Molecular imaging of cell death in tumors. Increasing annexin A5 size reduces contribution of phosphatidylserine-targeting function to tumor uptake

Abstract

Objective: Annexin A5 is a phosphatidylserine binding protein that binds dying cells in vivo. Annexin A5 is a potential molecular imaging agent to determine efficacy of anti-cancer therapy in patients. Its rapid clearance from circulation limits tumor uptake and, hence, its sensitivity. The aim of this study is to determine if non-invasive imaging of cell death in tumors will benefit from increasing circulation time of annexin A5 by increasing its size.

Procedures: Annexin A5 size was increased by complexation of biotinylated annexin A5 with Alexa-Fluor680-labeled streptavidin. The non-binding variant of annexin A5, M1234, was used as negative control. The HT29 colon carcinoma xenograft model in NMRI nude mice was used to measure tumor uptake in vivo. Tumor uptake of fluorescent annexin A5-variants was measured using non-invasive optical imaging.

Results: The annexin A5-streptavidin complex (4 ∶ 1, moles:moles, Mw ∼ 200 kDa) binds phosphatidylserine-expressing membranes with a Hill-coefficient of 5.7 ± 0.5 for Ca2+-binding and an EC50 of 0.9 ± 0.1 mM Ca2+ (EC50 is the Ca2+ concentration required for half maximal binding)(annexin A5: Hill-coefficient 3.9 ± 0.2, EC50 1.5 ± 0.2 mM Ca2+). Circulation half-life of annexin A5-streptavidin is ± 21 minutes (circulation half-life of annexin A5 is ± 4 min.). Tumor uptake of annexin A5-streptavidin was higher and persisted longer than annexin A5-uptake but depended less on phosphatidylserine binding.

Conclusion: Increasing annexin A5 size prolongs circulation times and increases tumor uptake, but decreases contribution of PS-targeting to tumor uptake and abolishes power to report efficacy of therapy.

Figure 1. Analyses of purity of anxA5 and M1234, and stoichiometry of anxA5-biotin and anxA5-AF680.

(A) 50 ng purified anxA5 and M1234 were run on an SDS-PAGE. The proteins were visualized by silver staining. (B) Maldi TOF/TOF spectrum of purified anxA5 showing a peak of the monomer at 35772 Da, the dimer at 71589 Da and the double protonated peak at 17826 Da. (C) Maldi TOF/TOF spectrum of anxA5 labeled with maleimide-biotin showing a peak at 36309 Da. Maleimide-biotin labeling resulted in a Mw increase of ±540 Da indicating 1∶1 stoichiometry. (D) Maldi TOF/TOF spectrum of anxA5 labeled with maleimide-AF680 showing a peak at 36672 Da. Maleimide-AF680 labeling resulted in a Mw increase of ±900 Da indicating 1∶1 stoichiometry. Comparable results were obtained with M1234-biotin and M1234-AF680 (not shown).

Figure 2. Three-dimensional (3-D) models of anxA5-Biotin (A) and anxA5-NP (B).

The 3-D structure was retrieved from the 1ANX entry of the Protein Data Bank (PDB). Residue 166 was replaced by a cysteine and coupled to maleimide-PEG2-Biotin using Yasara. The picture was generated using PyMOL. The structure of streptavidin was retrieved from the 1SWG entry of PDB. 4 anxA5-biotin monomers were docked to streptavidin’s biotin binding pockets and the picture was generated using ICM-Pro.

Figure 3. Binding of annexin-variants to phosphatidylserine containing membranes.

Panel A shows calcium-dependent binding of anxA5 (closed circles), M1234 (open circle), anxA5-NP (closed squares) and M1234-NP (open squares) to a synthetic phospholipid surface comprising 20 mole% phosphatidylserine and 80 mole% phosphatidylcholine. Binding was measured by ellipsometry and is expressed as change in degree of the analyser (Δ°) as described elsewhere . Panel B shows the % of apoptotic cells of a population of anti-Fas stimulated Jurkat cells that can be detected using fluorescently labeled annexin-variants and flow cytometry.

Figure 4. Red blood Cell (RBC) binding of anxA5 variants at low membrane density (1*10⁴ molecules/cell) as determined by calcium titration.

(A) Calcium titration curves of anxA5 (open circles) and anxA5-NP (open squares). Each point is the average of three experiments with bars indicating SEM. (B) Hill coefficient (black bars) and EC50 (grey bars) of anxA5 and anxA5-NP as determined from calcium titration curves.

Figure 5. Blood clearance and biodistribution of annexin-variants in NMRI nude mice.

2-labeled annexin-variant were injected intravenously. At various time-points 25 µl blood samples were collected to measure fluorescence. Blood concentration of annexin-variant was calculated using the constructed reference curves. Panel A shows the blood clearance of anxA5 (grey squares), M1234 (black circles), anxA5-NP (black triangles) and M1234-NP (grey triangles). Half-lives were calculated by mono-exponential fit (B). *p<0.05, **p<0.01, ***p<0.001. 2 nmoles of fluorescently labeled anxA5 (black bars, panel C), M1234 (grey bars, panel C), anxA5-NP (black bars, panel D) and M1234-NP (grey bars, panel D) were injected intravenously into tumor-bearing NMRI nude mice. 1 hour (panel C) and 24 hours (panel D) post-injection organs were collected, weighed and measured for fluorescence. Concentration of annexin-variant were calculated using the constructed reference curves.

Figure 6. Non-invasive optical imaging of kinetics of tumor-uptake of fluorescently labeled annexin-variants in tumor bearing NMRI nude mice untreated (–CYP) or treated with cyclophosphamide (+CYP).

Panels A and C show representative pictures of tumors of mice injected with anxA5 and M1234 (A), and anxA5-NP and M1234-NP (C). Panel B illustrates the time courses of tumor levels of anxA5 (closed circles, –CYP; open circles, +CYP) and M1234 (closed squares, –CYP; open squares, +CYP). The inset shows the time-courses during the first 10 hours. Panel D represents the time-courses of tumor levels of anxA5-NP (closed circles, –CYP; open circles, +CYP) and M1234 (closed squares, –CYP; open squares, +CYP).

Figure 7. Determination of apoptosis (A–C) and microvessel density (D) in tumors of NMRI nude mice: effects of cyclophosphamide (CYP) treatment.

Untreated (–CYP) and treated tumors (+CYP) were excised from tumor-bearing mice, frozen and sectioned. Sections were stained with TUNEL-assay and hematoxylin. % TUNEL-positive nuclei was determined as a measure of apoptosis (A–C). Panel A shows TUNEL-staining of a section of an untreated tumor and panel B of a CYP-treated tumor. Sections were stained with the endothelial specific antibody anti-CD31 and hematoxylin. Microvessel density was determined as % of tumor area that is CD31-positive (D). **p<0.01.

Figure 8. 3-compartment model and equations that were employed to fit the experimental data. k is constant, C is concentration (nM) and V is volume (L).

Figure 9. Computational compartmentalization of tumor uptake of anxA5 and M1234.

The experimental data of Fig. 6B were fit according to the model of Fig. 8. Panels A–C show representative fits for anxA5 uptake by untreated (A) and cyclophosphamide treated tumor (B) and for M1234 uptake by cyclophosphamide treated tumor (C.) The experimental data (red circles) were used to calculate concentrations in whole tumor (black line), tumor interstitium (Comp. 2, red line) and bound to the PS-target (Comp. 3, blue line). Panel D shows the concentrations of anxA5 and M1234 in compartment 3 (PS-target) of tumor that was either untreated or treated with cyclophosphamide (+CYP) 6 hours post-injection. *p<0.05, **p<0.01.