August 2, 2004
Glue for sealing the coverslip and drilled dish: Norland Optical Adhesive 71 (Lot 145)
Norland Products Inc., Cranbury, N.J. 08512
PP1 and PP2: Src family kinase inhibitors
U0126 and PD98059: ERK inhibitors
Cytochalasin D, Latrunculin B: Actin inhibitors
M7: MLCK inhibitor
BDM: myosin ATPase activity inhibitor
Staurosporine: PKC inhibitor
Cycloheximide: protein synthesis inhibitor
Actinomycin D: translation inhibitor
SB203580: p38 inhibitor
BIM: PKC inhibitor (bisindolylmaleimide I)
LY294002: PI3K inhibitor
Y27632: ROCK inhibitor
Nocodazole and taxol: microtubule inhibitors
1. Cancer epigenetics
2. Cell migration and tumor metastasis
1) Path generating or guidance? The role of ECM degradation. The
multiadhesive matrix protein: A laminin for basement membrane and
fibronectins for ECM of nonepithelial tissues.
2) Amoeboid cell movement and leukocytes extravasation? Constriction ring?
3) Track of recycle of actin?
4) Membrane ruffles, which are sites of active actin remodeling in motile cells?
5) Epithelial cells migrate in an opposite way, move from tightly adjacent
epithelial layer to soft tissue???
6) How the overlapped MT in axon formed?
7) Contact inhibition?
8) Fibroblast cell movement: a signal is elicited by tail retraction which
stimulates the leading edge to propagates, whereas in Dictyostelium (and
probably other fast-moving cells), a signal is generated by an active front
which causes other regions to retract? Refer to MBC 2001, Steven Munevar.
9) What determines the tumor cell invasion? What determines the invasive tumor
cells metastasis? The tumor cell motility is necessary for metastasis or not?
Metastases are tumor implants discontinuous with the primary tumor. Glioma
and basal cell carcinoma of the skin, both are highly invasive forms of
neoplasia, but they rarely metastasize. It is evident then that the properties of
invasion and metastasis are separable. Approximately 30% of newly
diagnosed patients with solid tumors present with metastases.
10) Focal adhesions: in vivo or in vitro? Annual Review Cell. Dev. 1995
Integrin belongs to CAMs (cell-adhesion molecules: the cadherins, Ig-suprefamily
CAMS, Integrins and Selectins)
Major adhesive interactions that bind cells to each other and to the extracellular
a. CAMs (Calcium switch assay and pan-cadherin antibody first, then E-
cad or N-cad or others)
b. Cell-matrix adhesions
c. Tight junction
d. Gap junction
e. Adherens junction
Beyond Matrigel: the method to dynamically monitor cell migration instead of
counting the cell number after fixation. Why not Matrigel? It is used for invasion
assay. Matrigel is a solubilized basement membrane preparation extracted from
the Engelbreth-Holm-Swarm mouse sarcoma, a tumor rich extracellular matrix
proteins. It mainly comprises laminin, collagen IV, heparan sulfate
proteoglycans, entactin, and nidogen, all components of basement membranes.
How about migration after invasion? Maybe not that complicated at first – Dr.
Epithelial cells or fibroblast? Sheet-like layer of epithelial tissue called an
epithelium (wound healing?) versus individual cell migration. Mostly, tumor
comes out as single cells during metastasis (Mercurio and Zetter).
Tug of war or communication to release cell tail for cell migration? How to
design the experiment? Block the calcium transportation but not interfere the
frontal calcium metabolism/influx?
Think carefully before move to next step. Don’t rush but make a good judge
which one is the critical way to go – Got movies from Steven Hanks (March 4,
2004) and watch for more than ten times: wt Cas cells move more active, they
search for way and move constantly. Vector cells move in a lazy way and looks
like being pushed out and move steadily. That’s the reason although vector cells
move more straight than wt cells but still move slower than wt. Try to look at
some certain cells instead of looking at the whole picture in the end to point the
In situ probe for detecting cancer (cells): microscopy- EM, IHC, anything new?
1. Protrusion: extension of the leading edge toward a motogenic stimulus– GTPase
Rac is a key regulator of protrusive activity.
Microtubule growth activates Rac1 to promote lamellipodial protrusion in
fibroblasts (Nature cell biology 1999)
Nocodazole: microtubule destabilizer
Taxol: microtubule stabilizer
New paper in JCS 2004, 117: 837-848 about lamellipodium and filopodium
formation and SCAR
Adhesion to matrix contacts (adhesion of the new protrusion to an extracellular
matrix leading to extension of a leading edge lamellipodium; formation, disassembly
and/or maturation). (Science review by A. F. Horwitz) Smilenov et al 1999 demonstrate
that focal adhesions are highly motile in stationary fibroblasts yet stationary in migrating
fibroblasts, suggesting the existence of a molecular clutch that couples traction and
contractile forces. Most adherent cells, although spread, are under tension. It is tension
that causes cells to round up when integrin-directed adhesions are perturbed. Migration is
initiated by polymerization of an actin network at the cell’s leading edge and is
maintained by contraction of myosin. The formation of new focal complexes is controlled
by the Rac signaling molecule, and their growth is determined by a Rho-dependent
process. Contractile forces behind the leading edge drive movement of the cell body. The
focal adhesion remains in place as the cell moves over them, confirming that these
adhesions serve as points of traction. In nonmotile cells, however, proved surprising. The
adhesion complexes move toward the center of the cell at a rate that varies with the
tension. … Thus, a key element in cell migration is the reciprocal regulation of Rac,
which appears active at cell’s leading edge where new protrusions and adhesions are
forming, and Rho, which appears to generate tension and stabilize adhesions more
centrally throughout the cell.
2. Contraction of the cytoplasm (strengthening of adhesions leading to generation of
contractile forces that combine to pull the cell body forward)
3. Release from contact sites (weakening and release of adhesions to permit
retraction of the cell rear; formation, disassembly and/or maturation)
4. Recycling the membrane receptors from the rear to the front of the cell
The rate-limiting step of metastasis formation is still unknown.
Metastasis-associated genes? Hox? Few? How metastasis is initiated and regulated?
1. Attachment of the tumor cell to the basement membrane (Down-regulation of E-
cadherin/catenin complex is a common feature in invasive carcinoma cells)
2. Degradation of the ECM/basement membranes / invasion?
3. Locomotion into the tissue (intravasation for some tumors) From the textbook of
Pathology, this step actually is much more complicated than just one process. It
includes intravasation, interaction with host lymphoid cells, tumor cell embolus
(within the circulation, tumor cells tend to aggregate in clumps, homotypic
adhesions among tumor cells as well as heterotypic adhesion between tumor
cells and blood cells, particularly platelets), adhesion to the basement membrane
of the blood, extravasation, metastatic deposit, angiogenesis again, and finally
metastatic tumor growth. Bruce R. Zetter: Intravasation and extravasation are
very easy to process. Migration as single cells? (Mercurio says so too) Migration
after extravasation?Yes, as single cells before initiation again and form
EMT (epithelial-mesenchymal transition): At metastases, migratory
fibroblasts sometimes revert to an epithelial phenotype, by a process involving
regulation of the E-cadherin--catenin complex.
Amoeboid cells are loosely attached to the ECM by focal complexes,
allowing rapid cell migration
Cells partially retain polarity still have a tail that contains retraction fibres and
a front that contains stress fibers and can form focal adhesion
Nat Med. 1997 October Quantification of tumor cell invasion using confocal
laser scan microscopy (can distinguish cell migration from matrix invasion)
Nature Reviews | Cancer, 2003 December
Intravital Imaging of Cell Movement in Tumors
Carcinoma cells in the primary tumor can move up to 10 times the velocity of
similar cells in vitro (Cell migration behavior differs between carcinoma cells
and normal cells/ metastatic and non-metastatic tumor cells: Protrusion,
Adhesion, Contraction and Release of adhesion, which one is the most
important stage) – There might be no common difference, cell dependent? Dr.
How about cell motility on soft substrates?
The highest velocities are observed for carcinoma cells in metastasis tumors
that are moving along linear paths in association with extracellular-matrix
(ECM) fibers. Same question as above: How about cell motility on soft
Carcinoma cells in non-metastatic tumors are fragmented during
intravasation as they squeeze through small pores in the basement
membrane/endothelium to gain access to the blood space???? Different from
Dr. Zetter’s point?
Treatment of cancer:
1. Minimize the growth of existing tumors
2. Limit their spread to new sites
Above 3 steps, which one is the most important? Invasion? Is there any
common difference or just cell dependent? Block of attachment, degradation
of the ECM and/or tumor cell migration/locomotion into the tissue
Methods in molecular medicine: Metastasis research protocols vol. II – Analysis of cell
behavior in vitro and in vivo
2. Immunoagnetic Cell Separation
3. Genetic Modification of Cell lines to enhance their metastatic capability
4. Cell aggregation assays (This assays have been set up to test the functionality of
the E-cadherin/catenin complex in epithelioid tumor cells. Functional integrity of
the complex is a prerequisite for cell-cell adhesion between epithelial cells, and
measuring cell aggregation in vitro has thus become another elegant tool to study
differences between invasive and non-invasive cell types. When did anoikis assay
in Kyoto, I saw this aggregation but didn’t realize the meaning of it. Can be used
to analyze the function of RUNX in tumor metastasis.
5. Cell migration and the Boyden Chamber
Two processes dependent upon cell migration speed metastasis by reducing the
distance between the primary tumor and these vessels. The first process is
invasion. The second one is angiogenesis, which is dependent on endothelial cell
migration. This assay is used to test the factors promote cell migration
6. Quantification of cell motility- Gold colloidal phagokinetic track assay and
wound healing assay
7. In vitro invasion assay using Matrigel
Basement membranes: Collagens, laminins, and proteoglycans. The basement
membranes become permeable during tissue development, repair, and at sites of
inflammation, to allow immune cells to reach the site.
Matirgel is considered as basement membrane and generated from EHS sarcoma.
Matrigel contains not only basement membrane components (collagens, laminin,
and proteoglycans)but also matrix degrading enzymes/their inhibitors and growth
factors. Invasion of tumor cells into Matrigel has been used to characterize
involvement of ECM receptors and matrix degrading enzymes which play roles in
8. Membrane invasion culture system
9. Collagen invasion assay
10. Chick heart invasion assay
11. Adhesion of tumor cells to matrices and endothelium
Integrin family possess numerous ligands that can be separated broadly into two
groups, the immunoglobulin supergene family of intracellular adhesion molecules
expressed on the membranes of counter-adhesive cells (e.g., ICAM-1) and ECM
(collagens, fibronetcin, vitronectin, etc.)
a. Adhesion to purified mixed elements
b. Adhesion to Endothelium
c. Adhesion to tissue sections (purpose of coat glass slides with lysine?)
d. Adhesion to Endothelium under conditions of flow
Methods in Cell-Matrix Adhesion Edited by Josephine C. Adams
Chapter 13 Functional Analysis of Cell adhesion: Quantitation of cell-matrix attachment
Steven K. Akiyama
The most commonly used assays for quantitating cell adhesion fall into two categories:
cell attachment assays and cell spreading assays.
Cell Attachment assay
1. Preload matrix-coated and blocked wells of a 96-well cluster with 50ul of serum-
free medium alone or with inhibitors/activators.
2. Harvest cells.
3. Quench trypsin and allow cells to recover for 20 min at 37oC.
4. Count the cells, centrifuge, and resuspend in serum-free medium at a
concentration of 2x105/ml.
5. Add 50ul of cells to wells prefilled with 50ul of serum free medium and incubate
in a humidified tissue culture incubator (For fibroblastic cells, the incubation
time for cell attachment is usually 15-30min).
6. Remove non-adherent cells by gently washing with serum-free medium and
aspirating the unbound cells.
7. Fix attached cells in 5%(w/v) glutaraldehyde in CMF-PBS for 30min.
8. Wash cells three times with CMF-PBS and stain cells, 0.1% crystal violet for 30-
9. Destain by washing extensively with DW. Use at least 200ul water per well.
10. Solubilize the bound dye with 10% (w/v) acetic acid or 2% SDS in PBS.
11. Read absorbance at 560-590nm.
Cell Spreading assay
1. Preload matrix-coated and blocked wells of a 96-well cluster with 50 ul of
serum free medium alone or with inhibitors.
2. Harvest cells.
3. Quench and allow cells to recover for 20 min at 37oC.
4. Count the cells, centrifuge and resuspend in serum free medium at a
concentration of 2x105/m.
5. Add 50 ul of cells to the previously prepared matrix coated surface and
incubate in a tissue culture incubator (The time chosen for the assay should
be long enough to get substantial cell spreading on the extracellular matrix
protein but not so long that more than about 2-3% of the cells begin to spread
on BSA. If allowed to incubate too long, many cells can modify the matrix-
coated surface by using membrane-bound proteases and synthesize and
secrete matrix proteins of their own, allowing them to appear to spread on
BSA. If long spreading times are necessary, it is possible to use
cyclohexamide to inhibit synthesis of new proteins. Generally, cells of
fibroblastic origin spread quite rapidly and 80-90% cell spreading can often
be reached in 30-60 min. Other cell types can take up to several hours.)
6. Terminate the assay by fixing the cells with 100 ul 6% glutaraldehyde/ 6%
formaldehyde in CMF-DPBS for 60 min.
7. Count sets of 100 cells from 3-8 randomly chosen fields. Alternatively, the
surface areas of randomly chosen cells from each well can be determined
using image analysis software.
Cell migration assay – Agarose Drop Migration assay
1. 1ul of 0.26% agarose
2. 1-3 days (check every 12hours)
Wound Healing Migration Assay
1. 0.5-50 ug/ul of extracellular matrix protein
2. wound each well of 6-well plate by using a sterile 200 ul yellow pipette tip to
mark a straight line down the center of the well.
3. Photo 4 fields each 24 hours.
4. Measure the rate at which cells close the wound using a calibrated eyepiece. It is
recommended that the cell nuclei be used to define the position of the cell.
Functional analysis of p130/Cas in cell migration in individual cells and cell
aggregate/sheet in 2D and 3D system.
Methods and Materials
Cell culture and Transfection
Mouse embryo fibroblast(MEF), derived from p130/Cas knockout mice, stably expressed
eGFP-vector, p130/Cas, or 15F mutant Cas were cultured using DME with 10% FBS at
5% CO2 incubator, 37oC. Cells were transfected with RFP-VASP by Nucleofection (x) .
Candidate Project 2:
Cancer cell metastasis is a complicated process including many steps: (Pathology
textbook). Our goal in this experiment is to analyze the cancer cell motility after
penetrating into interstitial connective tissue. These could happen when cancer cells
digest basement membrane [major components: laminin, entactin, heparin sulfate
proteoglycans, collagen IV; minor components: fibronectin, tenascin, SPARC
(osteonectin), and fibulin-1] or in some cancer (ovarian tumor), the degradation or loss of
basement membrane exists even before the morphologic transformation of epithelial cells
(Capo-Chichi et al, Cancer 2002; Yang DH, Xu XX et al Cancer 2002). Another reason is
in desmoplastic tumor, increased myofibroblasts around tumor cells.
We used 3D co-culture system and time-lapse microscope to analyze cell migration as
single cells or cell aggregate/sheet. Physiological thickness of basement membrane is 8
m. Transfected with GFP/RFP without sorting; Traction force? Rigidity of
matrix/concentration of collagen gel.
Methods and Materials:
1. DiI 546nm; 563nm
CM-Tracker (Molecular Probes, Eugene, Ore., USA)
2. DiOC6(3): 489nm; 499nm
Cell were stained for 5 min with 1 ug/ml of the fluorescent dye 3,3’-
dehexyloxacarbocyanine iodide [DiOC6(3), Molecular Probes, Eugene, Ore.,
USA] dissolved in PBS. This dye possesses a positively charged polycyclic
head domain and two hydrocarbon tails. It is cell-permeable, and readily
accumulates in intracellular membranes. At low concentrations it accumulates
in mitochondria, while at higher concentrations it labels the ER, which may be
identified by its tubular morphology. This dye is not fixable and is extracted
by detergents, and so is not particularly useful for double-labeling with
antibodies. Several laboratories have used it to study membrane dynamics in
living cells, however. DiOC6(3) is a very bright dye, but is not very
photostable, and I have found it useful to include N-propyl gallate in
specimen preparations to retard photobleaching. The dye may be made as a
0.5 mg/mL stock in ethanol and is stored in the dark below 0°C. It may be
diluted to 2.5 µg/mL into growth medium and used to stain living cells for 5
minutes. Alternatively, cells may be fixed with low (0.25%) concentrations of
glutaraldehyde and then stained with 2.5 µg/mL diluted into PBS for 10
seconds. At higher concentrations, the dye begins to label cytosolic structures
indiscriminately. Following labeling, samples should be mounted in PBS
supplemented with 3% N-propyl gallate and imaged immediately. Cellular
membranes will begin to bleb shortly thereafter, and the dye will redistribute.
3. PKH2(green)/26(red) fluorescence staining kit - Sigma
What is the ideal way to generate cell aggregates? Centrifuge (speed?)
1. Non-coated petri dish for more than 3 days
2. Spinner flask?
3. Simply centrifuge?
Candidate project 3
Wound healing and metastasis and mechanosensing of metastatic cancer cells
Cell migration of cancer cells is a critical process of the multiple steps for invasion and
metastasis of cancers. We analyze the locomotion of cancer cells behavior. A metastatic
breast caner cell line, MDA-435, was plated on coverslip and time-lapse microscopy.
Metastatic cancer cells don’t migrate too much either as single cells or cell islands. (On
other the hand, fibroblast are able to move through collagen gels in vitro and through the
extracellular matrix in vivo, at a ratio of about 1m/min). Wound closure or wound
healing by epithelial cell sheets involves two types: Purse strings contraction for small
wound (<0.008 mm2) and lamellae for larger wounds. The two processes may coordinate
to heal a certain wound in some case? In vitro analysis of wound healing (1,2)
demonstrates that edge cells form leader cell and following cells in a Rho dependent
mechanism. This is similar to the early stromal invasion occurring tin a cervical
intraepithelial neoplasm (Pathology textbook page 1053). Previously we report that
fibroblasts movement is guided by the rigidity of the substrate (Lo, C.-M. Biophys. J. 79,
144-1523,4). We suppose that wound healing in vitro of tumor cell sheets mimic invasion
in vivo (Stanford University report the same hypothesis this year) and tumor cell migrate
to interstitial connective tissues also regulated by flexibility of extracellular matrix. And
tumor metastasis is simply the result of loss or gain of mechanosensing ability. Cells
cannot survive and migrate at interstitial connective tissue unless they are transformed.
Methods and Materials
Cell culture and Microscopy
Wound healing assay
A modified wound healing assay was established. Cells were plated on coverslip to form
confluence at half of dish which separated by a coverslip from the substrate coated other
half with substrate flexibility/rigidity gradient. Once confluence formed, coverslip which
separates the cells from substrate was removed to generate a wound. Cancer cells were
able to move toward preferable substrate.
1. Cancer cells form an aggregate or disperse when plated on a soft/hard substrate.
2. Cancer cells move toward soft/hard substrate but avoid hard/soft substrate.
3. Traction forces of cancer cell aggregate (leader and follower cells)
4. Rho/Rac? Leading edge staining? FA staining?
5. Co-culture with fibroblast changes the mechanosensing of cancer cells?
Project 4: Flexibility of substrate regulates cell aggregates expanding
Introduction: The physical and properties of substrates underlying cells can have
profound effects on cell behavior and function. Previously, we reported that cells
growing on soft substrates formed tissue like aggregates after long period culture. To
investigate whether tissue/cells expand out through probing the rigidity of
Materials and Methods
Technical Program Menu
[261d] - Mechanistic Analysis of Hepatocyte Spheroid
Manolis S. Tzanakakis (speaker)
University of Minnesota
Dept of Chem Engi and Mat.
Susan Fugett Abu-Absi
University of Minnesota
Dept. of Chemical Engineering
University of Minnesota
Dept of Chem Engi and Mat.
Many cell types spontaneously self-assemble in vitro into three-dimensional
aggregates that exhibit structural and functional resemblance to the tissue of origin.
Examples include mammary gland cells, which form hollow spheroids accompanied by
enhanced secretion of milk proteins, and pancreatic cells, which organize in islet-like
clusters with high production of insulin. The course of formation of such tissue-like
structures demonstrates common characteristics including the dynamic reorganization of
the cytoskeleton and the intercellular adhesion structures as well as subsequent changes
in the force balance. Thus, the study of cell self-assembly is possible to provide a set of
directive principles with a great impact on the engineering of tissues. The present study
addressed the issue of cell self-assembly by utilizing the formation of rat hepatocyte
spheroids in static cultures as a model system. The self-assembly process was dissected in
different stages and analyzed. A hypothetical model explaining the phenomenon was
constructed and tested. As a self-organizing process, the formation of hepatocyte
spheroids requires the presence of feedback loops. An attempt was made to identify such
feedback loops from a mechanistic point of view. The changes in cell arrangement during
spheroid formation were followed by time-lapse confocal microscopy of binary
populations of hepatocytes marked with the DiI or DiO membrane dye. In the early
stages, hepatocytes exhibited high motility and the tendency to establish cell-cell contacts
whereas in the final stages the aggregates underwent compaction through active
contraction. Since the cell self-assembly is a dynamic phenomenon, a continuous
alteration in the force balance is inferred. The cytoskeletal reorganization as a
manifestation of force redistribution was examined using immunocytochemical staining
methods. Actin filaments transformed from stress fibers initially to cortical actin bundles
in the cell aggregates acting as contractile elements throughout the spheroid formation.
Furthermore, cytoskeleton-disrupting drugs were used to investigate the role of individual
type of filaments during self-assembly. The results suggest that the dynamic remodeling
of the actin filaments is necessary for efficient spheroid self-assembly.
Project 5: NMU treated fibroblasts vs. normal fibroblasts co-cultured with tumor cells
in 3D system and interfere the tumor cells motility/behavior?