Irradiated hosts gave rise to significantly more Trp53 null mammary cancers that grew more rapidly than those in sham-irradiated mice and exhibited an immunosuppressive tumor microenvironment. CAPE prevented the effect of host irradiation on tumor growth rate, immune signature and immunosuppression.

Densely ionizing radiation is a major component of the space radiation environment and has potentially greater carcinogenic effect compared to sparsely ionizing radiation that is prevalent in the terrestrial environment. It is unknown to what extent the irradiated microenvironment contributes to the differential carcinogenic potential of densely ionizing radiation. To address this gap 10-week old BALB/c mice were irradiated with 100 cGy sparsely ionizing g-radiation or 10 30 or 80 cGy of densely ionizing 350 MeV/amu Si particles and transplanted 3 days later with syngeneic Trp53 null mammary fragments. Tumor appearance was monitored for 600 days. Tumors arising in Si-particle irradiated mice had a shorter median time to appearance grew faster and were more likely to metastasize. Most tumors arising in sham-irradiated mice were ER-positive pseudo-glandular and contained both basal keratin 14 and luminal keratin 8/18 cells (designated K14/18) while most tumors arising in irradiated hosts were K8/18 positive (designated K18) and ER negative. Comparison of K18 vs K14/18 tumor expression profiles showed that genes increased in K18 tumors were associated with ERBB2 and KRAS while decreased genes overlapped with those down regulated in metastasis and by loss of E-cadherin. Consistent with this K18 tumors grew faster than K14/18 tumors and more mice with K18 tumors developed lung metastases compared to mice with K14/18 tumors. However K18 tumors arising in Si-particle irradiated mice grew even faster and were more metastatic compared to control mice. A K18 Si-irradiated host profile was enriched in genes involved in mammary stem cells stroma and Notch signaling. Thus systemic responses to densely ionizing radiation enriches for a ER-negative K18-positive tumor whose biology is more aggressive compared to similar tumors arising in non-irradiated hosts. Key Words: ionizing radiation; breast cancer; heavy ion radiation;initiation; promotion 3 different dose of Si were used. Total RNA was extracted from mammary tumors derived from transplantations of non-irradiated p53null mammary fragments into irradiated hosts. We analyzed a total of 45 Trp53-null tumors: 18 from sham-irradiated hosts 9 from 10 cGy Si-irradiated hosts 10 from 30 cGy Si-irradiated hosts and 8 from irradiated hosts.

To determine how host irradiation affects tumor profiles in 10 month aged mice treated with HZE or gamma irradiation.

Densely ionizing radiation is a major component of the space radiation environment and has potentially greater carcinogenic effect compared to sparsely ionizing radiation that is prevalent in the terrestrial environment. It is unknown to what extent the irradiated microenvironment contributes to the differential carcinogenic potential of densely ionizing radiation. To address this gap 10-week old BALB/c mice were irradiated with 100 cGy sparsely ionizing g-radiation or 10 30 or 80 cGy of densely ionizing 350 MeV/amu Si particles and transplanted 3 days later with syngeneic Trp53 null mammary fragments. Tumor appearance was monitored for 600 days. Tumors arising in Si-particle irradiated mice had a shorter median time to appearance grew faster and were more likely to metastasize. Most tumors arising in sham-irradiated mice were ER-positive pseudo-glandular and contained both basal keratin 14 and luminal keratin 8/18 cells (designated K14/18) while most tumors arising in irradiated hosts were K8/18 positive (designated K18) and ER negative. Comparison of K18 vs K14/18 tumor expression profiles showed that genes increased in K18 tumors were associated with ERBB2 and KRAS while decreased genes overlapped with those down regulated in metastasis and by loss of E-cadherin. Consistent with this K18 tumors grew faster than K14/18 tumors and more mice with K18 tumors developed lung metastases compared to mice with K14/18 tumors. However K18 tumors arising in Si-particle irradiated mice grew even faster and were more metastatic compared to control mice. A K18 Si-irradiated host profile was enriched in genes involved in mammary stem cells stroma and Notch signaling. Thus systemic responses to densely ionizing radiation enriches for a ER-negative K18-positive tumor whose biology is more aggressive compared to similar tumors arising in non-irradiated hosts. Key Words: ionizing radiation; breast cancer; heavy ion radiation;initiation; promotion 3 different dose of Si were used. Total RNA was extracted from mammary tumors derived from transplantations of non-irradiated p53null mammary fragments into irradiated hosts. We analyzed a total of 45 Trp53-null tumors: 18 from sham-irradiated hosts 9 from 10 cGy Si-irradiated hosts 10 from 30 cGy Si-irradiated hosts and 8 from irradiated hosts.

To investigate gene expression patterns in rat mammary carcinomas induced by ionizing radiation. Gene expression patterns were analyzed in radiation-induced rat mammary carcinomas and normal mammary gland tissues.

Proton irradiation is touted for its improved tumor targeting due to the physical advantages of ion beams for radiotherapy. Recent studies from our laboratory have shown that in addition to targeting advantages proton irradiation can inhibit angiogenic and immune factors and thereby modulate tumor progression. High-energy protons also constitute a principal component of the galactic cosmic rays to which astronauts are exposed. Increased understanding of the biological effects of proton exposure would thus contribute to both improved cancer therapy and carcinogenesis risk assessment for space travel. In addition age plays a major role in tumor incidence and is a critical consideration for estimating cancer risk. We investigated the effects of host age and proton exposure on tumor progression. Tumor lag time and growth dynamics were tracked following injection of murine Lewis lung carcinoma (LLC) cells into young (68 day) versus old (736 day) mice with or without coincident irradiation. Tumor progression was suppressed in old compared to young mice. Differences in progression were further modulated by proton irradiation (1GeV) with increased inhibition evident in old mice. Through global transcriptome analysis TGFB1 and TGFB2 were determined to be key players that contributed to the tumor dynamics observed. These findings point to older hosts providing decreased systemic tumor support which can be further inhibited by proton irradiation. Overall design: For genome-wide expression profiling of tumor tissue Mouse WG-6 BeadArray chips (Illumina San Diego CA) were used. Total RNA was amplified with the Ambion Illumina TotalPrep Amplification Kit (Ambion Austin TX) and labeled from all replicate biological samples for each condition. For tumor replicates thirty tumor samples from adolescent and thirty tumor samples from old mice for a total of 60 tumor samples were used. All replicate samples were run individually. For each age group ten tumor samples had received proton irradiation while twenty tumor samples were from unirradiated mice (as described above). Total RNA was isolated and purified using TRIzol (Invitrogen) and quantified using an Agilent Bioanalyzer. Samples were deemed suitable for amplification and hybridization if they had 28s/18s = 2:1 RIN >7. Total RNA of 500ng per sample was amplified using AmbionTotalPrep and 1.5ug of the product was loaded onto the chips. Following hybridization at 55C the chips were washed and then scanned using the Illumina iScan System. The data was checked with GenomeStudio (Illumina) for quality control. In GenomeStudio data was background subtracted and rank invariant normalization was applied. Data was imported into MultiExperiment Viewer MeV for statistical analysis. The statistically significant genes were determined using MeV by applying a one-way ANOVA analysis with standard Bonferroni correction with a FDR <0.05 that resulted in a list of significant genes. Average gene expression signals <10 were filtered out due to signal being

Proton irradiation is touted for its improved tumor targeting due to the physical advantages of ion beams for radiotherapy. Recent studies from our laboratory have shown that in addition to targeting advantages proton irradiation can inhibit angiogenic and immune factors and thereby modulate tumor progression. High-energy protons also constitute a principal component of the galactic cosmic rays to which astronauts are exposed. Increased understanding of the biological effects of proton exposure would thus contribute to both improved cancer therapy and carcinogenesis risk assessment for space travel. In addition age plays a major role in tumor incidence and is a critical consideration for estimating cancer risk. We investigated the effects of host age and proton exposure on tumor progression. Tumor lag time and growth dynamics were tracked following injection of murine Lewis lung carcinoma (LLC) cells into young (68 day) versus old (736 day) mice with or without coincident irradiation. Tumor progression was suppressed in old compared to young mice. Differences in progression were further modulated by proton irradiation (1GeV) with increased inhibition evident in old mice. Through global transcriptome analysis TGFB1 and TGFB2 were determined to be key players that contributed to the tumor dynamics observed. These findings point to older hosts providing decreased systemic tumor support which can be further inhibited by proton irradiation. Overall design: For genome-wide expression profiling of tumor tissue Mouse WG-6 BeadArray chips (Illumina San Diego CA) were used. Total RNA was amplified with the Ambion Illumina TotalPrep Amplification Kit (Ambion Austin TX) and labeled from all replicate biological samples for each condition. For tumor replicates thirty tumor samples from adolescent and thirty tumor samples from old mice for a total of 60 tumor samples were used. All replicate samples were run individually. For each age group ten tumor samples had received proton irradiation while twenty tumor samples were from unirradiated mice (as described above). Total RNA was isolated and purified using TRIzol (Invitrogen) and quantified using an Agilent Bioanalyzer. Samples were deemed suitable for amplification and hybridization if they had 28s/18s = 2:1 RIN >7. Total RNA of 500ng per sample was amplified using AmbionTotalPrep and 1.5ug of the product was loaded onto the chips. Following hybridization at 55C the chips were washed and then scanned using the Illumina iScan System. The data was checked with GenomeStudio (Illumina) for quality control. In GenomeStudio data was background subtracted and rank invariant normalization was applied. Data was imported into MultiExperiment Viewer MeV for statistical analysis. The statistically significant genes were determined using MeV by applying a one-way ANOVA analysis with standard Bonferroni correction with a FDR <0.05 that resulted in a list of significant genes. Average gene expression signals <10 were filtered out due to signal being

Age plays a crucial role in the interplay between tumor and host; with further perturbations induced by irradiation. Proton irradiation on tumors induces biological modulations including inhibition of angiogenic and immune factors critical to hallmark processes impacting tumor development in addition to physical targeting advantages. These advantages have provided promising results for proton therapy in cancer. Additionally protons have implications for carcinogenesis risk of space travel (due to the high proportion of high energy protons in space radiation). Through a systems biology approach we investigated how host tissue (i.e. splenic tissue) of tumor-bearing mice is altered with age with or without whole-body proton exposure. Transcriptome analysis was performed on splenic tissue from adolescent (68 day) versus old (736 day) C57BL/6 male mice injected with Lewis lung carcinoma cells with or without three fractionations of 0.5Gy (1GeV) proton irradiation. Global transcriptome analysis indicated that proton irradiation of adolescent hosts caused significant signaling changes within splenic tissues that support carcinogenesis within the mice as compared to old subjects. Increases in cell cycling and immunosuppression in irradiated adolescent hosts with CDK2 MCM7 CD74 and RUVBL2 as the key players were involved in the regulatory changes in host environment response (i.e. spleen). These results suggest a significant biological component to proton irradiation operative through host age that would indicate a modulation of host s ability to support carcinogenesis in adolescence and the bestowal of resistance to immunosuppression carcinogenesis and genetic perturbation by old age.

Age plays a crucial role in the interplay between tumor and host; with further perturbations induced by irradiation. Proton irradiation on tumors induces biological modulations including inhibition of angiogenic and immune factors critical to hallmark processes impacting tumor development in addition to physical targeting advantages. These advantages have provided promising results for proton therapy in cancer. Additionally protons have implications for carcinogenesis risk of space travel (due to the high proportion of high energy protons in space radiation). Through a systems biology approach we investigated how host tissue (i.e. splenic tissue) of tumor-bearing mice is altered with age with or without whole-body proton exposure. Transcriptome analysis was performed on splenic tissue from adolescent (68 day) versus old (736 day) C57BL/6 male mice injected with Lewis lung carcinoma cells with or without three fractionations of 0.5Gy (1GeV) proton irradiation. Global transcriptome analysis indicated that proton irradiation of adolescent hosts caused significant signaling changes within splenic tissues that support carcinogenesis within the mice as compared to old subjects. Increases in cell cycling and immunosuppression in irradiated adolescent hosts with CDK2 MCM7 CD74 and RUVBL2 as the key players were involved in the regulatory changes in host environment response (i.e. spleen). These results suggest a significant biological component to proton irradiation operative through host age that would indicate a modulation of host s ability to support carcinogenesis in adolescence and the bestowal of resistance to immunosuppression carcinogenesis and genetic perturbation by old age.

This is a genome-wide approach to identifying genes persistently induced in the mouse mammary gland by acute whole body low dose ionizing radiation (10cGy) 1 and 4 weeks after exposure. Gene expression that is modified under these parameters were compared between Tgfb1 wild type and heterozygote littermates in order to determine which genes induced or repressed by radiation were mediated via Tgfb1 status. Differential gene expression was analyzed in Tgfb1 heterozygote and wild type littermate 4th mammary glands after whole body exposure to an acute dose of 10cGy ionizing radiation. Estrus cycle was normalized in all mice two days prior to irradiation by injection with an estrogen and progesterone mixture. It is widely believed that the carcinogenic action of ionizing radiation is due to targeted DNA damage and resulting mutations but there is also substantial evidence that non-targeted radiation effects alter epithelial phenotype and the stromal microenvironment. Activation of transforming growth factor beta 1 (TGFbeta) is a non-targeted radiation effect that mediates cell fate decisions following DNA damage and regulates microenvironment composition; it could either suppress or promote cancer. Gene expression profiling shown herein demonstrates that low dose radiation (10 cGy) elicits persistent changes in Tgfb1 wild type and heterozygote murine mammary gland that are highly modulated by TGFbeta. We asked if such non-targeted radiation effects contribute to carcinogenesis by using a novel radiation chimera model. Unirradiated Trp53 null mammary epithelium was transplanted to the mammary stroma of mice previously exposed to a single low (10 -100 cGy) radiation dose. By 300 days 100% of transplants in irradiated hosts at either 10 or 100 cGy had developed Trp53 null breast carcinomas compared to 54% in unirradiated hosts. Tumor growth rate was also increased by high but not low dose host irradiation. In contrast irradiation of Tgfb1 heterozygote mice prior to transplantation failed to decrease tumor latency or increase growth rate at any dose. Host irradiation significantly reduced the latency of invasive ductal carcinoma compared to spindle cell carcinoma as well as those tumors negative for smooth muscle actin in wild type but not Tgfb1 heterozygote mice. However irradiation of either host genotype significantly increased the frequency of estrogen receptor negative tumors. These data demonstrate two concepts critical to understanding radiation risks. First non-targeted radiation effects can significantly promote the frequency and alter the features of epithelial cancer. Second radiation-induced TGFbeta activity is a key mechanism of tumor promotion. Keywords: Differential gene expression after low dose irradiation Two genotypes: TGBbeta1 heterozygote and wildtype mouse mammary glands. Two time points post-10cGy-irradiation per genotype (1 week 4 weeks); control time point was 1 week post-sham-irradiation. Two or three replicates per time point.