Reoxygenation has a significant impact on the tumor response to radiotherapy. With developments in radiotherapy technology, the relevance of the reoxygenation phenomenon in treatment efficacy has ...been a topic of interest. Evaluating the reoxygenation in the tumor microenvironment throughout the course of radiation therapy is important in developing effective treatment strategies. In the current study, we used electron paramagnetic resonance imaging (EPRI) to directly map and quantify the partial oxygen pressure (pO2) in tumor tissues. Human colorectal cancer cell lines, HT29 and HCT116, were used to induce tumor growth in female athymic nude mice. Tumors were irradiated with 3, 10, or 20 Gy using an x‐ray irradiator. Prior to each EPRI scan, magnetic resonance imaging (MRI) was performed to obtain T2‐weighted anatomical images for reference. The differences in the mean pO2 were determined through two‐tailed Student's t‐test and one‐way analysis of variance. The median pO2 60 min after irradiation was found to be lower in HCT116 than in HT29 (9.1 ± 1.5 vs. 14.0 ± 1.0 mmHg, p = 0.045). There was a tendency for delayed and incomplete recovery of pO2 in the HT29 tumor when a higher dose of irradiation (10 and 20 Gy) was applied. Moreover, there was a dose‐dependent increase in the hypoxic areas (pO2 < 10 mmHg) 2 and 24 h after irradiation in all groups. In addition, an area that showed pO2 fluctuation between hypoxia and normoxia (pO2 > 10 mmHg) was also identified surrounding the region with stable hypoxia, and it slightly enlarged after recovery from acute hypoxia. In conclusion, we demonstrated the reoxygenation phenomenon in an in vivo xenograft model study using EPRI. These findings may lead to new knowledge regarding the reoxygenation process and possibilities of a new radiation therapy concept, namely, reoxygenation‐based radiation therapy.
Electron paramagnetic resonance imaging (EPRI) maps the partial oxygen pressure (pO2) in tumor tissues. The median pO2 drops after irradiation in a dose‐dependent manner with a tendency for delayed and incomplete recovery (left panel). An area that shows pO2 fluctuation between hypoxia and normoxia—cycling hypoxia—is also depicted surrounding the region with stable hypoxia, and it slightly enlarges after recovery from acute hypoxia (right panel).
: Hypoxic zones in solid tumors contribute to radioresistance, and pharmacologic agents that increase tumor oxygenation prior to radiation, including antiangiogenic drugs, can enhance treatment ...response to radiotherapy. Although such strategies have been applied, imaging assessments of tumor oxygenation to identify an optimum time window for radiotherapy have not been fully explored. In this study, we investigated the effects of α-sulfoquinovosylacyl-1,3-propanediol (SQAP or CG-0321; a synthetic derivative of an antiangiogenic agent) on the tumor microenvironment in terms of oxygen partial pressure (pO
), oxyhemoglobin saturation (sO
), blood perfusion, and microvessel density using electron paramagnetic resonance imaging, photoacoustic imaging, dynamic contrast-enhanced MRI with Gd-DTPA injection, and T2*-weighted imaging with ultrasmall superparamagnetic iron oxide (USPIO) contrast. SCCVII and A549 tumors were grown by injecting tumor cells into the hind legs of mice. Five days of daily radiation (2 Gy) combined with intravenous injection of SQAP (2 mg/kg) 30 minutes prior to irradiation significantly delayed growth of tumor xenografts. Three days of daily treatment improved tumor oxygenation and decreased tumor microvascular density on T2*-weighted images with USPIO, suggesting vascular normalization. Acute effects of SQAP on tumor oxygenation were examined by pO
, sO
, and Gd-DTPA contrast-enhanced imaging. SQAP treatment improved perfusion and tumor pO
(ΔpO
: 3.1 ± 1.0 mmHg) and was accompanied by decreased sO
(20%-30% decrease) in SCCVII implants 20-30 minutes after SQAP administration. These results provide evidence that SQAP transiently enhanced tumor oxygenation by facilitating oxygen dissociation from oxyhemoglobin and improving tumor perfusion. Therefore, SQAP-mediated sensitization to radiation
can be attributed to increased tumor oxygenation. SIGNIFICANCE: A multimodal molecular imaging study evaluates pharmacological alteration of the tumor microenvironment to improve radiation response.
In vivo deuterated water (2H2O) labeling leads to deuterium (2H) incorporation into biomolecules of proliferating cells and provides the basis for its use in cell kinetics research. We hypothesized ...that rapidly proliferating cancer cells would become preferentially labeled with 2H and, therefore, could be visualized by deuterium magnetic resonance imaging (dMRI) following a brief period of in vivo systemic 2H2O administration. We initiated systemic 2H2O administration in two xenograft mouse models harboring either human colorectal, HT-29, or pancreatic, MiaPaCa-2, tumors and 2H2O level of ~ 8% in total body water (TBW). Three schemas of 2H2O administration were tested: (1) starting at tumor seeding and continuing for 7 days of in vivo growth with imaging on day 7, (2) starting at tumor seeding and continuing for 14 days of in vivo growth with imaging on day 14, and (3) initiation of labeling following a week of in vivo tumor growth and continuing until imaging was performed on day 14. Deuterium chemical shift imaging of the tumor bearing limb and contralateral control was performed on either day 7 of 14 after tumor seeding, as described. After 14 days of in vivo tumor growth and 7 days of systemic labeling with 2H2O, a clear deuterium contrast was demonstrated between the xenografts and normal tissue. Labeling in the second week after tumor implantation afforded the highest contrast between neoplastic and healthy tissue in both models. Systemic labeling with 2H2O can be used to create imaging contrast between tumor and healthy issue, providing a non-radioactive method for in vivo cancer imaging.
In addition to nitric oxide and carbon monoxide, hydrogen sulfide (H(2)S) is an endogenously synthesized gaseous molecule that acts as an important signaling molecule in the living body. ...Transcription factor hypoxia-inducible factor 1 (HIF-1) is known to respond to intracellular reduced oxygen (O(2)) availability, which is regulated by an elaborate balance between O(2) supply and demand. However, the effect of H(2)S on HIF-1 activity under hypoxic conditions is largely unknown in mammalian cells. In this study, we tried to elucidate the effect of H(2)S on hypoxia-induced HIF-1 activation adopting cultured cells and mice.
The H(2)S donors sodium hydrosulfide and sodium sulfide in pharmacological concentrations reversibly reduced cellular O(2) consumption and inhibited hypoxia- but not anoxia-induced HIF-1α protein accumulation and expression of genes downstream of HIF-1 in established cell lines. H(2)S did not affect HIF-1 activation induced by the HIF-α hydroxylases inhibitors desferrioxamine or CoCl(2). Experimental evidence adopting von Hippel-Lindau (VHL)- or mitochondria-deficient cells indicated that H(2)S did not affect neosynthesis of HIF-1α protein but destabilized HIF-1α in a VHL- and mitochondria-dependent manner. We also demonstrate that exogenously administered H(2)S inhibited HIF-1-dependent gene expression in mice.
For the first time, we show that H(2)S modulates intracellular O(2) homeostasis and regulates activation of HIF-1 and the subsequent gene expression induced by hypoxia by using an in vitro system with established cell lines and an in vivo system in mice.
We demonstrate that H(2)S inhibits hypoxia-induced HIF-1 activation in a VHL- and mitochondria-dependent manner.
Near-infrared photoimmunotherapy (NIR PIT) employs the photoabsorbing dye IR700 conjugated to antibodies specific for cell surface epidermal growth factor receptor (EGFR). NIR PIT has shown highly ...selective cytotoxicity in vitro and in vivo. Cell necrosis is thought to be the main mode of cytotoxicity based mainly on in vitro studies. To better understand the acute effects of NIR PIT, molecular imaging studies were performed to assess its cellular and vascular effects.
In addition to in vitro studies for cytotoxicity of NIR PIT, the in vivo tumoricidal effects and hemodynamic changes induced by NIR PIT were evaluated by 13C MRI using hyperpolarized 1,4-13C2 fumarate, R2* mapping from T2*-weighted MRI, and photoacoustic imaging. In vitro studies confirmed that NIR PIT resulted in rapid cell death via membrane damage, with evidence for rapid cell expansion followed by membrane rupture. Following NIR PIT, metabolic MRI using hyperpolarized fumarate showed the production of malate in EGFR-expressing A431 tumor xenografts, providing direct evidence for photosensitized tumor necrosis induced by NIR PIT. R2* mapping studies showed temporal changes in oxygenation, with an accompanying increase of deoxyhemoglobin at the start of light exposure followed by a sustained decrease after cessation of light exposure. This result suggests a rapid decrease of blood flow in EGFR-expressing A431 tumor xenografts, which is supported by the results of the photoacoustic imaging experiments. Our findings suggest NIR PIT mediates necrosis and hemodynamic changes in tumors by photosensitized oxidation pathways and that these imaging modalities, once translated, may be useful in monitoring clinical treatment response.
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•The mechanism of photoimmunotherapy relies on photo-oxidation.•Acute necrotic damage by photoimmunotherapy induces decreased blood flow.•13C MR spectroscopy with 1,4-13C fumarate is useful for an early detection of therapeutic effect of photoimmunotherapy.•Serum fumarase can serve as a treatment response biomarker for photoimmunotherapy.•Photoacoustic imaging provides information of real-time hemodynamic change during photoimmunotherapy.
Dynamic nuclear polarization (DNP) of 13C‐labeled substrates enables the use of magnetic resonance imaging (MRI) to monitor specific enzymatic reactions in tumors and offers an opportunity to ...investigate these differences. In this study, DNP‐MRI chemical shift imaging with hyperpolarized 1‐13C pyruvate was conducted to evaluate the metabolic change in glycolytic profiles after radiation of two glioma stem‐like cell‐derived gliomas (GBMJ1 and NSC11) and an adherent human glioblastoma cell line (U251) in an orthotopic xenograft mouse model. The DNP‐MRI showed an increase in Lac/Pyr at 6 and 16 h after irradiation (18% ± 4% and 14% ± 3%, respectively; mean ± SEM) compared with unirradiated controls in GBMJ1 tumors, whereas no significant change was observed in U251 and NSC11 tumors. Metabolomic analysis likewise showed a significant increase in lactate in GBMJ1 tumors at 16 h. An immunoblot assay showed upregulation of lactate dehydrogenase‐A expression in GBMJ1 following radiation exposure, consistent with DNP‐MRI and metabolomic analysis. In conclusion, our preclinical study demonstrates that the DNP‐MRI technique has the potential to be a powerful diagnostic method with which to evaluate GBM tumor metabolism before and after radiation in the clinical setting.
Orthotopic brain tumor mice were subjected to 13C‐MRI using hyperpolarized 1‐13C pyruvate to investigate the effect of radiation exposure on glucose metabolism by measuring lactate‐to‐pyruvate ratio in the tumor. Tissue sampling was conducted independently to quantify the lactate concentration and LDHA expression by metabolomic and immunoblot analyses, respectively. These indicated an acceleration of lactate‐to‐pyruvate conversion in one of the stem‐like cell‐derived gliomas after an irradiation during a 24‐h period of time.
Purpose
Spin‐lattice relaxation rate (R1)‐based time‐domain EPR oximetry is reported for in vivo applications using a paramagnetic probe, a trityl‐based Oxo71.
Methods
The R1 dependence of the trityl ...probe Oxo71 on partial oxygen pressure (pO2) was assessed using single‐point imaging mode of spatial encoding combined with rapid repetition, similar to T1‐weighted MRI, for which R1 was determined from 22 repetition times ranging from 2.1 to 40.0 μs at 300 MHz. The pO2 maps of a phantom with 3 tubes containing 2 mM Oxo71 solutions equilibrated at 0%, 2%, and 5% oxygen were determined by R1 and apparent spin–spin relaxation rate (
R2*) simultaneously.
Results
The pO2 maps derived from R1 and
R2* agreed with the known pO2 levels in the tubes of Oxo71. However, the histograms of pO2 revealed that R1 offers better pO2 resolution than
R2* in low pO2 regions. The SDs of pixels at 2% pO2 (15.2 mmHg) were about 5 times lower in R1‐based estimation than
R2*‐based estimation (mean ± SD: 13.9 ± 1.77 mmHg and 18.3 ± 8.70 mmHg, respectively). The in vivo pO2 map obtained from R1‐based assessment displayed a homogeneous profile in low pO2 regions in tumor xenografts, consistent with previous reports on
R2*‐based oximetric imaging. The scan time to obtain the R1 map can be significantly reduced using 3 repetition times ranging from 4.0 to 12.0 μs.
Conclusion
Using the single‐point imaging modality, R1‐based oximetry imaging with useful spatial and oxygen resolutions for small animals was demonstrated.
Imaging tumor microenvironments such as hypoxia, oxygenation, redox status, and/or glycolytic metabolism in tissues/cells is useful for diagnostic and prognostic purposes. New imaging modalities are ...under development for imaging various aspects of tumor microenvironments. Electron Paramagnetic Resonance Imaging (EPRI) though similar to NMR/MRI is unique in its ability to provide quantitative images of pO
in vivo. The short electron spin relaxation times have been posing formidable challenge to the technology development for clinical application. With the availability of the narrow line width trityl compounds, pulsed EPR imaging techniques were developed for pO
imaging. EPRI visualizes the exogenously administered spin probes/contrast agents and hence lacks the complementary morphological information. Dynamic nuclear polarization (DNP), a phenomenon that transfers the high electron spin polarization to the surrounding nuclear spins (
H and
C) opened new capabilities in molecular imaging. DNP of
C nuclei is utilized in metabolic imaging of 13C-labeled compounds by imaging specific enzyme kinetics. In this article, imaging strategies mapping physiologic and metabolic aspects in vivo are reviewed within the framework of their application in cancer research, highlighting the potential and challenges of each of them.
Abstract
Drastic sensitivity enhancement of dynamic nuclear polarization is becoming an increasingly critical methodology to monitor
real-time
metabolic and physiological information in chemistry, ...biochemistry, and biomedicine. However, the limited number of available hyperpolarized
13
C probes, which can effectively interrogate crucial metabolic activities, remains one of the major bottlenecks in this growing field. Here, we demonstrate 1-
13
C
N
-acetyl cysteine (NAC) as a novel probe for hyperpolarized
13
C MRI to monitor glutathione redox chemistry, which plays a central part of metabolic chemistry and strongly influences various therapies. NAC forms a disulfide bond in the presence of reduced glutathione, which generates a spectroscopically detectable product that is separated from the main peak by a 1.5 ppm shift. In vivo hyperpolarized MRI in mice revealed that NAC was broadly distributed throughout the body including the brain. Its biochemical transformation in two human pancreatic tumor cells in vitro and as xenografts differed depending on the individual cellular biochemical profile and microenvironment in vivo. Hyperpolarized NAC can be a promising non-invasive biomarker to monitor in vivo redox status and can be potentially translatable to clinical diagnosis.
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