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CR 75th Anniversary Commentary
Extracellular Matrix Invasion in Metastases and
Angiogenesis: Commentary on the Matrigel
"Chemoinvasion Assay"
Adriana Albini
Abstract
Invasive and metastatic cells must cross the basement membrane's extracellular matrix to disseminate to distant sites.
Although in the eighties the concept was well established, no
easy in vitro functional assay was available. Working in Hynda
Kleinman's and George Martin's laboratory at NIH (Bethesda,
MD), where the reconstituted basement membrane Matrigel was
discovered, I had the intuition that Matrigel coating of migration
filters could represent a valid tool to mimic in vitro biological
matrix barriers. The "chemoinvasion assay" using Matrigel in
Boyden blind-well chambers was developed in 1985–1986 and
published in Cancer Research in 1987. It was a rapid and easy
tool for studying invasion, a crucial step in cancer metastasis.
Since its conception, the assay has been employed for studies on
the metastatic process, angiogenesis, and for the screening of
drugs that are potentially able to decrease cell invasion. It was
adapted to be easily employed as a routine assay and commercialized. In that historical article, we also described the use
of thick layers of Matrigel for the study of morphogenesis of
invasive cells, a simple and visual assay, adaptable to reproduce collective cell migration in vitro. To date, in its diverse
optimized variants, the chemoinvasion assay is still widely
used, contributing to novel data production. In the era of
precision medicine and next-generation sequencing, the
cheap, fast, and reproducible chemoinvasion assay may have
further developments, including possible applications in the
investigations on cancer stem cells, immunity and immune
modulators, applications with siRNA silencing, selection of
aggressive cell populations, and phenotypes and genetic evaluations. Cancer Res; 76(16); 4595–7. 2016 AACR.
See related article by Albini A et al., Cancer Res 1987;47:3239–45.
Background and Historical Context
mation, tissue development and repair, and in invasion and
metastasis. Isolation and characterization of the molecular
components of these matrices was relatively difficult, as compared with that of many other connective tissues. Early assays to
study invasion employed tissues that contained BMs as barriers,
and they were sort of cumbersome; the tissues included chicken
chorioallantoic membrane, bladder wall, chick heart, and the
amnion (3).
When I arrived in the United States after three years of postdoctoral work on tumor cell chemotaxis at the Max Planck
Institute for Biochemistry in Martinsried (Germany), the laboratory of George R. Martin and Hynda Kleinman at NIH was
investigating the Engelbreth–Holm–Swarm (EHS) tumor. This
spontaneously arisen transplantable tumor produced multiple
layers of BM. As Lance Liotta states in his historical editorial (1):
"An explosion of new knowledge about the biochemistry of
basement membranes and the generation of reagents for cancer
research emanated from the discovery of the Engelbreth-HolmSwarm (EHS) sarcoma. The EHS sarcoma matrix, for the first time,
provided biochemical quantities of basement membrane collagen, proteoglycans, and glycoproteins."
The major protein and glycan components of BMs were characterized from this tumor and their cDNAs cloned (3). Extracts of
the EHS tumor had the ability to form a gel, which was named
Matrigel (from matrix and gel). Matrigel provided a BM matrix
with several unique characteristics, which became widely known
(4); it is liquid at 4 C but can polymerize into a gel when warmed,
producing a 3-dimensional matrix. The gel exerts biological
activity on numerous cell types, recapitulating several properties
of the tumor microenvironment.
In the late 1970s and early 1980s, the role of extracellular matrix
(ECM) and basement membranes (BM) in hindering cellular
migration within distant organs became widely accepted. The
pivotal work of Lance Liotta at NCI (Rockville, MD) and Josh
Fidler at MD Anderson Cancer Center (Houston, TX), describing
metastasis as a multistep phenomenon of which crossing ECM
compartments was a rate-limiting step, was crucial for further
studies on the topic (1, 2). BMs are continuous sheets of ECM
underlying most epithelial or endothelial cells. Several leaders in
the field over 30 years ago devoted their careers to study the
interactions of metastatic tumor cell with BMs. Laminin, its major
component, was first isolated by Pam Gehron-Robey and Rupert
Timpl in George Martin's laboratory at NIH (Bethesda, MD) and
published in 1979. The predominant components of BMs besides
laminin are type IV collagen, the proteoglycan perlecan, nidogen/
entactin, and several other factors (including collagen VII, collagen XVII, and collagen XVIII).
The network formed by these molecules creates a dense,
although thin, barrier that hinders the passage of cells and
macromolecules. BMs can become permeable during inflam-
Scientific and Technology Pole, IRCCS MultiMedica, Milan, Italy.
Corresponding Author: Adriana Albini, IRCCS MultiMedica, Via Fantoli 16/15,
Milan 20138, Italy. Phone: 3902-5540-6574; Fax: 3902-5540-6570; E-mail:
[email protected]
doi: 10.1158/0008-5472.CAN-16-1971
2016 American Association for Cancer Research.
www.aacrjournals.org
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 2016 American Association for Cancer Research.
4595
CR 75th Anniversary Commentary
Very shortly following my arrival at NIH as a Fogarty Visiting
Scientist, I had the idea to use diluted Matrigel to coat the porous
filters of the Boyden chambers, the common device for studying
cell migration, that I had extensively employed at the Max Planck
Institute to investigate the enhanced migratory capabilities of
cancer cells (5) and the delayed migration of senescent fibroblasts
(6). This latter study obtained the Max-Buerger award from the
German Society of Gerontology.
In the assay I developed at NIH, Nucleopore chemotaxis
filters with 8 or 12 mm diameter pores were coated with a 1:10
or 1:20 dilution of liquid Matrigel and allowed to dry. The
Matrigel was then reconstituted with culture media, creating a
thin, compact coating of reconstituted BM, as verified by
electron microscopy. The filter layered with rehydrated Matrigel
was placed between the two compartments of a Boyden chemotaxis chamber on a porous filter and proved to be a powerful
barrier to cell migration of "normal cells," while metastatic
ones were able to penetrate it in response to stimulatory factors,
mimicking metastasis to distant organs. The different times of
assay, Matrigel concentration, type of chemoattractant, cell
number, and cell types were tested and published in the Cancer
Research article (7). The Boyden chamber–Matrigel system was
proposed as a rapid and innovative assay to quantify the
invasive potential of tumor cells and named the "chemoinvasion assay."
I had the fortune and the privilege in the years from 1985 to
1987, when the article was conceived and then published, to meet
and discuss with giants in the field: Josh Fidler at MD Anderson
(Houston, TX), Lance Liotta, Stuart Aaronson, Mark Lippman,
and Mike Sporn, all at NCI (Bethesda, MD). From those scientists,
I received precious advice, insight, and material related to metastasis, invasion, cancer biology, extracellular matrix, and the
"tumor microenvironment" (the term was introduced later).
I obtained cancer cell cultures and normal or benign counterparts
from several laboratories and used a large variety of them, and
with the help of the Japanese postdoctoral fellow Yukihide
"Rocky" Iwamoto, to validate the method.
The vast international use and the "longevity" of the assay is
closely related to its properties: it is a simple method, reproducible, and it can be adapted to commercial chambers, such as the
transwell plates, with cross-laboratory consistency. It has allowed
and is allowing researchers to: (i) identify factors that can induce/
promote invasion; (ii) measure and discriminate different degrees
of "malignancy"; (iii) provide evidence about migratory/invasive
cellular functions that can then be verified in vivo; and (iv)
elucidate the role of molecules or pathways crucial for migratory/invasive properties.
In the Cancer Research article (7), we presented another application of Matrigel in cancer research, besides the chemoinvasion
assay with the Boyden chamber. This was the use of a thick layer of
Matrigel in culture wells for morphologic studies of tumor cell
behavior; the publication was the first showing the branching,
invasive morphology adopted by metastatic tumor cells on Matrigel. In that publication, as an example, a different morphology
was demonstrated by cells from benign prostate hyperplasia, and
benign and malignant prostate cancer cells, the latter forming
branching networks in a collective invasion modality. This second
application, a simple, fast, and visual assay, was later defined as
the "Matrigel morphogenesis assay." The assays received the
Doerenkamp Zbinden/John's Hopkins award as an alternative
to animal research.
4596 Cancer Res; 76(16) August 15, 2016
The Present and Future Perspectives
The chemoinvasion assay with Matrigel and its numerous
variations, 30 years after its publication, is still the most employed
in vitro system for testing cells with different invasive abilities, has
been applied to angiogenesis, and is able to provide insights into
numerous biological investigations. This model was optimized to
fit the needs of individual laboratories and to run several samples
at the same time, and it has been adapted to commercial packages.
Screening systems, first employed by Mary Hendrix as the MICS
assay (8), are also commercially available.
The chemoinvasion assay was improved for use under sterile
conditions, allowing the isolation of cells with a higher invasive
potential from mixed populations of cell lines or primary tumors.
We proposed the use of the tool for selection of highly migratory
cells (3), and it was employed by others, providing isolate cell
populations with a greater metastatic power in vivo and formation
of colonies in soft agar (9).
Concepts on metastasis have evolved since the introduction of
the chemoinvasion assay. Cancer stem cells (CSC) or cancerinitiating cells are able to recapitulate the tumor of origin when
transplanted in vivo. They are characterized by a slower proliferative rate or are relatively quiescent and undergo asymmetric
division. CSCs have been reported to be resistant to most chemotherapy agents. Various investigations have implicated that
metastasis comes from CSCs. Highly invasive CSC subpopulations can be selected in vitro using the chemoinvasion assay (9).
They show decreased differentiation, enhanced colony formation
in soft agar, and demonstrate the ability to from spheroids when
cultured in stemness conditions (serum deprivation in low attachment supports), higher invasive capabilities, increased tumor
take, and metastases formation in vivo. Matrigel has been shown
by us and others to support the take of even a few tumor cells in
vivo (10, 11), providing a microenvironment particularly convenient for CSCs. The chemoinvasion assay can represent an inexpensive cell-sorting apparatus to be applied in stem cell research
and to isolate rare cell populations, retaining high viability of the
selected cells.
The development of imaging techniques and cell-labeling
approaches also provides a new application for the 3-dimensional
assay of morphogenesis. Social networks can be studied. Evidence
shows that tumors often invade as cords of cells closely interacting
(12), a phenomenon termed "collective migration." These cell
societies may be easily studied in the morphogenesis assay in
Matrigel.
Novel tools have been envisaged that can be applied in the
chemoinvasion assays. The use of siRNA oligonucleotides and
the CRISPR/Cas9 system to silence expression of specific genes
provides molecular technologies that can elucidate the role of
new pathways in cancer, metastases, angiogenesis, and developmental biology. Cancer cells are stimulated to migrate as a
function of tumor size, hypoxia, and metabolism, the effects of
which can be tested in the chemoinvasion assay. Nanotechnology has been proposed for tracing and treating cells. We are
undergoing rapid evolution in technology that, combined with
the simplicity of the chemoinvasion assay, still offers a vast
range of applications.
The development of the chemoinvasion assay in the NIH
laboratory, the "house of Matrigel," led to life-long friendships
and to my wedding to next-door scientist Douglas Noonan that
was celebrated in the same year of the Cancer Research article
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 2016 American Association for Cancer Research.
Matrigel Chemoinvasion Assay
publication and was followed by the birth of our two children,
Thomas and Silvia Noonan, now university students.
(Bethesda, MD), for their encouragement as my mentors, and Douglas Noonan
for a life besides Matrigel.
Disclosure of Potential Conflicts of Interest
Grant Support
No potential conflicts of interest were disclosed.
Acknowledgments
I thank Antonino Bruno, Gabriele D'Uva, and Silvia Noonan for critical
reading of the manuscript. I thank George Martin and Hynda Kleinman,
formerly at Laboratory of Developmental Biology and Anomalies, NIH
The studies are supported by grants from the AIRC (Associazione Italiana per
la Ricerca sul Cancro) and the Health Ministry.
Received July 19, 2016; accepted July 19, 2016; published online August 15,
2016.
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Cancer Res; 76(16) August 15, 2016
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4597
Extracellular Matrix Invasion in Metastases and Angiogenesis:
Commentary on the Matrigel ''Chemoinvasion Assay''
Adriana Albini
Cancer Res 2016;76:4595-4597.
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