Ramipril reduces incidence and prolongates latency time of radiation-induced rat myelopathy after photon and carbon ion irradiation.

To test the hypothesis that the use of an angiotensin-converting enzyme inhibitor (ACEi) during radiotherapy may be ameliorative for treatment-related normal tissue damage, a pilot study was conducted with the clinically approved (ACE) inhibitor ramipril on the outcome of radiation-induced myelopathy in the rat cervical spinal cord model. Female Sprague Dawley rats were irradiated with single doses of either carbon ions (LET 45 keV/µm) at the center of a 6 cm spread-out Bragg peak (SOBP) or 6 MeV photons. The rats were randomly distributed into 4 experimental arms: (i) photons; (ii) photons + ramipril; (iii) carbon ions and (iv) carbon ions + ramipril. Ramipril administration (2 mg/kg/day) started directly after irradiation and was maintained during the entire follow-up. Complete dose-response curves were generated for the biological endpoint radiation-induced myelopathy (paresis grade II) within an observation time of 300 days. Administration of ramipril reduced the rate of paralysis at high dose levels for photons and for the first time a similar finding for high-LET particles was demonstrated, which indicates that the effect of ramipril is independent from radiation quality. The reduced rate of myelopathy is accompanied by a general prolongation of latency time for photons and for carbon ions. Although the already clinical approved drug ramipril can be considered as a mitigator of radiation-induced normal tissue toxicity in the central nervous system, further examinations of the underlying pathological mechanisms leading to radiation-induced myelopathy are necessary to increase and sustain its mitigative effectiveness.

Flow cytometric characterization of tumor subpopulations in three sublines of the Dunning R3327 rat prostate tumor model.

BACKGROUND: Subsets of tumor cells were characterized by mapping DNA ploidy patterns in correlation with established cell surface markers in three non-treated sublines of the Dunning R3327 prostate tumor system representing different progressional stages. METHODS: Flow cytometry was used to analyze DNA-index, cell cycle distribution as well as multiparametric aquisition of single and combined cell surface markers in single cell suspensions of frozen tumor tissues. RESULTS: The three Dunning prostate tumor sublines clearly differ in their ploidy status. In addition each tumor subline displays a characteristic cell surface marker profile, which is correlated with the cell cycle phase and the amount of genomic alterations. CONCLUSIONS: In a feasibility study we have shown that cross-reacting antibodies to human cell surface markers stain discrete tumor subpopulations in three sublines of the Dunning tumor model. Although it remains presently uncertain, which cell surface markers are most suitable for cell sorting to display cancer initiating (CIC) properties following subcutaneous or orthotopic grafting, the model may be useful for mechanistic investigations of putative stem-like tumor subpopulations and their significance in response to radio- or chemotherapy.

LY2109761 attenuates radiation-induced pulmonary murine fibrosis via reversal of TGF-ß and BMP-associated proinflammatory and proangiogenic signals.

PURPOSE: Radiotherapy is used for the treatment of lung cancer, but at the same time induces acute pneumonitis and subsequent pulmonary fibrosis, where TGF-ß signaling is considered to play an important role. EXPERIMENTAL DESIGN: We irradiated thoraces of C57BL/6 mice (single dose, 20 Gy) and administered them a novel small-molecule TGF-ß receptor I serine/threonine kinase inhibitor (LY2109761) orally for 4 weeks before, during, or after radiation. Noninvasive lung imaging including volume computed tomography (VCT) and MRI was conducted 6, 16, and 20 weeks after irradiation and was correlated to histologic findings. Expression profiling analysis and protein analysis was conducted in human primary fibroblasts. RESULTS: Radiation alone induced acute pulmonary inflammation and lung fibrosis after 16 weeks associated with reduced life span. VCT, MRI, and histology showed that LY2109761 markedly reduced inflammation and pulmonary fibrosis resulting in prolonged survival. Mechanistically, we found that LY2109761 reduced p-SMAD2 and p-SMAD1 expression, and transcriptomics revealed that LY2109761 suppressed expression of genes involved in canonical and noncanonical TGF-ß signaling and downstream signaling of bone morphogenetic proteins (BMP). LY2109761 also suppressed radiation-induced inflammatory [e.g., interleukin (IL)-6, IL-7R, IL-8] and proangiogenic genes (e.g., ID1) indicating that LY2109761 achieves its antifibrotic effect by suppressing radiation-induced proinflammatory, proangiogenic, and profibrotic signals. CONCLUSION: Small-molecule inhibitors of the TGF-ß receptor I kinase may offer a promising approach to treat or attenuate radiation-induced lung toxicity or other diseases associated with fibrosis.

Comparison of 1H blood oxygen level-dependent (BOLD) and 19F MRI to investigate tumor oxygenation.

Fluorine-19 [(19)F] MRI oximetry and (1)H blood oxygen level-dependent (BOLD) MRI were used to investigate tumor oxygenation in rat breast 13762NF carcinomas, and correlations between the techniques were examined. A range of tissue oxygen partial pressure (pO(2)) values was found in the nine tumors while the anesthetized rats breathed air, with individual tumor pO(2) ranging from a mean of 1 to 36 torr and hypoxic fraction (HF10) (<10 torr) ranging from 0% to 75%, indicating a large intra- and intertumor heterogeneity. Breathing oxygen produced significant increase in tumor pO(2) (mean DeltapO(2) = 50 torr) and decrease in HF(10) (P < 0.01). (1)H BOLD MRI observed using a spin echo-planar imaging (EPI) sequence revealed a heterogeneous response and significant increase in mean tumor signal intensity (SI) (DeltaSI = 7%, P < 0.01). R(2)* measured by multigradient-echo (MGRE) MRI decreased significantly in response to oxygen (mean DeltaR(2)* = -4 s(-1); P < 0.05). A significant correlation was found between changes in mean tumor pO(2) and mean EPI BOLD DeltaSI accompanying oxygen breathing (r(2) > 0.7, P < 0.001). Our results suggest that BOLD MRI provides information about tumor oxygenation and may be useful to predict pO(2) changes accompanying interventions. Significantly, the magnitude of the BOLD response appears to be predictive for residual tumor HFs.

Correlation of radiation response with tumor oxygenation in the Dunning prostate R3327-AT1 tumor.

PURPOSE: To investigate the application of pretreatment oxygenation to the AT1 subline of the Dunning R3327 prostate tumor, which is more hypoxic and faster growing than the H1 subline previously studied. METHODS AND MATERIALS: Dunning prostate R3327-AT1 tumors growing on Copenhagen rats were administered 30 Gy of X-ray radiation either with or without oxygen inhalation. Tumor oxygenation was sampled by (19)F nuclear magnetic resonance echo planar imaging relaxometry of the reporter molecule hexafluorobenzene, no more than 24 h before irradiation. RESULTS: Large tumors (>3.0 cm(3)) exhibited significantly greater hypoxic fractions and lower mean partial pressure of oxygen (pO(2)) than their smaller counterparts (<1.5 cm(3)). However, unlike the R3327-HI subline, large AT1 tumors generally did not respond to oxygen inhalation in terms of altered hypoxic fraction or response to irradiation. Although the tumors did not respond to oxygen inhalation, each tumor had a different pO(2), and there was a clear trend between level of oxygenation at time of irradiation and tumor growth delay, with considerably better outcome when mean pO(2) > 10 mm Hg. The comparatively small baseline hypoxic fraction in the group of small tumors was virtually eliminated by breathing oxygen, and the growth rate was significantly reduced for tumors on rats breathing oxygen during irradiation. CONCLUSIONS: These results further validate the usefulness of nuclear magnetic resonance oximetry as a predictor of response to radiation therapy.

Continuous low-dose (metronomic) chemotherapy on rat prostate tumors evaluated using MRI in vivo and comparison with histology.

Continuous low-dose (metronomic) therapy, based on cyclophosphamide (CTX) combined with thalidomide (Tha), was evaluated on Dunning prostate R3327-AT1 rat tumors. Significantly delayed tumor growth (P < .001) was observed with oral CTX alone at a low dose (metronomic cyclophosphamide or M-CTX; 30 mg/kg per day) or combined with Tha. To investigate dynamic changes in tumor physiology during early stages of treatment, magnetic resonance imaging (MRI) was applied before and during the M-CTX or M-CTX + Tha therapy. Dynamic contrast-enhanced MRI revealed significant changes in the tumor center by day 3 (P < .01); by day 7, only a thin peripheral tumor region showed high signal enhancement. There was a significant correlation between poorly enhancing fraction on day 7 and ultimate tumor growth delay (P < .02). The apparent transverse relaxation rate (R2*) showed similar baseline tumor heterogeneity, but no obvious changes with growth or therapy. Histology confirmed substantial necrosis in the tumor center, leaving a thin live peripheral rim. Immunohistochemistry showed a significant increase in vascular endothelial growth factor, and apoptotic tumor and vascular endothelial cells. These results show the efficacy of the metronomic CTX +/- Tha for delaying tumor growth and indicate that MRI provides insights into the mode of action and early indication of efficacy.

Tumor physiologic response to combretastatin A4 phosphate assessed by MRI.

PURPOSE: To evaluate the effect of the vascular targeting agent, combretastatin A4 phosphate, on tumor oxygenation compared with vascular perfusion/permeability. METHODS AND MATERIALS: (19)F MRI oximetry and dynamic contrast-enhanced (DCE)-MRI were used to monitor tumor oxygenation and perfusion/permeability in syngeneic 13762NF rat breast carcinoma. RESULTS: A significant drop was found in the mean tumor pO(2) (23 to 9 mm Hg, p <0.05) within 90 min after treatment (30 mg/kg of combretastatin A4 phosphate) and a further decrease was observed at 2 h (mean 2 mm Hg; p <0.01). The initial changes in pO(2) in the central and peripheral regions were parallel, but by 24 h after treatment, a significant difference was apparent: the pO(2) in the periphery had improved significantly, and the center remained hypoxic. These data are consistent with DCE-MRI, which revealed an approximately 70% decrease in perfusion/permeability (initial area under signal-intensity curve) at 2 h (p <0.001). The initial area under signal-intensity curve recovered fully after 24 h in a thin peripheral region, but not in the tumor center. CONCLUSION: The response observed by DCE-MRI, indicating vascular shutdown, paralleled the pO(2) measurements as expected, but quantitative pO(2) measurements are potentially important for optimizing the therapeutic combination of vascular targeting agents with radiotherapy.

Tumor oximetry: comparison of 19F MR EPI and electrodes.

We recently described a novel approach to measuring regional tumor oxygen tension. This approach is based on 19F pulse burst saturation recovery NMR echo planar imaging relaxometry of hexafluorobenzene or "FREDOM" (Fluorocarbon Relaxometry using Echo planar imaging for Dynamic Oxygen Mapping). We have now compared oxygen tension measurements using FREDOM with a traditional polarographic method (the Eppendorf Histograph) in a group of size matched Dunning prostate rat tumors R3327-AT1. We also compare MR and electrode approaches to monitoring dynamic changes with respect to interventions and demonstrate extension of the MR technique to rat breast tumors.

Tumor oxygen dynamics: correlation of in vivo MRI with histological findings.

Tumor oxygenation has long been recognized as a significant factor influencing cancer therapy. We recently established a novel magnetic resonance in vivo approach to measuring regional tumor oxygen tension, FREDOM (Fluorocarbon Relaxometry Using Echo Planar Imaging for Dynamic Oxygen Mapping), using hexafluorobenzene (HFB) as the reporter molecule. We have now investigated oxygen dynamics in the two Dunning prostate R3327 rat tumor sublines, AT1 and H. FREDOM revealed considerable intratumoral heterogeneity in the distribution of pO(2) values in both sublines. The anaplastic faster-growing AT1 tumors were more hypoxic compared with the size-matched, well-differentiated, and slower-growing H tumors. Respiratory challenge with oxygen produced significant increases in mean and median pO(2) in all the H tumors (P<.001), but no response in half of the larger AT1 tumors (>3 cm(3)). Immunohistochemical studies using the hypoxia marker, pimonidazole, and the vascular endothelial cell marker, CD31, confirmed that the H tumors had more extensive vasculature and less hypoxia than the AT1 tumors. These results further validate the utilization of FREDOM to monitor tumor oxygenation and concur with the hypothesis that the level of hypoxia is related to tumor growth rate and poor vascularity.

Correlation of tumor oxygen dynamics with radiation response of the dunning prostate R3327-HI tumor.

Our previous studies have shown that oxygen inhalation significantly reduces tumor hypoxia in the moderately well-differentiated HI subline of the Dunning prostate R3327 rat carcinoma. To test our hypothesis that modifying hypoxia could improve the radiosensitivity of these tumors, we performed experimental radiotherapy to compare the tumor response to ionizing radiation alone or in combination with oxygen inhalation. Tumor pO(2) measurements were performed on size-selected tumors several hours before radiotherapy using (19)F nuclear magnetic resonance echo planar imaging relaxometry (FREDOM) of the reporter molecule hexafluorobenzene. In common with our previous findings, the larger tumors (>3.5 cm(3)) exhibited greater hypoxia than the smaller tumors (<2 cm(3); P < 0.001), and oxygen inhalation reduced the hypoxic fraction (<10 Torr): In the larger tumors, hypoxic fraction dropped significantly from a mean baseline value of 80% to 17% (P < 0.001). The effect of oxygen administered 30 min before and during irradiation on tumor response to a single 30-Gy dose of photons was evaluated by growth delay. For the smaller tumors, no difference in growth delay was found when treatment was given with or without oxygen breathing. By contrast, breathing oxygen before and during irradiation significantly enhanced the growth delay in the larger tumors (additional 51 days). The differential behavior may be attributed to the low baseline hypoxic fraction (<10 Torr) in small tumors (20%) as a target for oxygen inhalation. There was a strong correlation between the estimated initial pO(2) value and the radiation-induced tumor growth delay (R > 0.8). Our histological studies showed a good match between the perfused vessels marked by Hoechst 33342 dye and the total vessels immunostained by anti-CD31 and indicated extensive perfusion in this tumor line. In summary, the present results suggest that the ability to detect modulation of tumor pO(2), in particular, the residual hypoxic fraction, with respect to an intervention, could have prognostic value for predicting the efficacy of radiotherapy.

Differential oxygen dynamics in two diverse Dunning prostate R3327 rat tumor sublines (MAT-Lu and HI) with respect to growth and respiratory challenge.

PURPOSE: Since hypoxia may influence tumor response to therapy and prognosis, we have compared oxygenation of tumors known to exhibit differential growth rate and tissue differentiation. METHODS AND MATERIALS: Regional tumor oxygen tension was measured using 19F nuclear magnetic resonance echo planar imaging relaxometry of hexafluorobenzene, which provided dynamic maps with respect to respiratory intervention. Investigations used two Dunning prostate R3327 rat tumor sublines: the fast growing, highly metastatic MAT-Lu and the moderately well-differentiated, slower growing HI. RESULTS: Both sublines showed significantly higher oxygen tension in smaller tumors (<2 cm(3)) than in larger tumors (>3.5 cm(3)). Pooled data showed that MAT-Lu tumors exhibited greater hypoxia compared with the size-matched HI tumors (p < 0.0001). Respiratory challenge (oxygen or carbogen) produced significant increases in mean pO(2) for tumors of both sublines (p < 0.0001). However, initially hypoxic regions displayed very different behavior in each subline: those in the HI tumors responded rapidly with significant elevation in pO(2), while those in the MAT-Lu tumors showed little response to respiratory intervention. CONCLUSIONS: These results concur with hypotheses that hypoxia is related to tumor growth rate and degree of differentiation. Under baseline conditions, the differences were subtle. However, response to respiratory intervention revealed highly significant differences, which, if held valid in the clinic, could have prognostic value.