PSMA_GFP

High accuracy prostate cancer detection system with PSMA promoter

PSMA promoter with GFP at its downstream.

Evaluation of detection ability theoretically

PSMA (prostate-specific membrane antigen) is a type II transmembrane glycoprotein consisting of 750 amino acids, encoded by the folate hydrolase 1 gene, which is situated on the short arm of chromosome 11. [1]

Histologically, PSMA is present at low levels in the epithelium of benign prostate tissue but shows a significant increase in expression—by 100 to 1,000 times—in prostate adenocarcinomas [2][3]. It is found in the majority of tumours (90-100%)[7], and higher levels of PSMA expression have been positively correlated with several indicators of tumour aggressiveness, such as Gleason grade[3], tumour stage[4], biochemical recurrence[5], and castration resistance[6].

Thus, PSMA has been proven as an effective biomarker for prostate cancer (PCa). Moreover, it shows fewer false-positive results than the commonly used PSA[8].

To produce PSMA, PCa cells contain substances that activate the promoter in its copy of the organism gene, which allows the transcription of PSMA to be facilitated. The same substance activates the PSMA promoter in this part and transcribes GFP to detect cancer cells. GFP is excited by light in the blue to ultraviolet range and exhibits green fluorescence [9], which can be easily performed.

Evaluation of detection ability experimentally

In our project, we transfected different concentrations of plasmids containing this composite part into various concentrations of the PSMA-positive cell line.(MLLB-2)

Firstly, to visualise the fluorescence given out by GFP, photographing of cells was performed after 8 hours of transfection. It was seen that fluorescence occurred inside the MLLB-2 cells, which confirmed that PSMA promoter is viable for detecting PSMA +ve cells.

①: GFP activity in PSMA +ve PCa cells (MLLB-2) (with blue light filter)
②: PSMA +ve PCa cells (without blue light filter)

Then, to quantify the fluorescence, we measured it with a plate reader with excitation at 470nm and emission at 510nm. At low concentrations of plasmids (3.33e-3 μg/uL), cancer concentration showed a positive concentration with the fluorescence.

At higher concentrations of plasmids (6.67e-3 μg/μL, 1.33e-2 ug/uL), cell concentrations show a lower correlation with fluorescence. Still, all fluorescence values are higher than the control cells with no plasmid transfected, showing that our part is an effective tool for the detection of prostate cancer. However, we recommend using lower concentrations of plasmids should you want to measure the stage of prostate cancer the patient has.

Advantages for our system

By using this promoter + reporter gene design, theoretically, this system can detect almost all kinds of diseases. As long as researchers find a suitable biomarker for the disease, a promoter in the genome can be isolated and used in all kinds of systems. Moreover, the reporter gene can be customised to any protein in your favour, like the bioluminescent Gluc which has a lower cytotoxicity.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1995
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 630
Illegal BsaI.rc site found at 1917

References

[1] Rinker-Schaeffer CW, Hawkins AL, Su SL, et al. Localization and physical mapping of the prostate-specific membrane antigen (PSM) gene to human chromosome 11. Genomics. 1995;30:105–108.

[2] Silver DA, Pellicer I, Fair WR, Heston WD, Cordon-Cardo C. Prostate-specific membrane antigen expression in normal and malignant human tissues. Clin Cancer Res. 1997;3:81–85.

[3] Bostwick DG, Pacelli A, Blute M, Roche P, Murphy GP. Prostate specific membrane antigen expression in prostatic intraepithelial neoplasia and adenocarcinoma. Cancer. 1998;82:2256–2261.

[4] Perner S, Hofer MD, Kim R, et al. Prostate-specific membrane antigen expression as a predictor of prostate cancer progression. Hum Pathol. 2007;38:696–701.

[5] Ross JS, Sheehan CE, Fisher HAG, et al. Correlation of primary tumor prostate-specific membrane antigen expression with disease recurrence in prostate cancer. Clin Cancer Res. 2003;9:6357–6362.

[6] Wright GL, Grob BM, Haley C, et al. Upregulation of prostate-specific membrane antigen after androgen-deprivation therapy. Urology. 1996;48:326–334.

[7] Ceci, F., & Fanti, S. (2019). PSMA-PET/CT imaging in prostate cancer: why and when. Clinical and Translational Imaging, 7(6), 377–379. https://doi.org/10.1007/s40336-019-00348-x

[8] Velonas, V., Woo, H., Remedios, C., & Assinder, S. (2013). Current status of biomarkers for prostate cancer. International Journal of Molecular Sciences, 14(6), 11034–11060. https://doi.org/10.3390/ijms140611034