Article Figures & Data
Figures
- Figure 1.
Illustration of the antibody fragment selection procedure and screening strategies. The target area for selection was chosen to be the middle part of a particular cancer nest due to clustered CD271+ staining present. The entire tissue on a consecutive formalin-fixed section was then incubated with a phage library. The target area was relocated and a minute disc (shadow stick) was positioned precisely above the target cells of interest. The target area was kept moist at all times. The shadow stick shielded the phage antibodies binding to cells of interest from UV-C irradiation. The phages were eluted, but only those protected by the shadow stick can replicate in bacteria and provide ampicillin resistance. Each colony represented an antibody fragment, which required screening for their specificity. They were picked and grown in separate wells of a master plate. 1) In the initial screening, all colonies were grown in microtiter plates and monoclonal phage antibodies produced. The phage antibodies were tested by phage ELISA on CD271+ cancer cells. 2) All phage antibodies bound with higher affinities than the negative control in the initial screening were produced monoclonally in 50-ml cultures. These were tested in different concentrations by a phage ELISA titration assay, which was performed simultaneously on CD271+ cancer cells and CD271+myoepithelial cells. This provided comparative results of each phage antibody. 3) Soluble antibody fragments were expressed and purified, and examined by IHC experiments on four different breast cancer biopsies to validate their specificity.
- Figure 2.
Immunohistochemistry showing breast cancer-specific staining with an antibody against fragment LH8. Immunohistochemistry was performed on three seperate sections of cryostat tissue from the luminal breast cancer patient 761. All pictures are merged with DAPI staining. Pictures A, C and E show staining against CK19, which indicate the presence of cancer cells. Picture B shows staining with the antibody fragment LH8 which consistently only binds cancer cells. The presence of cancer cells is confirmed by staining with the proliferation marker ki67 within these areas, as observed in picture D. Picture F shows staining with the mouse Cy3 conjugated anti-c-Myc antibody used for detection of the antibody fragments. This shows that the observed staining is not caused by up-regulated c-myc expression in cancer cells or unspecific binding by this secondary antibody.
- Figure 3.
Immunohistochemistry showing differential staining intensity in patient 761. The figure shows two different areas on the same section of a cryostat tissue from the luminal breast cancer patient 761. All pictures are merged with DAPI staining. Pictures A and C show staining against CK19. Picture B and D shows staining with the antibody fragment LH8. Although the pictures are from the same tissue section there exist variations in the staining pattern. Cancer cells are not uniformly stained but rather display different areas with different intensity. Some areas of cancer cells present very limited staining (Figure D, upper left and right) whereas others are very intense.
- Figure 4.
Immunohistochemistry showing differential staining intensity in patient 686. The figure shows two different areas on cryostat tissue from luminal breast cancer of patient 686 at higher magnification. All pictures are merged with DAPI staining. Pictures A and C show staining against CK19. Pictures B and D show staining with LH8. The pictures are representative and display an obvious difference in staining intensity in different cancer areas.
- Figure 5.
Immunohistochemistry performed on healthy breast tissue. For further characterization of LH8, immunohistochemistry was performed on tissue sections from three healthy donors. Picture A, D and G show DAPI staining. B, E and H show staining against CK19. Picture C, F and I show staining with the antibody fragment LH8. The upper, middle and lower rows represent the healthy donors P671, P820 and P923 respectively. No staining was observed in neither the healthy luminal cells or stroma.
