Table 1.
Patients’ cohort and tumor types used in enzymatic and conformational studies of their ACEs.
Fig 1.
ACE expression in lung cancer.
We performed immunostaining on the lung cancer specimens with anti-ACE mAbs CG2. We have analyzed 3 cases of SCLC (small cell lung cancer; pictures A-D), 9 cases of NSCLC (non-small lung cancer), here 3 squamous cell carcinoma (SCC; pictures E-H), 3 adenocarcinoma (ADC; pictures I-L) and 3 neuroendocrine carcinoma (NEC; pictures M-P). ACE expression and localization is shown by brown color. Compared to anti-CD68 (macrophages; pictures C, G, K, O) and AE1/3 (cancer cells; pictures D, H, L, P) ACE is expressed strongly in macrophages and endothelial cells of the tumor vascularization in all types of lung cancer analyzed (pictures B, F, J, N). A weak expression of ACE in cancer cells was only found in adenocarcinoma (picture J). ACE was not detected in cancer cells of SCLC, SCC and NEC (pictures B, F, N). A, E, I, M: H&E; B, F, J, N: CD143 (ACE); C, G, K, O: CD68; D, H, L, P: AE1/3. Magnification x100 (A-D, I-L, M-P), x200 (E-H).
Fig 2.
ACE expression sites in lung cancer.
Weak expression of ACE in lung adenocarcinoma cells (A). Strong expression of ACE in tumor vascular endothelial cells (arrows in A, C and D). Strong expression of ACE in macrophages and gigantic cell (B). Magnification: 100X (D), 200X (A, B, C).
Fig 3.
ACE activity in lung cancer tissues.
ACE activity in 12 homogenates (1:9 weight/volume ratio) of human lung cancer tissues (black dots) and in 10 homogenates from morphologically normal lung tissues (used as controls), (blue circles) was quantified using a spectrofluorometric assay with Z-Phe-His-Leu (2 mM) as substrates. Data expressed as individual values in mU/mg of protein (black dots and blue circles in A) and as mean values for control (blue bar- 28.6 ± 14.6 mU/mg) and for tumor lung tissue (9.4 ± 3.2 mU/mg). Data presented as a mean of at least 3 independent experiments (with intra-assay SD <10%).
Fig 4.
ACE conformation in lung cancer (conformational fingerprinting of ACE).
ACE activity was precipitated by 17 different mAbs from five individual homogenates of tumor lung tissues and their morphologically normal lung counterparts. Immunoprecipitated ACE activity is presented as a normalized value (Tumor/Normal ratio), to highlight differences in immunoprecipitation pattern of ACE from different homogenates by different antibodies. ACE activity in these 10 homogenates was quantified before immunocapture and ZPHL/HHL ratio was calculated and presented as a percentage of a value for morphologically normal tissues–values on the right side of the data for each patient. Data presented as a mean of at least 2–3 independent experiments in duplicates. Ratio increased more than 20% are highlighted with orange, more than 50% -with brown and more than 100%—with red. Bars highlighted with yellow- ratio decreased more than 20%, with blue—decreased more than 50%. Bars were highlighted if values were statistically significant (* p<0.05).
Fig 5.
ACE conformation in normal lung tissues.
Immunoprecipitated ACE activity from 5 homogenates of morphologically normal lung tissues from patients with lung cancer is presented as a normalized value to the pool of 4 homogenates of lung tissue from postmortem lung specimens. This highlights differences in immunoprecipitation pattern of ACE from different homogenates by different antibodies. Data are presented as mean of at least 2–3 independent experiments in duplicates. Bars color-coded—as in Fig 4. Blue boxes-mAbs which binding was changed in all lung cancer tissues in comparison to ACE from “normal” lung tissue.