BioengineeredTumorModel_DrJonasMoses_r2015

ENGINEERING
CANCER
PLATELETS, CYTOPLASTS AND
EXTRACELLULAR MATRIX IN THE
BIOENGINEERING OF A 3-DIMENSIONAL,
IN VITRO TUMOR MODEL
BY
JONAS MOSES, PhD
University of Illinois Press - Copyright 2007
Chicago, Illinois

This book is dedicated to my parents, Robert
Abram Moses, MD and Sylvia Greenfield Moses -
who instilled me in the love of all people, a respect
for our planet and a great passion for all things
medical and scientific; who encouraged my siblings
and me to question and explore… everything; who
supported their children in all manner of endeavors
lovingly and without judgment; who introduced
their children to innumerable cultures by traveling
the world together as a family and by welcoming
travelers into their home from every continent on
the planet; and who – by living their own lives as
giving, ethical, moral and modest human beings –
served as excellent role models for their children
(and many, many other people who met and knew
them, during their lives).
It is also dedicated to my brothers – Lloyd, Bruce,
Fred, Ed, Harsh, Joel and Tom
– who have been loving, patient, encouraging and
gentle good friends to me; who have been, at times,
my solace and support system; who often have
served as mirrors for my thoughts and ideas; and
who have always endeavored to provide me with an
unvarnished, and true reflection of Life’s realities,
when I most needed them.
Finally, this book is a gift to my son, Benjamin:
for he has been, since his birth (and continues to be)
the greatest of gifts to me. He is, first and foremost,
my “number one son,” and I am his biggest fan; he
has also been my inspiration, my guide, my sometime
sidekick and my friend. He has chosen, as his

grandparents and father before him, to pursue a
career in Medicine, and (his dad thinks) holds
within in his nature, and his gifts, the potential for
an ideal balance between intellect and instinct,
confidence and humility, drive and patience and
forthrightness and sensitivity.
Benjamin is, as many a performer has quipped
about the ‘star’ of any show, “a very tough act to
follow…”
Jonas Moses, PhD
June 29th
, 2007
At my usual table, in Starbucks, at North and Wells
Chicago, IL

TABLE OF CONTENTS
CHAPTER PAGE
I. INTRODUCTION……………………………………………………………..5
A. Bioengineering of a 3D, in vitro tumor model …………..……... 5
B. Tensegrity and the human body ……………………………….. 8
C. Differential digestion of DNA and chromatin organization by
ECM and cytoskeleton 9
D. Contact with laminin and other ECMPs are associated with
down-regulation and reversion of highly aggressive tumor cells
in VM patterns …………………………………………………….. 11
E. Formation of biofilms found in vasculogenic mimicry patterns
may be conferring special protection to highly invasive
tumorscells……….................................................................. 14
F. Is the Information for Cell Shape Derived from the Genome
or from the ECM? ………………………………………………... 16
G.References……………………………………………………………. 18
II. ECM AND CYTOPLASM DETERMINE SIMPLE MORPHOGENIC
RESPONSES IN THE ABSENCE OF NUCLEAR DNA ……………… 20
A. Materials and Methods ………………………………………………. 21
1. Cell culture ………………………………………………….... 21
2. Generation of cytoplasts ……………………………………. 22
3. Exposure of cytoplasts to ECM …………………………….. 25
4. Observation and data capture ………………………………. 25
B. Results ………………………………………………………………… 25
1. Comparing the morphogenetic response of nucleated cells
and cytoplasts derived from poorly invasive cells, and highly
invasive cancer cells on floating Matrigel rafts ………………… 25
2. Testing the relative contributions of the cytoplasm and
nucleus to simple ECM-directed pattern formation …………… 28
C.Discussion…………………………………………………………….. 29
D.References……………………………………………………………. 32

PLATELET CYTOKINESIS AND LACK OF THROMBOSIS IN
LAMININ- LINED VM PATTERNS………………………………………… 34
A. Materials and methods ………………………………………………. 38
1. Platelet acquisition and preparation ……………………… 38
2. Three dimensional matrix-containing cultures ………….. 38
3. Observation and data capture ……………………………. 38
B.Results…………………………………………………………….….. 38
1. Platelet adhesion/activation and ECM substrates ….….. 38
2. Platelet division …………………………………………….. 40
C.Discussion……………………………………………………………. 41
D. Acknowledgments …………………………………………………… 43
E. References …………………………………………………………… 44
IV.CONCLUSION………………………….……………………………………. 46
A. Highlights of observations, discoveries, outcomes
and implications………………………………………………………… 48
B. Potentialfutureinvestigation………………………………………….... 50
C. Cytoplast and Platelet Findings in the Context of the Bio-
engineeredTumorModel……………………………………………… 52
FIGURES
CHAPTER PAGE
I. Figure 1: “Halo Culture System” ……………………….…………… 12
Figure 2: “Low-density cultures of MUM2B cells
in Matrigel followed throughout 10 days”………….. 14
Figure 3: “Differential killing of metastatic versus spindle A
and B [?] cells in metastatic melanoma 3-dimenional
vasculogenic mimicry cultures ………………….. 16
II. Figure 1: “Methods for generating cytoplasts by centrifugation
And cytoplast exposure to ECM”………………………………… 22

Figure 2: “Phase micrographs of pattern formation by nucleated,
poorly-invasive and highly invasive cells seeded at equal
densities on floating rafts of Matrigel after 2 days” …. 26
Figure 3: “Pattern formation by cytoplasts derived from poorly-
invasive and highly invasive cells on floating rafts of
Matrigel after 36 hours” ………………………………. 27
Figure 4: “Testing the relative contributions of ECM and nuclei
to simple pattern formation” …………………………. 29
III. Figure 1: “Sequence of cytokinesis observed with platelet on
the upper right-hand corner when seeded
on plastic” ……………………………………………. 39
Figure 2: “Platelets behave differently when in contact with
various ECM protein substrata”………………….… 41
TABLES
CHAPTER PAGE
II. Table: “Generation of Cytoplasts from Non-invasive
and Invasive Cell Lines and Their Comparative
Morphogenic Responses” ………………………………………… 24

Chapter I: Introduction
Bioengineering of a 3D, In Vitro Tumor Model
Identifying the elements of a 3D in vitro tumor model --
and then assembling them in an organized manner -- must
begin with some directive questions, to guide the investigators
in meaningful ways down the path of experiments that will result
in a viable model. To build an in vitro 3D model, what kinds
of information would need to be gathered?
For example: 1) Since much previous investi-gation had
been undertaken in monolayer (2D) cultures, do cells behave
differently in a 3D environment than in a 2D environment?
2) Primary (or “normal”) vs. metastatic – do primary cells
behave differently than tumor cells in a 3D versus 2D
environment? 3) Since the human body is a unique 3D
environment, shouldn’t the emphasis in the development of a
3D tumor model be how cells behave in a 3D environment
closely approximating the human body? 4) What is the impact
of ECMP microenvironment (Extracellular Matrix Proteins) - do
cells behave differently in one 3D micro-environment than
another, and what is unique about tumor cells versus normal
cells - regarding cell behavior and shape? 5) Is the information
for cell shape derived solely from the genomic DNA or does
the ECM play a significant role? 6) If the cell is a kind of
tensegrity structure, is there a direct biomechanical
relationship between what occurs on the cell’s surface
(microenvironment) and what happens in the nucleus – i.e.
might the ECMPs play a directive role in gene expression?

This is but a partial list of the questions the investigators
in this study asked in the course of developing a global
model. As can be anticipated by the bioengineer involved in
such an investigative endeavor, with all such paths of research
exploration the effect is as of a cascading series of events: one
answered question leads to another question or series of
questions, and the effect is nearly exponential. The more
information gathered the better informed and more numerous
are the questions generated.
Thus was the case with this study and, though a good,
basic in vitro model has emerged from this line of
investigation, there are manifold refinements yet to be made,
based upon the new generation of questions the
investigators were able to articulate with the knowledge
gained. Even as this dissertation is being written, several new
research articles - by the some from the same group of
investigators - are in press, and new lines of investigation (on the
same theme) are underway.
While gathering information for this study – in developing
relevant lines of experimentation – the investigators had a
number of choices to make about the nature of the study. For
instance, since “vasculogenic mimicry” had been observed in
metastatic melanoma tumors for many years prior to this
team’s search for an in vitro model, it made sense to begin
by experimenting with metastatic melanoma.
Vasculogenic mimicry is a process whereby aggressive
tumors form extra-vascular perfusion channels made of
laminin, fibronectin, Type IV collagen, and other extracellular
matrix components, which circumscribe nests of typically
epithelioid tumor cells. These channels are loosely connected
to, or are contiguous with, blood vessels and are known

collectively as vasculogenic mimicry patterns (VM patterns), the
fluid-conducting meshwork or, more recently, extravascular
matrix patterns (ECMPs). An extremely aggressive form of
melanoma is metastatic uveal (ocular) melanoma; thus, this
cell line was chosen. The choice of metastatic uveal
melanoma made sense for other reasons, as well. There was
the consideration the eye is a unique environment within the
human body. The human eye is in some ways a closed system,
with no internal blood supply (in the mature eye) to either the
anterior or posterior chambers. There is neither any internal
mechanism for immune response to infiltration by disease
(no lymphatic supply) including cancers; thus, the tissues of
the eye are more readily observed (in the absence of any
acute, inflammatory processes). Indeed, when this closed
system is breached in some way – damage to the globe which
opens a wound to the air, for example – impressive infections
occur very rapidly, for the body’s natural immune system has
a difficult time reaching the inside of the eye to combat
that infection. This differential lack of immune response is
termed “immune privilege,” and there are several other
locations throughout the human body that also exhibit this. Over
the past 30 years, investigators have learned much about the
physiologic processes responsible for immune privilege in the
eye:
Special architectural features of the anterior chamber and
unique, immune-modulatory molecules – present in the
ocular fluids and expressed on ocular parenchymal cells.
Together they govern and modify the manner in which
antigenic material placed in the anterior chamber is
recognized by cells and molecules of the systemic immune
apparatus. In addition, these processes alter the ways in

which immune effector molecules and cells respond to foreign
and antigenic material that is present within the eye. The net
effect of these forces is to limit the intraocular development of
inflammation.
1,2,3
In addition, uveal melanoma metastasizes primarily to the
liver, which is a highly vascular and a readily accessible site
from which to derive tumor samples. It is also a site in which
these back-to-back looping patterns (vasculogenic mimicry
patterns) had been observed. While it is readily appreciated that
ease of access to the tumor site is a boon to the experimental
process, perhaps less obvious to the reader is the significance
and importance of choosing a site that exhibits VM patterns, as
we sought to develop a useful, in vivo solid tumor model.
Members of our investigative team had already demonstrated
that these patterns were a hallmark of highly invasive melanoma
tumors, and hypothesized that they also signaled end-stage
(terminal) disease. It appealed to them that in first seeking to
extend the specific experimental results achieved with this
cancer cell line to other cells lines, they may discover enough
commonalities to derive a model that would stand exemplar for
all highly invasive cells lines. The choice of uveal melanoma
proved an excellent one, and the model constructed from this
tumor cell line did, indeed, hold true for every other invasive cell
line tested.
4
Tensegrity and the Human Body
One model that has borne fruit, and which appears to hold
true both in the study of humans and other animals – from the
bio-molecular level to the whole organism – is that of the cell
as a tensegrity structure. Tensegrity (from “tensional
integrity”) was described by Buckminster Fuller as an

architectural system in which structures stabilize themselves by
balancing the counteracting forces of compression and
tension gives shape and strength to both natural and artificial
forms. Fundamentally, tensegrity is the pattern that results
when “push” and “pull” share an interdependent relationship
with each other. The pull is continuous and the push is
discontinuous. The continuous pull is balanced by the
discontinuous push- producing integrity of tension and com-
pression. Buckminster Fuller explained that these fundamental
phenomena were not opposites, but complements that could
always be found together. Tensegrity is a pair, like many co-
existing pairs, of fundamental physical laws -- push and pull;
compression and tension; repulsion and attraction.
5
The tensegrity model was further developed as a cellular
model, and a new word, “mechanogenomics,” was coined to
name the unique biomechanical relationship as observed and
described, between the ECM, cytoskeleton and genome.
4
However, long before members of our team began
investigating tensional integrity as it impacts cell shape and
behavior other scientists had recognized the significance of this
model.
In 1981, Donald Ingber described human cells (and tissues)
in terms of tensegrity structures
6
, and continued to develop this
analogy in a series of articles over the next twenty years. Dr.
Ingber asserted that the actin microfilament lattice of the
cytoskeleton behaves as if it depends on tensional integrity;
microtubules act as compression- resistant struts; and a third
layer of structural stability is contributed by the intermediate
filaments, as tensile stiffeners. This idea (and subsequent,
further investigations along the same line) has, in part, served
as the foundation for and inspiration behind much of the work

discussed in the body of this dissertation. For instance, the
suggestion that genes are regulated by higher order
chromatin structure, the cytoskeleton and extracellular matrix,
is an extension of Ingber’s proposition that not only at the
cellular level, but at every state from the molecular to the
systemic, there is tensegrity architecture at work, and this
mechanical aspect of structure fundamentally influences the
way in which biological processes occur.
7
Differential digestion of DNA and chromatin organization by
ECM and cytoskeleton
In the course of further expanding the tensegrity analogy as
it applies to internal and external biomechanics of the cell and
its surrounding micro-environment, our team began with the
genome itself – seeking to demonstrate that, even at the
molecular level, the DNA and protein organization of
chromosomes exist as a tensegrity structure. To illuminate this
structure, and demonstrate how it impacts the relationship
between the genome, the cytoskeleton and the extracellular
environment, I (and other members of our investigative team)
undertook a substantial amount of microsurgical genome
isolation experiments in conjunction with numerous enzyme
assays -- both broad- spectrum protein and DNA enzymes as
well as very specific and selective enzymes -- and developed
new isolation techniques. I (and, ultimately, others on our
team) then turned to differential digestion of intact, monolayer
cell cultures and cell cultures grown on various extracellular
matrix proteins.
I was the first to observe that the ECM differentially
stabilizes the sequestration and exposure of DNA in interphase
cells, in whole, permeabilized cell culture assays.

Subsequently, I (and others, since) demonstrated that there
are differences in chromatin digestion by the DNA-cutting
enzyme, Alu I, between the normal and malignant forms of
several cell types. These differences were shown to be
“independent of the cell cycle, as demonstrated by the identical
differential digestion of chromosomes extracted from cells and
nuclei, from intact cells of varying malignant behavior”4
. I also
developed an ECM assay chip, which – in concert with
additional studies within my research group (and, in other
laboratories) – led to the development of other useful cell- and
tissue-based assays.
C o l l e c t i v e l y , these generated several novel and
significant conclusions about both the increase in
sequestration of nuclear DNA seen with 3D cultures versus
2D cultures and the increase in sequestration of nuclear
DNA of highly aggressive/ invasive tumor cells versus poorly
invasive tumor cells and normal cells.
2
Further, my
colleagues and I established a clear and undeniable bio-
mechanical link between the ECM, the cytoskeleton and higher
order chromatin structure – ECM does play a role in the
exposure and sequestration of nuclear DNA and, thus, impacts
the expression of genes. The ways in which the ECM
induces and the cytoskeleton mediates such biomechanical
behavior within the cell is consistent with a model of the cell as
a tensegrity structure.
4,8
Finally, changes in DNA
sequestration and exposure that are mediated by the
cytoskeleton and induced by the ECM suggest mechanisms of
drug resistance
9
; this is of import in the bioengineering of
therapeutics directed at nuclear DNA and the means by
which these agents are delivered to the cell. It also suggests
some alternative approaches to drug therapy – perhaps

effecting local changes in the ECM, rather than seeking to
deliver drugs inside the cell.
Contact with laminin and other ECMPs are associated with
down-regulation and reversion of highly aggressive tumor cells
in VM patterns
Highly invasive tumors containing vasculogenic mimicry
patterns have often been associated with imminent death of the
host. It was theorized that the formation of such patterns
accompanied the up-regulation (expression) of genes for
highly metastatic behavior. In a series of experiments using
laminin I, collagen IV, fibronectin and other ECMPs as the
substrata for culturing of various highly invasive tumor cell
types, our investigative team learned that the opposite is true:
the presence of these VM patterns is instead associated with
the down-regulation and reversion of tumor cells to an
indolent cell type.
8
In one series of experiments, I showed that in a dense
colony or graft of highly invasive cells -- situated in the center
of a laminin or Matrigel substrate and then grown for several
days -- the culture first develops VM patterns at its center, and
then grows out onto the surrounding virgin matrix in what
appears to be VM pattern-forming cords. However, within a few
days, the cells that have grown out onto peripheral matrix begin
to retract and ultimately die off, leaving behind patterned trails
on the surface of the matrix. Of additional note was the
observation that cells close to the center of the original
colony demonstrate a different morphology than those in
growing out in the periphery. Many of the cells near the
center of the colony, which were not in direct contact with the
laminin or Matrigel substrate, exhibited epithelioid morphology,

whereas many of the cells growing in the periphery (as the
colony expanded outward) exhibited a spindle (or needle-
shaped) morphology (Figure 1).
Figure 1. Halo culture system. A: MUM2B metastatic melanoma cells
were constrained on plastic culture dishes by a 3-mm-diameter
cloning ring. B: Ten days after initial plating, the MUM2B cells did
not form vasculogenic mimicry patterns and expanded from the initial
constrained area to fill the well. C: MUM2B cells were seeded on a
raft of Matrigel until vasculogenic mimicry patterns formed. A 3-
mm-diameter punch was taken from the seeded raft and grafted
onto a virgin recipient bed of Matrigel. D: Ten days after the graft in
C was placed, MUM2B melanoma cells migrated only a short distance
onto the recipient Matrigel bed. E: Higher magnification of the edge of
the graft illustrated in D. The melanoma cells in the recipient bed are

elongated in contrast to the normal epithelioid shape of MUM2B cells.
The MUM2B cells formed reticulated looping patterns adjacent to the
graft. Note that the cells formed patterns and retracted back toward
the graft: impressions of the vacant patterns are visible in the top
right area of the picture, just below and to the left of the label. Original
magnifications: 50 (A-D); 200 (E).
Finally, while the initial graft of concentrated cells did
form VM patterns, and did send out satellite cells at its
periphery, little or no cell division was observed. These
findings appear to indicate that -- when in direct contact with
a laminin-rich environment – the laminin either causes highly
invasive tumor cells to become quiescent or is, in fact,
hostile to these cells and ultimately leads to invasive cell death.
In a second set of experiments, culturing a very
small number of cells – close enough to one another to be
observed in the same field at relatively high magnification, but
not touching one another so as to remove the variable of
cell-cell contact – I observed some of the same behaviors
as were seen in the densely-populated cultures. Some cells
would begin to elongate into a more spindle-shaped
morphology and traveled short distances across the matrix
much like a snail might, before shrinking and dying off. It is
suggested that this is due to poor adhesion to the laminin
substrate. Other cells rounded up into an epithelioid shape,
and remained in this way for days, without movement or
division (Figure 2).
8

Figure 2. A-H: Low-density cultures of MUM2B cells in Matrigel
followed throughout 10 days. The cell of interest appears in
boxes at low magnification in A, C, E and G and at higher
magnification in B, D, F, and H. Throughout the 10 days, the
elongated MUM2B cell retracted, leaving behind a footprint in the
Matrigel. Original magnifications: 50 (A, C, E, G); 100(B, D, F, H).
Formation of biofilms found in vasculogenic mimicry patterns
may be conferring special protection to highly invasive tumors
cells
Spermine and Spermidine are naturally-occurring
polyamines found in abundance throughout the human body.
These polyamines are known to confer stability in cells,
especially in the genome and, in large quantities can cause
tight sequestration of nuclear DNA.9-14
Also, already known is
that the DNA in highly aggressive tumor cells is highly
sequestered compared with normal or non-aggressive tumor
cells.4
There are several synthetic polyamine analogs (similar in
composition to spermine) that have been created in an effort to
capitalize on what is known about the action of spermine and
this tight sequestration of the DNA in highly invasive tumor cells;
the intent of these analogs being to interfere with the normal
action of spermine.9,15
When certain polyamine analogs are introduced into 3D cell
cultures of normal or poorly-invasive cell colonies, there is little or
no immediate effect, and there is little or no toxic effect
apparent.9,15
However, when cultures of highly invasive uveal
melanoma cells are drugged with these analogs, the killing
effect is relatively rapid and dramatic.9,15
In 3D cultures, which
have developed VM patterns, I observed an unusual effect

when drugging these cultures with certain analogs: the cells
closest to the perimeter of a VM channel (which appear to
have reverted from a metastatic morphology to a spindle A
type cell) are unaffected by the analog, while the cells furthest
away from the loop edge (and also furthest from the laminin or
Matrigel substrate), exhibiting a typically epithelioid morphology
(signal of highly invasive tumor cells) round up and die (Figure
3). Cell death was established by staining: based on
exclusion or incorporation of Trypan blue.9
A B
C D
Figure 3. A-D: Differential killing of metastatic versus spindle A and B
[?] cells in metastatic melanoma 3- dimensional vasculogenic mimicry
cultures. MUM2B spindle A and epithelioid cells form looping patterns
in mature cultures, and are then either untreated (A,B), or treated
(C,D) with CGC-11144. After 24 hours of incubation, Trypan dye is

added to all wells. Note that nearly all cells in A,B that were not
exposed to the analog exclude Trypan blue, while many cells
incorporate Trypan blue in the CGC-11144-treated cells (C,D). Black
arrows point to unaffected (non-stained), intact spindle A cells, at the
rim of one pattern loop; white arrow points to dead (trypan blue-
stained), epithelioid, metastatic cells.
Is the Information for Cell Shape Derived from the Genome or
from the ECM?
In this investigation, we sought to determine how much of
the control of normal versus tumor cell morphology is
conferred by nuclear DNA and how much by cytoplasm
and/or microenvironment (ECMPs). We reasoned that, in order
to appreciate cell behavior in the context of tissues, we
would need to create whole cultures of cells absent their
genomes, in order to observe their behavior in relation to one
another, in the formation of growth patterns – such as the
development into clumps (acinae), cord-like structures or VM
patterns. The study of individually enucleated cells has been
described in another paper.1
We explored several ways – both
as found in the literature and through our own innovation –
of enucleating cells in large numbers, to provide a large
enough population of “cytoplasts” (cells absent their nuclear
DNA) with which to create cell cultures. The enucleation of
large cell populations proved highly challenging, thus I sought
out alternative means of creating such populations, and in
the course of that research, proposed an alternative
cytoplast model (see Chapter III). Various members of our
team utilized both normal and tumor cell populations in the
creation of cytoplast cultures, for side-by-side comparison to

their nucleated counterparts. These comparisons demonstrated
that simple morphogenesis – change in cell shape and cell- cell
interaction in the development of cell colony arrangements or
patterns – does occur without the benefit of contributing
information from nuclear DNA. In the following chapter (Chapter
II), I present the details of this series of experiments, our
observations and findings, and I propose that the ECM
microenvironment does play a significant role in simple
morphogenesis, in normal cells, in non-aggressive and in
highly invasive tumor cell types. Further, I introduce the idea
that platelets may provide a suitable, natural analog to our
manufactured cytoplasts. Specific details of experiments
designed to explore this idea are discussed in Chapter III.
References
1. Ferguson TA, Green DR and Griffith TS: Cell death and
immune privilege. Int Rev Immunol. 2002 Mar-June; 21 (2-3):
153-72
2. Niederkorn JY: See no evil, hear no evil, do no evil: the
lessons of immune privilege. Nat Immunol. 2006
Apr;7(4):354-9.
3. Streilein JW: Immunoregulatory mechanisms of the eye.
Progress in Retinal and Eye Research, Volume 18, Number
3, July 1999, pp. 357-370
4. Maniotis AJ, Valyi-Nagy K, Karavitis J, Moses J, Boddipali V,
Nunez R, Bissell MJ, Folberg R: Chromatin sensitivity to Alu
I endonuclease is regulated by extracellular matrix and the
cytoskeleton. Am J Pathol 2005, 166:1187-1203
5. Fuller B: Tensegrity. Portfolio Artnews Annual 4, 112-127.
(1961)

6. Ingber DE, Madri JA and Jamieson JD: Role of basal lamina
in the neoplastic disorganization of tissue architecture. Proc
Nat Acad Sci USA; 78, 3901-3905. (1981).
7. Ingber DE: Cellular Tensegrity – defining new rules of
biological design that govern the cytoskeleton. Journal of Cell
Science 104, 613-627 (1993)
8. Folberg R, Arbieva Z,Moses J, Hayee A, Sandal T, Kadkol
S, Lin AY, Valyi-Nagy K, Setty S, Leach L, Chévez-Barrios
P, Larsen P, Majumdar D, Pe’er J and Maniotis AJ: Tumor
cell plasticity in uveal melanoma: micro-environment directed
dampening of the invasive and metastatic genotype and
phenotype accompanies the generation of vasculogenic
mimicry patterns. Am J Pathol 2006, 169:
9. Sandal T, Moses J, Valyi-Nagy K, Hayee A, Karavitis J,
Marton LJ, Folberg R and Maniotis AJ: Tumor biofilms and
cellular polarity control polyamine chemo- resistance and
genome sequestration in highly invasive tumors. (in
preparation)
10.Cohen SS: “A Guide to Polyamines” Oxford University Press,
New York, NY, 1998.
11.Tabor CW and Tabor H: Polyamines. Annual Review of
Biochemistry 53; 749- 790, 1984.
12.Pegg AE: Recent advances in the biochemistry of
polyamines in eukaryotes. Biochemistry Journal, 234; 249-
262, 1986.
13.Marton LJ, Morris DR 1987. Molecular and cellular functions
of the polyamines In Inhibition of Polyamine Metabolism:
Biological Significance and Basis for New Therapies, pp 79-
105. Eds PP McCann, AE Pegg and A Sjoerdsma. New
York: Academic press.

14.Porter CW and Janne J 1987. Modulation of anti-neoplastic
drug action by inhibitors of polyamine biosynthesis: In
Inhibition of Polyamine Metabolism: Biological Significance
and Basis for New Therapies, pp 203-248. Eds PP
McCann, AE Pegg and A Sjoerdsma. New York: Academic
press.
15.Marton LJ, Pegg AE: Polyamines as targets for therapeutic
intervention. Annual Review of Pharmacological Toxicology
1995. 35:55-91, 1995.

Chapter II
ECM and Cytoplasm Determine Simple Morphogenic
Responses in the Absence of Nuclear DNA
Maniotis et al. (and other investigative teams) have
previously shown that the morphogenetic programs of many
normal and neoplastic cells can be altered by varying the
composition of the extracellular matrix (ECM).1,2,3
Although
these ECM-induced changes may be associated with shifts in
gene expression, there is also evidence that the cytoskeleton
contributes to the control of cellular patterning independent
of the genotype. To separate the influence of gene
transcription from that of the cytoskeleton in ECM-regulated
pattern formation, our investigative team compared the
behavior of enucleated cytoplasts and nucleated cells from
which they were derived on floating rafts of Matrigel. Cytoplasts
derived from MCF10a breast epithelial cells, MDA-MB231
breast carcinoma, and M619 and MUM2B melanoma cells
formed the same patterns on Matrigel rafts as their nucleated
counterparts. Further, we compared the behavior of
cytoplasts derived from MCF10a breast epithelial cells on
Type I collagen and on Matrigel: enucleated cytoplasts
dispersed randomly on Type I collagen but formed tight
aggregates on Matrigel, identical to the behavior of nucleated
MCF10a cells. Therefore, the physical presence of nuclei is
not required to generate simple architectural patterning if the
appropriate ECM microenvironment is present. It was also
shown that tissue organization depends on interactions
between the extracellular matrix (ECM) and the cell.2,4
Highly
invasive melanoma cells form monolayers when cultured on

two-dimensional substrata (2D), but form complex looping
patterns when cultured on thick Matrigel or collagen Type I.3,5
N o r ma l breast cells form polarized, acini-like structures
when embedded in laminin rich reconstituted basement
membrane (Matrigel), but do not form correctly polarized
acini when grown in Type I collagen.6,7
Matrix-dependent
morphogenetic changes in breast cells have been associated
with profound changes in gene expression.8
Although it is generally assumed that tissue morphogenesis is
regulated principally by nuclear genes, there is evidence that the
extracellular matrix (ECM) microenvironment and the
cytoskeleton also contribute significantly to the control of cellular
phenotypes and tissue patterning in both normal and cancer
tissues, independently of the genotype.3,5,7,9-12
To assess the
relative contributions of nuclear and cytoplasmic control of simple
ECM-driven morphogenesis, we designed a method to generate
enucleated cells (cytoplasts). We then compared the
morphogenetic behavior of the enucleated cytoplasts to their
nucleated counterparts under different ECM conditions.
Materials and Methods
Cell Culture
Cell lines were derived from primary choroidal melanomas
and of low (OCM1a) and high (M619) invasive potential and
from highly invasive cells isolated or from a focus of metastatic
uveal melanoma to the liver (MUM2B); the characteristics of
these cells lines have been described in detail previously.
5
Melanoma cells were plated in DMEM (BioWhittaker, Inc.,
Walkersville, Maryland), and supplemented with 10% fetal

bovine serum (Fisher, Ontario, Canada) without the addition of
exogenous ECM molecules or growth factors. No antibacterial or
antifungal drugs were used in the maintenance of cell lines or in
experiments, as their chronic use has been shown by the
investigators, and others, to interfere with the differentiative
potential of other primary cell types or cell lines. MDA-MB231 cells
and MCF10a cells were obtained from the ATTC (Rockville,
Maryland), and were maintained on DMEM plus heat
inactivated calf serum. All cell cultures were determined to be
free of mycoplasma contamination using the GenProbe rapid
detection system (Fisher, Itasca, Illinois).
Generation of Cytoplasts
Coverslips 12 mm in diameter were pre-washed in 1N HCl for
1 hour and extensively washed with DD H20. The coverslips
were then placed into 60 mm Falcon tissue culture dishes, and
cells were seeded onto them in the presence of serum fibronectin
for up to 48 hours. After achieving confluence, the dishes
containing the coverslips were then exposed 10 mg/ml of
cytochalasin B (Sigma, St Louis, MO) for 1 hour to disrupt
actin filaments.
13
The coverslips were then removed and
placed upside down into 50 ml conical centrifuge tubes
containing pre-warmed media that also contained 10 mg/ml of
cytochalasin B. A swinging bucket rotor was used and pre-heated
to 37
o
C so that the angle of enucleation would be
perpendicular to the centrifugal field (Figure 1).

Figure 1. Methods for generating cytoplasts by centrifugation and
cytoplast exposure to ECM. A. Coverslips seeded with cells are
inverted in a tube containing the actin depolymerizing drug,
cytochalasin B.
12
B. Removal of nuclei are during the centrifugation
procedure. C. Appearance of spread M619 cytoplasts adhering to
the coverslip after removal from the centrifugation tube and after
cytochalasin B was washed out by 5 exchanges of media. Note black
arrow points to a cell that was not enucleated. D. Coverslip of
fibrosarcoma cytoplasts showing 100% enucleation rate. Note the
flat cytoplast morphology. E. Phase image of the appearance of 4
MCF 10a cytoplasts. F. Fluorescence micrograph of cytoplasts illustrated
in E. Preparation is stained with anti-β-tubulin antibody. Note the
normal distribution of microtubules central position of microtubule
organizing center. G. Floating raft of polymerized Matrigel in a 35 mm
Falcon Petri dish. H. Method for inverting coverslips containing the

cytoplasts onto the floating ECM rafts after the cytoplasts had
recovered from the enucleation procedure. The thin black arrow shows
the edge of the round coverslip, and the thick black arrow shows the
edge of the floating Matrigel raft. Reference bars: C-D 30 µm; E-F 20
µm.
The coverslips were spun variably for up to 80 minutes, depending
on the cell line. Optimal times for maximal cytoplast yield were
arrived at empirically for each cell line and are summarized in the
following Table.
Table. Generation of Cytoplasts from Non-invasive and Invasive Cell
Lines and Their Comparative Morphogenic Responses

Cytoplasts were motile and contained mitochondria, Golgi,
endoplasmic reticulum, microtubules, and microtubule organizing
centers but lacked a nucleus.14
Using this process, a 60-80% yield from MCF10a breast
epithelial cells and a 30- 60% yield from MDA-MB321 breast
carcinoma cells was generated. In addition, the highly invasive
MDA- MB231 breast cancer cells easily detached from the
coverslips during the enucleation centrifugation step, which
often resulted in 100% of the cells coming off except for small
torn pieces of cytoplasm (cellular footprints) that were left still
attached to the coverslips. A yield of 95% cytoplasts was
generated from all other cell lines. Regardless of cell line,
cytoplasts could be maintained in culture for up to 2 days before
they disintegrated.
Exposure of cytoplasts to extracellular matrices
We employed t wo methods of plating cytoplasts matrices.
In the first method, cytoplasts were trypsinized after the
cytochalasin was completely washed out by at least five media
replacements. Cytoplasts were then re-plated on matrix. This
method, however, was not as rapid or efficient in maintaining
viable cytoplast numbers as simply inverting the coverslip
containing the cytoplasts onto a transparent floating ECM made
of Matrigel. After the coverslips were inverted onto floating
Matrigel, the inverted coverslips on their rafts of ECM were
placed back into the incubator for 24 hours to allow the
cytoplasts time to have their apical surfaces contact and
respond to their new Matrigel environment (see Figure 1 G,H).

Observation and data capture
All experiment sets were observed using a Leica microscope
system. All cultures were observed at Bright Field, 20x, 40x
and 63x by phase microscopy, with a Leica inverted
microscope, and captured using a time-lapse video camera
(Sony Model H- SV1), attached to a PC-type desktop computer
equipped with Pinnacle Studio 8 Image software (Pinnacle
Systems, Modesto, CA).
Results
Comparing the morphogenetic response of nucleated cells
and cytoplasts derived from poorly invasive cells, and highly
invasive cancer cells on floating Matrigel rafts
Human cell lines were selected to demonstrate morpho-
genetic responses to different matrix conditions (see Table).
Nucleated poorly invasive cells (OCM1a uveal melanoma cells
and MCF10a breast epithelial cells) formed small aggregates
within 24 - 48 hours. However, highly invasive cells (M619 and
MUM2B uveal melanoma cells, and MDA-MB231 breast
carcinoma cells) formed networks of cellular cords within 24 - 48
hours under identical culture conditions (Figure 2).3
In addition to
networks of cellular cords, the highly invasive melanoma cells
formed packets of cells surrounded by loops of ECM
(vasculogenic mimicry patterns5
) after exposure to the Matrigel
rafts.

Figure 2. Phase micrographs of pattern formation by nucleated poorly
invasive and highly invasive cells seeded at equal densities on floating
rafts of Matrigel after 2 days. A. MCF 10a cells derived from fibrocystic
disease of breast forming compact aggregates. B. Highly invasive
MB231 breast carcinoma cells forming networks of cellular cords. C.
Poorly invasive OCM 1a cells forming aggregates. D. Highly invasive
M619 melanoma cells forming networks of cellular cords. E. Highly
invasive M619 melanoma cells forming immature vasculogenic
mimicry patterns. Asterisks identify packets of tumor cells surrounded by
loops of matrix (arrow). Reference bar: 20 µm.
When cytoplasts that were derived from poorly invasive
OCM1a melanoma cells were placed on thick floating rafts of
Matrigel, these non-nucleated forms generated spatially-
confined non-interconnected spherical cell aggregates within 3 -
24 hours (Figure 3 A-D), as was typical of nucleated poorly
invasive or normal-non-invasive cells (compare with Figure 2).
By contrast, under identical conditions, non-nucleated cytoplasts
derived from highly invasive M619 melanoma cells formed
networks of cellular cords Figure 3 E-H) resembling those
generated by nucleated cells of the same origin (compare
with Figure 2).

Figure 3. Pattern formation by cytoplasts derived from poorly invasive
and highly invasive cells on floating rafts of Matrigel after 36 hours. A.
Phase image of cytoplasts derived from poorly invasive OCM1a
melanoma cells forming compact aggregates. B. Same field as A labeled
with ethidium bromide. Only one of the 10 aggregates contains a
nucleated cell (arrow). C. Phase image of cytoplasts derived from highly
invasive M619 melanoma cells forming networks of cytoplast cords.
D. Same field as C labeled with ethidium bromide. Note the presence
of DNA in one cytoplast (arrow). Reference bar: 40 µm.
Testing the relative contributions of the cytoplasm and
nucleus to simple ECM-directed pattern formation
The behavior of cytoplasts on floating rafts (see Figure 3)
suggests that nuclei are not required for these cell types to

initiate simple pattern formation. However, this observation does
not separate the relative influence of the nucleus versus the
cytoplasm in controlling ECM-directed pattern formation. It is
known that MCF10a cells distribute randomly and fail to form
aggregates on Type I collagen, but characteristically do form
compact aggregates on Matrigel.7
Therefore, we tested the
behavior of nucleated and enucleated MCF10a cells on matrix
conditions permissive and non-permissive of pattern formation.
When nucleated MCF10a cells were placed on Type I
collagen rafts for 24 - 48 hours, they consistently distributed
randomly (Figure 4 A-B) and failed to form compact
aggregates. Similarly, cytoplasts derived from MCF10a cells failed
to form compact aggregates and were distributed randomly on
the collagen rafts in the same time period (Figure 4 C-D). By
contrast, MCF10a nucleated cells formed compact aggregates
on Matrigel in 24-48 hours, and cytoplasts derived from these
cells also formed compact aggregates in identical culture
conditions (Figure 4 E-H).

Figure 4. Testing the relative contributions of ECM and nuclei to simple
pattern formation. A. Phase image of poorly invasive MCF 10a
nucleated cells from fibrocystic disease of the breast dispersed on
collagen Type I. B. Fluorescence micrograph of same field in A labeled
with ethidium bromide. C. Phase image of poorly invasive MCF 10a
enucleated cytoplasts dispersed on collagen Type I. D. Fluorescence
micrograph of same field in C labeled with ethidium bromide. Only one
of the cells contains DNA. E. Phase image of poorly invasive MCF 10a
nucleated cells forming compact aggregates on Matrigel. F. Fluorescence
micrograph of same field in E labeled with ethidium bromide. [Ethidium
bromide, which fluoresces when exposed to UV light, binds to nuclear
DNA; and, both demonstrates the presence of DNA in the nucleus and
causes the bound DNA to become brittle.] G. Phase image of
poorly invasive MCF 10a enucleated cytoplasts forming compact
aggregates on Matrigel. H. Fluorescence micrograph of same field in G
labeled with ethidium bromide. One of the 8 aggregates contains two
cells with DNA. Reference bar a,b = 60 µm; c,d = 30 µm; e-h = 60 µm.
Discussion
This study was designed to identify the relative contributions
of the cytoplasm and the nucleus in the generation of simple
ECM-induced morphogenetic patterns (cord formation and
spherical aggregates) by poorly invasive cells and highly
invasive cells. Members of our investigative team,3
and others,7
had shown previously that nucleated cells of varying invasive
potential - normal fibroblasts and endothelial cells, poorly
invasive cells breast epithelial cells, poorly invasive melanoma
cells, and highly invasive fibrosarcoma, melanoma, and breast
carcinoma - do not form spheroidal nests, cords, or networks
when plated on fibronectin adsorbed to glass coverslips.

However, when these nucleated cells are placed on thick
matrices, they consistently form patterns characteristic of their
degree of invasive behavior: poorly invasive cells (the poorly
invasive OCM1a melanoma cell line, and MCF10a breast
epithelial cells) form small aggregates, while invasive cells
(M619 and MUM2B melanoma cells, and MDA-MB231 breast
carcinoma cells) form networks of cellular cords. In addition, the
highly invasive melanoma cells form vasculogenic mimicry
patterns (patterned amalgams of extracellular matrix surround-
ing packets of tumor cells) if they are permitted to invade into the
thick matrix.3,5
When enucleated cytoplasts were generated from each of
these cell lines, they reformed a monolayer devoid of patterning
after cytochalasin B (used to facilitate enucleation) was washed
out (see Figure 1). Next, the cytoplasts were placed on floating
rafts of Matrigel. Within only 24 hours, the cytoplasts
reorganized to generate patterns specific to the phenotype of
the corresponding nucleated cells: cytoplasts derived from
poorly invasive cells (OCM1a melanoma cells, and MCF10a
breast epithelial cells) formed aggregates, but cytoplasts from
the highly invasive cells (M619 and MUM 2B melanoma cells,
and MDA-MB231 breast cancer cells) all formed networks of
cords. These observations suggest that the cytoplasm plays a
critical role in regulating simple ECM-driven pattern formation.
To evaluate the relative contribution of the nucleus and
cytoplasm to ECM-regulated simple pattern formation, we tested
MCF10a breast epithelial cells which distribute randomly on
Type I collagen rafts but form compact aggregates on either
Matrigel. MCF10a enucleated cytoplasts dispersed randomly
on Type I collagen and formed compact aggregates on
Matrigel, completely recapitulating the behavior of their

nucleated counterparts. Therefore, the cytoplasm alone is
capable of generating ECM-driven simple pattern formation in
these cells. The mechanisms that orchestrate simple pattern
formation reside in the cytoplasm and are controlled by the
extracellular matrix. The cytoplasm - in the absence of gene
transcription - reorganizes its structure to generate a
morphogenetic response to laminin exposure.
Although tumor cell networks of cellular cords were
induced by exposing cytoplasts derived from highly invasive
melanoma cells to laminin, these cytoplasts did not form
vasculogenic mimicry patterns. Nucleated cells require as long
as 2 weeks to generate vasculogenic mimicry patterns; the life
span of enucleated cytoplasts is only 2-3 days. Additionally, it is
likely that the formation of vasculogenic mimicry patterns requires
active transcription.5,15
The fundamental importance of gene expression is not at
issue here. However, once proteins are expressed and are
functionally in place in the cytoplasm and at the cell surface, the
extracellular matrix and cytoplasm appear capable of
determining simple morphogenetic responses to the ECM
independent of new transcription or the presence or a nucleus.
Thus, strategies designed to modify cell behavior - especially
in cancer therapeutics - may benefit from consideration of both
active transcriptional regulation of cell behavior and cytoplasmic
responses to the ECM that function independent of active
nuclear control.
Finally, in the course of seeking a highly reproducible and
easily obtained source of cytoplasts – for use in the study of cell
behavior in different ECM environments in the absence of any
influence from nuclear DNA (see Chapter I) – I proposed that
blood platelets might serve as a cytoplast model, given the

absence of any DNA. In the following chapter (Chapter III), I
describe my investigation of platelets as a cytoplast model,
and how that investigation led to some significant
discoveries about platelet behavior – both as regards
cytokinesis and differentially, in the presence of various
ECMPs.

References
1. Maniotis AJ, Valyi-Nagy K, Karavitis J, Moses J, Boddipali V,
Nunez R, Bissell MJ, Folberg R: Chromatin sensitivity to
Alu I endonuclease is regulated by extracellular matrix
and the cytoskeleton. Am J Pathol 2005, 166:1187-1203
2. Bissell MJ, Hall HG, and Parry G: How does the extracellular
matrix direct gene expression? J Theor Biol 1982, 99: 31-68
3. Maniotis A, Chen C, and Ingber D: Demonstration of
mechanical interconnections between integrins, cytoskeletal
filaments, and nuclear scaffolds that stabilize nuclear
structure. Proc Nat Acad Sci USA 1997, 94: 849-854
4. Maniotis AJ, Folberg R, Hess A, Seftor EA, Gardner LMG,
Pe'er J, Trent JM, Meltzer PS, and Hendrix MJC: Vascular
channel formation by human melanoma cells in vivo and in
vitro: vasculogenic mimicry. Am J Pathol 1999, 155: 739-752
5. Maniotis AJ, Chen X, Garcia C, DeChristopher PJ, Wu D, Pe'er
J, and Folberg R: Control of melanoma morphogenesis,
endothelial survival, and perfusion by extracellular matrix.
Lab Invest 2002, 82: 1031-1043
6. Gudjonsson T, Ronnov-Jessen L, Villadsen R, Rank F, Bissell
MJ, and Petersen OW: Normal and tumor-derived
myoepithelial cells differ in their ability to interact with
luminal breast epithelial cells for polarity and basement
membrane deposition. J Cell Sci 2002, 115: 39-50
7. Weaver VM, Lelievre S, Lakins JN, Chrenek MA, Jones JC,
Giancotti F, Werb Z, and Bissell MJ: beta4 integrin-dependent
formation of polarized three-dimensional architecture confers
resistance to apoptosis in normal and malignant mammary
epithelium. Cancer Cell 2002, 2: 205-216

8. Bissell MJ, Weaver VM, Lelievre SA, Wang F, Petersen OW,
and Schmeichel KL: Tissue structure, nuclear organization,
and gene expression in normal and malignant breast.
Cancer Res 1999, 59: 1757-1763s
9. Folkman J and Moscona A: Role of cell shape in growth
control. Nature 1978, 273: 345-349
10. Ingber DE and Folkman J: Mechanochemical switching
between growth and differentiation during fibroblast growth
factor-stimulated angiogenesis in vitro: role of extracellular
matrix. J Cell Biol 1989, 198: 317-330
11. Strohman RC, Bayne E, Spector D, Obinata T, Micou-
Eastwood J, and Maniotis A: Myogenesis and histogenesis
of skeletal muscle on flexible membranes in vitro. In Vitro
Cell Dev Biol 1990, 25: 201-208
12. Chen CS, Mrksich M, Huang S, Whitesides GM, and Ingber
DE: Geometric control of cell life and death. Science 1997,
276: 1425-1428
13. Carter SB: Effects of cytochalasins on mammalian cells.
Nature 1967, 213: 261- 264
14. Maniotis A and Schliwa M: Microsurgical removal of centro-
somes blocks cell reproduction and centriole generation in
BSC-1 cells. Cell 1991, 67: 495-504
15. Bittner M, Meltzer P, Chen Y, Jiang Y, Seftor E, Hendrix M,
Radmacher M, Simon R, Yakhini Z, Ben-Dor A, Dougherty
E, Wang E, Marincola F, Gooden C, Leuders J, Glatfelter
A, Pollock P, Carpten J, Gillanders E, Leja D, Dietrich
K, Beaudry C, Berens M, Alberts D, Sondak V, Hayward
N, and Trent J: Molecular classification of cutaneous
melanoma by gene expression profiling. Nature 2000,
406: 536-540

Chapter III
Platelet Cytokinesis and Lack of Thrombosis in Laminin-lined VM
Patterns
Given the exceptionally complex and often-unsuccessful
nature of the process whereby large quantities of cytoplasts
might be derived from normal cells, I suggested an exploration
of alternative means for generating cytoplasts. I experimented
with several variations on the processes as described in the
available literature and, eventually, it occurred to me that
perhaps the very process of generating large quantities of
cytoplasts might be the wrong path to take. In re-thinking
the desired objective of this line of investigation – that of
determining if ECMPs were playing a significant (or any) role
in cell morphogenesis (in the absence of nuclear DNA and
transcription) – I turned to the consideration of one cell type
normally found in the human body that was always absent
any nucleus or DNA: platelets. I reasoned further that since
platelets never have nuclei, or undergo transcription, they might
be a suitable model for cytoplast behavior in different ECM
environments. It had been remarked on by members of our
investigative team – and further, there were no reports by
others in the literature – that platelets also never undergo
cytokinesis (or simple cell division), which might mean that
platelets were not such an appropriate model. Thus it was that I
set about more closely observing platelets in different
environments, to verify this. In fact, within a very short time,
not only did I observe cytokinesis, but I was able to capture
this process occurring. Furthermore, I (and a colleague) was
able to observe and capture platelet cytokinesis in multiple ECM

environments.
During the course of this experimentation, I also
discovered an important link between the behavior of platelets
in various ECM environments and the vasculogenic mimicry
patterns (alternatively called “extravascular microperfusion
channels” or “fluid- conducting meshwork”). Blood has been
found in solid tumors, both in gross-sectioning of specimens
and in microscopic histological examination, which has been
used by many researchers in supporting the idea of tumor
vasculogensis. However, there are few endothelial cell-lined
“blood vessels” found in solid tumors, most are leaky and
fragile – more likely remnants of once normal vessels and no
longer intact enough to be a reliable conduit for nutrients within
the solid tumor. Members of our team had already shown that
the VM patterns are fully capable of conveying nutrient-filled
fluids throughout the tumor. What the platelet work revealed is
that, contrary to initial expectations, these simple cytoplasts
can and do make their way through the fluid- conducting
meshwork of solid tumors.
Vasculogenic mimicry is a process whereby aggressive
tumors form extra-vascular perfusion channels made of
laminin, fibronectin, Type IV collagen, and other extracellular
matrix components that circumscribe nests of typically
epithelioid tumor cells. These channels connect to blood
vessels, and are known collectively as vasculogenic mimicry
patterns (VM patterns), the fluid conducting meshwork, or
more recently, extravascular matrix patterns (ECMPs). Despite
the fact that several groups have demonstrated these
channels transport plasma – and constitute a surface area
approximately 20 times that of intra-tumoral blood vessels or
capillaries within highly invasive tumors – it was previously

unknown how adhesion and activation of platelets and
(ultimately) thrombosis is prevented from occurring within these
extravascular channels.
Normally, platelet activation in blood vessels can be
induced by Collagen, von Willebrand factor, Thromboxane A2
(TxA2), Serotonin, Human Neutrophil Elastase, P- selectin, and
convulxin, (a purified protein from snake venom).1
Therefore,
I (and a colleague) tested how non-activated platelets behave
when they are incubated with the ECM components known to
comprise the VM patterns. When incubated on laminin I-rich
Matrigel (laminin I is a principal component of the VM patterns)
platelets did not adhere, nor did they become activated. The
same result was observed by incubating platelets on pure
Type I laminin.
By contrast, parallel cultures of platelets incubated on Type
I and Type IV collagens, fibrinogen plus thrombin, or on plastic
bottom Petri dishes adhered, remodeled ECM, and became
activated. Either with or without adherence, platelets were
observed to elongate, exhibit cytoplasmic movements in a
polarized fashion, and then undergo complete cytokinesis into
two distinct and completely separated pieces of cytoplasm.
Cytokinesis was observed in non-adherent cytoplasts
incubated on laminin I, which resisted their attachment, and on
substrata that promoted attachment and platelet flattening.
These data strongly suggest that laminin I, as a principal
component of the VM patterns, helps obviate platelet
adherence and subsequent coagulation in the extravascular
channels, and that platelets exhibit the ability to divide in a
polarized fashion.
For several decades, there has been much theoretical
promise of anti-angiogenic therapy for cancer. In recent years,

however, results from human clinical trials employing anti-
neoangiogenic agents have been less than encouraging.2
Members of our investigative team suggested that lack of
efficacy of anti-angiogenic strategies may in part be the result
of the ability of solid malignant tumors to destroy normal
vessels and form their own extravascular perfusion system;
they were the first to characterize this, and termed it
“vasculogenic mimicry.”3
Other laboratories have confirmed the
presence and perfusion function of these extravascular
channels in malignant solid tumors,4-11
and have termed them
“vasculogenic mimicry channels”12-15
or “the fluid-conducting
meshwork.”16
Despite the presence and verification of these perfused,
extravascular, plasma conducting matrix channels, an important
question remained as to how coagulation is obviated, once
plasma leaks from the coagulation-suppressed environment
that exists within the lumens of normal blood vessels and
capillaries. In this context, it has been shown that molecules
such as Factor XIII and IX, as well as tissue factor produced by
malignant tumor cells, helps keep coagulation in check in the
extravascular spaces.17,18
Alternatively, plasminogen activators
and deactivators also have been found in malignant tumors
that may also help antagonize extravascular coagulation within
tumors.18
After spending significant effort characterizing the molecules,
which comprise the extravascular matrix patterns in malignant
melanomas,19,20
I tested the ability of these molecules to
cause or prevent platelet adherence, which is thought to be a
first step in the normal coagulation reaction.
Herein, new evidence is introduced that the extracellular
matrix molecule, laminin I – which is not present in normal

vessels21
– may prevent the adhesion and subsequent activation
of platelets, and consequently also plays an important role
as a principal component of the extracellular matrix channels
within malignant solid tumors. Moreover, under these
experimental conditions, it was observed that platelets are
capable of exhibiting a process best described as
cytokinesis, despite their lack of chromatin, centrosomes,
mitotic spindle, and small cytoplasmic mass.
Taken together, these data suggest that the composition of
extravascular matrix channels is a critical factor in the genesis
of malignant tumors because blood coagulation is prevented
when an abundance of laminin I is present. These data also
provide new insights as to the importance of cytoplasm in the
process of cell division, in the absence of cellular organelles
normally attributed as key players for the generation of new
daughter cells.
Materials and Methods
Platelet acquisition and preparation
Freshly drawn, whole blood was collected in vacutainer
tubes (contribution of Dr. Amelia Bartholomew, Department of
Surgery at the University of Illinois, at Chicago IL). Platelets
were separated from other blood components either by low
speed (<1000 rpm) centrifugation, for one minute, or by
allowing to stand at room temperature (25
o
C) for two hours. In
either case, platelets and serum remained near the tops of the
tubes, while other blood components settled to the bottom.
All platelets utilized were suspended in serum (derived from
the whole blood) both when stored and during experimentation.

Three-Dimensional Matrix-containing Cultures
3D cultures were established by growing cells on matrix
components that were poured onto plastic tissue culture
dishes to a depth of about 0.2 mm followed by
polymerization for 1 hour at 37°C. Platelets were seeded at
saturating densities (50 million cells/60 mm dish) on the
polymerized 3D gel coatings.
Observation and data capture
All experiment sets were allowed to run for four days (96
hours) and observed using a Leica microscope system. All
cultures were observed at 20x, 40x and 63x by phase
microscopy, with a Leica inverted microscope, and captured
using a time-lapse video camera (Sony Model H-SV1), attached
to a PC-type desktop computer equipped with Pinnacle Studio 8
Image software (Pinnacle Systems, Modesto, CA).
Results
Platelet adhesion/activation and ECM substrates
As previously stated, there are many known platelet
activators.1
In my study, I focused upon the ECM proteins –
collagen IV, fibrinogen plus thrombin and Type I laminin – for
these have been associated either with the vessels seen in solid
tumors or the extravascular matrix channels within invasive
tumors.
I coated the bottoms of plastic Petri dishes with one of each
of the ECM protein substrates, with uncoated plastic dishes
as our control. In thirty repetitions of this experiment, I seeded
either serum containing only platelets, or serum containing

platelets and very low volumes of other blood components,
in the variously pre-treated (or untreated) dishes: four
controls, and four separate sets of collagen IV, fibrinogen
(with thrombin) and laminin I (Figure 1). When platelets (with or
without other blood components) were seeded on bare plastic,
the platelets both adhered to the plastic surface and flattened
out, indicating activation of the platelets (see Figure 1a).
Those platelets (with or without other blood components -
identified as fragments of RBCs and erythrocytes) seeded on
collagen, also completely adhered and spread out, as well as
shallowly embedding themselves into the collagen, indicating
activation (see Figure 1b). Further, these platelets were observed
to create traction lines in the substrate surface, due to
contraction of the collagen.
Platelets seeded (with or without other blood components)
on a thrombin-fibrinogen substrate also adhered and caused
contraction lines in the ECM surface, but the platelets did not
flatten out (see Figure 1c). Finally, the platelets seeded (with
or without other blood components) on laminin neither
adhered to the laminin nor flattened out. Rather, these
platelets continued to float in the serum, above the laminin
surface (see Figure 1d).
a b c d
Plastic Collagen IV Fibrin Laminin
(Thrombin-Fibrinogen)

Figure 1. Platelets behave differently when in contact with various
ECM protein substrata. Simian platelets seeded onto plastic
bottom Falcon Petri dishes (a), Type IV collagen (b), fibrin
(thrombin- fibrinogen) (c), and laminin (d) for 4 days. Note lines
of stress on the surface of collagen (asterisk-like lines) and
thrombin-fibrinogen, suggesting tension induced by the platelets
on these matrices. By contrast, note how the platelets in the
laminin-containing culture do not adhere (or activate).
Platelet Division
It is widely accepted that platelets are derived from the
fragmentation of megakaryocytes,1
whether by fragmentation of
long megakaryocyte processes forming on a solid substrate in
vitro, by fragmentation of long megakaryocyte pseudopods, so-
called proplatelets, protruding into the sinusoidal lumen in vivo,
or by more global fragmentation of megakaryocyte cytoplasm
into individual platelets as observed when the cells are kept
in suspension. Further, in recent years, there is a growing
body of evidence that a dominant site of platelet production is
within the pulmonary capillary bed.22
However, there are no
studies reporting the incidence of platelet division, after
complete fragmentation of the megakaryocyte.
In all experiments, save those performed using a thrombin/
fibrinogen substrate, I (and, later, several colleagues) observed
platelet division (Figure 2). On bare plastic, platelets first
adhered and flattened out (within two hours of seeding) into
several different shapes - some attaining a dog biscuit-like
appearance, others more triangular or starfish-like (see Figure
2a,b). Many of the dog biscuit-form and starfish-form platelets
then began shifting their cytoplasm back and forth from one end

to the other (in the dog biscuit-form) and from the center
to the arm ends (in the starfish-form). Over the course of
several minutes, many of these two platelet forms began to
split up and polarize their cytoplasm, concurrent with a
narrowing of the platelets middle. Ultimately, the “parent”
platelets divided into two (dog biscuit-form) or three (starfish-
form) “daughter” cells (see Figure 1a, Figure 2).
a b c d
Figure 2. Sequence of cytokinesis observed with platelet on the upper
right-hand corner (white arrow) when seeded on plastic. Zero time point
(17:07:14) (a); 1.6 minutes (17:08:54) (b); 2.5 minutes (c); and 4.5 minutes
division occurs and complete cytokinesis (d). Note in (a), white arrowheads
indicate platelets at different stages of cytokinesis.
When observed on collagen, platelets tended to divide into
three daughter cells, though two-daughter divisions were also
observed. While no complete divisions were observed when
platelets were adhered on thrombin/fibrinogen substrate, I
cannot say, definitively, that such divisions do not occur (see
Figure 1). Finally, platelets floating in serum, contiguous with a
laminin substrate, do not flatten out when dividing; rather, they
elongate, giving the appearance of two or three beads attached
to one another by a fine thread (see Figure 1), which ultimately
“breaks,” producing two to three daughter platelets (not shown).

Discussion
As demonstrated, previously, by members of this team of
investigators – and others – laminin-rich, extracellular matrix
constitutes the most abundant extracellular matrix component
that comprises the extravascular matrix channels. In
controlled experiments, using different matrix platforms
(plastic, collagen, laminin, fibronectin with thrombin), platelets
did not adhere, or become activated, on laminin I and
laminin-rich matrices, such as Matrigel (60% laminin-
containing). Thus it appears that laminin may resist platelet
adherence, which is a principal step in the coagulation cascade.
Unexpectedly, we also have found that the cytoplasm of
platelets can exhibit the typical characteristics of cytokinesis/
simple (non-mitotic) cell division, under certain experimental
conditions.
It is widely appreciated that special conditions exist within
the lumina of normal blood vessels and capillaries, and these
conditions resist coagulation. When the specialized surfaces of
normal blood vessels or capillaries are breeched or damaged,
the coagulation machinery becomes activated. The net result of
this complex activation process involves the adherence and
activation of plasma-borne platelets, followed by the
generation of a clot, and elaboration of repair mechanisms
that insure the prevention of plasma leakage into the
extravascular spaces.
The fact that particularly invasive tumors, such as
melanomas, invade and destroy tissues as resilient as skin or
bone, make it unlikely that structures as delicate as capillaries
formed by neo-angiogenesis can persist, or even form, in the
context of a highly invasive tumor, or its destructive path.
4,11,23

The discovery of vasculogenic mimicry provided an alternative
type of perfusion system erected by extracellular matrix
surrounding nests of what are believed to be invasive,
epithelioid, tumor cells, and theoretically, provided perfusion
into the tissue destructive environment of a tumor.
Subsequent analysis of volumes of blood vessels versus
vasculogenic mimicry perfusion spaces in 3-dimentional tumor
reconstructions revealed a 20/1 ratio of volume area in which
VM patterns serve as the predominant perfusion route.24-26
However, while making perfusion into such an environment
possible, the formation, existence, and perfusion of
extravascular matrix channels constituted a paradox within
the realm of coagulation biology. The results presented in this
study, therefore, provide a new mechanism whereby these
extravascular perfusion channels can function without sudden
and massive coagulation resulting, due to their laminin I-rich
composition, and abundant distribution compared to
endothelial cell-lined vessels that may transiently survive
within the malignant tumor.
These results suggest, in addition, that the viable biogenesis
of malignant tumors must proceed according to a rigid program
in which laminin I is synthesized and deposited by malignant
tumor cells,27
in order to provide a non-coagulated stream of
nutrients and plasma to enter the tumor's extravascular matrix
channels, as the tumor grows beyond a passively perfusable
size of a few millimeters.
Inadvertently, I had discovered that the cytoplasm of
platelets possess an autonomy to the extent that polarity
leading to simple cell division is possible, in the absence of
either chromatin or centrosomes, or indeed, in the absence
of a mitotic spindle (as far as has been previously recorded).

Thus, these studies have revealed that the self-assembly
of malignant tumor tissues depends upon the production and
self-association of a laminin I-rich matrix, and at the same time,
normal sub-cellular constituents such as platelets retain an
ability to generate two-ness,28
resulting in the self-disassembly
of cytoplasm itself into equivalent entities.
Acknowledgements
Platelets were kindly provided by Amelia Bartholomew,
M.D. – Associate Professor of Surgery, Department of
Surgery, University of Illinois (Chicago) School of Medicine.

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8

Chapter IV: Conclusion
Highlights of Observations, Discoveries, Outcomes and
Implications
What did emerge as a model of uveal melanoma,
moreover as a model for "highly invasive" (or "aggressive")
tumors, in general? What are some key facets of this
model? Is this limited only to the tumors themselves, or is
there a larger context, which includes micro-environmental
factors? Already reported, in publications to which I
contributed and/or in which I was the initial observer, in the
following, which led up to the series of experiments undertaken
in this book: 1) The nuclear DNA in tumor cells is more
sequestered than the DNA in normal and poorly invasive cells,
regardless of cell type. 2) This is seen to be the case whether
they are cultured in 2D or 3D environments. 3) 3D culture
environments confer more stability to nuclear DNA, regardless of
cell type, but the more invasive or aggressive the cell type, the
more a 3D environment stabilizes (sequesters) the cells’ DNA.
Also, the discovery that there is differential sequestration in
whole cell assays, between normal and invasive tumor cells,
independent of cell cycle. 4) Reverted and transformed cells
behave in a similar manner to their non-reverted and non-
transformed counterparts. 5) Cells behave differently in different
microenvironments, but highly invasive tumor cells, regard-
less of origin, behave the same in the same micro-
environments. 6) It does not appear likely that solid tumors
engage in angiogenesis, but they do have a micro-perfusion
system, a fluid-conducting meshwork of back- to- back looping
patterns (vasculogenic mimicry patterns), and that system is

comprised of certain extracellular matrix proteins
(polysaccharides), including laminin, collagen and fibronectin.
7) Highly invasive tumor cells are undifferentiated, though not
in the same manner as stem cells. Stem cells can, theoretically,
be programmed / manipulated to develop into most any tissue
in the body; whereas, when tumor cells revert, they
differentiate into the original cell type from which they
originated. When grown with Laminin or Matrigel, as the 3D
substrate, highly invasive tumor cell cultures form these fluid-
conduction meshwork patterns: which was believed to be a
hallmark of certain death in the host. However, the melanoma
tumor cells in direct contact with laminin or Matrigel will revert
to a spindle A (indolent) morphology, while the cells not in
contact with the ECM (extracellular matrix, in this case
laminin I) remain epitheliod (highly aggressive morphology). 8)
By bioengineering small tumors (in vitro), discovery that there
are at least two cell morphologies evident in so-called
vasculogenic mimicry patterns, and that melanoma tumor cells in
contact with the ECM (laminin) may revert to a spindle A
(indolent) morphology, and this is observed in dense culture
as well in cultures of small numbers of cells. 9) The reverted
cells in these extracellular matrix patterns (fluid-conducting
meshwork) resist toxic effects of polyamine analogs, while the
highly invasive epitheliod cells are killed. 10) The fluid-
conducting meshwork also appears to act as a biofilm,
perhaps protecting the most aggressive cells from toxic
effects of therapeutic drugs; this knowledge may be useful in the
development of drugs that are able to break down the biofilm,
or sidestep it. 11) There is a biomechanical relationship
between the ECM, the cytoskeleton and the genome, and
manipulation of the microenvironment has a measurable effect

on both exposure and sequestration of nuclear DNA.
Within the scope of the experimentation that forms the body
of this dissertation, I contributed to the observations and
discoveries that 1) the ECM and cytoskeleton appear to play
a significant role in simple changes in cell morphology and,
even in the absence of the nucleus, can apparently mediate
cell shape; 2) platelets do undergo simple division
(cytokinesis), and though this had never before been
documented, I captured the process both with video and still
camera; 3) platelets do not activate or clot in the presence of a
laminin I-rich environment, leading to an understanding of
how platelets could travel through the fluid-conducting meshwork,
without causing thrombosis.
Potential Future Investigation
Clearly, manufactured cytoplasts and platelets are not
identical, merely because they both lack a nucleus. Platelets
have less cytoplasm and lack the more involved cytoplasmic
machinery of normal cells that have been enucleated. Both are
fragile units, which survive only days in their final forms.
However, platelets – with their far simpler cytoplasmic
machinery – have been shown to undergo simple division,
while created cytoplasts have not been observed to do so. Are
there special conditions under which these cytoplasts might
also divide one time before dying? Is there an environment
wherein such division may occur, or is the enucleated cell
simply too complex in its other genome-dependent processes
to undergo simple cell division?
Platelet cytokinesis was observed and documented in
photographs and video, with platelets adhered to several

different ECM substrates, and platelets floating above a laminin
substrate. Because there is no apparent platelet adherence or
activation in the presence of laminin – which is a principle
component of the VM patterns seen in solid tumors – one line
of investigation, of some interest, is the determination of whether
or not there is something inherently different about the
cytokinesis platelets undergo when adhered to different ECM
substrates and that which occurs when not adhered but in the
presence of laminin I (as within these VM patterns). In a fairly
exhaustive search of the literature, I was not – nor others on my
research team – able to locate any descriptions of platelet
division (cytokinesis). Perhaps this has either not previously
been observed in normal human vasculature or it has,
inexplicably, gone unreported.
If free-floating, platelet cytokinesis is either rare, or non-
existent, in the human circulatory system, what is it about the
microenvironment of a bioengineered, metastatic, solid tumor’s
fluid-conducting meshwork that permits and/or promotes
platelet cytokinesis? Further, though perhaps not directly
related to metastatic tumors or tumor models, is there
platelet cytokinesis occurring during the clotting process in
normal vessels, and does such cytokinesis aid in clotting? From
the perspective of the cell-tissue engineer, what role does
platelet attachment to certain ECM substratum versus free-
floating in proximity to a laminin I-rich environment, have? In
the attached and activated platelet, is cytokinesis strictly a
function of biomechanical forces mediated by ECM attachment –
since there is no transcription to mediate such activity – and in
the free-floating platelets, is there a different type of purely
biomechanical force at play? Finally, can these biomechanical
forces be independently identified and quantified?

It is accepted fact that nucleated cells in the human body
require the presence of microtubules to undergo mitosis –
nuclear division plus cytokinesis – and yet platelets are not
nucleated and have limited cytoplasm. While this current study
did not pursue the identification of microtubule involvement in
platelet cytokinesis, it would seem likely that there is at least
one microtubule present and taking an active role in the
process. Further observation and experimentation could help to
identify the existence and action of one or more microtubules in
platelet division.
If platelets do not cause thrombosis within the tumor what, if
any, is their role as they pass through the tumor’s fluid-
conducting meshwork; or, is the presence of platelets in solid
tumors merely accidental: stray platelets find their way into the
tumor from leaky vessels at the normal tissue-tumor
boundary? Corollary to this, and again from the perspective of
the bioengineer, at what maximum density can platelets perfuse
these VM channels? Where a few stray platelets might pass,
unhindered through these perfusable micro-channels, would
they tolerate a constant flow of platelets – comparable to what
is seen in small, normal capillaries – or would such a flow
ultimately result in clotting, even though laminin I is present?
Plainly, further study is warranted, and anticipated. Of
course, there are other bioengineering questions yet to be
resolved, even in the further development and extension of the
tensegrity model to cell behavior, both in normal tissues and in
cancers. Demonstration of the various biomechanical forces at
work in mediating cell shape and malignancy, gene regulation
et al., is becoming widely accepted. However, there is, yet to
come, the quantification of such forces, and what role selective
interference with these forces may play in the treatment of

disease, especially cancer.
Cytoplast and Platelet Findings in the Context of the
Bioengineered Tumor Model
Documentation of new observations regarding the
behavior of cytoplasts, and platelets, may well advance the
field of Cell Biology, and help scientists and others better
appreciate the roles of these cells in the larger context of
modeling normal human tissues, organs and the circulatory
system. Clearly though, that platelets have been so recently
shown to undergo cytokinesis/simple division is – in the
multi-century exploration of the human body – as important
for reminding the community of Life Sciences and Medical
researchers there is still an abundance of basic science research
yet to be done as it is merely to document a novel behavior. In
this instance, it serves as well to point to the need for remaining
open-minded and ever questioning of the dogmas of Science
and Medicine, which often interfere with our objectivity, as
research scientists.
However, the study of cytoplasts and platelets, as
presented in this dissertation, is decidedly within the context of a
bioengineered, 3D, in vitro tumor model. As such the individual
observations and discoveries I – and other investigators with
whom I worked – have made about cytoplasts and platelets
are significant for their contribution in creating a viable in vitro
tumor model. Such a tumor model is not only useful as a basic
research tool; the potential diagnostic and therapeutic value
of this model is vast, indeed.
Knowing that the ECM plays a significant role in cell
morphogenesis may mean that affecting changes in the local

ECM environment of the invasive tumor can interfere selectively
with the highly aggressive cells and leave surrounding normal
tissues unharmed. Using the cellular tensegrity model
(mechanogenomics) as a guide, it may be possible to
selectively interfere with one or another structure of the
cytoskeleton (of highly invasive tumor cells) and cause
complete shutdown (total down-regulation) of these cells.
Appreciating that the DNA of highly invasive tumor cells is
more sequestered than that of poorly invasive or normal cells, in
part at least to local microenvironment (e.g. ECM proteins) can
help direct the nature of therapeutics developed as well as more
precisely identifying appropriate target sites.
It is now widely accepted in the literature that these VM
patterns are capable of supplying the internal cells of solid
tumors with the nutrients required for sustaining them, as
they are contiguous (if not continuous) with the blood vessels
in surrounding normal tissues. In fact, it makes sense to pursue
delivery of therapeutics through these micro-channels. Such a
therapeutic agent would need to be engineered so as to
permeate the “biofilm” barrier created by the laminin-lined VM
patterns.
As regards the role of platelets in the fluid-conducting
meshwork of invasive solid tumors, there is yet much to be
learned about the level of platelet perfusion through these
laminin-lined “micro-channels.” Another tactic – knowing that
platelets do not adhere to the surface of the laminin, therefore
do not cause thrombosis within the tumor – may be to
introduce a chemical compound into the local tumor
environment that caused the platelets within the tumor to
adhere and clot. I have been unable to find any literature
suggesting such an approach – though anti-angiogenesis tactics

abound – as to interfere with the perfusion of the fluid-
conducting meshwork. If the fluid-conducting meshwork – and
not any blood vessels within the tumor – is, indeed, the primary
means by which the tumor is “fed,” then blocking off these
micro-channels may cause the internal cells of the tumor to die.
I propose that, in addition to exploring how platelets may be
used in this manner, it is worth considering the bioengineering
of a platelet-like, platelet-sized nanosphere. These nano-
spheres would want to have the gross shape, surface
characteristics and plasticity of platelets; I suggest that as
platelet shape, texture, ability to deform and be compressed
are critical (aside from their non-adherence to the laminin I
substrata) aspects of their ability to pass through the
meshwork, thus must a bioengineered nanosphere share
these same characteristics and abilities. The nanospheres
would also want to be coated with something that selectively
adheres to laminin I. For instance, the sulfated HNK-1
carbohydrate (present on glycolipids and on several neural
recognition molecules) has been shown to mediate adhesion
to laminin I. Perhaps coating these nanospheres with HNK-1
or another, similar substance - adsorbed to the nanospheres in
a hydrogel or other co-polymer that time-releases the HNK-1
only in the presence of laminin I - would successfully cause
thrombosis in the solid tumors.
Thus, having created a viable 3D in vitro tumor model will
not only facilitate the further exploration of tumor/cell biology, it
can now be appreciated that this model will also be highly
useful in the testing of such treatment tactics as the
manipulation of tumor microenvironment, targeting key
cytoskeletal components of highly invasive tumor cells and
most efficacious sites within the tumor and agents specially-

engineered to penetrate the tumors’ biofilm barrier. Finally, this
in vitro model could prove an ideal means of experimenting
with the use of platelets or platelet-like nanospheres to
interfere with the perfusion of serum and other fluids through the
tumor.

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