Preparation of 68Ge/68Ga radionuclide generator

The generator was prepared using a borosilicate glass column. The dimensions of the column were 100 mm in height and 6 mm of internal radius; filters in the form of porous frits (20 µm) were placed at the top and at the bottom of the column in order to avoid the presence of particulate impurities and to prevent disturbance of the column bed during flow of eluent solution through it. For the purpose of radioprotection, the column was kept in an easily-transportable lead shielded cylindrical shape (250 × 10 × 55 mm; height × inner radius × external radius) and all operations were carried out in a closed system using connecting Teflon tubes. A sterile polyethylene 10 mL syringe was attached to the inlet tube via a Luer-lock connection to elute 68Ga from the generator with HCl (Fig. 2a).

Excess of tin dioxide, synthesized on demand by Keeling & Walker (UK) and previously characterized by our research group22, was calcined at 900 °C for 3 h in an oven and cooled later to room temperature. Next, it was sifted through a 100–150 µm sieve in a stainless steel frame. A borosilicate glass column was packed with 4.5 g of nano-SnO2 and preconditioned with 50 mL of 0.5 M HCl to remove fine particles. Subsequently the column was loaded with 740 MBq of 68Ge in no-carrier-added form (obtained from JSC Isotope, Russia) and washed with 50 mL of 1 M HCl solution in order to remove trace levels of non-adsorbed 68Ge. One day after loading 68Ge, enough time for in-growth of the 68Ga activity, the generator was ready to use.

Gallium-68 was eluted from the generator using 7 mL of hydrochloric acid (0.5–1 M HCl) prepared from a solution of 34% hydrochloric acid, Ultrex II ultrapure reagent (J. T. Baker) at a constant flow rate of 1 or 1.5 mL/min provided by an infusion pump. The generator was designed to operate either in the “dry” or “wet” mode of operation. In the “dry” mode, after each elution the rest of HCl was removed by passing air through the column; it often happens that some HCl remains in the column despite drying. In the “wet” mode, after each elution the column remains filled with HCl till the next elution. The performance of the generator was evaluated for 17 months, eluting it regularly at intervals of once, twice or three times per day (more than 300 elutions during the period of study). The elution profile was studied by collecting the eluates in fractions of 0.5 mL and determining their activity. A dose calibrator model VDC-405 (Veenstra Instruments) was used for the determination of 68Ga activity after elution. The elution yield was expressed as the percentage of the eluted 68Ga activity in relation to the theoretical activity of 68Ga in the generator column after decay correction for the elapsed time. As the half-life of the 68Ge is much greater than the 68Ga, the theoretical activity of 68Ga was calculated by using the equation corresponding to secular radiochemical equilibrium:

$${mathrm{A}}_{mathrm{d}}cong {A}_{p}left(1-{e}^{{lambda }_{d}t}right)$$

where Ap and Ad are the activity of 68Ge and 68Ga respectively and λd (0.0102 min−1)1 is the radioactive decay constant of 68Ga.

The activity of 68Ge in aliquots of eluates was analyzed by gamma-ray spectrometry using a calibrated coaxial High Purity Germanium (HPGe) detector model GEM 13,200 (Ortec) coupled to multichannel gamma-ray analyzer DSA1000 (Canberra). The generator eluates were analyzed after at least 48 h in order to allow the 68Ga to decay to a level that permits the detection of 68Ge since it can be detected indirectly as a decay product, 68Ga. All samples, which contained 1 mL, were measured using the same conditions: constant geometry using 1.5 mL conical bottom tubes made from polypropylene, placed at a distance of 5 cm from the detector and 6 h as minimum measurement time. The activity of each sample was calculated by quantifying the area of 511 keV peak in the spectrum obtained, which corresponds to radioactive decay of 68Ga. The Minimum Detectable Activity (MDA) for 68Ga was 1 Bq. Finally, the 68Ge activity was calculated by direct comparison with the results obtained from the measurement of a standard source of 68Ge, performed by Ionizing Radiations Metrology Laboratory belonging to the CIEMAT, under the same measurement conditions previously mentioned. The generator column was visually checked additionally by small animal PET/CT SuperArgus (SEDECAL, Spain) with the purpose of studying the possible spread of 68Ge zone along the column due to the number of elutions and the time of use of the generator.

The presence of potential chemical impurities (in the form of Ag, Al, As, Ba, Bi, Ca, Cd, Co, Cr, Cu, Fe, In, Mg, Mn, Ni, Pb, Sb, Sn, Ti and Zn) in the 68Ga eluate was determined in three groups according to the elution number: 24–61, 75–124 and 263–275. The trace level detection of the metal ion contamination in decayed samples were carried out by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) using a quadrupole single collector iCAP Q (Thermo Scientific) equipped with a collision cell. The water used was Milli-Q purity (Millipore) and the acids were purified by Sub-Boiling distillation using a duoPUR system (Milestone). The non-disposable materials used were previously washed with 5% HNO3 and distilled water. Quantification was carried out by external calibration and internal standard, preparing the standards by successive dilution of a certified standard (Alfa Aesar and Inorganic Ventures) to 2–5% (v/v) using 65% HNO3 (Sigma-Aldrich) for each of the elements.


Labelling properties of the 68Ge/68Ga generator were tested by a method based on the radiolabelling of DOTA-TOC (DOTA-[Tyr3]-octreotide), provided by ABX (Radeberg, Germany). This peptide, a DOTA derivatized somatostatin analog, shows high affinity to the SSTR2 subtype of somatostatin receptor expressing tumors and binds the trivalent Ga3+ with a high kinetic and thermodynamic stability. It is widely used in clinical routines for PET imaging of neuroendocrine tumors37. The procedure used to label, purify and determine the radiochemical purity of [68Ga]Ga-DOTA-TOC was essentially the same as that used by our group33 with some adjustments. A solution of 210 ± 5 mg HEPES (Sigma-Aldrich) dissolved in 1 mL of deionized water was transferred into a reaction vial which contained the peptide DOTA-TOC previously dissolved in water. The 68Ge/68Ga-generator was eluted with 7 ml of 1 M HCl solution (prepared from 34% HCl, Ultrex II ultrapure reagent, J. T. Baker) and 1 mL of the eluate was transferred to the reaction vial. The final pH of the reaction mixtu re was 3.5–4, measured by indicator paper strips (Merck). After very careful shaking, the mixture was heated for 5 min at 90 ± 5 ºC using a microwave with monomodal radiation and after cooled to room temperature with nitrogen.

The crude product was purified by solid-phase extraction cartridge Sep Pak light C18 cartridge (Waters) previously conditioned and equilibrated with 4 mL of pure ethanol and 4 mL of deionized water. The mixture was transferred onto Sep Pak where [68Ga]Ga-DOTA-TOC and colloidal 68Ga were retained, whereas free 68Ga passed through the cartridge. Next, the cartridge was rinsed with 4 mL of deionized water and the activity on the Sep Pak cartridge was reversely recovered with 0.5 mL ethanol 96% (v/v) (Scharlab); during this procedure all colloidal 68Ga was retained on the Sep Pak cartridge. Finally, ethanol was evaporated to dryness using a Speed Vac (Savant) and [68Ga]Ga-DOTA-TOC was dissolved with a 0.9% NaCl solution (Braun). The radiolabelling yield was calculated by comparing the fraction of 68Ga collected in ethanol with the activity in the initial radiolabelling reaction using decay-corrected radioactivity values.

The radiochemical purity of [68Ga]Ga-DOTA-TOC was determined by radio-High Performance Liquid Chromatography (radio-HPLC) (Jasco) equipped with a photo diode array detector MD-4015 (Jasco), a radioactivity detector LB 507A (Berthold) and the measurements performed under the following conditions. Column: Jupiter Proteo 90 Å column (Phenomenex), 4 µm, 250 × 4.6 mm. Eluents: 0.1% trifluoroacetic acid (TFA) in 5% acetonitrile (solvent A) and 0.1% TFA in 95% acetonitrile (solvent B). Gradient: 0–2 min (5% B), 2–17 min (5–100% B), 17–20 min (100% B), 20–22 min (100–5% B) and 22–24 min (5% B). Flow rate: 1 mL/min. Two aliquots, before and after purification step by Sep Pak, were taken to determine radiochemical purity. TFA was purchased from Sigma-Aldrich and Acetonitrile Ultra Gradient HPLC grade from J. T. Baker.

Preclinical model for pulmonary neuroendocrine tumors

All animal experiments were approved by the Animal Ethical Committee of the Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and conducted in compliance with institutional and national guidelines. A mouse model of high-grade neuroendocrine lung cancer (LCNEC) based on the ablation of four tumor suppressor genes (Rb, Rbl1,pTEN and Trp53) was used to perform imaging studies33, 34, 38.

PET/CT imaging

PET studies were performed in an Argus PET/CT (SEDECAL) scanner. The procedure used was as described by our group33. Briefly, PET studies (energy window 250–700 keV and 30 min static acquisition) and CT studies (voltage 45 kV, current 150  μA, 8 shots, 360 projections, and standard resolution) were performed at 90 min after intravenous injection of [68Ga]Ga-DOTA-TOC (2–5 MBq) in mice anesthetized by inhalation of 2–2.5% of 2–2.5% isoflurane (Esteve) in 100% oxygen (ALPHAGAZ™, Air Liquide) at a flow rate of 5% oxygen using a Fluovac System (Harvard Bioscience). PET images were corrected for random events and scatter with and without attenuation correction and reconstructed using the 2D–OSEM (Ordered Subset Expectation Maximization) algorithm (16 subsets and 2 iterations). For image-reading purposes PET images were fused with the corresponding CT images, which were used as a reference for image co-registration. Images were analyzed using the image analysis software AMIDE, version 1.0.4 (

Histology and Immunohistochemistry

Histology and Immunohistochemistry tests were performed to confirm the diagnosis of the tumor and the expression and localization of SSTR2. Lungs were perfused at necropsy with 4% formaldehyde, fixed and embedded in paraffin wax sections (5 μm) were stained with hematoxylin and eosin (H&E) for histological analysis or processed for immunohistochemistry, essentially as in previously described standard protocols39. For expression of SSTR2, a high temperature antigen unmasking technique consisting of 15-min microwaving of slides in 0.01 M citrate buffer (Antigen Retrieval Buffer, Abcam) was used after deparaffinization to enhance the staining. Sections were then incubated with 5% horse serum (Gibco) for 30 min to block the Fc receptor in tissue, and then washed three times with sterile PBS (Gibco, pH 7.5) prior to incubation with the anti-SSTR2 primary antibody (Sigma-Aldrich, diluted 1:100). Peroxidase-conjugated secondary antibody supplied by the manufacturer was used at 1:300 dilution and visualized using diaminobenzidine as a substrate (DAB kit Vector). Control slides were obtained by replacing primary antibodies with PBS (data not shown).

Statistical analyses

Continuous measurements are presented as mean ± standard deviation (μ ± σ), unless otherwise stated. Mean values were compared using one-way ANOVA followed by post hoc testing or custom contrasts. The difference was considered statistically significant when p value was < 0.05. Statistical analyses were performed using SPSS in 14.0.

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