[CANCER RESEARCH 55. 5085-5093,
November l, 1W5]
Invasion of Selectively Permeable Sea Urchin Embryo Basement Membranes by
Metastatic Tumor Cells, but not by Their Normal Counterparts1
Donna L. Livant,2 Stephanie Linn, Sonja Mark«ari. and Jill Shuster
DejiürÃ-ntenÃof Anattunv and Cell Biology, University of Michigan, Ann Arbor, Michigan 48109-0616
ABSTRACT
The selectively permeable basement membranes and the associated
extracellular matrix of sea urchin embryos can be obtained intact. Their
exterior surfaces have been used as invasion substrates for metastatic
melanoma, squamous cell carcinoma, and fibrosarcoma cells, for primary
squamous cell carcinoma cells, and for neonatal melanocv tes, fibroblasts,
and keratinocytes. About 18% of all metastatic tumor cells placed in
contact with sea urchin embryo basement membranes and their associated
extracellular matrix invaded them. About 4% of the cells of a primary
squamous cell carcinoma, which later metastasized, invaded these sub
strates. As expected, neonatal melanocytes. kcratinocytes, and fibroblasts
failed to invade; however, melanocytes treated with scatter factor (hepatocyte growth factor) invaded as efficiently as metastatic tumor cells. This
suggests that the lack of invasion by epidermal melanocytes is not due to
irreversible differentiation to a noninvasive phenotype. Invasion time
courses showed that the metastatic cells tested reached their maximal
invasion frequencies in 4 h: thus, invasion of these substrates is rapid and
efficient. This suggests that molecules participating in basement mem
brane recognition and invasion have been functionally conserved during
the time separating vertebrates from invertebrates and that their consti
tutive activity may allow metastatic cells to escape their tissues of origin.
INTRODUCTION
Migratory cells exhibit diverse hut highly regulated responses to the
basement membranes of different tissues. During development, for
example, melanocyte precursors differentiate in the neural crest and
then migrate extensively, crossing the basement membranes that sep
arate the epidermis from the dermis to occupy epidermal sites (Refs.
1 and 2; reviewed in Ref. 3). During immunosurveillance. lympho
cytes cross the walls of high endothelial venules. which include
basement membranes as they move from the circulatory to the lym
phatic system. Monocytes and polymorphonuclear neutrophils also
cross basement membranes to perform their functions (Refs. 4 and 5;
reviewed in Ref. 6). Thus, the regulated invasion of distant tissues by
migratory cells is fundamental to the formation of the pigmented
integument, as well as to the function of the immune system.
Tumor metastasis is a complex process during which cells detach
from a primary tumor and enter the circulation, eventually colonizing
distant tissues. To leave their tissue of origin and invade distant
tissues, metastatic tumor cells must also cross basement membranes
(reviewed in Ref. 7). Unlike normal, migratory cells that may invade
the basement membranes of distant tissues as part of their function,
metastatic cells indiscriminately cross basement membranes. Oppor
tunistic invasion enables them to colonize many tissues in a variety of
locations. Thus, the recognition of basement membranes as substrates
for invasion rather than as boundaries by metastatic cells is likely to
be necessary for distant metastasis to occur. The ability to migrate
Received 5/26/95: accepted 9/5/95.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked atlrenisemeni in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
' This work was supported by the Pfeiffer Foundation.
To whom requests for reprints should be addressed, at 3725 Medical Science II,
University of Michigan Medical Center, 115(1West Medical Center Drive, Ann Arbor. Ml
48109-0616.
across basement membranes indiscriminately is, therefore, likely to be
a fundamental feature of metastasis among a wide variety of cancers.
The invasion of basement membranes by tumor cells has been
studied extensively in vitra through the use of Matrigel, an artificial
basement membrane made by processing extracellular matrix constit
uents synthesized by the EHS1 tumor (8). Type IV procollagen,
laminin. and heparan sulfate proteoglycan have been isolated from the
EHS tumor (9), and laminin, type IV collagen, heparan sulfate pro
teoglycan, entactin, and fibronectin have been identified ¡mmunohistochemically in its secreted matrix (10). However, type IV collagenase and matrix metalloprotease have also been found to copurify with
laminin preparations from EHS tumor extracellular matrix (11), and
Matrigel is known to contain various growth factors including trans
forming growth factor ß,epidermal growth factor, fibroblast growth
factor, and platelet-derived growth factor (12). These components
may affect the results of experiments addressing the effects of EHS
laminin or Matrigel on cell behavior, particularly because Matrigel
has been shown to confer metastatic potential on otherwise nonmetastatic tumor cells when they are cotransplanted into mice (13). Also,
the invasiveness of normal and malignant epithelial and mesenchymal
cells on Matrigel has not always been observed to correlate with their
invasiveness in vivo (14, 15), suggesting that other regulatory inter
actions involving cells or their matrix may also function in determin
ing invasive behaviors.
Because artificial basement membranes are made from components
synthesized by cultured tumor cells (8-10), it is possible that they do
not completely replicate the structure of basement membranes syn
thesized by normal tissues. To circumvent this difficulty, a number of
naturally occurring basement membranes have been used to model in
vivo tumor cell invasion. Basement membranes can be obtained from
human amnions by exposure of their epithelial surfaces to detergent
(16). The thicknesses of amniotic basement membranes vary greatly
among different amnions and in different locations on the same
amnion (17), but small, variable numbers of highly invasive tumor
cells cross these basement membranes in 1-3 days (16-18) with
relative frequencies correlating to their metastatic potential in vivo
(16). However, the proximity of the developing fetus to the trophoblast may indicate that the amniotic basement membrane can function
as a protective barrier to invasion. The use of chick embryo chorioallantoic membranes allows the study of basement membrane pen
etration, as well as subsequent metastasis to organs of the developing
chick; but there is a requirement for disruption of the periderm before
invasion (19), and traumatized chorioallantoic membranes support the
invasion of mouse and chick embryo fibroblasts in the same assays.
Thus, the correlation of invasive behaviors observed on chorioallan
toic membranes with invasive behaviors exhibited by the cells in
intact organisms is limited. Bovine lens capsule basement membranes
are easy to isolate and function as a model of a capillary when bovine
endothelial cells are grown on them; but metastatic behaviors in vivo
correlate very incompletely with collagen or laminin-degrading activ
ities of tumor cells after placement on these basement membranes
(20).
3 The abbreviations used are: EHS, Engelbreth-Holm-Swarm;
MBM, melanocyte basal
medium.
5085
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1995 American Association for Cancer Research.
INVASION
OF SEA URCHIN EMBRYO
Artificial basement membranes or the naturally occurring basement
membranes used previously in invasion assays are not known to be
permeable to normal, migratory cells in intact organisms. Yet, many
of the basement membranes encountered by tumor cells during me
tastasis are potentially permeable to specific types of normal cells.
Because structures occurring either in basement membranes or dis
played on the surfaces of surrounding cells could potentially instruct
the migratory cells contacting them, selective permeability may result
from a combination of cues. Whether permeability is an instructional
function of some basement membranes or a passive attribute of many,
there could be significant structural differences between normally
impermeable and potentially permeable basement membranes that
result in distinct invasive mechanisms, even by metastatic cells.
Because of the difficulties in isolating intact, selectively permeable
basement membranes, with or without their associated extracellular
matrix, the role these structures play in invasion remains to be
elucidated.
Because they are easily prepared intact in large quantities, we have
utilized the subectodermal basement membranes of sea urchin em
bryos and their associated extracellular matrix as a model for the
basement membranes separating the epidermis from the dermis. In sea
urchin embryos, the ectoderm is surrounded by extracellular matrix.
The hyalin layer is located on the outer, apical surface of the ecto
derm. It consists of several proteins, including fibropellin, an epider
mal growth factor repeat-containing protein (21, 22). A thin basement
membrane is located on the inner, basal surface of the ectoderm. Its
structure and major constituents are similar to those of mammalian,
embryonic basement membranes. It is known to contain laminin,
fibronectin, collagen IV, and heparan and chondroitin sulfate proteoglycans (23-28).
After secretion by the ectoderm, the sea urchin embryo basement
membrane lines the blastocoel. Its inner surface functions specifically
as a migration substrate for primary mesenchyme cells as they secrete
the spicules of the larval skeleton (reviewed in Ref. 29), as well as for
secondary mesenchyme cells at the tip of the archenteron during
positioning of the invaginating primitive gut at the mouth site during
gastrulation (30). While functioning as a migration substrate for
primary mesenchyme cells and for other types of secondary mesen
chyme cells, this basement membrane also acts as an invasion sub
strate for pigment cells. Pigment cells begin differentiation in the
vegetal plate before gastrulation. As the archenteron invaginates from
the vegetal plate during gastrulation, pigment cells ingress, contact the
basement membrane lining the blastocoel, and migrate across it to
invade the ectoderm (31, 32).
Thus, the selectively permeable basement membrane lining the
sea urchin embryo blastocoel elicits several specific patterns of
migration from two disparate types of mesenchyme cells while
eliciting invasive behavior from pigment cells. Because its inner
surface is uniquely permeable to pigment cells during develop
ment, the sea urchin embryo subectodermal basement membrane is
functionally analogous to basement membranes that underlie mam
malian epidermis.
Sea urchin embryo basement membranes provide the first op
portunity to examine the molecular mechanism of invasion of a
naturally occurring, permeable, acellular substrate that may more
closely resemble many of the basement membranes encountered by
normal or metastatic migratory cells in intact organisms than
reconstituted or normally impermeable basement membranes do.
Here, we report the use of sea urchin embryo basement membranes
and their associated extracellular matrix as invasion substrates for
metastatic human melanoma, squamous cell carcinoma, and fibro
sarcoma cells, metastatic mouse melanoma cells, primary human
BASEMENT
MEMBRANES
squamous cell carcinoma cells, and neonatal human melanocytes,
keratinocytes, and fibroblasts.
MATERIALS
AND METHODS
Sources and Maintenance of Cells. SK-MEL-28 metastatic human mel
anoma cells (33), UM-SCC-10B metastatic laryngeal squamous cell carcinoma
cells, and UM-SCC-10A primary laryngeal squamous cell carcinoma cells (34)
were obtained from T. Carey (University of Michigan, Ann Arbor, Ml).
UM-MEL-1 metastatic human melanoma cells (35, 36), neonatal, differenti
ated keratinocytes, and neonatal fibroblasts were obtained from J. Varani
(University of Michigan, Ann Arbor, MI). B16F1 and B16F10 metastatic
mouse melanoma cells (37) were obtained from I. Fidler (University of Texas,
M. D. Anderson Cancer Center, Houston, TX). HT1080 human metastatic
fibrosarcoma cells (38) were obtained from the American Type Culture Col
lection (Rockville, MD). Neonatal melanocytes were obtained from Clonetics
(San Diego, CA). All cells except keratinocytes and melanocytes were cultured
in MEM supplemented with 10% FCS. Keratinocytes were cultured in keratinocyte growth medium (Clonetics). Melanocytes were cultured in melanocyte
growth medium-2 (Clonetics) until 48 h before their assay in an invasion
experiment. At this time, they were switched to MBM, supplemented with 10%
FCS. A penicillin-streptomycin
antibiotic was present in all cell cultures.
Growth of Sea Urchin Embryos and Preparation of Basement Mem
branes. Eggs and sperm were spawned from gravid Strongylocentrolus pur
púralas sea urchins obtained from Pacific BioMarine by intracoelomic injec
tion of 0.5 M KC1. Embryos (0.2-2.0 X IO6) were cultured in 4 liters of
artificial sea water obtained from Instant Ocean at 15°Cwith gentle stirring
and aeration for 72 h. The embryos were isolated by trapping them on a 63-fim
mesh, resuspending them in 50 ml of sea water, and pelleting them at 1500 rpm
for 2 min at 4°C.The embryos were then resuspended in 10 mM sodium
bicarbonate with 0.2% Triton for l h with gentle agitation. The resulting
basement membranes were pelleted at 1000 rpm for 10 min and resuspended
in 50 ml of sterile sea water. Three cycles of pelleting and resuspension in
sterile sea water, followed by three cycles of pelleting and resuspension in
MEM or MBM supplemented with 10% FCS and penicillin-streptomycin
(MEM10 and MBM10) were used to sterilize the basement membranes.
Basement membranes were finally resuspended at a density of about 500/ml,
and 0.5 ml of this suspension was placed in each well used for an invasion
assay. Twenty-four-well plates were used for invasion assays.
Preparation of Basement Membranes for Scanning Electron Micros
copy. Basement membranes were prepared with 0.2% Triton as described
above, rinsed once by pelleting and resuspension in sea water, then resus
pended in 1% gluteraldehyde and buffered with 100 mM cacodylate, pH 7.2.
During the fixation, the basement membranes were gently agitated at room
temperature for 30 min. Basement membranes were pelleted and resuspended
in 1% OsO4 and buffered with 100 mM cacodylate (pH 7.2) for 30 min. Then,
they were dehydrated through an ethanol series, resuspended twice in hexamethyldisilizane, and dried in a dessicator overnight. Finally, they were mounted
and sputter-coated with gold.
Preparation of Cells and Invasion Assays. Mammalian cells were iso
lated by a brief trypsin treatment, washed once, and resuspended in the
appropriate medium at a density of approximately 20,000/ml. In each well
used, 0.5 ml of suspended cells were placed over 0.5 ml of medium containing
about 250 basement membranes and allowed to settle without moving for 10
min. The basement membranes with added cells were then placed in an
incubator for the defined period of the invasion assay. For time courses, cells
on basement membranes were fixed at defined times with 2% formaldehyde in
PBS for 10 min at room temperature. For experiments requiring melanocyte
treatment with scatter factor, melanocytes were placed in MBM 10 with 50
ng/ml of scatter factor (hepatocyte growth factor) obtained from Collaborative
Biomedicai Products (Bedford, MA) for 15 min before placement on basement
membranes. The MBM10 medium also contained 50 ng/ml scatter factor
during the entire invasion assay.
Collection and Analysis of Data. Invasion assays were scored by micro
scopic examination under phase contrast at X400. Each cell in contact with a
basement membrane was scored for its position relative to the exterior or
interior of the basement membrane. A cell was judged to have invaded a
basement membrane if it was located below the focal plane passing through the
5086
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1995 American Association for Cancer Research.
INVASION
OF SEA URCHIN EMBRYO BASEMENT
basement membrane's upper surface bul above the focal plane passing through
the basement membrane's lower surface. The minimum viability of the cells in
each assay was always scored at the time of assay by determining the fraction
of spread, adherent cells on the bottom of each well that was scored.
An invasion frequency is the fraction of cells in contact with basement
membranes, which were located in their interiors at the time of assay. Thus, an
invasion frequency of one denotes invasion by 100% of the cells in contact
with basement membranes. Invasion frequencies were determined multiple
times for each cell-type assayed. For each type of cell assayed, the mean and
SD of the invasion frequencies were calculated.
To use the most general possible assumptions about the null hypothesis, that
normal and metastatic cells exhibit the same invasion frequencies, two-sample
rank testing by the Mann-Whitney test (39) was used to assess the significance
of the differences in the mean invasion frequencies of metastatic tumor cells,
primary tumor cells, and normal neonatal cells. This test assumes neither a
Gaussian probability distribution nor a value for SD.
RESULTS
Invasion of Sea Urchin Embryo Basement Membranes by Met
astatic and Primary Tumor Cells but not by Normally Noninvasive Neonatal Cells. The experiments described here employ the
subectodermal basement membranes of sea urchin embryos as intact
potential invasion substrates for metastatic mouse and human tumor
cells, for primary human tumor cells, and for their normal neonatal
human counterparts. The basement membranes were obtained from
early pluteus stage embryos that had recently completed gastrulation.
Fig. 1/4 shows a sea urchin embryo at the stage used for basement
membrane preparation. It consists of an ectoderm that is one-cell thick
surrounding a blastocoel. Blastocoelar structures include the primitive
gut, the spicules of the larval skeleton, and their associated mesenchymal cells. A basement membrane prepared for an invasion assay is
shown in Fig. Iß.The larval skeleton, as well as the basement
membrane lining the primitive gut, is visible in its interior.
In each well of an invasion assay, approximately 10,000 cells were
allowed to settle as single cells on the exteriors of about 250 wellseparated basement membranes with their associated extracellular
matrices. Fig. 1C shows basement membranes prepared for an inva
sion assay before the placement of cells on their outer surfaces. In a
typical invasion assay, one or two cells settle on approximately
one-half of the basement membranes and are subsequently assayed for
their invasive potential. Most of the cells placed in invasion assays
miss the basement membranes and adhere to the bottoms of the wells.
One to 2% of the cells placed in the invasion assays described adhered
to basement membrane exteriors in all experiments; thus, no signifi
MEMBRANES
cant differences in adhesion frequency were observed among the cells
tested. The fraction of cells spread and adherent to the bottoms of the
invasion dishes varied from 0.84 to 0.99 with 1 being 100% adherent;
thus, any observed lack of invasion was a property of the cells being
tested rather than a loss of viability as result of the procedure used to
subculture or assay the cells.
A diagram of a sea urchin embryo at the same stage and orientation
as the one shown in Fig. 1/4 is shown in Fig. ID. In addition to the
primitive gut and spicules, the subectodermal basement membrane
and the hyalin layer are indicated. The cells forming the wall of the
primitive gut and their associated extracellular matrix are also shown.
Metastatic human SK-MEL-28 (33) and UM-MEL-1 (35, 36) mel
anoma cells, metastatic mouse B16F1 and B16F10 melanoma cells
(37), metastatic human HT1080 fibrosarcoma cells (38), metastatic
human UM-SCC-10B squamous cell carcinoma cells, and primary
human UM-SCC-10A squamous cell carcimona cells (34) were as
sayed for their abilities to migrate to the interiors of sea urchin embryo
basement membranes after placement as single cells on their exteriors.
Fig. 2 shows the collected invasion frequencies for metastatic tumor
cells, primary tumor cells, and the corresponding normal cells after
placement on the exteriors of sea urchin embryo basement membranes
and incubation for 16 h. The cells chosen originated from tissues with
differing relationships to epithelial basement membranes in vivo.
SK-MEL-28 metastatic, human melanoma cells invaded sea urchin
embryo basement membranes with a mean frequency of 0.18 and a SD
of 0.027. UM-MEL-1 metastatic, human melanoma cells invaded sea
urchin embryo basement membranes with a mean frequency of 0.17
(± 0.051). B16F1 metastatic mouse melanoma cells invaded sea
urchin embryo basement membranes with a mean frequency of 0.17
(± 0.041). B16F10 metastatic mouse melanoma cells invaded sea
urchin embryo basement membranes with a frequency of 0.19.
HT1080 metastatic, human fibrosarcoma cells invaded sea urchin
embryo basement membranes with a mean frequency of 0.19
(±0.045). UM-SCC-10B metastatic, human carcinoma cells invaded
sea urchin embryo basement membranes with a mean frequency of
0.18 (±0.047). Thus, about 18% of metastatic tumor cells originating
from epithelial or connective tissue sources invaded subectodermal
sea urchin embryo basement membranes after placement on their
outer surfaces. As a control for the alteration of invasion frequencies
by loss of viability during isolation, the fraction of cells spread and
adherent to the bottom of each well used in the invasion assays was
determined at the time of invasion scoring. For metastatic cells, the
Fig. 1. A, Strongylocentrotus purpúralas embryo at Ihe early pluteus stage. This embryo was photographed in sea water at X400 by using phase contrast. <•,
ectoderm; g, stomach
of the primitive gut: .v. a spicule of the larval skeleton; p, a pigment cell in the ectoderm. The esophagus and the intestine are not visible from this side of the embryo. B, basement
membrane obtained from a sea urchin embryo, h, basement membrane and its associated extracellular matrix secreted by the ectoderm; .v,larval skeleton spicule; g, extracellular matrix
associated with the primitive gut. This basement membrane was photographed in MEM with 10% PCS at X40Ü.Bar, 20 ^.m. C, basement membranes prepared for an invasion assay.
These basement membranes were suspended in MEM with KKr PCS in the wells at the density normally used for invasion assays. These basement membranes were photographed
at x 100. Bar, 20 ¡an.D, Sirongylocemraius purpúralas embryo at the same age and in the same orientation as A. The spatial relationship of the ectoderm to its extracellular matrix
and to blastocoelar structures are shown, s, spicules; h, hyalin layer; e, ectoderm; h, subectodermal basement membrane; bl, blastocoel; g, stomach of the primitive gul; c, coelomic
pouches. The esophagus and intestine do not appear on the side of the embryo shown.
5087
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1995 American Association for Cancer Research.
INVASION
OF SEA URCHIN EMBRYO
BASEMENT
MEMBRANES
B16f1--I.•
.30.25.20ui31.«1>C.10.05.
*•99•
i:'•i;
•¿â€”_
»*
**UM-MEL-1«.*9"-t*HT1080;UM-SCC-g•ioe.*99UM-SCC-10A•
i.
•¿â€”---B16f10SK-MEL-28,
»
::Metastatic
«,
*KeratmocilesFibroblastsMelanocytesSF-Meianoc>1es•«
Tumor Cells
Primary
Tumor Cells
Normal
Cells
Fig. 2. Collected invasion frequencies of metastatic tumor cells, primary tumor cells, and normal cells. The positions of at least 100 cells were scored for each data point. Each point
represents invasion frequency for cells in an invasion assay. At least two wells were assayed for each cell type in an invasion assay, and the data were pooled. All of the experiments
involving metastatic tumor cells are grouped together and so designated on the abscissa, as are the experiments involving primary tumor cells. Hi'tiv\ dotted lint's separate the invasion
frequencies of metastatic tumor cells from those of primary tumor cells and those of normal cells. Lìghl
dotted lines separate the invasion frequencies observed for each metastatic cell
line. The names of the cell lines appear above. All of the experiments involving normal cells are grouped together and are designated on the abscissa. Light dotted lines separate the
data obtained for keratinocytes, fibroblasts. melanocytes, and scatter factor-treated melanocytes, and they are labeled above. Mean invasion frequency for all metastatic cells is indicated
with a horizontal solid line, and the first SD with horizontal dashed lines. The mean invasion frequency for the primary tumor cells tested is indicated with a horizontal solid line, and
the first SD is indicated with horizontal unshed lines. The mean invasion frequency for all untreated normal cells coincides with the abscissa, and the first SD above the abscissa is
indicated with a horizontal dashed line. The mean invasion frequency for alt scatter factor-treated melanocytes tested is indicated with a horizontal solid line-, and the first SD is indicated
with horizontal dashed lines.
mean fraction of viable, spread, and adherent cells on the well bottoms
was 0.92 (±0.09).
Melanocytes, differentiated keratinocytes, and fibroblasts, cultured
from neonatal human foreskins, were allowed to settle as single cells on
the exteriors of sea urchin embryo basement membranes and incubated
for 16 h. The mean invasion frequency for normal neonatal melanocytes
was 0.0014 (±0.0038). The mean invasion frequencies observed for
differentiated, neonatal keratinocytes and for neonatal fibroblasts were
O.(X).For all normal cells tested, the mean fraction of cells on the well
bottoms that were viable, spread, and adherent was 0.93 (±0.06). As a
control for batch-specific differences in basement membranes, which
might affect invasion frequencies, normal cells were always assayed in
parallel with metastatic cells. Thus, the failure of normal cells to invade
is due to neither cellular damage nor abnormalities in the batches of
basement membranes used in the invasion assays.
Two-sample rank testing by the Mann-Whitney test (39) shows that
the chance probability of the differences in invasion frequencies
observed between the metastatic cell types and the untreated neonatal
cell types tested is <0.0005. Thus, the differences in invasion frequen
cies observed between all of the metastatic cell types and all of the
untreated neonatal cell types are significant to greater than 99%
confidence. The mean of all of the metastatic mean invasion frequen
cies is 0.181 (±0.017). The mean invasion frequency observed for
each metastatic cell line tested falls within the first SD of the mean of
all of the mean invasion frequencies. Thus, there are no quantitative
differences in the mean invasion frequencies of the metastatic tumor
cell lines tested.
UM-SCC-10A cells that were cultured from a primary, poorly
invasive squamous cell carcinoma, which later metastasized (34),
were also tested for invasion on sea urchin embryo basement mem
branes. Their mean invasion frequency was 0.042 (±0.0084). The
mean UM-SCC-10A primary carcinoma invasion frequency is over
four times less than that observed for UM-SCC-10B cells
(0.18 ±0.047), which were cultured from its metastasis. Two-sample
rank testing shows that the chance probability of the differences in
invasion frequencies observed between the metastatic cell types and
the UM-SCC-10A primary carcinoma cells is <0.0005. By the same
test, the chance probability of the differences in invasion frequencies
observed between UM-SCC-10A primary carcinoma cells and all
untreated neonatal cells tested is also <0.0()05. Thus, the differences
in invasion frequencies observed between all of the metastatic cell
types and UM-SCC-10A primary carcinoma cells, as well as the
differences in invasion frequencies observed between all of the nor
mal cell types and UM-SCC-10A cells are significant to >99%
confidence.
Because scatter factor is known to promote melanocyte motility
(40) and to cause a variety of epithelial cells to lose adhesion, divide,
and differentiate (41-43), melanocytes were treated with scatter factor
during their incubation on sea urchin embryo basement membranes.
Scatter factor-treated melanocytes were found to exhibit a mean
invasion frequency of 0.185 (±0.05), which is indistinguishable from
those of the metastatic melanoma, fibrosarcoma, and carcinoma cells
tested. Two-sample rank testing shows that the chance probability of
the difference in invasion frequencies observed between scatter fac
tor-treated and untreated neonatal melanocytes is <0.0005; thus, this
difference is significant to >99% confidence.
Fig. 3 shows examples of cells that have invaded sea urchin embryo
basement membranes. SK-MEL-28, UM-SCC-10B, HT1080, and
scatter factor-treated neonatal melanocytes located inside of basement
membranes are each shown in two views. The invaded basement
50KS
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1995 American Association for Cancer Research.
INVASION
OF SEA URCHIN EMBRYO
BASEMENT
MEMBRANES
Fig. 3. Basement membranes invaded by melastatic and normal cells. A and B, invasion by SK-MEL-28. b, basement membrane invaded by an SK-MEL-28 cell shown with the
focal plane passing through its surface in A. In B, the same basement membrane is shown with the focal plane passing through its interior, m. an SK-MEL-28 cell, which has extended
a long process appears between the spicules of the larval skeleton. The basement membranes shown in A and B were photographed at X400 by using phase contrast. C and D. invasion
by UM-SCC-10B. b, basement membrane invaded by a UM-SCC-10B cell; shown with the focal plane passing through its surface in C. In D, the same basement membrane is shown
with the focal plane passing through its interior, c, a UM-SCC-10B cell located inside the basement membrane. The basement membranes shown in C and D were photographed at X400
by using phase contrast. E and F. invasion by HT1080. b, basement membrane invaded by an HT1080 cell; shown with the focal plane passing through its surface in E. An HT1080
cell appears on its surface near the apex. In F, the same basement membrane is shown with the focal plane passing through its interior./, an HT108Ãœcell located inside the basement
membrane. The basement membranes shown in E and F were photographed at X400 by using phase contrast. G and H, invasion by scatter factor-treated melanocytes. b, basement
membrane invaded by two melanocytes; shown with the focal plane passing through its surface in G. In H, the same basement membrane is shown with the focal plane passing through
its interior. Two melanocytes are located inside between the spicules (mei). The basement membranes in G and H were photographed at X400 by using phase contrast. Bar. 20 jim
for all panels in Fig. 3; shown in G.
membrane is first shown with the focal plane adjusted to show its
upper exterior surface, then with the focal plane passing through its
interior in which the cells may be seen. In these examples, as in all of
the basement membranes scored for invasion, tears, holes, or loss of
tension in the basement membranes stretched over the spicules were
not observed by microscopic examination at X400. Invaded basement
membranes thus appeared to be grossly indistinguishable from those
which were not invaded.
Fig. 4 shows an example of an SK-MEL-28 metastatic human
melanoma cell invading a sea urchin embryo basement membrane
after 2 h of incubation. The majority of the cell body appeared to be
localized on a lateral, outer surface of the basement membrane,
whereas a broad front of cytoplasm appeared to extend across the
basement membrane, wrapping around a spicule in the blastocoel.
Fibrous extensions of the basement membrane or its associated matrix
appeared to be stretched over the surface of the invading cell, but
gaping holes or loss of tension in the basement membrane was not
apparent. Actual cell movement was easily visible.
5089
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1995 American Association for Cancer Research.
INVASIONoÃ--SI:A l RI HIN IMBKVIIHASIMINT MIMIWANIS
Fig. 4. SK-MEL- 28 cell invading a basement membrane, h. basement membrane; m,
melanoma cell. This cell was photographed at X4(X) by using phase contrast. Bar. 10 (¿m.
Fig. 5. Normal cells on the surfaces of basement membranes. A, melanocyte. A
melanocyte (mei) is shown 16 h after placement on the surface of a basement membrane.
The enlargement at the end of its process suggests that it is actively migrating, b, surface
of the basement membrane. This basement membrane was photographed at X400 using
phase contrast. Bar, 20 fj.m for all panels in Fig. 4. B, a differentiated keratinocyte (ker)
is shown 16 h after placement on the surface of a basement membrane, b, surface of the
basement membrane. This basement membrane was photographed at X400 using phase
contrast. C, a fibroblast (fib) is shown 16 h after placement on the surface of a basement
membrane, b, surface of the basement membrane. This basement membrane was photo
graphed at X400 using phase contrast.
Wells containing the cells added to the basement membranes were
fixed at the times indicated.
The time courses of SK-MEL-28, UM-MEL-1, B16F1, HT108Ü,
and UM-SCC-10B invasion are shown in Fig. 6. Basement mem
branes prepared from two unrelated sets of embryos were used in
these experiments. As can be seen from these time courses, the
invasion frequencies of each metastatic cell tested reached their max
imal values in 4 h, irrespective of where the cells originated in vivo.
Neonatal melanocytes made invasive by scatter factor treatment also
exhibited similar invasion kinetics (data not shown).
Intactness of Sea Urchin Embryo Basement Membrane Inva
sion Substrates. Although the correspondence between in vivo inva
sive behaviors and the invasion frequencies observed for normal,
primary tumor, and metastatic tumor cells in invasion assays and the
results of invasion time courses suggest that the sea urchin embryo
subectodermal basement membranes used are intact; the numbers and
sizes of defects present in preparations of these basement membranes
through which cells could migrate into their interiors without active
invasion were characterized independently by light and scanning
electron microscopy.
A typical preparation of sea urchin embryo basement membranes
and their associated hyalin layer extracellular matrix was examined by
both light and scanning electron microscopy. Three hundred basement
membranes were examined by light microscopy for visible defects.
Four of these basement membranes, or about 1%, had tears whose
largest dimensions ranged from 4 to 7 firn (data not shown). Because
the cells to be tested for invasion have diameters of approximately 30
p,m, these defects are likely to be the smallest possible, which could
potentially admit cells into basement membrane interiors by passive
movement.
The points of the spicules often appeared to protrude at the apices
of the basement membranes observed by light microscopy. Although
.20
.18
ID
.16
n
s-14
0)
Fig. 5 shows examples of normal neonatal melanocytes, fibroblasts,
.12
and keratinocytes after placement as single cells on basement mem
brane exteriors and incubation for 16 h. Unlike tumor cells, normal
.10
cells were unable to migrate from the exteriors to the interiors of
basement membranes, but they adhered well to the outer surfaces of
.08
the basement membranes. In general, they appeared to spread more on
O
the basement membrane surfaces than did the metastatic tumor cells.
.06
Sometimes, as for the melanocyte shown in Fig. 5/4, melanocytes
appeared to be actively migrating along the basement membrane
.04
surfaces. Basement membranes in contact with normal, uninvasive
cells also always appeared to be intact when viewed for invasion assay
.02
scoring.
Time Course of Basement Membrane Invasion by Metastatic
1
2345678
Tumor Cells. The speed of invasion by single metastatic cells after
Hours
placement on the outer surfaces of sea urchin embryo basement
Fig.
6.
Invasion
time
courses.
The
number
of hours after placement on the surfaces of
membranes was assessed for SK-MEL-28 melanoma, HT10KO fibro
the basement membranes is plotted on the abscissa. The invasion frequency is plotted on
sarcoma, and UM-SCC-10B squamous cell carcinoma human cells
the ordinale. •¿.
•¿,
and A. invasion experiments performed with a preparation of basement
membranes from embryos unrelated to those used to prepare the basement membranes
and for B16F1 mouse melanoma cells. The cells assayed were allowed
used in experiments depicted with D, O, and A. •¿.
UM-MEL-1 invasion frequencies. •¿
to settle as single cells on the exteriors of sea urchin embryo basement
and O, SK-MEL-28 invasion frequencies. D, UM-SCC-IOB invasion frequencies. A,
membranes briefly before being moved to an incubator at time zero.
HT 1080 invasion frequencies. A, BlnFI invasion frequencies.
5090
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1995 American Association for Cancer Research.
INVASION OF SEA URCHIN EMBRYO BASEMENT MEMBRANES
DISCUSSION
Fig. 7. Sou urchin embryo basement membrane and its associated extracellular matrix
viewed by scanning electron microscopy. The apical surface is visible. This basement
membrane was photographed at XI KM).Inset, surface of a similarly prepared basement
membrane photographed at X30.800.
the basement membranes appeared to be stretched over them, the
resolution of light microscopy is insufficient to evaluate intactness in
this region. To address this issue, measure the size distribution of
smaller potential basement membrane defects, and examine the ap
pearance of their outer surfaces, the same preparation of basement
membranes was fixed and mounted for scanning electron microscopy.
Fig. 7 shows a typical basement membrane with its associated extra
cellular matrix viewed by scanning electron microscopy at a low
magnification with a higher magnification view of the fibrous apical
surface as an inset. Neither the extracellular matrix preparation shown
nor any of the 100 others examined at this time had spicules protrud
ing at their apices. In all instances, they appeared to be intact in this
region. The visible, outer surfaces of 12 of these basement membranes
were examined, and the sizes of the potential defects were measured.
An average of six potential defects having a mean largest dimension
of 1 pirn were observed per visible basement membrane side. The
single largest defect observed was 2.5 pim. All of these potential
defects are very likely too small to permit passive movement through
the basement membranes. Thus, only 1% of the basement membranes
in a typical preparation contained defects that might be large enough
to permit the passive migration of the human or mouse cells placed on
their exteriors to their interiors.
The primitive gut lumen might be the largest potential pathway to
the basement membrane interior. In the invasion assays described
here, basement membranes were obtained from embryos about 2 days
before the formation of the mouth opening (44); thus, the extracellular
matrix associated with the primitive gut and extending across its end
should be intact in these preparations. However, it is difficult to
observe its condition by either light or scanning electron microscopy.
To evaluate the role of primitive gut lumens as pathways to basement
membrane interiors, basement membranes were obtained from living
embryos that failed to gastrulate and form primitive guts. These
embryos were of the same age as those used to prepare normal
basement membranes. Metastatic human melanoma SK-MEL-28 cells
and neonatal melanocytes placed on the exteriors of these basement
membranes exhibited invasion frequencies of 0.23 and ().()(), respec
tively. These invasion frequencies, plotted in Fig. 2, were indistin
guishable from those obtained when SK-MEL-28 cells or melanocytes
were placed on normal basement membranes; thus, the absence of
primitive guts affects neither the absolute nor the relative invasion
frequencies observed for these cells.
The results of experiments described here show that metastatic
human and mouse tumor cells, primary human tumor cells, and
untreated neonatal human cells normally contacting basement mem
branes underlying epithelia exhibited the expected invasive or noninvasive behaviors when placed on the outer surfaces of sea urchin
embryo subectodermal basement membranes and their associated
extracellular matrix. The results also show that the observed differ
ences in their invasion frequencies are highly significant. Further
more, the metastatic melanoma cells of humans and mice, metastatic
human fibrosarcoma cells, and metastatic human squamous cell car
cinoma cells invaded sea urchin embryo basement membranes with
mean invasion frequencies that are statistically indistinguishable. The
invasion frequencies of the metastatic cells tested were maximal
within 4 h. Because both the mean invasion frequencies and the
invasion time courses are indistinguishable for the metastatic cells
tested, it is possible that they utilize very similar invasive mechanisms
to invade these basement membranes.
The experiments described here also show that a cell line cultured
from a relatively uninvasive primary human carcinoma exhibited a
greatly reduced mean invasion frequency relative to those of meta
static cells, and that its mean invasion frequency was greatly elevated
when compared to untreated neonatal cells. Because the average
viabilities of all tumor cells and neonatal cells tested were very similar
and because invasive and uninvasive cells were always tested in
parallel, the reduced ability of primary squamous cell carcinoma cells
and the failure of normal neonatal cells to invade was due to neither
the loss of viability nor abnormalities in the batches of basement
membranes used in the invasion assays.
Although melanocytes migrate through the dermis to colonize the
epidermis by passing through the basement membrane separating
them, they do not appear to cross this basement membrane from its
outer surface once in the epidermis (1). Thus, the noninvasive behav
ior of epidermal neonatal melanocytes in the invasion assays de
scribed here is fully consistent with their behavior in vivo. The failure
of melanocytes to invade could be explained by their irreversible
differentiation to a noninvasive phenotype after arrival in the epider
mis. Alternatively, their invasive phenotype could be influenced by
the ligands displayed by the cells or the substrate in their new location.
Irreversible differentiation to a noninvasive phenotype after arrival in
the epidermis does not account for the failure of epidermal neonatal
melanocytes to cross sea urchin basement membranes when placed on
their outer surfaces because treatment of neonatal melanocytes with
scatter factor or hepatocyte growth factor rendered them as invasive as
metastatic melanoma cells. Thus, inability to recognize the outer
surfaces of these basement membranes as invasion substrates may
account for their lack of invasion, which can be induced by scatter
factor treatment.
Sea urchin embryo subectodermal
basement membranes and
their associated extracellular matrices appear to be intact invasion
substrates by several independent criteria, (a) The complete cor
respondence with in vivo invasive behaviors of the invasion fre
quencies observed for normal cells and for primary and metastatic
tumor cells, originating in epithelia and connective tissues, sug
gests that passive movement is not likely to account for a signif
icant fraction of the cells found inside the basement membranes
after placement on their exterior surfaces, (b) The speed and
efficiency of invasion, as observed by invasion time courses for
metastatic melanoma, carcinoma, and fibrosarcoma cells, suggest
that invasion does not occur at only a few randomly located sites
or defects because of the rapidity and highly directed nature of the
cellular migration that would be required, (c) As measured by
5091
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1995 American Association for Cancer Research.
INVASION
OF SEA URCHIN EMBRYO BASEMENT
scanning electron microscopy, the exterior surfaces of the base
ment membranes and their associated extracellular matrices have
potential defects very likely too small to permit the cells assayed
access to the interiors of basement membranes by passive migra
tion, (d) Scanning electron microscopy also showed that the base
ment membranes and their associated extracellular matrices were
intact in the regions near the points of the spicules. (e) Basement
membranes obtained from embryos that failed to gastrulate were
invaded by metastatic SK-MEL-28 human melanoma cells as fre
MEMBRANES
ACKNOWLEDGMENTS
The authors gratefully acknowledge helpful suggestions made by Drs.
Thomas Carey, James Varani, and Donald MacCallum at the University of
Michigan and by Dr. Lance Liotta at the NIH.
REFERENCES
quently as were preparations from normal embryos; thus, primitive
gut lumens did not appear to be significant pathways to their
interiors. Obvious defects in the invaded basement membranes
were not apparent as they were scored. Instead, they were indistingiushable in appearance from uninvaded basement membranes
in the same assay. During invasion, fibers of extracellular matrix
appeared to stretch over the surface of the invading cell. Highly
localized and regulated proteolysis could well be involved in
invasion; but the defects introduced by the invading cell might not
be easily visible by light microscopy or might be repaired by the
cell as part of the invasive process.
The experiments reported here show that metastatic tumor cells
derived from melanocytes, epithelial cells, and fibroblasts rapidly
invaded sea urchin embryo basement membranes and their associated
extracellular matrices with indistinguishable invasion frequencies.
The similar invasion frequencies exhibited by metastatic tumor cells
deriving from different tissues may result because the molecules that
function in invasion are present on the surfaces of invading cells for
a relatively constant fraction of the cell cycle.
Thus, the molecules participating in the recognition of basement
membranes as invasion substrates seem to have been functionally
conserved during at least the evolutionary time separating vertebrates
from invertebrates. Because UM-SCC-10A primary carcinoma cells
invaded from the outer surfaces of sea urchin embryo basement
membranes at an intermediate frequency, acquisition of the ability to
recognize highly conserved basement membrane molecules as inva
sion substrates may be a necessary step in the process by which
primary tumor cells become metastatic.
The time required for metastatic tumor cells to invade sea urchin
embryo basement membranes is consistent with the time required for
lymphocyte transit from blood to lymph in mice and rats. To move
from the blood to the lymph, lymphocytes must invade the endothelium and underlying basement membrane of lymph node high endothelial venules (4, 5). Four h after labeled thoracic duct lymphocytes
were transfused into mice or rats, a significant fraction of them had
crossed the walls of high endothelial venules and were found in the
paracortexes of lymph nodes (45, 46). Thus, the invasion of subectodermal basement membranes of sea urchin embryos by human and
mouse cells mimics invasive behaviors in vivo with respect to both
timing and relative frequencies.
The results of the invasion assays described here demonstrate
that the abilities of mammalian cells to recognize acellular, echinoderm embryo basement membranes as invasion substrates in
vitro correlate completely with their invasive phenotypes in vivo,
rather than with their tissues of origin. The correspondence be
tween the invasive behaviors of mouse and human cells placed on
these heterologous invasion substrates and their invasive behaviors
in intact organisms suggests that invasive behavior can be elicited
by the recognition of specific basement membrane molecules in the
absence of the cells that secreted them and that these molecules are
functionally conserved across the evolutionary time separating
vertebrates from invertebrates.
1. Hulley, P. A., Stander, C. S., and Kidson, S. H. Terminal migration and early
differentiation of melanocytes in embryonic chick skin. Dev. Biol., 145: 182—194,
1991.
2. Erickson, C. A., Duong, T. D., and Tosney, K. W. Descriptive and experimental
analysis of the dispersion of neural crest cells along the dorsolateral path and their
entry into the ectoderm in the chick embryo. Dev. Biol., 151: 251-272, 1992.
3. Holbrook, K. A., Vogel, A. M., Underwood, R. A., and Foster, C. A. Melanocytes in
human embryonic and fetal skin: a review and new findings. Pigm. Cell Res. /
(Suppl.).- 6-17, 1988.
4. Freemont, A. J., Stoddart, R. W., Steven, F., Jones, C. J., and Matthews, S. The
structure of the basement membrane of human lymph node high endothelial venules:
an ultrastructural, histochemical, and immunocytochemical study. Histochem. J., 18:
421-428, 1986.
5. Anderson, A. O., and Anderson, N. D. Lymphocyte emigration from high endothelial
venules in rat lymph nodes. Immunology, 31: 731-748. 1976.
6. Springer, T. A. Traffic signals for lymphocyte recirculation and leukocyte emigration:
the multistep paradigm. Cell, 76: 301-314, 1994.
7. Liotta, L. A., Stetler-Stevenson, W. G., and Steeg, P. S. Cancer invasion and
metastasis: positive and negative regulatory elements. Cancer Invest., 9: 543-551,
1991.
8. Kleinman, H. K., McGarvey, M. L, Hassell, J. R., Star, V. L, Cannon, F. B., Laurie,
G. W., and Martin, G. R. Basement membrane complexes with biological activity.
Biochemistry. 25: 312-318, 1986.
9. Kleinman, H. K.. McGarvey. M. L., Liotta, L. A., Robey, P. G., Tryggvason, K., and
Martin, G. R. Isolation and characterization of type IV procollagen, laminin, and
heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry, 21: 6188-6193,
1982.
10. Grant, D. S., Kleinman, H. K., Leblond, C. P., Inoue, S., Chung, A. E., and Martin, G. R.
The basement membrane-like matrix of the mouse EHS tumor: II. Immunohistochemical
quantitation of six of its components. Am. J. Anal., 174: 387-398, 1985.
11. MacKay, A. R., Gomez, D. E., Cottam, D. W., Rees, R. C., Nason, A. M., and
Thorgeirsson, U. P. Identification of the 72-kDa (MMP-2) and 92-kDa (MMP-9)
gelatinase/type IV collagenase in preparations of laminin and Matrigel. Biotech
niques, /5: 1048-1051, 1993.
12. Vukicevic, S., Kleinman, H. K., Luyten, F. P., Roberts, A. B., Roche, N. S., and
Reddi, A. H. Identification of multiple active growth factors in basement membrane
Matrigel suggests caution in interpretation of cellular activity related to extracellular
matrix components. Exp. Cell Res., 202: 1-8, 1992.
13. Fridman, R., Sweeney, T. M., Zain, M., Martin, G. R., and Kleinman. H. K.
Malignant transformation of NIH-3T3 cells after subcutaneous co-injection with a
reconstituted basement membrane (Matrigel). Ini. J. Cancer, 51: 740-744, 1992.
14. Noel, A. C., Calle, A., Emonard, H. P., Nusgens, B. V., Simar, L.. Foidart, J.. Lapiere,
C. M., and Foidart, J-M. Invasion of reconstituted basement membrane matrix is not
correlated lo the malignant metastatic cell phenotype. Cancer Res., 51: 405-414,
1991.
15. Simon, N., Noel, A., and Foidart, J-M. Evaluation of in vitro reconstituted basement
membrane assay to assess the invasiveness of tumor cells. Invasion Metastasis, 12:
156-167, 1992.
16. Liotta, L. A., Lee, C. W., and Morakis, D. J. A new method for preparing large
surfaces of intact human basement membrane for tumor invasion studies. Cancer
Lett., //: 141-152, 1980.
17. Hendrix, M. J., Seftor, E. A., Seftor, R. E. B., Misiorowski, R. L., Saba, P. Z.,
Sundareshan, P., and Welch, D. R. Comparison of tumor cell invasion assays: human
amnion versus reconstituted basement membrane barriers. Invasion Metastasis. 9:
278-297, 1989.
18. Cresson, D. H., Beckman, W. C., Jr., Tidwell, R. R., Geratz, J. D., and Siegal, G. P.
In vitro inhibition of human sarcoma cells1 invasive ability by bis (5-amidino-2-
19.
20.
21.
22.
23.
24.
benzimidazolyl) methane-a novel esteroprotease inhibitor. Am. J. Pathol., 123:
46-56, 1986 .
Armstrong, P. B., Quigley, J. P., and Sidebottom, E. Transepithelial invasion and
intramesenchymal infiltration of the chick embryo chorioallantois by tumor cell lines.
Cancer Res., 42: 1826-1837, 1982.
Slarkey, J. R., Hosick, H. L., Stanford, D. R., and Liggil, H. D. Interaction of
metastatic tumor cells with bovine lens capsule basement membrane. Cancer Res., 44:
1585-1594, 1984.
Bisgrove, B. W., Andrews, M. E., and Raff, R. A. Fibropellins, products of an EOF
repeat-containing gene, form a unique extracellular matrix structure that surrounds the
sea urchin embryo. Dev. Biol.. 146: 89-99, 1991.
Bisgrove, B. W., and Raff. R. A. The SpEGF 111gene encodes a member of the
fibropellins: EGF repeat-containing proteins that form the apical lamina of the sea
urchin embryo. Dev. Biol., 757: 526-538, 1993.
McCarthy, R. A., Beck, K., and Burger, M. M. Laminin is structurally conserved in
the sea urchin basal lamina. EMBO J., 6: 1587-1593. 1987.
Amemiya. S. Development of the basal lamina and its role in migration and pattern
formation of primary mesenchyme cells in sea urchin embryos. Dev. Growth &
Differ., 31: 131-145, 1989.
5092
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1995 American Association for Cancer Research.
INVASION
OF SEA URCHIN EMBRYO BASEMENT
25. Wcssel, G. M., and McC'lay. D. R. Gastrulation in the sea urchin embryo requires the
deposition of crosslinked collagen wilhin the extracellular matrix. Dev. Biol., 121:
149-165, 1987.
26. Lane, M. C., and Solursh. M. Primary mesenchyme cell migration requires a chondroitin sulfale/dermatan sulfate proteoglycan. Dev. Biol., 143: 389-397, 1991.
27. Solursh, M., and Katow, II. Initial characterization of sulfated macromolecules in
the blastocoels of mesenchyme blastulae of Stnm^\'l<tceiitnnii\ pttrpurntu.\ and
Lylechinus piclus. Dev. Biol., 94: 326-336, 1992.
28. DeSimone, D. W., Spiegel, E., and Spiegel. M. The biochemical identification of
fibronectin in the sea urchin embryo. Biochem. Biophys. Res. Commun., 133:
183-188, 1985.
29. Decker. G. L-, and Lcnnarz, W. J. Skeletogenesis in the sea urchin embryo. Devel
opment, 103: 231-247, 1988.
3(1. Mardin, J., and McClay, D. R. Target recognition by the archenteron during sea urchin
gastrulation. Dev. Biol., 142: 86-102. 1990.
31. Gibson, A. W., and Burke. R. D. The origin of pigment cells in embryos of the sea
urchin Stronfìvloceritmlu*purpurara*. Dev. Biol.. 107: 414-419. 1985.
32. Gibson. A. W.. and Burke. R. D. Migratory and invasive behavior of pigment cells in
normal and animali/ed sea urchin embryos. Exp. Cell Res.. 173: 546-557. 1987.
33. Carey, T. E., Takahashi, T.. Resnick, L. A.. Oettgen, H. F., and Old, L. J. Cell surface
antigens of human malignant melanoma. Proc. Nati. Acad. Sci. USA. 73: 3278-3282,
1976.
34. Virolainen, E., Vanharanta. R., and Carey. T. E. Steroid hormone receptors in human
squamous carcinoma cell lines. Int. J. Cancer, 33: 19-25. 1984.
35. Varani, J.. Mitra, R. S., McCIenic, B. J., Fligiel, S. E., Inman, D. R.. Dixit, V. M., and
Nickloloff. B. J. Modulation of fibroneclin production in normal human melanocytes
and malignant melanoma cells by intcrfcron--y and tumor necrosis factor-a. Am. J.
Pathol., 134: 827-836, 1989.
36. Varani, J., Carey, T. E., Fligiel, S. E., McKeever. P. E., and Dixit. V. Tumor
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
MEMBRANES
type-specific differences in cell-substrate adhesion among human tumor cell lines. Int.
J. Cancer, 39: 397-403, 1987.
Fidlcr. I. J. Selection of successive tumour lines for metastasis. Nature (Lond.), 242:
148-149, 1973.
Rasheed, S., Nelson-Rees, W. A., Toth, E. M., Arnstein, P., and Gardner, M. B.
Characterization of a newly derived human sarcoma line (HT 10KO).Cancer (Phila.),
33: 1027-1033, 1974.
Zar. J. H. Two-sample rank testing. In: Biostatistical Analysis, pp. 138-142. Englewood Cliffs, NJ: Prentice-Hall, 1984.
Halaban, R., Rubin, J. S., Funasaka, Y., Cobb, M., Boulton, T., Falcilo, D., Rosen, E.,
Chan. A.. Yoko. K.. White, W.. Cook. C., and Moellmann. G. Mel and hepatocyte
growth factor/scalier factor signal transduction in normal melanoctes and melanoma
cells. Oncogene, 7: 2195-2206, 1992.
Matsumoto. K., Tajima, H., and Nakamura, T. Hepatocyte growth factor is a potent
stimulator of human melanocyte DNA synthesis and growth. Biochem. Iliophys. Res.
Commun., 176: 45-51, 1991.
Gherardi, E. Hepatocyte growth factor/scatter factor and the behavior of epithelial
cells. Symp. Soc. Exp. Biol., 47: 163-181. 1993.
Sonnenberg. E.. Meyer. D.. Weidner. K. M.. and Birchmeier, C. Scatter factor/
hepatocyte growth factor and its receptor, the c-met tyrosine kinase, can mediate a
signal exchange between mesenchyme and cpithelia during mouse development. J.
Cell Biol., 123: 223-235. 1993.
Leahy, P. S. Laboratory culture of Stnmxylm-entronu purpúralas adults, embryos,
and larvae. Methods Cell Biol., 7: 1-13, 1986.
Sprent, J. Circulating T and B lymphocytes of the mouse I. Migratory proterties. Cell.
Immunol., 7: 10-39, 1973.
Gowans, J. L.. and Knight. E. J. Roule of re-circulation of lymphocytes in the rat.
Proc. R. Soc. Ser. Biol. Sei., I5VB: 257-282. 1969.
5093
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1995 American Association for Cancer Research.
Invasion of Selectively Permeable Sea Urchin Embryo Basement
Membranes by Metastatic Tumor Cells, but not by Their Normal
Counterparts
Donna L. Livant, Stephanie Linn, Sonja Markwart, et al.
Cancer Res 1995;55:5085-5093.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/55/21/5085
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1995 American Association for Cancer Research.