18F-C2Am: a targeted imaging agent for detecting tumor cell death in vivo using positron emission tomography

Abstract

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.

Keywords: C2Am; Cell death; PET; Synaptotagmin-I; TRAILR2; Tumor.

Fig. 1

Radiochemical labeling of C2Am, using N-(5-[¹⁸F]fluoropentyl) maleimide, to yield ¹⁸F-C2Am. The rapid Michael addition reaction takes place under mild conditions (20 °C, pH5, HBS buffer) in the presence of ascorbic acid. The secondary structure of C2Am is shown, highlighting the single cysteine residue (top, yellow) and the active site (lower) containing three calcium ions (yellow spheres)

Fig. 2

C2Am binds to dead cells in vitro. Flow cytometric analysis of MEDI3039-treated MDA-MB-231 (a) and Colo205 (b) cells shows three distinct populations (first and third plots, from left), which were gated based on their levels of UV_A autofluorescence (NADH content) and plasma membrane integrity (Sytox R) as: viable (v%), apoptotic (a%) and necrotic (n%) cells. C2Am-AF750, a near infrared fluorophore-labeled derivative of C2Am labels (second and fourth plots) preferentially bound apoptotic (orange) and necrotic (red) cells, in comparison with viable (green) cells (c, d). Plots of C2Am-AF750 mean fluorescence intensity (MFI) ratios for necrotic/viable, apoptotic/viable and dead/viable MDA-MB-231 (e) and Colo205 (f) cells following treatment with MEDI3039 (mean ± SD, n = 3), error bars lie within the symbols when not shown. MEDI3039-treated MDA-MB-231 (g) and Colo205 (h) cells were incubated with ¹⁸F-C2Am at different concentrations (1–1000 nM) and the fraction of activity retained in the cell pellets (%) after three washes was measured. The signal-to-background ratio (SBR, black bars) of treated (grey bars) versus vehicle-treated (open bars, control) cells is also shown

Fig. 3

Biodistribution profile of ¹⁸F-C2Am, 2 h post-administration. Mice bearing Colo205 tumors were treated with MEDI3039 (closed bars; 0.4 mg/kg, i.v., 24 h) or vehicle (open bars). ¹⁸F-C2Am was injected (1 MBq, i.v.) and tissues collected post-mortem 2 h following administration. Fraction of injected activity per gram of tissue (IA/g, %) (mean ± SEM, n = 5 per treatment group). Two-tailed, t test, unequal variance. *P < 0.05

Fig. 4

PET images of ¹⁸F-C2Am in tumor-bearing mice, pre- and post-treatment. Maximum intensity projections of the PET signal in sagittal sections of representative mice, bearing MDA-MB-231 (a) and Colo205 (b) tumors. Projections are shown from immediately before (0) and up to 120 min after injection of ¹⁸F-C2Am. Signal is shown as injected activity per gram of tissue (IA/g, %) and overlaid on a CT-derived skeleton mask. White arrows indicate tumor location. Time–activity (IA/g, %) curves for the indicated tissues in treated (MEDI3039, 0.4 mg/kg, 24 h) MDA-MB-231 (c) and Colo205 (d) tumor-bearing mice. Also shown are tumor-to-blood (T/b) and tumor-to-muscle (T/m) ratios for both tumor models. e, f Pairwise analysis of PET signal pre- and 1 h (first and third columns) or 2 h (second and fourth columns) post-treatment, expressed as IA/g (%) (e) or SUV (f). Pairwise analysis (g) of tumor bioluminescence pre- and post-treatment (first and third plots) and correlation analysis (Pearson) of tumor PET signal with the reduction in BLI signal (second and fourth plots), 1 h after ¹⁸F-C2Am administration. Data (c, d) is mean ± SEM, n = 5−6 per treatment group. Black and red symbols (e, g) correspond to MEDI3039 treatment at 0.4 or 0.1 mg/kg (for 24 h), respectively. e, g Two-tailed, pairwise t test, unequal variance. *P < 0.05, **P < 0.01

Fig. 5

Reduced tumor uptake of (inactive) ¹⁸F-iC2Am versus (active) ¹⁸F-C2Am in MEDI3039-treated mice (0.4 mg/kg, 24 h, i.v.) bearing Colo205 tumors. Red lined triangles (¹⁸F-C2Am); black lined squares (¹⁸F-iC2Am)

Fig. 6

Effect of pre-blocking of PS using (× 10) molar excess of unlabeled C2Am on ¹⁸F-C2Am tumor contrast in MEDI3039-treated mice (0.4 mg/kg, 24 h, i.v.) bearing MDA-MB-231 tumors. Red lined triangles (¹⁸F-C2Am; no pre-blocking); black lined squares (¹⁸F-C2Am; 10x pre-blocking with C2Am)

Fig. 7

Pearson correlation analysis of tumor PET signal with histological markers of cell death. a Pearson P (left) and R (right) correlation values for tumor signal, expressed as different contrast metrics, 1 h and 2 h post-administration of ¹⁸F-C2Am, with cell death markers (CC3, open bars, or TUNEL, closed bars) estimated from two sections (200 µm apart) from the same tumors, from both Colo205 and MDA-MB-231 models. (b) Pearson correlation analysis of tumor contrast, expressed as IA/g, % (left), SUV (middle) and T/b ratio (right), 1 h (top row) or 2 h (lower row) after ¹⁸F-C2Am administration, with tumor cell death (CC3, %). Tumor contrast is expressed as the signal ratio, post/pre-treatment. Pooled data (a, b) from Colo205 (n = 9, red symbols) and MDA-MB-231 (n = 9, blue symbols) tumor-bearing mice, treated with MEDI3039 (0.1 or 0.4 mg/kg, 24 h, i.v., filled symbols), 5FU (Colo205, 100 mg/kg, i.p., 24 h, open red symbols) or doxorubicin (MDA-MB-231, 100 mg/kg, i.p., 24 h, open blue symbols). Two-tailed, Pearson P and R values are shown (a). CC3 cleaved caspase 3, TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. T/b tumor-to-blood ratio, Tx treatment

Fig. 8

Correlation of ¹⁸F-C2A signal with immunohistochemical markers of cell death for representative MDA-MB-231 and Colo205 tumors. a Cleaved caspase-3 (CC3). Tumor section positive CC3 pixel count values (%) are shown. b Maximum intensity projection (MIP) of PET signal (IA/g, %). c Autoradiography of tumor sections acquired immediately after imaging (% of area with signal). Tumors were collected from different mice, bearing either Colo205 or MDA-MB-231, 2 h following administration of ¹⁸F-C2Am. MEDI3039 (MEDI) used at 0.4 mg/kg, i.v., for 24 h. 5FU and Doxorubicin (Dox) were both used at 100 mg/kg, i.p., for 24 h. UT untreated tumors