S.A. Krueger

Role of Apoptosis in Low-Dose Hyper-radiosensitivity

Abstract Krueger, S. A., Joiner, M. C., Weinfeld, M., Piasentin, E. and Marples, B. Role of Apoptosis in Low-Dose Hyper-radiosensitivity. Radiat. Res. 167, 260–267 (2007). Little is known about the mode of cell killing associated with low-dose hyper-radiosensitivity, the radiation response that describes the enhanced sensitivity of cells to small doses of ionizing radiation. Using a technique that measures the activation of caspase 3, we have established a relationship between apoptosis detected 24 h after low-dose radiation exposure and low-dose hyper-radiosensitivity in four mammalian cell lines (T98G, U373, MR4 and 3.7 cells) and two normal human lymphoblastoid cell lines. The existence of low-dose hyper-radiosensitivity in clonogenic survival experiments was found to be associated with an elevated level of apoptosis after low-dose exposures, corroborating earlier observations (Enns et al., Mol. Cancer Res. 2, 557–566, 2004). We also show that enriching populations of MR4 and V79 cells with G1-phase cells, to minimize the numbers of G2-phase cells, abolished the enhanced low-dose apoptosis. These cell-cycle enrichment experiments strengthen the reported association between low-dose hyper-sensitivity and the radioresponse of G2-phase cells. These data are consistent with our current hypothesis to explain low-dose hyper-radiosensitivity, namely that the enhanced sensitivity of cells to low doses of ionizing radiation reflects the failure of ATM-dependent repair processes to fully arrest the progression of damaged G2-phase cells harboring unrepaired DNA breaks entering mitosis.

Pulsed low-dose irradiation of orthotopic glioblastoma multiforme (GBM) in a pre-clinical model: Effects on vascularization and tumor control

BACKGROUND AND PURPOSE To compare dose-escalated pulsed low-dose radiation therapy (PLRT) and standard radiation therapy (SRT). METHODS AND MATERIALS Intracranial U87MG GBM tumors were established in nude mice. Animals received whole brain irradiation with daily 2-Gy fractions given continuously (SRT) or in ten 0.2-Gy pulses separated by 3-min intervals (PLRT). Tumor response was evaluated using weekly CT and [(18)F]-FDG-PET scans. Brain tissue was subjected to immunohistochemistry and cytokine bead array to assess tumor and normal tissue effects. RESULTS Median survival for untreated animals was 18 (SE±0.5) days. A significant difference in median survival was seen between SRT (29±1.8days) and PLRT (34.2±1.9days). Compared to SRT, PLRT resulted in a 31% (p<0.01), 38% (p<0.01), and 53% (p=0.01) reduction in normalized tumor volume and a 48% (p<0.01), 51% (p<0.01), and 70% (p<0.01) reduction in tumor growth rate following the administration of 10Gy, 20Gy, and 30Gy, respectively. Compared to untreated tumors, PLRT resulted in similar tumor vascular density, while SRT produced a 40% reduction in tumor vascular density (p=0.05). Compared to SRT, PLRT was associated with a 28% reduction in degenerating neurons in the surrounding brain parenchyma (p=0.05). CONCLUSIONS Compared to SRT, PLRT resulted in greater inhibition of tumor growth and improved survival, which may be attributable to preservation of vascular density.

Pulsed Versus Conventional Radiation Therapy in Combination With Temozolomide in a Murine Orthotopic Model of Glioblastoma Multiforme

PURPOSE To evaluate the efficacy of pulsed low-dose radiation therapy (PLRT) combined with temozolomide (TMZ) as a novel treatment approach for radioresistant glioblastoma multiforme (GBM) in a murine model. METHODS AND MATERIALS Orthotopic U87MG hGBM tumors were established in Nu-Foxn1(nu) mice and imaged weekly using a small-animal micropositron emission tomography (PET)/computed tomography (CT) system. Tumor volume was determined from contrast-enhanced microCT images and tumor metabolic activity (SUVmax) from the F18-FDG microPET scan. Tumors were irradiated 7 to 10 days after implantation with a total dose of 14 Gy in 7 consecutive days. The daily treatment was given as a single continuous 2-Gy dose (RT) or 10 pulses of 0.2 Gy using an interpulse interval of 3 minutes (PLRT). TMZ (10 mg/kg) was given daily by oral gavage 1 hour before RT. Tumor vascularity and normal brain damage were assessed by immunohistochemistry. RESULTS Radiation therapy with TMZ resulted in a significant 3- to 4-week tumor growth delay compared with controls, with PLRT+TMZ the most effective. PLRT+TMZ resulted in a larger decline in SUVmax than RT+TMZ. Significant differences in survival were evident. Treatment after PLRT+TMZ was associated with increased vascularization compared with RT+TMZ. Significantly fewer degenerating neurons were seen in normal brain after PLRT+TMZ compared with RT+TMZ. CONCLUSIONS PLRT+TMZ produced superior tumor growth delay and less normal brain damage when compared with RT+TMZ. The differential effect of PLRT on vascularization may confirm new treatment avenues for GBM.

TMPRSS2/ERG fusion gene expression alters chemo- and radio-responsiveness in cell culture models of androgen independent prostate cancer

The androgen regulated transmembrane serine protease (TMPRSS2) and ETS transcription factor (ERG) gene fusion is a strong prognostic factor for disease recurrence following prostatectomy. Expression of TMPRSS2/ETS‐related gene (ERG) fusion gene transcripts is linked with tumor proliferation, invasion, and an aggressive phenotype. The aim of this study was to define the effect of TMPRSS2/ERG fusion gene expression on chemo‐ and radiosensitivity in prostate tumor cell lines.

Determining if low dose hyper-radiosensitivity (HRS) can be exploited to provide a therapeutic advantage: A cell line study in four glioblastoma multiforme (GBM) cell lines

Abstract Purpose: To determine if ultra-fractionation using repeated pulses of radiation (10 × 0.2 Gray [Gy]) would be more cytotoxic than continuously-delivered radiation to the same total dose (2 Gy) in four glioma cell lines. Materials and methods: Human T98G, U373, U87MG and U138MG cells were conventionally X-irradiated with 0.1–8 Gy and clonogenic survival assessed. Next, cells were treated with either a single dose of 2 Gy or 10 pulses of 0.2 Gy using a 3-min inter-pulse interval and DNA (Deoxyribonucleic acid) repair (pHistone H2A.X), G2-phase cell cycle checkpoint arrest (pHistone H3) and apoptosis (caspase-3) compared between the two regimens. A dose of 0.2 Gy was selected as this reflects the hyper- radiosensitivity (HRS)/increased radioresistance (IRR) transition point of the low-dose cell survival curve. Results: T98G, U87MG and U138MG exhibited distinct HRS responses and survival curves were well-described by the Induced Repair model. Despite the prolonged delivery time, ultra-fractionation (10 × 0.2 Gy) was equally effective as a single continuously-delivered 2 Gy dose. However, ultra-fractionation was more effective when given for five consecutive days to a total dose of 10 Gy. The increased effectiveness of ultra-fractionation could not be attributed directly to differences in DNA damage, repair processes or radiation-induced apoptosis. Conclusions: Ultra-fractionation (10 × 0.2 Gy) is an effective modality for killing glioma cell lines compared with standard 2 Gy dosing when multiple days of treatment are given.

The Effects of Pulsed Radiation Therapy on Tumor Oxygenation in 2 Murine Models of Head and Neck Squamous Cell Carcinoma

PURPOSE To evaluate the efficacy of low-dose pulsed radiation therapy (PRT) in 2 head and neck squamous cell carcinoma (HNSCC) xenografts and to investigate the mechanism of action of PRT compared with standard radiation therapy (SRT). METHODS AND MATERIALS Subcutaneous radiosensitive UT-SCC-14 and radioresistant UT-SCC-15 xenografts were established in athymic NIH III HO female mice. Tumors were irradiated with 2 Gy/day by continuous standard delivery (SRT: 2 Gy) or discontinuous low-dose pulsed delivery (PRT: 0.2 Gy × 10 with 3-min pulse interval) to total doses of 20 Gy (UT14) or 40 Gy (UT15) using a clinical 5-day on/2-day off schedule. Treatment response was assessed by changes in tumor volume, (18)F-fluorodeoxyglucose (FDG) (tumor metabolism), and (18)F-fluoromisonidazole (FMISO) (hypoxia) positron emission tomography (PET) imaging before, at midpoint, and after treatment. Tumor hypoxia using pimonidazole staining and vascular density (CD34 staining) were assessed by quantitative histopathology. RESULTS UT15 and UT14 tumors responded similarly in terms of growth delay to either SRT or PRT. When compared with UT14 tumors, UT15 tumors demonstrated significantly lower uptake of FDG at all time points after irradiation. UT14 tumors demonstrated higher levels of tumor hypoxia after SRT when compared with PRT as measured by (18)F-FMISO PET. By contrast, no differences were seen in (18)F-FMISO PET imaging between SRT and PRT for UT15 tumors. Histologic analysis of pimonidazole staining mimicked the (18)F-FMISO PET imaging data, showing an increase in hypoxia in SRT-treated UT14 tumors but not PRT-treated tumors. CONCLUSIONS Differences in (18)F-FMISO uptake for UT14 tumors after radiation therapy between PRT and SRT were measurable despite the similar tumor growth delay responses. In UT15 tumors, both SRT and PRT were equally effective at reducing tumor hypoxia to a significant level as measured by (18)F-FMISO and pimonidazole.

Hematopoietic Stem and Progenitor Cell Migration After Hypofractionated Radiation Therapy in a Murine Model

PURPOSE To characterize the recruitment of bone marrow (BM)-derived hematopoietic stem and progenitor cells (HSPCs) within tumor microenvironment after radiation therapy (RT) in a murine, heterotopic tumor model. METHODS AND MATERIALS Lewis lung carcinoma tumors were established in C57BL/6 mice and irradiated with 30 Gy given as 2 fractions over 2 days. Tumors were imaged with positron emission tomography/computed tomography (PET/CT) and measured daily with digital calipers. The HSPC and myelomonocytic cell content was assessed via immunofluorescent staining and flow cytometry. Functionality of tumor-associated HSPCs was verified in vitro using colony-forming cell assays and in vivo by rescuing lethally irradiated C57BL/6 recipients. RESULTS Irradiation significantly reduced tumor volumes and tumor regrowth rates compared with nonirradiated controls. The number of CD133(+) HSPCs present in irradiated tumors was higher than in nonirradiated tumors during all stages of regrowth. CD11b(+) counts were similar. PET/CT imaging and growth rate analysis based on standardized uptake value indicated that HSPC recruitment directly correlated to the extent of regrowth and intratumor cell activity after irradiation. The BM-derived tumor-associated HSPCs successfully formed hematopoietic colonies and engrafted irradiated mice. Finally, targeted treatment with a small animal radiation research platform demonstrated localized HSPC recruitment to defined tumor subsites exposed to radiation. CONCLUSIONS Hypofractionated irradiation resulted in a pronounced and targeted recruitment of BM-derived HSPCs, possibly as a mechanism to promote tumor regrowth. These data indicate for the first time that radiation therapy regulates HSPC content within regrowing tumors.

Preclinical Models for Translational Research Should Maintain Pace With Modern Clinical Practice

The technical evolution of radiation therapy for brain cancer has improved both the efficacy of individual radiation treatments and patient safety. The advent of computed tomography (CT)-based planning marked an important initial shift toward target-directed treatment (1). Treatment planning accuracy was further increased by fusing CT planning images with positron emission tomography (PET) and magnetic resonance imaging (MRI) scans (2, 3). Additionally, cone beam CT images acquired before each fraction were added to compensate for any deviations from the simulation CT (4). These innovations, both in target delineation and in image guidance, have translated into better tumor control and fewer treatment related toxicities (5). Future advances are likely to come from emerging pharmaceutical approaches (6) or direct targeting of biological mechanisms that drive tumor radiation resistance (7). To best facilitate the rapid translation of this new radiobiology, novel approaches need to be validated in preclinical animal models using established clinical procedures. Cell culture experiments provide basic radiobiological information, such as defining the time course of radiation damage processing and repair. However, the translational relevance of in vitro data to the clinical practice of radiation oncology is limited. Preclinical animal models offer greater potential, particularly when the conditions of clinical practice are mimicked by the use of an orthotopic tumor model or target-directed radiation therapy using a relevant treatment regimen. Innovative radiation therapy devices specifically developed for preclinical models are making this type of research approach now possible (8-10), along with noninvasive methods to define in situ tumor response such as bioluminescence (10-12), use of reporter transgenes (13), optical imaging (14), and microMR/PET/CT imaging (15-18). For example, Baumann and colleagues (10) developed a genetically modified tumor cell line for a bioimageable intracranial tumor,

Low Dose Hyper-Radiosensitivity: A Historical Perspective

This chapter discusses the biology of low-dose hyper-radiosensitivity (HRS) with reference to radiation-induced DNA damage and cellular repair processes. Particular attention is paid to the significance of G2-phase cell cycle check-points in overcoming low-dose hyper-radiosensitivity and the impact of HRS on low-dose rate radiobiology. The history of HRS from the original in vivo discovery to the most recent in vitro and clinical data are examined to present a unifying hypothesis concerning the molecular control and regulation of this important low-dose radiation response. Finally, pre-clinical and clinical data are discussed, from a molecular viewpoint, to provide theoretical approaches to exploit HRS biology for clinical gain.

OC-0048: Tumor microenvironment response and bone marrow cell migration after pulsed radiotherapy

Results: Nineteen men and 6 women, with a median age of 69 years, were included. Ten were current smokers, 14 exsmokers (stopped more than 4 weeks before surgery) and 1 non-smoker. Fourteen patients had adenocarcinoma, eleven patients had squamous cell carcinoma. When compared to distant lung tissue, gene expression of PD-L2, HGF, VEGFR2 and VEGFR3 were downregulated in tumor tissue. PD-L1 expression was also downregulated in tumor tissue, but only in active smokers. For PD-L2, HFG, VEGFR2 and VEGFR3, methylation data shows a clear hypermethylation pattern in the promoter and enhancer regions of tumor tissue, which is conform the results of the transcriptome sequencing. Qualitative results of the expression and methylation data are depicted in figure 1.