Increased PDT Efficacy When Associated with Nitroglycerin: A Study on Retinoblastoma Xenografted on Mice.

PURPOSES: The aim of the study was to assess the efficacy of a treatment protocol that combines photodynamic therapy (PDT) and nitroglycerin (NG) on human retinoblastoma tumors xenografted on mice. We aimed to increase the PDT efficiency (in our least treatment-responsive retinoblastoma line) with better PS delivery to the tumor generated by NG, which is known to dilate vessels and enhance the permeability and retention of macromolecules in solid tumors. METHODS: In vivo follow-up of the therapeutic effects was performed by sodium MRI, which directly monitors variations in sodium concentrations non-invasively and can be used to track the tumor response to therapy. NG ointment was applied one hour before PDT. The PDT protocol involves double-tumor targeting, i.e., cellular and vascular. The first PS dose was injected followed by a second one, separated by a 3 h interval. The timelapse allowed the PS molecules to penetrate tumor cells. Ten minutes after the second dose, the PS was red-light-activated. RESULTS: In this study, we observed that the PDT effect was enhanced by applying nitroglycerin ointment to the tumor-bearing animal's skin. PDT initiates the bystander effect on retinoblastomas, and NG increases this effect by increasing the intratumoral concentration of PS, which induces a higher production of ROS in the illuminated region and thus increases the propagation of the cell death signal deeper into the tumor (bystander effect).

AsiDNA Is a Radiosensitizer with no Added Toxicity in Medulloblastoma Pediatric Models.

PURPOSE: Medulloblastoma is an important cause of mortality and morbidity in pediatric oncology. Here, we investigated whether the DNA repair inhibitor, AsiDNA, could help address a significant unmet clinical need in medulloblastoma care, by improving radiotherapy efficacy without increasing radiation-associated toxicity. EXPERIMENTAL DESIGN: To evaluate the brain permeability of AsiDNA upon systemic delivery, we intraperitoneally injected a fluorescence form of AsiDNA in models harboring brain tumors and in models still in development. Studies evaluated toxicity associated with combination of AsiDNA with radiation in the treatment of young developing animals at subacute levels, related to growth and development, and at chronic levels, related to brain organization and cognitive skills. Efficacy of the combination of AsiDNA with radiation was tested in two different preclinical xenografted models of high-risk medulloblastoma and in a panel of medulloblastoma cell lines from different molecular subgroups and TP53 status. Role of TP53 on the AsiDNA-mediated radiosensitization was analyzed by RNA-sequencing, DNA repair recruitment, and cell death assays. RESULTS: Capable of penetrating young brain tissues, AsiDNA showed no added toxicity to radiation. Combination of AsiDNA with radiotherapy improved the survival of animal models more efficiently than increasing radiation doses. Medulloblastoma radiosensitization by AsiDNA was not restricted to a specific molecular group or status of TP53. Molecular mechanisms of AsiDNA, previously observed in adult malignancies, were conserved in pediatric models and resembled dose increase when combined with irradiation. CONCLUSIONS: Our results suggest that AsiDNA is an attractive candidate to improve radiotherapy in medulloblastoma, with no indication of additional toxicity in developing brain tissues.

Cellular density, a major factor involved in PDT cytotoxic responses: Study on three different lines of human retinoblastoma grafted on nude mice.

BACKGROUND: PDT represents a very localized and non-mutagen antitumoral treatment using a photosensitive molecule (porphyrin family) light activated. The first way of cell damage is a direct one, active on the very site where ROSs have been produced. The second one is indirect by activating and transmitting the processes of cellular death signaling. In order to seek for a better characterization of the photo-biology involved in in vivo PDT and to better understand the differences on the treatment outcome, we have used three different human retinoblastomas xenografted on mice. METHODS: Mice were treated according to the double targeting protocol exposed in a previous paper. One i.v. dose (0.6 mg/kg) of PS was followed by a second dose, separated by a 3 h interval (double targeting PDT). As a consequence both cancer cells and blood vessels were targeted. The treatment was repeated two times, at 4 days interval. RESULTS: First of all, sodium MRI revealed qualitative differences in the sodium average content of the three retinoblastoma lines before treatment. After the PDT treatments the tumor responses were different between the lines as revealed by sodium MRI and later on by histology. CONCLUSIONS: We have put into evidence that PDT is accompanied by a bystander effect that may propagate the cellular death triggered by the initial photoreaction. This effect is highly dependent on the cellular density of the tissue; therefore this factor is to be taken into account in clinical PDT protocols.

In vivo magnetic resonance microscopy of Drosophilae at 9.4 T.

In preclinical research, genetic studies have made considerable progress as a result of the development of transgenic animal models of human diseases. Consequently, there is now a need for higher resolution MRI to provide finer details for studies of small animals (rats, mice) or very small animals (insects). One way to address this issue is to work with high-magnetic-field spectrometers (dedicated to small animal imaging) with strong magnetic field gradients. It is also necessary to develop a complete methodology (transmit/receive coil, pulse sequence, fixing system, air supply, anesthesia capabilities, etc.). In this study, we developed noninvasive protocols, both in vitro and in vivo (from coil construction to image generation), for drosophila MRI at 9.4 T. The 10 10 80-µm resolution makes it possible to visualize whole drosophila (head, thorax, abdomen) and internal organs (ovaries, longitudinal and transverse muscles, bowel, proboscis, antennae and optical lobes). We also provide some results obtained with a Drosophila model of muscle degeneration. This opens the way for new applications of structural genetic modification studies using MRI of drosophila.

Radial multigradient-echo DCE-MRI for 3D K(trans) mapping with individual arterial input function measurement in mouse tumor models.

The purpose of this study was to provide proof of concept for a new three-dimensional (3D) radial dynamic contrast enhanced MRI acquisition technique, called "Radial Entire Tumor with Individual Arterial input function dynamic contrast-enhanced MRI" (RETIA dynamic contrast-enhanced MRI), which allows for the simultaneous measurement of an arterial input function in the mouse heart at 2 s temporal resolution and coverage of the whole tumor. Alternating 2D and 3D projections contribute to the 2D heart image or 3D tumor data with a 3-cm field of view. Sixty-four 2D images of the heart are obtained during acquisition of each 3D tumor dataset. In a pilot study, global K(trans) and ve values were measured in four mice, in a respiratory motion-animated subcutaneously implanted breast tumor model. This technique is expected to be most useful for the characterization of microvasculature in motion-animated orthotopic tumors.

PDT induced bystander effect on human xenografted colorectal tumors as evidenced by sodium MRI.

BACKGROUND: Previous in vivo studies on photodynamic therapy (PDT)-treated, high cellular density tumors showed evidences of a bystander effect accompanying the therapy, cellular death continuing beyond the limits of the photochemical reactions in time and space. This process is generated by the initially damaged cells on the light pathway. The aim of this study was to determine if the bystander effect may be induced as well in colorectal xenografted tumors (less compact structure) and if the cellular signaling depends primarily on cellular proximity or not. METHODS: The photosensitizer was a glycoconjugated, meso substituted porphyrin derivative synthesized at Institut Curie. The longitudinal follow-up of the tumors was carried out by (23)Na/(1)H MRI, ideal imaging modality for mapping the extracellular compartment. Two regimens were followed in order to target either blood vessels alone or blood vessels and cancer cells simultaneously. RESULTS: The antivascular PDT did not succeed to arrest the tumors growth at the end of the follow-up. For double targeting PDT, we managed to stop the tumoral evolution. Sodium MRI evidenced a bystander effect. CONCLUSION: The results obtained showed that the bystander effect is more difficult to induce for the type of colorectal tumors used in this work. It needs a double treatment, 4 days apart, in order to be promoted.

2D and 3D radial multi-gradient-echo DCE MRI in murine tumor models with dynamic R*2-corrected R1 mapping.

Dynamic contrast-enhanced MRI is extensively studied to define and evaluate biomarkers for early assessment of vasculature-targeting therapies. In this study, two-dimensional and three-dimensional radial multi-gradient-echo techniques for dynamic R*(2)-corrected R(1) mapping based on the spoiled gradient recalled signal equation were implemented and validated at 4.7 T. The techniques were evaluated on phantoms and on a respiratory motion animated tumor model. R(1) measurements were validated with respect to a standard inversion-recovery spin-echo sequence in a four-compartment phantom covering a range of relaxation rates typically found in tumor tissue. In the range of [0.4, 3] sec(-1), R(1) differences were less than 10% for both two-dimensional and three-dimensional experiments. A dynamic contrast-enhanced MRI pilot study was performed on a colorectal tumor model subcutaneously implanted in mice at the abdominal level. Low motion sensitivity of radial acquisition allowed image recording without respiratory triggering. Three-dimensional K(trans) maps and significantly different mean K(trans) values were obtained for two contrast agents with different molecular weights. The radial multi-gradient-echo approach should be most useful for preclinical experimental conditions where the tissue of interest experiences physiologic motion, like spontaneous extracerebral tumors developed by transgenic mice, and where dynamic contrast-enhanced MRI is performed with high-relaxivity contrast agents.

23Na MRI longitudinal follow-up of PDT in a xenograft model of human retinoblastoma.

BACKGROUND: Photodynamic therapy is an established cancer treatment in which a photosensitizing agent is activated by exposure to light thus generating cytotoxic reactive oxygen species that cause cellular damage. METHODS: A new photosensitizer synthesized at Curie Institute was used to treat retinoblastoma xenografts in mice, a glycoconjugated meso substituted porphyrin derivative, that showed some retinoblastoma cell affinity. The longitudinal follow-up of the tumors was carried out by (23)Na MRI (without adding exogenous contrast agents) to map the extracellular compartment and to characterize cell packing. Two regimens were followed to target either blood vessels alone or blood vessels and cancer cells simultaneously. RESULTS AND CONCLUSIONS: Only the protocol targeting both cancer cells and blood vessels effectively induces cellular death, confirmed by histology at the end of the experiment. Sodium MRI evidences a huge change in the cellular density of tumors only 24h after a double targeting (vascular and cellular) PDT treatment. We suggest that this change was possibly due to a bystander effect that can be promoted by the intercellular signaling favored by the high cellular density of retinoblastoma. These results indicate that non-invasive (23)Na imaging (which detects the tumor response to treatment from very early stages) in association with non-mutagenic therapies represents an effective option for tailored and individualized clinical treatments.

Simultaneous two-voxel localized (1)H-observed (13)C-edited spectroscopy for in vivo MRS on rat brain at 9.4T: Application to the investigation of excitotoxic lesions.

(13)C spectroscopy combined with the injection of (13)C-labeled substrates is a powerful method for the study of brain metabolism in vivo. Since highly localized measurements are required in a heterogeneous organ such as the brain, it is of interest to augment the sensitivity of (13)C spectroscopy by proton acquisition. Furthermore, as focal cerebral lesions are often encountered in animal models of disorders in which the two brain hemispheres are compared, we wished to develop a bi-voxel localized sequence for the simultaneous bilateral investigation of rat brain metabolism, with no need for external additional references. Two sequences were developed at 9.4T: a bi-voxel (1)H-((13)C) STEAM-POCE (Proton Observed Carbon Edited) sequence and a bi-voxel (1)H-((13)C) PRESS-POCE adiabatically decoupled sequence with Hadamard encoding. Hadamard encoding allows both voxels to be recorded simultaneously, with the same acquisition time as that required for a single voxel. The method was validated in a biological investigation into the neuronal damage and the effect on the Tri Carboxylic Acid cycle in localized excitotoxic lesions. Following an excitotoxic quinolinate-induced localized lesion in the rat cortex and the infusion of U-(13)C glucose, two (1)H-((13)C) spectra of distinct (4x4x4mm(3)) voxels, one centred on the injured hemisphere and the other on the contralateral hemisphere, were recorded simultaneously. Two (1)H bi-voxel spectra were also recorded and showed a significant decrease in N-acetyl aspartate, and an accumulation of lactate in the ipsilateral hemisphere. The (1)H-((13)C) spectra could be recorded dynamically as a function of time, and showed a fall in the glutamate/glutamine ratio and the presence of a stable glutamine pool, with a permanent increase of lactate in the ipsilateral hemisphere. This bi-voxel (1)H-((13)C) method can be used to investigate simultaneously both brain hemispheres, and to perform dynamic studies. We report here the neuronal damage and the effect on the Tri Carboxylic Acid cycle in localized excitotoxic lesions.

MRI methodological development of intervertebral disc degeneration: a rabbit in vivo study at 9.4 T.

Intervertebral disc (IVD) degeneration is a complex process characterized by biochemical and structural changes in both the nucleus pulposus and the anulus fibrosus. In this study, we were able to obtain in vivo magnetic resonance (MR) images of the rabbit spine, with several MR imaging (MRI) contrasts (rho, T(1) and T(2)). We quantified several parameters (T(2), apparent diffusion coefficient, disc height and area) to differentiate between healthy and degenerative IVDs and to characterize the degeneration process. To our knowledge, there has not been any previous in vivo study of rabbit IVDs at high-field MRI (9.4 T). A custom radio frequency (RF) coil for 9.4 T was designed to match rabbit IVD morphology, to study the degeneration in vivo on a model of human lumbar disease. Our new probe, a custom half-birdcage-type coil, obtains the necessary exploration depth while meeting the requirements for signal homogeneity and sensitivity of the study. This design addresses some of the difficulties with constructing RF coils at high field strengths.

Simultaneous dynamic T1 and T2* measurement for AIF assessment combined with DCE MRI in a mouse tumor model.

A double-delay SR-MGE-SNAP sequence allowing simultaneous T1 and T2* measurement was developed for integrating arterial input function (AIF) measurement into DCE MRI. Implemented on a 4.7-T animal MR system, this technique was applied to mice with colorectal tumor xenografts. AIF, measured in the mouse heart, was modeled by a bi-exponential function, whereas tumor K(trans) and v(e) parameter maps were obtained from analysis with a two- compartment model using an individually measured AIF. AIF analysis of T2*-corrected data yielded A1 = 9.2 +/- 4.3 kg/l, A(2) = 4.2 +/- 0.8 kg/l, m1 = 2.3 +/- 1.1 min(-1), and m2 = 0.05 +/- 0.02 min(-1). The mean initial plasma concentration C ( p )(t = 0) = 8.0 +/- 2.7 mM was compatible with estimated 8.6 mM. Without T2*-correction distribution phase parameters A1, m1, and C(p)(t = 0) were underestimated. In tumors, neglect of T2* effects yielded mean K(trans) values which were reduced by 14% (P < 0.05), whereas v(e) showed only a slight non-significant reduction. Simultaneous measurement of DeltaR1 and DeltaR2* studied in highly and poorly vascularized and (pre-)necrotic tumor regions revealed complementary behavior of both parameters with respect to vascular properties. In conclusion, the presented measurement technique is a promising tool for dynamic MRI applications studied in animal models at high field strengths and/or with CA of high relaxivities, as it combines classical DCE MRI integrating AIF assessment with dynamic T2* measurement.

Sodium magnetic resonance imaging of diuresis: spatial and kinetic response.

Renal function is highly correlated with the sodium concentration gradient along the corticomedullary axis. The application of 3D high-resolution sodium magnetic resonance imaging (MRI) provided a means to quantify in vivo the spatial and temporal changes in renal tissue sodium concentration under normal and diuretic conditions. A detailed, pixel-by-pixel analysis of the intact rat kidney sodium MR images yielded a quantitative measure of the corticomedullary sodium gradient before and at early and later times after the administration of two distinct diuretic agents, furosemide and mannitol. Furosemide, a loop diuretic, induced a fivefold reduction in the cortical-outer medullary sodium gradient, whereas mannitol, an osmotic diuretic, did not affect this gradient. Both diuretics induced a 50% decrease in the sodium concentration of the inner medulla; however, mannitol exerted its effect twice as fast as furosemide with a 2.5-min exponential decay constant. These specific changes were attributed to the different mechanism of action and site of activity of each diuretic agent. Thus, high-resolution (23)Na MRI offers a unique, noninvasive tool for functional imaging of the kidney physiology.

Molecular dynamics assignment of NMR correlation times to specific motions in a "basket-handle porphyrin" heme.

Iron (II) basket-handle porphyrins (BHP) are a series of encumbered heme models designed several years ago to mimic the ligand binding site of hemoproteins. Contrary to expectations, kinetic investigations have revealed that the k(on) rates for CO and/or O2 binding were only marginally affected by the assumed central steric hindrance of the iron atom. Thus, it was hypothesized that the internal dynamics of the molecule might be at the origin of the poor steric protection. To address this issue, measurements of nuclear magnetic resonance relaxation rates, fluorescence anisotropy experiments, and molecular dynamics simulations were undertaken. The size of BHP is small enough to allow the simulation in explicit chloroform with an almost complete sampling of the conformational space. The order parameters calculated from the MD trajectory compare well with the NMR experimental data and the predicted rotational correlation time corresponding to the Brownian motion of the molecule is in good agreement with the fluorescence measurements. Moreover, combining the results obtained using the three techniques allows the attribution of each internal NMR correlation time to a particular internal motion, revealing that even such medium-sized molecules are able to display quite complex internal dynamics. In particular, the handle phenyls that were assumed to sandwich the porphyrin have in fact a vanishing probability to be found in the proximity of the iron atom. They are therefore unable to reduce ligand accessibility significantly, which may explain the behavior of the k(on) rates.

Functional sodium magnetic resonance imaging of the intact rat kidney.

BACKGROUND: Renal fluid homeostasis depends to a large extent on the sodium concentration gradient along the corticomedullary axis. The spatial distribution and extent of this gradient were previously determined by invasive methods, which yielded a range of results. We demonstrate here the capacity of sodium magnetic resonance imaging (MRI) to quantify non-invasively renal sodium distribution in the intact kidney. METHODS: Sodium MRI was applied to study normal, diuretic, and obstructed rat kidneys in vivo. The images were recorded at 4.7 Tesla using a 3-dimensional gradient echo sequence, with high spatial and temporal resolution. The tissue sodium concentration (TSC) was obtained by taking into account the measured nuclear relaxation rates and MRI visibility relative to a reference saline solution. RESULTS: The corticomedullary sodium gradient increased linearly from the cortex to the inner medulla by approximately 31 mmol/L/mm, from a TSC of approximately 60 mmol/L to approximately 360 mmol/L. Furosemide induced a 50% reduction in the inner-medulla sodium and a 25% increase in the cortical sodium. The kinetics of these changes was related to the specific site and mechanism of the loop diuretic. Distinct profiles of the sodium gradient were observed in acute obstructed kidneys, as well as spontaneously obstructed kidneys. The changes in the sodium gradient correlated with the extent of damage and the residual function of the kidneys. CONCLUSION: Quantitative assessment of the renal corticomedullary sodium gradient by high resolution sodium MRI may help verify new aspects of the kidney concentrating mechanism and serve as a non-invasive diagnostic method of renal function.

Parametric imaging of tumor perfusion using flow- and permeability-limited tracers.

PURPOSE: To quantitatively evaluate the spatial distribution of flow- and permeability-limited perfusion in MCF7 human breast cancer tumors orthotopically implanted in CD1-NU mice. MATERIALS AND METHODS: Flow-limited perfusion was derived from (2)H-MRI recorded before and after infusion of deuterated water. Permeability-limited perfusion was evaluated from GdDTPA-enhanced (1)H-MRI. RESULTS: The dominant processes in tumor perfusion, namely blood flow and capillary permeability, were mapped in orthotopically implanted MCF7 human breast cancer tumors. The dynamic data were processed according to physiological models, yielding parametric maps of intravascular volume fraction, water perfusion rate, GdDTPA permeability rate constant, and extracellular volume fraction accessible to GdDTPA. The maps exhibited the heterogeneous distribution of each perfusion parameter. Most of the tumor tissue (> or =95%) was perfused with HDO, while GdDTPA was perfused in only about 50% of it. In most loci the perfusion rate was limited by capillary permeability to GdDTPA. CONCLUSION: The results demonstrated the instructive value of tracers with different properties used in conjunction to achieve a deeper understanding of tumor perfusion capacity. This study offers tools for the accurate, noninvasive evaluation of drug delivery efficacy.

Parametric imaging of tumor perfusion with deuterium magnetic resonance imaging.

Imaging of the vasculature and its functioning over the entire lesion may significantly aid in cancer diagnosis, assessment of prognosis, and therapeutic evaluation. In the current study we present a dynamic three-dimensional deuterium magnetic resonance imaging method that determines the intravascular volume fraction and water perfusion rate at a resolution of 2 mm(2)/pixel. The method was tested and utilized to characterize the vasculature of orthotopic MCF7 human breast cancer tumors in CD1-NU athymic mice. A new algorithm based on Patlak's kinetic model was developed to analyze the dynamic images acquired during and after termination of infusion with deuterated water. The resulting parametric maps spanned a wide range from 0.4 to 35.2% for the intravascular volume fraction and from 4 x 10(-6) to 3.9 x 10(-3) min(-1) for the perfusion rate and exhibited high intratumoral and intertumoral heterogeneity at both parameters. The intravascular volume fraction did not correlate with the corresponding perfusion rate, demonstrating the irregular outgrowth of tumor neovascularization. Averaging the data or analyzing at spatially degraded resolution completely masked the presence of both "hot spots" and hypoxic loci, highlighting the critical importance of high spatial resolution. The method is applicable to other types of tumors and animal models and may be extended to humans.