MT/ret^+/- transgenic mice (C57BL/6 background, called RET mice, litter one) expressing the rfp-ret oncogene develop a spontaneous melanoma 2730. Constitutively activated rfp-ret enhances cRET protein expression in the process of melanomagenesis in RET mice 31. Non-transgenic littermates (MT/ret^-/-) were used for transplantation experiments. Mice were maintained in our own animal facilities corresponding to a pathogen free environment. All experiments were performed in compliance with French Ministry of Agriculture regulations for animal experimentation (number 75-510).

HB-19 was synthesized as described previously 1026. Although it is readily soluble in water, HB-19 was dissolved in PBS for the purpose of treatment of mice. Ten days old RET mice were treated intraperitoneally with HB-19 at 5 injections/week during week 1-3 and 2 injections/week during week 4-42. The dose of HB-19 was 50, 100, and 200 μg for the first, second and the rest of the weeks, respectively. Control mice were injected with PBS at the different time points. Clinical signs of mice treated (n = 9) and untreated (n = 11) were assessed once a week on vigil mice and once a month on anesthetized mice. Development of facial or dorsal tumor nodules was recorded. At the end of the treatment (day 300), all mice were sacrificed and autopsied to monitor for distant metastasis.

Flow cytometry. Cutaneous tumors were pooled, mechanically dissociated and digested with 1 mg/mL collagenase A and 0.1 mg/mL DNase I (Roche, Mannheim, Germany) for 25 min at 37°C. Single cell suspensions were filtered, washed in PBS, 5% FCS, 0.5 mM EDTA and resuspended in RPMI 1640. After incubation with anti-FcgII/IIIR antibody (clone 2.4G2), cell suspensions were stained at 4°C, for 15 min with the following combinations of monoclonal antibodies (all from Pharmingen): PerCP-conjugated anti-CD45.2/APC-conjugated anti-CD11b and APC-conjugated anti-CD45.2/PE-conjugated anti-TcRab. Flow cytometric analyses were performed on a FacsCalibur cytofluorometer (BD Biosciences) and data were analyzed using CellQuestPro Software (BD).

The TIII cell-line was derived from the neck cutaneous nodule that developed in a RET mouse. Cells were cultured in RPMI 1640 with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin. In addition to the nucleus, nucleolin is also expressed in the cytoplasm and at the cell surface of TIII cells, as it is the case in different types of tumor cells and other melanoma cell lines 332.

TIII (1×10⁶) cells cultured in the presence or not of HB-19 (10 μM; 12 passages) were transplanted subcutaneously in 10 weeks old MT/ret^-/- mice. Fourteen days later, mice were sacrificed and tumor mass was determined with a caliper. Alternatively, mice were injected in the tail vein with TIII cells (5×10⁵) in the absence or presence of 10 μM of HB-19. Twenty hours later and then daily during two weeks, mice were treated by intraperitoneal injections of PBS or PBS containing HB-19 (5 mg/kg). Mice were then sacrificed, and the number of black macro-metastases on the lung surface was counted.

Cells were plated 24 hours before the experiment in eight-well glass slides (Lab-Tek Brand; Nalge Nunc International, Naperville, IL). Cells were fixed with paraformaldehyde (PFA; 3.7%, 10 min), permeabilized by Triton X-100 (0.5%, 15 min) and stained for the intracellular actin cytoskeleton using FITC-conjugated phalloidin (Sigma) 1026. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI).

TIII cells (2×10⁴) were mixed in 0.35% top agar diluted in RPMI containing 10% FCS in the absence or presence of different concentrations of HB-19 before plating onto 0.8% bottom agar in 12-multiwell plates. Cells were treated every two days during 14 days. Colonies with diameter superior to 100 μm were scored as positive using a phase contrast microscope equipped with a measuring grid at magnification 50×. The number of colonies was determined by analyzing 5 fields/well from 3 wells 10.

Tumors were fixed in FineFix (Milestone, Bazainville, France) for paraffin inclusion. Sections of 8 μm thickness were re-hydrated and saturated in PBS containing 5% goat serum. Sections were incubated with 1:20 dilution of a rat anti-mouse CD34 monoclonal antibody (Abcam, Cambridge, UK) for 2 hours at room temperature. After two washes in PBS, sections were incubated for 1 hour at room temperature with biotin-conjugated goat anti-rat IgG (Chemicon International Inc., Temecula, CA) diluted at 1:500, followed by three washes in PBS and incubation with avidin/peroxidase complex (Vector Laboratories, Burlingame, CA). The horseradish-Peroxidase activity was revealed by incubating the sections with 3,3'-Diaminobenzidine substrate kit (Vector Laboratories). Finally, the sections were counterstained with haematoxylin, followed by water wash and cover slipped with Mowiol medium. Five microscopic fields (at 200-fold magnification) were selected randomly for analysis using the Image analysis. The density of endothelial cells in each field was expressed as the ratio of cell area/total area examined × 100 (%). These values were then averaged for the tumors recovered from control and HB-19 treated mouse.

TIII cells were cultured in RPMI medium containing 10% FCS in the absence or presence of HB-19. Total RNA was prepared from cells (5×10⁵) and fresh tumors isolated from control and HB-19 treated RET mice using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. RT was carried out with oligo(dT) and 1-4 μg of total RNA using Superscript II RNase H- Reverse Transcriptase (Gibco BRL). The expression of specific mRNAs was investigated by RT-PCR using primers for matrix metalloproteinase-2 and -9 (MMP-2, MMP-9), vascular endothelial growth factor (VEGF-A), tumor necrosis factor alpha (TNF-α), signal transducer and activator of transcription 1 (STAT-1), melanoma inhibitory activity (MIA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PCR was performed in a RoboCycler 96 (Stratagene) with the following primers: MMP-2 sense 5'-GAGTTGGCAGTGCAATACCT-3' and antisense 5'-GCCGTCCTTCTCAAAGTTGT-3'; MMP-9 sense 5'-AGTTTGGTGTCGCGGAGCAC-3' and antisense 5'-TACATGAGCGCTTCCGGCAC-3'; VEGF-A sense 5'-AGAGCAACATCACCATGCAG-3' and antisense 5'-AGGAATCCCAGAAACAACCC-3'; TNF-α sense 5'-ACTCCCAGAAAAGCAAGCAA-3' and antisense 5'-TGGAAGACTCCTCCCAGGTA-3'; STAT-1 sense 5'-CGTGGGAACGGAAGCATTTG-3 and antisense 5'-GAGACATCATAGGCAGCGTG-3'; MIA sense 5'-ATCCTATCTCCATGGCTGT-3' and antisense 5'-ACTGGCAGTAGAAATCCCA-3'; GAPDH sense 5'-CGTCCCGTAGACAAAATGGT-3' and antisense 5'-CCTTCCACAATGCCAAAGTT-3'. PCR amplification conditions were 95°C for 2 min, 30 cycles at 95°C for 30 sec, 53°C (for MMP-2, VEGF, Mia-1 and GAPDH) or 55°C (for STAT-1) or 57°C (MMP-9) for 30 sec and 72°C for 45 sec, and this was followed by 5-min incubation at 72°C. The expected RT-PCR product for MMP-2, MMP-9, VEGF-A, TNF-α, STAT-1, MIA-1 and GAPDH was 666, 754, 663, 688, 425, 267 and 527 base pairs, respectively.

Statistical significance was determined by ANOVA unpaired T test or the Wilcoxon log-rank test using the GraphPad Prism 4.0 software (San Diego, CA). Values of p < 0.05 were considered significant.

To evaluate the in vivo anti-tumor effect of HB-19 on the natural course of melanoma progression, RET mice were treated in a prophylactic setting that extended 300 days as described in Methods. The particular aspects of the clinical diagnosis of control and HB-19 treated RET mice are presented individually in Table 1. HB-19 treatment significantly delayed the development of measurable cutaneous tumors that occurred at day 50 and 75 in control and treated mice, respectively (Figure 1A and 1B). The difference between these two groups was even more significant when the incidence of large cutaneous tumors (over 60 mm²) was compared (Log-rank test; p < 0.001). Indeed, large tumors were observed from day 75 onward in control mice, whereas they started to develop at day 190 in HB-19 treated mice (Figure 1B). We next examined the effect of HB-19 on all cutaneous nodules taking account their location, since our previous observations indicated that the severity grade of melanoma is associated with the location of skin tumors, and that in particular the occurrence of nodules in the posterior part of the body corresponds to a more aggressive disease 28. Both facial and dorsal cutaneous nodules developed later and were less frequent in HB-19 treated compared to control mice (Figure 1C and 1D: Log-rank Wilcoxon test, p < 0.001; Figure 1E and 1F p < 0.05). In addition, tumor vascularization was evaluated by CD34 antigen staining in sections of tumors recovered from control and HB-19 treated RET mice (Figure 2). Clearly, numerous vessels remained with an open lumen in the control samples 'angiogenic hot spots' localized heterogeneously within the tumor as compared to HB-19 treated samples. Angiogenesis quantified by image analysis of CD34-labeled endothelial cells showed that HB-19 induced a reduction of 51% in microvessel density compared to control tumors.

It is worthwhile to note that two regressions were observed in the HB-19 treated mouse N°2 and concern tumors localized under the eye and thigh (Table 1), while no regression occurred in the control group. Nevertheless, we cannot conclude that these regressions are induced by the treatment although spontaneous regressions are extremely rare in the RET model. Interestingly, at the end of the experiment two out of nine HB-19 treated mice (mouse N°4 and 6) were still tumor free at autopsy, and that mouse N°5 displayed no melanoma symptom at 7.5 months of age, whereas all control mice bore tumors (Table 1). Of note, the absence of cutaneous tumors is extremely infrequent in the RET mouse model. Indeed, in a similar group of 10 month old RET mice in our animal house facility, only one mouse out of 73 was free of cutaneous nodules. Finally, 8 out of 11 control mice had either retroperitoneal metastasis or mediastinal adenopathies, whereas only 3 out of 9 HB-19 treated animals (mouse N°1, 2 and 3) displayed visceral or lung metastasis (Table 1). Therefore, distant metastasis tends to be less frequent in the HB-19 treated compared to the untreated group (Table 1; p = 0.09). We have recently observed that myeloid (CD11b⁺) and T (TCRαβ⁺) cells represent the two major hematopoietic cell populations that infiltrate spontaneous tumors in the RET model (Lengagne et al., in preparation). However, HB-19 treatment did not exert an apparent effect on the proportion of myeloid and T cell populations infiltrating tumors in the RET mice (Table 2).

During this study, no apparent behavior modifications were noted in response to HB-19 treatment. Moreover, when mice were sacrificed at the end of the study, autopsy revealed no apparent effect on various tissues (including lungs, liver, spleen, and kidney) of HB-19 treated compared to control mice injected with PBS. One out of nine mice treated with HB-19 (mouse N°5) died at day 219 in the course of treatment (Table 1). This mouse displayed no clinical signs of melanoma, thus suggesting a spontaneous death by an unexplained mechanism that might occur although rarely even in control mice.

HB-19 treatment of melanoma-derived tumor cells impairs their tumorigenic potential. Multiple passages of TIII cells in the presence of HB-19 resulted in profound effects on cell morphology without affecting significantly the multiplication index of cells. Intracellular staining of actin allowed visualization of such morphological modifications between untreated and HB-19 treated cells (Figure 3). As expected, control TIII cells proliferated without contact inhibition by crawling over each other and with extensions characteristic of migration and/or invasion of tumorigenic cells as compared to the HB-19 treated cells. On the other hand, HB-19 cultured TIII cells appeared to be smaller in size with rounded morphology, thus suggesting that HB-19 treatment could affect their malignant phenotype. In accord with this, the capacity of HB-19 precultured TIII cells to form colonies in soft agar was significantly impaired (Figure 4A). For this purpose, cells were cultured in the absence or presence of different concentrations of HB-19, and then assayed for colony formation without further addition of HB-19. Preculturing cells with HB-19 resulted in a dose dependent reduction of the number of colonies, reaching 56% inhibition when cells were precultured at 10 μM of HB-19.

To assess whether HB-19 can modify the tumorigenic potential of TIII melanoma cells in vivo, MT/ret^-/- mice were injected subcutaneously with control and HB-19 precultured TIII cells. After 14 days, most of the mice that received control TIII cells developed larger tumors than mice that received HB-19 precultured cells (Figure 4B). The mean tumor mass in the control and HB-19 treated group was 91.4 ± 22.5 and 59.1 ± 15.8 mm², respectively. In order to illustrate the action of HB-19 treatment on metastasis, TIII cells were injected intravenously in MT/ret^-/- mice. The presence of lung metastasis was assessed after 14 days during which time control and HB-19 treated mice received daily intraperitoneal injections of PBS or HB-19, respectively. The results presented in Figure 4C indicate that HB-19 treatment significantly reduced the number of lung metastasis; the mean number of lung macro-metastases in the control and HB-19 treated group was 71 ± 5 and 41 ± 8, respectively.

An altered nuclear nucleolin pattern in cutaneous melanocytic lesions observed by immunohistochemistry has been associated with melanoma progression in patients 33. Indeed, immunofluorescence laser confocal microscopy studies on the melanoma TIII cells revealed the presence of high number of nucleoli, which could account for the observed nucleolin pattern in melanoma lesions. Using biotinylated HB-19, we demonstrated that HB-19 binds surface nucleolin in a dose dependent manner in the melanoma TIII cells reaching saturation at 4 μM concentration. Following binding, HB-19 is internalized and is concentrated in the cytoplasm without translocation into the nucleus (data not presented). During this process, there is specific reduction of surface/cytoplasmic nucleolin without any apparent effect on the level of nuclear nucleolin or its nucleolar distribution (similar to the data presented in human breast cancer cells 10). This latter and the lack of an apparent effect on anchorage dependent cell proliferation suggest that the reduced tumorigenicity of HB-19 treated TIII cells is not due to a potential toxic effect of HB-19.

We investigated the expression of transcripts coding MMP-2 and MMP-9 that degrade components of the extracellular matrix 34, VEGF-A that triggers neovascularization 35, TNF-α that contributes to all stages of the malignant process 36, STAT-1 that regulates major cellular events including tumorigenesis 37, MIA that is associated with progression and metastasis of malignant melanoma 38, and the housekeeping gene GAPDH as a control. Expression of various transcripts was investigated at 3-4 days post seeding of cells, since preliminary experiments indicated that expression of MMP-2 and MMP-9 mRNA is dependent on the cell density as it has been reported previously 39. Twenty-four hours after HB-19 treatment, the level of transcripts coding MMP-2, MMP-9, and TNF-α was markedly reduced in TIII cells at 10 μM HB-19, whereas these transcripts were completely abolished at 25 μM HB-19. This is a selective effect, since the expression of transcripts coding VEGF-A, STAT1, MIA, and GAPDH seemed not to be affected by HB-19 treatment (Figure 5A). By quantitative RT-PCR, the reduction of transcripts coding MMP-2, MMP-9, and TNF-α was estimated to be > 90 and > 95% at 10 and 25 μM HB-19, respectively (data not shown). We next investigated the level of transcripts in HB-19 cultured TIII cells after twelve passages, and in such HB-19 precultured cells after further seven passages in the absence of HB-19. The expression of transcripts coding MMP-2, MMP-9, and TNF-α was selectively reduced in HB-19 cultured cells, whereas expression of the other genes was not significantly affected (Figure 5B). Interestingly, the expression of transcripts coding MMP-2, MMP-9, and TNF-α remained markedly reduced in HB-19 precultured TIII cells that were further passaged for 7-times in the absence of HB-19 (Figure 5C). Consistent with these in vitro results, the expression of transcripts coding MMP-2, MMP-9, and TNF-α is similarly inhibited in the melanoma tumors recovered from HB-19 treated RET mice. A typical example is presented in Figure 6 showing the expression of various genes in tumors recovered from HB-19 treated RET mice at the eye, ear and intraperitoneum compared to tumors recovered from control RET mice at similar locations, respectively.