Synthesis and radiolabeling
CPCR4.3 was synthesized as described [10]. The corresponding 3-iodo-Tyr-analog was obtained by iodination of CPCR4.3 with N-iodo-succinimide [11].
The reference ligand [68Ga] Pentixafor was prepared according to a previously established protocol [2]. Radioiodination of CXCR4.3 was carried out using the IodoGen® method. Briefly, 100–200 μg of peptide were dissolved in 0.5 mL TRIS iodination buffer (25 mM Tris·HCl, 0.4 M NaCl, pH 7.5) and transferred to an Eppendorf reaction tube coated with 150 μg of IodoGen®. Upon addition of [125I] NaI (18–20 MBq, Hartmann Analytik, Braunschweig, Germany), the reaction vessel was briefly vortexed and the labeling reaction was allowed to proceed for 15 min at RT. The peptide solution was then removed from the insoluble oxidizing agent. Separation of [125I]CPCR4.3 from unlabeled precursor was achieved using gradient RP-HPLC (column: Nucleosil 100 C18 (5 μm, 125 × 4.0 mm; CS GmbH, Langerwehe, Germany), gradient: 22–42% ethanol (0.5% acetic acid) in water (0.5% acetic acid) within 20 min, flow: 1 mL/min).
For in vitro binding and uptake studies, the HPLC product fraction was used as such and diluted to the required concentration using the respective assay medium. For biodistribution experiments, excess ethanol was removed by bubbling an argon stream through the product fraction at 90 °C for 20 min. [125I]CPCR4.3 was then reconstituted to an activity concentration of app. 1 MBq/100 μL using PBS and was then used for the in vivo study.
Lipophilicity
The lipophilicity (log P) of [125I]CPCR4.3 was determined via a modified shake-flask method as described previously [12].
Cell culture
Unless stated otherwise, the cell lines used in this study were purchased from ECACC (European Collection of Authenticated Cell Cultures, Salisbury, UK), DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) or ATCC (American Type Culture Collection, Wesel, Germany). Jurkat human T lymphocyte cells (DSMZ: ACC282), SH-SY5Y human neuroblastoma cells (ECACC: 94030304), and 4 T1 mouse mammary carcinoma cells (ATCC: CRL-2539) were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS). Daudi human Burkitt lymphoma cells (ECACC: 85011437) were cultured in RPMI-1640 medium supplemented with 10% FCS, 2 mM l-glutamine, 1% non-essential amino acids, 50 μM β-mercaptoethanol, and 100 units/mL of penicillin/streptomycin (P/S). Eμ-myc 1080 mouse B cell lymphoma cells (kindly provided by Prof. Ulrich Keller, Charité, Berlin) [13] were grown in RPMI 1640 medium supplemented with 20% FCS, 1% NEA, 100 units/mL of P/S, and 0.1% 2-mercaptoethanol. The human colon carcinoma cell line HT-29 (ECACC: 91072201), the breast cancer cell line MCF-7 (ECACC: 86012803), and LNCaP human prostate carcinoma cells (DSMZ: ACC256) were cultured in DMEM/F-12 medium with Glutamax-I (1:1) supplemented with 10% FCS. CHO-K1 cells (Chinese hamster ovary cells, ECACC: 85051005) were cultured in RPMI-1640 medium supplemented with 10% FBS, 2 mM l-glutamine, and 100 units/mL of P/S. All cell lines were maintained at 37 °C in a humidified 5% CO2 atmosphere. Media and supplements were obtained from Biochrom (Berlin, Germany) or Gibco (life technologies, Darmstadt, Germany).
In the assay medium used for internalization studies, FCS was replaced by 5% bovine serum albumin (BSA; Sigma, St. Louis, USA). For cell counting, a Countesse automated cell counter (Invitrogen, Carlsbad, USA) was used.
Comparative binding studies using various cell lines
CXCR4-mediated cellular uptake of [125I]CPCR4.3 and the reference [68Ga] Pentixafor was investigated in various cell lines with different endogenous hCXCR4 (Jurkat, Daudi, HT-19, MCF-7, SH-5YSY, LNCaP) and mCXCR4 (Eμ-myc 1080, 4 T1) expression levels. For data validation, CHO-K1 cells transiently transfected with hCXCR4 and mCXCR4 [2] were also included, with non-transfected CHO-K1 cells serving as CXCR4 negative controls.
In the case of suspension cells (Daudi, Jurkat, Eμ-myc 1080), samples containing 2 × 105 cells in assay medium were incubated with [125I]CPCR4.3 (0.1 nM) or the reference [68Ga] Pentixafor (1 nM) at 37 °C for 60 min in the presence (non-specific binding) or absence (control) of 100 μM AMD3100 (n = 3 per concentration, total sample volume: 250 μL). After incubation, the tubes were centrifuged (3 min, 1300 rcf, Megafuge 1.0, Heraeus Thermo Scientific) and the supernatant was carefully removed. After washing twice with 200 μL of cold HBSS, the amount of bound radioligand in the cell pellet was quantified using a γ-counter (WALLAC; 1480 WIZARD™ 3″).
In the case of the adherent cell lines (CHO, 4 T1, HT-19, MCF-7, SH-5YSY, LNCaP), app. 150,000 cells/well were seeded into PLL-coated 24-well plates (Greiner, Bio One, Frickenhausen, Germany) on the day prior to the experiment. On the day of the experiment, the culture medium was removed, and the cells were left to equilibrate in 200 μL of assay medium (RPMI + 5% BSA) at 37 °C for a minimum of 15 min before the experiment. Then, the cells were incubated with [125I]CPCR4.3 (0.1 nM) or the reference [68Ga] Pentixafor (1 nM) at 37 °C for 60 min in the presence (non-specific binding) or absence (control) of 100 μM AMD3100 (n = 3 per concentration, total sample volume: 250 μL). Upon incubation, the incubation medium was removed, and cells were rinsed twice with 250 μL of HBSS and lysed using 300 μL of 1 N NaOH. The lysate was transferred to vials and combined with 250 μL of HBSS used for rinsing the wells. Quantification of the amount of free and bound activity was performed in an Automatic Gamma Counter.
Determination of IC50
Affinities for human and murine CXCR4 (hCXCR4 and mCXCR4) were determined in competitive binding assays (IC50) [14] using either Jurkat or Eμ-Myc 1080 mouse lymphoma cells (2 × 105 cells/sample) in Hank’s buffered salt solution (1% BSA) and [125I]CPCR4.3 as radioligand. To allow data normalization, [natGa] Pentixafor and FC-131 were included as references in this study. Experiments were performed in triplicate with n = 3 per concentration in each experiment. IC50 values were calculated using GraphPad Prism 6.01 (Graph Pad Software, San Diego, USA).
Internalization and externalization
The internalization of [125I]CPCR4.3 and [68Ga] Pentixafor into the human cancer cell lines HT-29, SH-5YSY, MCF-7, and LNCaP was investigated in analogy to a previously published protocol [15]. Non-specific internalization was determined in the presence of 100 μM AMD3100.
To determine ligand washout and recycling kinetics, cells (CHO-hCXCR4, CHO-mCXCR4, as well as HT-29 and MCF-7 cells as representative cell lines endogenously expressing hCXCR4) were first incubated with [125I]CPCR4.3 (0.1 nM) at 37 °C for 30 min and washed with HBSS. In the experiment allowing ligand recycling, 250 μL of assay medium were added to the wells (n = 3). In the experiment inhibiting ligand recycling, 250 μL of assay medium containing 100 μM AMD3100 were added to the wells (n = 3). Subsequently, cells were incubated at 37 °C for 5, 15, 30, and 60 min, respectively. The supernatant was removed and combined with 250 μL of HBSS used for rinsing the cells. This fraction represents the amount of externalized ligand at the respective time point. The following lysis of the cells was performed as described for the radioligand binding study.
In vivo biodistribution studies
Animal experiments were performed in accordance with the current animal welfare regulations in Germany (approval #55.2-1-54-2532-71-13).
To investigate the biodistribution of [125I]CPCR4.3, approximately 550 kBq (15 μCi) of [125I]CPCR4.3 in a total volume of 100 μL of PBS (pH 7.4) were injected intravenously (i.v.) into the tail vein of immunocompetent Black Six mice (n = 4) under isoflurane anesthesia. To determine mCXCR4 specificity of ligand accumulation, 50 μg AMD3100/mouse were coinjected (n = 4). The animals were sacrificed 60 min post injection (p.i.), and the organs of interest were dissected. The radioactivity was measured in weighted tissue samples using a γ-counter. Data are expressed in % ID/g tissue (mean ± SD). For a comparative biodistribution study in an immunodeficient mouse strain, CD-1 nu/nu mice were used. Statistical analysis (one-tailed t test) of the separate biodistribution data sets was performed using Microsoft Excel.