Murine B16F10 melanoma cell line was obtained from the American Type Culture Collection (Rockville, MD). Tumor cells were maintained in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 1.0% sodium pyruvate, glutamate, and Pen-Strep (Life Technologies, Inc.). Cells were maintained as subconfluent cultures before use and harvested with trypsin-ethylenediaminetetraacetic acid (Life Technologies, Inc.).

Cell adhesion assays were performed as described previously with some modifications.²² Briefly, 48-well nontissue culture plates were coated with native triple helical or thermally denatured collagen types I and IV (10.0 μg/ml) for 12 hours at 4°C. The plates were next washed with PBS and nonspecific binding sites were blocked by incubation with 1.0% bovine serum albumin (BSA) in PBS for 1 hour at 37°C. Tumor cells (B16F10) from subconfluent cultures were harvested, washed, and resuspended in adhesion buffer containing RPMI 1640, 1 mmol/L MgCl₂, 0.2 mmol/L MnCl₂, and 0.5% BSA in the presence or absence of function-blocking antibodies (0 to 100 μg/ml) or an isotype-matched control antibody. Tumor cells were added to the coated plates in a total volume of 200 μl and allowed to attach for 15 to 30 minutes. Nonattached cells were removed by washing and attached cells were stained with crystal violet as described previously.²² Cell adhesion was quantified by measuring the optical density of eluted crystal violet from attached cells at a wavelength of 600 nm.²² In cell proliferation assays, microtiter wells were coated with either native or denatured collagen type I or IV (10 μg/ml). Tumor cells (B16F10) were resuspended in proliferation buffer containing 1.0% serum in the presence or absence of mAb HUIV26 or an isotype-matched control antibody (0 to 100 μg/ml) and allowed to proliferate throughout a 3-day time course. Cellular proliferation was measured with a WST-1 tetrazolium salt cleavage assay kit (Chemicon International) according to the manufacturer’s instructions. Cell proliferation was monitored using a microplate reader at a wavelength of 490 nm. Experiments were performed in triplicates and repeated twice with similar results.

Cell migration assays were performed as described previously with some modifications.²² Briefly, membranes (8.0-μm pore size) from transwell migration chambers were coated with native triple helical or thermally denatured collagen type I or IV (10.0 μg/ml) for 12 hours at 4°C. The transwells were next washed with PBS and nonspecific binding sites were blocked by incubation with 1.0% BSA in PBS for 1 hour at 37°C. Tumor cells from subconfluent cultures were harvested, washed, and resuspended in migration buffer containing RPMI 1640, 1 mmol/L MgCl₂, 0.2 mmol/L MnCl₂, and 0.5% BSA in the presence or absence of function-blocking antibody (0 to 100 μg/ml) or an isotype-matched control. Tumor cells were allowed to migrate to the underside of the coated transwell membranes for 2 to 4 hours. Tumor cells remaining on the top-side of the membrane were removed and cells that had migrated to the under side were stained with crystal violet as described previously.²² Cell migration was quantified by direct cell counts per microscopic field.

Twelve-day-old fertilized chick eggs were obtained from SPAFAS (North Franklin, CT) and maintained in a 48-place tabletop egg incubator (Lyon Electric, Chula Vista, CA) as described previously.^23,24 Prominent blood vessels were visualized through the eggshell of the 12-day-old chick embryos with the aid of an egg candle.^23,24 The area of the outer egg shell where prominent blood vessels are located close to the inner shell surface was swabbed with 70% ethanol and a small window was cut through the egg shell with a hobby grinding wheel (Dremel Emerson Electric Co., Racine, WI). The embryos were returned to the incubator until tumor cells were prepared for injection. Subconfluent cultures of B16F10 melanoma cells were washed with sterile PBS and harvested with trypsin ethylenediaminetetraacetic acid. Tumor cells were washed with serum containing Dulbecco’s modified Eagle’s medium and resuspended in sterile PBS at concentrations ranging from 0.5 to 5.0 × 10⁶ per ml. Next, the small windows cut through the egg shell were carefully removed and a drop of mineral oil was added to the shell membrane to enhance visualization of the underlying blood vessels.^23,24 Tumor cell suspensions were injected intravenously in a total volume of 100 μl per embryo. The embryos were allowed to incubate undisturbed for 24 hours or a total of 7 days. To quantify experimental B16F10 lung metastasis, embryos were sacrificed at day 19 and both lobes of the chick lungs were dissected. The lungs were analyzed with the aid of a stereo microscope set at a defined magnification. The total number of isolated and discrete pigmented lung surface lesions was carefully counted on each side of each lobe for each embryo. A typical experiment included at least six embryos per condition. Experimental metastasis was described as the mean number of surface B16F10 melanoma lesions per lung per experimental condition.

To detect the presence of B16F10 melanoma cells at early time points (24 hours) semiquantitative RT-PCR was performed using primers specific for B16 M562 melanoma antigen and control primers for chick 18s ribosome as previously described.^25,26 Briefly, chick lungs from each experimental condition were harvested at the end of a 24-hour incubation period after tumor cell inoculation and mRNA prepared as previously described.^25,26 To examine the relative levels of B16F10 melanoma cells in the lungs of chick embryos at early time points, we used PCR primers directed to the B16-specific M562 melanoma antigen (forward) 5′-TGGGCTTGTAGTACTGGACTC-3′ and (reverse) 5′-TTCACGTGATTTACAATCTCCTATTG-3′.^25,26 In control experiments, PCR primers specific for chick 18S ribosome were used that included (forward) 5′-TTCGTATTGTGCCGCTAGAG-3′ and (reverse) 5′-GCATCGTTAATGGTCGGAAC-3′.

The experimental metastasis assay was performed essentially as described with some modifications.^24,27 Briefly, subconfluent cultures of B16F10 melanoma cells were harvested, washed, and resuspended in sterile PBS in the presence or absence of mAb HUIV26 or an isotype-matched control antibody (100.0 μg/ml). Female BALB/c mice were injected intravenously (100 μl) with tumor cells (2 × 10⁵). After injection of the tumor cells, the mice were untreated or treated daily (7 days) (intraperitoneally) or treated with a single injection of mAb HUIV26 or normal mouse IgM control antibody (100 μg) in a total volume of 100 μl of sterile PBS. At the end of the treatment periods, the mice were sacrificed and the lungs were removed for analysis. To quantify experimental B16F10 lung metastasis, lungs were dissected and placed in 35-mm culture plates. The lungs were analyzed with the aid of a stereomicroscope set at a defined magnification (×30). The total number of isolated and discrete pigmented lung surface lesions was carefully counted on each lobe for each specimen. Experimental metastasis is described as the mean number of surface tumor lesions per lung per experimental condition. Presence of tumor lesions within the lungs was confirmed by histological analysis.

Lungs from chick embryos or mice were dissected and embedded in OTC, snap-frozen, and 4.0-μm sections were cut with a cryostat as described previously.^28,29 For histological analysis, frozen sections of lung tissue were fixed in 10% formalin and stained with hematoxylin and eosin. Immunohistochemical analysis was performed as previously described with some modifications.^30–32 Briefly, lung sections (4.0 μm) were fixed for 30 seconds in 50% methanol and 50% acetone. The tissue sections were washed three times and incubated with 2.5% BSA in PBS to block nonspecific binding sites. mAb HUIV26 was diluted in 2.5% BSA in PBS to a final concentration of 10 μg/ml and 100 μl was added to the tissue sections. Tissues were incubated for a total of 2 hours at 37°C. The tissues were next washed five times with PBS for 5 minutes each followed by incubation with horseradish peroxidase-labeled goat anti-mouse secondary antibody (1:300) for 1 hour. The tissue sections were washed as before and photographed at a magnification of ×200. For immunofluorescence co-staining, tissue sections were prepared as described above and incubated with goat anti-mouse rhodamine-labeled secondary antibody (1:400) and fluorescein isothiocyanate-labeled L. esculentum lectin (10 μg/ml) for detection of blood vessels. The tissue sections were washed as before and a drop of anti-fade mounting medium added. The tissue sections were then photographed at a magnification of ×630 under oil emersion.

Studies have suggested proteolytic remodeling of ECM components such as collagen type IV can regulate cellular invasion and migration. Thus, we examined the effects of mAb HUIV26 on malignant tumor cell migration in vitro. Membranes from transwell migration chambers were coated with native or denatured collagen type I or IV and B16F10 tumor cells were resuspended in migration buffer in the presence or absence of mAb HUIV26 or an isotype-matched control antibody. Tumor cell suspensions were added to the upper chambers of the migration wells and allowed to migrate to the under side of the coated membranes for 4 hours. Migration was measured by counting the number of tumor cells that migrated to the underside of the wells as described in the Materials and Methods section. As shown in Figure 2, A and B, B16F10 melanoma cells migrated on both denatured collagen type I and IV. Importantly, migration of B16F10 tumor cells was inhibited dose dependently as compared to an isotype-matched control antibody with maximal inhibition (50%) observed at 100 μg/ml. Moreover, mAb HUIV26 had little effect on tumor cell migration on either native or denatured collagen type I (Figure 2, C and D). These data are consistent with our previous studies and suggest that cellular interactions with the HUIV26 cryptic site play a role in regulating tumor cell migration on structurally altered collagen type IV.

Our previous studies have shown that cryptic epitopes within collagen are specifically exposed within basement membrane collagen type IV of malignant human and murine tumors as well as the subendothelial basement membrane of angiogenic blood vessels.^18–21 Importantly, our current studies suggest that malignant B16F10 melanoma cells can interact with the HUIV26 cryptic site and function-blocking antibody directed to this site inhibits cellular adhesion and migration. Given these findings, the possibility exists that inhibiting cellular interactions with the HUIV26 site may impact tumor cell metastasis. Therefore, we established a rapid experimental metastasis assay to study the potential role of the HUIV26 epitope in this process. To facilitate these studies, we used the chick embryo model in conjunction with metastatic B16F10 melanoma cells. Subconfluent B16F10 melanoma cells were resuspended in sterile PBS. Twelve-day-old chick embryos were injected intravenously with 100 μl of B16F10 cell suspension, and the embryos were allowed to incubate for a total of 7 days. At the end of the 7-day incubation period, the embryos were sacrificed and the lungs were resected, washed, and placed in OTC embedding compound and snap-frozen as described in the Materials and Methods section. Frozen sections (4.0 μm) were cut and nonspecific binding sites were blocked with BSA. The tissue sections from each experimental condition were next incubated with control buffer or mAb HUIV26 and immunoreactivity was visualized by incubation with horseradish peroxidase-labeled goat anti-mouse secondary antibody. As shown in Figure 3A, left, no specific exposure of the HUIV26 cryptic epitope was detected within control lungs whereas lungs from embryos injected with B16F10 melanoma cells stained positive (brown) for the HUIV26 cryptic epitope (Figure 3A, right), indicating the exposure of this cryptic collagen epitope (brown). To further examine the distribution of this cryptic epitope we performed immunofluorescence co-localization analysis. Frozen tissue sections from lungs from chick embryos previously injected with B15F10 tumor cells were stained with mAb HUIV26 followed by incubation with fluorescein isothiocyanate-labeled L. esculentum lectin, which has previously been shown to mark blood vessels.²⁰ As shown in Figure 3B, the HUIV26 cryptic epitope (red) was detected in close association with blood vessels (green) at both early (24 hours) and late (7 days) time points after tumor cell inoculation. These findings are in agreement with previously published reports and indicate that the HUIV26 cryptic epitope is generated within B16F10 lung lesions in the chick embryo. Taken together, these findings confirm the suitability of this model to assess the potential anti-metastatic effects of mAb HUIV26.

To examine the effects of mAb HUIV26 on B16F10 experimental metastasis, subconfluent B16F10 cells were harvested, washed, and resuspended in sterile PBS in the presence or absence of mAb HUIV26 or an isotype-matched control antibody. The B16F10 cell suspensions were injected intravenously (100 μl per embryo) and 7 days later the embryos were sacrificed and the lungs removed for analysis. As shown in Figure 4A, injection of untreated B16F10 melanoma cells resulted in the formation of numerous B16F10 lung tumor foci (black lesions). In contrast, lungs from chick embryos treated with mAb HUIV26 exhibited a reduction in B16F10 lung surface lesions. Histological examination of the lungs confirmed a reduction in infiltration of B16F10 melanoma cells into the lung tissue. To quantify the effects of mAb HUIV26 on B16F10 experimental metastasis, the number of B16F10 surface lesions was counted on each side of the lung for each lung. As shown in Figure 4B, in the presence of mAb HUIV26 (100 μg), the mean number of B16F10 lung foci was significantly (P < 0.001) reduced by ~65% as compared to no treatment or control antibody, whereas 1.0 μg of mAb HUIV26 per embryo exhibited little effect. To confirm the specificity of these findings and exclude any nonspecific effects of the buffer, the antibody preparation was immune-depleted with denatured collagen type IV (depleted) or the control protein denatured collagen type I (cont-depleted). As shown in Figure 5C, the denatured collagen type IV-depleted antibody preparation failed to inhibit B16F10 experimental metastasis (P > 0.200). In contrast, the control depleted antibody preparation significantly (P < 0.005) inhibited experimental metastasis by ~60% as compared to controls providing additional evidence for specific effects of mAb HUIV26. To further examine the temporal effects of mAb HUIV26 on B16F10 cell metastasis in this model, we harvested chick lungs 24 hours after tumor cell injection and analyzed them for the presence of B16F10 tumor cells by semiquantitative RT-PCR using primers specific for B16 M562 melanoma antigen as has been previously described.^25,26 As shown in Figure 4D, B16F10 melanoma cells could be detected within the lungs of untreated (Figure 4D, lanes 5 to 8) or control antibody-treated (Figure 4D, lanes 9 to 12) chick embryos 24 hours after tumor cells injection. In contrast, the relative levels of B16F10 tumor cells as measured by the B16 melanoma antigen RNA was reduced (Figure 4D, lanes 1 to 4) as compared to controls. Importantly, although the semiquantitative RT-PCR failed to detect signals from two of the lungs shown after a fixed number of cycles, lower intensity signals (Figure 4D, lanes 3 and 4) as compared to controls were detected in lung samples under identical conditions. These findings are consistent with the reduction in metastatic lung lesions observed at later time points. In addition, these results suggest an early reduction of experimental metastasis when angiogenesis is unlikely to play a significant role. Taken together, these findings suggest that mAb HUIV26 inhibits experimental metastasis of B16F10 to the lungs of chick embryos and that this inhibitory effect can occur early in the metastatic cascade.