Mechanisms Underlying the Development of Murine T-Cell Lymphoblastic Lymphoma/Leukemia Induced by Total-Body Irradiation.

Exposure to ionizing radiation is associated with an increased risk of hematologic malignancies in myeloid and lymphoid lineages in humans and experimental mice. Given that substantial evidence links radiation exposure with the risk of hematologic malignancies, it is imperative to deeply understand the mechanisms underlying cellular and molecular changes during the latency period between radiation exposure and the emergence of fully transformed malignant cells. One experimental model widely used in the field of radiation and cancer biology to study hematologic malignancies induced by radiation exposure is mouse models of radiation-induced thymic lymphoma. Murine radiation-induced thymic lymphoma is primarily driven by aberrant activation of Notch signaling, which occurs frequently in human precursor T-cell lymphoblastic lymphoma (T-LBL) and T-cell lymphoblastic leukemia (T-ALL). Here, we summarize the literature elucidating cell-autonomous and non-cell-autonomous mechanisms underlying cancer initiation, progression, and malignant transformation in the thymus following total-body irradiation (TBI) in mice.

Imaging Assessment of Radiation Therapy-Related Normal Tissue Injury in Children: A PENTEC Visionary Statement.

The Pediatric Normal Tissue Effects in the Clinic (PENTEC) consortium has made significant contributions to understanding and mitigating the adverse effects of childhood cancer therapy. This review addresses the role of diagnostic imaging in detecting, screening, and comprehending radiation therapy-related late effects in children, drawing insights from individual organ-specific PENTEC reports. We further explore how the development of imaging biomarkers for key organ systems, alongside technical advancements and translational imaging approaches, may enhance the systematic application of imaging evaluations in childhood cancer survivors. Moreover, the review critically examines knowledge gaps and identifies technical and practical limitations of existing imaging modalities in the pediatric population. Addressing these challenges may expand access to, minimize the risk of, and optimize the real-world application of, new imaging techniques. The PENTEC team envisions this document as a roadmap for the future development of imaging strategies in childhood cancer survivors, with the overarching goal of improving long-term health outcomes and quality of life for this vulnerable population.

Impact of cancer therapy on clonal hematopoiesis mutations and subsequent clinical outcomes.

ABSTRACT: Exposure to cancer therapies is associated with an increased risk of clonal hematopoiesis (CH). The objective of our study was to investigate the genesis and evolution of CH after cancer therapy. In this prospective study, we undertook error-corrected duplex DNA sequencing in blood samples collected before and at 2 time points after chemoradiation in patients with esophageal or lung cancer recruited from 2013 to 2018. We applied a customized workflow to identify the earliest changes in CH mutation count and clone size and determine their association with clinical outcomes. Our study included 29 patients (87 samples). Their median age was 67 years, and 76% (n = 22) were male; the median follow-up period was 3.9 years. The most mutated genes were DNMT3A, TET2, TP53, and ASXL1. We observed a twofold increase in the number of mutations from before to after treatment in TP53, which differed from all other genes examined (P < .001). Among mutations detected before and after treatment, we observed an increased clone size in 38% and a decreased clone size in 5% of TP53 mutations (odds ratio, 3.7; 95% confidence interval [CI], 1.75-7.84; P < .001). Changes in mutation count and clone size were not observed in other genes. Individuals with an increase in the number of TP53 mutations after chemoradiation experienced shorter overall survival (hazard ratio, 7.07; 95% CI, 1.50-33.46; P = .014). In summary, we found an increase in the number and size of TP53 CH clones after chemoradiation that were associated with adverse clinical outcomes.

p53 promotes revival stem cells in the regenerating intestine after severe radiation injury.

Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced acute GI syndrome. Through single-cell RNA-sequencing of the irradiated mouse small intestine, we find that p53 target genes are specifically enriched in regenerating epithelial cells that undergo fetal-like reversion, including revival stem cells (revSCs) that promote animal survival after severe damage of the GI tract. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce fetal-like revSCs. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells and is controlled by an Mdm2-mediated negative feedback loop. Together, our findings reveal that p53 suppresses severe radiation-induced GI injury by promoting fetal-like reprogramming of irradiated intestinal epithelial cells.

Histological assessment of intestinal injury by ionizing radiation.

Given the potential risk of radiological terrorism and disasters, it is essential to develop plans to prepare for such events. In these hazardous scenarios, radiation-induced gastrointestinal (GI) syndrome is one of the many manifestations that may happen after the organism is exposed to a lethal dose of ionizing radiation. Therefore, it is critical to better understand how the intestinal tissues initiate and orchestrate regeneration following severe radiation injury. In this chapter, we aimed to provide several key considerations for researchers who utilize histological assessment to study radiation-induced intestinal injury. Rigor and reproducibility are critical in experimental design and can be achieved by maintaining proper radiation administration, maintaining consistency in sample collection, and selecting and using appropriate controls. We also provided technical details of histological preparation of the intestines with tips on dissecting, cleaning, fixing, and preserving. Step-by-step descriptions of both bundling and Swiss rolling are provided with discussion on how to choose between the two approaches. In the following section, we detailed several histological assessment methods and then provided suggestions on how to use histological assessment to study cellular dynamics in the small intestines. Finally, we touched on some non-histological assessments. We hope that the information provided in this chapter will contribute to the research society of radiation-induced intestinal injury with an ultimate goal of promoting the development of radiation countermeasures against the GI acute radiation syndrome.

Characterizing the role of Phlda3 in the development of acute toxicity and malignant transformation of hematopoietic cells induced by total-body irradiation in mice.

The tumor suppressor p53 is a transcriptional factor that plays a crucial role in controlling acute toxicity and long-term malignant transformation of hematopoietic cells induced by genotoxic stress such as ionizing radiation. Among all transcriptional targets of p53, one gene that is robustly induced by radiation is the pleckstrin homology domain-only protein Phlda3. However, the role that Phlda3 plays in regulating the response of hematopoietic cells to radiation is unknown. Here, using isogenic cell lines and genetically engineered mouse models, we showed that radiation induces Phlda3 in human leukemia cells and mouse normal hematopoietic cells in a p53-dependent manner. However, deletion of the Phlda3 gene did not ameliorate radiation-induced acute hematologic toxicity. In addition, distinct from mice that lose p53, loss of Phlda3 did not alter the latency and incidence of radiation-induced thymic lymphoma in mice. Remarkably, whole-exome sequencing data showed that lymphomas in irradiated Phlda3+/+ mice harbor a significantly higher number of single nucleotide variants (SNVs) and indels compared to lymphomas in irradiated Phlda3+/- and Phlda3-/- littermates. Together, our results indicate that although deletion of Phlda3 does not accelerate the development of radiation-induced thymic lymphoma, fewer SNVs and indels are necessary to initiate lymphomagenesis after radiation exposure when Phlda3 is silenced.

Mis-splicing Drives Loss of Function of p53E224D Point Mutation.

Background: Tp53 is the most commonly mutated gene in cancer. Canonical Tp53 DNA damage response pathways are well characterized and classically thought to underlie the tumor suppressive effect of Tp53. Challenging this dogma, mouse models have revealed that p53 driven apoptosis and cell cycle arrest are dispensable for tumor suppression. Here, we investigated the inverse context of a p53 mutation predicted to drive expression of canonical targets, but is detected in human cancer. Methods: We established a novel mouse model with a single base pair mutation (GAG>GAC, p53E221D) in the DNA-Binding domain that has wild-type function in screening assays, but is paradoxically found in human cancer in Li-Fraumeni syndrome. Using mouse p53E221D and the analogous human p53E224D mutant, we evaluated expression, transcriptional activation, and tumor suppression in vitro and in vivo. Results: Expression of human p53E224D from cDNA translated to a fully functional p53 protein. However, p53E221D/E221D RNA transcribed from the endogenous locus is mis-spliced resulting in nonsense mediated decay. Moreover, fibroblasts derived from p53E221D/E221D mice do not express a detectable protein product. Mice homozygous for p53E221D exhibited increased tumor penetrance and decreased life expectancy compared to p53 WT animals. Conclusions: Mouse p53E221D and human p53E224D mutations lead to splice variation and a biologically relevant p53 loss of function in vitro and in vivo.

Temporary Knockdown of p53 During Focal Limb Irradiation Increases the Development of Sarcomas.

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.

p53 promotes revival stem cells in the regenerating intestine after severe radiation injury.

Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced GI injury. Through single-cell RNA-sequencing of the irradiated mouse intestine, we find that p53 target genes are specifically enriched in stem cells of the regenerating epithelium, including revival stem cells that promote animal survival after GI damage. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce revival stem cells. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells that is controlled by an Mdm2-mediated negative feedback loop. These results suggest that p53 suppresses severe radiation-indued GI injury by promoting intestinal epithelial cell reprogramming. One-Sentence Summary: After severe radiation injury to the intestine, transient p53 activity induces revival stem cells to promote regeneration.

Intraoperative detection and removal of microscopic residual sarcoma using wide-field imaging.

BACKGROUND: The goal of limb-sparing surgery for a soft tissue sarcoma of the extremity is to remove all malignant cells while preserving limb function. After initial surgery, microscopic residual disease in the tumor bed will cause a local recurrence in approximately 33% of patients with sarcoma. To help identify these patients, the authors developed an in vivo imaging system to investigate the suitability of molecular imaging for intraoperative visualization. METHODS: A primary mouse model of soft tissue sarcoma and a wide field-of-view imaging device were used to investigate a series of exogenously administered, near-infrared (NIR) fluorescent probes activated by cathepsin proteases for real-time intraoperative imaging. RESULTS: The authors demonstrated that exogenously administered cathepsin-activated probes can be used for image-guided surgery to identify microscopic residual NIR fluorescence in the tumor beds of mice. The presence of residual NIR fluorescence was correlated with microscopic residual sarcoma and local recurrence. The removal of residual NIR fluorescence improved local control. CONCLUSIONS: The authors concluded that their technique has the potential to be used for intraoperative image-guided surgery to identify microscopic residual disease in patients with cancer.

The p53 Transactivation Domain 1-Dependent Response to Acute DNA Damage in Endothelial Cells Protects against Radiation-Induced Cardiac Injury.

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.

Transplantation of Unirradiated Bone Marrow Cells after Total-Body Irradiation Prevents the Development of Thymic Lymphoma in Mice through Niche Competition.

Mouse models of radiation-induced thymic lymphoma are commonly used to study the biological effects of total-body irradiation (TBI) on the formation of hematologic malignancies. It is well documented that radiation-induced thymic lymphoma can be inhibited by protecting the bone marrow (BM) from irradiation; however, the mechanisms underlying this phenomenon are poorly understood. Here, we aimed to address this question by performing transplantation of BM cells from genetically engineered mice that have defects in tumor immunosurveillance or occupying different thymic niches. We found that BM cells from mice that have impaired tumor immunosurveillance, by deleting tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ) or perforin-1 (PRF1), remained sufficient to suppress the formation of radiation-induced thymic lymphoma. On the other hand, BM cells from Rag2-/-; γc-/- mice and Rag2-/- mice, which have defects in occupying thymic niches beyond double negative (DN2) and DN3, respectively, failed to inhibit radiation-induced lymphomagenesis in the thymus. Taken together, based on our findings, we propose a model where unirradiated BM cells suppress radiation-induced lymphomagenesis in the thymus by competing with tumor-initiating cells for thymic niches beyond the DN3 stage.

Investigating the Role of Inflammasome Caspases 1 and 11 in the Acute Radiation Syndrome.

Exposure to high dose radiation causes life-threatening acute and delayed effects. Defining the mechanisms of lethal radiation-induced acute toxicity of gastrointestinal and hematopoietic tissues are critical steps to identify drug targets to mitigate and protect against the acute radiation syndrome (ARS). For example, one rational approach would be to design pharmaceuticals that block cell death pathways to preserve tissue integrity in radiation-sensitive organ systems including the gastrointestinal tract and hematopoietic compartment. A previous study reported that the inflammasome pathway, which mediates inflammatory cell death through pyroptosis, promotes ARS. However, we show that mice lacking the inflammatory executioner caspases, caspase-1 and caspase-11, are not protected from ARS when compared directly to littermates expressing caspase-1 and caspase-11. These results suggest that alternative pathways will need to be targeted by drugs that successfully mitigate and protect against the ARS.

Epithelial Regeneration After Doxorubicin Arises Primarily From Early Progeny of Active Intestinal Stem Cells.

BACKGROUND & AIMS: aISCs (aISCs) are sensitive to acute insults including chemotherapy and irradiation. Regeneration after aISC depletion has primarily been explored in irradiation (IR). However, the cellular origin of epithelial regeneration after doxorubicin (DXR), a common chemotherapeutic, is poorly understood. METHODS: We monitored DXR's effect on aISCs by enumerating Lgr5-eGFP+ and Olfm4+ crypts, cleaved caspase-3 (CASP3+) immunofluorescence, and time-lapse organoid imaging. Lineage tracing from previously identified regenerative cell populations (Bmi1+, Hopx+, Dll1+, and Defa6+) was performed with DXR damage. Lineage tracing from aISCs was compared with lineage tracing from early progeny cells (transit-amplifying cells arising from aISCs 1 day predamage) in the context of DXR and IR. We compared stem cell and DNA damage response (DDR) transcripts in isolated aISCs and early progeny cells 6 and 24 hours after DXR. RESULTS: Epithelial regeneration after DXR primarily arose from early progeny cells generated by aISCs. Early progeny cells upregulated stem cell gene expression and lacked apoptosis induction (6 hours DXR: 2.5% of CASP3+ cells, p<0.0001). aISCs downregulated stem cell gene expression and underwent rapid apoptosis (6 hours DXR: 63.4% of CASP3+ cells). There was minimal regenerative contribution from Bmi1+, Hopx+, Dll1+, and Defa6+-expressing populations. In homeostasis, 48.4% of early progeny cells were BrdU+, and expressed low levels of DDR transcripts. CONCLUSIONS: We show that DXR effectively depleted aISCs in the small intestine and subsequent epithelial regeneration depended on nonquiescent early progeny cells of aISCs. The chemoresistant phenotype of the early progeny cells may rely on a dampened DDR in contrast to aISCs' robust DDR, which facilitates expeditious apoptosis.

Selective ERBB2 and BCL2 Inhibition Is Synergistic for Mitochondrial-Mediated Apoptosis in MDS and AML Cells.

The ERBB2 proto-oncogene is associated with an aggressive phenotype in breast cancer. Its role in hematologic malignancies is incompletely defined, in part because ERBB2 is not readily detected on the surface of cancer cells. We demonstrate that truncated ERBB2, which lacks the extracellular domain, is overexpressed on primary CD34+ myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) cells compared with healthy hematopoietic cells. This overexpression of ERBB2 is associated with aberrant, oncogenic signaling with autophosphorylation of multiple tyrosine sites. Like in breast cancers, ERBB2 can exist as truncated isoforms p95ERBB2 and p110ERBB2 in MDS and AML. Neutralization of ERBB2 signaling with ERBB2 tyrosine kinase inhibitors (i.e., lapatinib, afatinib, and neratinib) increases apoptotic cell death and reduces human engraftment of MDS cells in mice at 21 weeks posttransplantation. Inhibition of ERBB2 modulates the expression of multiple pro- and anti-apoptotic mitochondrial proteins, including B-cell lymphoma 2 (BCL2). Dual blockade with ERBB2 and BCL2 inhibitors triggers additional reductions of BCL2 phosphorylation and myeloid cell leukemia-1 (MCL1) expression compared with single drug treatment. Dual therapy was synergistic at all tested doses, with a dose reduction index of up to 29 for lapatinib + venetoclax compared with venetoclax alone. Notably, these agents operated together and shifted cancer cells to a pro-apoptotic phenotype, resulting in increased mitochondrial cytochrome c release and activated caspase-3-mediated cell death. IMPLICATIONS: These findings warrant study of ERBB2 and BCL2 combination therapy in patients with MDS and AML. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/5/886/F1.large.jpg.

Whole-Exome Sequencing of Radiation-Induced Thymic Lymphoma in Mouse Models Identifies Notch1 Activation as a Driver of p53 Wild-Type Lymphoma.

Mouse models of radiation-induced thymic lymphoma are widely used to study the development of radiation-induced blood cancers and to gain insights into the biology of human T-cell lymphoblastic leukemia/lymphoma. Here we aimed to identify key oncogenic drivers for the development of radiation-induced thymic lymphoma by performing whole-exome sequencing using tumors and paired normal tissues from mice with and without irradiation. Thymic lymphomas from irradiated wild-type (WT), p53+/-, and KrasLA1 mice were not observed to harbor significantly higher numbers of nonsynonymous somatic mutations compared with thymic lymphomas from unirradiated p53-/- mice. However, distinct patterns of recurrent mutations arose in genes that control the Notch1 signaling pathway based on the mutational status of p53. Preferential activation of Notch1 signaling in p53 WT lymphomas was also observed at the RNA and protein level. Reporter mice for activation of Notch1 signaling revealed that total-body irradiation (TBI) enriched Notch1hi CD44+ thymocytes that could propagate in vivo after thymocyte transplantation. Mechanistically, genetic inhibition of Notch1 signaling in immature thymocytes prevented formation of radiation-induced thymic lymphoma in p53 WT mice. Taken together, these results demonstrate a critical role of activated Notch1 signaling in driving multistep carcinogenesis of thymic lymphoma following TBI in p53 WT mice. SIGNIFICANCE: These findings reveal the mutational landscape and key drivers in murine radiation-induced thymic lymphoma, a classic animal model that has been used to study radiation carcinogenesis for over 70 years.

Characterization of cardiovascular injury in mice following partial-heart irradiation with clinically relevant dose and fractionation.

BACKGROUND AND PURPOSE: Late cardiac toxicity is a major side effect of radiation therapy (RT) for breast cancer. We developed and characterized a mouse model of radiation-induced heart disease that mimics the dose, fractionation, and beam arrangement of left breast and chest wall RT. MATERIAL AND METHODS: Female wild-type (C57BL6/J) and atherosclerosis-prone apolipoprotein E-deficient (ApoE-/-) mice (on a C57BL/6J background) on regular chow were treated with 2 Gy × 25 fractions of partial-heart irradiation via opposed tangential beams to the left chest wall. The changes in myocardial perfusion and cardiac function of C57BL/6J mice were examined by single-photon emission computed tomography (SPECT) and echocardiography, respectively. In addition to SPECT and echocardiography, the formation of calcified plaques and changes in cardiac function of ApoE-/- mice were examined by dual-energy microCT (DE-CT) and pressure-volume (PV) loop analysis, respectively. The development of myocardial fibrosis was examined by histopathology. RESULTS: Compared to unirradiated controls, irradiated C57BL/6J mice showed no significant changes by SPECT or echocardiography up to 18 months after 2 Gy × 25 partial-heart irradiation even though irradiated mice exhibited a modest increase in myocardial fibrosis. For ApoE-/- mice, 2 Gy × 25 partial-heart irradiation did not cause significant changes by SPECT, DE-CT, or echocardiography. However, PV loop analysis revealed a significant decrease in load-dependent systolic and diastolic function measures including cardiac output, dV/dtmax and dV/dt min 12 months after RT. CONCLUSIONS: Following clinically relevant doses of partial-heart irradiation in C57BL/6J and ApoE-/- mice, assessment with noninvasive imaging modalities such as echocardiography, SPECT, and DE-CT yielded no evidence of decreased myocardial perfusion and cardiac dysfunction related to RT. However, invasive hemodynamic assessment with PV loop analysis indicated subtle, but significant, changes in cardiac function of irradiated ApoE-/- mice. PV loop analysis may be useful for future preclinical studies of radiation-induced heart disease, especially if subtle changes in cardiac function are expected.

Sensitization of Endothelial Cells to Ionizing Radiation Exacerbates Delayed Radiation Myelopathy in Mice.

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.

Inhibiting Glycogen Synthase Kinase-3 Mitigates the Hematopoietic Acute Radiation Syndrome in a Sex- and Strain-dependent Manner in Mice.

The Radiation and Nuclear Countermeasures Program at the National Institute of Allergy and Infectious Diseases (NIAID) mandated that medical countermeasures for treating Acute Radiation Syndrome (ARS) must have efficacy when administered at least 24 h after radiation exposure. At this time point, many cells within key target tissues, such as the hematopoietic system and the gastrointestinal (GI) tract, will already be dead. Therefore, drugs that promote the regeneration of surviving cells may improve outcomes. The serine/threonine kinase glycogen synthase kinase-3 (GSK-3) regulates stem and progenitor cell self-renewal and regeneration in the hematopoietic and GI compartments. We tested inhibition of GSK-3ß by SB216763 24 h after total body irradiation (TBI) and sub-total body irradiation (SBI). Here, we show that subcutaneous administration of SB216763 promotes the regeneration of surviving hematopoietic stem/progenitor cells (HSPCs), including myeloid progenitor cells, and improves survival of C57Bl/6 male mice when administered 24 h after TBI. However, these results were not recapitulated in female C57Bl/6 animals, suggesting a sex difference in GSK-3ß signaling in HSPCs. Subcutaneous administration of SB216763 in male mice stimulated activation of Sox2 transcription but failed to induce Sox2 transcription in female C57Bl/6 mice. Using TCF/lef-GFP reporter mice, we examined Wnt signaling in HSPCs of irradiated male and female mice treated with SB216763. GSK-3 inhibition elevated Wnt reporter activity in HSPCs isolated from male but not female mice. SB216763 did not mitigate hematopoietic ARS in males or females of a second strain of wild-type mice, C3H. In addition, administration of SB216763 did not mitigate hematopoietic ARS beyond the currently available standard approved therapy of ciprofloxacin and granulocyte-colony stimulating factor (G-CSF) in male C57Bl/6 mice. Further, SB216763 did not mitigate GI-ARS after SBI in C57Bl/6 male mice. The lack of efficacy in both sexes and multiple strains of mice indicate that SB216763 is not suitable for further drug development as a mitigator of ARS. Our studies demonstrate that activation of Wnt signaling in HSPCs promotes hematopoietic regeneration following radiation exposure, and targeting this pathway downstream of GSK-3ß may mitigate ARS in a sex- and strain-independent manner.

Photon-counting cine-cardiac CT in the mouse.

The maturation of photon-counting detector (PCD) technology promises to enhance routine CT imaging applications with high-fidelity spectral information. In this paper, we demonstrate the power of this synergy and our complementary reconstruction techniques, performing 4D, cardiac PCD-CT data acquisition and reconstruction in a mouse model of atherosclerosis, including calcified plaque. Specifically, in vivo cardiac micro-CT scans were performed in four ApoE knockout mice, following their development of calcified plaques. The scans were performed with a prototype PCD (DECTRIS, Ltd.) with 4 energy thresholds. Projections were sampled every 10 ms with a 10 ms exposure, allowing the reconstruction of 10 cardiac phases at each of 4 energies (40 total 3D volumes per mouse scan). Reconstruction was performed iteratively using the split Bregman method with constraints on spectral rank and spatio-temporal gradient sparsity. The reconstructed images represent the first in vivo, 4D PCD-CT data in a mouse model of atherosclerosis. Robust regularization during iterative reconstruction yields high-fidelity results: an 8-fold reduction in noise standard deviation for the highest energy threshold (relative to unregularized algebraic reconstruction), while absolute spectral bias measurements remain below 13 Hounsfield units across all energy thresholds and scans. Qualitatively, image domain material decomposition results show clear separation of iodinated contrast and soft tissue from calcified plaque in the in vivo data. Quantitatively, spatial, spectral, and temporal fidelity are verified through a water phantom scan and a realistic MOBY phantom simulation experiment: spatial resolution is robustly preserved by iterative reconstruction (10% MTF: 2.8-3.0 lp/mm), left-ventricle, cardiac functional metrics can be measured from iodine map segmentations with ~1% error, and small calcifications (615 µm) can be detected during slow moving phases of the cardiac cycle. Given these preliminary results, we believe that PCD technology will enhance dynamic CT imaging applications with high-fidelity spectral and material information.