20 research outputs found
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Targeted Imaging Agents for Detecting Tumour Cell Death following Therapy
Cell death is an important target for imaging the early response of tumours to treatment. In this study a phosphatidylserine-binding protein (C2Am) has been derivatised with a fluorine-18 containing maleimide for imaging tumour cell death in a xenograft murine advanced colorectal cancer.
A one-pot, two-step automated synthesis of N-(5-[18F]fluoropentyl)maleimide using a GE TRACERlab FXFN automated module (within 58±5.8 min (n = 12), >98% radiochemical purity and 12±3% decay corrected yield) has been developed. This was used to label the single cysteine present on C2Am within 30 min in PBS (Am=212000±30000 MBq/µmol (n = 3)). Using xenograft models of breast and colorectal cancer, and a TRAIL-R2 agonist for inducing cell death, the binding of [18F]FPenM-C2Am was tested in vitro and in vivo using biodistribution and dynamic PET imaging studies. Cell death detection was validated by cell death histology assays CC3 and TUNEL. For colorectal cancer, there was a positive correlation between [18F]FPenM-C2Am signal in tumours post treatment and the corresponding histologic markers of cell death CC3 (Pearson r = 0.82) and TUNEL (Pearson r = 0.95).
[18F]FPenM-C2Am showed a favourable biodistribution profile, with predominantly renal clearance and minimal retention in spleen (0.79±0.05 %ID/g), liver (1.18±0.13 %ID/g), small intestine (0.97±0.25 %ID/g) and kidney (6.90±0.56 %ID/g) at 2 h after probe administration. In a xenograft model of colorectal cancer treated with a TRAIL-R2 agonist, for 24h, at 0.2-0.4 mg/kg, i.v. [18F]FPenM-C2Am generated tumour-to-muscle and tumour-to-blood ratios following treatment of 6.7±0.8-fold and 1.89±0.23-fold, respectively, at 2 h after administration. A statistically significant pairwise difference was obtained between the tumour-to-muscle contrast prior to and following therapy (P=0.0137, unpaired two-tailed t-test).
Given the favourable biodistribution profile of [18F]FPenM-C2Am, and its ability to produce rapid and cell death-specific image contrast, this agent has potential for clinical translation. We have initiated cGMP manufacture and toxicology studies required for a Phase 1 trial.
Aberrant cell surface glycosylation has been described as one of the key hallmarks of cancer. Monitoring glycosylation could provide an insight into tumour progression, proliferation and ultimately could potentially be used for monitoring treatment response.
Aberrant glycosylation can be observed by harnessing the cell’s metabolism to incorporate into its glycome unnatural sugars bearing bioorthogonal chemical reporters. These reporters are targeted subsequently by fluorescent, magnetic or optoacoustic probes that allow imaging.
As part of this work, we have demonstrated in vitro that a peracetylated cyclopropene mannosamine (Ac4ManNCCp)-modified sugar can be used as a tool for imaging hypersialylation in an advanced colorectal cancer model. Further optimisation of the probe to improve solubility is required to facilitate transitioning from in vitro to in vivo imaging. Nevertheless, overcoming this solubility issue would allow for facile labelling of surface glycans using PET radionuclides
Monitoring tumor cell death in murine tumor models using deuterium magnetic resonance spectroscopy and spectroscopic imaging.
2H magnetic resonance spectroscopic imaging has been shown recently to be a viable technique for metabolic imaging in the clinic. We show here that 2H MR spectroscopy and spectroscopic imaging measurements of [2,3-2H2]malate production from [2,3-2H2]fumarate can be used to detect tumor cell death in vivo via the production of labeled malate. Production of [2,3-2H2]malate, following injection of [2,3-2H2]fumarate (1 g/kg) into tumor-bearing mice, was measured in a murine lymphoma (EL4) treated with etoposide, and in human breast (MDA-MB-231) and colorectal (Colo205) xenografts treated with a TRAILR2 agonist, using surface-coil localized 2H MR spectroscopy at 7 T. Malate production was also imaged in EL4 tumors using a fast 2H chemical shift imaging sequence. The malate/fumarate ratio increased from 0.016 ± 0.02 to 0.16 ± 0.14 in EL4 tumors 48 h after drug treatment (P = 0.0024, n = 3), and from 0.019 ± 0.03 to 0.25 ± 0.23 in MDA-MB-231 tumors (P = 0.0001, n = 5) and from 0.016 ± 0.04 to 0.28 ± 0.26 in Colo205 tumors (P = 0.0002, n = 5) 24 h after drug treatment. These increases were correlated with increased levels of cell death measured in excised tumor sections obtained immediately after imaging. 2H MR measurements of [2,3-2H2]malate production from [2,3-2H2]fumarate provide a potentially less expensive and more sensitive method for detecting cell death in vivo than 13C MR measurements of hyperpolarized [1,4-13C2]fumarate metabolism, which have been used previously for this purpose.Cambridge European Scholarship from the Cambridge Trus
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18F-C2Am: a targeted imaging agent for detecting tumor cell death in vivo using positron emission tomography.
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
Preclinical PET Imaging of Tumor Cell Death following Therapy Using Gallium-68-Labeled C2Am
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
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Research data supporting "Assessment of the sensitivity of ²H MR spectroscopy measurements of [2,3-²H₂]fumarate metabolism for detecting tumor cell death"
Zip folder with 8 Excel files containing the raw measurements of main RAW_Figure_1 (1A-C, G-I - Spectra were zero- and first-order phase corrected, and the peaks modelled with the AMARES toolbox), RAW_Figure_2 (Apparent malate production rates were calculated between 0 and 20 min following fumarate administration and plotted against the tumor fumarate concentration at 20 minutes. The curves can be fitted to the Michaelis-Menten equation to obtain an estimate of Km and Vmax), RAW_Figure_3 (3M,3N - Images were analyzed using a CytoNuclear v1.6 algorithm on HALO v3.0.311.293. Shown are percentage of positive cells), RAW_Figure_4 (4A-L - Pearson correlation analysis of histological markers of cell death, CC3 (A – F) and TUNEL (G – L) % positive cells, with the malate/fumarate ratio and malate concentration (mM) obtained by summing the spectra between 20 and 60 min after fumarate injection.)
Supplementary_Figure_1 (S1A-C, G-I - Spectra were zero- and first-order phase corrected, and the peaks modelled with the AMARES toolbox, shown are ²H MR spectroscopic measurements of labeled fumarate, malate and water concentrations in MDA-MB-231 tumors following injection of increasing concentrations of [2,3-²H₂]fumarate),
RAW_Supplementary_Figure 3 (Tumor malate concentrations before and after treatment with 0.1, 0.4, and 0.8 mg/kg MEDI3039 between 20 and 60 minutes after injecting increasing fumarate concentrations (0.1, 0.3 and 0.5 g/kg)), RAW_Supplementary_Figure_4 (S4A-C - Ratios were obtained by summing the fumarate and malate signals between 20 and 60 min after injection of [2,3-²H₂]fumarate at 0.1, 0.3 and 0.5 g/kg. The malate/fumarate ratio is the dependent variable and was assessed at increasing concentrations of fumarate at each of the MEDI3039 drug concentrations.), Supplementary_Figure_5 (S5A-C - Representative time course data for labeled fumarate, malate and water concentrations with the corresponding coefficients of variance, following injection of increasing concentrations of [2,3-²H₂]fumarate.)This work was supported by grants supported by grants from Cancer Research UK (C197/A29580, C197/A17242, C9685/A25177). F. Hesse received a Cambridge European Scholarship from the Cambridge Trust
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Assessment of the sensitivity of 2 H MR spectroscopy measurements of [2,3-2 H2 ]fumarate metabolism for detecting tumor cell death.
Funder: Cambridge Trust; doi: http://dx.doi.org/10.13039/501100005370Imaging 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. 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 nontoxic 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.Cambridge Trus
Deuterium magnetic resonance spectroscopic imaging of tumor cell death in vivo following oral delivery of 2Hlabeled fumarate
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
Imaging glioblastoma response to radiotherapy using 2H magnetic resonance spectroscopy measurements of fumarate metabolism
Early detection of tumor cell death in glioblastoma following treatment with chemoradiation has the potential to distinguish between true disease progression and pseudoprogression. Tumor cell death can be detected non-invasively in vivo by imaging the production of [2,3-2H2]malate from [2,3-2H2]fumarate using 2H magnetic resonance (MR) spectroscopic imaging. We show here that 2H MR spectroscopy and spectroscopic imaging measurements of [2,3-2H2]fumarate metabolism can detect tumor cell death in orthotopically implanted glioblastoma models within 48 hours following the completion of chemoradiation. Following the injection of [2,3- 2H2]fumarate into tumor-bearing mice, production of [2,3-2H2]malate was measured in a human cell line-derived model and in radio-sensitive and radio-resistant patient- derived models of glioblastoma that were treated with temozolomide followed by targeted fractionated irradiation. The increase in the [2,3-2H2]malate/[2,3- 2H2]fumarate signal ratio post-treatment, which correlated with histological assessment of cell death, was a more sensitive indicator of treatment response than diffusion-weighted and contrast agent-enhanced 1H MRI measurements, which have been used clinically to detect responses of glioblastoma to chemoradiation. Overall, early detection of glioblastoma cell death using 2H MRI of malate production from fumarate could help improve the clinical evaluation of response to chemoradiation
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Deuterium magnetic resonance imaging of serine metabolism in mouse models of glioblastoma
Abstract
Purpose
Serine is a major source of one-carbon units needed for the synthesis of nucleotides
and for the production of intramitochondrial NADPH and plays an important role in
cancer cell proliferation. The aim of this study was to develop a 2H magnetic
resonance spectroscopic imaging method for imaging tumor serine metabolism.
Methods
Sequential 2H spectra and spectroscopic images were used to monitor the metabolism
of [2,3,3-2H3]serine in patient-derived glioblastoma cells in vitro and in tumors obtained
by their orthotopic implantation in mouse brain.
Results
[14,14-2H2] 5,10-methylene tetrahydrofolate, [2H]glycine, [2H]formate and labelled
water were detected in cell suspensions and water labelling in spectroscopic images
of tumors. Studies in cells and tumors with variable mitochondrial content and inhibitor
studies in cells demonstrated that most of the labelled serine was metabolized in the
mitochondria. Water labelling in the cell suspensions was correlated with formate
labelling and therefore water labelling observed in tumors could be used to provide a
surrogate measure of flux in the pathway of one-carbon metabolism in vivo.
Conclusion
The method has the potential to be used clinically to select patients for treatment with
inhibitors of one carbon metabolism and subsequently to detect their early responses
to such treatment
Deuterium MRSI of tumor cell death in vivo following oral delivery of 2 H ‐labeled fumarate
Purpose: There is an unmet clinical need for direct and sensitive methods to detect cell death in vivo, especially with regard to monitoring tumor treatment response. We have shown previously that tumor cell death can be detected in vivo from 2H MRS and MRSI 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‐labeled fumarate. Methods: Mice with subcutaneously implanted EL4 tumors were fasted for 1 h before administration (200 μl) of [2,3‐2H2]fumarate (2 g/kg bodyweight) via oral gavage without anesthesia. The animals were then anesthetized, and after 30 min, 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 min. 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). Labeled malate was undetectable in spectroscopic images acquired before treatment and increased in the tumor area following treatment. The increase in the malate/fumarate signal ratio was similar to that observed previously following intravenous administration of labeled fumarate. Conclusion: Orally administered [2,3‐2H2]fumarate can be used to detect tumor cell death noninvasively following treatment with a sensitivity that is similar to that obtained with intravenous administration