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From the Wistar Institute,*
Philadelphia, Pennsylvania;
the Department of Surgery,
Children’s
Hospital of Philadelphia, Philadelphia, Pennsylvania; the Department of
Pathology,
University of Pennsylvania,
Philadelphia, Pennsylvania; and the Department of
Dermatology,§
University of Tuebingen,
Tuebingen, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods for the isolation and culture of human melanocytes14,15 are now routine and have been useful in studying melanocyte function. Such studies indicate the role of cell-cell and cell-matrix interactions in the regulation of melanocytic phenotype and function.10,16,17 In monolayer culture, human melanocytes proliferate, display a bi- or tripolar morphology, and express melanoma-associated antigens.18 However, in cocultures with keratinocytes, melanocytes more closely resemble the phenotype seen in situ, maintaining a constant ratio with keratinocytes, exhibiting a multidendritic morphology, and expressing no melanoma-associated antigens.19-22 Melanocytes in coculture are functional, and melanosomes from melanocytes have been successfully transferred to keratinocytes in a skin equivalent model.23,24 More recently, several skin reconstruction models have been used to investigate the biological properties of normal human melanocytes.9,10,25-27
Tissue culture of melanoma cells also induces a different phenotype due to the two-dimensional growth conditions, and the biological properties of cultured melanoma cells only partially resemble those in situ.28 Cultured cells from radial growth phase (RGP) melanoma have characteristics of both malignant and non-malignant cells: they are immortal but do not grow anchorage independently in soft agar nor are they tumorigenic in mice.29 In addition, RGP cells require exogenous growth factors30 for continuous growth in culture due to limited autoproduction of mitogens. In a typical RGP lesion, melanoma cells predominantly reside in the epidermis, with little invasion into the dermis. RGP primary melanomas are considered metastasis-incompetent, ie, they do not invade lymphatics and capillaries. Melanoma cells from the biologically advanced vertical growth phase (VGP) of primary lesions have an infinite lifespan31 they proliferate independently of exogenous growth factors, with the exception of insulin,32 because they produce a variety of growth factors.33 Of these, basic fibroblast growth factor (bFGF) appears to be the most important, because inhibition of bFGF or its receptor in melanoma cells with antisense oligodeoxynucleotides inhibits their growth.34-37
In this study, we analyzed the growth patterns of melanoma cells representing different stages of tumor progression. As a model, we used human skin reconstructs with normal melanocytes, RGP and VGP primary and metastatic melanomas, which were all incorporated into the epidermis to simulate the physiological context. We found distinct growth patterns that mirrored the lesions from which the cells were originally derived, as well as a remarkable stability in the biological phenotype of melanoma cells over time in culture. With melanoma progression, there was an aberrant formation of the basement membrane and the ability of VGP and metastatic melanoma cells to invade the dermis and proliferate there. RGP melanomas survived in the dermal in vitro environment and were tumorigenic in vivo only when cells were transduced to overexpress the bFGF gene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Keratinocytes, fibroblasts, and melanocytes were isolated from neonatal foreskins (obtained from Cooperative Human Tissue Network, Philadelphia, PA) after routine circumcision, and cultured as described.21 All reagents were obtained from Sigma Chemical Co. (St. Louis, MO) except for recombinant human epidermal growth factor (EGF) and bovine pituitary extract (BPE), which were obtained from Gibco-BRL (Grand Island, NY). Keratinocyte cultures were maintained in keratinocyte growth medium (KGM) composed of MCDB 153 medium supplemented with amino acids,38 0.1 mmol/L ethanolamine, 0.1 mmol/L O-phosphorylethanolamine, 5 x 10-7 mol/L hydrocortisone, 5 µg/ml insulin, 5 ng/ml recombinant human EGF, and 100 µg/ml BPE. Keratinocytes at passage 2–5 were used for skin reconstructs.
Human primary (RGP: WM35; VGP: WM793 and WM115) and metastatic melanoma (WM852) cells were isolated and cultured as described.29-31 Sbcl2 RGP-like cells were a gift of Dr. B. Giovanelli (Stehlin Foundation for Cancer Research, St. Joseph Hospital, Houston, TX). The highly aggressive melanoma cell lines 451Lu and 1205Lu were selected from lung metastatic lesions in mice after subcutaneous (s.c.) injection of WM164 and WM793 melanoma cells, respectively.39,40 Melanoma cells were maintained in melanocyte growth medium in the absence of EGF, phorbol ester, and BPE. All cell lines were used between 50 and 100 passages in culture except Sbcl2 cells, which were at more than 200 passages. All cultures were tested twice a year for absence of mycoplasma contamination.
In Vitro Reconstruction of Human Skin and Melanoma
Reconstructs were generated using described techniques and media
formulations3,30,41
with modifications. For dermal
reconstruction, 1 ml of a cell-free buffered collagen solution
consisting of rat tail collagen, type I (Collaborative Biomedical,
Bedford, MA), at a final concentration of 1.35 mg/ml in DMEM with 10%
FCS was added to tissue culture inserts (Transwell, Costar, Cambridge,
MA) in 6-well plates (Figure 1)
. This
precoated acellular layer was then overlaid with 3 ml of
fibroblast-containing collagen (1.125 x
105/ml). After a 5-day incubation at 37°C,
fibroblasts had contracted the collagen gel, which formed a concave
central area for subsequent seeding of epidermal cells. For epidermal
reconstruction, the mature dermal reconstructs were rinsed and
equilibrated with 2 ml of epidermal growth medium (EGM) as
described.41
Minor modifications included the addition of
2% dialyzed FCS and the omission of linoleic acid. After 1 hour, EGM
was removed, and the surface of the dermal reconstructs was allowed to
dry. Neonatal foreskin keratinocytes (1.5 x
105/reconstruct, in a total volume of 50 µl)
were seeded onto the concave center of the dermal reconstructs and
incubated at 37°C for 2 hours to allow attachment of the seeded
cells. Composite cultures were then submerged by adding 3 ml of EGM
outside and 2 ml inside of the insert. Seeded keratinocytes were
allowed to attach and proliferate. After 4 to 6 days of submerged
culture, with regular feedings every 2 days, skin reconstructs were
lifted to the air-liquid interface and medium was switched to
maintenance medium41
with similar modifications described
for EGM above. After 10 to 14 days of air exposure, skin reconstructs
were harvested. Skin reconstructs were fixed overnight with 4%
paraformaldehyde, dehydrated, and embedded in paraffin. Sections were
stained with hematoxylin and eosin (H&E). For incorporation of
melanocytes or melanomas, cells were seeded together with keratinocytes
onto dermal reconstructs at a 1:5 ratio of melanocytic cells to
keratinocytes. Culture conditions were the same as for reconstructs
containing keratinocytes alone. Human melanoma cell lines derived from
RGP (WM35, Sbcl2) and VGP primary (WM793, WM115) and metastatic
melanomas (451Lu, 1205Lu, WM852) were tested in the reconstructs. All
experiments were performed in duplicate. For testing tumorigenicity of
Sbcl2 cells, 5 x 106
cells were injected
s.c. into 5 SCID mice per group. Tumor growth was monitored for 4
weeks. Paraffin sections of reconstructs and tumor tissues were stained
immunohistochemically with antibodies detecting type IV collagen,
laminin, Ki67 proliferation marker, or S100 melanocytic markers all
using standard methods. Apoptosis in paraffin sections was evaluated
using an in situ apoptosis detection kit (ApopTag, Oncor,
Gaithersburg, MD).
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The adenoviral vector carrying the lacZ reporter gene (LacZ/Ad5) was obtained from the Vector Core, University of Pennsylvania (Philadelphia, PA). The adenoviral vector carrying the bFGF gene (bFGF/Ad5) has been described in detail elsewhere.42 bFGF/Ad5 induces in normal melanocytes, at 20 plaque forming units (PFU)/cell, at 48 hours, a biologically active 18-kd protein that is found predominantly in the cytoplasm but that can also be found in the culture supernatant. Through the use of adenoviral vectors expressing ß galactosidase or green fluorescent protein we showed that, in preliminary experiments, 100% of melanoma cells were transduced after 48 hours when infected at 20 PFU/cell. Melanocytes and melanoma cells transduced with bFGF/Ad5 express the protein in vitro and in vivo.42
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human skin reconstructs resembled the architecture of skin
in situ (Figure 2)
. The dermal
equivalent contained extracellular matrix material (collagen) with
interspersed fibroblasts. The fibroblasts were single, bipolar, and
horizontally oriented, and migrated into the acellular collagen gel,
forming a homogeneously structured dermis. The epidermal equivalent
comprised proliferating basal cells and sequentially differentiated
stratified cell layers (Figure 2A)
. The basal cells were vertically
oriented, well-organized and cuboidal to columnar, containing
oval-shaped nuclei. Numerous layers of progressively flattening
keratinocytes with less prominent nuclei constituted the stratum
spinosum. A stratum granulosum, approximately 3 layers thick, formed
above the stratum spinosum. A stratum corneum consisting of flattened,
anucleate corneocytes developed on top. Compared to human skin, skin
reconstructs lacked rete ridges. When melanocytes were mixed with
keratinocytes, they settled at the interface between dermis and
epidermis (Figure 2, B and C)
, where they remained singly with a
multidendritic morphology throughout the life span of the reconstructs
of 28 to 35 days.
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To study the biological properties of melanoma cells from
different stages of progression, we incorporated seven human melanoma
cell lines derived from RGP and VGP primary and metastatic melanomas
into skin reconstructs. RGP melanoma cells Sbcl2 formed nests within
the epidermis and band-like tumor cell aggregates at the
epidermal-dermal junction (Figure 2D)
. RGP primary melanoma cells, WM35
(Figure 2E)
, were disposed as individual cells and as small nests
within the epidermis and at the epidermal-dermal junction. Staining for
S-100 protein (Figure 2F)
demonstrated the lack of competence for
invasive growth into the dermis. VGP primary melanoma cells WM793
(Figure 2G)
and WM115 (Figure 2H)
formed nests and clusters at the
epidermal-dermal junction, and exhibited invasive growth into the
dermis as demonstrated by staining for S-100 protein (Figure 2I)
.
Whereas WM793 VGP melanoma cells invaded the dermis in clusters with
only a few individual cells preceding the cluster, WM115 cell invaded
the dermis more individually.
When metastatic melanoma cells were incorporated into skin
reconstructs, they displayed rapid proliferation and aggressive
invasive growth deep into the dermis. As shown in Figure 2J
, 451Lu
metastatic melanoma cells formed tumor cell clusters at the
epidermal-dermal junction, and exhibited growth of tumor cell nests in
the dermis. Figure 2K
reflects that 1205Lu metastatic cells were
disposed as tumor cell nests at the epidermal-dermal junction and
showed vertical invasive growth of tumor cell strands deep into the
dermis. WM852 metastatic cells (Figure 2L)
also displayed rapid
vertical growth of individual tumor cells deep into the dermis.
Basement Membrane Development
Staining for type IV collagen (a component of the lamina densa) of
normal human skin reconstructs revealed linear deposition along the
epidermal-dermal junction (Figure 3A)
,
indicating that in a physiological context, keratinocytes and
fibroblasts synthesize and deposit basement membrane proteins in
vitro. Laminin was also deposited in the basement membrane zone,
with diffuse and weak staining in the epidermis (not shown). When RGP
primary melanoma WM35 were seeded together with keratinocytes, they
produced collagen type IV within the epidermal nests (Figure 3, B and C)
. At the same time, a seemingly intact basement membrane was formed.
Seeding of VGP primary melanoma cells WM115 together with keratinocytes
resulted in epidermal and dermal growth (Figure 3, D and E)
. The
basement membrane was irregular and collagen type IV was scattered
throughout the lesion. When metastatic melanomas were seeded together
with keratinocytes, collagen type IV distribution was similar to VGP
melanomas within and surrounding tumor cell nests.
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RGP melanomas either cannot penetrate the basement membrane for
migration into the dermis (see Figure 2, D
-F), or on entering the
dermis they undergo apoptosis.4
Because bFGF is a major
growth and survival factor for melanocytes and melanomas, we determined
whether bFGF could induce proliferation of RGP melanoma cells in the
dermis in the absence of an overlaying keratinocyte layer. Sbcl2
transduced with the control vector LacZ/Ad5 and then embedded together
with fibroblasts remained singly distributed in the dermal matrix
(Figure 4A)
, and the cells did not
proliferate (Figure 4C)
. Parental Sbcl2 cells or cells transduced with
control do not produce bFGF as determined by immunostaining and Western
blotting.42
On the other hand, when the melanoma cells
were transduced with the bFGF gene before incorporation into
reconstructs, they expressed bFGF and formed small nests within the
matrix (Figure 4B)
. The bFGF-overexpressing Sbcl2 cells also migrated
outside of the dermis and adhered to its surface, where they
proliferated and formed nests (Figure 4D)
. The Sbcl2 cells transduced
with bFGF were also tumorigenic when injected s.c. into SCID mice,
whereas LacZ transduced control cells were not. The control Sbcl2 cells
all died within 4 days after injection and the injected cells were no
longer detectable. On the other hand, bFGF-transduced cells developed
into palpable lesions within 10 days. After 14 days a tumor nodule had
formed (Figure 4E)
with numerous cells expressing the proliferation
marker Ki67 (Figure 4F)
. Due to the transient nature of
adenovirus-induced gene expression, tumors regressed after 3 to 4
weeks.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Melanocytes adhere to keratinocytes through E-cadherin.47
This close interaction allows keratinocytes to regulate expression of
cell surface antigens on melanocytes and to control cell
growth.21,22
Melanoma cells do not express
E-cadherin48
and thus grow independently of keratinocytes.
Although RGP melanomas grow separated from the basement membrane in the
upper layers of the epidermis, they remain dependent on keratinocytes.
When RGP melanoma cells are transduced with the ß3 subunit of the
vß3 vitronectin receptor, they grow invasively into the
dermis4
and are tumorigenic in immunodeficient mice. It
remains unknown which genes are activated to trigger the increased
growth and invasiveness after overexpression of an adhesion receptor.
The present study suggests that RGP primary melanoma cells require activation of the gene bFGF for survival, proliferation, and migration into the dermis. bFGF is apparently the most important growth factor in melanoma.28 bFGF has autocrine growth stimulatory functions because cells cannot survive in vitro and in vivo without it.17,34-37 RGP melanomas do not express bFGF protein when cell extracts are tested by Western blotting, although RNA can be detected after 30 cycles by reverse transcriptase-polymerase chain reaction.49 The low level production of bFGF by RGP melanomas is apparently insufficient for survival of those RGP melanoma cells that begin to enter the dermis from the epidermis. In the epidermis, keratinocytes provide the necessary bFGF for melanocytes17 and apparently also for RGP cells. bFGF appears to stimulate the expression of an enzyme(s) that degrades collagen I which is tightly constricted by the embedded fibroblasts. The up-regulation of metalloproteinases for collagens by bFGF has been reported.50
Reconstructs displayed the basement membrane that separates the epidermis and dermis. Apparently both keratinocytes and fibroblasts contribute to its formation in normal skin reconstructs.51 In RGP melanoma reconstructs, fibroblasts and keratinocytes can still form an intact basement membrane, but the melanoma cells begin to synthesize their own collagen IV. The synthesis of collagen type IV and laminin by melanomas has also been demonstrated in patients’ lesions.52 VGP melanomas traversing into the dermis no longer allow the formation of an intact basement membrane. Instead, collagen type IV appears to be randomly distributed throughout the lesion. It is not clear whether this reflects increased degradation or decreased production of the collagen type IV. However melanoma cells producing their own collagen type IV are unlikely to increase the production of degradative enzymes. It is possible that the malignant cells transmit signals to fibroblasts in the stroma to produce less collagen. Further studies are needed to determine the cross-talk between normal and malignant cells for the production of proteolytic enzymes and their activators. The human skin reconstruct model should be ideally suited to investigate the relative contribution of individual genes for invasion. Molecular engineering of each normal skin constituent and of the tumor cells will allow a better dissection of each step of invasion.
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Footnotes |
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Supported in part by Natinal Institutes of Health grants CA47159, CA25874, CA76674, and CA10815 and by NASA Grant NAG 9–832.
Accepted for publication September 29, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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rfi T, Shih I-M, Valyi-Nagy I, Levengood G, Menssen H-D, Halpern AC, Elder DE, Herlyn M: Growth regulation of cultured human nevus cells. J Invest Dermatol 1993, 100:281S-287S
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