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

Cyclin-Dependent Kinase-Associated Proteins Cks1 and Cks2 Are Essential during Early Embryogenesis and for Cell Cycle Progression in Somatic Cells ▿

Cks proteins associate with cyclin-dependent kinases and have therefore been assumed to play a direct role in cell cycle regulation. Mammals have two paralogs, Cks1 and Cks2, and individually deleting the gene encoding either in the mouse has previously been shown not to impact viability. In this study we show that simultaneously disrupting CKS1 and CKS2 leads to embryonic lethality, with embryos dying at or before the morula stage after only two to four cell division cycles. RNA interference (RNAi)-mediated silencing of CKS genes in mouse embryonic fibroblasts (MEFs) or HeLa cells causes cessation of proliferation. In MEFs CKS silencing leads to cell cycle arrest in G2, followed by rereplication and polyploidy. This phenotype can be attributed to impaired transcription of the CCNB1, CCNA2, and CDK1 genes, encoding cyclin B1, cyclin A, and Cdk1, respectively. Restoration of cyclin B1 expression rescues the cell cycle arrest phenotype conferred by RNAi-mediated Cks protein depletion. Consistent with a direct role in transcription, Cks2 is recruited to chromatin in general and to the promoter regions and open reading frames of genes requiring Cks function with a cell cycle periodicity that correlates with their transcription

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

Additional file 1: Figure S1. of Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

PET image, summed 40–60 min, of the uptake of targeting [methyl-11C]-ZEGFR:2377-ST-CH3 in one SCID mouse (prone) bearing one s.c. FaDu tumor (1 × 106 cells, 13 days). Only 16-μg protein was injected and nearly all radioactivity localized very quickly in the liver giving SUV mean >2.5 times larger than for animals receiving spiked tracer injections. The tumor (white arrow) was barely discernible with a faint vascular signal. Even radioactivity distributing to the kidneys and urinary bladder during the 60 min was markedly reduced. Thereafter, the amount of protein administered was adjusted to 50–100 mg to partially block the hepatic uptake and free the ligand for tumor targeting, as discussed in the text. (PPTX 422 kb