Molecular imaging probes such as PET-tracers have the potential to improve the accuracy of tumor characterization by directly visualizing the biochemical situation. Thus, molecular changes can be detected early before morphological manifestation. The A3 adenosine receptor (A3AR) is described to be highly expressed in colon cancer cell lines and human colorectal cancer (CRC), suggesting this receptor as a tumor marker. The aim of this preclinical study was the evaluation of
Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths in men and women worldwide [
An essential characteristic of most solid tumors is hypoxia, which inevitably leads to accumulation of adenosine within the tumor microenvironment as a result of the breakdown of adenine nucleotides, which has been recognized in the 1990s [
High expression of A3AR mRNA and protein has been reported in colon and breast carcinoma compared to adjacent nonneoplastic tissue by Madi et al. Remarkably, even higher levels of A3AR mRNA have been found in lymph node metastases than in primary tumor tissue, suggesting A3AR-overexpression as a marker for tumor progression [
The A3AR antagonist
HT-29 cells and CHO-K1 cells were purchased from ATCC. HT-29 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 10
Six-week-old male BALB/c mice (BALB/cAnNRj, Division of Laboratory Animal Science and Genetics, Himberg, Austria) were kept under conventional housing conditions, with food and water supply ad libitum and a 12 h day/night cycle. Male, immunodeficient CB17-SCID mice (CB-17/Icr-
After 10 to 14 days upon arrival, CB17-SCID mice were injected subcutaneously with 2 × 106 HT-29 cells into one flank and 2 × 106 CHO-K1 cells in the opposite flank (
Colorectal carcinoma tissue and adjacent healthy colon tissue were obtained directly after tumorectomy from two patients after full informed consent and quick-frozen in 2-methylbutane (−40°C). Tissue was sliced into 16
Competitive binding assays were performed using hA1AR,
For the flow cytometric evaluation of hA3AR expression, single-cell suspensions of HT-29 cells (2 × 105 per tube) were fixed and permeabilized using Cytofix/Cytoperm™ kit (BD Biosciences). Cells were incubated with mouse monoclonal anti-human A3AR (100
Cell lysates were prepared from 75 cm2 cell culture flasks when cells reached 80% confluency using radioimmunoprecipitation assay (RIPA) buffer and protease inhibitor cocktail according to the manufacturer’s instructions. Tissue lysates from HT-29 xenografts were prepared according to a standard protocol using RIPA buffer (according to sample size approx. 4 times of lysis buffer), protease inhibitor, and Ultra-Turrax® for homogenization. The protein concentration of cell lysates was determined using Pierce™ BCA Protein Assay Kit (Thermo Scientific), and 20
HT-29 cells were seeded on chamber slides (3 × 105/mL, 200
Tissue slices were thawed and reconstituted in assay buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM EDTA, 1% BSA, 1 unit adenosine deaminase/100 mL) for 30 min at RT. Radiosynthesis of
Vicinal cryosections of colorectal carcinoma and healthy colon tissue were stained to identify regions with hA3AR expression following a standard protocol. In brief, cryosections were fixed (96% ethanol, 10 min), permeabilized (0.2% Triton X-100 in PBS, 5 min), and blocked using Bloxall™ Blocking Solution and a dedicated avidin/biotin blocking kit (Invitrogen, Thermo Fisher Scientific). Additionally, sections were incubated with goat serum (1 : 10 in PBS) to reduce nonspecific binding. Rabbit polyclonal anti-A3AR (1 : 100, ab203298; Abcam) was used 1 : 100 in PBS + 0.1% BSA for 1 h in a humid, dark chamber. Purified rabbit IgG (Life technologies) was used as an isotype control likewise. Cryosections were washed 3 times for 5 min (PBS + 0.1% Tween-20) and incubated with biotinylated anti-rabbit IgG (1 : 200, PBS + 5% goat serum) for 30 min. After washing, further detection was performed with the Vectastain® ABC kit (Vector Laboratories) according to the manufacturer’s instructions. DAB substrate kit (Abcam) was used as a chromogen to detect peroxidase, and haematoxylin was used for counterstaining of cell nuclei. Immunohistochemically stained slides were acquired on an automated TissueFAXS microscope system (TissueGnostics, Vienna, Austria) at a 5-fold and 20-fold magnification.
Stability of
Radiosynthesis was performed as described elsewhere [
Ex vivo biodistribution of
Xenograft-bearing CB17-SCID mice were anesthetized using isoflurane (2.5%) mixed with oxygen (1.5 L/min) to avoid movement during the imaging. Blocking agents (2 mg/kg BW FE@SUPPY or MRS1523) or the respective vehicle control (Tween-20/EtOH/0.9% physiological saline 1/9/90) was administered retroorbitally 2 min prior to the radiotracer administration (
Affinity and selectivity of FE@SUPPY and its potential metabolites upon cleavage by carboxylesterases, DFE@SUPPY, and FE@SUPPY:11 [
Affinity and selectivity data of FE@SUPPY and metabolites towards adenosine receptor subtypes (
Compound | hA1AR |
|
hA3AR | hA1/hA3AR |
|
---|---|---|---|---|---|
FE@SUPPY |
|
|
|
669 | 285 |
DFE@SUPPY |
|
|
|
324 | 112 |
FE@SUPPY:11 | ≥57 |
|
|
≥20 | 2 |
The human colorectal adenocarcinoma cell line (HT-29) was characterized regarding its hA3AR protein expression using flow cytometry and western blot. Flow cytometric analysis resulted in mean fluorescence intensity (
Flow cytometric analysis of HT-29 cells revealed expression of hA3AR protein (green). The isotype control did not show fluorescence signal (blue).
Fluorescence microscopy of HT-29 cells showed cell membrane-specific staining, pointing at the expression of hA3AR on the cell surface, which is typical for GPCRs (Figure
Immunofluorescent staining of HT-29 cells; left: DAPI, middle: FITC, right: merge. Upper row: mouse isotype control, lower row: anti-human A3AR staining.
In all investigated regions of the two CRC patients,
Upper row: immunohistochemical staining of hA3AR. Lower row: autoradiographic analysis of the corresponding vicinal tissue slices using
Biodistribution of
Biodistribution was assessed 70 min after tracer application in healthy BALB/c mice and revealed a high accumulation of radioactivity in fat-rich regions (brown adipose tissue, BAT) likely due to the tracer’s lipophilicity [
Western blot analysis of HT-29 cell lysate and tissue lysate derived from HT-29 xenograft tumors. CHO-K1-hA3AR cell lysate was loaded as a positive control. An additional bond of unknown identity was detected in HT-29 cell lysate.
For a detailed analysis of the pharmacokinetics, volumes of interest were also generated for mouse body liquids including blood, urine, and bile fluid (Figure
Adenosine concentrations of ~0,5
We found a favorable binding profile of
The authors declare that they have no conflicts of interest.
This work was supported by the Austrian Science Fund (FWF P26502-B24, M. Mitterhauser) and was performed with the support of the Molecular Imaging Cluster of the Medical University of Vienna, Austria. J. Fazekas-Singer was supported by Grant CCHD W1205-B09 awarded to Dr. E. Jensen-Jarolim. The authors want to thank Petra Fürlinger for her collaboration in the autoradiography experiments and Markus Zeilinger for his support in statistics.
Table S1: binding values of FE@SUPPY are expressed as fmol/mm2 tissue.