Table 1.
Figure 1.
Schematic Diagram of the Self-Assembly Sequence for the Nanodevice (NP-Trx3).
The fluorescein-labeled nanodevice (ND-Trx3) displays three copies of the bacterial thioredoxin (Trx) as a cellular targeting ligand. It is assembled by annealing three synthetic oligodeoxynucleotides containing fluoresein (*) at a centrally-located site and 5-Fluorocytosine (F) at each of the three methyltransferase recognition sites, followed by covalent linkage of methyltransferase fusion proteins as previously described [19]. Fluorescence labeling permits visualization of the bound device with fluorescence microscopy. Multivalency improves the avidity of the device since many of the known thioredoxin interacting partners (e.g. the human thioredoxin reductases) are dimeric [9].
Figure 2.
ND-Trx3 Binding to PCA and BPH in Frozen Tissue Sections.
Representative frozen tissue sections were incubated for 1 minute with 20nM of the fluorescence-labeled ND-Trx3 in 200 µl of ice cold PBS or with 200 µl of PBS alone as control. Adjacent sections were also stained with H&E and Masson's Trichrome. The slides were then analyzed by fluorescent microscopy and a tiled image of the entire tissue slice was obtained to identify regions of ND-Trx3 binding. Tiled images photographed at 100X magnification allowed visualization of the entire tumor specimen after size reduction. Tumor (T) and Stromal (S) regions are indicated in each panel. PBS: Phosphate Buffered Saline, H&E: Hematoxylin and Eosin. Trichrome-stained sections were counter stained with hematoxylin (blue nuclear stain). The pattern of fluorescence observed with the nanodevice in the cancer specimen (A) was confined to the stromal region identified as the violet region in the adjacent section stained with Masson's Trichrome (C). Control BPH sections (B) did not bind the nanodevice significantly and yielded very low levels of fluorescence, and only weak staining with Masson's Trichrome (D). H&E stained sections containing tumor gave blue color in the tumor regions and light brown staining in regions of reactive stroma (E) and with BPH (F).
Figure 3.
Comparison of the Pattern of Nanodevice Binding with that of Immunohistochemical Staining with Antibodies to TXNRD1, TXNRD2 and PRDX1 in Representative Tumor Specimens.
Adjacent sections (5 µm-thick) of resected tissue specimens containing prostate cancer or BPH were incubated with ND-Trx3, or immunohistochemically (IHC) stained with antibodies to TXNRD1, TXNRD2, or Peroxiredoxin. Each immunohistochmically stained section was also counterstained with hematoxylin (blue nuclear staining). The fluorescence pattern observed with the nanodevice (A) was similar to the brown staining in the stromal regions (S) and at the nterface between stroma (S) and tumor (T) observed TXNRD2 (E). Fluorescence due to the nanodevice was essentially absent from BPH specimens (B). This pattern was similar to that of antibody to TXNRD2, which did not effecively stain BPH (F). Anti-TXNRD1 stained the cancer regions (T) more effectively than the stromal regions (S) (C). Anti-TXNRD1 gave only light brown staining with BPH (D). Antibody to PRDX1 gave strong brown staining in the cancer (T) with somewhat less staining in the reactive stroma (S) (G). Anti-PRDX1 gave uniform brown staining with BPH (H).
Figure 4.
Distinguishing PCA from BPH Based on Nanodevice Binding to Thioredoxin Interacting Proteins.
Surgically resected tissue specimens were obtained from 35 patients. Serial sections of the tissue specimens (5 µm thick) were incubated with 20 nM of the nanodevice in PBS and 1% BSA. Fluorescence binding was observed and a numerical grading system was given as the level of nanodevice fluorescence in the stroma by two independent investigators (the author EMS and GB from acknowledgements). 0- No visible signal, 1- weakly visible signal, 2- a moderately intense visible signal and 3- a bright and intense signal within the reactive stroma. The binding level for PCA-associated stroma was significantly greater than that of BPH (p = 0.0127) based on the t-test.