Gy Irradiation Research Articles

Abstract A050: Ferritin, associated with worse overall survival in recurrent GBM, is reduced by combination of HDACi, ONC206 and radiation

Abstract Background: GBM is the most common primary brain malignancy with dismal prognosis. Current standard of care is chemoradiation&surgery. The main issue with GBM is the resistance & recurrence. GBM can have high Ferritin levels associated with disease progression & immunosuppression in the GBM TME. Ferritin light chain (FTL), a key protein in iron metabolism, is mutated in GBM leading to high ferritin levels (both light& heavy chain) & is associated with poor survival of (GBM) patients in part due to inhibition of ferroptosis. Panobinostat is a histone deacetylase inhibitor with antineoplastic & antiangiogenic effects in glioma. Imipridone ONC206 is under Phase I clinical development in treatment of adult & pediatric patients with newly diagnosed and/or recurrent primary brain tumors (NCT04732065 and NCT04541082).We investigated the novel combination of irradiation, Panobinostat and ONC206 with regard to GBM cell line sensitivity&ferritin levels. Materials & methods: We investigated cell viability and drug synergies of ONC206 plus Panobinostat with or without radiation in 4 human GBM cell lines at 72 hours. Changes in Ferritin level treatment in U87 & U251 cells were measured using cytokine profiling at 24 h time point. Information regarding recurrent cases of adult GBM from TCGA &GlioVis were used for survival data. Results: We previously have shown the synergy between Panobinostat, Irradiation and ONC206 (2023 AACR meeting). Treatment of U87 cells with Panobinostat and ONC206 at their IC50s +/- 4 Gy radiation at 24h time point led to reduction in secreted ferritin levels (Panobinostat alone: 26.3% decrease; ONC206 alone: 14.7% decrease; RT 4Gy alone 14.7% decrease; triple therapy: 32.2% decrease). In panobinostat treated U251 cells there was a 10% increase in ferritin at 24 h. ONC206 resulted in a reduction by 34.71%, 4 Gy RT caused a slight increase of 4% in ferritin amount while the triple showed 49.96% reduction in ferritin levels. mRNA expression of FTL gene (Ferritin Light Chain) is significantly higher in GBM tumors compared to normal brain tissue. From TCGA 17 cases of recurrent gliomas with FTL mutation were available with median survival of less than a year. In data from GlioVis of recurrent GBM cases 263 patients had high FTL with median survival of 12.6 months compared to 262 patients with low FTL with median survival of 14.9 months. Conclusions: GBM with high ferritin correlates with poor patient survival. FTL is associated with cell proliferation &angiogenesis of GBM cells & is protective from ferroptosis in glioma cells. ferritin may be a therapeutic target in recurrent & therapy resistant cases of GBM and ferroptosis modulating drugs &can be a novel treatment option. ONC206 & Panobinostat with irradiation is a novel combination & can be considered for temozolomide or radiation-resistant & recurrent cases. More studies are needed to investigate the mechanism & impact of ferritin in disease progression, therapy resistance& ferroptosis targeting therapeutics. We are currently exploring these directions including in vivo studies. Citation Format: Vida Tajiknia, Wafik El Deiry, Lanlan Zhou, William Macdonald, Praveen Srinivasan. Ferritin, associated with worse overall survival in recurrent GBM, is reduced by combination of HDACi, ONC206 and radiation [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A050.

SOX2 and OCT4 mediate radiation and drug resistance in pancreatic tumor organoids

Pancreatic cancer has a five-year survival rate of only 10%, mostly due to late diagnosis and limited treatment options. In patients with unresectable disease, either FOLFIRINOX, a combination of 5-fluorouracil (5-FU), oxaliplatin and irinotecan, or gemcitabine plus nab-paclitaxel combined with radiation are frontline standard regimens. However, chemo-radiation therapy has shown limited success because patients develop resistance to chemotherapy and/or radiation. In this study, we evaluated the role of pancreatic cancer stem cells (CSC) using OCT4 and SOX2, CSC markers in mouse pancreatic tumor organoids. We treated pancreatic tumor organoids with 4 or 8 Gy of radiation, 10 μM of 5-FU (5-Fluorouracil), and 100 μM 3-Bromopyruvate (3BP), a promising anti-cancer drug, as a single treatment modalities, and in combination with RT. Our results showed significant upregulation of, OCT4, and SOX2 expression in pancreatic tumor organoids treated with 4 and 8 Gy of radiation, and downregulation following 5-FU treatment. The expression of CSC markers with increasing treatment dose exhibited elevated upregulation levels to radiation and downregulation to 5-FU chemotherapy drug. Conversely, when tumor organoids were treated with a combination of 5-FU and radiation, there was a significant inhibition in SOX2 and OCT4 expression, indicating CSC self-renewal inhibition. Noticeably, we also observed that human pancreatic tumor tissues exhibited heterogeneous and aberrant OCT4 and SOX2 expression as compared to normal pancreas, indicating their potential role in pancreatic cancer growth and therapy resistance. In addition, the combination of 5-FU and radiation treatment exhibited significant inhibition of the β-catenin pathway in pancreatic tumor organoids, resulting in sensitization to treatment and organoid death. In conclusion, our study emphasizes the crucial role of CSCs in therapeutic resistance in PC treatment. We recommend using tumor organoids as a model system to explore the impact of CSCs in PC and identify new therapeutic targets.

Radiation-induced effects on TGF-β and PDGF receptor signaling in cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) consist of heterogeneous connective tissue cells and are often constituting the most abundant cell type in the tumor stroma. Radiation effects on tumor stromal components like CAFs in the context of radiation treatment is not well-described. This study explores potential changes induced by ionizing radiation (IR) on platelet-derived growth factor (PDGF)/PDGFRs and transforming growth factor-beta (TGF-β)/TGFβRs signaling systems in CAFs. Experiments were carried out by employing primary cultures of human CAFs isolated from freshly resected non-small cell lung carcinoma tumor tissues. CAF cultures from nine donors were treated with one high (1 × 18 Gy) or three fractionated (3 × 6 Gy) radiation doses. Alterations in expression levels of TGFβRII and PDGFRα/β induced by IR were analyzed by western blots and flow cytometry. In the presence or absence of cognate ligands, receptor activation was studied in nonirradiated and irradiated CAFs. Radiation exposure did not exert changes in expression of PDGF or TGF-β receptors in CAFs. Additionally, IR alone was unable to trigger activation of either receptor. The radiation regimens tested did not affect PDGFRβ signaling in the presence of PDGF-BB. In contrast, signaling via pSmad2/3 and pSmad1/5/8 appeared to be down-regulated in irradiated CAFs after stimulation with TGF-β, as compared with controls. Our data demonstrate that IR by itself is insufficient to induce measurable changes in PDGF or TGF-β receptor expression levels or to induce receptor activation in CAFs. However, in the presence of their respective ligands, exposure to radiation at certain doses appear to interfere with TGF-β receptor signaling.

185 Long-Term Results of Induction Chemotherapy Followed by 50 Gy Radiation Therapy Alone for Low-Risk HPV-Positive Oropharynx Cancer

185 Long-Term Results of Induction Chemotherapy Followed by 50 Gy Radiation Therapy Alone for Low-Risk HPV-Positive Oropharynx Cancer

Proapoptotic effects of ceranib-2 in combination with radiation therapy on human breast cancer cells.

Strategies for cancer therapy involve radiation therapy (RT), which accounts for about 40% of all cancer treatment types. As to current chemotherapeutics, cancer cells also develop resistance that remains a clinical problem, such as disease recurrence. Recent studies focused on understanding the molecular mechanisms of radiation-induced cell death. Conventional RT aims at treatment with a single fraction per day of 8-30 Gy per fraction. Radiotherapy increases intracellular ceramide levels that trigger cell death. Additionally, increasing intracellular ceramide by radiation may restore therapeutic sensitivity to cancer treatments. Drugs that inhibit ceramide-metabolizing enzymes like ceramidases are expected to be radiotherapy sensitizers. In this research, we investigated the proapoptotic effects of SRS alone and in combination with ceranib-2, a ceramidase inhibitor in human breast adenocarcinoma cells. The molecular mechanism of action of RT and ceranib-2 was investigated on MCF-7 cells exposed to 13 µM ceranib-2 for 24 hours following 20 Gy radiation using MTT, radiotherapy, and annexin-V analyses. Results indicated that the dose of 20 Gy radiation induces apoptosis on human breast cancer cells with and without co-treatment with ceranib-2 by causing cytotoxicity in the cells. Based on the results of ceranib-2 exposure, it can be concluded that the mechanism of action may rely on an increase of intracellular ceramides, also called apoptotic lipids. The study results suggest that co-treatment of human breast adenocarcinoma cells with a ceramidase inhibitor, ceranib-2, and a high dose of radiation of 20 Gy exerted cytotoxicity and apoptosis and might be a solid, potent alternative to current therapy strategies.

The ATR inhibition by Elimusertib enhances the radiosensitivity of MDA-MB-231 triple negative breast cancer in vitro

Purpose DNA damage response (DDR) is the principal mechanism regulating genomic stability and cell cycle checkpoint activation by coordinating DNA repair and apoptotic pathways. Ataxia telangiectasia and Rad3-related protein (ATR) play a significant role in the DDR due to its capability to detect a wide spectrum of DNA damage. Therefore, targeting DDR, specifically ATR, is a promising therapeutic strategy in cancer treatment. Furthermore, the inhibition of ATR sensitizes cancer cells to radiotherapy (RT). Herein, we, for the first time, investigated the synergistic effects of Elimusertib (BAY-1895344) as a highly potent selective ATR inhibitor with RT combination in triple-negative breast cancer (TNBC), in vitro. Methods MDA-MB-231 TNBC cells were firstly treated with different concentrations of Elimusertib for 24 h and then exposed to 4 and 8 Gy of X-ray irradiation. After post-irradiation for 72 h, WST-1, Annexin V, cell cycle, acridine orange/propidium iodide, mitochondria staining and western blot analysis were conducted. Results Our findings showed that 4 Gy irradiation and lower doses (especially 2 and 4 nM) of Elimusertib combination exerted a considerable anticancer activity at 72 h post-irradiation through apoptotic cell death, marked nuclear and mitochondrial damages and the suppression of ATR-Chk1 based DDR mechanism. Conclusion ATR inhibition by Elimusertib in combination with RT may be a promising new treatment strategy in the treatment of TNBC. However, further experiments should be performed to elucidate the underlying molecular mechanisms of the therapeutic efficacy of this combination treatment and its association with DNS repair mechanisms in TNBC, in vitro and in vivo.

Histopathological evaluation of the effects of Flattening Filter (FF) and Flattening Filter Free (FFF) X-ray: In vivo malignant melanoma cancer models

The objective of this study was to investigate the radiobiological effects of Flattening Filter (FF) and Flattening Filter Free (FFF) beams on Malignant Melanoma cancer (MMe Ca) modeled NOD-Prkdc IL2rg tm1 (NSG) mice in vivo, with a specific focus on necrotic and immunofluorescent changes.Thirty-five adult NSG mice models were randomly divided into four groups; MMe Ca, FF with a low dose rate of 400 MU/min (FF-400) and FFF with a high dose rate of 1400 MU/min (FFF-1400), and Control. The NSG mice in FF-400 and FFF-1400 groups (MMe Ca plus RT groups) were irradiated with a single dose of 20 Gy at 400 MU/min and 1400 MU/min. Radiotherapy was administered to NSG mice 20 days after tumor transplantation. The mice were then sacrificed 2 days after radiotherapy for assessment of the necrotic area, cell proliferation and apoptosis.The Ki-67 proliferation index showed a significant increase (p < 0.05) in the MMe Ca group (G1) compared to the FF-400 and FFF-1400 radiotherapy groups (G2 and G3) and the control group (G4), whereas a significant decrease was observed in the radiotherapy groups due to single-dose 20Gy irradiation (p < 0.05). The apoptotic index increased with irradiation in the FF-400 and FFF-1400 radiotherapy groups (p < 0.05). The necrotic area values of FF-400 and FFF-1400 at different dose rates (G2 and G3) increased statistically significantly (p < 0.05) compared to the MMe Ca group (G1). In addition, the Ki-67 cell proliferation, apoptotic index, and necrotic area parameters exhibited statistically significant differences between the FF-400 and FFF-1400 beams (p < 0.05). The FFF-high dose rate beams were found to be more effective on MMe Ca compared to the FF conventional beams (p < 0.05).It was concluded that FF and FFF beams caused significant radiobiological changes in cell proliferation (Ki-67), apoptosis (TUNEL) and necrosis histopathological parameters and the FFF beam was more effective on MMe Ca.

Efficacy of Immediate Postoperative Single Fractional 10Gy Radiotherapy for Earlobe Keloids.

The ear is a common site for keloid formation, typically due to ear piercing or trauma. Auricular keloids are always difficult to treat and pose a challenging situation. The purpose of this study is to present our experience treating earlobe keloids through complete surgical excision followed by immediate postoperative single fraction 10Gy radiation therapy either on the same day or the next day. The authors retrospectively evaluated 48 patients with 71 earlobe keloids treated with complete surgical excision followed by single-fractional 10Gy radiotherapy within 24 hours from May 2021 to December 2022. The outcome was reported with a recurrence-free rate and side effects. The mean follow-up period was 20.8 months. All patients tolerated the treatments well. The overall recurrence-free rate was 100%. There were only 2 grade III radiation dermatitis. There were no reports of second malignancies or severe complications. The authors have obtained excellent outcomes from treating earlobe keloids through complete excision followed by postoperative radiotherapy with a single fractional dose of 10Gy on the same day or the next day.

Abstract B032: Compromised TGFβ signaling and DNA repair primes response to immune checkpoint inhibition by activating NK cells in immunologically cold breast cancers

Abstract We investigated the interplay between immunosuppressive TGFβ signaling and immunogenic DNA repair in response to immune checkpoint inhibition (ICI) in a new model of breast cancer. Transforming growth factor beta (TGFβ) is a canonical suppressor. Cancer cells evade TGFβ either by selectively overcoming TGFβ proliferative barrier or completely shutting down signaling. Loss of TGFβ signaling is accompanied by high immunosuppressive TGFβ activity. We have shown that loss of TGFβ is critical to genomic integrity. It endorses classical DNA repair and inhibits error-prone repair. We described a score, βAlt, that reports the TGFβ/DNA repair functional relationship. Pan-cancer analysis showed that high βAlt cancers, in which the TGFβ target signature is low and DNA repair signature is high, use error-prone DNA repair, are sensitive to chemoradiation and have a greater fraction of the genome altered compared to low βAlt cancers, which maintain TGFβ signaling. We established a novel murine tumor derived transplant model (mTDT) of triple-negative breast cancer, similar to a human PDX, in immunocompetent BALB/cJ mice. Transcriptomics analysis showed that mTDT range from high to low βAlt and from immune poor to immune rich, which was confirmed by multiplex immunstaining. Unsupervised clustering of mTDT based on βAlt score demonstrated that low βAlt was associated with immune rich tumors while high βAlt was associated with immune poor tumors. A similar relationship is evident in the TCGA breast cancers. We then asked how mTDT respond to ICI. Mice bearing mTDT were randomized to monotherapy consisting of single fraction 10 Gy radiation, anti-PD-L1 or TGFβ inhibition, dual or triple treatment. Neither anti-PD-L1 or TGFβ inhibition affected survival, alone or in combination, whereas radiation significantly increased survival. Unexpectedly immune poor, high βAlt mTDT were the most responsive. To evaluate biological correlates, mice were stratified as a function of treatment as responders, in which tumor growth was controlled for 7 days post-treatment, or non-responders, in which tumor growth was uncontrolled. Tumor infiltrating CD45+ cells increased (P=0.02) as did circulating CD8 T cells (P&amp;lt;0.001) in responders. Natural killer (NK) cells were also significantly expanded in responders (P&amp;lt;0.001). TGFβ broadly downregulates chemokine receptor expression necessary to recruit NK cells and impairs NK cell cytotoxic effector function. We depleted NK cells to test whether they were necessary for the pronounced response to triple treatment. NK depletion was completely abrogated response (P&amp;lt;0.00001) indicating NK cells can provoke an ICI response in high βAlt mammary tumors devoid of lymphocytes. This preclinical model and human breast cancer shows that loss of TGFβ signaling resulting in error-prone DNA damage response is strongly associated with immune poor tumors, and suggests that TGFβ inhibition can override NK cell dysfunction to create vulnerability to ICI and anti-tumor immunity. Citation Format: Jade Moore, Jim Gkantalis, Ines Guix, Colin Foster, Mary Helen Barcellos-Hoff. Compromised TGFβ signaling and DNA repair primes response to immune checkpoint inhibition by activating NK cells in immunologically cold breast cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B032.

Extracellular miRNAs in the serum and feces of mice exposed to high‑dose radiation.

Exposure to high-dose radiation causes life-threatening intestinal damage. Histopathology is the most accurate method of judging the extent of intestinal damage following death. However, it is difficult to predict the extent of intestinal damage. The present study investigated extracellular microRNAs (miRNAs or miRs) in serum and feces using a radiation-induced intestinal injury mouse model. A peak of 25-200 nucleotide small RNAs was detected in mouse serum and feces by bioanalyzer, indicating the presence of miRNAs. Microarray analysis detected four miRNAs expressed in the small intestine and increased by >2-fold in serum and 19 in feces following 10 Gy radiation exposure. Increased miR-375-3p in both serum and feces suggests leakage due to radiation-induced intestinal injury and may be a candidate for high-dose radiation biomarkers.

Alleviation of radiation combined skin injury in rat model by topical application of ascorbate formulation

Purpose This research endeavor was undertaken to elucidate the impact of an innovative ascorbate formulation on the regeneration process of full-thickness excision wounds in a rat model exposed to whole-body gamma irradiation, replicating conditions akin to combat or radiation emergency scenarios. Materials and Methods We established a comprehensive rat model by optimizing whole body γ-radiation doses (5–9 Gy) and full-thickness excision wound sizes (1–3 cm2) to mimic radiation combined injury (RCI). The developed RCI model was used to explore the healing potential of ascorbate formulation. The study includes various treatment groups (i.e., sham control, radiation alone, wound alone, radiation + wound, and radiation + wound + formulation). The ascorbate formulation was applied twice daily, with a 12-hour gap between each application, starting 1 hour after the initiation of the wound. The healing potential of the formulation in the RCI context was evaluated over 14 days through hematological, molecular, and histological parameters. Results The combination of a 5 Gy radiation dose and a 1 cm2 wound was identified as the optimal setting to develop the RCI model for subsequent studies. The formulation was used topically immediately following RCI, and then twice daily until complete healing. Treatment with the ascorbate formulation yielded noteworthy outcomes and led to a substantial reduction (p < .05) in the wound area, accelerated epithelialization periods, and an increased wound contraction rate. The formulation’s localized healing response improved organ weights, normalized blood parameters, and enhanced hematopoietic and immune systems. A gene expression study revealed the treatment up-regulated TGF-β and FGF, and down-regulated PDGF-α, TNF-α, IL-1β, IL-6, MIP-1α, and MCP-1 (p < .05). Histopathological assessments supported the formulation’s effectiveness in restoring cellular architecture and promoting tissue regeneration. Conclusion Topical application of the ascorbate formulation in RCI resulted in a significant improvement in delayed wound healing, leading to accelerated wound closure by mitigating the expression of inflammatory responses.

ALA-RDT in GBM: protocol of the phase I/II dose escalation trial of radiodynamic therapy with 5-Aminolevulinic acid in patients with recurrent glioblastoma

BackgroundDespite improvements in surgical as well as adjuvant therapies over the last decades, the prognosis for patients with glioblastoma remains poor. Five-Aminolevulinic acid (5-ALA) induced porphyrins are already used for fluorescence-guided resection and as photosensitizer for photodynamic therapy. New findings reveal their potential use as sensitizing agents in combination with ionizing radiation.MethodsWe initiated a phase I/II dose escalation study, treating patients with recurrence of glioblastoma with oral 5-ALA concurrent to radiotherapy (RT). This prospective single-center study based in the University Hospital Münster aims to recruit 30 patients over 18 years of age with histologically verified recurrence of supratentorial glioblastoma in good performance status (KPS ≥ 60). Following a 3 + 3 dose-escalation design, patients having undergone re-resection will receive a 36 Gy RT including radiodynamic therapy fractions (RDT). RDT constitutes of oral administration of 5-ALA before the irradiation session. Two cohorts will additionally receive two fractions of neoadjuvant treatment three and two days before surgery. To determine the maximum tolerated dose of repeated 5-ALA-administration, the number of RDT-fractions will increase, starting with one to a maximum of eight fractions, while closely monitoring for safety and toxicity. Follow-up will be performed at two and five months after treatment. Primary endpoint will be the maximum tolerated dose (MTD) of repeated ALA-administration, secondary endpoints are event-free-, progression-free-, and overall-survival. Additionally, 5-ALA metabolites and radiobiological markers will be analysed throughout the course of therapy and tissue effects after neoadjuvant treatment will be determined in resected tissue. This protocol is in accordance with the SPIRIT guidelines for clinical trial protocols.DiscussionThis is the protocol of the ALA-RDT in GBM-study, the first-in-man evaluation of repeated administration of 5-ALA as a radiosensitizer for treatment of recurrent glioblastoma.Trial registrationThis study was approved by the local ethics committee of the Medical Association of Westphalia-Lippe and the University of Münster on 12.10.2022, the German federal institute for Drugs and medical devices on 13.10.2022 and the federal office for radiation protection on 29.08.2022. This trial was registered on the public European EudraCT database (EudraCT-No.: 2021-004631-92) and is registered under www.cliniclatrials.gov (Identifier: NCT05590689).

Circ_0026134 regulates the miR-1270/GRB2 pathway to affect the radiosensitivity of hepatoma cells

Objective: To investigate whether circular RNA 0026134 (circ_0026134) affects the radiosensitivity of hepatoma cells by regulating the miR-1270/growth factor receptor-bound protein 2 (GRB2) pathway. Methods: Real-time quantitative PCR (RT-qPCR) was used to detect the expression levels of circ_0026134, miR-1270, and GRB2 in liver cancer tissues and cells. Bioinformatics analysis, a dual-luciferase gene reporter assay, RT-qPCR, and western blot were used to analyze the targeting relationships between circ_0026134 and miR-1270 and miR-1270 and GRB2. The effects of circ_0026134, miR-1270, and GRB2 expression combined with 6 Gy on the proliferation, invasion, migration, and apoptosis of Huh7 and SK-HEP-1 cells were detected by a cell counting kit, a transwell assay, a scratch assay, and flow cytometry. The tumorigenesis experiment was used to detect the effect of silencing circ_0026134 in nude mice. Measurement data are expressed as the mean ± standard deviation. The independent sample t-test was used for comparison between two groups, and the one-way analysis of variance and SNK-q test were used for comparison between multiple groups. P < 0.05 was considered statistically significant. Results: The expression levels of circ_0026134 and GRB2, Huh7, and SK-HEP-1 cells in liver cancer tissues were significantly increased, while the expression levels of miR-1270 were significantly decreased (P < 0.05). The expression of circ_0026134 in Huh7 and SK-HEP-1 decreased significantly after radiotherapy (P < 0.05). circ_0026134 binds directly to miR-1270 and negatively regulates miR-1270 expression (P < 0.05). miR-1270 binds directly to GRB2 and negatively regulates GRB2 expression (P < 0.05). 6 Gy radiation significantly inhibited the proliferation, migration, and invasion of Huh7 and SK-HEP-1 cells and induced apoptosis (P < 0.05). Silencing circ_0026134 or overexpression of miR-1270 significantly enhanced the anti-proliferation, anti-migration, invasion, and pro-apoptosis effects of 6 Gy treatment on hepatoma cells (P < 0.05). Inhibition of miR-1270 significantly weakened the effects of silencing circ_0026134 combined with 6 Gy radiation on proliferation, migration, invasion, and apoptosis of hepatoma cells (P < 0.05). Overexpression of GRB2 significantly weakened the effects of miR-1270 overexpression combined with 6 Gy radiation on proliferation, migration, invasion, and apoptosis of hepatoma cells (P < 0.05). circ_0026134 knockdown significantly delayed tumor growth in vivo (P < 0.05). Conclusion: Silencing circ_0026134 strengthens radiation treatment's anti-proliferation, anti-migration, invasion, and pro-apoptotic effects in hepatoma cells by negatively regulating the miR-1270/GRB2 pathway, thereby enhancing radiosensitivity.

Neoadjuvant anti-programmed death 1 (PD-1) therapy in patients (pts) with resectable mismatch repair–deficient (dMMR) locally advanced rectal cancer (LARC): EPOC2201.

TPS226 Background: Neoadjuvant radiotherapy and chemotherapy followed by surgical resection is a standard treatment for LARC. As anti-PD-1 therapy for patients with dMMR colorectal cancer is responsive in the context of metastatic disease, several clinical trials which evaluate the efficacies of anti-PD-1 therapy in patients with dMMR LARC are on-going and suggested the outstanding responses (Cercek A. et al., N Engl J Med 2022). Methods: We initiated the EPOC 2201 VOLTAGE-2 study, which is an ongoing, non-randomized, single-arm phase II trial. The major eligibility criteria were as follows: treatment-naïve with rectal cancer located 12 cm from the anal verge; confirmed dMMR; clinical stage II/III in primary cohort (N=35) and stage I in exploratory cohort (N=20); age ≥ 18; Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1; and presence of sufficient organ function. The study treatments include 12 cycles of nivolumab (NIVO) monotherapy (240 mg every 2 weeks) followed by assessment using Memorial Sloan Kettering (MSKCC) Regression Schema (Smith J.J. et al., BMC Cancer 2015). If clinical complete response (cCR) or near complete response (nCR) are observed, non-operative management with additional 12 cycles of NIVO will be performed. If incomplete response (IR) is observed, standard chemoradiotherapy with 50.4Gy of radiation plus capecitabine followed by surgery will be performed. The primary endpoint is the 2-year cCR rate in primary cohort. Various survival endpoints including progression-free survival (PFS) and overall survival (OS) are assessed as secondary endpoints. Safety analyses are performed using the Common Terminology Criteria for Adverse Events (CTCAE) ver. 5.0, and the Clavien‒Dindo classification (ver. 2.0) is added for postoperative complications. Assuming null and alternative hypotheses, cCR rates of 23% and 45%, the planned sample size for primary cohort was set at 35, with a one-sided alpha of 5% and power of 80%. An exploratory biomarker study was also conducted using serially collected tumor and blood samples. Whole genome-based minimal residual disease (MRD) assay and whole exome/whole transcriptome-based tumor and immune microenvironment analyses are performed. As of August 2023, patient’s enrollment is ongoing. Clinical trial information: jRCT2031220484 .

Reactive oxygen species may be involved in the distinctive biological effects of different doses of 12C6+ ion beams on Arabidopsis.

Heavy ion beam is a novel approach for crop mutagenesis with the advantage of high energy transfer line density and low repair effect after injury, however, little investigation on the biological effect on plant was performed. 50 Gy irradiation significantly stimulated the growth of Arabidopsis seedlings, as indicated by an increase in root and biomass, while 200 Gy irradiation significantly inhibited the growth of seedlings, causing a visible decrease in plant growth. The Arabidopsis seeds were irradiated by 12C6+. Monte Carlo simulations were used to calculate the damage to seeds and particle trajectories by ion implantation. The seed epidermis received SEM detection and changes in its organic composition were detected using FTIR. Evidence of ROS and antioxidant systems were analyzed. RNA-seq and qPCR were used to detect changes in seedling transcript levels. Monte Carlo simulations revealed that high-dose irradiation causes various damage. Evidence of ROS and antioxidant systems implies that the emergence of phenotypes in plant cells may be associated with oxidative stress. Transcriptomic analysis of the seedlings demonstrated that 170 DEGs were present in the 50 Gy and 200 Gy groups and GO enrichment indicated that they were mainly associated with stress resistance and cell wall homeostasis. Further GO enrichment of DEGs unique to 50 Gy and 200 Gy revealed 58 50Gy-exclusive DEGs were enriched in response to oxidative stress and jasmonic acid entries, while 435 200 Gy-exclusive DEGs were enriched in relation to oxidative stress, organic cyclic compounds, and salicylic acid. This investigation advances our insight into the biological effects of heavy ion irradiation and the underlying mechanisms.

Abstract A030: Fusion between macrophages and cancer cells up-regulates signal regulatory protein pathway and contributes to malignant progression of pancreatic cancer after radiation

Abstract Introduction: Fusion between tumor cells with leukocytes contributes to tumor heterogeneity, resulting in tumorigenesis, metastasis, and treatment resistance. Recent studies demonstrate that an inflammatory microenvironment enhances tumor-macrophage fusion. We hypothesize that tumor-macrophage fusion hybrids contribute to radiation induced pancreatic cancer progression. Materials and Methods: Tumor tissues of female patients receiving previous bone marrow transplantation (BMT) from male donors due to leukemia/lymphoma were collected for Y chromosome and immunofluorescence study. Murine subcutaneous tumor models of cre-loxP or dual fluorescence system were established to demonstrate fusion between Pan02, a murine pancreatic adenocarcinoma cell line, and stromal cells of ROSA (Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo/Jnarl) mice with or without local radiation exposure. KC (pdx-1-cre-KrasLSL-G12D) mice, were given whole body irradiation followed by BMT from ROSA mice to measure fusion between pancreatic cancer and hematopoietic cells. Liposomal chlodronate, a macrophage depleting agent, and immunofluorescence staining of fusion hybrids in ROSA mice tumors were used to demonstrate macrophage as a fusion partner. Fusion hybrid clones of murine/human pancreatic adenocarcinoma cells with macrophages were selected from co-culture and sorting followed by studies including ploidy, clonogenicity, migration and radio-sensitivity. Affymetrix microarray analysis was used to find differentially expressed genes of fusion hybrids. Results: Fusion phenomenon was demonstrated by showing colocalization of Y chromosomes with epithelial markers in cancer tissues from female patients receiving prior BMT from male donors. Fusion hybrids were found in pancreas tumors of KC mice after BMT from ROSA mice. Fusion hybrids within tumors increased by time, three folds within 6 weeks; and 2.5 folds after 20Gy radiation exposure. Liposomal clodronate reduced fusion hybrids and tumor growth. Immunofluorescence stain of tumor tissues demonstrated CD11b(+) macrophage to be the major fusion partner. Purified Fusion hybrids showed enhanced migration, proliferation, aneuploidy and radio-resistance. Model based gene analysis of microarray study disclosed signal regulatory protein (SIRP) signal pathway was significantly upregulated in fusion hybrids. Upregulated SIRP signal molecules, including DAP12, PyK2, were validated by immunoblots. Silencing DAP12 in myeloid cells from human peripheral blood showed decreased fusion rate with Panc-1 cells. DAP12mRNA depletion in Panc-1 fusion hybrids disclosed enhanced radio-sensitivity. Defactinib (VS6063), a PyK2 inhibitor, reduced fusion phenomenon and suppressed pancreatic tumor growth of mice after local radiation. Conclusions: Fusion between pancreatic cancer cells and macrophages contribute to radiation enhanced malignant progression of pancreatic cancer. SIRP signal pathway is a potential target to disrupt fusion phenomenon and ameliorate pancreatic cancer progression after radiation. Citation Format: Hui Ju Ch'ang, Yi-Chih Tsai, Ting Wei Li, Rui-Jun Liu, Tze-Sing Huang, Sse-Pei Wan, Su-Liang Chen, Shih-Sheng Jiang. Fusion between macrophages and cancer cells up-regulates signal regulatory protein pathway and contributes to malignant progression of pancreatic cancer after radiation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A030.

Acute Large Dose Irradiation Sensitizes Surviving Cells to Subsequent Irradiation; Implications for Stereotactic Body Radiation Therapy.

To determine if the radiation sensitivity of cells that survive acute high-dose radiation exposure used in stereotactic body radiation therapy (SBRT), differs from the sensitivity of non-irradiated cells and cells that survive multiple 2 Gy doses of radiation. Isogenic rodent and two human tumor cell lines were exposed to 14 × 2 Gy of radiation, or a single acute dose of 12 Gy. The most resistant cell line was also exposed to an acute dose of 15 Gy. One week after 12 Gy, and 4 days after 14 × 2 Gy, surviving cells were exposed to 0-8 Gy in 2 Gy doses and cell survival was assessed by colony formation. In addition, the colony forming efficiency of 12 Gy survivors was evaluated for 1 month postirradiation. For cells exposed to 15 Gy, the response of surviving cells to 6 Gy was determined for up to 35 days postirradiation and compared to the 6 Gy surviving fraction of control cells. The radiation sensitivity of cells that survived 12 Gy exposure, and cells that survived 14 fractions of 2 Gy irradiation did not differ from the response of unirradiated control cells. However, the growth rate and colony forming efficiency of 12 Gy survivors was transiently reduced for greater than 2 weeks postirradiation. In contrast to the unchanged sensitivity of 12 Gy surviving cells at day 7 postirradiation, 15 Gy survivors exhibited enhanced sensitivity to radiation for up to 21 days postirradiation and suggests a biological basis for SBRT.

Abstract A001: Discovery of imidazo [4,5-c]pyridin-2-ones as selective inhibitors of DNA-dependent protein kinase and effective radiosensitizers

Abstract Inhibition of the repair of radiation-induced DNA double strand breaks (DSB) offers the potential to sensitize tumors to radiation therapy. The dominant role of non-homologous end-joining in the repair of radiation-induced DSBs indicates that DNA-dependent protein kinase (DNA-PK) is an excellent target for the development of radiosensitizers. We report the discovery of a new chemical class of kinase inhibitor based on the imidazo[4,5-c]pyridin-2-one scaffold. The class was developed by scaffold-hopping from the pan phosphoinositide 3-kinsase (PI3K) and PI3K-like kinase (PIKK) inhibitor dactolisib. Iterative development culminated in the identification of SN39536 as a low nM DNA-PK inhibitor with excellent selectivity. Further modification led to the discovery of the more potent and more selective SN40905. Both SN39536 and SN40905 were effective inhibitors of DNA-PK kinase activity in a biochemical assay with substantial selectivity for DNA-PK across the PIKK and PI3K families. Both compounds selectively inhibited growth of HAP1 PRKDC wild-type cells when combined with radiation, but not the corresponding PRKDC-/- cells. SN39536 and SN40905 were effective radiosensitizers of colorectal carcinoma (HCT116), non-small cell lung cancer (H460, H1299, and A549), pancreatic (BxPC-3, PANC-1, and MiaPaCa-2) and head and neck squamous cell carcinoma (FaDu and UT-SCC-74B) cells determined using a clonogenic survival endpoint. Both SN39536 and SN40905 displayed high oral bioavailability. When administered PO at a range of non-toxic doses, both SN39536 and SN40905 provided significant additional tumor cell killing of HCT116, UT-SCC-74B, and BxPC-3 tumor xenografts in combination with a single (13 Gy) radiation treatment determined by ex vivo clonogenic survival assays. SN39536 also provided substantial tumor growth inhibition of HCT116 tumor xenografts in combination with RAD (10 Gy). SN39536 and SN40905 represent a new, potent, and selective class of DNA-PK inhibitors with significant potential as radiosensitizers for the treatment of human cancers. Citation Format: Michael P. Hay, Cho R. Hong, Lydia P. Liew, Benjamin D. Dickson, Way W. Wong, Sophia F. O'Brien-Gortner, Rebecca Airey, Jagdish Jaiswal, William R. Wilson, Stephen M. Jamieson. Discovery of imidazo [4,5-c]pyridin-2-ones as selective inhibitors of DNA-dependent protein kinase and effective radiosensitizers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A001.

The Predictive Value of 8-Hydroxy-Deoxyguanosine (8-OHdG) Serum Concentrations in Irradiated Non-Small Cell Lung Carcinoma (NSCLC) Patients.

Ionizing radiation is strongly linked to direct or indirect DNA damage, as with the production of reactive oxygen species (ROS), which in turn produce DNA damage products, such as 8-hydroxy-2-deoxyguanosine (8-OHdG). In this study, we aimed to investigate the formation of 8-OHdG after irradiation in patients with non-small cell cancer (NSCLC) and its use as a biomarker. Sixteen patients with squamous and thirty-six patients with non-squamous pathology were included. An enzyme-linked-immunosorbent assay (ELISA) was performed before and after radiation. A dose-dependent relationship was confirmed: 8-OHdG plasma concentrations, increased in the total of NSCLC patients and specifically with a linear correlation in non-squamous pathology; in squamous histology, after an initial increase, a significant decrease followed after 20 Gy dose of irradiation. The pretreatment total irradiated tumor volume (cm3) was positively correlated with 8-OHdG levels in patients with squamous histology. When plotting the 8-OHdG plasma concentration at a 10 Gy irradiation dose to the baseline, the AUC was 0.873 (95% CI 0.614-0.984), p < 0.0001, with an associated criterion value of >1378 as a cutoff (sensitivity 72.7%, specificity 100%). When normalizing this ratio to BSA, the associated criterion cutoff value was >708 (sensitivity of 100%, specificity 80%). Lastly, 8-OHdG levels were closely related with the development of radiation-induced toxicities.

Oxidant-Dependent Sensitizing, Protective, and Mitigative Effects in X-Ray-Irradiated Pulmonary Endothelial Cells.

The primary response of proliferating bovine pulmonary artery endothelial cells (BPAECs) after X-ray irradiation [≤10 gray (Gy)] is shown to be transient cell-cycle arrest. Accompanying oxidant-linked functional changes within the mitochondria are readily measured, but increased autophagy is not. Radiation-induced apoptosis is negligible in this line-important because cells undergoing apoptosis release oxygen-derived species that can overwhelm/mask the radiation-associated species and their effects that we wish to investigate. Cells irradiated and cultured at 3% oxygen exhibited delayed cell-cycle arrest (6-8 hours after 10 Gy irradiation) compared with those maintained at 20% oxygen (2-4 hours after 10 Gy irradiation). At 3% oxygen, either only during or only after irradiation, results intermediate between 20% and 3% oxygen throughout were obtained. No variability in cell-cycle distribution was observed for unirradiated cells cultured under different prevailing oxygen levels. Mitochondrially localized manganese superoxide dismutase delayed the X-ray-induced cell-cycle changes when over-expressed in BPAEC, indicating superoxide to be one of the key oxygen-derived cytotoxic species involved in the radiobiological response. Also, the peroxynitrite biomarker 3-nitrotyrosine was elevated, whereas hydrogen peroxide levels were not. Lastly, the utility of the BPAEC for screening potential countermeasures to ionizing radiation is demonstrated with some quinoline derivatives. Three of the five compounds appeared mitigative, and all were protective. It is suggested that the oxidation-reduction chemistry of these compounds probably offers a reasonable explanation for their observed ameliorative properties. Furthermore, the results suggest a promising new direction in the search for lead compounds as countermeasures to the effects of ionizing radiation. SIGNIFICANCE STATEMENT: The primary radiological response of proliferating bovine pulmonary artery endothelial cells is cell-cycle arrest, starting soon after X-ray irradiation (1-10 Gy) at 20% O2 but delayed by 4 hours at systemic (3%) O2. Oxygen/superoxide is found to be radio-sensitizing in at least two distinct time windows, during and after the irradiation, with both responses antagonized by various hydroxyquinoline derivatives. Similar responses in many other cell lines are likely to be masked by elevated oxidants associated with apoptosis.