Article Figures & Data
Figures
- Figure 1.
J8H antibody prevents ITGA6 cleavage and inhibits migration. A, PC3B1a cells were incubated with ITGA6 function-blocking antibodies for 48 hours and whole cell lysates assayed for the full length (A6), membrane-bound cleaved fraction (A6p), or amino terminal domain (A6N). Lanes represent duplicate-treated samples. B, cell migration through laminin-coated transwell inserts was assayed in the presence of the indicated neutralizing antibody specific for ITGA6 (J8H, J1B5, GoH3) or ITGB1 (AIIB2). Data are presented as % of control, which is isotype matched, nonspecific immunoglobulin (IgG). The non-antibody negative control is heparin sulfate proteoglycan (HSPG) and the non-antibody positive control is EDTA. C, PC3B1a cells were assayed for adhesion to a laminin 511–coated plate in the presence or absence of the indicated reagent. Data are presented as % control, where control is IgG as above.
- Figure 2.
Study design. A, schematic illustrating the animal protocol including intracardiac injection (I.C. injection) with prostate cancer cells (PC3B1a) and weekly tail vein injections of antibody (J8H, 4 mg/kg). B, representative micro-CT scans of control vs. J8H-treated animals are shown before injection (Wk 0) or weekly beginning with week 3, which is the first point that radiographic lesions could be detected. The ROI used for lesion volume analysis is shown (white dotted circle). Animals were removed from the study (X) when imminent fracture was indicated by the radiologist (*).
- Figure 3.
Quantitative analysis of tumor progression. A, metastatic bone lesions were recorded if detected in 3 views by micro-CT (axial, coronal, and sagittal) as lytic lesions. The osteolyic lesions (n = 69) were followed weekly by micro-CT analysis in untreated animals (solid line, diamonds, n = 9) or J8H-treated animals (dashed line, squares, n = 13). The untreated lesions (n = 29) and treated lesions (n = 40) were scored for progression by the radiologist without knowledge of the treatment groups. B, serial lesion volumes were used to determine the rate of tumor progression over time. Significance was determined using the Kruskal–Wallis nonparametric analysis (P < 0.01).
- Figure 4.
Survival advantage of J8H-treated animals. Survival advantage of the J8H-treated animals was determined using log rank analysis of the Kaplan–Meier survival time. Animals were followed for up to 8 weeks and were sacrificed if an imminent fracture in any lesion was detected by the radiologist, as dictated by the protocol.
- Figure 5.
J8H-treated animals demonstrate a higher frequency of sclerotic lesions. A, axial, coronal, and sagittal views demonstrate reactive bone surrounding the osteolytic lesions. B, matched histologic sections obtained at the end of the study of the lesion in A. The black dotted circle defines PC3B1a prostate tumor growth abutting the epiphyseal plate. Increased magnification of the lesion demonstrates a sclerotic type lesion, encircled by vessels and new bone formation. C, the frequency of lytic or sclerotic lesions was scored by a radiologist using micro-CT images from untreated (n = 29) or J8H-treated (n = 40) animals. Individual lesions were evaluated at the end of the study, and data are presented as % of total lesions.
- Figure 6.
Soluble factors produced by PC3B1a cells with extracellular engagement of ITGA6. A, PC3B1a cells were incubated with J8H antibody for the indicated time, and the conditioned media collected and analyzed for Cyr61 concentration by ELISA assay. Concentration was calculated based on standard curve. B, PC3B1a CM or serum-deprived media (SDM) prepared in the presence or absence of J8H antibody engagement was incubated with HFF cells for 72 hours, followed by incubation with β-galactosidase substrate for 4 hours. Cells were then harvested and analyzed for SA-β-Gal activity by flow cytometry.
Volume 13, Issue 6
