Immunohistochemistry of melanoma tissue microarrays for VEGFR-2
To estimate the frequency of VEGFR-2 expression on human melanoma cells, we stained matched TMAs containing 43 metastatic melanoma samples with commercially-available antibodies: A-311–13
. The results are summarized in , and representative microscopic images are shown in . Endothelial cells, which serve as an internal positive control for staining of endogenous VEGFR-2 (reviewed in17
), stained with antibody 55B11, but showed no staining or very weak staining with antibody A-3 (). On the other hand, antibody A-3 showed strong staining of vascular smooth muscle (panel a) and histiocytes (panel c) whereas antibody 55B11 did not stain either histiocytes or smooth muscle cells (panels b and d). For clarity, higher magnification images of melanoma cells stained positive for VEGFR-2 with antibodies A-3 and 55B11 are shown in , panels e and f. Antibody A-3 stained melanoma cells in 79% of the specimens, but antibody 55B11 only stained 9% (). The staining intensity on melanoma cells with antibody A-3 varied from 1+ to 3+ whereas the positive samples with antibody 55B11 only showed 1+ staining. These results revealed disparities in staining by these commercial antibodies and prompted a broader census of melanomas and more thorough examination of antibody specificity.
To analyze VEGFR-2 expression on melanoma cells among melanoma metastases, we used antibody 55B11 to stain additional TMA blocks for a total of 167 melanoma samples from 133 patients (58 female, 75 male, ages 23 – 90; mean 59±16). The tumor samples on the TMAs included 9 primary cutaneous and 158 metastatic melanomas. Metastatic melanomas were located in lymph node (57), skin and soft tissue (93), small intestine (7), and peritoneum (1). Melanoma cell morphology was epithelioid in 133 samples, spindle in 7, and mixed (epithelioid and spindle) in 27.
Melanoma cells from only 11 of the 167 specimens immunostained for VEGFR-2 (7%; 95% exact binomial confidence interval: 3.3, 11.5). These 11 specimens were from 11 different patients (11/133 = 8%) and all had only 1+ staining (). The mean age and sex of the 11 patients with VEGFR-2+ melanomas, as well as the location and histology of the melanomas are listed in . Our conclusion was that VEGFR-2 is rarely expressed by melanoma cells in tumors, and only at relatively low levels. This contrasts with previous reports that a large majority of melanomas express VEGFR-2.
VEGFR-2 immunohistochemical staining of melanoma specimens and demographics of patients positive for VEGFR-2 from 8 TMAs using VEGFR-2 55B11 Antibody
Immunoblotting melanoma cell extracts for VEGFR-2
To validate the specificity of the anti-VEGFR-2 antibodies, we performed immunoblotting with A-3 and 55B11. Extracts of melanoma cell lines that either express or do not express VEGFR-2 (VMM18 and DM122,3
respectively) were analyzed by immunoblotting (). There was no obvious difference between VMM18 and DM122 cells in the overall pattern of bands prominently stained by antibody A-3. Antibody A-3 does stain a band corresponding to the size of VEGFR-2 in the VMM18 extracts. However, most of the immunostaining intensity with antibody A-3 was in other bands between 25 and 100 kDa that were common to VMM18 and DM122 cells. In contrast, with antibody 55B11 a dominant triplet of proteins (210–230 kDa) corresponding to VEGFR-2 was immunostained in extracts of VMM18 and these bands were not seen with DM122 cells.3
Figure 2 Immunoblotting VEGFR-2 in melanoma cell extracts. a), Triplicate samples of 20 ug protein of VMM18 (VEGFR-2+) and DM122 (VEGFR-2neg) melanoma cell line extracts were resolved on the same gel and transferred to Immobilon. Separate sections of the filter (more ...)
We transfected VMM18 cells with siRNA to knockdown endogenous VEGFR-2. Nonspecific siRNA was transfected into a separate sample of VMM18 cells as a control. Cell extracts were prepared and split into 3 aliquots that were resolved on the same gel, transferred onto a filter that was cut into strips and these were immunoblotted with different antibodies. Immunoblotting showed knockdown of VEGFR-2 dramatically reduced staining of the 210–230 kDa bands with antibody 55B11, whereas immunoblotting of the several bands by antibody A-3 was not affected (). This indicated that the major bands stained by antibody A-3 were not fragments of VEGFR-2 and instead were cross-reactive proteins endogenous to the melanoma cells. This experiment was independently replicated with identical results. We concluded that antibody 55B11 primarily stained VEGFR-2 whereas antibody A-3 primarily reacted with several other proteins.
We transiently transfected VEGFR-2neg DM122 melanoma cells with a plasmid to express full-length VEGFR-2. Cell extracts were prepared and split into 3 aliquots that were resolved on the same gel and immunoblotted with the A-3 and 55B11 VEGFR-2 antibodies (). Blotting for beta-actin was used as a loading control (not shown). Immunoblotting revealed multiple bands with the A-3 antibody with 95% of the total staining intensity in bands <150 kDa. However, immunoblotting with the 55B11 antibody showed a pair of bands at approximately 210 and 230 kDa corresponding to VEGFR-2, accounting for > 90% of the total staining intensity. These data demonstrate that the 55B11 antibody has high specificity for VEGFR-2, whereas the A-3 antibody predominantly reacts with other endogenous protein in melanoma cells.
Immunohistochemistry of melanoma cell extracts for VEGFR-2
We compared immunostaining of the VEGFR-2+ VMM18 melanoma cell line transiently transfected with siRNA to knockdown VEGFR-2 () and the DM122 VEGFR-2-null melanoma cell line expressing ectopic full-length VEGFR-2 (). Cytospins were prepared to deposit the cells, which were stained with the 55B11 antibody ( and , panels a, e, and g) vs. the A-3 antibody ( and , panels b, f, and h). As negative controls, samples were stained the same, but omitting incubation with primary antibody ( and , panels c and d). Untreated cells were used as additional control ( and , panels a and b). In , VMM18 cells transfected with the non-specific control siRNA stained positive with both the 55B11 and A-3 antibodies, as expected (, panels e and f). VMM18 cells transfected with siRNA to target VEGFR-2 were no longer stained with the 55B11 antibody (, panel g) but remained positive for staining with the A-3 antibody (, panel h). In , the VEGFR-2 negative DM122 cells that were mock transfected (panels e and f) did not stain with the 55B11 antibody, but did stain positive with the A-3 antibody. However, DM122 cells expressing full-length VEGFR-2 showed a difference from control and now stained positive with the 55B11 antibody. In contrast, there was no difference in staining with the A-3 antibody between DM122 cells (, panels g and h). Thus, immunohistochemical staining of the melanoma cell lines ( and ) matched the immunoblotting results () and showed that only staining with 55B11 antibody and not staining with the A-3 antibody was affected by either knockdown or over expression of VEGFR-2.
Figure 3 Immunohistochemistry of VMM18 melanoma cells. Cytospins were processed and stained as described under materials and methods with antibody 55B11 (panels a, c, e, and g), or antibody A-3 (panels b, d, f, and h). VMM18 melanoma cells alone (positive control) (more ...)
Figure 4 Immunohistochemistry of DM122 melanoma cells. Cytospins were processed and stained as described under materials and methods with antibody 55B11 (panels a, c, e, and g), or antibody A-3 (panels b, d, f, and h). DM122 melanoma cells alone (positive control) (more ...)