significantly lower in cells receiving 20 mW laser than those treated with 40 and 80 mW (p < 0.05). None of the laser groups showed significant changes in BcL-2, but Bax was significantly lower in cells receiving 40 and 80 mW (p < 0.05), compared to controls. Laser irradiation at 660 nm (2 J/cm2, 30 s) significantly reduced the viability of oral cancer cells when using 20 mW power. These specifications align with the recommendation that the lowest possible laser dose should be applied for treating cancer patients. The exact mechanism of cell death following laser therapy with these specifications requires further investigation.

High-frequency low-intensity semiconductor laser irradiation enhances osteogenic differentiation of human cementoblast lineage cells

PurposeLaser irradiation activates a range of cellular processes in the periodontal components and promotes tissue repair. However, its effect on osteogenic differentiation of human cementoblast lineage cells remains unclear. This study aimed to examine the effects of high-frequency semiconductor laser irradiation on the osteogenic differentiation of human cementoblast lineage (HCEM) cells.MethodsHCEM cells were cultured to reach 80% confluence and irradiated with a gallium-aluminum-arsenide (Ga-Al-As) semiconductor laser with a pulse width of 200 ns and wavelength of 910 at a dose of 0–2.0 J/cm2. The outcomes were assessed by analyzing the mRNA levels of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and type I collagen (COLL1) using real-time polymerase chain reaction (PCR) analysis 24 h after laser irradiation. Cell mineralization was evaluated using ALP activity, calcium deposition, and Alizarin Red staining.ResultsThe laser-irradiated HCEM cells showed significantly enhanced gene expression levels of ALP, RUNX2, and COLL1 as well as ALP activity and calcium concentration in the culture medium compared with the non-irradiated cells. In addition, enhanced calcification deposits were confirmed in the laser-irradiated group compared with the non-irradiated group at 21 and 28 days after the induction of osteogenic differentiation.ConclusionHigh-frequency semiconductor laser irradiation enhances the osteogenic differentiation potential of cultured HCEM cells, underscoring its potential utility for periodontal tissue regeneration.

The extended transmission of cold atmospheric plasma-induced anticancer effects between cells

The application of cold atmospheric plasma (CAP) in cancer therapy has recently received extensive attention. However, researchers have mainly studied the effects of CAP on plasma-irradiated cancer cells and have not determined whether CAP affects surrounding or distant non-irradiated cancer cells. Here, the viability and intracellular reactive oxygen species levels of plasma-irradiated and non-irradiated cancer cells were monitored in vitro. Our results showed that CAP may induce plasma-irradiated cancer cells (A549 cells, A375 cells, J82 cells and HT29 cells) to secrete soluble factors into the cell culture medium, which can transmit plasma-induced anticancer effects from plasma-irradiated cancer cells to non-irradiated cancer cells. Our research confirmed that plasma-induced anticancer effects can be transmitted between cancer cells.

Potential effect of fruit and flower extracts of Arbutus unedo L. on Tetrahymena pyriformis exposed to a cobalt-60 source

Exposure of Tetrahymena pyriformis cultures to cobalt-60 for 72 h significantly impacted the cells' growth, appearance, and physiology. This study aims to investigate the protective effects of Arbutus unedo L flowers and fruit extracts on T. pyriformis against gamma radiation. Initially, aqueous and 50% ethanolic extracts of the fruits and flowers were prepared, and their cytotoxicity on the ciliate was evaluated. The irradiated ciliate's cellular viability and morphological aspect improved when a non-toxic concentration of 25 µg/mL was added to the growth medium. The addition of extracts restored glyceraldehyde-3-phosphate dehydrogenase and succinate dehydrogenase activities to their initial levels, similar to non-irradiated cells. In addition, the extracts reduced oxidative stress markers, such as lipid peroxidation, and decreased the activities of antioxidant defence enzymes, catalase, and superoxide dismutase. This may be attributed to the antioxidant properties of the extracts. Results of this study revealed that the flower extracts exhibited better protective effects than the fruit extracts, with superior antioxidant activity in the in-vitro DPPH scavenging assay. These results suggest that A. unedo flower extracts may have potential as exogenous radioprotective agents.

Irradiated tumour cell-derived microparticles upregulate MHC-I expression in cancer cells via DNA double-strand break repair pathway

Radiotherapy (RT) is used for over 50 % of cancer patients and can promote adaptive immunity against tumour antigens. However, the underlying mechanisms remain unclear. Here, we discovered that RT induces the release of irradiated tumour cell-derived microparticles (RT-MPs), which significantly upregulate MHC-I expression on the membranes of non-irradiated cells, enhancing the recognition and killing of these cells by T cells. Mechanistically, RT-MPs induce DNA double-strand breaks (DSB) in tumour cells, activating the ATM/ATR/CHK1-mediated DNA repair signalling pathway, and upregulating MHC-I expression. Inhibition of ATM/ATR/CHK1 reversed RT-MP-induced upregulation of MHC-I. Furthermore, phosphorylation of STAT1/3 following the activation of ATM/ATR/CHK1 is indispensable for the DSB-dependent upregulation of MHC-I. Therefore, our findings reveal the role of RT-MP-induced DSBs and the subsequent DNA repair signalling pathway in MHC-I expression and provide mechanistic insights into the regulation of MHC-I expression after DSBs.

Profile of miRNAs in small extracellular vesicles released from glioblastoma cells treated by boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is a tumor cell-selective particle-radiation therapy. In BNCT, administered p-boronophenylalanine (BPA) is selectively taken up by tumor cells, and the tumor is irradiated with thermal neutrons. High-LET α-particles and recoil 7Li, which have a path length of 5-9μm, are generated by the capture reaction between 10B and thermal neutrons and selectively kill tumor cells that have uptaken 10B. Although BNCT has prolonged the survival time of malignant glioma patients, recurrences are still to be resolved. miRNAs, that are encapsulated in small extracellular vesicles (sEVs) in body fluids and exist stably may serve critical role in recurrence. In this study, we comprehensively investigated microRNAs (miRNAs) in sEVs released from post-BNCT glioblastoma cells. Glioblastoma U87 MG cells were treated with 25 ppm of BPA in the culture media and irradiated with thermal neutrons. After irradiation, they were plated into dishes and cultured for 3 days in the 5% CO2 incubator. Then, sEVs released into the medium were collected by column chromatography, and miRNAs in sEVs were comprehensively investigated using microarrays. An increase in 20 individual miRNAs (ratio > 2) and a decrease in 2 individual miRNAs (ratio < 0.5) were detected in BNCT cells compared with non-irradiated cells. Among detected miRNAs, 20 miRNAs were associated with worse prognosis of glioma in Kaplan Meier Survival Analysis of overall survival in TCGA. These miRNA after BNCT may proceed tumors, modulate radiation resistance, or inhibit invasion and affect the prognosis of glioma.

Live Imaging to Quantify Cellular Radiosensitivity in Patient-Derived Tumor Organoids.

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, often (but not always) because of differences in the ability of malignant cells to repair oxidative DNA damage as elicited by ionizing rays. Clonogenic assays have been employed for decades to assess the sensitivity of cultured cancer cells to ionizing irradiation, largely because irradiated cancer cells often die in a delayed manner that is difficult to quantify with short-term flow cytometry- or microscopy-assisted techniques. Unfortunately, clonogenic assays cannot be employed as such for more complex tumor models, such as patient-derived tumor organoids (PDTOs). Indeed, irradiating established PDTOs may not necessarily abrogate their growth as multicellular units, unless their stem-like compartment is completely eradicated. Moreover, irradiating PDTO-derived single-cell suspensions may not properly recapitulate the sensitivity of malignant cells to RT in the context of established PDTOs. Here, we detail an adaptation of conventional clonogenic assays that involves exposure of established PDTOs to ionizing radiation, followed by single-cell dissociation, replating in suitable culture conditions and live imaging. Non-irradiated (control) PDTO-derived stem-like cells reform growing PDTOs with a PDTO-specific efficiency, which is negatively influenced by irradiation in a dose-dependent manner. In these conditions, PDTO-forming efficiency and growth rate can be quantified as a measure of radiosensitivity on time-lapse images collected until control PDTOs achieve a predefined space occupancy.

Abstract 567: Fisetin sensitizes triple negative breast cancer cells to radiation

Abstract Triple negative breast cancer (TNBC) represents a highly aggressive subtype, constituting approximately 20% of all breast cancer cases. Y-box binding protein-1 (YB-1) is often overexpressed in various tumor types and has been implicated in conferring resistance to cell death induced by radiotherapy. Fisetin, a flavonoid compound with demonstrated anti-cancer properties, operates in part through inhibiting phosphorylation of YB-1 by p90 ribosomal S6 kinase (RSK). In this study, we investigated the potential synergistic effects of combining fisetin with radiotherapy in TNBC. The activation status of the RSK signaling pathway was assessed in both total cell lysate and subcellular fractions using Western blotting. A standard clonogenic assay was employed to evaluate post-irradiation cell survival. To investigate the frequency of double-strand breaks (DSBs) and chromosomal aberrations, γH2AX foci assay and 3-color fluorescence in situ hybridization analyses were conducted, respectively. The DSB repair pathways targeted by fisetin were examined in cells expressing genomically integrated reporter constructs by quantifying green fluorescence protein expression through flow cytometry. Apoptosis and autophagy were measured using flow cytometric quantification of sub-G1 cells and the assessment of LC3-II protein expression, respectively. Kinase array and phosphoproteomics analyses were carried out to study the potential impact of fisetin on the signaling involved in the DNA damage response (DDR) after irradiation. We demonstrated that fisetin mimics the effects of RSK pharmacological inhibitors in terms of effect on YB-1 phosphorylation. Treatment with fisetin increased frequency of DSB in non-irradiated cells and impaired repair of DSB after irradiation. Fisetin attenuated DSB repair by suppressing the classical non-homologous end-joining and homologous recombination repair pathways. The frequency of chromosomal aberration was enhanced by fisetin treatment. The effect of fisetin on inhibiting DSB repair was partially YB-1 dependent. Phosphoproteomic analysis revealed that fisetin inhibits DDR signaling, which leads to radiosensitization in TNBC cells, as shown in combination with single dose or fractionated doses irradiation. Fisetin neither inhibited DSB repair nor radiosensitized normal human fibroblast HSF7 cells. The radiosensitizing effect of fisetin was not attributed to enhancing apoptosis or modulating autophagy. In summary, combining fisetin with radiotherapy may enhance TNBC radiotherapy outcomes. -This research was supported by a grant from the German Research Council (DFG,TO 685/2-3). Citation Format: Shayan Khozooei, Soundaram Veerappan, Mahmoud Toulany. Fisetin sensitizes triple negative breast cancer cells to radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 567.

Abstract 2886: Osteoclast and neuronal cross-talk as a mechanism for chest-wall pain caused by thoracic radiosurgery for lung cancer

Abstract Radiation therapy (RT)-induced chest wall pain (CWP) is a severe complication after thoracic stereotactic body radiation therapy (SBRT) for lung tumors; with increased survivorship, ~30% of patients develop severe CWP. The causes of CWP are unknown, but could involve: 1] damage to intercostal peripheral nerves; 2] bone loss from affected osteoclast activity, or 3] both (secreted factors from activated osteoclasts can affect nerves). This study tested the potential for radiation to affect osteoclasts and/or sensory neurons to induce pain. Mature osteoclasts differentiated from RAW264.7 cells plated on Osteoassay surfaces were exposed to an acute 10 Gy Cs137 γ-rays and bone resorption was assessed. We collected conditioned media (CM) from these cells at 48h and applied to cultured primary mouse dorsal root ganglia neurons (irradiated and non-irradiated neuron), assessing neurite growth and pain-associated marker (CGRP, SP) expression through immunohistochemistry and RT-qPCR. Risedronate (50µM), an anti-resorptive agent, was administered pre-radiation to inhibit osteoclast activity. Unpaired t-tests were utilized for statistical analyses (α=0.05). Osteoclast resorption activity was increased by 48h after irradiation. These irradiated, activated osteoclasts exhibited increased mRNA expression of differentiation (RANK) and activity (CTSK, MMP9) markers. Neurons treated with CM from irradiated osteoclasts exhibited elevated levels of CGRP (+38.5%) and SP (+29.5%) compared to cells treated with CM from non-irradiated cells. Consistently, in co-cultures of osteoclasts and neurons exposed to radiation, irradiated neurons exhibited significantly increased neuropeptide levels. Risedronate not only reduced osteoclast activity but also inhibited neuropeptide expression in irradiated neurons within these co-cultures. Moreover, neuropeptide levels were similar between directly irradiated neurons and non-irradiated neurons. However, irradiated neurons exposed to CM from either irradiated or non-irradiated osteoclasts showed a marked 4-fold increase in neuropeptide levels vs neurons that were non-irradiated and then received osteoclast CM. Our findings indicate that radiation activates osteoclasts and leads to direct bone loss in vitro and signaling between irradiated osteoclasts and non-irradiated neurons increases pain responses. Importantly, this response is enhanced when neurons were also previously irradiated. During SBRT, intercostal nerves may be influenced by adjacent irradiated osteoclasts, increasing expression of pain-related neuropeptides as the osteoclasts break down bone, thus enhancing the risk of CWP as treatment progresses across time. Understanding these interactions could reveal potential targets for preventing CWP, allowing higher tumor radiation doses with less toxicity. Support: The Milton Raben Foundation. Citation Format: Sun Park, Megan Peters, Joseph Moore, Kaitlyn Reno, Michael Farris, Christopher Peters, Jeffery Willey. Osteoclast and neuronal cross-talk as a mechanism for chest-wall pain caused by thoracic radiosurgery for lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2886.

Abstract 480: Nanotherapeutic strategies to improve targeted radionuclide therapy

Abstract Cancer is a leading cause of death worldwide. Several multidisciplinary approaches exist for cancer treatment, including radiotherapy and chemotherapy. Radiotherapy uses high-energy radiation to kill cancer cells; chemotherapy inhibits cancer cell proliferation and often kills cells by targeting the cell cycle. Resistance to radiotherapy and chemotherapy is a key determining factor in the outcome of therapeutic efficacy. Conventional nontargeted radiotherapy also affects distant nonirradiated cells, leading to DNA damage and changes in the cell cycle that are often linked to secondary carcinogenesis in patients. In recent years, targeted radionuclide therapy (TRT) has emerged as a promising personalized treatment strategy that delivers cytotoxic levels of radiation directly and specifically to cancer cells. Among radioisotopes,225Ac exhibits desirable properties for TRT: multiple α-particle emission and high cytotoxicity. One of the challenges with 225Ac is the nonspecific toxicity caused by the release and relocation of its decay daughters. Different approaches have been proposed to prevent the relocation of decay daughters, including nanoparticles. Nanoparticles have been pursued as a promising delivery vehicle of α-emitting radioisotopes to the tumor site. Here, we show specific targeting of Her2-positive breast cancer cells with 225Ac-radiolabeled lanthanum orthovanadate (LaVO4) nanoparticles. Nanoparticles’ surfaces were functionalized to improve biocompatibility and conjugated to target cancer cells, respectively. Cellular uptake and localization of these engineered nanoparticles were analyzed by a Confocal microscope and IncuCyte live cell analysis system. Results confirm its localization in the nucleus following the endolysosomal path, enhancing the nanoparticles’ effectiveness as a delivery vehicle for α-emitting radioisotopes with the potential of increased treatment efficacy. Upon addition of cancer-targeting ligands, these nanoparticles can achieve higher efficiency by delivering the radioisotope to the tumor cells with a potential of safe encapsulation of all α-emitters in the decay chain. The anticancer efficiency of 225Ac-radiolabeled nanoparticles was demonstrated by a dose-dependent effective killing of 3D breast cancer spheroids using both IncuCyte live imaging and the cell survival assays. Ultimately, the results of this study will be crucial in determining the future use of targeted radiolabeled nanoparticle-based delivery systems as an approach for more efficacious cancer treatment. Citation Format: Debjani Pal, Miguel Toro Gonzáleza, Amber N. Bibleb, Brian Sanders, Anna Plechaty, Owee Kirpekar, Mircea Podar, Sandra M. Davern. Nanotherapeutic strategies to improve targeted radionuclide therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 480.