Fig 2: B1R expression in normal cerebral cortical and tumoral tissues of F98 glioma-bearing rats.(A) Left: Representative electropherograms and gel-like images (insets) of RT-PCR products amplified from one tumor and autologous controlateral tissue (normal); LM and UM correspond to lower and upper internal markers, respectively. Right: Histographic representation of B1R expression from multiple normal and tumoral tissues. B1R mRNA level was normalized to the corresponding 18S level for each biopsy. n = 5 rats. *p<0.05 vs normal. (B) Western blot of rat brain soluble protein extracts probed with the anti-B1R antiserum AS434. Left: Rat brain tumor shows a robust single immunoreactive band around 60 kD. In the absence of antiserum, no band was seen in the rat brain samples (not shown). Right: Histographic representation of B1R expression from multiple normal and tumoral tissues. B1R protein level was normalized to the corresponding ß-actin level for each biopsy. n = 4 rats. *p<0.05 vs normal. (C) The antiserum used for Western blot was also used to characterize the location of the proteins in rat brain cortical samples by IHC, along with HRP (panels i–v), and TEM immunogold labeling (panel vi). Photomicrographs illustrating positive B1R immunoreactivity in tumor (C, i–iii) and microvascular endothelial cells (ii) adjacent to the tumor (black arrowheads). Note the chromosomal staining on all tumor cells under mitosis (black arrowheads, iii). Negative control with preimmune serum showed no staining (v). Weak positive B1R staining of glial cells from both implanted (ii) and contralateral hemispheres (iv) is also shown. Magnification as indicated. EM photomicrograph (vi) showing subcellular localization of B1R in a glioma cancer cell (white arrowheads). Insert: digital enlargement of the delineated area (white rectangle) showing B1R immunoreactivity at both the inner and outer leaflets of the nuclear envelope. Scale bar = 500 nm. Photomicrographs of HRP labeling were equalized in terms of contrast, brightness and gamma using ImagePro Plus 5.1.

Fig 3: Overexpression of B1R in human glioma tissues.(A) Detection of B1R in various human glioma cell lines and nontransformed counterparts, normal human astrocytes, by WB analysis using the anti-B1R antibody RC72. ß-actin serves as a loading control in the lower panel. The doublet band in U138-MG cells may indicate degradation of B1R. (B) Comparative quantification of B1R mRNA levels among normal and glioma brain tissue samples was normalized against that of the corresponding ß-actin. (C) Expression of B1R in normal versus tumoral tissue specimens (left panel) or in paired primary glioma (T) and peritumoral tissue biopsies (P) (right panel), with each pair obtained from a same patient. Western blot analysis was performed using the anti-B1R antiserum AS434. (D) Representative images from IHC assay of paraffin-embedded specimens of primary glioma tissue biopsies (WHO grades II–IV) exposed to pre-immune serum or AS434 antiserum. Optical magnification is indicated in the bottom-right corner of each image.

Fig 4: NG29 induces changes in local concentration of gadolinium and platinum in tumoral cerebral tissues.Direct measures of drug concentration (ng/g of tissue) by ICP-MS in three different tissue extracts (tumor, tumor periphery and contralateral) from the brain of F98 glioma-bearing Fischer rats were made following intraarterial (carotid artery) or intravenous (femoral vein) injections of Gd-DTPA (Gd) (143 mM i.v.) and carboplatin (Pt) (20 mg/kg i.a. or i.v.) with the B1R agonist NG29 (250 nmol/kg i.a.; 5 µmol/kg i.v.) or saline (CTL). Note the difference in ordinate scaling between drug concentrations in contralateral and tumoral tissues. Data are mean ± S.E.M. of 7–10 rats in each group. *p<0.05 versus respective control; N.S.: non significant.