Whole abdominal pencil beam scanned proton FLASH increases acute lethality.

PURPOSE: Ultra-high dose-rate FLASH radiotherapy (RT) has emerged as a modality which promises to reduce normal tissue toxicity while maintaining tumor control. Previous studies of gastrointestinal toxicity using passively scattered FLASH proton therapy (PRT) have, however, yielded mixed results, suggesting that the requirements for gastrointestinal sparing by FLASH are an open question. Furthermore, the more clinically relevant pencil beam scanned (PBS) FLASH PRT has not yet been assessed in this context, despite differences in the spatiotemporal dose-rate distributions compared to passively scattered PRT. Here, we provide the first report on the effects of PBS FLASH PRT on acute gastrointestinal injury in mice after whole abdominal irradiation. METHODS AND MATERIALS: Whole abdominal irradiation was performed on C57BL/6J mice using the entrance channel of the Bragg curve of a 250 MeV PBS proton beam at field-averaged dose-rates of 0.6 Gy/s for conventional (CONV) and 80-100 Gy/s for FLASH PRT. A 2D strip ionization chamber array was used to measure the dose and dose rate for each mouse. Survival was assessed at 14 Gy. Intestines were harvested and processed as Swiss rolls for analysis using a novel artificial intelligence (AI)-based crypt assay to quantify crypt regeneration 4 days post-irradiation. RESULTS: Survival was significantly reduced following 14 Gy FLASH PRT compared to CONV (P<0.001). Our AI-based crypt assays demonstrated no significant difference in intestinal crypts/cm or crypt depth between groups 4 days post-irradiation. Furthermore, we found no significant difference in EdU+ cells/crypt or Olfactomedin4+ intestinal stem cells with FLASH relative to CONV PRT. CONCLUSIONS: Overall, our data demonstrate significantly impaired survival following abdominal PBS FLASH PRT without apparent differences in intestinal histology 4 days post-irradiation.

Thrombopoietin mimetic stimulates bone marrow vascular and stromal niches to mitigate acute radiation syndrome.

BACKGROUND: Acute radiation syndrome (ARS) manifests after exposure to high doses of radiation in the instances of radiologic accidents or incidents. Facilitating regeneration of the bone marrow (BM), namely the hematopoietic stem and progenitor cells (HSPCs), is key in mitigating ARS and multi-organ failure. JNJ-26366821, a PEGylated thrombopoietin mimetic (TPOm) peptide, has been shown as an effective medical countermeasure (MCM) to treat hematopoietic-ARS (H-ARS) in mice. However, the activity of TPOm on regulating BM vascular and stromal niches to support HSPC regeneration has yet to be elucidated. METHODS: C57BL/6J mice (9-14 weeks old) received sublethal or lethal total body irradiation (TBI), a model for H-ARS, by 137Cs or X-rays. At 24 h post-irradiation, mice were subcutaneously injected with a single dose of TPOm (0.3 mg/kg or 1.0 mg/kg) or PBS (vehicle). At homeostasis and on days 4, 7, 10, 14, 18, and 21 post-TBI with and without TPOm treatment, BM was harvested for histology, BM flow cytometry of HSPCs, endothelial (EC) and mesenchymal stromal cells (MSC), and whole-mount confocal microscopy. For survival, irradiated mice were monitored and weighed for 30 days. Lastly, BM triple negative cells (TNC; CD45-, TER-119-, CD31-) were sorted for single-cell RNA-sequencing to examine transcriptomics after TBI with or without TPOm treatment. RESULTS: At homeostasis, TPOm expanded the number of circulating platelets and HSPCs, ECs, and MSCs in the BM. Following sublethal TBI, TPOm improved BM architecture and promoted recovery of HSPCs, ECs, and MSCs. Furthermore, TPOm elevated VEGF-C levels in normal and irradiated mice. Following lethal irradiation, mice improved body weight recovery and 30-day survival when treated with TPOm after 137Cs and X-ray exposure. Additionally, TPOm reduced vascular dilation and permeability. Finally, single-cell RNA-seq analysis indicated that TPOm increased the expression of collagens in MSCs to enhance their interaction with other progenitors in BM and upregulated the regeneration pathway in MSCs. CONCLUSIONS: TPOm interacts with BM vascular and stromal niches to locally support hematopoietic reconstitution and systemically improve survival in mice after TBI. Therefore, this work warrants the development of TPOm as a potent radiation MCM for the treatment of ARS.

Thrombopoietin mimetic stimulates bone marrow vascular and stromal niches to mitigate acute radiation syndrome.

Background: Acute radiation syndrome (ARS) manifests after exposure to high doses of radiation in the instances of radiologic accidents or incidents. Facilitating the regeneration of the bone marrow (BM), namely the hematopoietic stem and progenitor cells (HSPCs), is a key in mitigating ARS and multi-organ failure. JNJ-26366821, a PEGylated thrombopoietin mimetic (TPOm) peptide, has been shown as an effective medical countermeasure (MCM) to treat hematopoietic-ARS (H-ARS) in mice. However, the activity of TPOm on regulating BM vascular and stromal niches to support HSPC regeneration has not yet been elucidated. Methods: C57BL/6J mice (9-14 weeks old) received sublethal or lethal total body irradiation (TBI), a model for H-ARS, by 137Cs or X-rays. At 24 hours post-irradiation, mice were subcutaneously injected with a single dose of TPOm (0.3 mg/kg or 1.0 mg/kg) or PBS (vehicle). At homeostasis and on days 4, 7, 10, 14, 18, and 21 post-TBI with and without TPOm treatment, BM was harvested for histology, BM flow cytometry of HSPCs, endothelial (EC) and mesenchymal stromal cells (MSC), and whole-mount confocal microscopy. For survival, irradiated mice were monitored and weighed for 30 days. Lastly, BM triple negative cells (TNC; CD45-, TER-119-, CD31-) were sorted for single-cell RNA-sequencing to examine transcriptomics after TBI with or without TPOm treatment. Results: At homeostasis, TPOm expanded the number of circulating platelets and HSPCs, ECs, and MSCs in the BM. Following sublethal TBI, TPOm improved BM architecture and promoted recovery of HSPCs, ECs, and MSCs. Furthermore, TPOm elevated VEGF-C levels in normal and irradiated mice. Following lethal irradiation, mice improved body weight recovery and 30-day survival when treated with TPOm after 137Cs and X-ray exposure. Additionally, TPOm reduced vascular dilation and permeability. Finally, single-cell RNA-seq analysis indicated that TPOm increased the expression of collagens in MSCs to enhance their interaction with other progenitors in BM and upregulated the regeneration pathway in MSCs. Conclusions: TPOm interacts with BM vascular and stromal niches to locally support hematopoietic reconstitution and systemically improve survival in mice after TBI. Therefore, this work warrants the development of TPOm as a potent radiation MCM for the treatment of ARS.

PEGylated thrombopoietin mimetic, JNJ­26366821 a novel prophylactic radiation countermeasure for acute radiation injury.

Thrombopoietin (TPO) is the primary regulator of platelet generation and a stimulator of multilineage hematopoietic recovery following exposure to total body irradiation (TBI). JNJ­26366821, a novel PEGylated TPO mimetic peptide, stimulates platelet production without developing neutralizing antibodies or causing any adverse effects. Administration of a single dose of JNJ­26366821 demonstrated its efficacy as a prophylactic countermeasure in various mouse strains (males CD2F1, C3H/HeN, and male and female C57BL/6J) exposed to Co-60 gamma TBI. A dose dependent survival efficacy of JNJ­26366821 (- 24 h) was identified in male CD2F1 mice exposed to a supralethal dose of radiation. A single dose of JNJ­26366821 administered 24, 12, or 2 h pre-radiation resulted in 100% survival from a lethal dose of TBI with a dose reduction factor of 1.36. There was significantly accelerated recovery from radiation-induced peripheral blood neutropenia and thrombocytopenia in animals pre-treated with JNJ­26366821. The drug also increased bone marrow cellularity and megakaryocytes, accelerated multi-lineage hematopoietic recovery, and alleviated radiation-induced soluble markers of bone marrow aplasia and endothelial damage. These results indicate that JNJ­26366821 is a promising prophylactic radiation countermeasure for hematopoietic acute radiation syndrome with a broad window for medical management in a radiological or nuclear event.

Orthovoltage X-Rays Exhibit Increased Efficacy Compared with γ-Rays in Preclinical Irradiation.

Radionuclide irradiators (137Cs and 60Co) are commonly used in preclinical studies ranging from cancer therapy to stem cell biology. Amidst concerns of radiological terrorism, there are institutional initiatives to replace radionuclide sources with lower energy X-ray sources. As researchers transition, questions remain regarding whether the biological effects of γ-rays may be recapitulated with orthovoltage X-rays because different energies may induce divergent biological effects. We therefore sought to compare the effects of orthovoltage X-rays with 1-mm Cu or Thoraeus filtration and 137Cs γ-rays using mouse models of acute radiation syndrome. Following whole-body irradiation, 30-day overall survival was assessed, and the lethal dose to provoke 50% mortality within 30-days (LD50) was calculated by logistic regression. LD50 doses were 6.7 Gy, 7.4 Gy, and 8.1 Gy with 1-mm Cu-filtered X-rays, Thoraeus-filtered X-rays, and 137Cs γ-rays, respectively. Comparison of bone marrow, spleen, and intestinal tissue from mice irradiated with equivalent doses indicated that injury was most severe with 1-mm Cu-filtered X-rays, which resulted in the greatest reduction in bone marrow cellularity, hematopoietic stem and progenitor populations, intestinal crypts, and OLFM4+ intestinal stem cells. Thoraeus-filtered X-rays provoked an intermediate phenotype, with 137Cs showing the least damage. This study reveals a dichotomy between physical dose and biological effect as researchers transition to orthovoltage X-rays. With decreasing energy, there is increasing hematopoietic and intestinal injury, necessitating dose reduction to achieve comparable biological effects. SIGNIFICANCE: Understanding the significance of physical dose delivered using energetically different methods of radiation treatment will aid the transition from radionuclide γ-irradiators to orthovoltage X-irradiators.

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity.

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrinα6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.