Radiolabeled RGD tracer kinetics annotates differential αvβ3 integrin expression linked to cell intrinsic and vessel expression

Israt S. Alam, Timothy H. Witney, Giampaolo Tomasi, Laurence Carroll, Frazer J. Twyman Quang-Dé Nguyen and Eric O. Aboagye.

Comprehensive Cancer Imaging Centre, Faculty of Medicine, Imperial College London, London W12 0NN, UK.

Correspondence to: Eric Aboagye; e-mail:

Electronic Supplementary Materials, Molecular Imaging and Biology:

Materials and Methods

1)  Flow cytometry

Cell pellets (0.5 x 106) were washed in ice-cold phosphate buffered saline (PBS) with 1% BSA and resuspended in 100μl of the same buffer containing anti-Integrin αvβ3 antibody clone LM609 conjugated to fluorescein isothiocyanate (FITC; Millipore, M.A., U.S.A.). Cells were incubated with the antibody for 30 minutes at 4°C. Cells were subsequently washed twice, resuspended in PBS and kept briefly on ice and then analyzed in an LSRII flow cytometer (BD Biosciences, Rockville, MD USA), with 10, 000 events/analysis recorded. Data were analyzed using FlowJo software Tree Star, Inc. Ashland, OR, U.S.A).

2) Western blot

For whole-cell lysate preparation, cells were harvested and lysed in RIPA buffer (Thermo Fisher Scientific Inc., Rockford, IL, USA). Plasma membrane proteins were fractionated using a membrane protein extraction kit from Biovision (Mountain View, CA) [18]. The final protein pellet was resuspended in 0.5% SDS in PBS. Whole cell and plasma membrane proteins were resolved by SDS-PAGE and transferred onto polyvinylidene fluoride membranes using a standard western blotting protocol. Membranes were probed using a rabbit anti-β3 integrin antibody (Cell Signalling Technology, Danvers, Massachusetts, USA; 1:1000). Rabbit anti-actin antibody (Sigma-Aldrich Co. Ltd, Poole, Dorset, UK; 1:5000) was used as loading control and a peroxidase-conjugated donkey anti-rabbit IgG antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; 1:2000) as the secondary antibody.

Proteins were visualized using the Amersham ECL kit (GE Healthcare, Chalfont St Giles, Bucks, UK). Blots were scanned (Bio-Rad GS-800 Calibrated Densitometer; Bio-Rad, Hercules, CA, USA) and signal quantification was performed by densitometry using scanning analysis software (Quantity One; Bio-Rad).

3) Radiochemistry

In this study we radiolabeled DOTA-cyclic RGDfK dimer (DOTA-[c(RGDfK)]2), with 68GaCl3 eluted from a TiO2-based 370 MBq 68Ge/68Ga generator (Eckert & Ziegler Isotope Products IGG100, Berlin, Germany) using 0.1 M HCl (Sigma-Aldrich). 10ml of acid was used to elute the generator, with the first 1.5ml bolus containing the majority of the activity collected and used for labelling. DOTA-[c(RGDfK)] dimer acetate was purchased from ABX advanced biochemical compounds GmbH (Radeberg, Germany). 30µl (1mg/ml H2O) of DOTA-[c(RGDfK)]2 dimer acetate was added to 120µl sodium acetate (160mg/ml) in a reactor vessel. Then, 1.5 ml of 68GaCl3 (315-370 MBq) was added. The reaction was heated at 100oC for 420 seconds and subsequently trapped on tC18 cartridge (pre-conditioned with 2ml ethanol and then 2ml water). The cartridge was washed with water (5 ml), before the labelled compound was eluted (in 100 µl fractions) with 25% ethanol in sterile PBS (v/v).

4) In vitro cell uptake of 68Ga-DOTA-[c(RGDfK)]2

Cells (3 x 105) were plated into 6-well plates the night prior to analysis. On the day of the experiment, fresh growth medium, containing 40 µCi 68Ga-DOTA-[c(RGDfK)]2, was added to individual wells. Cell uptake was measured following incubation at 37°C in a humidified atmosphere of 5% CO2 for 30 minutes. Plates were subsequently placed on ice, washed 3 times with ice-cold PBS and lysed in RIPA buffer (Thermo Fisher Scientific Inc., Rockford, IL, USA; 0.5 ml, 10 min). For the M21L suspension cell line, the cells were washed by spinning down in Eppendorf tubes for 3 minutes at 600g. Cell lysates were transferred to counting tubes and decay-corrected radioactivity was determined on a gamma counter (Cobra II Auto-Gamma counter, Packard Biosciences Co, Pangbourne, UK). Aliquots were snap-frozen and used for protein determination following radioactive decay according to a BCA 96-well plate assay (Thermo Fisher Scientific Inc., Rockford, IL, USA). Data were expressed as percent of total radioactivity per mg protein. For blocking studies, cells were incubated with a cold monomer RGD (10mM; Sigma-Aldrich) for 30 minutes prior to addition of radioactivity and for the duration of the uptake time course.

5) Biodistribution study

For the biodistribution study, mice were maintained under anesthesia following their PET scan and sacrificed by exsanguination via cardiac puncture at 60 min post radiotracer injection to obtain blood, plasma, urine, heart, lung, liver, kidney, muscle and tumour. Tissue radioactivity was determined on a gamma counter (Cobra II Auto-Gamma counter, Packard Biosciences Co, Pangbourne, UK) and decay corrected. Data were expressed as percent injected dose per gram of tissue.

6) In vivo tracer metabolism

Briefly, female BALB/c mice under general anaesthesia (2.5% isofluorane; non-recovery anaesthesia) were administered a bolus i.v. injection of 68Ga-DOTA-[c(RGDfK)]2 (~ 3.7 MBq) and sacrificed by exsanguination via cardiac puncture at 30 min post radiotracer injection. Aliquots of heparinized blood were rapidly centrifuged (14000 g, 5 min, 4oC) to obtain plasma. Plasma samples were subsequently snap-frozen in liquid nitrogen and kept on dry ice prior to analysis. Kidney and liver samples were immediately snap-frozen on dry ice.

To process the samples for HPLC analysis, samples were thawed and kept at 4°C immediately before use. Ice cold methanol (1.5 ml) was added to the ice cold plasma (200 μl) and the resulting suspension centrifuged (14000 g; 4°C; 3 min). The supernatant was then decanted and evaporated to dryness on a rotary evaporator (bath temperature, 40°C), then resuspended in HPLC mobile phase (Solvent A: acetonitrile/water/ethanol/acetic acid/1.0 M ammonium acetate/0.1 M sodium phosphate [800/127/68/2/3/10]; 1.1 ml). Samples were filtered through a hydrophilic syringe filter (0.2 μm filter; Millex PTFE filter, Millipore, MA., USA) and the sample (∼1ml) then injected via a 1 ml sample loop onto the HPLC for analysis.

Tissues were homogenized in ice-cold methanol (1.5 ml) using an Ultra-Turrax T-25 homogenizer (IKA Werke GmbH and Co. KG, Staufen, Germany) and subsequently treated as per plasma samples. Samples were analyzed on an Agilent 1100 series HPLC system (Agilent Technologies, Santa Clara, CA, USA), configured as described above, using the method of [23]. A Gemini C18 HPLC column (Phenomenex, CA, USA.; 4.6 x 150 mm) stationary phase and a mobile phase comprising of Solvent A (water/0.1% TFA) and Solvent B (acetonitrile/0.1% TFA) delivered at a flow rate of 1 ml/min were used for analyte separation. The gradient was set as follows: 95% A for 2 mins; 95% to 5% A in 10 min; 5% A for 2 mins; 5% to 95% A in 2 min.

7) Calculating microvessel density

Microvessel density (MVD) was assessed from CD31 stained sections using a method originally described by Weidner et al [21]. Briefly, MVD was measured by microscopic examination of most vascularized areas of the tumour. Areas of greatest staining were primarily assessed at a low magnification of x100 (x10 objective lens and x10 ocular lens). After selection of representative zones, MVD was counted at a higher magnification of x400 (x40 objective lens and x10 ocular lens). A single microvessel was defined as a discrete cluster of cells positive for CD31 staining, with no requirement for the presence of a lumen. Mean vessel density represents the mean number of vessels counted on four separate microscopic fields performed at x400 magnification.

Results

Fig. S1. Biodistribution of 68Ga-DOTA-[c(RGDfK)]2 in M21, M21L and MDA-MB-231 tumour bearing mice 60 minutes post injection of tracer. SI, small intestine; LI, large intestine. High activity in the urine and kidney is suggestive of renal excretion of the radiotracer. (Data points represent mean ± SEM, n = 6, *p<0.05, **p<0.01, ***p<0.001).

Fig. S2. Representative radio-chromatograms of 68Ga-DOTA-[c(RGDfK)]2 standard and of plasma, kidney and liver samples taken from mice 30 minutes post-administration of the tracer. Graphs depict activity in counts per second (CPS) against time. The dimeric RGD tracer is metabolically stable in these samples (n=3).

Fig. S3. Immunohistochemical staining of β3 integrin expression in two different M21 tumours showing high (a and b) and low (c and d) levels of staining. Total magnification: x200 (left column, a and c) and x600 (right column, b and d). Scale bar represents 100 µm and 50 µm at x200 and x600 magnification respectively.

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