ABSTRACT
Radiation therapy (RT) is frequently used to treat cancers, including soft-tissue sarcomas. Prior studies established that the toll-like receptor 9 (TLR9) agonist cytosine-phosphate-guanine oligodeoxynucleotide (CpG) enhances the response to RT in transplanted tumors, but the mechanisms of this enhancement remain unclear. Here, we used CRISPR/Cas9 and the chemical carcinogen 3-methylcholanthrene (MCA) to generate autochthonous soft-tissue sarcomas with high tumor mutation burden. Treatment with a single fraction of 20 Gy RT and 2 doses of CpG significantly enhanced tumor response, which was abrogated by genetic or immunodepletion of CD8+ T cells. To characterize the immune response to CpG+RT, we performed bulk RNA-Seq, single-cell RNA-Seq, and mass cytometry. Sarcomas treated with 20 Gy and CpG demonstrated increased CD8 T cells expressing markers associated with activation and proliferation, such as Granzyme B, Ki-67, and IFN-γ. CpG+RT also upregulated antigen presentation pathways on myeloid cells. Furthermore, in sarcomas treated with CpG+RT, TCR clonality analysis suggests an increase in clonal T cell dominance. Collectively, these findings demonstrate that CpG+RT significantly delays tumor growth in a CD8 T cell-dependent manner. These results provide a strong rationale for clinical trials evaluating CpG or other TLR9 agonists with RT in patients with soft-tissue sarcoma.
Subject(s)
CD8-Positive T-Lymphocytes , Oligodeoxyribonucleotides , Toll-Like Receptor 9 , Animals , Toll-Like Receptor 9/agonists , Mice , Oligodeoxyribonucleotides/pharmacology , Oligodeoxyribonucleotides/administration & dosage , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , Sarcoma/radiotherapy , Sarcoma/therapy , Sarcoma/pathology , Injections, Intralesional , CRISPR-Cas Systems , Sarcoma, Experimental/pathology , Sarcoma, Experimental/radiotherapy , FemaleABSTRACT
Diverse tumor metabolic phenotypes are influenced by the environment and genetic lesions. Whether these phenotypes extend to rhabdomyosarcoma (RMS) and how they might be leveraged to design new therapeutic approaches remains an open question. Thus, we utilized a Pax7Cre-ER-T2/+; NrasLSL-G12D/+; p53fl/fl (P7NP) murine model of sarcoma with mutations that most frequently occur in human embryonal RMS. To study metabolism, we infuse 13C-labeled glucose or glutamine into mice with sarcomas and show that sarcomas consume more glucose and glutamine than healthy muscle tissue. However, we reveal a marked shift from glucose consumption to glutamine metabolism after radiation therapy (RT). In addition, we show that inhibiting glutamine, either through genetic deletion of glutaminase (Gls1) or through pharmacological inhibition of glutaminase, leads to significant radiosensitization in vivo. This causes a significant increase in overall survival for mice with Gls1-deficient compared to Gls1-proficient sarcomas. Finally, Gls1-deficient sarcomas post-RT elevate levels of proteins involved in natural killer cell and interferon alpha/gamma responses, suggesting a possible role of innate immunity in the radiosensitization of Gls1-deficient sarcomas. Thus, our results indicate that glutamine contributes to radiation response in a mouse model of RMS.
Subject(s)
Glutaminase , Glutamine , Sarcoma , Animals , Glutamine/metabolism , Mice , Glutaminase/metabolism , Glutaminase/genetics , Glutaminase/antagonists & inhibitors , Sarcoma/metabolism , Sarcoma/radiotherapy , Sarcoma/genetics , Glucose/metabolism , Disease Models, Animal , Radiation ToleranceABSTRACT
PURPOSE: Despite aggressive multimodal treatment that typically includes definitive or adjuvant radiation therapy (RT), locoregional recurrence rates approach 50% for patients with locally advanced human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC). Thus, more effective therapeutics are needed to improve patient outcomes. We evaluated the radiosensitizing effects of ataxia telangiectasia and RAD3-related (ATR) inhibitor (ATRi) BAY 1895344 in preclinical models of HNSCC. METHODS AND MATERIALS: Murine and human HPV-negative HNSCC cells (MOC2, MOC1, JHU-012) were treated with vehicle or ATRi with or without 4 Gy. Checkpoint kinase 1 phosphorylation and DNA damage (γH2AX) were evaluated by Western blot, and ATRi half-maximal inhibitory concentration was determined by MTT assay for HNSCC cells and immortalized murine oral keratinocytes. In vitro radiosensitization was tested by clonogenic assay. Cell cycle distribution and mitotic catastrophe were evaluated by flow cytometry. Mitotic aberrations were quantified by fluorescent microscopy. Tumor growth delay and survival were assessed in mice bearing MOC2 or JHU-012 transplant tumors treated with vehicle, ATRi, RT (10 Gy × 1 or 8 Gy × 3), or combined ATRi + RT. RESULTS: ATRi caused dose-dependent reduction in checkpoint kinase 1 phosphorylation at 1 hour post-RT (4 Gy) and dose-dependent increase in γH2AX at 18 hours post-RT. Addition of RT to ATRi led to decreased BAY 1895344 half-maximal inhibitory concentration in HNSCC cell lines but not in normal tissue surrogate immortalized murine oral keratinocytes. Clonogenic assays demonstrated radiosensitization in the HNSCC cell lines. ATRi abrogated the RT-induced G2/M checkpoint, leading to mitosis with unrepaired DNA damage and increased mitotic aberrations (multinucleated cells, micronuclei, nuclear buds, nucleoplasmic bridges). ATRi and RT significantly delayed tumor growth in MOC2 and JHU-012 in vivo models, with improved overall survival in the MOC2 model. CONCLUSIONS: These findings demonstrated that BAY 1895344 increased in vitro and in vivo radiosensitivity in HPV-negative HNSCC preclinical models, suggesting therapeutic potential warranting evaluation in clinical trials for patients with locally advanced or recurrent HPV-negative HNSCC.
Subject(s)
Carcinoma, Squamous Cell , Head and Neck Neoplasms , Morpholines , Papillomavirus Infections , Pyrazoles , Radiation-Sensitizing Agents , Humans , Animals , Mice , Squamous Cell Carcinoma of Head and Neck/radiotherapy , Squamous Cell Carcinoma of Head and Neck/drug therapy , Checkpoint Kinase 1/metabolism , Neoplasm Recurrence, Local/drug therapy , Radiation-Sensitizing Agents/pharmacology , Carcinoma, Squamous Cell/pathology , Head and Neck Neoplasms/radiotherapy , Head and Neck Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , G2 Phase Cell Cycle Checkpoints/radiation effects , Cell Line, Tumor , Ataxia Telangiectasia Mutated Proteins/metabolismABSTRACT
Approximately half of patients with cancer receive radiotherapy and, as cancer survivorship increases, the low rate of radiation-associated sarcomas is rising. Pharmacologic inhibition of p53 has been proposed as an approach to ameliorate acute injury of normal tissues from genotoxic therapies, but how this might impact the risk of therapy-induced cancer and normal tissue injuries remains unclear. We utilized mice that express a doxycycline (dox)-inducible p53 short hairpin RNA to reduce Trp53 expression temporarily during irradiation. Mice were placed on a dox diet 10 days prior to receiving 30 or 40 Gy hind limb irradiation in a single fraction and then returned to normal chow. Mice were examined weekly for sarcoma development and scored for radiation-induced normal tissue injuries. Radiation-induced sarcomas were subjected to RNA sequencing. Following single high-dose irradiation, 21% of animals with temporary p53 knockdown during irradiation developed a sarcoma in the radiation field compared with 2% of control animals. Following high-dose irradiation, p53 knockdown preserves muscle stem cells, and increases sarcoma development. Mice with severe acute radiation-induced injuries exhibit an increased risk of developing late persistent wounds, which were associated with sarcomagenesis. RNA sequencing revealed radiation-induced sarcomas upregulate genes related to translation, epithelial-mesenchymal transition (EMT), inflammation, and the cell cycle. Comparison of the transcriptomes of human and mouse sarcomas that arose in irradiated tissues revealed regulation of common gene programs, including elevated EMT pathway gene expression. These results suggest that blocking p53 during radiotherapy could minimize acute toxicity while exacerbating late effects including second cancers. SIGNIFICANCE: Strategies to prevent or mitigate acute radiation toxicities include pharmacologic inhibition of p53 and other cell death pathways. Our data show that temporarily reducing p53 during irradiation increases late effects including sarcomagenesis.
Subject(s)
Radiation Injuries , Sarcoma , Humans , Mice , Animals , Tumor Suppressor Protein p53/genetics , Sarcoma/genetics , Cell Cycle , DNA DamageABSTRACT
Radiotherapy remains a common treatment modality for cancer despite skeletal complications. However, there are currently no effective treatments for radiation-induced bone loss, and the consequences of radiotherapy on skeletal progenitor cell (SPC) survival and function remain unclear. After radiation, leptin receptor-expressing cells, which include a population of SPCs, become localized to hypoxic regions of the bone and stabilize the transcription factor hypoxia-inducible factor-2α (HIF-2α), thus suggesting a role for HIF-2α in the skeletal response to radiation. Here, we conditionally knocked out HIF-2α in leptin receptor-expressing cells and their descendants in mice. Radiation therapy in littermate control mice reduced bone mass; however, HIF-2α conditional knockout mice maintained bone mass comparable to nonirradiated control animals. HIF-2α negatively regulated the number of SPCs, bone formation, and bone mineralization. To test whether blocking HIF-2α pharmacologically could reduce bone loss during radiation, we administered a selective HIF-2α inhibitor called PT2399 (a structural analog of which was recently FDA-approved) to wild-type mice before radiation exposure. Pharmacological inhibition of HIF-2α was sufficient to prevent radiation-induced bone loss in a single-limb irradiation mouse model. Given that ~90% of patients who receive a HIF-2α inhibitor develop anemia because of off-target effects, we developed a bone-targeting nanocarrier formulation to deliver the HIF-2α inhibitor to mouse bone, to increase on-target efficacy and reduce off-target toxicities. Nanocarrier-loaded PT2399 prevented radiation-induced bone loss in mice while reducing drug accumulation in the kidney. Targeted inhibition of HIF-2α may represent a therapeutic approach for protecting bone during radiation therapy.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors , Bone Diseases, Metabolic , Humans , Animals , Mice , Basic Helix-Loop-Helix Transcription Factors/physiology , Receptors, Leptin , Mice, Knockout , Stem Cells , Hypoxia-Inducible Factor 1, alpha SubunitABSTRACT
Medulloblastoma is the most common malignant brain tumor of children. Although standard of care radiotherapy for pediatric medulloblastoma (PM) can lead to long-term remission or cure in many patients, it can also cause life-long cognitive impairment and other adverse effects. The pathophysiological mechanisms involved in radiation-induced cerebral damage are incompletely understood, and their elucidation may lead to interventions that mitigate radiation toxicity. To explore the mechanisms of radiation-induced cerebral damage, transgenic mouse models of PM and non-tumor-bearing controls were exposed to radiation doses that ranged from 0 to 30 Gy. Between 0-20 Gy, a significant dose-dependent reduction in tumor-associated hydrocephalus and increase in overall survival were observed. However, at 30 Gy, hydrocephalus incidence increased and median overall survival fell to near-untreated levels. Immunohistochemistry revealed that both tumor-bearing and non-tumor-bearing mice treated with 30 Gy of radiation had significantly more reactive astrocytes and microvascular damage compared to untreated controls. This effect was persistent across mice that were given 1 and 2 weeks of recovery time after irradiation. Our data suggest that radiation therapy promotes neural death by inducing long-term neuroinflammation in PM, suggesting radiation delivery methods that limit inflammation may be effective at widening the therapeutic window of radiation therapy in PM patients.
Subject(s)
Brain Neoplasms , Cerebellar Neoplasms , Hydrocephalus , Medulloblastoma , Radiation Injuries , Humans , Child , Mice , Animals , Medulloblastoma/genetics , Medulloblastoma/radiotherapy , Brain Neoplasms/radiotherapy , Radiation Injuries/etiology , Cerebellar Neoplasms/genetics , Cerebellar Neoplasms/radiotherapy , Cerebellar Neoplasms/complications , Hydrocephalus/complicationsABSTRACT
Diffuse midline gliomas (DMGs) are lethal brain tumors characterized by p53-inactivating mutations and oncohistone H3.3K27M mutations that rewire the cellular response to genotoxic stress, which presents therapeutic opportunities. We used RCAS/tv-a retroviruses and Cre recombinase to inactivate p53 and induce K27M in the native H3f3a allele in a lineage- and spatially-directed manner, yielding primary mouse DMGs. Genetic or pharmacologic disruption of the DNA damage response kinase Ataxia-telangiectasia mutated (ATM) enhanced the efficacy of focal brain irradiation, extending mouse survival. This finding suggests that targeting ATM will enhance the efficacy of radiation therapy for p53-mutant DMG but not p53-wildtype DMG. We used spatial in situ transcriptomics and an allelic series of primary murine DMG models with different p53 mutations to identify transactivation-independent p53 activity as a key mediator of such radiosensitivity. These studies deeply profile a genetically faithful and versatile model of a lethal brain tumor to identify resistance mechanisms for a therapeutic strategy currently in clinical trials.
ABSTRACT
PURPOSE: Tumor hypoxia contributes to aggressive phenotypes and diminished therapeutic responses to radiation therapy (RT) with hypoxic tissue being 3-fold less radiosensitive than normoxic tissue. A major challenge in implementing hypoxic radiosensitizers is the lack of a high-resolution imaging modality that directly quantifies tissue-oxygen. The electron paramagnetic resonance oxygen-imager (EPROI) was used to quantify tumor oxygenation in two murine tumor models: E0771 syngeneic transplant breast cancers and primary p53/MCA soft tissue sarcomas, with the latter autochthonous model better recapitulating the tumor microenvironment in human malignancies. We hypothesized that tumor hypoxia differs between these models. We also aimed to quantify the absolute change in tumor hypoxia induced by the mitochondrial inhibitor papaverine (PPV) and its effect on RT response. PROCEDURES: Tumor oxygenation was characterized in E0771 and primary p53/MCA sarcomas via EPROI, with the former model also being quantified indirectly via diffuse reflectance spectroscopy (DRS). After confirming PPV's effect on hypoxic fraction (via EPROI), we compared the effect of 0 versus 2 mg/kg PPV prior to 20 Gy on tumor growth delay and survival. RESULTS: Hypoxic sarcomas were more radioresistant than normoxic sarcomas (p=0.0057, 2-way ANOVA), and high baseline hypoxic fraction was a significant (p=0.0063, Cox Regression Model) hazard in survivability regardless of treatment. Pre-treatment with PPV before RT did not radiosensitize tumors in the sarcoma or E0771 model. In the sarcoma model, EPROI successfully identified baseline hypoxic tumors. DRS quantification of total hemoglobin, saturated hemoglobin, changes in mitochondrial potential and glucose uptake showed no significant difference in E0771 tumors pre- and post-PPV. CONCLUSION: EPROI provides 3D high-resolution pO2 quantification; EPR is better suited than DRS to characterize tumor hypoxia. PPV did not radiosensitize E0771 tumors nor p53/MCA sarcomas, which may be related to the complex pattern of vasculature in each tumor. Additionally, understanding model-dependent tumor hypoxia will provide a much-needed foundation for future therapeutic studies with hypoxic radiosensitizers.
ABSTRACT
The main deterrent to long-term space travel is the risk of Radiation Exposure Induced Death (REID). The National Aeronautics and Space Administration (NASA) has adopted Permissible Exposure Levels (PELs) to limit the probability of REID to 3% for the risk of death due to radiation-induced carcinogenesis. The most significant contributor to current REID estimates for astronauts is the risk of lung cancer. Recently updated lung cancer estimates from Japan's atomic bomb survivors showed that the excess relative risk of lung cancer by age 70 is roughly four-fold higher in females compared to males. However, whether sex differences may impact the risk of lung cancer due to exposure to high charge and energy (HZE) radiation is not well studied. Thus, to evaluate the impact of sex differences on the risk of solid cancer development post-HZE radiation exposure, we irradiated Rb fl/fl ; Trp53 fl/+ male and female mice infected with Adeno-Cre with various doses of 320 kVp X-rays or 600 MeV/n 56 Fe ions and monitored them for any radiation-induced malignancies. We observed that lung adenomas/carcinomas and esthesioneuroblastomas (ENBs) were the most common primary malignancies in X-ray and 56 Fe ion-exposed mice, respectively. In addition, 1 Gy 56 Fe ion exposure compared to X-rays led to a significantly higher incidence of lung adenomas/carcinomas (p=0.02) and ENBs (p<0.0001). However, we did not find a significantly higher incidence of any solid malignancies in female mice as compared to male mice, regardless of radiation quality. Furthermore, gene expression analysis of ENBs suggested a distinct gene expression pattern with similar hallmark pathways altered, such as MYC targets and MTORC1 signaling, in X-ray and 56 Fe ion-induced ENBs. Thus, our data revealed that 56 Fe ion exposure significantly accelerated the development of lung adenomas/carcinomas and ENBs compared to X-rays, but the rate of solid malignancies was similar between male and female mice, regardless of radiation quality.
ABSTRACT
ATRX is one of the most frequently altered genes in solid tumors, and mutation is especially frequent in soft tissue sarcomas. However, the role of ATRX in tumor development and response to cancer therapies remains poorly understood. Here, we developed a primary mouse model of soft tissue sarcoma and showed that Atrx-deleted tumors were more sensitive to radiation therapy and to oncolytic herpesvirus. In the absence of Atrx, irradiated sarcomas had increased persistent DNA damage, telomere dysfunction, and mitotic catastrophe. Our work also showed that Atrx deletion resulted in downregulation of the CGAS/STING signaling pathway at multiple points in the pathway and was not driven by mutations or transcriptional downregulation of the CGAS/STING pathway components. We found that both human and mouse models of Atrx-deleted sarcoma had a reduced adaptive immune response, markedly impaired CGAS/STING signaling, and increased sensitivity to TVEC, an oncolytic herpesvirus that is currently FDA approved for the treatment of aggressive melanomas. Translation of these results to patients with ATRX-mutant cancers could enable genomically guided cancer therapy approaches to improve patient outcomes.
Subject(s)
Herpesviridae , Sarcoma , Animals , Mice , Humans , Signal Transduction , Sarcoma/genetics , Sarcoma/radiotherapy , X-linked Nuclear Protein/genetics , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Immunity, InnateABSTRACT
The adaptive immune system plays an essential anti-tumor role through immunosurveillance and response to immunotherapies. Characterizing phenotypic features and mechanisms of dysfunction of tumor-specific T cell populations may uncover novel immunotherapeutic targets and biomarkers of response. To study tumor-specific T cell responses in vivo, a tumor model must express a known neoantigen. While transplant models with known neoantigen expression are widely available, autochthonous tumor models in which the tumor coevolves with the immune system are limited. In this study, we combined CRISPR/Cas9 and sleeping beauty transposase technology to develop an autochthonous orthotopic murine sarcoma model with oncogenic KrasG12D, functionally impaired p53, and expression of known MHCI and MHCII sarcoma neoantigens. Using MHC tetramer flow cytometry, we identified a tumor-specific immune response in the peripheral blood as early as 10 days after tumor induction leading to tumor clearance. Tumors developed at high penetrance after co-depletion of CD8 and CD4 T cells, but depletion of either CD8 or CD4 T cells alone was insufficient to permit tumor growth. These results suggest that CD8 and CD4 T cells can independently contribute to immunosurveillance leading to clearance of sarcomas expressing MHCI and MHCII neoantigens.
ABSTRACT
Genetically engineered mice are commonly used to model brainstem gliomas in pre-clinical research. One technique for inducing primary tumors in these genetically engineered mice involves delivering viral vectors containing the code for gene-editing proteins. We present a protocol for generating primary brainstem gliomas using the RCAS-TVA retroviral delivery system and the Cre/loxP gene editing system. We describe steps for transfecting and harvesting chicken fibroblast cells, intracranially injecting cells into mice, imaging primary tumors, and treating primary tumors with focal, image-guided brain irradiation. For complete details on the use and execution of this protocol, please refer to Deland et al. (2021).1.
Subject(s)
Brain Stem Neoplasms , Glioma , Mice , Animals , Retroviridae/genetics , Brain Stem Neoplasms/genetics , Brain Stem Neoplasms/therapy , Glioma/genetics , Glioma/therapy , Recombination, GeneticABSTRACT
This study aims to investigate whether adding neoadjuvant radiotherapy (RT), anti-programmed cell death protein-1 (PD-1) antibody (anti-PD-1), or RT + anti-PD-1 to surgical resection improves disease-free survival for mice with soft tissue sarcomas (STS). We generated a high mutational load primary mouse model of STS by intramuscular injection of adenovirus expressing Cas9 and guide RNA targeting Trp53 and intramuscular injection of 3-methylcholanthrene (MCA) into the gastrocnemius muscle of wild-type mice (p53/MCA model). We randomized tumor-bearing mice to receive isotype control or anti-PD-1 antibody with or without radiotherapy (20 Gy), followed by hind limb amputation. We used micro-CT to detect lung metastases with high spatial resolution, which was confirmed by histology. We investigated whether sarcoma metastasis was regulated by immunosurveillance by lymphocytes or tumor cell-intrinsic mechanisms. Compared with surgery with isotype control antibody, the combination of anti-PD-1, radiotherapy, and surgery improved local recurrence-free survival (P = 0.035) and disease-free survival (P = 0.005), but not metastasis-free survival. Mice treated with radiotherapy, but not anti-PD-1, showed significantly improved local recurrence-free survival and metastasis-free survival over surgery alone (P = 0.043 and P = 0.007, respectively). The overall metastasis rate was low (â¼12%) in the p53/MCA sarcoma model, which limited the power to detect further improvement in metastasis-free survival with addition of anti-PD-1 therapy. Tail vein injections of sarcoma cells into immunocompetent mice suggested that impaired metastasis was due to inability of sarcoma cells to grow in the lungs rather than a consequence of immunosurveillance. In conclusion, neoadjuvant radiotherapy improves metastasis-free survival after surgery in a primary model of STS.
Subject(s)
Sarcoma , Soft Tissue Neoplasms , Mice , Animals , Neoadjuvant Therapy , Tumor Suppressor Protein p53/genetics , Sarcoma/radiotherapy , Progression-Free Survival , Disease-Free Survival , Soft Tissue Neoplasms/pathology , Soft Tissue Neoplasms/surgery , Retrospective Studies , Radiotherapy, Adjuvant , Neoplasm Recurrence, Local/pathologyABSTRACT
Diffuse midline gliomas arise in the brainstem and other midline brain structures and cause a large proportion of childhood brain tumor deaths. Radiation therapy is the most effective treatment option, but these tumors ultimately progress. Inhibition of the phosphoinositide-3-kinase (PI3K)-like kinase, ataxia-telangiectasia mutated (ATM), which orchestrates the cellular response to radiation-induced DNA damage, may enhance the efficacy of radiation therapy. Diffuse midline gliomas in the brainstem contain loss-of-function mutations in the tumor suppressor PTEN, or functionally similar alterations in the phosphoinositide-3-kinase (PI3K) pathway, at moderate frequency. Here, we sought to determine if ATM inactivation could radiosensitize a primary mouse model of brainstem glioma driven by Pten loss. Using Cre/loxP recombinase technology and the RCAS/TVA retroviral gene delivery system, we established a mouse model of brainstem glioma driven by Pten deletion. We find that Pten-null brainstem gliomas are relatively radiosensitive at baseline. In addition, we show that deletion of Atm in the tumor cells does not extend survival of mice bearing Pten-null brainstem gliomas after focal brain irradiation. These results characterize a novel primary mouse model of PTEN-mutated brainstem glioma and provide insights into the mechanism of radiosensitization by ATM deletion, which may guide the design of future clinical trials.
ABSTRACT
Thoracic radiation therapy can cause endothelial injury in the heart, leading to cardiac dysfunction and heart failure. Although it has been demonstrated that the tumor suppressor p53 functions in endothelial cells to prevent the development of radiation-induced myocardial injury, the key mechanism(s) by which p53 regulates the radiosensitivity of cardiac endothelial cells is not completely understood. Here, we utilized genetically engineered mice that express mutations in p53 transactivation domain 1 (TAD1) (p5325,26) or mutations in p53 TAD1 and TAD2 (p5325,26,53,54) specifically in endothelial cells to study the p53 transcriptional program that protects cardiac endothelial cells from ionizing radiation in vivo. p5325,26,53,54 loses the ability to drive transactivation of p53 target genes after irradiation while p5325,26 can induce transcription of a group of non-canonical p53 target genes, but not the majority of classic radiation-induced p53 targets critical for p53-mediated cell cycle arrest and apoptosis. After 12 Gy whole-heart irradiation, we found that both p5325,26 and p5325,26,53,54 sensitized mice to radiation-induced cardiac injury, in contrast to wild-type p53. Histopathological examination suggested that mutation of TAD1 contributes to myocardial necrosis after whole-heart irradiation, while mutation of both TAD1 and TAD2 abolishes the ability of p53 to prevent radiation-induced heart disease. Taken together, our results show that the transcriptional program downstream of p53 TAD1, which activates the acute DNA damage response after irradiation, is necessary to protect cardiac endothelial cells from radiation injury in vivo.
Subject(s)
Endothelial Cells , Heart , Radiation Injuries , Tumor Suppressor Protein p53 , Animals , Apoptosis/genetics , Apoptosis/radiation effects , DNA Damage , Endothelial Cells/metabolism , Heart/radiation effects , Mice , Radiation Injuries/metabolism , Transcriptional Activation , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Objective. To develop and characterize novel methods of extreme spatially fractionated kV radiation therapy (including mini-GRID therapy) and to evaluate efficacy in the context of a pre-clinical mouse study.Approach. Spatially fractionated GRIDs were precision-milled from 3 mm thick lead sheets compatible with mounting on a 225 kVp small animal irradiator (X-Rad). Three pencil-beam GRIDs created arrays of 1 mm diameter beams, and three 'bar' GRIDs created 1 × 20 mm rectangular fields. GRIDs projected 20 × 20 mm2fields at isocenter, and beamlets were spaced at 1, 1.25, and 1.5 mm, respectively. Peak-to-valley ratios and dose distributions were evaluated with Gafchromic film. Syngeneic transplant tumors were induced by intramuscular injection of a soft tissue sarcoma cell line into the gastrocnemius muscle of C57BL/6 mice. Tumor-bearing mice were randomized to four groups: unirradiated control, conventional irradiation of entire tumor, GRID therapy, and hemi-irradiation (half-beam block, 50% tumor volume treated). All irradiated mice received a single fraction of 15 Gy.Results. High peak-to-valley ratios were achieved (bar GRIDs: 11.9 ± 0.9, 13.6 ± 0.4, 13.8 ± 0.5; pencil-beam GRIDs: 18.7 ± 0.6, 26.3 ± 1.5, 31.0 ± 3.3). Pencil-beam GRIDs could theoretically spare more intra-tumor immune cells than bar GRIDs, but they treat less tumor tissue (3%-4% versus 19%-23% area receiving 90% prescription, respectively). Bar GRID and hemi-irradiation treatments significantly delayed tumor growth (P < 0.05), but not as much as a conventional treatment (P < 0.001). No significant difference was found in tumor growth delay between GRID and hemi-irradiation.Significance. High peak-to-valley ratios were achieved with kV grids: two-to-five times higher than values reported in literature for MV grids. GRID irradiation and hemi-irradiation delayed tumor growth, but neither was as effective as conventional whole tumor uniform dose treatment. Single fraction GRID therapy could not initiate an anti-cancer immune response strong enough to match conventional RT outcomes, but follow-up studies will evaluate the combination of mini-GRID with immune checkpoint blockade.
Subject(s)
Neoplasms , Radiometry , Animals , Dose Fractionation, Radiation , Mice , Mice, Inbred C57BL , Neoplasms/radiotherapy , Tumor BurdenABSTRACT
PURPOSE: Diffuse intrinsic pontine gliomas (DIPGs) arise in the pons and are the leading cause of death from brain tumors in children. DIPGs are routinely treated with radiation therapy, which temporarily improves neurological symptoms but generally fails to achieve local control. Because numerous clinical trials have not improved survival from DIPG over standard radiation therapy alone, there is a pressing need to evaluate new therapeutic strategies for this devastating disease. Vascular damage caused by radiation therapy can increase the permeability of tumor blood vessels and promote tumor cell death. METHODS AND MATERIALS: To investigate the impact of endothelial cell death on tumor response to radiation therapy in DIPG, we used dual recombinase (Cre + FlpO) technology to generate primary brainstem gliomas which lack ataxia telangiectasia mutated (Atm) in the vasculature. RESULTS: Here, we show that Atm-deficient tumor endothelial cells are sensitized to radiation therapy. Furthermore, radiosensitization of the vasculature in primary gliomas triggered an increase in total tumor cell death. Despite the observed increase in cell killing, in mice with autochthonous DIPGs treated with radiation therapy, deletion of Atm specifically in tumor endothelial cells failed to improve survival. CONCLUSIONS: These results suggest that targeting the tumor cells, rather than endothelial cells, during radiation therapy will be necessary to improve survival among children with DIPG.
Subject(s)
Brain Stem Neoplasms , Glioma , Animals , Brain Stem Neoplasms/pathology , Brain Stem Neoplasms/radiotherapy , Endothelial Cells/pathology , Glioma/pathology , Glioma/radiotherapy , MiceABSTRACT
Delayed radiation myelopathy is a rare, but significant late side effect from radiation therapy that can lead to paralysis. The cellular and molecular mechanisms leading to delayed radiation myelopathy are not completely understood but may be a consequence of damage to oligodendrocyte progenitor cells and vascular endothelial cells. Here, we aimed to determine the contribution of endothelial cell damage to the development of radiation-induced spinal cord injury using a genetically defined mouse model in which endothelial cells are sensitized to radiation due to loss of the tumor suppressor p53. Tie2Cre; p53FL/+ and Tie2Cre; p53FL/- mice, which lack one and both alleles of p53 in endothelial cells, respectively, were treated with focal irradiation that specifically targeted the lumbosacral region of the spinal cord. The development of hindlimb paralysis was followed for up to 18 weeks after either a 26.7 Gy or 28.4 Gy dose of radiation. During 18 weeks of follow-up, 83% and 100% of Tie2Cre; p53FL/- mice developed hindlimb paralysis after 26.7 and 28.4 Gy, respectively. In contrast, during this period only 8% of Tie2Cre; p53FL/+ mice exhibited paralysis after 28.4 Gy. In addition, 8 weeks after 28.4 Gy the irradiated spinal cord from Tie2Cre; p53FL/- mice showed a significantly higher fractional area positive for the neurological injury marker glial fibrillary acidic protein (GFAP) compared with the irradiated spinal cord from Tie2Cre; p53FL/+ mice. Together, our findings show that deletion of p53 in endothelial cells sensitizes mice to the development of delayed radiation myelopathy indicating that endothelial cells are a critical cellular target of radiation that regulates myelopathy.
Subject(s)
Spinal Cord Injuries/radiotherapy , Animals , Dose-Response Relationship, Radiation , Endothelial Cells , Female , Glial Fibrillary Acidic Protein/radiation effects , Humans , Male , Mice , Radiation Injuries, Experimental , Radiation, Ionizing , Spinal Cord/drug effects , Time Factors , Tumor Suppressor Protein p53/radiation effectsABSTRACT
Diffuse intrinsic pontine glioma (DIPG) kills more children than any other type of brain tumor. Despite clinical trials testing many chemotherapeutic agents, palliative radiotherapy remains the standard treatment. Here, we utilized Cre/loxP technology to show that deleting Ataxia telangiectasia mutated (Atm) in primary mouse models of DIPG can enhance tumor radiosensitivity. Genetic deletion of Atm improved survival of mice with p53-deficient but not p53 wild-type gliomas after radiotherapy. Similar to patients with DIPG, mice with p53 wild-type tumors had improved survival after radiotherapy independent of Atm deletion. Primary p53 wild-type tumor cell lines induced proapoptotic genes after radiation and repressed the NRF2 target, NAD(P)H quinone dehydrogenase 1 (Nqo1). Tumors lacking p53 and Ink4a/Arf expressed the highest level of Nqo1 and were most resistant to radiation, but deletion of Atm enhanced the radiation response. These results suggest that tumor genotype may determine whether inhibition of ATM during radiotherapy will be an effective clinical approach to treat DIPGs.
Subject(s)
Brain Stem Neoplasms , Gene Deletion , Genotype , Glioma , Radiation Tolerance , Animals , Ataxia Telangiectasia Mutated Proteins/deficiency , Ataxia Telangiectasia Mutated Proteins/metabolism , Brain Stem Neoplasms/genetics , Brain Stem Neoplasms/metabolism , Brain Stem Neoplasms/radiotherapy , Cell Line, Tumor , Chickens , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Glioma/genetics , Glioma/metabolism , Glioma/radiotherapy , Mice , Mice, Transgenic , NAD(P)H Dehydrogenase (Quinone)/genetics , NAD(P)H Dehydrogenase (Quinone)/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Immunotherapy fails to cure most cancer patients. Preclinical studies indicate that radiotherapy synergizes with immunotherapy, promoting radiation-induced antitumor immunity. Most preclinical immunotherapy studies utilize transplant tumor models, which overestimate patient responses. Here, we show that transplant sarcomas are cured by PD-1 blockade and radiotherapy, but identical treatment fails in autochthonous sarcomas, which demonstrate immunoediting, decreased neoantigen expression, and tumor-specific immune tolerance. We characterize tumor-infiltrating immune cells from transplant and primary tumors, revealing striking differences in their immune landscapes. Although radiotherapy remodels myeloid cells in both models, only transplant tumors are enriched for activated CD8+ T cells. The immune microenvironment of primary murine sarcomas resembles most human sarcomas, while transplant sarcomas resemble the most inflamed human sarcomas. These results identify distinct microenvironments in murine sarcomas that coevolve with the immune system and suggest that patients with a sarcoma immune phenotype similar to transplant tumors may benefit most from PD-1 blockade and radiotherapy.