A dual-reporter, diagnostic vector for prostate cancer detection and tumor imaging

Subjects

Abstract

Detection of prostate-specific antigen (PSA) as a screening strategy for prostate cancer is limited by the inability of the PSA test to differentiate between malignant cancer and benign hyperplasia. Here, we report the use of a cancer-specific promoter, inhibition of differentiation-1 (Id1), to drive a dual-reporter system (Ad5/3-Id1-SEAP-Id1-mCherry) designed for detection of prostate cancer using a blood-based reporter-secreted embryonic alkaline phosphatase (SEAP) and tumor visualization using a fluorescent reporter protein, mCherry. In human prostate tumors, Id1 levels are correlated with increased Gleason grade and disease progression. To evaluate the performance of the dual-reporter system, a prostate cell panel with varying aggressive phenotypes was tested. Following infection with the Ad5/3-Id1-SEAP-Id1-mCherry vector, expression of the SEAP and mCherry reporters was shown to increase with increasing levels of cellular Id1. No correlation was observed between Id1 and PSA. To evaluate in vivo performance, flank tumors were grown in athymic male mice using three prostate cancer cell lines. Following intra-tumoral injection of the vector, tumors formed by cells with high Id1 had the greatest reporter expression. Interestingly, tumors with the lowest levels of Id1 and reporter expression produced the greatest amounts of PSA. These data support the use of Ad5/3-Id1-SEAP-Id1-mCherry as a predictor of prostate cancer malignancy and as a strategy for tumor localization.

Introduction

Since 1986, screening for prostate cancer has relied heavily on the detection of prostate-specific antigen (PSA) in the blood. The relative levels of PSA predict the abnormal presence of hyperplasia in the prostate. However, the PSA test cannot distinguish between lethal and non-lethal disease, owing to the low specificity of the test to differentiate aggressive cancer.1, 2, 3, 4, 5 Considering these limitations, an improved method for prostate cancer screening is needed to accurately distinguish between aggressive and indolent cancers.

Inhibitor of differentiation (Id1) is a member of the inhibitor of differentiation family of transcription factors. They form inactive heterodimers with the basic helix–loop–helix family of transcription factors that control cellular processes, such as cell-fate determination, proliferation, cell-cycle regulation, angiogenesis, invasion and migration.6,7 Id1 gene expression is cancer-specific and has been demonstrated to have increasing levels in human prostate tumors, which correlate with increasing Gleason grade and progression.8,9 Several studies10, 11, 12, 13, 14 have shown expression of Id1 to be an indicator of malignancy, with no expression found in cases of benign prostate hyperplasia and normal prostate tissue. The correlation between increased Id1 expression and cancer aggressiveness supports the use of the Id1 promoter for controlling diagnostic reporter function.

Recently, the diagnostic vector Ad5/3-Id1-SEAP-Id1-mCherry was constructed and shown to be highly specific and sensitive for breast cancer detection in both in vitro and in vivo models.15,16 With this vector, dual-reporter expression of secreted embryonic alkaline phosphatase (SEAP) and the fluorescent protein mCherry is driven by the cancer-specific promoter Id1 and allows for both blood-based screening and localized visualization of cancer. Introduction of two Id1 promoters upstream of each reporter allows for equal and effective promoter control of SEAP and mCherry expression, which can be used in collaboration or independently. Furthermore, this diagnostic adenovirus (Ad) is designed to shuttle the reporter genes more effectively to cancer cells by replacement of the Ad5 fiber knob domain with that of the Ad3 fiber. This modification overcomes the limited availability of the native coxsackievirus and Ad receptor, which is commonly downregulated in cancer cells.17

SEAP is a truncated and secreted form of human embryonic alkaline phosphatase that is extremely stable and nonimmunogenic. Owing to its heat stability and resistance to the phosphatase inhibitor L-homoarginine, reporter SEAP levels can be measured independently from endogenous alkaline phosphatase activity with high sensitivity. The imaging reporter mCherry, has an excitation peak of 587 nm and emission peak of 610 nm, and is a mutated variant of the widely used mRFP1. The mCherry protein matures more quickly and completely than mRFP1, yielding a higher extinction coefficient and brightness, yet bleaches 10 times more slowly.18 The longer wavelength of mCherry decreases the interference from tissue auto-fluorescence and allows for greater tissue penetration. mCherry can be detected by fluorescence imaging in the surgical setting using laparoscopic techniques, including robotic surgery (e.g., daVinchi), to improve surgical treatment of relevant prostate cancer. Importantly, as both the SEAP and mCherry reporters are under the control of Id1, reporter expression using the diagnostic vector Ad5/3-Id1-SEAP-Id1-mCherry is predicted to be an indicator of cancer prognosis.

In the present study, Ad5/3-Id1-SEAP-Id1-mCherry was evaluated as a diagnostic system for screening prostate cancer using normal cells and cancer cell lines of differing aggressive phenotypes. By correlating Id1 expression with SEAP levels and mCherry fluorescence, the effectiveness of Ad5/3-Id1-SEAP-Id1-mCherry in predicting cancer cell phenotype was compared with PSA results. In addition, the applicability of the diagnostic vector for in vivo cancer detection and localization was determined. The dual-reporter vector represents a novel method for non-invasively measuring cancer aggressiveness and visually monitoring prostate cancer, ultimately overcoming the limitations associated with PSA-based diagnoses.

Results

Id1 expression, but not PSA level, is an indicator of prostate cancer cell aggressiveness

Six prostate cell lines were categorized based on their reported behaviors19, 20, 21, 22, 23, 24 and measured levels of PSA secretion (Table 1). Normal prostate cells (WPMY-1) secreted a baseline amount of 314.1±8.1 pg ml−1 PSA. Two of the four cell lines with reportedly aggressive phenotypes (Du145 and PC3) had relatively lower PSA levels, ranging from 230 to 266 pg ml−1, whereas the remaining aggressive cells lines (VCaP and MDA-PCA-2b) had significantly (P<0.001) greater levels of PSA as compared with WPMY-1. The one non-aggressive cell type, LNCaP, also secreted significantly increased amounts of PSA in comparison with baseline levels. Id1 expression was elevated in all aggressive prostate cancer cell types compared with normal prostate cells and less-aggressive cells (Figure 1a). There was no statistical correlation between Id1 expression and PSA level (P=0.65; Figure 1b).

Table 1 Description of cell lines used for analyses of Ad5/3-Id1-SEAP-Id1-mCherry diagnostic efficacy
Figure 1
figure1

Id1 expression of the prostate cell panel and its lack of correlation with PSA. (a) Id1 expression was evaluated in cell lysates by western blot and quanitified with densitometry. Id1 intensity was normalized to the corresponding level of of β-actin. (b) Linear regression analysis was used to demonstrate no correlation between cellular Id1 levels and the PSA levels reported in Table 1. PSA data are shown as mean±s.e.m. (n=3).

SEAP and mCherry reporter expression correlate with Id1 levels and are indicators of prostate cell aggressiveness

A quantitative luciferase (luc) assay was performed to account for differences between cell lines in their susceptibility to infection with the tropism-modified Ad5/3 vector caused by possible differences in Ad3 receptor levels. The relative levels of infectivity of the Ad5/3-CMV-Luc vector for each cell type are shown in Figure 2a. These luciferase counts were subsequently used to normalize diagnostic reporter expression. SEAP and mCherry reporter expressions were therefore only a reflection of Id1 promoter activity. After infection with Ad5/3-Id1-SEAP-Id1-mCherry, the prostate cancer lines with aggressive phenotypes (VCaP, MDA-PCA-2b, PC3 and Du145) had increased levels of SEAP reporter compared with non-aggressive (LNCaP) and normal (WPMY-1) cells (Figure 2b). For PC3 and Du145, Ad3-normalized SEAP expression was significantly elevated at 4 and 6 days post infection compared with non-aggressive LNCaP cells at the corresponding time points. On the basis of the quantification of cellular Id1 presented in Figure 1a, the cell types were grouped based on low (<0.25 a.u.), moderate (0.25–1.5 a.u.) or high (>1.5 a.u.) Id1 levels in order to assess the relationship between SEAP reporter and Id1 promoter levels. Cells with moderate Id1 levels had increased SEAP reporter expression as compared with cells with low Id1. Cells with high Id1 levels had significantly increased SEAP reporter expression as compared with cells with moderate and low Id1 (Figure 2c). Likewise, representative fluorescent images of the mCherry reporter confirmed diagnostic vector efficiency (Figure 3). mCherry intensity corresponded to cellular Id1 levels, with the greatest fluorescence observed in the aggressive Du145 cells (49.1±5.2 fluorescent counts), followed by VCaP cells (35.6±7.5 fluorescent counts) and WPMY-1 cells (16.4±3.5 fluorescent counts).

Figure 2
figure2

SEAP reporter expression in the prostate cell panel and its relationship with cellular Id1. (a) Susceptibility for infection via the Ad3 fiber serotype was determined for each cell type using Ad5/3-CMV-Luc (multiplicity of infection=1). Luciferase activity was used to normalize diagnostic reporter expression for differences in vector infectivity due to varying levels of Ad3 receptor expression. (b) SEAP reporter was measured in culture medium 2, 4 and 6 days post infection with Ad5/3-Id1-SEAP-Id1-mCherry (multiplicity of infection=1) and normalized with luciferase activity. *P<0.01 vs LNCap at corresponding time point. All data are reported as mean±s.e.m. (n=4). (c) Cells types were group based on low (<0.25 a.u.), moderate (0.25–1.5 a.u.) or high (>1.5 a.u.) Id1 levels and SEAP reporter expression averaged for each group. The low-Id1 group consisted of two cell types (WPMY-1 and LNCaP), the moderate-Id1 group consisted of three cells types (PC3, VCaP and MDA-PCA-2b) and the high-Id1 group consisted of one cell type (Du145). *P<0.01 vs low and **P<0.001 vs all groups.

Figure 3
figure3

mCherry reporter fluorescence in normal prostate cells and cancerous cells with moderate and high levels of Id1. Representative fluorescence images of (a) normal prostate cells (WPMY-1) and cancerous cells with (b) moderate Id1 expression (VCaP) and (c) high Id1 expression (Du145). Inserts are corresponding bright-field images. All images were acquired using a × 10 objective.

In vivo reporter expression correlates with prostate cancer malignancy despite PSA levels

To evaluate the potential of the vector to diagnose prostate cancer in vivo, flank tumors were formed using three different prostate cancer cell lines with low (LNCaP), moderate (PC3) and high (Du145) expression levels of Id1. At the time of vector injection, there were no significant differences in tumor size between any of the groups (LNCaP: 207±32 mm3; PC3: 203±37 mm3; Du145: 171±29 mm3). Elevated plasma levels of PSA were detected in mice bearing LNCaP tumors (1860±144 pg ml−1), whereas mice with PC3 and Du145 tumors did not have detectable amounts of PSA.

SEAP and mCherry reporter expression were monitored in all mice over a 14-day time period (Figures 4a and 5a). For SEAP analyses, post-treatment levels were compared with baseline SEAP expression measured at day 0 prior to vector injection. Two days after intra-tumoral (IT) injection of Ad5/3-Id1-SEAP-Id1-mCherry, SEAP reporter expression was significantly elevated over baseline levels for the Du145 group, and mCherry reporter fluorescence permitted visual localization of these tumors. SEAP reporter expression was detectable in mice bearing PC3 tumors beginning 2 days after IT injection, and tumor fluorescence was detected at day 6. Post-treatment SEAP levels in mice with LNCaP tumors were slightly elevated above baseline levels beginning at day 6, and mCherry fluorescence was not observed in these tumors at any time point. The SEAP measured in plasma over the entire 14-day time course following vector injection was totaled to represent the effect of making multiple post-treatment diagnostic readings (Figure 4b). The combined effect of successive SEAP measurements revealed an elevated post-treatment SEAP level for each of the tumor types that was significantly greater than the baseline level. Furthermore, a proportional relationship between total amount of measured SEAP and tumor Id1 was observed, with tumors formed by Du145 cells leading to the highest plasma levels of the SEAP reporter and LNCaP tumors having the least. This trend was also observed with mCherry reporter expression, as Du145 tumors had the brightest tumor fluorescence at all time points followed by PC3 tumors and finally LNCaP tumors, which showed negligible mCherry expression (Figure 5). A representative image of the tumor fluorescence visualized at day 6 post vector injection is shown in Figure 5b.

Figure 4
figure4

In vivo SEAP reporter expression following IT injection of the diagnostic vector. (a) Plasma levels of the SEAP reporter were monitored over a 14-day period for tumors formed by prostate cancer cells with high (Du145), moderate (PC3) and low (LNCaP) levels of Id1. All data are reported as mean±s.e.m. (n=5). *P<0.001 vs baseline and +P<0.01 vs baseline (the order of stacked symbols correspond to order of data points). (b) SEAP amounts measured over the entire time course were totaled and compared with baseline levels measured before vector injection. *P<0.001 vs baseline and +P<0.001 vs LNCaP and PC3.

Figure 5
figure5

mCherry tumor fluorescence following IT injection of the diagnostic vector. (a) mCherry reporter expression for tumors formed by prostate cancer cells with high (Du145), moderate (PC3) and low (LNCaP) levels of Id1 was monitored and quantified over a 14-day time period. All data are reported as mean±s.e.m. (n=5). +P<0.05 vs PC3; *P<0.01 vs LNCaP; P<0.05 vs LNCaP. (b) Representative images of mCherry fluorescence at day 6 post IT injection of the diagnostic vector for LNCaP, PC3 and Du145 tumors.

Discussion

Gene therapies have great potential for both treatment and diagnosis of cancer. In particular, the use of adenoviral vectors for molecular imaging of cancer offers opportunities to non-invasively gain information regarding tumor location as well as disease-specific information with regard to metabolism, receptor expression or tumor vascularity.25 Much of the development and clinical application of viral-based vector strategies, however, has been limited by their associated immunogenicity, pathogenicity and natural tropism. The diagnostic Ad Ad5/3-Id1-SEAP-Id1-mCherry investigated in this work overcomes many of the challenges commonly associated with viral vectors, as it is replication defective and engineered to demonstrate enhanced cancer-specific infectivity. The hybrid Ad5/3 fiber of this vector ablates tropism of coxsackievirus and Ad receptor and overcomes its limited availability in cancer by introducing the Ad3 serotype fiber. Thus, the hybrid Ad5/3 fiber allows for improved and selective infectivity. In addition, as the dual-reporter system is under the control of the cancer-specific promoter Id1, reporter expression is designed to be cancer-specific and an indicator of prognosis.

The current work evaluated the ability of the dual-reporter vector, Ad5/3-Id1-SEAP-Id1-mCherry, to non-invasively detect and monitor prostate cancer using expression of a SEAP reporter for blood-based detection and mCherry reporter for fluorescence imaging. Using a panel of prostate cancer cells and normal prostate cells, it was demonstrated that, unlike PSA, cellular expression of both the SEAP and mCherry reporters was directly proportional to cellular levels of Id1. Given the direct relationship between the aggressive nature of cancer and Id1 expression, these findings support the use of Ad5/3-Id1-SEAP-Id1-mCherry for evaluating prostate cancer aggressiveness. In vivo studies confirmed the ability to measure blood-based levels of the SEAP reporter and to identify mCherry tumor fluorescence in situ. These results also confirmed that reporter expression correlated with Id1 levels and not the level of vector infectivity, as tumors formed by Du145 cells with high Id1 expression had the highest reporter levels and tumors formed by LNCaP cells with low Id1 expression had the lowest levels of reporter expression, despite a higher level of vector infectivity observed in LNCaP cells (Figure 2a). Furthermore, these data demonstrate that PSA was an inaccurate measure of cancer malignancy as the non-aggessive LNCaP tumors led to elevated plasma PSA, whereas the aggressive Du145 and PC3 cells had non-detectable levels of PSA. Together, these data suggest that the Ad5/3-Id1-SEAP-Id1-mCherry diagnostic vector could serve as a sensitive and non-invasive strategy for diagnosing prostate cancer that would overcome the current limitations associated with the PSA test by providing measures that are based on tumor cell aggressiveness.

Our previous work demonstrated the detection sensitivity of the Ad5/3-Id1-SEAP-Id1-mCherry diagnostic system, showing that the threshold value for detecting the SEAP reporter could be achieved with as little as 7000 infected breast cancer cells.15 The present work is proof of principle that this same diagnostic vector can be applied for detection of prostate cancer and that, similarly to the breast cancer model, reporter activity is driven by the Id1 promoter. Using IT injections of the Ad vector, this work demonstrates that tumor infectivity leads to production of the SEAP and mCherry reporters that are sufficiently distinguishable above background levels to aid in cancer detection and tumor visualization. Future studies will address the need for targeted delivery of the diagnostic vector to the tumor following systemic injection.

The in vivo time course analyses of SEAP and mCherry reporter expression following IT injection of the diagnostic vector revealed differences in the temporal translation of the dual-reporter system. Whereas SEAP levels appeared to peak at 3 days post injection, maximum mCherry tumor fluorescence was not observed until day 6 post treatment. These temporal differences in reporter expression could be potentially explained by the continual intracellular accumulation of the mCherry reporter accompanied by its slower degradation versus the immediate secretion of SEAP into the bloodstream, followed by its degradation and clearance. These data suggest that in cases of tumors with low to moderate levels of Id1, accumulation of mCherry is necessary to achieve a fluorescent signal that can be detected above background. This is evidenced by the fact that PC3 tumors with moderate Id1 levels were not visually detected before 6 days post injection, whereas Du145 tumors with high Id1 expression were visualized at the earliest time point. Given the unknown half-life of the blood-based SEAP reporter, the time course analyses do not delineate between SEAP accumulation and the production of newly made and expressed SEAP. However, it is clear from the LNCaP data presented in Figure 4 that, for potential clinical applications successive measurements over a series of several days may be required to assess reporter function and accurately define tumor behavior, especially for less-aggressive cancers.

The lower limit of detection for tumor visualization using mCherry fluorescence was not surpassed with LNCaP tumors. As reporter expression was dependent on cellular Id1 expression and LNCaP cells have relatively low levels of Id1, mCherry expression was not sufficient to produce a detectable fluorescent signal. The dependency of the diagnostic vector on adequate expression of Id1 represents a limitation of the current system for visually locating less-aggressive cancers. In future applications, non-aggressive prostate cancers could be diagnosed by non-elevated SEAP levels, however, visual localization of the cancer based on tumor fluorescence would be virtually impossible in the absence of sufficient Id1 activation. Likewise, tumor size (i.e., the number of cells available for transduction) is an important consideration for the prognostic application of this Ad vector, as reporter expression is proportional to cellular levels of Id1 within the tumor.

The present work introduces a novel strategy for detection and localization of prostate cancer that overcomes the current limitations of the PSA test to distinguish between aggressive cancer and indolent conditions, such as benign prostate hyperplasia. The correlation between reporter expression and cellular Id1 enables SEAP levels to be used as a predictive measure of prostate cancer aggressiveness, and mCherry fluorescence as an aid for tumor visualization. A major challenge of viral-based delivery systems for gene therapy applications is achieving targeted tumor delivery and sufficient infectivity. Future studies will develop strategies to target vector delivery and infectivity of both aggressive and non-aggressive prostate cancer. This strategy would assist clinicians in the detection and treatment of prostate cancer and ultimately reduce the mortality associated with this disease.

Materials and methods

Cell culture

The efficacy of the diagnostic vector was evaluated using six prostate cancer cell lines: WMPY-1, MDA-PCa-2b, VCaP, PC3, Du145 and LNCaP (American Type Culture Collection, Manassas, VA, USA). Two commonly used prostate cell lines (RWPE-1 and Ca-HPV-10) were excluded from the cell panel, due to immortalization vector HPV-18 interaction with endogenous Id1 protein.26, 27, 28 LNCaP cells were maintained in RPMI-1640 with 10% fetal bovine serum (FBS) and 1% L-glutamine. Du145 cells were grown in Eagle's minimum essential medium with 10% FBS and 1% L-glutamine. VCap cells were maintained in Dulbecco's modified Eagle's medium containing 10% FBS and 1% L-glutamine. WMPY-1 cells were grown in Dulbecco's modified Eagle medium with 5% FBS and 1% L-glutamine. PC3 cells were grown in F12k basal medium with 10% FBS. MDA-PCa-2b cells were maintained in F12k basal medium containing 20% FBS, 25 ng ml−1 cholera toxin, 10 ngml−1 mouse epidermal growth factor, 5 nM phosphoethanolamine, 100 pgml−1 hydrocortisone, 45 nM selenious acid and 5 μg ml−1 bovine insulin. All cells were cultured at 37 °C and 5% CO2. Cells were allowed to reach 75–90% confluency before passaging.

In vitro experiments

Cells (1.0 × 105 cells cm−2) were plated in quadruplicate 24 h before infection with Ad5/3-Id1-SEAP-Id1-mCherry. Cells were infected at a multiplicity of infection ratio of one. Virus concentration (PFU ml−1) was calculated based on the plaque-forming units using a standard agarose-overlay plaque assay. Media was collected at 2, 4 and 6 days post infection and SEAP levels measured using the Great EscAPe SEAP Fluorescence Detection kit (Clontech Laboratories, Mountain View, CA, USA). Media was also collected from uninfected cells to measure background fluorescence. In addition, PSA levels were quantified in the culture medium of each cell type by an enzyme-linked immunosorbent assay specific for human PSA (AbCam, Cambridge, MA, USA).

In order to qualify SEAP reporter expression as a singular function of Id1 promoter activity, a normalization assay was performed using an Ad5/3-CMV-Luc Ad to quantify vector infectivity. This Ad was generated using the same Ad5/3 adenoviral backbone used to construct the Ad5/3-Id1-SEAP-Id1-mCherry Ad. For all cell lines, 1.0 × 105 cells were infected with Ad5/3-CMV-Luc (multiplicity of infection=1). Forty-eight hours post infection, cells were imaged with an IVIS-100 CCD imaging system (Caliper Life Sciences, Mountain View, CA, USA). Matched region of interest analysis was performed using instrument software (Living Image 4.2, Xenogen, Hopkinton, MA, USA) to quantify total luciferase counts per well. Luciferase counts represented a relative level of Ad5/3 infectivity for each cell type and were subsequently used to normalize the SEAP measurements.

For data analysis, background fluorescence from uninfected cells was subtracted from the measured fluorescence of infected cells and SEAP levels were normalized to corresponding vector infectivity. To evaluate the relationship between SEAP reporter expression and cellular Id1, cells were grouped based on their level of Id1 expression as determined by western blot (low Id1 level: <0.25 a.u.; moderate Id1 level: 0.25–1.5 a.u.; high Id1 level: >1.5 a.u.). On the basis of these guidelines, the low-Id1 group consisted of WPMY-1 and LNCaP cells, the moderate-Id1 group consisted of PC3, VCaP and MDA-PCA-2b cells and the high-Id1 group consisted of Du145 cells. Day 4 SEAP reporter expression was averaged for the cell types in each group and compared.

Western blot

Protein lysates from all cell lines were collected with RIPA-modified buffer (Sigma-Aldrich, St Louis, MO, USA) with 1% SDS and phosphatase inhibitors (1 mM sodium orthovanadate, 25 mM b-glycerophosphate and 100 mM sodium fluoride) and protease inhibitors (10 mg ml−1 leupeptin, 10 mg ml−1 aprotinin and 1 mM phenylmethylsulfonyl fluoride). Protein lysates (15 μg, determined by the Lowry assay) were separated with 4–12% bis-Tris electrophoresis gel (Life Technology, Carlsbad, CA, USA), followed by transfer to polyvinylidene fluoride membranes (Millipore Immobilon, Billerica, MA, USA). Membranes were blocked with 5% bovine serum albumin and probed with rabbit monoclonal anti-mouse Id1, clone 195-14 (CalBioreagents, San Mateo, CA, USA), followed by horseradish peroxidase-conjugated goat anti-rabbit Ig (SouthernBiotech, Birmingham, AL, USA). All membranes were washed three times with tris-buffered saline and tween 20 buffer for 20 min per wash. Id1 protein was visualized using chemiluminescent substrate (SuperSignal West Pico Chemiluminescent Substrate, ThermoScientific, Rockford, IL, USA). Densitometry was performed using ImageJ software (version 1.47t, National Institutes of Health, Bethesda, MD, USA), and Id1 levels were normalized to the respective β-actin controls.

In vivo analyses

Animal studies were performed in accordance with the National Institutes of Health recommendations and the approval of the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham. Athymic male nude mice were obtained from Frederick Cancer Research (Hartford, CT, USA). Flank tumors were grown over a 6–8-week time period following implantation of 4 × 106 prostate cancer cells (LNCaP, PC3 or Du145). For implantation of PC3 and Du145 cells, the cells were harvested, resuspended in cold phosphate-buffered saline and injected subcutaneously into the left flank. The gelatin sponge Vetspon (Novartis Animal Health, Greensboro, NC, USA) was used to facilitate LNCaP tumor development. For inoculation of LNCaP cells with Vetspon, the material was cut to 0.5 cm3 and 4 × 106 cells in 0.15 ml cold phosphate-buffered saline were seeded into the Vetspon by gently pipetting until the cells were completely absorbed. A small incision was created on the left flank and the cell-soaked Vetspon placed under the skin. The wound was closed with two prolene sutures. Mature tumors were injected intra-tumorally with 1 × 109 PFU of the diagnostic vector Ad5/3-Id1-SEAP-Id1-mCherry. To measure SEAP reporter expression, blood was collected retro-orbitally into heparinized capillary tubes and plasma levels of SEAP measured using the Great EscAPe Detection kit (Clontech Laboratories). SEAP levels and mCherry tumor fluorescence were monitored on days 2, 3, 6, 10 and 14 post injection.

Fluorescence imaging

Representative in vitro fluorescent images were acquired on day 2 post infection with Ad5/3-Id1-SEAP-Id1-mCherry. Cell images ( × 100) were rendered using an mCherry 587 nm excitation/600-nm-long pass filter on an inverted microscope with a halogen light source and Nuance multi-spectral camera (CRi, Woburn, WA, USA). A liquid-crystal tunable wavelength filter in the camera was set for collection of emission images from 600 to 720 nm in 5-nm increments. Composite images (unmixed composites) were generated for each image cube by unmixing the spectral signature of the mCherry reporter from those of background auto-fluorescence. For in vivo images, mice were anesthetized with isoflurane and tumors were imaged with a Leica stereomicrosope (Model MZ-FLIII, Vashaw Scientific, Norcross, GA, USA). Filter, camera and image processing from the in vitro imaging methods were used in coordination with the stereomicroscope. Quantitative region of interest analysis for mCherry fluorescence with background subtraction was performed with ImageJ software and total counts from size-matched regions of interest were recorded.

Statistical analyses

Results are reported as the mean plus or minus s.e.m. Data were analyzed for statistical significance using the Student’s t-test or analysis of variance with Bonferroni’s multiple comparison test where appropriate using Prism (version 6.0, GraphPad Software, La Jolla, CA, USA). For statistical analyses of PSA data, values for the normal prostate cells (WPMY-1) were used as a baseline PSA level and comparisons with baseline were made using the Student’s t-test. Linear regression analysis was used to evaluate the correlation between PSA levels and cellular Id1. For in vivo quantification of the mCherry reporter, background fluorescence was subtracted from the fluorescence intensity of the infected tumors. Student’s t-tests were used to make pairwise comparisons of in vivo mCherry tumor fluorescence and SEAP expression.

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Acknowledgements

This work was supported the UAB Small Animal Imaging Shared Facility NIH Research Core Grant (P30CA013148) and the DOD Prostate Cancer Research Program (PC111230).

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Correspondence to K R Zinn.

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Richter, J., Mahoney, M., Warram, J. et al. A dual-reporter, diagnostic vector for prostate cancer detection and tumor imaging. Gene Ther 21, 897–902 (2014). https://doi.org/10.1038/gt.2014.68

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