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The Mucosal Invasion Model
A Novel In Vitro Model for Evaluating the Invasive Behavior of Mucocutaneous Malignancies
Eben L. Rosenthal, MD;
Mark K. Wax, MD;
Peter Anderson, MD;
Molly Kulecz-Martin, PhD
Arch Otolaryngol Head Neck Surg. 2001;127:1467-1470.
ABSTRACT
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Background Prevention of regional and metastatic spread of cutaneous malignancies
requires understanding the physiologic mechanism of tumor cell invasion. In
vitro models are convenient for studying the in vitro invasive phenotype of
normal cells, tumor cell lines, or genetically altered cells in a 3-dimensional
matrix, but they should attempt to recapitulate the complex in vivo submucosal
environment. A new acellular extracellular matrix, porcine submucosal matrix
(PSM), is thought to accurately recapitulate the submucosal matrix. A novel
in vitro model using PSM to assess mucocutaneous tumor cell invasion was studied.
Methods The morphologic characteristics, growth, and invasive behavior of human
head and neck squamous cell carcinoma (UM-SCC-1, UM-SCC-5, UM-SCC-17B, and
OSC-19) cell lines were assessed on the PSM gel and compared with commonly
used in vitro invasion models (type I collagen and Matrigel matrices). The
invasive phenotype of canine kidney cells was also assessed on each matrix,
because this cell line is known to demonstrate a characteristic in vitro invasive
phenotype.
Results The PSM-supported head and neck squamous cell carcinoma tumor cell line
growth and single cell invasion were seen under stimulated conditions, similar
to type I collagen gels. The invasive phenotype of canine kidney cells behaved
similarly on PSM and collagen. Matrigel did not support growth well, and invasion
occurred only superficially in isolated areas.
Conclusion The PSM is a good in vitro model for assessment of pharmacologic and
genetic manipulations of head and neck squamous cell carcinoma tumor cell
lines and has several advantages over other commonly used matrices.
INTRODUCTION
PATIENT SURVIVAL in cutaneous and mucocutaneous malignant tumors of
the head and neck is largely limited by failure to control regional and distant
metastatic disease. Central to tumor cell invasion and metastasis is the ability
of malignant cells to migrate through the normal barriers presented by components
of the extracellular matrix (ECM).1 Determining
the mechanisms tumor cells use to traverse the ECM may lead to therapies directed
against regional and metastatic spread of disease, thereby improving patient
survival.
Understanding of the mechanisms of tumor cell invasion and metastasis
has been expanded by use of in vitro models that allow subtle manipulations
of tumor cell environments. Currently, there is not a model to assess the
submucosal matrix that head and neck tumor cells must traverse in vivo. In
vitro models used to assay malignant cell invasion usually use isolated ECM
components, such as type I collagen, fibrin, or mixtures of ECM components
such as the extract Matrigel (Becton Dickinson and Company, Bedford, Mass).2-5 A matrix
of porcine submucosal intestine has recently been introduced that retains
the convenience of other commonly used in vitro invasion materials but more
closely recapitulates the in vivo stroma milieu of mucosal surfaces.6-9
The porcine submucosal matrix (PSM), also called small intestinal submucosa, has been used in numerous preclinical animal
studies to restore damaged tissue structures.10-11
In its most basic compositional form, small intestinal submucosa consists
primarily of type I collagen and other typical components of the submucosal
ECM including fibronectin, glycosaminoglycans, and growth factors.7-9
Many common in vitro substrates used to study mechanisms of tumor cell
invasion and metastasis fail to adequately reproduce the complexity of the
submucosal matrix. In vivo, metastatic epithelial tumor cells must migrate
through a stromal barrier composed of collagen (types I and III), elastin,
fibronectin, glycoproteins, proteoglycans, and many other molecules. Matrigel
is largely composed of basement membrane components, including a predominant
basement membrane protein, laminin, obtained by excretion of transformed cultured
murine fibroblasts, and is used in hundreds of studies each year (577 English-language
publications identified on MEDLINE between 1998 and 2001).12-15
It lacks components of the submucosal ECM through which tumor cells must migrate
to gain access to the bloodstream. In addition, type I collagen gels represent
only a fraction of the components found in vivo within the stromal matrix.
A single component in isolation does not allow for cell-matrix interactions
that are considered to be important for phenotype expression in vitro.1, 14-16
We describe herein a novel model that uses porcine intestine submucosal
ECM for the evaluation of cutaneous tumor cell invasion. This acellular model
can be useful to study adhesion and cell-matrix interactions, to assess the
invasive potential of genetically manipulated (transfected) cell lines, and
to evaluate the effects of growth factors and/or inhibitors on tumor cell
invasion. We outline the advantages, disadvantages, and technical considerations
in the use of this model.
MATERIALS AND METHODS
CELL CULTURE
The human head and neck squamous cell carcinoma (HNSCC) lines UM-SCC-1,
UM-SCC-5, and UM-SCC-17B (provided by Thomas Carey, PhD, University of Michigan,
Ann Arbor), OSC-19 (provided by Toshio Yokoi, MD, Kanazawa University, Kanazawa,
Japan) and MDCK cells (provided by Stephen Weiss, MD, University of Michigan,
Ann Arbor) were routinely maintained in minimal essential medium supplemented
with L-glutamine (2mM), penicillin (100 U/mL), and streptomycin (100 µg/mL;
all from Life Technologies Inc, Gaithersburg, Md) and 10% heat-inactivated
fetal bovine serum (Hyclone Laboratories Inc, Logan, Utah). Cells were cultured
at 37°C in humidified 5% carbon dioxide–95% air.
CELL GROWTH AND MOTILITY
Dishes were coated with 0.8 mL of either type I rat tail collagen, 3.0
mg/mL (Sigma-Aldrich Corp, St Louis, Mo), Matrigel (both normal and growth
factor reduced; Becton Dickinson and Company), or PSM (vivoSIS Gel, 3.0-mg/mL
collagen concentration; a gift of Cook Biotech, West Lafayette, Ind). Growth
assays were performed with cells plated at 1 x 104 in 6-well
dishes coated with matrix (0.8 mL) and assessed during a 6-day incubation
period (at 1, 3, and 6 days) by count of cells per high-power field (x40).
CELL INVASION ASSAY
Matrigel was placed in the 12-mm upper portion of the 2-chambered culture
system (Transwell dishes, pore size, 3 µm; Corning Costar Corp, Cambridge,
Mass) and gelled for 45 minutes at 37°C. The PSM was provided as a neutralized
solution of 3.0-mg/mL collagen concentration that was gelled at 37°C for
45 minutes. Type I collagen (0.9 mL) was added to the upper chamber dishes
and gelled for 45 minutes at 37°C. The collagen was prepared by means
of rat-tail type I collagen dissolved in 0.2% acetic acid at 3.2 mg/mL and
gelled by neutralizing the acid with 0.3N sodium hydroxide containing phenol
red as a pH indicator. A final concentration of 3.0 mg/mL was chosen to closely
match the type I collagen concentration of PSM. Medium was then added to the
upper and lower chambers before the addition of 2 x 105 cells
per well. All invasion assays were performed in this serum-containing medium.
Cells were grown to confluence (12-24 hours), and if growth factors were used,
either recombinant human scatter factor (SF, 0.5nM; Becton Dickinson and Company)
or epidermal growth factor (EGF, 3.5nM; Becton Dickinson and Company) was
added to the lower chamber. Media were changed every 3 days, and the respective
growth factor was re-added to the lower chamber. Invasion occurred during
a 7-day incubation period, at which time samples were harvested for histologic
analysis and cellular invasion was assessed.
SAMPLE FIXATION AND PROCESSING
Gels were removed from the upper chamber with minimal manipulation and
then placed in 2.7% formaldehyde and embedded in paraffin. Sections (3 µm
wide) were cut and stained with hematoxylin-eosin. Tumor cell invasion was
assessed by light microscopy in a minimum of 4 randomly selected sections
for each experimental sample.
RESULTS
MORPHOLOGY AND GROWTH
The HNSCC cell lines (UM-SCC-1, UM-SCC-5, UM-SCC-17B, and OSC-19) were
sparsely plated on gels of collagen, Matrigel, and PSM grown on collagen.
Tumor cell lines plated on Matrigel consistently failed to reach confluence,
and instead cells developed a weblike pattern that persisted throughout the
7-day culture period with some stacking of cells (Figure 1A). The HNSCC tumor cell lines on collagen and PSM displayed
single or multiple layering of tumor cells (depending on the tumor cell line)
with very similar morphologic characteristics and time to confluence (Figure 1). For all HNSCC cell lines tested,
the cell growth rates were lower for Matrigel than collagen or PSM (Figure 2).
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Figure 1. Inverted light microscopy of head
and neck squamous cell carcinoma tumor cell line UM-SCC-1 cultured for 4 days
on Matrigel (gel form; Becton Dickinson and Company, Bedford, Mass) (A), collagen
(2.8 mg/mL) (B), and porcine submucosal matrix (C). For all parts, hematoxylin-eosin,
original magnification x20.
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Figure 2. Cell growth on in vitro substrates.
Results were averaged from 3 head and neck squamous cell carcinoma cell lines
(OSC-19, UM-SCC-1, and UM-SCC-17B) in duplicate experiments. Growth rates
were based on daily counts per high-power field (HPF) during 6-day culture
period on Matrigel (Becton Dickinson and Company, Bedford, Mass), collagen,
and porcine submucosal matrix.
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TUMOR CELL INVASION
Invasion studies of HNSCC and melanoma cell lines were conducted under
EGF-stimulated conditions by means of a 2-chambered culture system during
a 7-day incubation period. Invasion was assessed by phase microscopy and confirmed
by histologic analysis. Certain cell lines (UM-SCC-1 and OSC-19) displayed
a single cell invasive phenotype beginning at 48 hours into the collagen and
PSM throughout the surface of the gel (Figure
3). The UM-SCC-5 and UM-SCC-17B tumor cell lines invaded the matrix
at the end of the 7-day incubation period but did not penetrate as deeply
into the matrix as UM-SCC-1 and OSC-19. On Matrigel, the HNSCC tumor cell
lines under EGF-stimulated conditions demonstrated small, rounded clusters
of cell that penetrated into the matrix as small groups of cells after 72
to 96 hours in culture (Figure 3).
Tumor cell invasion on Matrigel was limited to distinct areas over the culture
surface. The isolated areas of cells on cross-sectional histologic examination
were consistent with the inverted light microscopic view (Figure 1A).
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Figure 3. Photomicrographs of head and neck
squamous cell carcinoma tumor cell invasion (OSC-19). Histologic cross-sectional
analysis was performed on tumor cells after a 7-day epidermal growth factor–stimulated
(3.5nM) incubation period atop Matrigel (Becton Dickinson and Company, Bedford,
Mass) (A), collagen (B), or porcine submucosal matrix (C). Arrows illustrate
tumor cells invading the matrix. Note the separation artifact that occurs
during processing of Matrigel for histologic analysis. For all parts, hematoxylin-eosin,
original magnification x40.
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MDCK CELL INVASION
The MDCK cells have a well-defined invasive phenotype on collagen when
stimulated with SF–hepatocyte growth factor (SF/HGF).17-18
To assess the influence of PSM on the invasive pattern, subconfluent cultures
were maintained for 24 hours on Matrigel (normal and growth factor reduced),
collagen (3.0 mg/mL), and PSM and then stimulated with SF/HGF. On Matrigel
the cells formed a weblike pattern and the invasive phenotype, and there was
no significant evidence of invasion at the conclusion of the 7-day incubation
period. No invasion occurred in the absence of SF/HGF. Stimulated MDCK cells
behaved similarly on collagen and PSM: focal areas of single cell invasion
occurred throughout the matrix after 5 days (data not shown). The morphologic
character of the invading tumor cells was similar on collagen and PSM.
COMMENT
In vitro models of tumor cell invasion are commonly used to evaluate
the phenotype of genetically altered (transfected) tumor cells, or tumor cell
response to growth factors and/or inhibitors.3-5
The invasion models evaluated in this study all have several advantages compared
with in vivo models. First, evaluation can be done by inverted phase light
microscopy throughout the culture period and cross-sectional histologic analysis
can be performed for the purposes of graphic representation. Second, the assays
are practical. They are relatively inexpensive and can be conducted in a relatively
short time course (2 weeks). Last, these experiments can be conducted with
the use of commercially available reagents, which improve reproducibility
between laboratories. Currently, there is no invasion model specific for the
submucosal matrix. We describe a novel invasion model for HNSCC cell lines
using components from porcine submucosal intestine.
All the HNSCC tumor cells attached and formed a monolayer on PSM, and
under EGF-stimulated conditions the tumor cells invaded over the course of
72 hours in a single cell fashion. The morphologic features and growth of
HNSCC cell lines on PSM were consistent with the initial report of this matrix
that described the growth and morphologic characteristics of epithelial cell
lines cultured atop PSM.6 Matrigel (as a gel
form) did not allow variations in morphologic features or support cell growth
in the same manner as PSM. The HNSCC cells consistently displayed a web pattern
of growth that was not consistent with morphologic findings on the other ECM
components assessed in this report. The HNSCC and MDCK cells had little invasive
potential when cultured on Matrigel.
Because of its high type IV collagen content, Matrigel is considered
a model of the basement membrane,19 whereas
type I collagen and PSM represent the stromal or submucosal matrix, which
is predominantly type I collagen.6-7
Although investigations often focus on the mechanism by which tumor cells
degrade and migrate through the basement membrane, invasion through the stromal
ECM is more relevant to development of new therapeutic modalities, since at
the time of cancer diagnosis the tumor has already progressed beyond the basement
membrane.20 Although PSM or type I collagen
can be used to assess tumor cell invasion, the PSM contains the matrix components
specific to the submucosal matrix. The value of in vitro models is in comparing
phenotypic changes that occur with perturbations in the system, such as addition
of a growth factor or transfection with a gene. Moreover, the mechanism of
novel molecular and pharmacologic interventions during the HNSCC tumor cell
invasive program can be assessed by this model.
In summary, the PSM model has several advantages over other in vitro
invasion matrices. Compared with collagen, PSM better recapitulates aspects
of the physiologic matrix that HNSCC cells must traverse in vivo. It supports
tumor cell growth better, is less expensive, and allows single cell invasion
compared with Matrigel. Therefore, the PSM should be considered as an in vitro
model for assessment of new pharmacologic molecular genetic modalities of
altering HNSCC cell invasion and metastasis.
AUTHOR INFORMATION
Accepted for publication August 24, 2001.
Corresponding author and reprints: Eben L. Rosenthal, MD, Division
of Otolaryngology–Head and Neck Surgery, University of Alabama at Birmingham,
1501 Fifth Ave S, Birmingham, AL 35233 (e-mail: oto{at}uab.edu).
From the Division of Otolaryngology–Head and Neck Surgery, University
of Alabama at Birmingham (Dr Rosenthal); and Section of Head and Neck Oncology,
Department of Otolaryngology–Head and Neck Surgery (Drs Wax and Anderson)
and Department of Dermatology (Dr Kulecz-Martin), Oregon Health Sciences University,
Portland.
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