Search

Peer reviewed: True, Simple Summary: There is an unmet clinical need for imaging agents capable of detecting the presence, extent, and distribution of tumor cell death following treatment. We describe here a gallium-68-labeled derivative of the C2A domain of Synaptotagmin-I (68Ga-C2Am), which binds to the phosphatidylserine exposed by dying cells, for imaging tumor cell death in vivo using positron emission tomography (PET). Since PET is a highly sensitive tomographic imaging technique that is widely used in clinical oncology and gallium-68 has emerged as a cost-effective radiotracer that can be eluted on site from a benchtop generator, 68Ga-C2Am could find clinical application for the rapid assessment of tumor responses to treatment. Abstract: There is an unmet clinical need for imaging agents capable of detecting early evidence of tumor cell death, since the timing, extent, and distribution of cell death in tumors following treatment can give an indication of treatment outcome. We describe here 68Ga-labeled C2Am, which is a phosphatidylserine-binding protein, for imaging tumor cell death in vivo using positron emission tomography (PET). A one-pot synthesis of 68Ga-C2Am (20 min, 25 °C, >95% radiochemical purity) has been developed, using a NODAGA-maleimide chelator. The binding of 68Ga-C2Am to apoptotic and necrotic tumor cells was assessed in vitro using human breast and colorectal cancer cell lines, and in vivo, using dynamic PET measurements in mice implanted subcutaneously with the colorectal tumor cells and treated with a TRAIL-R2 agonist. 68Ga-C2Am showed predominantly renal clearance and low retention in the liver, spleen, small intestine, and bone and generated a tumor-to-muscle (T/m) ratio of 2.3 ± 0.4, at 2 h post probe administration and at 24 h following treatment. 68Ga-C2Am has the potential to be used in the clinic as a PET tracer for assessing early treatment response in tumors.

Imaging the metabolism of [2,3-2 H2 ]fumarate to produce malate can be used to detect tumor cell death post-treatment. Here we assess the sensitivity of the technique for detecting cell death by lowering the concentration of injected [2,3-2 H2 ]fumarate and by varying the extent of tumor cell death through changes in drug concentration. Mice were implanted subcutaneously with human triple negative breast cancer cells (MDA-MB-231) and injected with 0.1, 0.3 and 0.5 g/kg [2,3-2 H2 ]fumarate before and after treatment with a multivalent TRAlL-R2 agonist (MEDI3039) at 0.1, 0.4 and 0.8 mg/kg. Tumor conversion of [2,3-2 H2 ]fumarate to [2,3-2 H2 ]malate was assessed from a series of 13 spatially localized 2 H MR spectra acquired over 65 min using a pulse-acquire sequence with a 2 ms BIR4 adiabatic excitation pulse. Tumors were then excised and stained for histopathological markers of cell death; cleaved caspase 3 (CC3) and DNA damage (terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)). The rate of malate production and the malate/fumarate ratio plateaued at tumor fumarate concentrations of 2 mM, which were obtained with injected [2,3-2 H2 ]fumarate concentrations of 0.3 g/kg and above. Tumor malate concentration and the malate/fumarate ratio increased linearly with the extent of cell death determined histologically, which was varied by changing drug concentration. At an injected [2,3-2 H2 ]fumarate concentration of 0.3 g/kg, 20% CC3 staining corresponded to a malate concentration of 0.62 mM and a malate/fumarate ratio of 0.21. Extrapolation indicated that there would be no detectable malate at 0% CC3 staining. The use of low and non-toxic fumarate concentrations and the production of [2,3-2 H2 ]malate at concentrations that are within the range that can be detected clinically suggest this technique could translate to the clinic.

Cell death is an important imaging target for assessing early tumour treatment response and the effectiveness of therapy. Traditionally, anatomical imaging modalities measure changes in tumour size (Response Evaluation Criteria in Solid Tumours (RECIST)). More recently these assessments have also included functional and molecular information, as these parameters can show earlier responses to chemotherapies, e.g. using PET measurements of 2-[¹⁸F]-fluoro-2-deoxy D-glucose uptake (FDG-PET). PET Response Criteria in Solid Tumours (PERCIST) was introduced as a potentially more sensitive method of assessing treatment response, particularly with therapies that stabilise disease. While metabolic imaging may indicate drug target engagement, and in some cases tumour cell death, there is a need for imaging methods that detect tumour cell death more directly post-treatment and that can give an indication of longer term treatment outcomes. In this project ²H magnetic resonance spectroscopic imaging with ²H-fumarate and PET-CT with ¹⁸F-C2Am are investigated as methods for the early detection of tumour cell death post-treatment. Fumarate, an intermediate in the tricarboxylic acid cycle, is hydrated in the reaction catalysed by the enzyme fumarase to produce malate. With loss of plasma membrane integrity in dying cells fumarate can rapidly gain access to the enzyme. By measuring increased malate production in treated tumours after fumarate injection, cell necrosis can be assessed. It could be established that ²H MRS and MRSI measurements at 7 T of [2,3-²H₂]fumarate conversion to ²H-labelled malate can be used to detect early cell death in various tumour models. ¹⁸F-C2Am is an ¹⁸F-labelled derivative of a phosphatidylserine-binding protein, the C2A domain of synaptotagmin-I (C2Am), which can be used to image tumour cell death in vivo using PET. C2Am binds in a Ca²⁺ -dependent manner with nanomolar affinity to phosphatidylserine (PS) on the outer leaflet of the plasma membrane of apoptotic cells or to PS on the inner leaflet following disruption of plasma membrane integrity in necrotic cells. The agent was tested in mice bearing human xenografts treated with a TRAIL-R2 agonist, MEDI3039, or with conventional chemotherapy. Both imaging markers may provide more specific and sensitive methods for detecting cell death, which can be used to rapidly assess treatment response and effectiveness at an early stage., Cancer Research UK Cambridge Institute Studentship (core funding); Cambridge European Scholarship from the Cambridge Trust

Hyperpolarized (HP) 13 C magnetic resonance enables non-invasive probing of metabolism in vivo. To date, only 13 C-molecules hyperpolarized with persistent trityl radicals have been injected in humans. We show here that the free radical photo-induced in alpha-ketoglutaric acid (α-KG) can be used to hyperpolarize photo-inactive 13 C-molecules such as [1-13 C]lactate. α-KG is an endogenous molecule with an exceptionally high radical yield under photo-irradiation, up to 50 %, and its breakdown product, succinic acid, is also endogenous. This radical precursor therefore exhibits an excellent safety profile for translation to human studies. The labile nature of the radical means that no filtration is required prior to injection while also offering the opportunity to extend the 13 C relaxation time in frozen HP 13 C-molecules for storage and transport. The potential for in vivo metabolic studies is demonstrated in the rat liver following the injection of a physiological dose of HP [1-13 C]lactate.

Hyperpolarized (HP) 13 C magnetic resonance enables non-invasive probing of metabolism in vivo. To date, only 13 C-molecules hyperpolarized with persistent trityl radicals have been injected in humans. We show here that the free radical photo-induced in alpha-ketoglutaric acid (α-KG) can be used to hyperpolarize photo-inactive 13 C-molecules such as [1-13 C]lactate. α-KG is an endogenous molecule with an exceptionally high radical yield under photo-irradiation, up to 50 %, and its breakdown product, succinic acid, is also endogenous. This radical precursor therefore exhibits an excellent safety profile for translation to human studies. The labile nature of the radical means that no filtration is required prior to injection while also offering the opportunity to extend the 13 C relaxation time in frozen HP 13 C-molecules for storage and transport. The potential for in vivo metabolic studies is demonstrated in the rat liver following the injection of a physiological dose of HP [1-13 C]lactate.

Purpose: The performance of pulse sequences in vivo can be limited by fast relaxation rates, magnetic field inhomogeneity, and nonuniform spin excitation. We describe here a method for pulse sequence optimization that uses a stochastic numerical solver that in principle is capable of finding a global optimum. The method provides a simple framework for incorporating any constraint and implementing arbitrarily complex cost functions. Efficient methods for simulating spin dynamics and incorporating frequency selectivity are also described., Methods: Optimized pulse sequences for polarization transfer between protons and X-nuclei and excitation pulses that eliminate J-coupling modulation were evaluated experimentally using a surface coil on phantoms, and also the detection of hyperpolarized [2-¹³C]lactate in vivo in the case of J-coupling modulation-free excitation., Results: The optimized polarization transfer pulses improved the SNR by ~50% with a more than twofold reduction in the B₁ field, and J-coupling modulation-free excitation was achieved with a more than threefold reduction in pulse length., Conclusion: This process could be used to optimize any pulse when there is a need to improve the uniformity and frequency selectivity of excitation as well as to design new pulses to steer the spin system to any desired achievable state., Magnetic Resonance in Medicine, 87 (5), ISSN:0740-3194, ISSN:1522-2594

Introduction: Trialing novel cancer therapies in the clinic would benefit from imaging agents that can detect early evidence of treatment response. The timing, extent and distribution of cell death in tumors following treatment can give an indication of outcome. We describe here an 18F-labeled derivative of a phosphatidylserine-binding protein, the C2A domain of Synaptotagmin-I (C2Am), for imaging tumor cell death in vivo using PET. Methods: A one-pot, two-step automated synthesis of N-(5-[18F]fluoropentyl)maleimide (60 min synthesis time, > 98% radiochemical purity) has been developed, which was used to label the single cysteine residue in C2Am within 30 min at room temperature. Binding of 18F-C2Am to apoptotic and necrotic tumor cells was assessed in vitro, and also in vivo, by dynamic PET and biodistribution measurements in mice bearing human tumor xenografts treated with a TRAILR2 agonist or with conventional chemotherapy. C2Am detection of tumor cell death was validated by correlation of probe binding with histological markers of cell death in tumor sections obtained immediately after imaging. Results: 18F-C2Am showed a favorable biodistribution profile, with predominantly renal clearance and minimal retention in spleen, liver, small intestine, bone and kidney, at 2 h following probe administration. 18F-C2Am generated tumor-to-muscle (T/m) ratios of 6.1 ± 2.1 and 10.7 ± 2.4 within 2 h of probe administration in colorectal and breast tumor models, respectively, following treatment with the TRAILR2 agonist. The levels of cell death (CC3 positivity) following treatment were 12.9–58.8% and 11.3–79.7% in the breast and colorectal xenografts, respectively. Overall, a 20% increase in CC3 positivity generated a one unit increase in the post/pre-treatment tumor contrast. Significant correlations were found between tracer uptake post-treatment, at 2 h post-probe administration, and histological markers of cell death (CC3: Pearson R = 0.733, P = 0.0005; TUNEL: Pearson R = 0.532, P = 0.023). Conclusion: The rapid clearance of 18F-C2Am from the blood pool and low kidney retention allowed the spatial distribution of cell death in a tumor to be imaged during the course of therapy, providing a rapid assessment of tumor treatment response. 18F-C2Am has the potential to be used in the clinic to assess early treatment response in tumors.

PURPOSE: Imaging tumor metabolism in vivo using hyperpolarized [1-13 C]pyruvate is a promising technique for detecting disease, monitoring disease progression, and assessing treatment response. However, the transient nature of the hyperpolarization and its depletion following excitation limits the available time for imaging. We describe here a single-shot multi spin echo sequence, which improves on previously reported sequences, with a shorter readout time, isotropic point spread function (PSF), and better signal-to-noise ratio. METHODS: The sequence uses numerically optimized spectrally selective excitation pulses set to the resonant frequencies of pyruvate and lactate and a hyperbolic secant adiabatic refocusing pulse, all applied in the absence of slice selection gradients. The excitation pulses were designed to be resistant to the effects of B0 and B1 field inhomogeneity. The gradient readout uses a 3D cone trajectory composed of 13 cones, all fully refocused and distributed among 7 spin echoes. The maximal gradient amplitude and slew rate were set to 4 G/cm and 20 G/cm/ms, respectively, to demonstrate the feasibility of clinical translation. RESULTS: The pulse sequence gave an isotropic PSF of 2.8 mm. The excitation profiles of the optimized pulses closely matched simulations and a 46.10 ± 0.04% gain in image SNR was observed compared to a conventional Shinnar-Le Roux excitation pulse. The sequence was demonstrated with dynamic imaging of hyperpolarized [1-13 C]pyruvate and [1-13 C]lactate in vivo. CONCLUSION: The pulse sequence was capable of dynamic imaging of hyperpolarized 13 C labeled metabolites in vivo with relatively high spatial and temporal resolution and immunity to system imperfections.

Additional file 1. Supporting methods and data.

Purpose There is an unmet clinical need for direct and sensitive methods to detect cell death in vivo, especially in regard to monitoring tumor treatment response. We have shown previously that tumor cell death can be detected in vivo from 2H magnetic resonance spectroscopy and spectroscopic imaging measurements of increased [2,3-2H2]malate production following intravenous injection of [2,3-2H2]fumarate. We show here that cell death can be detected with similar sensitivity following oral administration of the 2H-labelled fumarate. Methods Mice with subcutaneously implanted EL4 tumors were fasted for 1 h before administration (200 µl) of [2,3-2H2]fumarate (2g/kg bodyweight) via oral gavage without anesthesia. The animals were then anaesthetized and after 30 minutes tumor conversion of [2,3-2H2]fumarate to [2,3-2H2]malate was assessed from a series of 13 2H spectra acquired over a period of 65 minutes. The 2H spectra and 2H spectroscopic images were acquired using a surface coil before and at 48 h after treatment with a chemotherapeutic drug (etoposide, 67 mg/kg). Results The malate/fumarate signal ratio increased from 0.022  0.03 before drug treatment to 0.12  0.04 following treatment (P=0.023, n=4). Labelled malate was undetectable in spectroscopic images acquired prior to treatment and increased in the tumor area post-treatment. The increase in the malate/fumarate signal ratio was similar to that observed previously following intravenous administration of labelled fumarate. Conclusion Orally administered [2,3-2H2]fumarate, can be used to detect tumor cell death non-invasively post treatment with a sensitivity that is similar to that obtained with intravenous administration.