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


CONCLUSIONS
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.
Johnson
Mai
M
Department of Molecular, Cellular and Integrative Physiology, UCLA, Los Angeles, CA, USA.
Karanikolas
Breanne D W
BD
Priceman
Saul J
SJ
Powell
Russell
R
Black
Margaret E
ME
Wu
Hsiao-Ming
HM
Czernin
Johannes
J
Huang
Sung-Cheng
SC
Wu
Lily
L
D000818:Animals; D045744:Cell Line, Tumor; D016503:Drug Delivery Systems / Q000379:methods; D015870:Gene Expression; D006147:Guanine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics; D008297:Male; D051379:Mice; D015336:Molecular Probe Techniques; D049268:Positron-Emission Tomography / Q000379:methods; D011471:Prostatic Neoplasms / Q000000981:diagnostic imaging* / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics; D013937:Thymidine Kinase / Q000235:genetics / Q000493:pharmacokinetics*; D014161:Transduction, Genetic / Q000379:methods
C113308:9-(4-fluoro-3-hydroxymethylbutyl)guanine; D019275:Radiopharmaceuticals; D006147:Guanine; D013937:Thymidine Kinase