Purpose: Melanoma is one of the most aggressive types of cancer with currently no chance of cure once the disease has spread to distant sites. Therapeutic options for advanced stage III and IV are very limited, mainly palliative, and show response in only ∼20% of all cases. The presented preclinical study was done to investigate the influence of a combined treatment of the epidermal growth factor receptor inhibitor erlotinib and the vascular endothelial growth factor monoclonal antibody bevacizumab in melanoma.

Experimental Design and Results: The epidermal growth factor receptor was expressed in all cell lines tested, and treatment with erlotinib did inhibit the activation of the MEK/extracellular signal-regulated kinase and AKT signaling pathways. Whereas in vitro no influence on tumor cell proliferation was seen with erlotinib or bevacizumab monotherapy, a decreased invasive potential on erlotinib treatment in a three-dimensional Matrigel assay was observed. Furthermore, both drugs inhibited proliferation and sprouting of endothelial cells. In vivo, in a severe combined immunodeficient mouse xenotransplantation model, reduction in tumor volume under combined treatment with erlotinib and bevacizumab was superior to the additive effect of both single agents. This was associated with reduced cell proliferation, increased apoptosis, and a reduction in tumor angiogenesis compared with control or single treatment groups. Likewise, the reduction in the extent of lymph node and lung metastasis was most pronounced in animals treated with both drugs.

Conclusion: The presented data strongly support the use of a combination of erlotinib and bevacizumab as a novel treatment regimen for metastatic melanoma.

Translational Relevance

Metastatic melanoma is a highly treatment-resistant tumor with currently only palliative treatment options. Therefore, novel therapeutic strategies are of utmost importance. Erlotinib and bevacizumab are currently approved for the treatment of several solid tumors but have not yet been tested in melanoma. We present novel data on the efficacy of a combination of erlotinib and bevacizumab in preclinical models of melanoma and find the combination of these drugs to be significantly active against melanoma growth and metastasis in a severe combined immunodeficient mouse xenotransplantation model. Treatment with erlotinib and bevacizumab did influence cell proliferation, tumor cell apoptosis, and tumor angiogenesis. Taken together, our data provide the preclinical rationale for clinical studies using erlotinib and bevacizumab in melanoma.

Malignant melanoma is the most aggressive type of skin cancer. It accounts for 3% of all skin cancers but is responsible for at least 80% of skin cancer-related deaths (1). The incidence of melanoma, especially among Caucasians, has been rapidly increasing within the last decades. The disease outcome of melanoma patients depends on depth of invasion of the primary tumor, tumor burden, ulceration, and sites of metastasis. Wide excision of malignant melanoma in early stages leads to complete cure and a 5-year survival rate of 95%. Involvement of the regional lymph node basin reduces the 5-year survival rates to as low as 26% and spreading to distant sites (lung, liver, and brain) decreases the 1-year survival to 41% and the 5-year survival rates to 14% (2). Despite many efforts to find improved therapeutic regimens, none was proven to be superior to cytostatic therapy with dacarbazine, which itself is associated with response rates of only 10% to 15% and rarely leads to complete remissions.

Recently, novel targeted drugs directed against the epidermal growth factor (EGF) receptor (EGFR; erbB1) or the vascular endothelial growth factor (VEGF) have been shown to have significant clinical activity in different types of cancer (37).

Signaling through the EGFR affects cellular functions from proliferation to differentiation as well as programmed cell death in normal cells (8). In tumor cells, it contributes to tumor metastasis via increased cell motility and intravasation (9). Overexpression or disturbed regulations have been found in breast cancer, non-small cell lung cancer, and clear cell renal carcinoma as well as in melanoma and glioblastoma (10). Eighty-nine percent of primary cutaneous melanomas and 91% of melanoma metastases show either EGF or EGFR expression (11, 12), making it a possible therapeutic target for the treatment of metastatic melanoma.

Erlotinib, a small molecule inhibitor specific for the EGFR kinase, is an orally available, quinazoline based-agent competing with ATP for binding to the intracellular catalytic domain of the receptor kinase, thereby inhibiting autophosphorylation of the receptor critical for binding to downstream signaling proteins (13). Erlotinib has shown activity in the treatment of non-small cell lung, colon, and pancreatic cancer as well as in glioblastoma and is currently approved for the treatment of locally advanced or metastatic non-small cell lung cancer and locally advanced, unresectable, or metastatic pancreatic cancer (14).

VEGF has been identified as a potent contributor to angiogenesis, tumor proliferation, and lymphangiogenesis in malignant melanoma (15).

Bevacizumab is a humanized monoclonal antibody directed against VEGF in humans (16). It neutralizes all isoforms of VEGF, thereby preventing the interaction of VEGF with its receptors (17). Bevacizumab inhibits VEGF-induced endothelial cell proliferation, tumor angiogenesis, and subsequently tumor growth as well as access of metastatic tumor cells to the vasculature (16, 18). Furthermore, treatment with VEGF inhibiting drugs was shown to be associated with a normalization of aberrant tumor vessels, thereby facilitating the delivery of other drugs to the tumor (19, 20). Bevacizumab has shown clinical activity in colorectal, breast, non-small cell lung, and metastatic renal cell carcinoma. It is currently approved for the treatment of metastatic colorectal cancer in combination with chemotherapy (7), metastatic HER-2-negative breast cancer (21), and recurrent, locally advanced, unresectable, or metastatic non-small cell lung cancer (22).

Currently, no preclinical evaluation on the activity of these two drugs in malignant melanoma is available. The aim of this study was therefore to identify the influence of erlotinib and bevacizumab on melanoma cell proliferation in vitro and to further identify possible additive or synergistic effects in a severe combined immunodeficient (SCID) mouse/human melanoma xenotransplantation model.

Cells. 518A2, 607B (courtesy of Dr. Peter Schrier), SK-Mel-28, A375 (American Type Culture Collection), Mel-Juso (German Collection of Microorganisms and Cell Culture), M24met (courtesy of R.A. Reisfeld, Scripps Institute), and 6F (isolated from an ovaric metastasis) melanoma cells were maintained in DMEM (Invitrogen) supplemented with 8% heat-inactivated FCS. Human umbilical vein endothelial cells (HUVEC) were prepared from umbilical cords by incubation with a 5% collagenase solution; mouse aortic endothelial cells were obtained from Pharmakine. Endothelial cells were cultured in Endothelial Cell Medium (Promo Cell) containing 15% FCS.

Western blot. Cells were detached from culture plates in a lysis buffer containing 1% NP-40, 0.1% SDS, 100 mmol/L NaCl, 50 mmol/L Tris (pH 7.4-7.7), 10 mmol/L EDTA complemented with 10 mmol/L p-nitrophenylphosphate, 250 units/mL aprotinin, 40 μg/mL leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride, 10 mmol/L NaF, and 40 mmol/L β-glycerophosphate. Micro BCA Protein Assay kit (Pierce) was used to determine protein concentration. Equal amounts of protein were separated by SDS-PAGE and transferred electrophoretically onto a nitrocellulose membrane. Ponceau red stain and an antibody directed against β-actin were used as loading controls. Antibodies used were directed against EGFR (Lab Vision), p44/42 mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and phospho-p44/42 mitogen-activated protein kinase, AKT and phospho-AKT, and VEGF receptor (VEGFR1; Santa Cruz Biotechnology) and VEGFR2 (Cell Signaling Technology). Secondary antibodies used were horseradish peroxidase conjugated (Santa Cruz Biotechnology). Blots were developed using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology).

Cell proliferation assay. Cells (2,500 per well) were plated into 96-well plates and subsequently treated with either increasing concentrations of erlotinib (300 nmol/L-30 μmol/L) or bevacizumab (1-100 μg/mL) or recombinant human VEGF (PeproTech). The erlotinib concentrations used correlate with a phase I study by Hidalgo et al., where after oral ingestion maximum plasma concentrations of 0.219 to 2.13 μg/mL were achieved (3). Proliferation was measured after 48 h (melanoma cells) to 72 h (mouse aortic endothelial cells) using the Cell Titer 96 assay (Promega) on a 1420 multilabel counter (Victor2, Wallac) at 490 nm.

Invasion chamber assays. Invasion chamber assays were done using Matrigel invasion chambers (BD Biosciences) with a pore size of 8 μm in the top well. Cells (5 × 104) were plated into the top chamber in serum-free medium and 5% FCS was used as chemoattractant in the bottom well. After 12, 24, or 48 h, the top chamber was removed, the interior was swapped with a cotton bud, and transmigrated cells were fixed with ethanol for 15 min. Transmigrated cells were counted after staining with gentian violet. The area covered by stained cells was counted using an AxioVision Digital Imaging system (Carl Zeiss) with the membrane surface set as 100%.

Three-dimensional spheroid-based angiogenesis assay. A confluent cell monolayer of HUVECs was trypsinized and cells were suspended in 10 mL endothelial cell medium containing 20% methylcellulose (Sigma). Cells (400 per drop) were distributed to Petri dishes and turned upside down. The plates were incubated for 24 h at 37°C. Afterwards, spheroids were collected by centrifugation and overlaid with methylcellulose. Spheroid/methylcellulose solution was mixed with rat-tail collagen, 10× concentrated M199 medium (Sigma), 1 mol/L HEPES, and 0.2 N NaOH and dispensed in 24-well plates. After polymerization, spheroids were overlaid with the corresponding conditioned medium containing 10% FCS in the absence or presence of erlotinib (1 or 2 μmol/L), bevacizumab (1 μg or 50 μg/mL), or their combination (1 μmol/L and 1 μg) and incubated for 24 h. Conditioned medium was obtained by incubating 518A2 or M24met cells with basal endothelial cell medium for 48 or 72 h. Endothelial cell medium containing 10% FCS was used as a negative control and stimulation with 25 ng recombinant human VEGF as a positive control. Images were taken with an Olympus ε 330 mounted on the microscope and sprout length was measured using a standardized scale (23).

ERK and AKT activity assays. Cells were grown to 80% confluence. Subsequently, the cell culture medium was changed to serum-free medium for 24 h and then replaced by serum-free medium with or without 1 μmol/L erlotinib for 1 h, after which cells were stimulated for 10 min with 100 nmol/L EGF. The experiment was stopped by snap freezing with liquid nitrogen and subsequent addition of lysis buffer.

Animals. Six- to 8-week-old female C.B-17 scid/scid (SCID-) mice were purchased from Charles River Laboratories. The animals were kept in a pathogen-free environment and every procedure was done in a laminar airflow cabinet. The experiments were done according to the regulations of the Ethics Committee for the Care and Use of Laboratory Animals at the Medical University Vienna.

Mouse experiments. For the local tumor growth experiment, 2 × 106 518A2 melanoma cells in 100 μL physiologic saline were injected subcutaneously into the right flank. Animals were randomly assigned to treatment groups and therapy started when tumor nodules reached a mean size of 50 mm3. Animals were treated with 500 μg erlotinib orally every day, 5 mg/kg bevacizumab intraperitoneally twice weekly, or with a combination of both drugs. Animals in the control group received solvent orally or physiologic saline intraperitoneally. Animals were controlled for distress development every day and tumor size was assessed regularly by caliper measurement. Tumor volume was calculated using the formula: (length × width2) / 2. Treatment was continued for 20 days and tumors were removed at the end of the experiment and partially cryopreserved in OCT or fixed in 7.5% formaldehyde for paraffin embedding.

For the metastasizing melanoma model, 2 × 106 M24met melanoma cells were injected into the right flank of SCID mice. Animals were randomly assigned to treatment groups and treatment was started when tumors had reached a mean volume of 50 mm3. After 10 days, animals were anesthetized, tumors were removed, and the wounds were sutured. Treatment was continued until day 20. Axillary and inguinal lymph nodes as well as the lungs were removed to assess metastatic spread. Primary tumors were processed as above; lungs and lymph nodes were paraffin embedded. Lymph node diameter was assessed by caliper measurement and presence of metastases in lymph nodes was verified by H&E staining. Lung metastases were assessed by measurement of the vimentin-positive area in three representative sections per lung using an AxioVision Digital Imaging system. Five different fields of view at ×20 magnification were assessed per section.

Immunohistochemistry. Ki-67: Paraffin-embedded sections from mouse tumors were deparaffinized according to routine procedures and the sections were incubated in citrate buffer for 15 min at 85°C. Subsequently, samples were incubated with mouse anti-human Ki-67 antibody (Immunotech) and a biotinylated anti-mouse antibody (Vector Laboratories).

Vimentin: Paraffin-embedded lung sections were deparaffinized and the tissue sections were then incubated in citrate buffer overnight at 85°C. Staining was done with an anti-human vimentin antibody (DAKO) as described previously (24).

CD31, F4/80, and SMA: Frozen tumor sections were treated with 0.1% Triton X-100 in PBS and subsequently incubated overnight with an antibody directed against murine CD31 (BD Pharmingen), F4/80 (Serotec), or SMA. After incubation with the respective biotinylated second step antibodies, staining was done using AEC reagent (DAKO).

Terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Terminal deoxynucleotidyl transferase-mediated nick end labeling was done on cryosections from xenotransplanted tumors using the In situ Cell Death Detection Kit from Roche according to the manufacturer' instructions.

Statistical analysis. Statistical analysis was done by ANOVA with Bonferroni post-test using GraphPad Prism software (GraphPad Software). P values < 0.05 were considered significant.

Immunoblot results showed a strong signal at 170 kDa correlating with the molecular weight of the EGFR in all melanoma cell lines (Fig. 1A).

Fig. 1.

A, melanoma cell lines were tested for the expression of the EGFR. Expression of the EGFR was positive in all cell lines. Human vascular endothelial cells (HUVEC) were used as positive control; an antibody against β-actin was used as loading control. B and C, melanoma cell lines 518A2, SK-Mel-28, and M24met were serum starved for 24 h (Ctrl.) and subsequently treated with 100 nmol/L EGF in the presence (Erlotinib) or absence (EGF) of 1 μmol/L erlotinib. A reduction of (B) ERK phosphorylation and (C) AKT phosphorylation (p-AKT) was seen in all cell lines.

Fig. 1.

A, melanoma cell lines were tested for the expression of the EGFR. Expression of the EGFR was positive in all cell lines. Human vascular endothelial cells (HUVEC) were used as positive control; an antibody against β-actin was used as loading control. B and C, melanoma cell lines 518A2, SK-Mel-28, and M24met were serum starved for 24 h (Ctrl.) and subsequently treated with 100 nmol/L EGF in the presence (Erlotinib) or absence (EGF) of 1 μmol/L erlotinib. A reduction of (B) ERK phosphorylation and (C) AKT phosphorylation (p-AKT) was seen in all cell lines.

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We first tested the ability of erlotinib to block the activation of the MEK/ERK and AKT signaling pathways, which are both known to be activated via the EGFR and are involved in the regulation of growth, motility, and apoptosis. As shown in Fig. 1B, a clear reduction in the EGF-induced phosphorylation of ERK was observed in 518A2 cells on treatment with erlotinib, whereas only a modest reduction was seen in SK-Mel-28 and M24met cells. In contrast, AKT phosphorylation was reduced in all three cell lines under the same conditions, with the strongest reduction in SK-Mel-28 and M24met (Fig. 1B). No change in ERK or AKT protein expression was observed in any of the cell lines used.

Despite these results, treatment with erlotinib up to a concentration of 10 μmol/L erlotinib and bevacizumab up to a concentration of 100 μg was not associated with a reduction in proliferation in any of five melanoma cell lines tested in vitro (data not shown).

In contrast to the growth assays, a significant difference was observed in the three-dimensional invasion chamber assay. 518A2, SK-Mel-28, and M24met cells were chosen for these experiments and incubated either with or without erlotinib or bevacizumab. Transmigrated cells were counted after 12, 24, or 48 h, respectively, depending on their migrational behavior in serum-free conditions as published previously (25). Erlotinib did reduce transmigration by up to 60 ± 6% in 518A2, 64 ± 7% in M24met, and 58 ± 3% in SK-Mel-28 cells (P < 0.001 versus control for all cell lines), showing a clear effect on the migrational behavior in a three-dimensional assay (Fig. 2A).

Fig. 2.

A, melanoma cell lines 518A2, SK-Mel-28, and M24met were plated into Matrigel invasion chambers in combination with 0.5 or 1 μmol/L erlotinib; 5% serum was used as a chemoattractant. Numbers of transmigrated cells were counted after 12 to 48 h depending on the migrational speed of the respective cell line. A reduction in the number of transmigrated cells was observed in all three cell lines on treatment with erlotinib. Bars, SD. *, significant differences compared with the respective control value (n = 3 experiments). B to D, HUVEC spheroids were suspended in rat collagen and plated into 24-well plates. Spheroids were incubated with conditioned medium (CM) from either M24met (C) or 518A2 cells (D) in the absence or presence of erlotinib (E), bevacizumab (B), or the combination of both drugs. Medium with 10% FCS was used as negative control (Control) and stimulation with 25 ng recombinant human VEGF as positive control (VEGF). Numbers of sprouts and sprout length were measured after 24 h and the total sprout length per spheroid was calculated. A reduction of total sprout length was observed in all treatment groups, with the most significant reduction in the combined treatment arm. B, representative micrographs from all treatment groups. C and D, bars, SD (n = 3 experiments).

Fig. 2.

A, melanoma cell lines 518A2, SK-Mel-28, and M24met were plated into Matrigel invasion chambers in combination with 0.5 or 1 μmol/L erlotinib; 5% serum was used as a chemoattractant. Numbers of transmigrated cells were counted after 12 to 48 h depending on the migrational speed of the respective cell line. A reduction in the number of transmigrated cells was observed in all three cell lines on treatment with erlotinib. Bars, SD. *, significant differences compared with the respective control value (n = 3 experiments). B to D, HUVEC spheroids were suspended in rat collagen and plated into 24-well plates. Spheroids were incubated with conditioned medium (CM) from either M24met (C) or 518A2 cells (D) in the absence or presence of erlotinib (E), bevacizumab (B), or the combination of both drugs. Medium with 10% FCS was used as negative control (Control) and stimulation with 25 ng recombinant human VEGF as positive control (VEGF). Numbers of sprouts and sprout length were measured after 24 h and the total sprout length per spheroid was calculated. A reduction of total sprout length was observed in all treatment groups, with the most significant reduction in the combined treatment arm. B, representative micrographs from all treatment groups. C and D, bars, SD (n = 3 experiments).

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A sprouting assay using HUVECs and conditioned medium from 518A2 and M24met melanoma cells revealed that erlotinib and bevacizumab are both able to reduce endothelial cell sprouting. Erlotinib (1 μmol/L) reduced the total sprout length by 51% (P > 0.05), whereas erlotinib (2 μmol/L) did reduce sprout length by 74% (P < 0.01) in HUVECs conditioned with 518A2 medium. Sprout length was reduced by 85% (P < 0.001) and 89% (P < 0.001) in the cells conditioned with M24met medium on treatment with 1 or 2 μmol/L erlotinib, respectively. Bevacizumab reduced the total sprout length by 76% (P < 0.001) in HUVECs conditioned with 518A2 medium and by 75% (P < 0.001) in the cells conditioned with M24met medium. Combined treatment at a concentration of 1 μmol/L erlotinib and 1 μg bevacizumab showed the strongest deregulation with a reduction of sprout length by 84% (P < 0.001) and 96% (P < 0.001), respectively (Fig. 2B, C and D).

Based on these in vitro results, we used a human melanoma-SCID mouse xenotransplantation model to test the influence of erlotinib, bevacizumab, and the combination of both drugs in vivo. To test if these agents have any influence on tumor growth in SCID mice, 518A2 melanoma cells were injected subcutaneously and treated with vehicle, erlotinib, bevacizumab, or a combination of both drugs. Tumor take was comparable in all four groups and treatment was started when tumors in all groups had reached a mean tumor volume of 50 mm3 and continued for 20 days. Tumor volume in the erlotinib-treated population was reduced by 16% (P > 0.05) and bevacizumab-treated animals showed a decrease of 52% (P < 0.001) compared with the control group. Most importantly, the conjoint treatment with erlotinib and bevacizumab showed the highest reduction in tumor volume of 85% compared with the control arm (P < 0.001; Fig. 3A).

Fig. 3.

A, 518A2 melanoma cells were injected into the right flank of female C.B-17 SCID mice. Mice were treated with erlotinib (orally, daily, 50 mg/kg), bevacizumab (intraperitoneally, twice weekly, 5 mg/kg), or a combination of both drugs. Bars, SD. B and C, M24met cells were injected into the right flank of CB17 SCID treated as mentioned above. Tumors were removed after 10 d and treatment was continued for further 10 d. Metastases in inguinal and axillary lymph nodes (B) and lungs (C) were assessed by measuring volume or vimentin-positive area in an immunohistochemical staining, respectively. Bars, SD. *, significant differences compared with the respective control group (n = 10 mice per group).

Fig. 3.

A, 518A2 melanoma cells were injected into the right flank of female C.B-17 SCID mice. Mice were treated with erlotinib (orally, daily, 50 mg/kg), bevacizumab (intraperitoneally, twice weekly, 5 mg/kg), or a combination of both drugs. Bars, SD. B and C, M24met cells were injected into the right flank of CB17 SCID treated as mentioned above. Tumors were removed after 10 d and treatment was continued for further 10 d. Metastases in inguinal and axillary lymph nodes (B) and lungs (C) were assessed by measuring volume or vimentin-positive area in an immunohistochemical staining, respectively. Bars, SD. *, significant differences compared with the respective control group (n = 10 mice per group).

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We furthermore assessed the influence of erlotinib and bevacizumab on the formation of locoregional lymph node and on lung metastases by using the metastasizing cell line M24met (26). Cells were injected into the right flank and treatment was started when tumors reached 50 mm3. Ten days after onset of therapy, the primary tumors were removed and treatment continued until day 20. As the primary tumors in this model were removed at an earlier time point, the differences between treatment groups had not yet reached significance, but trends were comparable with the observations in the 518A2-derived subcutaneous tumors (data not shown). To elucidate the influence of erlotinib and bevacizumab on regional metastatic spread in SCID mice, the diameters of axillary and inguinal lymph nodes were measured. Although we did see a clear trend toward a reduction in the size of lymph node metastasis by treatment with erlotinib (40.3%; P > 0.05) or bevacizumab (49.3%; P > 0.05), only the combined treatment with both drugs did show a statistically significant reduction in lymph node diameter (68.2%; P < 0.01; Fig. 3B). Analysis of distant metastasis to the lungs substantiated this result, as only the combination of erlotinib and bevacizumab was capable of significantly reducing metastasis by 71.4% (P < 0.01), whereas erlotinib and bevacizumab as single agents did only achieve a modest reduction of 30.5% (P > 0.05) and 23.6% (P > 0.05), respectively (Fig. 3C).

Immunohistochemical staining of subcutaneous mouse tumors with the proliferation marker Ki-67 showed a decrease of proliferation activity in the erlotinib, bevacizumab, and combination group compared with the control group. As expected, the least staining was observed in the tumors of animals treated with erlotinib alone or in combination with bevacizumab (Figs. 4A and 5A). Staining of murine CD31 did show the expected reduction of vascularization in the bevacizumab group but did reveal the strongest reduction in the conjoint treatment arm, showing that the treatment with erlotinib did augment the antiangiogenic effect of bevacizumab (Figs. 4B and 5B). No difference was seen in the number of SMA-positive pericytes, which were located along larger postcapillary vessels or in the number of F4/80-positive tumor-infiltrating macrophages (data not shown). Terminal deoxynucleotidyl transferase-mediated nick end labeling of tumor sections did furthermore show a strong induction of apoptosis under combined treatment with erlotinib and bevacizumab compared with control or single drug-treated animals (Fig. 5C). Although all melanoma cell lines used in the in vivo experiments did express VEGFR1 (Fig. 6A), none of the melanoma cell lines used in the in vivo experiments did express VEGFR2 (Fig. 6B). Neither did we see increased proliferation of melanoma cells on stimulation with recombinant human VEGF-A nor a decrease of proliferation under treatment with bevacizumab in vitro (data not shown). This excludes that the recently described, VEGFR2-mediated autocrine loop (27) is responsible for the observed antitumor effects. To ensure that the implication that tumor cell-derived human VEGF could be a stimulant for mouse endothelial cells in our model system, we incubated mouse aortic endothelial cells with either recombinant human VEGF or conditioned medium from M24met melanoma cells. Under both conditions, we did observe a clear stimulation of murine endothelial cell proliferation (Fig. 6C).

Fig. 4.

A, sections from 518A2-derived mouse tumors were stained with an antibody against the proliferation marker Ki-67. Representative micrographs. B, sections from M24met-derived mouse tumors were stained with an antibody against murine CD31. Representative micrographs.

Fig. 4.

A, sections from 518A2-derived mouse tumors were stained with an antibody against the proliferation marker Ki-67. Representative micrographs. B, sections from M24met-derived mouse tumors were stained with an antibody against murine CD31. Representative micrographs.

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Fig. 5.

A, tumor sections were treated as in Fig. 4A and the relative area positive for Ki-67 per field of view was measured (n = 10 sections per group). B, tumor sections were treated as in Fig. 4B and the relative area of CD31+ vessels per field of view was measured (n = 30 sections per group). C, DNA strand breaks were stained by terminal deoxynucleotidyl transferase-mediated nick end labeling on sections from M24met-derived mouse tumors. Columns, mean number of positive cells from five ×100 fields of view per section (n = 10 sections per group; A-C). Bars, SD. *, significant differences compared with the respective control group.

Fig. 5.

A, tumor sections were treated as in Fig. 4A and the relative area positive for Ki-67 per field of view was measured (n = 10 sections per group). B, tumor sections were treated as in Fig. 4B and the relative area of CD31+ vessels per field of view was measured (n = 30 sections per group). C, DNA strand breaks were stained by terminal deoxynucleotidyl transferase-mediated nick end labeling on sections from M24met-derived mouse tumors. Columns, mean number of positive cells from five ×100 fields of view per section (n = 10 sections per group; A-C). Bars, SD. *, significant differences compared with the respective control group.

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Fig. 6.

A and B, melanoma cell lines were tested for the expression of the VEGFR1 or VEGFR2 by Western blotting. Human vascular endothelial cells (HUVEC) were used as positive control; β-actin was used as loading control. C, murine endothelial cells were incubated in either endothelial cell medium (Control) or medium containing 25 ng VEGF (VEGF) or medium conditioned by incubation with M24met melanoma cells (CM).

Fig. 6.

A and B, melanoma cell lines were tested for the expression of the VEGFR1 or VEGFR2 by Western blotting. Human vascular endothelial cells (HUVEC) were used as positive control; β-actin was used as loading control. C, murine endothelial cells were incubated in either endothelial cell medium (Control) or medium containing 25 ng VEGF (VEGF) or medium conditioned by incubation with M24met melanoma cells (CM).

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Therapy of advanced stages of malignant melanoma has thus far been limited to palliative measures and has not been able to improve the patients overall survival. Because metastasis in solid tumors is a multistep process involving the tumor itself, the tumor microenvironment, and their interaction (28), it is obvious that the treatment of metastatic melanoma is urgently needing therapeutic strategies aiming at multiple tumor sites (29, 30) with improved response rates better than those observed with standard cytotoxic drugs.

Thus far, no preclinical data on the effectivity of the EGFR inhibitor erlotinib or its combination with the monoclonal antibody against VEGF bevacizumab for the treatment of melanoma exist. The purpose of this study was to investigate the influence of erlotinib and bevacizumab on growth and invasiveness of malignant melanoma cell lines and endothelial cell sprouting in vitro and in a SCID mouse-human melanoma xenotransplantation model in vivo regarding melanoma growth and metastasis.

We found the EGFR to be expressed in all melanoma cell lines tested irrespective of their origin (primary tumor, lymph node, and subcutaneous or visceral metastasis). In growth assays, we did not see an influence of erlotinib on melanoma cell proliferation.

Interestingly, in a three-dimensional assay mimicking the basal membrane penetration of metastasizing tumor cells, we detected that erlotinib at varying concentrations was capable of significantly reducing the number of transmigrating melanoma cells. These findings are consistent with recent publications that melanoma cells react differently to pharmacologic interventions in two-dimensional assays compared with three-dimensional assays (31). We further assessed the kinase activity of ERK and AKT in three melanoma cell lines and observed a distinct decrease of phosphorylation on erlotinib treatment. ERK and AKT are both involved in tumor cell proliferation, growth, survival, and apoptosis in malignant melanoma and can be constitutively activated in malignant melanoma (32, 33). The observed reduction in transmigration on treatment with erlotinib can be linked to AKT or ERK inhibition. Phosphorylation of AKT leads to Rac activation resulting in a promotion of microtubule formation and stabilization at the leading edge of migrating fibroblasts (34, 35). ERK can phosphorylate focal adhesion kinase, calpain, microtubule-associated proteins, paxillin, and myosin light chain kinase, all of which are associated with cell spreading, lamellopodia extension, and tail retraction (36). Separate inhibition of either the ERK or the AKT pathway, however, did not show significant improvement in inhibiting melanoma progression. In contrast, dual targeting recently led to encouraging results in vitro (29). As shown in our experiments, erlotinib has the potential to target both pathways simultaneously, although the extent of inhibition especially of ERK activation was highly cell line dependent. Activating mutations downstream of the EGFR, as are frequently present in melanoma, would circumvent this inhibition.

Based on our in vitro results and in accordance to recent publications on the additive effects of dual targeting of the EGFR and VEGF in other solid tumors (4, 6, 37, 38), we tested erlotinib alone or in combination with bevacizumab in a SCID mouse-human melanoma xenotransplantation model in vivo. In contrast to the known side effects of both drugs in humans, no notable drug-related toxicity was observed in the murine treatment groups compared with animals in the control group.

Tumor take was delayed in the erlotinib-treated animals compared with control. Both single agents did delay tumor take, with the higher activity seen in the bevacizumab group. However, the combination of both drugs did show a superadditive reduction in tumor volume. This was associated with reduced expression of the proliferation marker Ki-67 as well as a reduction in tumor vascularization that superceded the antiangiogenic effect of single bevacizumab treatment. Endothelial cells in tumor vasculature have recently been found to express the EGFR and to be responsive to tyrosine kinase inhibitors, whereas their normal counterparts express different receptor family members (39). Furthermore, EGF signaling is well known to control the expression of VEGF (40). The results observed in the in vivo assays are substantiated by the in vitro endothelial cell sprouting assay, where the combination of bevacizumab and erlotinib did result in a highly significant inhibition of sprout formation in a collagen matrix.

It is well established that bevacizumab is a humanized monoclonal antibody directed against human VEGF. Therefore, the effects seen with this drug in preclinical in vivo models in various tumors cannot be attributed to the blocking of murine VEGF but rather blocking the effects of tumor-secreted VEGF on the murine vasculature (41). That this could well be the case in our model system is supported by the observation that recombinant human VEGF as well as conditioned medium from melanoma cells did stimulate the growth of murine endothelial cells in vitro, supporting a species-independent angiogenic effect of human VEGF, which has also been observed in a previous publication (42).

VEGFR2-positive melanoma cells have been recently found to be dependent on VEGF secreted by melanoma cells. Disruption of this activating loop by either blocking VEGF or its receptor resulted in an inhibition of proliferation. Furthermore, inhibition of mammalian target of rapamycin, a downstream target of AKT, in combination with blocking VEGF did induce apoptosis selectively in VEGFR2-positive melanoma cells. (27). In vitro, we did not observe increased proliferation of melanoma cells on treatment with recombinant VEGF-A or inhibition under treatment with bevacizumab. We did see a limited reduction of Ki-67 staining under single treatment with bevacizumab and an increased level of apoptosis in tumors treated with bevacizumab and erlotinib, which also inhibited the AKT-mammalian target of rapamycin signaling pathway but did not find VEGFR2 expressed in the cell lines used in our experiments. A possible explanation would be that the selective VEGFR2-mediated growth inhibition and apoptosis are less restricted to a single receptor subtype in vivo compared with in vitro experiments, although we do not have unequivocal evidence supporting this hypothesis. It is, however, interesting to note that we did find VEGFR1 expressed in all melanoma cell lines used in our experiments.

The combined treatment with erlotinib and bevacizumab did not only reduce tumor take but did also show a significant reduction of locoregional lymph node as well as lung metastases. Notably, neither of both single drugs did show a statistically significant influence on disease extent in both metastatic sites. This is of prime interest as melanoma typically metastasizes to locoregional lymph nodes before progressing to involvement of parenchymal organs.

In conclusion, we show that the combination of erlotinib and bevacizumab is highly active against growth and metastasis of malignant melanoma in preclinical models. This study substantiates the importance of dual targeting in malignant melanoma and provides a rationale for clinical investigations of these drugs in this highly treatment-resistant tumor.

P. Pilarski, employment, Roche Austria; H. Pehamberger, commercial research grant, Roche Austria.

Grant support: “Jubiläumsfonds” of the Austrian National Bank grant 12687 (A. Swoboda).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. T. Valero and M. Mikula for support with the immunohistochemical analysis and K. Neumüller for the isolation of HUVEC.

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