Dianthin-30 or gelonin versus monomethyl auristatin E, each configured with an anti-calcitonin receptor antibody, are differentially potent in vitro in high-grade glioma cell lines derived from glioblastoma.

We have reported that calcitonin receptor (CTR) is widely expressed in biopsies from the lethal brain tumour glioblastoma by malignant glioma and brain tumour-initiating cells (glioma stem cells) using anti-human CTR antibodies. A monoclonal antibody against an epitope within the extracellular domain of CTR was raised (mAb2C4) and chemically conjugated to either plant ribosome-inactivating proteins (RIPs) dianthin-30 or gelonin, or the drug monomethyl auristatin E (MMAE), and purified. In the high-grade glioma cell line (HGG, representing glioma stem cells) SB2b, in the presence of the triterpene glycoside SO1861, the EC50 for mAb2C4:dianthin was 10.0 pM and for mAb2C4:MMAE [antibody drug conjugate (ADC)] 2.5 nM, 250-fold less potent. With the cell line U87MG, in the presence of SO1861, the EC50 for mAb2C4:dianthin was 20 pM, mAb2C4:gelonin, 20 pM, compared to the ADC (6.3 nM), which is >300 less potent. Several other HGG cell lines that express CTR were tested and the efficacies of mAb2C4:RIP (dianthin or gelonin) were similar. Co-administration of the enhancer SO1861 purified from plants enhances lysosomal escape. Enhancement with SO1861 increased potency of the immunotoxin (>3 log values) compared to the ADC (1 log). The uptake of antibody was demonstrated with the fluorescent conjugate mAb2C4:Alexa Fluor 568, and the release of dianthin-30:Alexa Fluor488 into the cytosol following addition of SO1861 supports our model. These data demonstrate that the immunotoxins are highly potent and that CTR is an effective target expressed by a large proportion of HGG cell lines representative of glioma stem cells and isolated from individual patients.

A novel population of α-smooth muscle actin-positive cells activated in a rat model of stroke: an analysis of the spatio-temporal distribution in response to ischemia.

In a rat model of stroke, the spatio-temporal distribution of α-smooth muscle actin-positive, (αSMA+) cells was investigated in the infarcted hemisphere (ipsilateral) and compared with the contralateral hemisphere. At day 3 postischemia, αSMA+ cells were concentrated in two main loci within the ipsilateral hemisphere (Area A) in the medial corpus callosum and (Area B) midway through the striatum adjacent to the lateral ventricle. By day 7 and further by day 14, fewer αSMA+ cells remained in Areas A and B but a steady increase in the peri-infarct was observed. αSMA+ cells also expressed glial acidic fibrillary protein [GFAP: αSMA+/GFAP+ (29%); αSMA+/GFAP- (71%) phenotypes] and feline leukemia virus C receptor 2 (FLVCR2), but not ED1(microglia) and established markers of pericytes normally located in vascular wall. αSMA+ cells were also located close to the subventricular zones (SVZ) adjacent to Areas A and B. In conclusion, αSMA+ cells have been identified in a spatial and temporal sequence from the SVZ, at intermediate loci and in the vicinity of the peri-infarct. It is hypothesized that novel populations of αSMA+ precursors of pericytes are born on the SVZ, migrate into the peri-infarct region and are incorporated into new vessels of the peri-infarct regions.

The expression of calcitonin receptor detected in malignant cells of the brain tumour glioblastoma multiforme and functional properties in the cell line A172.

AIM: Previous studies have indicated that expression of calcitonin receptor (CTR) could be induced in a proinflammatory environment. In the present study, CTR-immunoreactivity (CTR-ir) was investigated in brain tissue from patients with glioblastoma multiforme (GBM). METHODS AND RESULTS: In immunohistochemical analysis of GBM samples, tissues with complex glomeruloid structures surrounded by malignant cells were analysed for CTR-ir using anti-human CTR antibodies generated against two separate epitopes of CTR. CTR-ir was associated predominantly with glial cells. Regions with CTR-ir cells were found in 12 of 14 GBM tumours (P < 0.05). Using confocal microscopy, CTR-ir cells were identified that were also positive for glial fibrillary acidic protein, nestin and CD133. Antibodies were verified using immunoblots and confocal microscopy of the Cercopithecus aethiops(COS)-7 transfectants. Immunoblots of membrane preparations from the CTR-positive cell lines demonstrated a major band (≈ 67 kDa) and minor band (≈ 52 kDa), but the intensity was reversed for the GBM cell line A172. In cultured A172 cells, functional studies demonstrated calcitonin stimulation of adenylyl cyclase and inhibition of extracellular-regulated kinase (ERK)1/2 phosphorylation. CONCLUSIONS: The findings that (i) CTR was expressed by glioma cells in a majority of GBM tumours tested, (ii) CTR(+) /CD133(+) cells were identified and (iii) second messenger systems were functionally modified by calcitonin in A172 cells suggest that CTR might be a useful therapeutic target in GBM.