Titration of variant HSV1-tk gene expression to determine the sensitivity of 18F-FHBG PET imaging in a prostate tumor

Abstract

Because of its high selectivity and specificity for the imaging reporter probe 9-(4-(18)F-fluoro-3-[hydroxymethyl]butyl)guanine ((18)F-FHBG), the herpes simplex virus type 1 thymidine kinase (HSV1-tk) variant sr39tk is actively being studied as a PET reporter gene. We recently demonstrated the capability of using a prostate-specific transcriptional amplification PET reporter vector, AdTSTA-sr39tk, to target prostate cancer lymph node metastasis. However, one area that warrants further study is the examination of the sensitivity of PET by determining the minimum percentage of cells expressing the sr39tk transgene needed for detection. Addressing this question could determine the sensitivity of vector-mediated sr39tk PET in cancer-targeting strategies.

Methods: DU-145, PC-3, and CWR22Rv.1 prostate cancer cell lines (a total of 1 x 10(6) cells) were studied, of which 7%, 10%, 25%, 50%, or 70% were transduced with the lentiviral vector constitutively expressing HSV1-sr39tk-IRES-enhanced green fluorescent protein (EGFP). Cells were subcutaneously implanted into the left shoulder of severe combined immunodeficient mice and evaluated. Tumor cells comparably transduced with an EGFP control vector were implanted on the right shoulder. Mice were imaged using PET with (18)F-FHBG at 8, 15, and 22 d after tumor implant. On day 23, tumors were isolated and analyzed for sr39tk transgene expression by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR), Western blotting, immunohistochemistry, and flow cytometry for EGFP expression.

Results: Results showed a linear relationship between the level of sr39tk expression and the quantity of tracer accrual in DU-145, with the minimal value for PET detection at 10%. The magnitude of tracer retention in sr39tk-expressing cells was amplified over time as the tumor grew. Protein levels in the stepwise titration increased with the percentage of sr39tk-transduced cells.

Conclusion: The stepwise titration of prostate cancer cells transduced with the lenti-CMV-sr39tk-IRES-EGFP determined the minimum number of sr39tk-expressing tumor cells necessary to be detected by PET using the (18)F-FHBG reporter probe. Furthermore, PET signal correlated well with traditional methods of protein evaluation such as flow cytometry, quantitative RT-PCR, Western blotting, and immunohistochemistry. Unlike the traditional methods, however, the use of PET is noninvasive and will be more advantageous in clinical situations.

FIGURE 1

Differential uptake of ¹⁸FFHBG in DU-145, PC-3, and CWR22Rv.1 PCa cell lines. (A) Total of 7% or 70% of cells stably transduced with lentiviral vector expressing HSV1-sr39tk-IRES-EGFP or IRES-EGFP as control were implanted on left or right shoulder, respectively. Tracer uptake seen in abdominal area of all mice is indicative of clearance route but is cropped out in DU-145 and PC-3 for clarity. Arrows indicate tumor position. (B) DU-145 cells transduced with increasing levels of HSV1-sr39tk have higher ¹⁸F-FHBG uptake (SUV), and intensity of signal increased over time with tumor volume (n = 4). (C) Tumor volume measure by CT was in range of 2.7–3.4 mm³ on day 8, 26.9–38.1 mm³ on day 15, and 152.8–212.8 mm³ on day 22.

FIGURE 2

HSV1-sr39tk transgene expression by flow, quantitative RTPCR, Western blotting, and immunohistochemistry. Tissues for these analyses were obtained from mice (n = 4) on day 23. (A) Single-cell suspensions of tumors were made, and percentage GFP-positive cells were obtained by flow analysis. (B) Quantitative RT-PCR analysis of sr39tk expression relative to β-actin levels. GFP tumor was used as a control. (C) Western blotting of sr39tk expression in tumor extracts. Sr39tk ratio to β-actin was analyzed using ImageJ (4 representative tumors per group shown). (D) Immunohistochemistry staining for tk protein in tumor sections. Number of cells positive for sr39tk expression increases with increasing percentage of lentiviral transduction. IHC+ = immunohistochemistry-positive; R, L, B, N = different animals within groups.

FIGURE 3

Correlation of sr39tk expression among various methods (Table 1). (A) PET signal (SUV) and sr39tk-expressing groups. (B) Western blotting and PET signal. (C) Flow cytometry and PET signal. (D) Flow cytometry and IHC-positive cell count. IHC+ = immunohistochemistry-positive.