Immune Response following FLASH and Conventional Radiation in Diffuse Midline Glioma.

PURPOSE: Diffuse midline glioma (DMG) is a fatal tumor traditionally treated with radiation therapy (RT) and previously characterized as having a noninflammatory tumor immune microenvironment (TIME). FLASH is a novel RT technique using ultra-high dose rate that is associated with decreased toxicity and effective tumor control. However, the effect of FLASH and conventional (CONV) RT on the DMG TIME has not yet been explored. METHODS AND MATERIALS: Here, we performed single-cell RNA sequencing (scRNA-seq) and flow cytometry on immune cells isolated from an orthotopic syngeneic murine model of brainstem DMG after the use of FLASH (90 Gy/sec) or CONV (2 Gy/min) dose-rate RT and compared to unirradiated tumor (SHAM). RESULTS: At day 4 post-RT, FLASH exerted similar effects as CONV in the predominant microglial (MG) population, including the presence of two activated subtypes. However, at day 10 post-RT, we observed a significant increase in the type 1 interferon α/ß receptor (IFNAR+) in MG in CONV and SHAM compared to FLASH. In the non-resident myeloid clusters of macrophages (MACs) and dendritic cells (DCs), we found increased type 1 interferon (IFN1) pathway enrichment for CONV compared to FLASH and SHAM by scRNA-seq. We observed this trend by flow cytometry at day 4 post-RT in IFNAR+ MACs and DCs, which equalized by day 10 post-RT. DMG control and murine survival were equivalent between RT dose rates. CONCLUSIONS: Our work is the first to map CONV and FLASH immune alterations of the DMG TIME with single-cell resolution. Although DMG tumor control and survival were similar between CONV and FLASH, we found that changes in immune compartments differed over time. Importantly, although both RT modalities increased IFN1, we found that the timing of this response was cell-type and dose-rate dependent. These temporal differences, particularly in the context of tumor control, warrant further study.

The Tumorigenic Effect of lncRNA AFAP1-AS1 is Mediated by Translated Peptide ATMLP Under the Control of m6 A Methylation.

Long noncoding RNAs (lncRNAs) in eukaryotic transcripts have long been believed to regulate various aspects of cellular processes, including carcinogenesis. Herein, it is found that lncRNA AFAP1-AS1 encodes a conserved 90-amino acid peptide located on mitochondria, named lncRNA AFAP1-AS1 translated mitochondrial-localized peptide (ATMLP), and it is not the lncRNA but the peptide that promotes the malignancy of nonsmall cell lung cancer (NSCLC). As the tumor progresses, the serum level of ATMLP increases. NSCLC patients with high levels of ATMLP display poorer prognosis. Translation of ATMLP is controlled by m6 A methylation at the 1313 adenine locus of AFAP1-AS1. Mechanistically, ATMLP binds to the 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) and inhibits its transport from the inner to the outer mitochondrial membrane, which antagonizes the NIPSNAP1-mediated regulation of cell autolysosome formation. The findings uncover a complex regulatory mechanism of NSCLC malignancy orchestrated by a peptide encoded by a lncRNA. A comprehensive judgment of the application prospects of ATMLP as an early diagnostic biomarker for NSCLC is also made.

Single-Cell Transcriptome Analysis of Radiation Pneumonitis Mice.

Radiation-induced lung injury (RILI), especially radiation pneumonitis (RP), is a common clinical complication associated with thoracic radiotherapy for malignant tumors. However, the specific contributions of each cell subtype to this process are unknown. Here, we provide the single-cell pathology landscape of the RP in a mouse model by unbiased single-cell RNA-seq (scRNA-seq). We found a decline of type 2 alveolar cells in the RP lung tissue, with an expansion of macrophages, especially the Fabp4low and Spp1high subgroup, while Fabp4high macrophages were almost depleted. We observed an elevated expression of multiple mitochondrial genes in the RP group, indicating a type 2 alveolar cell (AT2) response to oxidative stress. We also calculated the enrichment of a cGAS-STING signaling pathway, which may be involved in regulating inflammatory responses and cancer progression in AT2 cells of PR mice. We delineate markers and transcriptional states, identify a type 2 alveolar cell, and uncover fundamental determinants of lung fibrosis and inflammatory response in RP lung tissue of mice.

Smurf2 inhibition enhances chemotherapy and radiation sensitivity in non-small-cell lung cancer.

Lung cancer has been the most common cancer worldwide for several decades. The outcomes of patients with locally advanced lung cancer remain dismal, and only a minority of patients survive more than 5 years. However, tumor therapeutic resistance mechanisms are poorly studied. Identification of therapeutic resistance pathways in lung cancer in order to increase the sensitivity of lung tumor cells to therapeutic agents is a crucial but challenging need. To identify novel genes that modulate the response to platinum-based therapy, we performed a genome-wide high-throughput ribonucleic acid interference (RNAi) screen via transfection of human lung cancer (PC9) cells with a viral short hairpin RNA (shRNA) library. We further validated a potential target via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic survival assays on PC9 and A549 lung tumor cells transfected with small interfering RNAs (siRNAs) to successfully downregulate protein expression and then treated with increasing doses of cisplatin or X-ray radiation. We determined protein expression by immunohistochemistry (IHC) after chemoradiotherapy and analyzed gene expression-based survival outcomes in two cohorts of human non-small-cell lung cancer (NSCLC) patients. The screen identified several targets involved in epithelial-to-mesenchymal transition (EMT), including Smurf1, Smurf2, YAP1, and CEBPZ, and glycolytic pathway proteins, including PFKFB3. Furthermore, we found that the small molecule proteasome inhibitor bortezomib significantly downregulated Smurf2 in lung cancer cells. The addition of bortezomib in combination with cisplatin and radiation therapy in PC9 and A549 cells led to an increase in deoxyribonucleic acid (DNA) double-strand breaks with increased numbers of γ-H2AX-positive cells and upregulation of apoptosis. Finally, we found that Smurf2 protein expression was upregulated in situ after treatment with cisplatin and radiation therapy in a relevant cohort of patients with stage III NSCLC. Additionally, Smurf2 gene expression was the strongest predictor of survival in patients with squamous NSCLC after chemotherapy or chemoradiotherapy. We successfully identified and validated Smurf2 as both a common modulator of resistance and an actionable target in lung cancer. These results suggest the urgent need to investigate clinical Smurf2 inhibition via bortezomib in combination with cisplatin and radiation for patients with locally advanced NSCLC.

Mitochondrial Damage Response and Fate of Normal Cells Exposed to FLASH Irradiation with Protons.

Radiation therapy (RT) plays an important role in cancer treatment. The clinical efficacy of radiation therapy is, however, limited by normal tissue toxicity in areas surrounding the irradiated tumor. Compared to conventional radiation therapy (CONV-RT) in which doses are typically delivered at dose rates between 0.03-0.05 Gy/s, there is evidence that radiation delivered at dose rates of orders of magnitude higher (known as FLASH-RT), dramatically reduces the adverse side effects in normal tissues while achieving similar tumor control. The present study focused on normal cell response and tested the hypothesis that proton-FLASH irradiation preserves mitochondria function of normal cells through the induction of phosphorylated Drp1. Normal human lung fibroblasts (IMR90) were irradiated under ambient oxygen concentration (21%) with protons (LET = 10 keV/µm) delivered at dose rates of either 0.33 Gy/s or 100 Gy/s. Mitochondrial dynamics, functions, cell growth and changes in protein expression levels were investigated. Compared to lower dose-rate proton irradiation, FLASH-RT prevented mitochondria damage characterized by morphological changes, functional changes (membrane potential, mtDNA copy number and oxidative enzyme levels) and oxyradical production. After CONV-RT, the phosphorylated form of Dynamin-1-like protein (p-Drp1) underwent dephosphorylation and aggregated into the mitochondria resulting in mitochondria fission and subsequent cell death. In contrast, p-Drp1 protein level did not significantly change after delivery of similar FLASH doses. Compared with CONV irradiation, FLASH irradiation using protons induces minimal mitochondria damage; our results highlight a possible contribution of Drp1-mediated mitochondrial homeostasis in this potential novel cancer treatment modality.

Ionizing radiation-induced long noncoding RNA CRYBG3 regulates YAP/TAZ through mechanotransduction.

Mechanotransduction sensing of tissue architecture and cellular microenvironment is a fundamental regulator of cell fate, including cancer. Meanwhile, long noncoding RNAs (lncRNAs) play multifunctions during cancer development and treatment. However, the link between lncRNAs and cellular mechanotransduction in the context of cancer progression has not yet been elucidated. In this study, using atomic force microscopy (AFM), we find that ionizing radiation reduces tumor stiffness. Ionizing radiation-induced lncRNA CRYBG3 can blunt YAP/TAZ activity through interference with mechanotransduction, resulting in the inhibition of cell proliferation, invasion, and metastasis of lung cancer cells. In vivo, we found that loss of lncRNA CRYBG3 could power the tumor initiation and metastasis ability, but this was abolished by concomitant deplete TAZ. At the molecular level, lncRNA CRYBG3 that in turn dysregulates F-actin organization, activates the LATS1/2 kinase, all in all resulting in YAP/TAZ nuclear exclusion. Our research proposes that lncRNA CRYBG3 is a mediator of radiotherapy through its control of cancer-tissue mechanotransduction and wiring YAP/TAZ activity to control tumor growth and metastasis.

A proposed change to astronaut exposures limits is a giant leap backwards for radiation protection.

Addressing the uncertainties in assessing health risks from cosmic ray heavy ions is a major scientific challenge recognized by many previous reports by the National Academy of Sciences (NAS) and the National Council on Radiation Protection and Measurements (NCRP) advising the National Aeronautics and Space Administration (NASA). These reports suggested a series of steps to pursue the scientific basis for space radiation protection, including the implementation of age and sex dependent risk assessments and exposure limits appropriate for a small population of radiation workers, the evaluation of uncertainties in risk projections, and developing a vigorous research program in heavy ion radiobiology to reduce uncertainties and discover effective countermeasures. The assessment of uncertainties in assessing risk provides protection against changing assessments of risk, reveals limitations in information used in space mission operations, and provides the impetus to reduce uncertainties and discover the true level of risk and possible effectiveness of countermeasures through research. However, recommendations of a recent NAS report, in an effort to minimize differences in age and sex on flight opportunities, suggest a 600 mSv career effective dose limit based on a median estimate to reach 3% cancer fatality for 35-year old females. The NAS report does not call out examples where females would be excluded from space missions planned in the current decade using the current radiation limits at NASA. In addition, there are minimal considerations of the level of risk to be encountered at this exposure level with respect to the uncertainties of heavy ion radiobiology, and risks of cancer, as well as cognitive detriments and circulatory diseases. Furthermore, their recommendation to limit Sieverts and not risk in conjunction with a waiver process is essentially a recommendation to remove radiation limits for astronauts. We discuss issues with several of the NAS recommendations with the conclusion that the recommendations could have negative impacts on crew health and safety, and violate the three principles of radiation protection (to prevent clinically significant deterministic effects, limit stochastic effects, and practice ALARA), which would be a giant leap backwards for radiation protection.

Androgen Receptor, Although Not a Specific Marker For, Is a Novel Target to Suppress Glioma Stem Cells as a Therapeutic Strategy for Glioblastoma.

Targeting androgen receptor (AR) has been shown to be promising in treating glioblastoma (GBM) in cell culture and flank implant models but the mechanisms remain unclear. AR antagonists including enzalutamide are available for treating prostate cancer patients in clinic and can pass the blood-brain barrier, thus are potentially good candidates for GBM treatment but have not been tested in GBM orthotopically. Our current studies confirmed that in patients, a majority of GBM tumors overexpress AR in both genders. Enzalutamide inhibited the proliferation of GBM cells both in vitro and in vivo. Although confocal microscopy demonstrated that AR is expressed but not specifically in glioma cancer stem cells (CSCs) (CD133+), enzalutamide treatment significantly decreased CSC population in cultured monolayer cells and spheroids, suppressed tumor sphere-forming capacity of GBM cells, and downregulated CSC gene expression at mRNA and protein levels in a dose- and time-dependent manner. We have, for the first time, demonstrated that enzalutamide treatment decreased the density of CSCs in vivo and improved survival in an orthotopic GBM mouse model. We conclude that AR antagonists potently target glioma CSCs in addition to suppressing the overall proliferation of GBM cells as a mechanism supporting their repurposing for clinical applications treating GBM.

Silver nanoparticles protect against arsenic induced genotoxicity via attenuating arsenic bioaccumulation and elevating antioxidation in mammalian cells.

Arsenic (As) and its compounds have been classified as Group I carcinogenic agents by the International Agency for Research on Cancer (IARC); however, there is few specific and efficient antidotes used for As detoxification. The present study aimed to investigate the protective effects of silver nanoparticles (AgNPs) at non-toxic concentrations on As(Ⅲ) induced genotoxicity and the underlying mechanism. Our data showed that AgNPs pretreatment significantly inhibited the generation of phosphorylated histone H2AX (γ-H2AX, marker of nuclear DNA double strand breaks) and the mutation frequencies induced by As(Ⅲ) exposure. Atomic fluorescence spectrometer (AFS) and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) analysis revealed that the intracellular accumulation of As(Ⅲ) in human-hamster hybrid AL cells was declined by AgNPs via suppressing the expression of specific As(Ⅲ)-binding protein (Gal-1). Moreover, the activities of antioxidant enzymes were greatly up-regulated by AgNPs, which eventually inhibited the generation of reactive oxygen species (ROS) induced by As(Ⅲ) and the downstream stress-activated protein kinases/c-Jun amino-terminal kinases (SAPK/JNK) signaling pathway. These results provided clear evidence that AgNPs dramatically suppressed the genotoxic response of As(Ⅲ) in mammalian cells via decreasing As(Ⅲ) bioaccumulation and elevating intracellular antioxidation, which might provide a new clue for AgNPs applications in As(Ⅲ) detoxification.

The long noncoding RNA CRYBG3 induces aneuploidy by interfering with spindle assembly checkpoint via direct binding with Bub3.

Aneuploidy is a hallmark of genomic instability that leads to tumor initiation, progression, and metastasis. CDC20, Bub1, and Bub3 form the mitosis checkpoint complex (MCC) that binds the anaphase-promoting complex or cyclosome (APC/C), a crucial factor of the spindle assembly checkpoint (SAC), to ensure the bi-directional attachment and proper segregation of all sister chromosomes. However, just how MCC is regulated to ensure normal mitosis during cellular division remains unclear. In the present study, we demonstrated that LNC CRYBG3, an ionizing radiation-inducible long noncoding RNA, directly binds with Bub3 and interrupts its interaction with CDC20 to result in aneuploidy. The 261-317 (S3) residual of the LNC CRYBG3 sequence is critical for its interaction with Bub3 protein. Overexpression of LNC CRYBG3 leads to aneuploidy and promotes tumorigenesis and metastasis of lung cancer cells, implying that LNC CRYBG3 is a novel oncogene. These findings provide a novel mechanistic basis for the pathogenesis of NSCLC after exposure to ionizing radiation as well as a potential target for the diagnosis, treatment, and prognosis of an often fatal disease.

Radiation Exposure-Induced Changes in the Immune Cells and Immune Factors of Mice With or Without Primary Lung Tumor.

Recent studies have demonstrated that radiation activates in situ antitumor immunity and consequently induced a synergistic effect of radiotherapy and immunotherapy. However, studies related to radiation-induced changes in immune system of tumor-bearing mice are limited, which are of great significance to improve the efficacy of radioimmunotherapy. In this study, we first established a primary lung tumor mouse model using urethane. Then part of the right lung of the mouse was exposed to X-ray irradiation with a computed tomography-guided small animal irradiator and the changes of immune cells in both peripheral blood and spleen were determined by flow cytometry. Besides, the levels of both cytokines and immunoglobulins in mouse serum were detected by a protein chip. We found that B lymphocytes increased while CD8+ T lymphocytes reduced significantly. Interleukin-3 (IL-3), IL-6, regulated upon activation, normally T-expressed, and presumably secreted factor (RANTES), and vascular endothelial growth factor (VEGF) were found to be decreased after tumor formation, and the similar results have also been observed with kappa, IgG3, IgE, IgM, and IgG2a. After irradiation, lower concentrations of IgD, kappa, and IgM were found in the serum. Our findings indicate that localized tumor irradiation caused some obvious changes like inhibiting the ability of innate immunity, and these changes may be useful in predicting prognosis.