Clare Lamont

Proton Irradiation Suppresses Angiogenic Genes and Impairs Cell Invasion and Tumor Growth

The energy deposition characteristics of proton radiation have attracted considerable attention in light of its implications for carcinogenesis risk in space travel, as well for application to cancer treatment. In space, it is the principle component of the galactic cosmic radiation to which astronauts will be exposed. For treatment, an increasing number of proton facilities are being established to exploit the physical advantages of this radiation type. However, the possibility that there may also be biologically based advantages to proton exposure has not been considered in either context. We demonstrate here that high-energy proton irradiation can inhibit expression of major pro-angiogenic factors and multiple angiogenesis-associated processes, including invasion and endothelial cell proliferation, which is prominent in cancer progression. Dose-dependent suppression of angiogenic signaling was demonstrated for both cancer and nontransformed cells. Pan-genomic microarray analysis and RT-PCR revealed that post-irradiation (0.5, 1.0 and 2.0 Gy), critical pro-angiogenic signaling factors including: vascular endothelial growth factor (VEGF), interleukin 6 and 8 (IL-6, IL-8) and hypoxia-inducible factor-1 alpha (HIF-1A), were significantly downregulated. Co-culture studies demonstrated that endothelial cell proliferation and invasion were inhibited by culturing with irradiated cancer or fibroblast cells, which suggests that proton irradiation may, in addition to direct action, contribute to angiogenesis suppression through modulation of paracrine signalings from targeted cells. Addition of recombinant IL-8 or VEGF partially restored these functions in vitro, while in vivo, an attenuated tumor growth rate was demonstrated for proton-irradiated human lung cancer cells. Taken together, these findings provide novel pre-clinical evidence that proton irradiation may, in addition to its physical targeting advantages, have important biological ramifications that should be a consideration in the optimization of proton therapy.

Tumor-host signaling interaction reveals a systemic, age-dependent splenic immune influence on tumor development

The concept of age-dependent host control of cancer development raises the natural question of how these effects manifest across the host tissue/organ types with which a tumor interacts, one important component of which is the aging immune system. To investigate this, changes in the spleen, an immune nexus in the mouse, was examined for its age-dependent interactive influence on the carcinogenesis process. The model is the C57BL/6 male mice (adolescent, young adult, middle-aged, and old or 68, 143, 551 and 736 days old respectively) with and without a syngeneic murine tumor implant. Through global transcriptome analysis, immune-related functions were found to be key regulators in the spleen associated with tumor progression as a function of age with CD2, CD3ε, CCL19, and CCL5 being the key molecules involved. Surprisingly, other than CCL5, all key factors and immune-related functions were not active in spleens from non-tumor bearing old mice. Our findings of age-dependent tumor-spleen signaling interaction suggest the existence of a global role of the aging host in carcinogenesis. Suggested is a new avenue for therapeutic improvement that capitalizes on the pervasive role of host aging in dictating the course of this disease.

Proton irradiation impacts age-driven modulations of cancer progression influenced by immune system transcriptome modifications from splenic tissue

Age plays a crucial role in the interplay between tumor and host, with additional impact due to irradiation. Proton irradiation of tumors induces biological modulations including inhibition of angiogenic and immune factors critical to ‘hallmark’ processes impacting tumor development. Proton irradiation has also provided promising results for proton therapy in cancer due to targeting advantages. Additionally, protons may contribute to the carcinogenesis risk from 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 was 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 0.5 Gy (1-GeV) 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 with older subjects. Increases in cell cycling and immunosuppression in irradiated adolescent hosts with CDK2, MCM7, CD74 and RUVBL2 indicated these were the key genes involved in the regulatory changes in the host environment response (i.e. the spleen). Collectively, these results suggest that a significant biological component of proton irradiation is modulated by host age through promotion of carcinogenesis in adolescence and resistance to immunosuppression, carcinogenesis and genetic perturbation associated with advancing age.

Mathematical modeling of tumor-tumor distant interactions supports a systemic control of tumor growth

Interactions between different tumors within the same organism have major clinical implications, especially in the context of surgery and metastatic disease. Three main explanatory theories (competition, angiogenesis inhibition, and proliferation inhibition) have been proposed, but precise determinants of the phenomenon remain poorly understood. Here, we formalized these theories into mathematical models and performed biological experiments to test them with empirical data. In syngeneic mice bearing two simultaneously implanted tumors, growth of only one of the tumors was significantly suppressed (61% size reduction at day 15, P < 0.05). The competition model had to be rejected, whereas the angiogenesis inhibition and proliferation inhibition models were able to describe the data. Additional models including a theory based on distant cytotoxic log-kill effects were unable to fit the data. The proliferation inhibition model was identifiable and minimal (four parameters), and its descriptive power was validated against the data, including consistency in predictions of single tumor growth when no secondary tumor was present. This theory may also shed new light on single cancer growth insofar as it offers a biologically translatable picture of how local and global action may combine to control local tumor growth and, in particular, the role of tumor-tumor inhibition. This model offers a depiction of concomitant resistance that provides an improved theoretical basis for tumor growth control and may also find utility in therapeutic planning to avoid postsurgery metastatic acceleration. Cancer Res; 77(18); 5183-93. ©2017 AACR.

Proton irradiation augments the suppression of tumor progression observed with advanced age.

Proton radiation is touted for improved tumor targeting, over standard gamma radiation, due to the physical advantages of ion beams for radiotherapy. Recent studies from our laboratory demonstrate that in addition to these targeting advantages, proton irradiation can inhibit angiogenic and immune factors critical to “hallmark” processes that impact cancer progression, thereby modulating tumor development. Outside the therapeutic utilization of protons, high-energy protons constitute a principal component of galactic cosmic rays and thus are a consideration in carcinogenesis risk for space flight. Given that proton irradiation modulates fundamental biological processes known to decrease with aging (e.g. angiogenesis and immunogenicity), we investigated how proton irradiation impacts tumor advancement as a function of host age, a question with both therapeutic and carcinogenesis implications. Tumor lag time and growth dynamics were tracked, after injection of murine Lewis lung carcinoma (LLC) cells into syngeneic adolescent (68 day) vs. old (736 day) C57BL/6 mice with or without coincident irradiation. Tumor growth was suppressed in old compared to adolescent mice. These differences were further modulated by proton irradiation (1 GeV), with increased inhibition and a significant radiation-altered molecular fingerprint evident in tumors grown in old mice. Through global transcriptome analysis, TGFβ1 and TGFβ2 were determined to be key players that contributed to the tumor dynamics observed. These findings suggest that old hosts exhibit a reduced capacity to support tumor advancement, which can be further reduced by proton irradiation.

56Fe ion irradiation enhances angiogenesis and other inter-cellular determinants of carcinogenesis risk

In the assessment of radiogenic cancer risk from space flight, it is imperative to consider effects not only on the creation of cancer cells (initiation) but also on cell–cell interactions that play an important and often decisive role in the promotion and progression phases. Autopsy results confirm that most adults carry fully malignant tumors that are held in check at a small size and will never become symptomatic [ 1, 2]. This introduces the possibility that cosmic radiation may significantly influence cancer risk through alteration of the bottleneck inter-tissue interactions responsible for maintaining this dormant state. One such bottleneck is the growth limitation imposed by the failure of the tumor to induce blood vessels (angiogenesis). Other deciding events are the ability of a tumor to proliferate and invade. We have previously shown that proton radiation, the most prevalent radiation in space, has a suppressive effect on all three of these functional responses. It down-regulates angiogenic genes like VEGF and HIF-1α and impairs cell invasion and tumor growth [ 3]. We decided to test these responses after 56Fe irradiation, an HZE radiation type present in the cosmic environment with presumably high carcinogenic potential [ 4]. Human microvascular endothelial cells (HMVEC) and normal human dermal fibroblast (NHDF) cells were irradiated with different doses of 56Fe ion radiation (1 GeV/n) at Brookhaven National Laboratory and RNA was extracted 6 h later. Genomic-wide array analysis was done on the isolated RNA through the Agilent Platform. It was observed that several pro-angiogenic genes like VEGF, IL-6 and HIF-1α were significantly up-regulated after treatment with 56Fe ion radiation (Fig. 1). These results were also confirmed at the mRNA and protein levels with the human and murine lung cancer lines, A549 and LLC, respectively. Additional verification of modulation of these key genes was also observed when lungs of C57BL/6 mice treated with 56Fe ion radiation showed an increase in VEGF and MMP9 mRNA and protein expression 6 h post-irradiation (Fig. 2). Cell invasion was shown to be increased by 56Fe ion radiation in various cell types, including fibroblast, tumor and endothelial progenitor cells. 56Fe ion irradiation also modulated functional processes crucial to angiogenesis. It enhanced the ability of untargeted (bystander) endothelial cells to invade and proliferate in response to factors produced by targeted fibroblast or cancer cells in vitro. Results also carry over to in vivo. C57BL/6 mice exposed to whole-body irradiation with 0.2 Gy dose of 56Fe and injected subcutaneously with LLC tumor cells showed a significant augmentation in tumor growth and growth rate in the irradiated group. Additionally, nude mice exposed to whole-body 56Fe radiation and injected intravenously with A549 cancer cells 3 h post-irradiation demonstrated a significant enhancement in lung colonization capacity when compared with the sham-irradiated control mice injected. These results together suggest cell and tissue-level responses to 56Fe irradiation may act to overcome major cancer progression-level bottlenecks including those related to angiogenesis, cell proliferation and invasion. This is of significant concern for cancer risk estimations pertinent to NASA as achieving these cancer hallmark processes can make the difference between a radiation-induced cancer cell progressing to a clinically detectable cancer in astronauts or not. In conclusion, we demonstrate a strong radiation quality dependence for space radiation carcinogenesis risk manifested through influences on intercellular interactions in the progression phase of carcinogenesis. Fig. 1. Heatmaps of selected differentially regulated major angiogenesis genes after proton and 56Fe ion radiation in HMVECs and NHDF. Cells were treated with either 0, 0.5, 1 or 2 Gy of proton radiation or 0, 0.2, 0.4 or 1 Gy of 56Fe ion dose. Among the major regulated genes were VEGF, HIF-1A and IL-6; they were down-regulated by proton radiation and up-regulated by iron radiation. Fig. 2. Immunofluorescence images of lungs of C57BL/6 mice treated with 0, 0.2 or 1 Gy of 56Fe ion dose and stained 6 h later. Pro-angiogenic factors VEGF and MMP9 were increased in mice that received the 56Fe ion treatment.

Abstract 4340: Increased cytokine and chemokine expression in U87MG glioblastoma cells after large clinically relevant single doses of ionizing radiation

BACKGROUND: Stereotactic radiosurgery is an established treatment option that uses high, single fraction doses of radiation to target tumors. Despite the best clinical strategies to eliminate brain tumors using this technique or others (surgery, chemotherapy and/or other radiotherapy techniques), post-therapy recurrence of brain tumors remains a major challenge in patients. To better understand the radiation response, we investigated how such high doses modulate the molecular signaling within glioblastoma cell populations from several days to months after irradiation. METHODS: The glioblastoma cell line, U87MG, was treated with single doses of ionizing radiation between 0 to 16Gy in vitro followed by: examination of cell proliferation; clonogenic survival; extraction of RNA; and subcutaneous injection into nude mice to monitor tumor growth 24 hours after cell irradiation. Genome-wide expression profiling was run on the Illumina HT-12 bead array platform and analysis for significant expressed genes was performed along with network analysis using Ingenuity Pathway Analysis and GenePattern platforms. RESULTS: As expected, high doses of radiation stunted growth of the U87MG cells from 0 to 14 days. Long term, single cell clonogenic survival of the irradiated cells was approximately 3% and 0.01% following 8Gy or 16Gy, respectively. Genome-wide gene expression at 1, 4 and 6 days after irradiation of 8 or 16Gy has revealed a dose-dependent increase in inflammatory cytokines and chemokines. Increased cytokine expression for IL-6 and IL-8 was validated in vitro using qRT-PCR. In vivo, 100% of the untreated U87MG cells formed tumors, whereas less than half of all irradiated treatment groups failed to form tumors. In those samples that did grow despite irradiation, the average time to tumor formation (volume = 300mm 3 ) was 13, 32, 58 and 88 days for 0, 8, 12, and 16Gy respectively while the exponential fits to tumor growth rate was shown to be accelerated after 8Gy but diminished after 12 and 16Gy. IL-6 gene expression was found to be elevated in tumors formed from irradiated cells versus control. CONCLUSION: The data presented here provides novel insight into the cellular and molecular modulation of glioblastoma cell populations by large, clinically relevant, single doses of ionizing radiation. A salient finding is the expression of inflammatory cytokines as a major player in the radiation response at periods long after the irradiation insult. This study was supported by NIH ICBP 1U54CA149233 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4340. doi:1538-7445.AM2012-4340

Abstract 1319: Proton irradiation exerts antiangiogenic and other tumor progression-limiting effects

The precise targeting and energy deposition characteristics of proton theapy have attracted considerable attention in the clinical oncology community. An increasing number of proton facilities are being established to exploit the physical advantages of this radiation for cancer treatment. However, the fact that there may also be biologically-based advantages for the use of protons has essentially been overlooked. We here demonstrate that proton irradiation inhibits expression of major pro-angiogenic factors and multiple angiogenesis-associated processes, including invasion and endothelial cell proliferation, which are critical to cancer progression. Dose-dependent suppression of angiogenic signaling was demonstrated for human tumor, fibroblasts and microvascular endothelial cells. Pan-genomic microarray analysis and RT-PCR revealed that post-irradiation (0.5, 1.0 2.0 Gy) critical pro-angiogenic signaling factors, including vascular endothelial growth factor (VEGF), interleukin 6 and 8 (IL-6, IL-8) and the hypoxia-inducible factor-1 alpha (HIF-1α), were significantly downregulated. Co-culture studies demonstrated proliferation and invasion of endothelial cells was inhibited by irradiation of both tumor and fibroblast cells, suggesting that proton irradiation may contribute to angiogenesis suppression through paracrine signalings from targeted cells as well as by direct action. Addition of recombinant IL-8 or VEGF partially restored these functions, once again indicating that suppression of these factors plays a functional role in inhibition of tumor progression. Finally an attenuated growth rate and a reduced invasive capacity was demonstrated in vivo for proton-irradiated human lung cancer, A549. Taken together, these findings provide novel preclinical evidence that proton irradiation may, in addition to its physical advantages, have important biological ramifications that should be a consideration in the optimization of proton therapy. This work was supported by NASA NSCOR grant #NNJ06HA28G Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1319.