Targeting EGFR and VEGFR2 tyrosine kinases with positron emission tomography : evaluation of two radiotracers

Receptor tyrosine kinases (RTKs) are commonly involved in the development, growth and spread of cancer. Targeted therapy with tyrosine kinase inhibitors (TKIs) has proven a successful treatment strategy against cancers in which growth is dependent on the expression of these receptors. Increased effectiveness of treatment can potentially be achieved by individual characterizations of the disease, enabling tailoring of the therapy directed at the specific targets found. Positron emission tomography (PET) is a non-invasive imaging technique that allows characterization of biochemical processes and quantification of targets such as RTKs. In PET, the distribution of radiolabeled molecules in the body is traced by the emission of photons produced after the decay of the radionuclide that is incorporated in the tracer molecule. Drugs and other xenobiotics are often metabolized in the body to facilitate excretion. A PET tracer can be metabolized into radioactive metabolites, which often display pharmacokinetic behaviors different than that of the parent molecule. It is therefore pivotal to characterize the metabolism of novel PET tracers before accurate estimations of biological target levels, based on radioactivity uptakes, can be performed. This doctoral thesis focuses on the preclinical evaluation of two tracer molecules, [11C]PD153035 and [11C]PAQ, each targeting a specific RTK known to play important roles in cancerogenesis. Both tracers are based on TKIs and have been proven to be potent RTK inhibitors in vitro. In papers I and III, the in vitro and in vivo metabolism, respectively, of [11C]PD153035 was investigated. We found that the tracer was extensively metabolized by cytochrome P450 enzymes into several different radioactive metabolites. Furthermore, the results indicate that the metabolism impairs quantification of the target RTK, the epidermal growth factor receptor (EGFR). In papers II and IV the pharmacokinetics and angiogenesis detection properties of (R,S)-[11C]PAQ and (R)-[11C]PAQ were assessed in various models of cancer in mice. The results show that the tracer is metabolically stable and that areas with increased angiogenic activity, based on vascular endothelial growth factor receptor 2 expression (VEGFR2), can be visualized with PET. Uptake of radioactivity correlated well to areas with high expression of the receptor both with the labeled racemate and the R-isomer. In addition, high focal uptake was observed with (R)-[11C]PAQ in lungs with metastases that exhibited high expression levels of the VEGFR2. In summary, we conclude that [11C]PD153035 is metabolized very rapidly in rat and that a similar metabolism in humans would imply serious limitations if the tracer is used in patient stratification for EGFR targeted therapy. (R)-[11C]PAQ, on the other hand, is a promising tracer that, pending positive results in further validation studies, can prove to be a valuable tool for personalizing cancer treatment based on expression levels of VEGFR2

Glycolytic Response to Inflammation Over Time: Role of Myeloid HIF-1alpha

The in vivo response to lipopolysaccharide (LPS) occurs rapidly and has profound physiological and metabolic effects. The hypoxia inducible (HIF) transcription factor is an intrinsic and essential part of inflammation, and is induced by LPS. To determine the importance of the HIF response in regulating metabolism following an LPS response, glucose uptake was quantified in a time dependent manner in mice lacking HIF-1α in myeloid cells. We found that deletion of HIF-1α has an acute protective effect on LPS-induced hypoglycemia. Furthermore, reduced glucose uptake was observed in the heart and brown fat, in a time dependent manner, following loss of HIF-1α. To determine the physiological significance of these findings, cardiovascular, body temperature, and blood pressure changes were subsequently quantified in real time using radiotelemetry measurements. These studies reveal the temporal aspects of HIF-1α as a regulator of the metabolic response to acute LPS-induced inflammation

Combining [(11)C]-AnxA5 PET imaging with serum biomarkers for improved detection in live mice of modest cell death in human solid tumor xenografts

BACKGROUND: In vivo imaging using Annexin A5-based radioligands is a powerful technique for visualizing massive cell death, but has been less successful in monitoring the modest cell death typically seen in solid tumors after chemotherapy. Here we combined dynamic positron emission tomography (PET) imaging using Annexin A5 with a serum-based apoptosis marker, for improved sensitivity and specificity in assessment of chemotherapy-induced cell death in a solid tumor model. METHODOLOGY/PRINCIPAL FINDINGS: Modest cell death was induced by doxorubicin in a mouse xenograft model with human FaDu head and neck cancer cells. PET imaging was based on (11)C-labeled Sel-tagged Annexin A5 ([(11)C]-AnxA5-ST) and a size-matched control. 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]-FDG) was utilized as a tracer of tissue metabolism. Serum biomarkers for cell death were ccK18 and K18 (M30 Apoptosense® and M65). Apoptosis in tissue sections was verified ex vivo for validation. Both PET imaging using [(11)C]-AnxA5-ST and serum ccK18/K18 levels revealed treatment-induced cell death, with ccK18 displaying the highest detection sensitivity. [(18)F]-FDG uptake was not affected by this treatment in this tumor model. [(11)C]-AnxA5-ST gave robust imaging readouts at one hour and its short half-life made it possible to perform paired scans in the same animal in one imaging session. CONCLUSIONS/SIGNIFICANCE: The combined use of dynamic PET with [(11)C]-AnxA5-ST, showing specific increases in tumor binding potential upon therapy, with ccK18/K18 serum measurements, as highly sensitive markers for cell death, enabled effective assessment of modest therapy-induced cell death in this mouse xenograft model of solid human tumors.VetenskapsrådetPublishe

Glycolytic Response to Inflammation Over Time: Role of Myeloid HIF-1alpha.

The in vivo response to lipopolysaccharide (LPS) occurs rapidly and has profound physiological and metabolic effects. The hypoxia inducible (HIF) transcription factor is an intrinsic and essential part of inflammation, and is induced by LPS. To determine the importance of the HIF response in regulating metabolism following an LPS response, glucose uptake was quantified in a time dependent manner in mice lacking HIF-1α in myeloid cells. We found that deletion of HIF-1α has an acute protective effect on LPS-induced hypoglycemia. Furthermore, reduced glucose uptake was observed in the heart and brown fat, in a time dependent manner, following loss of HIF-1α. To determine the physiological significance of these findings, cardiovascular, body temperature, and blood pressure changes were subsequently quantified in real time using radiotelemetry measurements. These studies reveal the temporal aspects of HIF-1α as a regulator of the metabolic response to acute LPS-induced inflammation.Funded by a PRF award from the Wellcome Trust and by the Swedish Research Council (Vetenskapsrådet), Swedish Cancer Fund (Cancerfonden) and Swedish Children's Cancer Fund (Barncancerfonden)

Optimized, automated and cGMP-compliant synthesis of the HER2 targeting [68Ga]Ga-ABY-025 tracer

Abstract Background The Affibody molecule, ABY-025, has demonstrated utility to detect human epidermal growth factor receptor 2 (HER2) in vivo, either radiolabelled with indium-111 (111In) or gallium-68 (68Ga). Using the latter, 68Ga, is preferred due to its use in positron emission tomography with superior resolution and quantifying capabilities in the clinical setting compared to 111In. For an ongoing phase II study (NCT05619016) evaluating ABY-025 for detecting HER2-low lesions and selection of patients for HER2-targeted treatment, the aim was to optimize an automated and cGMP-compliant radiosynthesis of [68Ga]Ga-ABY-025. [68Ga]Ga-ABY-025 was produced on a synthesis module, Modular-Lab PharmTracer (Eckert & Ziegler), commonly used for 68Ga-labelings. The radiotracer has previously been radiolabeled on this module, but to streamline the production, the method was optimized. Steps requiring manual interactions to the radiolabeling procedure were minimized including a convenient and automated pre-concentration of the 68Ga-eluate and a simplified automated final formulation procedure. Every part of the radiopharmaceutical production was carefully developed to gain robustness and to avoid any operator bound variations to the manufacturing. The optimized production method was successfully applied for 68Ga-labeling of another radiotracer, verifying its versatility as a universal and robust method for radiosynthesis of Affibody-based peptides. Results A simplified and optimized automated cGMP-compliant radiosynthesis method of [68Ga]Ga-ABY-025 was developed. With a decay corrected radiochemical yield of 44 ± 2%, a radiochemical purity (RCP) of 98 ± 1%, and with an RCP stability of 98 ± 1% at 2 h after production, the method was found highly reproducible. The production method also showed comparable results when implemented for radiolabeling another similar peptide. Conclusion The improvements made for the radiosynthesis of [68Ga]Ga-ABY-025, including introducing a pre-concentration of the 68Ga-eluate, aimed to utilize the full potential of the 68Ge/68Ga generator radioactivity output, thereby reducing radioactivity wastage. Furthermore, reducing the number of manually performed preparative steps prior to the radiosynthesis, not only minimized the risk of potential human/operator errors but also enhanced the process’ robustness. The successful application of this optimized radiosynthesis method to another similar peptide underscores its versatility, suggesting that our method can be adopted for 68Ga-labeling radiotracers based on Affibody molecules in general. Trial registration: NCT, NCT05619016, Registered 7 November 2022, https://clinicaltrials.gov/study/NCT05619016?term=HER2&cond=ABY025&rank=

Optimisation of the Synthesis and Cell Labelling Conditions for [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS: a Direct In Vitro Comparison in Cell Types with Distinct Therapeutic Applications

Erratum inCorrection: Optimisation of the Synthesis and Cell Labelling Conditions for [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS: a Direct In Vitro Comparison in Cell Types with Distinct Therapeutic Applications.Friberger I, Jussing E, Han J, Goos JACM, Siikanen J, Kaipe H, Lambert M, Harris RA, Samén E, Carlsten M, Holmin S, Tran TA.Mol Imaging Biol. 2022 Jun;24(3):510. doi: 10.1007/s11307-022-01709-1.PMID: 35174429 Free PMC article. No abstract available.International audienceAbstract Background There is a need to better characterise cell-based therapies in preclinical models to help facilitate their translation to humans. Long-term high-resolution tracking of the cells in vivo is often impossible due to unreliable methods. Radiolabelling of cells has the advantage of being able to reveal cellular kinetics in vivo over time. This study aimed to optimise the synthesis of the radiotracers [ 89 Zr]Zr-oxine (8-hydroxyquinoline) and [ 89 Zr]Zr-DFO-NCS (p-SCN-Bn-Deferoxamine) and to perform a direct comparison of the cell labelling efficiency using these radiotracers. Procedures Several parameters, such as buffers, pH, labelling time and temperature, were investigated to optimise the synthesis of [ 89 Zr]Zr-oxine and [ 89 Zr]Zr-DFO-NCS in order to reach a radiochemical conversion (RCC) of >95 % without purification. Radio-instant thin-layer chromatography (iTLC) and radio high-performance liquid chromatography (radio-HPLC) were used to determine the RCC. Cells were labelled with [ 89 Zr]Zr-oxine or [ 89 Zr]Zr-DFO-NCS. The cellular retention of 89 Zr and the labelling impact was determined by analysing the cellular functions, such as viability, proliferation, phagocytotic ability and phenotypic immunostaining. Results The optimised synthesis of [ 89 Zr]Zr-oxine and [ 89 Zr]Zr-DFO-NCS resulted in straightforward protocols not requiring additional purification. [ 89 Zr]Zr-oxine and [ 89 Zr]Zr-DFO-NCS were synthesised with an average RCC of 98.4 % (n = 16) and 98.0 % (n = 13), respectively. Cell labelling efficiencies were 63.9 % (n = 35) and 70.2 % (n = 30), respectively. 89 Zr labelling neither significantly affected the cell viability (cell viability loss was in the range of 1–8 % compared to its corresponding non-labelled cells, P value > 0.05) nor the cells’ proliferation rate. The phenotype of human decidual stromal cells (hDSC) and phagocytic function of rat bone-marrow-derived macrophages (rMac) was somewhat affected by radiolabelling. Conclusions Our study demonstrates that [ 89 Zr]Zr-oxine and [ 89 Zr]Zr-DFO-NCS are equally effective in cell labelling. However, [ 89 Zr]Zr-oxine was superior to [ 89 Zr]Zr-DFO-NCS with regard to long-term stability, cellular retention, minimal variation between cell types and cell labelling efficiency

Combining [¹¹C]-AnxA5 PET Imaging with Serum Biomarkers for Improved Detection in Live Mice of Modest Cell Death in Human Solid Tumor Xenografts

<div><h3>Background</h3><p><em>In vivo</em> imaging using Annexin A5-based radioligands is a powerful technique for visualizing massive cell death, but has been less successful in monitoring the modest cell death typically seen in solid tumors after chemotherapy. Here we combined dynamic positron emission tomography (PET) imaging using Annexin A5 with a serum-based apoptosis marker, for improved sensitivity and specificity in assessment of chemotherapy-induced cell death in a solid tumor model.</p> <h3>Methodology/Principal Findings</h3><p>Modest cell death was induced by doxorubicin in a mouse xenograft model with human FaDu head and neck cancer cells. PET imaging was based on ¹¹C-labeled Sel-tagged Annexin A5 ([¹¹C]-AnxA5-ST) and a size-matched control. 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]-FDG) was utilized as a tracer of tissue metabolism. Serum biomarkers for cell death were ccK18 and K18 (M30 Apoptosense® and M65). Apoptosis in tissue sections was verified <em>ex vivo</em> for validation. Both PET imaging using [¹¹C]-AnxA5-ST and serum ccK18/K18 levels revealed treatment-induced cell death, with ccK18 displaying the highest detection sensitivity. [¹⁸F]-FDG uptake was not affected by this treatment in this tumor model. [¹¹C]-AnxA5-ST gave robust imaging readouts at one hour and its short half-life made it possible to perform paired scans in the same animal in one imaging session.</p> <h3>Conclusions/Significance</h3><p>The combined use of dynamic PET with [¹¹C]-AnxA5-ST, showing specific increases in tumor binding potential upon therapy, with ccK18/K18 serum measurements, as highly sensitive markers for cell death, enabled effective assessment of modest therapy-induced cell death in this mouse xenograft model of solid human tumors.</p> </div

Response to doxorubicin at 72 h is revealed with [¹¹C]-AnxA5-ST but not [¹⁸F]-FDG or [¹¹C]-mTrx-GFP-ST.

<p>Representative MIP and transaxial images of radioactivity (summed over 35–55 min) after tail vein injection in mice with FaDu xenografts of <b><i>A</i></b><i>)</i> [¹¹C]-AnxA5-ST or <b><i>B</i></b><i>)</i> [¹¹C]-mTrx-GFP-ST, 72 h after treatment with a single dose of doxorubicin (5 mg/kg). Also shown are representative images of [¹⁸F]-FDG, at <b><i>C</i></b><i>)</i> baseline or, alternatively, <b><i>D</i></b><i>)</i> after treatment with doxorubicin and 90 min after the image with [¹¹C]-AnxA5-ST in <b><i>A</i></b><i>)</i>. <b><i>E</i></b><i>)</i> Time activity curves of single tumors showing uptake of [¹¹C]-AnxA5-ST (solid triangles and squares) or [¹⁸F]-FDG (lines) before (solid triangle and dashed line) or after doxorubicin treatment (solid squares and solid line). <b><i>F</i></b><i>)</i> Corresponding experiments showing a comparison between of [¹¹C]-mTrx-GFP-ST (empty triangles and squares) and [¹⁸F]-FDG (lines) tumor uptake in the same mouse before (empty triangles and dashed line) or after doxorubicin treatment (empty squares and solid line).</p

Increased tumor uptake upon doxorubicin treatment using [¹¹C]-AnxA5-ST but not [¹¹C]-mTrx-GFP-ST.

<p><b><i>A</i></b><i>)</i> MIP and transaxial images of radioactivity (summed over 10–55 min) after tail vein injection of [¹¹C]-AnxA5-ST (left) or [¹¹C]-mTrx-GFP-ST (right) in a mouse bearing a FaDu xenograft without doxorubicin treatment. <b><i>B</i></b><i>)</i> Time activity curves for tumor uptake of [¹¹C]-AnxA5-ST (solid squares or solid line) and [¹¹C]-mTrx-GFP (empty squares or dashed line) in untreated mice (lines) or 72 h after a single-dose treatment with doxorubicin (squares). Note the similar uptake of [¹¹C]-AnxA5-ST and [¹¹C]-mTrx-GFP-ST in untreated mice, due to the non-specific EPR effect, while upon doxorubicin treatment an increase in [¹¹C]-AnxA5-ST uptake was seen in contrast to a non-statistically significant tendency to decrease of [¹¹C]-mTrx-GFP-ST. <b><i>C</i></b><i>)</i> Logan plot analyses of distribution volumes of [¹¹C]-AnxA5-ST or [¹¹C]-mTrx-GFP-ST in FaDu xenograft tumors in untreated and doxorubicin treated mice (symbols as in panel <i>B</i>). <b><i>D</i></b><i>)</i> Diagram summarizing tumor binding potential in PET imaging using either [¹¹C]-AnxA5-ST (solid symbols) or the size-matched control protein [¹¹C]-mTrx-GFP-ST (open symbols) with the mice carrying FaDu xenograft tumors, either with (squares, <i>n</i> = 9 for [¹¹C]-AnxA5-ST and <i>n</i> = 6 for [¹¹C]-mTrx-GFP-ST) or without (triangles, <i>n</i> = 12 for [¹¹C]-AnxA5-ST and <i>n</i> = 8 for [¹¹C]-mTrx-GFP-ST), with asterisks indicating statistical significance between pair of groups. The <i>p</i> values were as follows: [¹¹C]-AnxA5-ST in untreated tumors <i>vs.</i> the same ligand in doxorubicin treated: <i>p = 0.0173</i>; [¹¹C]-AnxA5-ST in doxorubicin treated tumors <i>vs.</i> [¹¹C]-mTrx-GFP-ST in untreated tumors: <i>p = 0.0152</i>; [¹¹C]-AnxA5-ST in doxorubicin treated tumors <i>vs.</i> [¹¹C]-mTrx-GFP-ST in doxorubicin treated tumors: <i>p = 0.0048</i>; remaining pair-wise comparisons showed a lack of statistically significant differences, as indicated for [¹¹C]-mTrx-GFP-ST with or without treatment (n.s. = no significance, <i>p>0.1</i>).</p