Background
β8-Integrin was first described in 1991 [
1] as one of five β-integrins (β1, -3, -5, -6, and -8) pairing with the αv monomer, which is the only α integrin it dimerizes with. Although αvβ8 is comparable to other αv integrins (particularly αvβ3) in that it recognizes the extracellular matrix (ECM) protein vitronectin (Vn) by the arginine-glycine-aspartate (RGD) sequence contained therein, its uniqueness is manifested by the fact that unlike αvβ3, αvβ5, or αvβ1, transmembrane αvβ8 does not exert adhesive forces, i.e., does not promote cell binding to Vn [
2]. Hence, unlike other integrins which transmit physical forces and thereby enable the adhesion of cells to ECM proteins, αvβ8 appears to be involved mainly into signaling.
Mounting experimental evidence suggested that explaining the biological role and significance of αvβ8-integrin requires, in essence, a closer view on a family of mammalian cytokines called transforming growth factor beta (TGF-β1 and -3, herein referred to as TGF-β) [
3]. TGF-β is produced by almost any cell type and secreted into the extracellular space, albeit not as a free protein ligand capable of binding to its respective receptor, but as a "protected", i.e., inactive, aggregate with another inhibitory protein referred to as latency-associated peptide (LAP). This aggregate, called small latency complex (SLC), is often covalently linked to the ECM by another protein called latent TFG-β binding protein (LTBP), forming the so-called large latent complex (LLC). An essential functionality of αv-integrins (above all, αvβ8, and αvβ6) is their ability to release TGF-β from its complex with LAP by binding to a RGD sequence contained therein [
4,
5]. Again, αvβ8-integrin displays a unique mode of action. While a pulling force exerted by αvβ6-integrin distorts the structure of LAP and exposes TGF-β to its receptor [
6,
7], αvβ8 achieves a similar result by dragging the SLC into close proximity of matrix metalloprotease 14 (MMP14, synonym: MT1-MMP), which cleaves LAP and thereby generates free TGF-β [
8]. Taken together, expression of αvβ8-integrin first and foremost enables cells to liberate TGF-β from its latent complexes in the extracellular space, and αvβ8 expression is therefore closely connected to TGF-β-related signaling and its role in pathogenesis, particularly of fibrosis and cancer [
9].
Generally, TGF-β acts as a growth suppressor onto normal cells and may function as a tumor suppressor [
10]. For instance, compared to normal airway epithelium, a low αvβ8-integrin expression was found in epithelial lung cancers, which apparently supports its progression because low αvβ8 results in reduced TGF-β levels and consequently derogated tissue homeostasis [
11]. However, tumor cells may also escape the growth-inhibiting effect of TGF-β by means of altered downstream pathways, for instance, a Ser-15 mutation on p53 [
12] or a loss of Smad4 [
13]. Hence, any means of increasing the concentration of activated TGF-β in their surrounding, for instance by αvβ8-integrin upregulation, is assumed to give such cells a growth advantage over normal cells, turning TGF-β into a tumor growth promoter [
9]. For TGF-β-resistant tumors, a concomitant αvβ8 upregulation must therefore be expected to promote invasion and metastasis, particularly because TGF-β also stimulates angiogenesis, epithelial-mesenchymal transition (EMT), cell motility, tumor cell stemness, and colonization of the metastatic niche [
14]. Such pathogenic mechanisms have, for example, been detailed for glioblastoma (GBM) [
15] and prostate cancer [
16] cell lines, and for astrocytes which may control the angiogenic activity of adjacent endothelial cells by αvβ8-integrin expression-mediated regulation of local TGF-β levels [
17]. In addition, other αvβ8-dependent signalling axes, e.g., involving RhoGDI1, may also be relevant for pathogenesis [
18].
In view of its multifaceted role in human pathology and oncogenesis, we anticipate a substantial scientific and clinical value for in-vivo mapping of physiological and pathological αvβ8-integrin expression patterns. For this purpose, we developed
68Ga-Triveoctin, a
68Ga-labeled trimer of the αvβ8 selective octapeptide c(GLRGDLp(
NMe)K) [
19] suitable for imaging of αvβ8-integrin expression by means of positron emission tomography (PET).
Methods
General
Synthesis and characterization of Triveoctin is described in the Additional file
1. The integrin affinities were determined by a solid-phase binding assay, applying a previously described protocol [
20]. β8 immunohistochemistry stainings were done as described [
18]. All animal studies have been performed in accordance with general animal welfare regulations in Germany and the institutional guidelines for the care and use of animals.
68Ga radiolabeling [
21], cultivation of MeWo cells and generation of respective subcutaneous xenografts [
18], determination of
n-octanol/phosphate-buffered saline (PBS) distribution coefficients (log
D) and ex-vivo biodistribution studies [
22], and µPET imaging [
23] were done as described previously in detail (a brief summary is provided in the following).
Radiochemistry and preclinical studies
For fully automated 68Ga labeling, non-processed eluate of a 68Ge/68Ga-generator with a SnO2 matrix (by IThemba LABS, SA; 1.25 mL, 1 M HCl, eluted 68Ga activity approx. 700 MBq) was adjusted to pH 2 by adding HEPES buffer (400 µL of a 2.7 M solution), used to label 2 nmol of Triveoctin or TRAP-AvB8 for 3 min at 95 °C, which was purified by solid-phase extraction using a SepPak® C8 light cartridge (Waters). Radiochemical yields were 95.8 ± 1.3% (n = 10), referring to the final product after purification. Quality control was performed by radio-TLC with citrate buffer, confirming > 99% purity (referring to absence of non-complexed 68Ga, see Figure S3). Distribution coefficients (log D) were determined by shake-flask method using n-octanol and PBS.
MeWo cells (ATCC®, HTB-65™) were grown at 37 °C under 5% CO2 atmosphere in DMEM/HAM (Biochrom, Berlin, Germany) with 10% fetal calf serum (Thermo Fisher). Approx. 107 cells were subcutaneously injected with Matrigel® (Corning, #354262) into the right shoulder of 6–8-week-old female CB17 severe combined immunodeficiency (SCID) mice, which were used about 2–3 weeks later for PET and biodistribution.
For ocular autoradiography and histology, a mouse was sacrificed 50 min after injection of 12 MBq 68Ga-Triveoctin. The eyes were excised, rinsed with PBS and embedded in Sakura Tissue-Tek®. After equilibrium had been reached at − 20 °C, lateral 50-μm cross sections were cut on a cryostat microtome (Leica CM1950) and thaw-mounted on SuperFrost Plus microscope slides. The slides were air-dried on a heating plate and exposed to an imaging plate from 45 min after sacrifice onwards overnight. The imaging plate was read out by a CR-35 Bio Scanner (Raytest). Gaussian smoothing was applied to the final images. The same sections were subsequently HE-stained in a standard automated process.
PET imaging in human
Using a fully automated synthesis module, the eluate of a 68Ge/68Ga generator (by Eckert & Ziegler, Berlin, Germany; 0.1 M HCl, approx. 500 MBq 68Ga) was directly eluted into the reaction vial containing Triveoctin (35 µg) and adjusted to pH 4.5 with sodium acetate. After heating for 4 min at 95 °C, the mixture was passed over a solid-phase extraction cartridge (Waters Sep-Pak®light tC-18), which was purged with water (10 mL). Thereafter, 68Ga-Triveoctin was eluted with ethanol/water mixture (1:1 by volume, 1 mL), followed by isotonic saline (9 mL). The formulation containing approx. 5% ethanol was passed through a 0.22-µm filter into a sterile injection vial and dispensed for injection. Quality control was done by radio-HPLC using a Shimadzu system equipped with a column Chromolith® Performance RP18e (Phenomenex, Aschaffenburg, Germany), gradient 0–100% acetonitrile in water within 15 min, flow rate 1.4 mL/min, tR = 8.53 min, and met the in-house specifications for 68Ga-labeled compounds (> 95%).
Application of
68Ga-Triveoctin was done according to §13/2b of the German Drug Act (Arzneimittelgesetz). A human subject received a single intravenous injection of
68Ga-Triveoctin (173 MBq, approx. 25 µg; for radiolabeling and QC, see Additional file
1). There were no adverse or clinically detectable pharmacologic effects. No significant changes in vital signs or the results of laboratory studies or electrocardiograms were observed. 25 min p.i., the patient underwent a list-mode PET/CT imaging protocol on a Biograph Vision 600 (
Siemens Healthineers, Knoxville, USA). A standard low-dose CT was acquired from the whole body (X-ray tube current 10 mA, tube voltage 100 kV, spiral pitch factor 1.5, 3.0 mm slice thickness) and used for absolute scatter correction of the subsequent PET scan. The emission PET scan was acquired over 19 min using continuous bed motion with a speed of 2.2 mm s
–1 for the legs and 1.4 mm s
–1 for the remaining body. The PET scan was repeated 45 min p.i. without another CT scan. Another PET/CT imaging sequence was acquired 90 min p.i. as the subject had to leave the scanner for voiding. All scans were obtained during normal breathing. PET images were reconstructed using the TRueX algorithm with 4 iterations, 5 subsets, time-of-flight (TOF) application and without filtering. Resulting PET images had an image matrix size of 440 × 440 with a voxel size of 1.65 × 1.65 × 3.0 mm. The dosimetry values were calculated using OLINDA V1.1 [
24] on the basis of the human PET data.
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