Requirements for the Transformation and Immortalization of Melanocytes
To immortalize primary human melanocytes, we used retrovirus infection to introduce the SV40ER and hTERT cDNAs into the cells. Following this genetic modification, the melanocytes proliferated indefinitely in culture (data not shown) but failed to form either anchorage-independent colonies in soft agar or tumors when injected subcutaneously into immunocompromised mice (; ). We subsequently introduced an oncogenic Ras protein (RasG12V) into the immortalized melanocytes, yielding Mel-STR cells, and observed that RasV12 efficiently transformed these cells, as demonstrated by their ability to form colonies in suspension culture and tumors in vivo (Mel-STR; ; ).
Figure 1 Characterization of retrovirus-transduced primary human melanocytes. a, Schematic of the transformation protocol. b, Expression of the LT, Ras, and TPR-met proteins in primary and engineered cell lines. c, Anchorage-independent colony formation of engineered (more ...)
Table 1 Primary tumor formation and secondary metastasis. Incidence of tumor formation and metastasis in mice subcutaneously injected with melanocyte (Mel-STR), mammary epithelial cell (MEC-STR) and fibroblast (BJ-STR) cells engineered to express the SV40ER, (more ...)
Several tyrosine kinase receptors (RTKs) that signal through the Ras-MAPK pathway have also been implicated in melanomagenesis. In particular, melanoma cell lines frequently exhibit autocrine stimulation provoked by hepatocyte growth factor (HGF) secretion 16
, and c-Met, the receptor for HGF, is frequently upregulated during melanoma progression 17,18
. To test whether activation of the HGF-c-Met signaling loop would also suffice to provide the proliferative signal for transformation, we introduced a constitutively active form of the c-Met receptor (termed TPR-Met) 19
into the immortalized human melanocytes.
TPR-met was able to substitute for RasV12 in enabling the melanocytes to become transformed, as indicated by the ability of these Mel-STM cells to form suspension colonies in vitro and tumors in vivo (). In contrast, ectopic over-expression of HGF, which generates a c-Met autocrine growth-stimulatory loop, failed to transform the immortalized melanocytes (). Hence, while HGF over-expression increased the in vitro proliferation of the melanocytes (data not shown) and increased survival of minute colonies in soft agar (), tumor formation was not observed in vivo ().
Histology and Immunohistochemistry of Mel-STR Melanomas
Examination of histological sections of primary tumors generated by the Mel-STR and Mel-STM cells revealed growths exhibiting characteristics of high-grade, well-vascularized, epithelioid, amelanotic melanomas (). Clinical tumor samples are routinely diagnosed as melanomas by immunohistochemical staining for melanoma-specific markers as well as the absence of staining for cytokeratins. Indeed, the Mel-STR tumors stained positively for the melanoma markers MART-1 and vimentin, and lacked expression of cytokeratins ( and Figure S1). In contrast, human mammary epithelial cells transformed through the introduction of the identical set of genes (MECSTR cells) were negative for MART-1 expression and stained positively for cytokeratins ().
Since the disruption of adherens junctions frequently accompanies melanoma progression, we examined the expression and localization of E-cadherin and β-catenin in the melanoma tumor sections. We observed that the Mel-STR melanomas, unlike normal melanocytes, did not express E-cadherin, and exhibited weak membranous and cytoplasmic staining for β-catenin (Supplementary Fig.2 online). Staining for the SV40 LT antigen revealed that significant regions of the tumors contained cells that did not express the LT protein and were therefore composed of recruited stromal cells of murine origin (Supplementary Fig.2 online). Histological examination revealed that these latter cells were frequently associated with regions of inflammation and necrosis and were, by several criteria, largely neutrophils (data not shown).
Transformed Melanoma Cells Form Metastatic Tumors In Vivo
We undertook an extensive anatomical analysis at necropsy of the organs of melanoma-bearing mice in order to determine whether macroscopically visible metastases developed following subcutaneous injection of Mel-STR melanoma cells. To facilitate visualization of metastases in these animals, we used Mel-STR cells into which the green fluorescent protein (GFP) gene had been introduced through use of a retroviral vector. Mice were sacrificed when they appeared moribund, which occurred in the majority of mice approximately 6-8 weeks after the initial injection of transformed cells.
We observed numerous mice with widespread metastatic dissemination. Metastatic nodules were observed in 92% of mice bearing Mel-STR tumors, most commonly in the lungs (92%) and lymph nodes (29%), as well as in the liver (27%), spleen (22%), and small bowel (12%) (; ). In general, the histology of metastatic growths resembled that of primary tumors. In cases where mice bore metastases to the liver, spleen, or small bowel, metastatic burden was also invariably present in the lungs, raising the possibility that lung metastases may serve to further disseminate additional metastases to other organs in these animals.
Figure 2 Primary Mel-STR melanomas give rise to widespread metastases in vivo. Immunocompromised NOD/SCID mice were injected subcutaneously with 5×105 GFP-labeled Mel-STR cells. Organs were harvested at necropsy and were immediately visualized for GFP (more ...)
Histological examination of metastasis-bearing lungs revealed that metastatic melanomas frequently encapsulated and grew along the lung vasculature (). Metastasis-associated lung vessels invariably demonstrated vasculitis and fibrinoid necrosis. In certain cases, melanoma cells could also be observed within the lumina of blood vessels. In such cases, tumor fibrin thrombi were frequently observed within the vessel lumina. The vascular association of metastatic growths suggested a hematogenous route of dissemination.
The lymph node metastases mentioned above were located adjacent to the growing tumor mass, suggesting that the affected nodes were associated with the lymphatic drainage from the site of implantation. In a subset of mice, we also observed Mel-STR metastases in the axillary lymph nodes of the animals. Furthermore, in cases of axillary lymph node metastasis, the growths were observed exclusively (8/8 cases; ) on the side of the mouse ipsilateral to the original site of injection. This pattern of growth parallels the flow of the lymphatic fluid, suggesting that the melanoma cells reach the axial lymph node through a lymphatic rather than hematogenous route.
While intestinal metastases are rarely observed in cancers that arise outside of the peritoneal cavity, a peculiar aspect of melanoma is its tendency to metastasize to the small bowel 20,21
. We found that this behavior is recapitulated in our experimental model, and that 11% of injected mice had visible metastatic nodules on the surface of the small bowel (; ). Moreover, in a small number of cases, metastatic melanoma cells could be observed that were invading into the small bowel lumen (). Thus, with high penetrance, the metastatic spectrum of the Mel-STR cells paralleled to a remarkable extent that observed in human melanoma patients.
In stark contrast to these observations, metastatic nodules were very rarely observed in mice bearing subcutaneous fibroblast (BJ-STR) or mammary epithelial cell (MEC-STR) tumors () 12,13
. In addition, orthotopic injection of MEC-STR cells into the fourth inguinal mammary gland did not enable the resultant tumors to metastasize. Since these primary human cell types were all transformed in vitro
through the introduction of an identical set of genes, these observations indicate that the unique ability of transformed melanocytes to efficiently generate metastatic nodules is in part a consequence of their pre-existing differentiation program.
Clonal and Genomic Analysis of Metastatic Nodules
The short latency with which metastases were seen after introduction of the melanoma cells into mice, together with the large number of microscopic and macroscopic metastases observed, suggested that many cells in the polyclonal population of transformed melanocytes already possessed the ability to form metastases prior to their introduction into murine hosts. Nonetheless, it remained formally possible that metastasis only occurred in vivo after rare metastasis-prone cells arose within the primary tumor cell population. To address this issue, we undertook a clonal analysis of the disseminated metastases arising in single animals in vivo.
We dissected individual metastatic nodules arising in single Mel-STR-injected mice and dissociated the tissue fragments into single cells by enzyme treatment. We then plated the heterogeneous cell populations (comprised of both metastatic human tumor cells and murine stromal cells) on tissue culture dishes and selected for the melanoma cells using a drug marker that had introduced by retroviral infection into the tumor cells during the initial transformation process. This procedure allowed us to obtain pure tumor cell populations, each of which was derived from a single metastatic nodule in vivo
. Because retrovirus infection leads to quasi-random insertion of proviral sequences into host cell genomes, we were able to use Southern blot analysis22
to identify the provirus integration sites in the ancestral clone(s) from which the various Mel-STR populations were derived. We therefore isolated genomic DNA from each of the metastatic nodule-derived tumor cell populations and performed Southern blot analysis, probing for GFP sequence.
The continuous distribution in the sizes of cleaved DNA fragments derived from the parental transformed cells demonstrated the extensive polyclonality of the Mel-STR population immediately prior to injection in vivo (). Upon examination of metastatic Mel-STR nodules, at least 10 distinct cell clones were present among the metastases of two animals that had been injected with the same polyclonal population of transformed melanocytes (, #305,7). Since these various clones were identified through examination of a small number of nodules relative to the total metastastic burden, it is apparent that a large number of additional cell clones were present among the metastases of these animals. In support of this notion, genomic DNA obtained directly from the entire lung of one metastasis-laden animal demonstrated a continuous distribution of DNA fragments, indicating the presence of numerous distinct clones of metastatic tumor cells in the lung (, #305Lu, Pr). This continuous distribution of integrant sizes was reminiscent of that observed in the starting population of injected cells, indicating extensive clonal heterogeneity among the metastatic Mel-STR cells.
Figure 3 Primary Mel-STR melanomas rapidly seed distinct metastatic clones to secondary organs. a, Southern blot analysis of the metastases arising in single Mel-STR-injected mice. Lanes were loaded with BamHI-cleaved genomic DNA extracted either from organs (#305Lu, (more ...)
In addition to the clonal analyses, we used array CGH to directly interrogate the genomes of metastatic nodules to determine whether alterations in DNA copy number were observed relative to the primary tumors. No significant alterations in DNA copy number were observed in six independent nodules isolated from three distinct organs, relative to the primary tumor DNA. This result indicates that within the level of resolution afforded by the array CGH assay, genomic alterations sustained at the site of primary tumor growth were not responsible for enabling Mel-STR cell metastasis.
Mel-STR Tumors Rapidly Seed Metastatic Cells to Secondary Organs
To further examine the metastatic phenotype of the transformed melanoma cells, we determined the stage of tumor growth at which metastatic Mel-STR cells were seeded into secondary organs. Accordingly, we surgically resected primary Mel-STR tumors from age-matched cohorts of mice at specific times after injection (18, 28 or 44 days), and examined the mice for metastatic growths at the endpoint of the experiment (44 days). To ensure full resection of the primary melanomas, we removed a 0.5cm margin of skin during the surgery and confirmed the absence of melanoma cells by histological examination of the adjacent skin (data not shown). We found that even in mice in which the primary tumor was removed just 18 days after injection (mean wt. = 400mg), numerous metastatic nodules could be seen in the lungs of the animals (5/5) at the endpoint of the experiment (). Similarly, mice whose primary tumors were resected 28 days after injection (mean wt.= 600mg) also developed extensive lung metastases (4/4).
Taken together with the results of the clonal analyses, these observations enabled us to estimate a lower bound on the frequency with which metastasis-enabling alterations must be occurring in vivo if the Mel-STR cells must acquire the ability to metastasize subsequent to their introduction in vivo. This frequency, ~1/100, is several orders of magnitude more frequent than the estimated frequencies per cell generation of gene mutation (see Supplementary Note online). Thus, taken in conjunction with the array CGH data, these data suggest that it is unlikely that additional genetic alterations beyond those initially introduced during the transformation protocol were required in vivo to enable the injected Mel-STR cells to metastasize.
The Neural Crest Cell Factor Slug is Expressed in Melanocytes Prior to Transformation
The results above indicated that the differentiation program of melanocytes could cooperate with oncogenic lesions to uniquely predispose their transformed counterparts to forming tumors that metastasize, when compared to fibroblasts and epithelial cells transformed with identical genes. Since the process of cancer cell invasion bears numerous cellular and molecular similarities to neural crest cell migration in the developing embryo, and given the known derivation of dermal melanocytes from the neural crest, we hypothesized that elements of the motility-associated molecular program mediating neural crest cell migration may contribute to melanoma's metastasis.
Genes of the Snail-superfamily have previously been implicated in both promoting cancer cell invasiveness 23
and governing neural crest cell migration 24
. While Snail was not significantly expressed in the Mel-STR cells, the related Slug transcription factor was expressed at both the mRNA and protein levels (data not shown; ). Interestingly, Slug expression was also observed in non-tumorigenic, immortalized melanocytes. This finding led us to use expression arrays to examine Slug mRNA levels in benign nevus samples obtained from human patients. We observed that benign nevi expressed Slug mRNA, and moreover, expression of the Slug transcript in these samples correlated strongly and significantly (p < 0.01) with genes known to be essential for neural crest cell and/or melanoblast migrations during development (). Thus, expression of the neural crest migration-associated genes Slug (SLUGH), Endothelin Receptor B (EDNRB), ErbB3, and CD44 was highly correlated in adult nevus samples 24-28
. These findings indicated that components of an embryonic differentiation program involved in neural crest cell motility and migration were expressed in benign melanocytic lesions prior to neoplastic transformation.
Figure 4 Suppression of Slug expression inhibits melanoma metastasis in vivo. a, Western blot analysis of Slug protein expression. Slug protein levels were examined in melanocyte cell lines engineered with LT (SVV), LT, hTERT & HGF (STH), LT, hTERT & (more ...)
Slug is Essential for Mel-STR Melanoma Metastasis in vivo
We utilized retrovirus siRNA-mediated inhibition to examine whether the expression of Slug, a master regulator of neural crest cell specification and migration, was in fact contributing to the metastatic ability of the Mel-STR melanoma cells. Accordingly, we designed three independent retrovirus siRNAs directed against SLUGH (siSlug1-3) and stably introduced them into the Mel-STR cells. As two independent controls, we also introduced siRNAs against the GFP (siGfp2) and luciferase (siLuc) proteins into the same cell population. Using quantitative RT-PCR, we determined that siSlug1 and siSlug2 reduced endogenous SLUGH mRNA levels by ~66%, whereas siSlug3 reduced endogenous mRNA levels by 80% (). Essentially identical levels of relative SLUGH mRNA transcript reduction were observed when these same vectors were introduced into the Mel-STV cells ().
To test whether a reduction in Slug levels affected melanoma growth or progression, we introduced the Mel-STR+siSlug3 cells, as well as two control lines (Mel-STR +siGfp2 or +siLuc), subcutaneously into immunodeficient mice. SLUGH inhibition resulted in a slight decrease in primary tumor growth rates when compared to the control melanoma lines (). No apparent differences were observed in the histologies of the primary tumors that developed in the Mel-STR+siSlug3 line versus control lines (data not shown).
In contrast, there was a marked reduction in the incidence of metastasis when primary tumor cells experiencing Slug inhibition were seeded in host mice. Metastatic lung tumor burden in the Mel-STR+siSlug3 tumors was reduced by more than 10-fold when compared to control siGfp2 or siLuc tumors (). This was evaluated by staining lung tissue sections for the Mel-STR-specific marker human vimentin, and subsequently quantifying the stained area in random fields with NIH Image software (see Supplementary Fig.1 online). Importantly, to control for differences in primary tumor growth rates, mice were sacrificed at times such that the average primary tumor burden in the two experimental groups was comparable (). These results provide strong indication that Slug is an important component of the metastatic program in the Mel-STR melanoma cells.