PMC

Biotinyl-BSA-Gd-DTPA-based molecular targeting agent. Anti-c-Met Ab was conjugated to the albumin via a sulfo-link (B). A similar procedure was used for a normal rat IgG probe used as a control, in addition to the (A) non-Ab control (biotinyl-BSA-Gd-DTPA without Ab). Diagram for biotinyl-BSA-Gd-DTPA modified from ref..

(A) Western blot of c-Met in normal rat brain tissue, and a C6 glioma (18 days after implantation). (B) Immunofluorescence detection of c-MET (red florescence, 20x magnification) in (i) contralateral control brain tissue, and (ii) a glioma region in a C6-glioma-bearing rat (18 days after implantation of C6 cells). Nuclei are stained blue (Dapi).

T1 values in glioma and normal brain tissue from representative rats administered anti-c-Met-biotinyl-albumin-Gd-DTPA (anti-c-Met1 and anti-c-Met2), or normal rat IgG-biotinyl-albumin-Gd-DTPA, in normal (N) and tumour (T) tissues. Note a decrease in T1 values over a period of 3 hrs in glioma (T) regions of C6 rats administered the anti-c-Met targeting agent, whereas T1 values for normal brain regions return to pre-contrast T1 values after 3 hrs post-contrast.

(i) Morphological images of rats with C6 gliomas treated with (A) the anti-c-Met probe (T1w image, 3 hrs after anti-c-Met probe), (B) the non-Ab non-specific control biotinyl-albumin-Gd-DTPA contrast agent (FLASH image) and (C) the non-specific control normal rat IgG probe (T2w image). (ii) Apparent probe/contrast agent concentration maps derived from T1 maps at 3 hrs after probe/contrast agent administration (subtracted from the pre-contrast T1 map), for the same treatment groups as in (i).

T2-weighted MR images of rats with gliomas administered with either (A) albumin-Gd-DTPA-biotin, or (B) normal rat IgG-albumin-Gd-DTPA-biotin. Outlined regions depict areas where fluorescence images were obtained following in vivo administration of probes. These control probes depict a lack of non-specific binding in normal (i) and glioma (ii) tissues that have undergone fluorescent staining (Cy3-labelled streptavidin) for the biotin moiety on each probe. Fluorescence staining of the biotin group on albumin-Gd-DTPA (non-specific, non-Ab) and the normal rat IgG (non-specific) probes in C6-treated rats with gliomas (Aii and Bii, respectively), and their respective normal brain tissues (Ai and Bi) on the contralateral side (magnification 20x). For a comparison see Fig.3I, which shows specificity of the anti-c-Met probe in glioma tissue.

T1-weighted MR images (T1w) obtained at 3 hrs after administration of the anti-c-Met molecular targeting agent (c-Met) in rat C6 gliomas (at 23 and 17 days, respectively). Similar images have also been acquired at 3 hrs after administration of control non-Ab-Gd-DTPA-BSA (no Ab) in C6 gliomas (21 days after implantation), and control normal rat IgG-Gd-DTPA-BSA (IgG; 21 days after implantation) in C6 glioma-bearing rats. T₂-weighted images (T2w) (with the exception of the central image in the bottom panel (no Ab; FLASH), which is a FLASH image) are displayed to help depict the gliomas, since they depict a stronger contrast between the tumour and the surrounding healthy tissue in comparison to T₁-weighted images. Regions of interest used for signal intensity (SI) and T1 measurements in , are displayed as red circles and labelled N for normal tissue, TI for tumour interior and TP for tumour periphery regions.

T1-weighted MR images pre-(A) and post-administration (B) 25 and (C) 175 min.) of anti-c-Met molecular targeting agent in a C6 glioma (23 days after implantation). (D) Intensity difference image between (A) and (C). Histological slices were taken from the same rat that was imaged in within normal brain tissue (E), a tumour border region (G; displayed rotated by 90°), and within a tumour necrotic centre (H). Fluorescence staining (Cy3-labelled streptavidin) of the biotin moiety of the anti-c-Met targeting agent (red fluorescence) is shown in corresponding histological slices (tumour side, I, which is the same region as histological slice H; and control brain tissue, F, which is the same region as histological slice E; 20x magnification). Nuclei are stained with Dapi (blue fluorescence). Note specific binding of the anti-c-Met probe in glioma tissue shown in frame I.

Relative differences in regional (A) MR signal intensities and (B) relative probe concentrations (differences in relaxation rates [1/T₁][10⁻⁶ M]), as outlined in each T₁-weighted image in , following pre-and post-administration after 3 hrs, of the anti-c-Met molecular targeting agent (anti-c-Met) (region-of-interests [ROIs] shown in T1w c-Met images; in rat C6 gliomas at 23, and 17 days, respectively); pre- and post-administration, after 3 hrs, of non-Ab Gd-DTPA-BSA (non-Ab) in C6 gliomas at 21 days (from ROIs depicted in T1w no Ab images), and pre- and post-administration, after 3 hrs, of normal rat IgG-Gd-DTPA-BSA (rat IgG) in a C6 glioma at 21 days (from ROIs shown in T1w IgG images). Mean ± S. D. values for normal brain (N), tumour interior (TI) and tumour periphery (TP) are displayed in histograms. Significant differences were obtained if P-values were (*) <0.05, or (**) <0.01, between groups highlighted. For the normal rat IgG probe the number of regions sampled for TP and N was n = 1, that is no statistical data was obtained.