Preclinical Evaluation of the Novel Monoclonal Antibody H6-11 for Prostate Cancer Imaging

The biological properties of the novel monoclonal antibody (mAb) H6-11 and its potential utility for oncological imaging studies were evaluated using <i>in vitro</i> and <i>in vivo</i> assays. Immunoreactivity of H6-11 to the human prostate cancer PC-3 cell line and solid tumor xenografts was initially demonstrated using immunofluorescence staining; the specificity of H6-11 for prostate cancer was further evaluated using a commercial array of human prostate cancer and normal tissue samples (<i>n</i> = 49) in which H6-11 detected 95% of prostate adenocarcinomas. The <i>K</i>_d value of 61.7 ± 30 nM was determined using ¹²⁵I-labeled H6-11. Glycosylation analysis suggested the antigenic epitope of the glycan is an O-linked β-<i>N</i>-acetylglucoside (<i>O</i>-GlcNAc) group. Imaging studies of PC-3 tumor-bearing mice were performed using both optical imaging with NIR fluorescent dye-labeled H6-11 and microPET imaging with ⁸⁹Zr-labeled H6-11. These <i>in vivo</i> studies revealed that the labeled probes accumulated in PC-3 tumors 48–72 h postinjection, although significant retention in liver was also observed. By 120 h postinjection, the tumors were still evident, although the liver showed significant clearance. These studies suggest that the mAb H6-11 may be a useful tool to detect prostate cancer <i>in vitro</i> and <i>in vivo</i>

Rapamycin blocks CXCL12 induced migration and actin polymerization of T cells.

<p>(A) Primary human T cells were labeled with calcein (5 µM) for 1 h in media and washed. Pretreatment of labeled cells was done with or without rapamaycin (100 ng/ml) for 1 h. Cells (1×10⁵ in 100 µl) were placed in the upper wells of 24-well transwell migration chambers with 5 µm pores (Corning, Corning, NY). In the lower wells, either medium alone or CXCL12 (100 ng/ml) was added to a total volume of 600 µl, and the chambers were incubated for 2 hours at 37°C in 5% CO2 incubator. Triplicate well determinations were performed for each treatment. The level of fluorescence of cells migrating across the chamber was assessed using a microfluorimeter. * indicates the value is statistically significant at p<0.05 level (n = 4). PMI indicates ‘percent migration index’. (B) Primary human T cells were treated with CXCL12 for 30 minutes in the presence or absence of pretreated rapamycin. Confocal microscopy was done according to the procedure mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024667#s3" target="_blank">Materials and Methods</a> to monitor actin polymerization. Red color indicates the actin polymerization. Representative images are shown from each treatment group. (C) Effect of rapamycin and KU-0063794 (KU) on CXCL12-induced cell migration, and effect of MIP3β and TARC in the presence or absence of rapamycin on the migration of resting T cells were performed as described in panel A. (D) Migration assay for CEM cells was performed similarly as primary T cells described above. (E) Dose response curve for rapamycin effect.</p

Role of mTOR in CXCL12-induced signaling and cell migration.

<p>(A) Whole cell lysates from CEM stable clones carrying either empty vector (EV) or shRNA constructs (sh-222 and sh-224) were analyzed by western blot analysis to determine the levels of total p70^S6K1. (B) Whole cell lysates from EV and sh-222 clones treated with CXCL12 for 30 minutes were analyzed by western blot analysis to determine the levels of phospho-p70^S6K1. (C) CEM cells were pretreated with rapamycin (100 ng/ml) and KU-0063794 (1 µM) for 1 hour, and then CXCL12 treatment was done for 30 minutes. Equal amounts of whole cell lysates were analyzed by western blot analysis. (D) Migration assay for EV, sh-222 and sh-224 clones were performed similarly as primary T cells described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024667#pone-0024667-g001" target="_blank">Fig. 1A</a>. (E) Surface expression of CXCR4 for EV, sh-222, and sh-224 were determined by FACS analysis, and the levels are expressed as mean fluorescence intensity (MFI). (F) Confocal microscopy to monitor CXCL12-induced actin polymerization in EV, sh-222, and sh-224 clones were performed similarly as primary T cells described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024667#pone-0024667-g001" target="_blank">Fig. 1B</a>. (G) In vivo migration of EV and sh-222 clone mediated by CXCL12 was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024667#s3" target="_blank">Materials and Methods</a>. Tissue staining from individual mice is shown here.</p

Case-control analysis on non-synonymous <i>PALB2</i> variants.

<p>Case-control analysis on non-synonymous <i>PALB2</i> variants.</p

Co-segregation analysis of <i>PALB2</i> variants in two high-risk CMM families.

<p>Individuals that have melanoma (MM) are represented by black circles (female) and black boxes (male). The age of diagnosis of each cancer is indicated in brackets. A line through a symbol indicates that the person is deceased. Individuals carrying a <i>PALB2</i> mutation are indicated by an ‘M’, while those wild-type for the variant are indicated by ‘WT’. Other cancer types are also indicated on the pedigree. Unaffected siblings are represented by a diamond with the number indicating the number of siblings. The arrow indicates the proband in each family.</p

Effects of GDC0941-AZD6244 combination therapy on PI3K/AKT and MEK/ERK pathways.

<p>All four pancreatic cancer cell lines were treated with the GDC0941-AZD6244 combination for 24 hours, Cell lysates were then harvested to detect p-AKT (S473)/AKT, p-ERK (T202/Y204)/ERK, p-S6 (S240/244)/S6 and p-4E-BP1 (S65)/4E-BP1.</p

Effects of GDC0941 and AZD6244 on PI3K/AKT and MEK/ERK pathways of MIA PaCa-2 and PANC-1 cell lines.

<p>Both MIA PaCa-2 and PANC-1 cells were treated with 400 nm GDC0941, 200 nM AZD6244 or a combination at these doses for 24 hours. Cell proteins were then harvested to detect p-AKT(S473)/AKT, p-ERK(T202/Y204)/ERK, p-S6(S240/244)/S6 and p-4E-BP1(S65)/4E-BP-1.</p

<i>PALB2</i> variants detected through Sanger sequencing and exome sequencing.

<p>*European American population.</p><p>na is not available.</p

Cell viability effect of GDC0941 and AZD6244 in BxPC-3, MIA PaCa-2, PANC-1 and Capan-2 pancreatic cancer cell lines.

<p>Cells were treated with varying concentrations of GDC0941 (A) or AZD6244 (B) alone for 72 hours. Doses ranged from 1 nM to 10 µM.</p

Effects of GDC0941 and AZD6244 therapy on cell apoptosis in pancreatic cancer cell lines.

<p>Pancreatic cancer cell lines were treated with GDC0941 alone, AZD6244 alone or GDC0941-AZD6244 in combination for 72 hours. Cell apoptosis was detected by flow cytometry. A, B: combinations vs. untreated groups (*p values <0.05), combinations vs. single agents (*p values <0.05), single agents vs. untreated groups (*p values <0.05). C, D: combinations vs. untreated groups (*p value <0.05) and combinations vs. single agents (*p value <0.05).</p