NOTE: End date changed to 5/31/2023 per JSC Grants Office and NSSC information (Ed., 6/2/22)

NOTE: End date changed to 5/31/2022 per NSSC information (Ed., 3/3/2020)

Two addenda were added to the NSCOR in the April 2017 – March 2018 reporting period. The first was designed to determine if a low dose aspirin regimen could be an effective countermeasure to high LET radiation-induced hepatocellular carcinoma. Two exposure conditions are included, and acute exposure to 0.2 Gy 300 MeV 28Si ions and a chronic exposure to 0.4 Gy of 252Cf neutrons. The second was designed to compare fractioned exposures to a simulated GCR (galactic cosmic radiation) beam to an acute exposure for the induction of hepatocellular carcinoma.

Sequencing of Radiation-Induced Tumors: During the funding period covered by this report, we selected 55 hepatocellular carcinomas (HCCs) and 15 thymic lymphomas for genomic sequencing. These tumor samples were archived in the course of previous NASA-funded research.

The rationales for selecting HCC and thymic lymphomas are that both tumor types are strongly radiation-induced but have sufficient spontaneous incidences to provide tumors from unexposed mice for controls. Tumors of both types grow large enough in mice to provide material for DNA collection without having to resort to extraction from paraffin blocks. We already know a considerable amount about these tumor types. In addition to genomic sequencing, for the thymic lymphomas we could also perform RNAseq on thymus tissue from unirradiated mice that could be integrated into the data analysis.

A total of 70 tumor samples were selected for sequencing: 15 HCCs from unirradiated male mice 15 HCCs from gamma ray irradiated male mice 25 HCCs from HZE ion irradiated male mice 5 thymic lymphomas from unirradiated mice (both males and females) 5 thymic lymphomas from gamma ray irradiated mice (both males and females) 5 thymic lymphomas from HZE ion irradiated mice (both males and females)

DNA was extracted from the tumor samples by using DNeasy Blood and Tissue Kit from Qiagen following the manufacturer’s protocol. The DNA quality was good as assessed by the 260/280 and 260/230 ratios calculated from the absorbance values measured by using a Nanodrop spectrophotometer. The DNAs from the samples were submitted to the sequencing core in DNAlink Inc., Los Angeles, CA.

For RNAseq we harvested thymuses from unirradiated 6-week old male mice from the 8 founder strains of the HS/Npt stock that was used as a source of tumors for the genomic sequencing. We collected 3 thymuses from each strain. High quality RNA was prepared using a DNase I on-column digestion protocol. Paired end next generation Illumina sequencing was done on a library prepared from poly-A selected RNA by the Genomics and Microarray Core at the University of Colorado Anschutz Medical Campus. The sequencing data have been submitted to the Sequence Read Archive, archived, alignments done, and analysis is now underway.

Acute neutron exposures: Project 3 originally included carcinogenesis studies of male and female mice irradiated acutely with neutrons at doses of 0.1, 0.2, and 0.4 Gy. These groups were removed from the study at the direction of the NASA Space Radiation (SR) Element and then added back in the previous funding period. Acute exposures were done at the Radiological Research Accelerator Facility (RARAF) at Columbia University. The female mice were irradiated at the end of 2020. Because of routine maintenance and accelerator downtime at RARAF, we were unable to irradiate the male mice until now. A group of 108 male C3H mice was irradiated with a dose of 0.1 Gy in November 2021 and a further group of 106 mice was irradiated with a dose of 0.2 Gy in December 2021. These mice are currently being monitored for tumor development.

Two addenda were added to the NSCOR in the April 2017 – March 2018 reporting period. The first was designed to determine if a low dose aspirin regimen could be an effective countermeasure to high LET radiation-induced hepatocellular carcinoma. Two exposure conditions are included, and acute exposure to 0.2 Gy 300 MeV 28Si ions and a chronic exposure to 0.4 Gy of 252Cf neutrons. The second was designed to compare fractioned exposures to a simulated GCR (galactic cosmic radiation) beam to an acute exposure for the induction of hepatocellular carcinoma.

In Project 2, we found no evidence that high LET radiation exposure leads to enhanced metastatic potential in ether of the two model systems tested. However, we did identify serum biomarkers for preclinical detection of hepatocellular carcinoma. These results have been published online in the International Journal of Radiation Biology (see Bibliography section for Udho et al.).

In Project 3, we calculated survival data for C3H male and BALB/c female mice that received acute exposures to 0.4 Gy of neutrons and compared those results to mice of the same strain and sex irradiated with 0.4 Gy of neutrons at low dose rate (1mGy/day). Acute exposure led to considerable life shortening in female BALB/c mice with a small improvement in survival if the same dose is delivered at low dose rate. In male C3H mice there is also considerable life shortening from the acute exposure, but low dose rate sparing is substantial. Whether the greater detrimental effect of the low dose rate exposure in BALB/c female mice compared to male C3H mice is due to the strain difference or the sex difference is unknown. Note that these results are for overall survival out to 800 days of age; histopathology review of the tumors that arose in these groups is in progress.

In Project 4, Drs. Raber, Emmett, and computational expert Jessica Minnier focused on analyzing the lipid data sets which had been recompiled with a s/n of 5:1. Drs. Emmett and Yu (University of Texas Medical Branch-UTMB) then assigned lipid i.d.s to the significant m/z values identified by computational analysis. Further correlations were made between the behavioral data (Dr. Raber), the miRNA data (Dr. Story), and the lipid data (Dr. Emmett). At this point, biological correlations between all data sets were assigned. The results were reported in the manuscript entitled: "Associations between lipids in selected brain regions, plasma miRNA, and behavioral and cognitive measures following 28Si ion irradiation," which has been published online in Scientific Reports (see Bibliography section for Minnier et al.).

Two Addenda were in progress during the period covered by this report: a study of low dose aspirin as a radiation countermeasure and a comparison of the effects of acute exposure and fractionated exposure to GCRsim. We observed no difference in survival between male C3H mice that received acute GCRsim exposures and those that received fractionated exposures.

Two addenda were added to the NSCOR in the April 2017 – March 2018 reporting period. The first was designed to determine if a low dose aspirin regimen could be an effective countermeasure to high LET radiation-induced hepatocellular carcinoma. Two exposure conditions are included, and acute exposure to 0.2 Gy 300 MeV 28Si ions and a chronic exposure to 0.4 Gy of 252Cf neutrons. The second was designed to compare fractioned exposures to a simulated GCR (galactic cosmic radiation) beam to an acute exposure for the induction of hepatocellular carcinoma.

In Project 2, we performed comparative analysis of gene expression profiles between mouse and human HCC. The human HCC gene expression profile was obtained from The Cancer Genome Atlas (TCGA) published data in Cell. 2017 Jun 15; 169(7):1327–1341.e23. Our analysis identified subgroups of human HCC patients that resembled gene expression of high-LET radiation-induced HCC or low-LET radiation-induced / spontaneous HCC in mouse model. The high-LET HCC group showed better survival in Kaplan-Meier analysis and were enriched with more tumor-infiltrating immune cells that function as tumor suppressors when compared with other types of HCC.

We also developed and validated the C3H Afp-mCherry mouse model for HCC. This model consists of the C3H susceptible strain with the addition of an mCherry fluorescent reporter driven by the alpha fetoprotein (Afp) promoter. The reporter is designed to fluorescently label cancer cells for simple and unambiguous identification of liver tumors and metastasis. Using this mouse model, we have identified biomarkers for early detection of liver disease, many of which are also established biomarkers used for diagnosis of human liver disease (e.g., AFP and ALT). Cholesterol and albumin serum biomarkers were elevated in an HCC specific manner, a finding in humans that indicates a steatohepatitic HCC subtype. Interestingly, the vast majority of HCCs observed in the C3H Afp-mCherry congenic mice are of the steatohepatitic subtype. HCC prognosis is rather poor due to late diagnosis and poor sensitivity of current testing methods. We have shown that elevated plasma AFP is capable of identifying mice that will eventually develop HCC, at least 6 months in advance of when tumors are typically detectible in C3H mice. In combination with other biomarkers being identified in this NSCOR, there is the potential to develop a panel of biomarkers for a minimally invasive blood test to identify early stage liver disease in mice that could potentially be translatable for use in humans. Lung lesions arising in our irradiated mice are currently being examined to determine if they are metastasized from the liver. Results of these analyses will help us answer the question of whether there is an increased HCC malignancy from HZE Ions.

In Project 3, we calculated survival data for C3H male and BALB/c female mice irradiated with 252Cf neutrons at low dose rate (1mGy/day). Histopathology review on these groups is in progress.

In Project 4, BALB/c and C3H female and male mice and their F2 hybrid progeny were irradiated with 28Si ions (350 MeV/n, 0.2 Gy) and tested for behavioral and cognitive performance 1, 6, and 12 months following irradiation. The plasma of the mice was collected for analysis of miRNA levels. Select, pertinent brain regions were dissected for lipidomics analyses and analyses of levels of select biomarkers shown to be sensitive to effects of space radiation in previous studies. We analyzed the relationships between behavioral and cognitive measures, plasma miRNAs, lipid in pertinent brain regions, and biomarkers in brain sensitive to effects of radiation in previous studies. A manuscript with these data is currently being finalized.

The miRNA expression patterns developed in Project 1 have been applied to the endpoints for cognitive function being developed in Project 4. There are numerous miRNA, already identified as associated with a number of psychologic disturbances, fear for example, whose expression is altered in mice responding to different cognitive stimuli. These data are being analyzed now.

Raw lipid data sets collected on the brain samples provided by Dr. Raber have been recompiled with a s/n of 5:1. These files were sent to Dr. Raber’s group on 3/5/2020 and his computational mathematician (Jessica Minnier) is analyzing the data now. When complete we will correlate to the lipid i.d.s and determine biological significance correlated to the behavioral data.

Two Addenda were in progress during the period covered by this report: a study of low dose aspirin as a radiation countermeasure and a comparison of the effects of acute exposure and fractionated exposure to GCRsim. We saw no difference in either survival or tumor-free survival for mice on a low dose aspirin regimen during and following acute 28Si ion exposure. A similar study of aspirin efficacy against low dose rate neutron exposure is in progress.

Major Milestones (April 2019 – March 2020)

- A new mouse model for HCC has been developed and validated for studying HCC. 264 F1 and 264 C3H Afp-mCherry congenic male and female mice have been irradiated and blood samples collected at 3 to 6 month intervals and liver and lung tissues at terminal collections (21 to 24 months post irradiation).

- We have shown that the new mouse model containing the mCherry reporter for labeling cancer cells allows for unambiguous identification of liver tumors and metastases.

- Plasma and tumor biomarkers have been identified that detect liver disease can predict the development of HCC before any visible sign of a tumor using simple blood tests.

- Low dose rate (1 mGy/day) neutron irradiation leads to dose dependent life shortening in female mice.

- A low dose aspirin regimen dose not improve overall survival or tumor-free survival following acute 28Si ion exposure.

- Plasma miRNAs that distinguish between tumor-bearing and non-tumor mice have been identified.

Two addenda were added to the NSCOR in the April 2017 – March 2018 reporting period. The first was designed to determine if a low dose aspirin regimen could be an effective countermeasure to high LET radiation-induced hepatocellular carcinoma. Two exposure conditions are included, and acute exposure to 0.2 Gy 300 MeV 28Si ions and a chronic exposure to 0.4 Gy of 252Cf neutrons. The second was designed to compare fractioned exposures to a simulated GCR (galactic cosmic radiation) beam to an acute exposure for the induction of hepatocellular carcinoma.

We monitored the F2 mice until they became moribund or reached 800 days of age. All of the F2 mice have now been necropsied and any tissue lesions detected were prepared for histopathology. Histopathology has been completed on about half of these mice.

In Project 2, we completed the sample collection from the (B6C3H)F1 Tg::Apf-mCherry mice. Liver and lung tissue sections were examined by a pathologist and we have found a variety of pathologies in the liver (HCC, dysplastic nodules, hepatitis, and steatosis) and lung (peribronchial chronic inflammation and carcinoma), though no major difference among treatment groups (sham, gamma-irradiated, and silicon-irradiated) was observed. Our mouse model was validated and found to be an excellent model for detecting early alpha fetoprotein (AFP) expression in liver disease as well as for unambiguous identification of metastasis from the liver to other tissues. Among the approximately 30 measurements obtained from plasma and whole blood (liquid biopsy-accessible measurements), we have found AFP and alanine aminotransferase (ALT) to be good predictors of the presence of liver disease (sensitivity 60-70%, specificity 85-90%).

Our second cohort of mice, the C3H Tg::Afp-mCherry, which also include a pilot study with the radioprotectant PrC-210, were irradiated a tNASA Space Radiation Laboratory (NSRL) NSRL18A and we completed plasma collections at two timepoints (6 and 9 months post irradiation) for our longitudinal study.

Also ongoing in Project 2 is the genomic and epigenomic characterization of HCC tumors in C3H mice. At this juncture we have started to develop approaches that use other HCC data sets from mice based upon exposures to chemicals (7 datasets) or in genetically engineered mouse strains (4 datasets) to compare to low LET, high LET, or spontaneous tumors generated in the prior NSCOR. Furthermore, we have begun making comparisons of these mouse datasets to datasets for human HCC.

In Project 3 we completed the low dose rate neutron irradiations and performed acute neutron exposures at Columbia University. We also completed low dose neutron exposures of mice and rats for the CNS (central nervous system) NSCOR, rats for a cardiovascular effects study (Marjan Boerma, Principal Investigator-PI), and mice for an in utero effects study (Jon Steller, PI). The facility is currently being used to irradiate mice for the GI (Gastronintestinal) NSCOR (Albert Fornace, PI), and for a countermeasure addendum and an immunological effects pilot study. In Project 4, we finalized behavioral and cognitive testing of BALB/c, C3H, and F2 mice 1, 6, and 12 months following acute 28Si ion irradiation. At the three time points, plasma was collected for miRNA analyses by Dr. Story’s laboratory at UTSW (University of Texas Southwestern Medical Center) (Project 1). At the three time points, selected mice of the parental strains were sacrificed by cervical dislocation and their hypothalamus and amygdala dissected. The tissues of one hemisphere were analyzed for lipidomics by Dr. Emmett’s laboratory at UTMB (University of Texas Medical Branch at Galveston). Six lipid measures of the mice from the three time points were analyzed. From the hypothalamic and amygdaloid tissues of the other hemisphere, ELISAs were performed to assess levels of the specific biomarkers. For the parental strains, we compiled a large excel file with all the behavioral data for the open field, object recognition, and contextual and cued fear learning and morning, all the plasma miRNA data, and the lipidomics data on the six lipids. The data are currently analyzed for several manuscripts. This year, we also performed a countermeasure study with aspirin. An independent cohort of C3H male mice were tested 6 and 18 months following 28Si ion irradiation as part of an aspirin treatment study. In addition, we started to behaviorally test C3H and BALB/c mice as part of Project 3, following chronic neutron exposure. Mice were tested at 600 days of age in March of 2019. The data are currently being analyzed.

For the Addenda, we began neutron irradiation of mice for the low dose rate arm of the aspirin countermeasure study. We irradiated mice for the GSAM (Galactic Cosmic Ray Simulation and Mitigation) study in November of 2018 (NSRL-18C). 135 male C3H mice were irradiated with 0.4 Gy of simulated GCR split into 19 fractions delivered over 4 weeks. An additional 135 mice received an acute exposure of 0.4 Gy.

Major Milestones (April 2018 – March 2019)

* Lipid data completed for liver, lung and serum for all time points (1, 6, 12 and 18-momth) for Project 1.

* Completed necropsies and histopathology slide preparation for all F2 mice in Project 1.

* Completed low dose rate neutron exposures for Project 3 and performed acute neutron exposures at Columbia University.

* Provide low dose rate neutron exposures for several NASA funded investigators.

* Lipid data completed for amygdala and hypothalamus samples for all time points provided for Project 4.

* Irradiated mice at NSRL for the GSAM study.

* Began the low dose rate exposure aspirin countermeasure study.

* Completed behavioral and cognitive testing of BALB/c, CRH, and F2 mice 1, 6, and 12 months following acute 28Si ion irradiation, along with plasma miRNA, brain lipidomics on 6 lipids.

* Completed behavioral testing of mice treated with aspirin as a contermeasure.

* Completed behavioral testing of C3H and BALB/c following chronic neutron exposure at 600 days of age.

Two addenda were added to the NSCOR in the April 2017 – March 2018 reporting period. The first was designed to determine if a low dose aspirin regimen could be an effective countermeasure to high LET radiation-induced hepatocellular carcinoma. Two exposure conditions are included, and acute exposure to 0.2 Gy 300 MeV 28Si ions and a chronic exposure to 0.4 Gy of 252Cf neutrons. The second was designed to compare fractioned exposures to a simulated GCR (galactic cosmic radiation) beam to an acute exposure for the induction of hepatocellular carcinoma.

In Project 2, gene expression profiles were re-analyzed using pathologically confirmed archival samples. The analysis identified common signaling pathways in high-LET induced, low-LET induced, and spontaneous hepatocellular carcinoma (HCC). These include activation of PI3K/AKT and suppression of PTEN (phosphatase and tensin homolog deleted from chromosome 10) signaling, which are also the molecular mechanisms in human HCC. Other identified signaling pathways are highly involved in ROS (reactive oxygen species) signaling, inflammatory responses, cell proliferation, and apoptosis. Despite the common molecular pathways, a subset of genes including 815 probes emerged as a signature to separate not only tumor and non-tumor, but also radiation-type in each group. The signature does not have a statistically significant pathway enrichment. We are currently investigating the driver genes and functional impact of this signature on different types of HCC.

We completed experimental metastasis studies with MMTV-PyMT mice and continued studies with Afp-mCherry mice. In the MMTV-PyMT model, we found that high LET exposures do not increase metastatic efficacy.

Ninety Afp-mCherry mice were irradiated or sham irradiated at NASA Space Radiation Laboratory (NSRL). These mice are used to study HCC metastasis to the lung. The mice are being monitored for circulating tumor cells, and some have been sacrificed 3 months post-irradiation to detect the earliest stages of tumorigenesis using newly developed tissue clearing and optical approaches.

Project 3 involves the commissioning of a neutron irradiator in a shielded vivarium that meets the animal care requirements for long term mouse housing. Over the past year we renovated the building, calculated the activity of radioactive material required, and acquired the source and the irradiator. In Project 4, we are determining the effects of space radiation on brain function by performing neurobehavioral tests on mice exposed to simulated space radiation or neutron radiation. This year we completed the behavioral and cognitive testing at three time points following 28Si irradiation (300 MeV/n, 0.2 Gy): 1, 6, and 12 months. A striking observation was that irradiated BALB/c mice and irradiated F2 mice showed more hippocampus-contextual fear memory and a wider range in individual variability in contextual fear memory than irradiated C3H mice. The cognitive data illustrate the individual performance differences in 28Si ion-irradiated mice, especially in irradiated F2 mice and data support the potential to identify biomarkers for radiation-induced cognitive deficits. Eight mice of each treatment and sex were sacrificed and pertinent brain regions dissected for lipidomics analyses in the laboratory by Dr. Mark Emmett at University of Texas Medical Branch (UTMB). 192 samples were extracted and 191 (one sample was lost) were analyzed for comprehensive lipid composition by ultra high-resolution mass spectrometry methods. All samples were block randomized for consistency. Data analysis is in progress.

In addition, Dr. Michael Story at UTSW (University of Texas Southwestern Medical Center) is analyzing plasma miRNA in the same mice. Finally, we are assessing levels of some specific markers shown to be sensitive to effects of aging and space radiation on the brain. In addition to the study described above, an independent cohort of C3H male mice were tested 6 months following 28Si ion irradiation as part of an aspirin treatment study. The dose of aspirin used has CNS (central nervous system) effects in C3H/HeNCrl mice. This is an important finding as the dosing was based on aspirin studies in C57BL/6J mice. We are currently analyzing these data and consider testing at later time points.

For the addenda, we irradiated the mice for the acute 28Si exposure condition in the aspirin countermeasure study. The chronic neutron exposures are scheduled to begin in mid-May or early June of 2018. Work on the GCR simulator addendum is slated to begin in November of 2018 (NSRL-18C).

In Project 2 we have reviewed the pathology on 208 cases of mouse hepatocellular carcinomas (HCC) from the previous Carcinogenesis NSCOR to allow correct classification of cases as metastatic and nonmetastatic. Each case has been assigned a histologic subtype and mitotic index, and semi-quantitatively scored for local invasion, vascular invasion, cellular pleomorphism, nuclear pleomorphism, degree of differentiation, and presence of fibrosis. We have also begun to look for mutations in the tumors by sequencing DNA from five of them (whole exome sequencing). We have detected mutations in these tumors that are also common in human HCC.

We have begun experimental metastasis studies with MMTV-PyMT and Afp-mCherry transgenic mice. MMTV-PyMT mice are a model for mammary tumor metastasis to the lungs. We have irradiated forty-four of these mice on two different strain backgrounds and followed them for mammary tumor development. We are now quantifying the extent of metastases in the mice. Viable tumors have been cryopreserved and serially transplanted for further work.

Ninety Afp-mCherry mice were irradiated or sham irradiated at NSRL. These mice are used to study HCC metastasis to the lung. The mice are being monitored for circulating tumor cells, and some have been sacrificed 3 months post-irradiation to detect the earliest stages of tumorigenesis using newly developed tissue clearing and optical approaches.

Project 3 involves the commissioning of a neutron irradiator in a shielded vivarium that meets the animal care requirements for long term mouse housing. Over the past year we renovated the building, calculated the activity of radioactive material required, and acquired the source and the irradiator.

For Project 4, we completed neurobehavioral testing for the one month time point and collected tissues from the mice that were tested.

In Project 1 we have optimized a lipidomic screen for use with mouse samples. In Project 2 we have reviewed the pathology on 210 cases of mouse hepatocellular carcinomas (HCC) and characterized them for histological features that may be relevant to their propensity to metastasize to the lung, including fibrosis, local invasion, vascular invasion, cellular and nuclear pleomorphism, tumor subtype, and mitotic index. One feature, high microvessel density, appears to be more common in metastatic HCC. We have also begun to look for mutations in the tumors by sequencing DNA from five of them (whole exome sequencing). We have detected mutations in these tumors that are also common in human HCC.

Project 3 requires us to install a neutron irradiator in a heavily shielded building that also meets the animal care requirements for long term mouse housing. We have secured a suitable building and begun the process of renovating it. We are also drawing up the specifications for the radioactive source in the neutron irradiator.

For Project 4 we have identified the neurobehavioral assays that will be most sensitive to radiation effects.