PSMA imaging as a non-invasive tool to monitor inducible gene expression in vivo

Tetracycline (Tet)-On and Tet-Off systems have been widely used in biomedical research as an important tool allowing for controlled gene expression in eukaryotic cells and organisms [ 1 , 2 , 3 , 4 ]. Those systems become effective upon binding of a tetracycline class antibiotic [ 5 ], either by switching off gene expression in a so-called Tet-Off system or by inducing gene expression in a Tet-On system, wherein a mutant reverse Tet-regulated trans-activator is employed [ 6 , 7 ]. This non-invasive approach has found wide application in gene therapy and signal cascade activation monitoring, as well as in cell motility tracking studies [ 8 ]. Ideally, such system should allow for expression of potentially antigenic proteins in immune competent hosts without triggering an immune response. However, inducible systems require high degrees of biocompatibility and target-to-background contrast and sensitivity [ 9 ]. The prostate-specific membrane antigen (PSMA; also known as glutamate-carboxypeptidase II—GCPII; NAALADase; folate hydrolase I—FOLH1) has been widely studied as a drug target for prostate cancer imaging and therapy [ 9 , 10 , 11 , 12 ]. This enzyme is highly expressed in most metastatic prostate cancers. Favourable results in a phase 3 therapeutic trial, using [ Lu]Lu-PSMA-617, were recently reported [ 13 ], and several F- and Ga-labelled agents are finding widespread use for diagnostic imaging, with high tumour-to-background ratios allowing for disease detection in most cases of prostate cancer (> 90%) [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ]. PSMA imaging benefits from enzyme internalization upon binding radiotracers, allowing cellular retention to improve imaging contrast [ 28 , 29 , 30 , 31 , 32 ]. Castanares et al. reported favourable results using PSMA as a reporter gene using adenoviral gene transfer using HCT116 cells, with improved target-to-background ratios over the human sodium iodide transporter and the mutant herpes simplex virus type I thymidine kinase [ 33 ]. The aim of this study was to evaluate the feasibility of non-invasive monitoring of inducible gene expression using PSMA as a reporter probe. Such a reporter system could be useful to track inducible gene expression in vivo for research applications. For this purpose, we studied the doxycycline-induced expression of human prostate-specific membrane antigen (hPSMA) in a murine TRAMP-C2 cell line, which does not constitutively express PSMA [ 34 , 35 , 36 , 37 ]. TRAMP-C2 cells acquired from American Type Culture Collection (ATCC) and cultured according to ATCC specifications. The TRAMP-C2 cells were transduced with a customized lentiviral vector carrying tetracycline (syn. doxycycline-; “DOX”) inducible PSMA expression system. Creating a DOX-inducible expression system was performed as follows: First, PSMA was sub-cloned out of an expression plasmid EX-G0050-Lv205 (GeneCopoeia, Inc.) with EcoRI and BamH1 and ligated into the target pLVX-TRE3G vector. Next, the ligated product was transformed into E. coli strain DH5alpha for plasmid amplification and verification using restriction digests and gel electrophoresis. To produce lentiviruses, the protocol provided by Clonetech was employed. The lentiviral vector plasmid DNA (PSMA pLVX-TRE3G and pLVX-Tet3G) was diluted with water and added to a tube of Lenti-X Packaging Single Shots provided by Clonetech, and vortexed at high speed. After incubating at room temperature, samples were added dropwise to the 293 T (HEK 293 T) cell culture dishes at 70% confluence. Following 12 h of incubation at 37 °C, 20% O 2 , and 5% CO 2 in a water-jacketed incubator, fresh complete growth medium was replaced and incubated at 37 °C and 5% CO 2 . At 72 h after the start of transfection, the lentiviral supernatants were harvested and filtered through 0.45-µm filter to remove cellular debris. The filtered pLVX-Tet3G and PSMA pLVX-TRE3G supernatants were stored at -80 C, thawed slowly on ice, and added to the TRAMP-C2 cells at 70% confluence, at a 1:1 ratio with 4-µg/mL polybrene. The cells were transduced for 12 h at 37 °C and 5% CO 2 in a water-jacketed incubator, after which the culture medium was discarded and replaced with fresh growth medium. The resulting bulk PSMA TRAMP-C2 population was incubated by adding doxycycline at varying concentrations up to 1 µg/ml (2 µM) and incubating for 18 h. For the purpose of flow cytometry analysis, the cells were seeded in V-bottom 96-well plates and pretreated with 1-μg/ml doxycycline hydrochloride (Sigma-Aldrich, St. Louis MO, USA), 18–24 h before the flow cytometry study. Media were removed, and cells were washed with DPBS (Gibco, Carlsbad, CA, USA) supplemented with 2% foetal calf serum and 0.02% NaN 3 , prior to and following a 1-h-long incubation in the dark with 1 ug/ml of Alexa Fluor® 488 anti-human PSMA Antibody (BioLegend, San Diego, CA, USA) per 10 cells. LNCaP cells (maintained in RPMI media supplemented with 10% FBS and 1% penicillin/streptomycin) and wild-type TRAMP-C2 cells were used as positive and negative controls, respectively. Additional negative controls were “unstained” cells—treated with 100–200 μl/well of DPBS with 2% foetal calf serum and 0.02% NaN 3 , and isotype controls, incubated for 1 h with 1 µg/ml Alexa Fluor® 488 Mouse IgG1, κ Isotype Ctrl (FC; Biolegend, San Diego, CA, USA), as well as cells without doxycycline pre-treatment. Flow cytometry runs were performed on q FACScalibur flow cytometer (Becton, Dickinson and Co., Franklin Lake, NJ, USA). Data were analysed using FlowJo software (FlowJo LLC, Ashland, OR, USA). The “bulk” PSMA TRAMP-C2 cells were then isolated into single-cell clones by limiting dilution in multiple 96-well plates. This method was used to generate a clonal populations, each arising from a single cell. Over the next 2 weeks, these single cells were cultured with G418 (500 µg/mL) and puromycin (3 µg/mL) to isolate clones that have “medium”- and “high”-level PSMA expression. Out of the 25 clones that were tested, four highly expressing populations (designated as clones 1, 14, 16, and 19) were isolated for further experiments. Clones 14 and 19 were validated to be “intermediate” expressors while clones 1 and 16 were high expressors. Protein expression was validated through immunoblotting, using [ Lu]Lu-PSMA-617. The four isolated clones were initially maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% heat-inactivated FBS, 5% Nu-Serum IV, 1% penicillin/streptomycin, 0.005-mg/mL bovine insulin, and 10-nmol/L dehydroisoandrosterone (DHEA) [ 41 ], and after 1 week, 300 ug/ml of geneticin (G418) was introduced to the media. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin. The cells were confirmed pathogen-free using the IMPACT I PCF profile test (IDEXX BioAnalytics). All mouse experiments were approved by the Animal Care Committee of the University of British Columbia. 10 × 10 cells of each of the four clones 1, 14, 16, and 19 in 100 μl of media and Matrigel (1:1) were subcutaneously inoculated over the left shoulder of male NOD.Cg-Rag1 Il2rg SzJ (NRG) mice of 12 weeks of age and older (Jackson Laboratory, Bar Harbor, ME, USA), using a 25-gauge needle. The mice were maintained in a pathogen-free animal facility with restricted access on a 12:12 light cycle, monitored for tumour size, weight, and general signs of illness. Five–8 weeks post-inoculation, mice with tumour volume of at least 200 mm were selected for in vivo imaging and biodistribution studies with the F-labelled radiotracer DCFPyL [ 11 , 20 , 21 , 22 , 42 ]. A group of mice was pretreated with 50-µg doxycycline per g body weight in 100–200-μl DPBS intraperitoneally, every 24 h for 3 days prior to the study. Additional mice were used as controls and did not receive the antibiotic prior to the study. All animals were randomized to the various groups without considering any other variable than the minimum tumour size. The weight of the mice was 33 ± 3 g at the time of biodistribution. For each clone, a minimum of four mice without administered doxycycline and nine mice with administered doxycycline were used for activity biodistribution in organs. The investigators were not blinded as to which group the animals belonged to during the experimental procedures or data analysis. For each clone, one mouse was randomly selected for micro-PET-CT imaging before and after doxycycline induction. The mice were intravenously injected with 1.47 ± 0.28 MBq of [ F]DCFPyL for biodistribution studies. Ex vivo biodistribution studies were performed immediately post-CO 2 euthanasia (following anaesthesia in 2% isoflurane in oxygen). Organs were harvested, weighed, and counted on a PerkinElmer WIZARD 2480 gamma counter (PerkinElmer Inc., Waltham, MA, USA). Organ uptake was calculated in per cent injected activity per gram of tissue (%ID/g), and an unpaired Student’s t-test was performed using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA) with tumour uptake post-doxycycline induction in the test group and tumour uptake without doxycycline induction in the control group. The cutoff for significance was a p -value under 0.05. For imaging, at least one mouse from each group was randomly selected to be used before and after tetracycline induction so that the animal served as its own control. For both imaging sessions, the mice received and 5 ± 0.86 MBq of [ F]DCFPyL. Following baseline imaging in the absence of tetracycline, the mice recovered from anaesthesia and were treated with 50-µg doxycycline per g body weight in 100–200-μl dPBS intraperitoneally, every 25 h for 3 days prior to the subsequent study. PET-CT images were acquired one hour post-tracer injection using Siemens Inveon micro-PET-CT scanner (Siemens Medical Solutions, Knoxville, TN, USA) and analysed using the Inveon Research Workplace (Siemens Medical Solutions, Ann Arbor, MI, USA). PET-CT images were compared side by side for the same mouse before and after induction using the same uptake bar with colour spectrum corresponding to the percentage of injected activity per gram tissue. The results of in vitro flow cytometry of a representative example of a parental clone are shown in Fig. 1 as histogram plots of cell count versus FITC fluorescence. The results of flow cytometry from the four clones are available in Additional file 1 : Fig. S1. Except for the LNCaP cells as positive control, doxycycline-untreated cells showed identical negative PSMA-expressing profiles for unstained, isotype-stained, and anti-PSMA-stained tests. Upon doxycycline induction, all four clones showed increase in PSMA expression upon doxycycline induction with a shift in mean channel fluorescence signal for anti-PSMA-stained populations, except the negative control (wild-type TRAMP-C2 cells). Flow cytometry results of PSMA expression. PSMA-expressing clones were isolated from the “bulk” population. The histogram shows a representative clone (clone 16) with high expression under doxycycline induction (blue) compared to non-induced control (red) Uptake of [ F]DCFPyL in NRG mice followed a similar pattern of distribution in different organs as in studies using LNCaP cells [ 43 ], with higher degrees of variability in uptake in adrenal (2.17 ± 2.3% ID/g) and seminal glands (5.04 ± 12.32% ID/g) due to possible urine contamination of these organs during harvesting. Pre-treatment with doxycycline significantly impacted the level of [ F]DCFPyL uptake in tumours (Fig. 2 ). Clone 19 showed significantly lower uptake (Additional file 1 : Fig. S2). An unpaired t-test resulted in p -values under 0.0001 for all four clones, when comparing uptake levels without induction versus post-induction. PET-CT images of four mice, each inoculated with a different clone, confirmed this finding when images before and after induction were compared. In addition, tumours could only be visualized with high tumour-to-background ratio after undergoing induction with intraperitoneal doxycycline injection (Fig. 3 and Additional file 1 : Fig. S3). In vivo PSMA expression levels in different clones before and after doxycycline induction. Transfected TRAMP-C2 clones express PSMA upon doxycycline induction in vivo . Unpaired t -tests for all four clones showed a p -value of < 0.0001 when comparing radioactivity uptake of [ F]DCFPyL in mice with or without pre-treatment with doxycycline. The figure shows the %ID/g of tumour uptake in the induced (doxycycline) mice compared to the control group for clone 16 Comparison of PET-CT images before and after doxycycline induction. Comparison of PET-CT images before (left) and after (right) doxycycline induction in a representative mouse (clone 16). Uptake in the tumours was only visualized after doxycycline administration. The spectrum bar has a range 0–7.1%ID/g for PET (yellow/red tones). The tumour is indicated with a blue arrow

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