1. The study developed a novel 2.5D in vitro dot migration assay to better understand the invasive behaviors of proneural and mesenchymal glioblastoma tumor cells. 2. The assay incorporated both a surface (glass coverslip) and extracellular matrix components (Matrigel and FBS) to model the in vivo tumor microenvironment. 3. Preliminary results showed that the inflammatory cytokine TNF-alpha promoted greater cellular adhesion and dissemination of proneural PBT003 tumor cells in the assay, supporting one of the study's hypotheses. Further testing of additional cell lines was needed to fully evaluate the hypotheses.

1. Studies of Glioblastoma Subtype and Differentiation Dependence of Tumor Cell
Dissemination in a Novel 2.5D In Vitro Assay
Joy Cai, Raina Siegal Alicia Mizes, Patrick Le, Negin Baghadachi, Easun Aranachalam, Christine Brown, and Michael Barish
Department of Neuroscience
BACKGROUND
ABSTRACT DATA AND RESULTS
QUESTIONS/HYPOTHESIS
Dot Migration Assay Protocol
(Further improved from N. Baghdadchi, June 2014)
1. Begin experiment 4-5 days after tumor cells have been passaged. Make sure cells are about
70% confluent, with mostly tumor spheres suspended in culture rather than single cells.
2. Follow defined passaging protocol (depending on cell line in question). [More cell lines used]
3. Re-suspend cells in 1 mL of supplemented DMEM-F12 media with EGF and FGF (1:2000), 3%
BD Matrigel basement membrane matrix, and 2% Fetal Bovine Serum (enrich to a density of 5
x 106
cells per mL). [Addition of FBS]
4. Transfer 200ul of cell suspension into a microtube.
5. Perform serial dilutions to create cell concentrations of 5 x 106
, 2.5 x 106
, 1.25 x 106
, 0.63 x
106
, and 0.31 x 106
all in separate microtubes.
6. Transfer coverslips into 20 of the 24 wells.
a. Leave last row of the plate empty to add just media into. This will help to keep the inside of
the plate moist, preventing the evaporation of media.
7. Take 10 uL of mixture suspension from 5 x 106
cell concentration and apply to middle of
coverslip at 45º angle. Repeat 3 more times to fill an entire row with the same concentration.
a. Do the same procedure for each cell concentration, allotting each its own row. This will
give the option of 4 different study groups (each within a different column).
8. Incubate cells in incubator (37º C and 5% CO2) for 4-5 hours. [Increased incubation time to
5-6 hours for suspension cells]
9. After 4-5 hours of incubation, add 500 uL of supplemented DMEM-F12 media with EGF and
FGF (1:2000), 3% BD Matrigel basement membrane matrix, 2% Fetal Bovine Serum, and
preferred concentrations of TNF-alpha into each well.
a. Add the media very gently towards the edge of the coverslip, preventing the media from
going under. Streak the coverslip with media leading up the dot of cells and proceed to
slowly fill the well with media.
10. After three days of incubation, refresh the medium by removing the existing media and adding
500 uL of fresh supplemented DMEM-F12 media with EGF and FGF (1:2000), 3% BD Matrigel
basement membrane matrix, 2% Fetal Bovine Serum, and preferred concentrations of TNF-
alpha, into each well.
[Addition of Matrigel and FBS to ensure consistent cell environment]
11. Three days later (after 6 days of incubation), fix the cells and stain with different primary
antibodies for ICC.
**The dot migration assay presented above was performed using patient-derived glioblastoma multiforme cells from short-term
cultures (PBT003, PBT008, PBT017, and PBT030).**
Figure 5. Layout of an individual 24-
well plate Figure 6. Mapping of 24-well plate used in migration assay
Figure 4. Dot migration visualization
2.5 D Migration Assay
To better visualize both collective and individual cell movements, we have developed a novel 2.5 D migration assay. This assay
models in vivo structures and promotes the ability to study cell migration and invasion by incorporating surface and extracellular matrix
(ECM) components. The incorporation of both components is crucial as tumor cells are found to both adhere to a surface (ex. blood
vessel) and float in the ECM. Using Matrigel and FBS as an ECM and the glass coverslip as a vascular surface, we can control substrate
adhesion and the ECM. We can also better visualize cell morphology as well as permit the probing of cell physiology and gene
expression patterns in vitro.1
Figure 3. Dot migration assay mimicking the in vivo condition
MATERIALS/METHODS
Glioblastoma Multiforme (GBM), or grade IV, is one of the most malignant types of primary brain tumors.
Due to its highly infiltrative and invasive nature, therapeutic resistance and tumor recurrence after surgical
removal is common. Consequently, it is one of the deadliest human cancers with a median survival time of just 12
to 14 months after diagnosis. Despite the robust advances in surgery, radiotherapy, and chemotherapy, this
aforementioned life span has remained the same for the past several decades. Gaining a better understanding of
the cellular and molecular heterogeneity of glioma tumors, one of GBMs most important features, may help to
produce new strategies for therapeutic intervention.4
Tumor heterogeneity can be classified into two subtypes: intertumor heterogeneity and intratumor
heterogeneity. Intertumor heterogeneity is characterized by the genetic differences between tumors originating in
the same organ, while intratumor heterogeneity is classified as the diversity within cancer cells of the same
tumor. Both have become a recent focus in GBM research over the past several years contributing to advances
in molecular technology while providing a more detailed understanding of the molecular landscape of GBM as a
whole.6
This landscape includes 4 different subtypes: proneural (PN), mesenchymal (MES), neural, and classical,
all of which differ by the type of genetic abnormalities they carry out and by the patients clinical characteristics.
Proneural tumors are characterized as to having the most mutations in the IDH1 gene, which contributes to
abnormal cell growth. TP53, the most common mutation in GBM, occurs in 54% of all proneural tumors.
Proneural tumors also have the highest expression and number of mutations within the PDGFRA gene, which
leads to uncontrolled tumor growth.8
The MES subtype has the most number of mutations in the NF1 tumor
suppressor gene, occurring more than 37% in all MES tumors. The MES subtype also has mutations in the PTEN
and TP53 tumor suppressor genes. The MES subtype is associated with greater aggressiveness and low survival
in comparison to GBMs enriched with proneural genes. Moreover, tumors exhibiting PN phenotypes have been
found to undergo transition into mesenchymal phenotype during recurrence.7
The heterogeneity that is found within and amongst GBM cells is impacted heavily by the heterogeneity of
the microenvironment in which the cells are located and surrounded by. The formation of a tumor involves the
evolution of a myriad of cell types including neoplastic cells with extracellular matrix, vascular endothelial,
stromal, and immune cells. The topography of this tumor niche can differ drastically among glioblastoma patients
due to a tumor’s access to growth factors, structural support, immune cell interactions, and vascular supply. The
vascular supply can vary from the tumor’s tissue of origin, functionality, and interstitial pressure. The immune
response of each person creates differentiation between anti-tumor resistance, tumor infiltration, and activation.4
These microenvironments can pose as a challenge when mimicking in vivo conditions in studies that use in vivo
assays.
Tumor necrosis factor (TNF-alpha), an endogenous pyrogen, is a cytokine that is involved in cellular
processes such as tumorigenesis inhibition, cellular proliferation, apoptosis, coagulation, and necrosis. Produced
by macrophages, the primary role of TNF-alpha is to regulate the monocytes, or the immune response against a
tumor. TNF-alpha is a chemoattractant for neutrophils, a type of white blood cells, promoting the expression of
adhesion molecules on endothelial cells and migrating neutrophils. It has been proven that TNF-alpha promotes
an increased expression of VCAM-1 in PBT003 cells.1
1. Do all of the cell lines proliferate under our conditions at the same rate and under the same spatial pattern?
a. We hypothesize that PBT017 and PBT030 mesenchymal cells, will migrate farther and proliferate more frequently
under the dot migration assay conditions than PBT003 and PBT008 proneural cells.
2. Are these proliferation and migration patterns affected by TNF-alpha exposure?
a. We hypothesize that TNF-alpha will promote greater cellular adhesion and dissemination among all 4 cell types.
Immunofluorescence utilizes fluorescent-labeled antibodies to detect specific target antigens. This allows for
further characterization of the cell lines used and provides a method to quantify the effects of TNF-alpha used
in the dot migration assay.
Figure 1. Fluorescent Staining. The primary antibody, made in
animal A, detects a specific antigen and binds to it. The secondary
antibody, made in animal B (anti-animal A), binds to the primary
antibody. The secondary antibody emits excitation light at a certain
wavelength through fluorescence imaging.
Figure 2. Fluorescence Imaging Technique. The fluorescent
molecules, fluorophores, absorb a photon and emit another
photon of longer wavelength-light a nanosecond later. Modern
detection devices can detect these photons and transform
these detection events into quantifiable electrical signals.
CONCLUSIONS
FUTURE DIRECTIONS BIBLIOGRAPHY
As characterized by the National Institute of Health, there are four subtypes of Glioblastoma
Multiforme (GBM): proneural (PN), mesenchymal (MES), neural, and classical. Tumor cells of the
mesenchymal subtype are the most genetically unique from tumor cells of the proneural subtype. A
better understanding of what sets these two groups apart will lead to patient-specific treatments
tailored to the particular pattern of genomic changes within each tumor at question.
In this study, patient derived brain tumor (PBT) cells of the mesenchymal (PBT017/PBT030)
and proneural (PBT008/PBT003) subtypes were used. The proneural subtype was found to express
genes associated with glial cells and neurogenesis, while the mesenchymal subtype expressed
genes associated with angiogenesis and mesenchymal gain.7
To better understand the invasive
nature of PN and MES cancer cells, as dissemination provides the seed for tumor recurrence, we
devised a novel migration assay to model the invasive behaviors of PN and MES tumor cells in
vitro. In order to characterize these individual cell lines and their dissemination patterns, we used
fluorescent staining and imaging to quantify the expression of several molecular markers.
Furthermore, we incorporated the use of tumor necrosis factor-alpha (TNF-alpha) to view and
compare its effects on proliferation and migration. TNF-alpha has been shown to induce the
expression of adhesion molecules and contribute to inflammatory responses.5
Therefore, TNF-alpha
was additionally observed to see its effects on vascular cell adhesion molecule (VCAM-1)
expression.
Abbreviation Name Structure Purpose
GFAP Glial Fibrillary Acidic
Protein
Intermediate Filament Protein
expressed in CNS
To stain for an astrocyte marker on the patient derived brain tumor (PBT)
cells.
KI-67 MKI67 Protein Encoded by the MK167 Gene To mark cellular proliferation and determine cell growth fraction of a given
cell population.
VCAM 1 Vascular Cell
Adhesion Protein 1
Protein Encoded by the VCAM1 gene To observe cellular adhesion in endothelial cells.
ANXA2 Annexin A2 Calcium-Dependent Phospholipid-
Binding Protein
To help organize exocytosis of intracellular proteins to the extracellular
domain.
MMP2 Matrix
Metalloproteinase-2
Enzyme encoded by the MMP2 Gene To breakdown the extracellular matrix and degrade type IV collagen.
VLA-4 Very Late Antigen-4 Integrin Dimer composed of CD49D
and CD29
To bind to VCAM1 molecule located on the endothelial cells.
CD44 Homing Cell
Adhesion Molecule
Cell-surface glycoprotein To involve in cell-cell interactions, cell adhesion, and migration and to
signal for cell survival.
CLCN3 Chloride Channel 3 H+
/Cl−
exchange transporter 3 protein To catalyze the selective flow of CL- ions across the cell membranes.
Hoechst Hoechst Stain Bisbenzimide To fluorescent stain (blue) for DNA.
TRADITIONAL INVASION ASSAYS
What Makes Our Assay Different?
In vitro 2D and 3D migration assays have been used to display various mechanisms associated with invasion and metastasis. Common methods
currently employed to investigate the invasive potentials of tumor cells are: (a) wound healing or “scratch” assay, (b) Boyden Chamber migration assay
using Transwell inserts (with an optional upper chamber ECM coating) and supplemented or conditioned tissue culture media as a lower chamber
chemoattractant, (c) fence assay (ring assay), (d) spheroid migration assay, (e) sedimentation assay, and others. The relevance of these models, however,
for in vivo behavior is limited by their underlying commitment to surface matrix migration, each of which is limited in its ability to reveal interactions of
multiple motility and adhesion mechanisms operating in complex environments. Our novel in vitro 2.5D migration assay incorporates both surface and
extracellular matrix (ECM) components which better reflects the in vivo configuration of surface and environs.1
= Our developments
1. Dot Migration Barrier and Time-Lapse Imaging
a. To further improve the accuracy of the dot migration assay, it would be beneficial to implement a physical barrier
between the initial 10 uL of cells and the rest of the coverslip. A migration assay that incorporates a barrier will help
to prevent cells from entering a defined area or from dispersing when the initial flood of media is presented. Cells of
interest are seeded around or within this barrier, and after the formation of a peripheral monolayer the barrier is
removed and migration into the cell-free area is monitored. The barrier configuration in conjunction with time-lapse
imaging enables the quantitative assessment of individual cell migration, total migration, net displacement,
migration efficiency, migration velocity, and cell polarization.3
A.
2. Epithelial-Mesenchymal Transition (EMT) and Mesenchymal-Epithelial Transition (MET)
a. During the 6-day incubation period, we have observed the formation of satellite neurospheres from the original
plated dot of cells. We hypothesize that epithelial-mesenchymal transition (EMT) occurred from the cells losing
their cellular polarity and cell-cell adhesion, and gaining migratory properties to become mesenchymal cells. Due
to the formation of satellites, we hypothesize that mesenchymal-epithelial transition happened from the cells re-
gaining epithelial properties and settling in a different area of the coverslip. In the future, it would be interesting to
delve deeper into these two transitions as to gain a better understanding of each.
TNF-alpha
TNF-alpha
Control
Control
Extracellular Matrix Components
Matrigel: A solubilized basement membrane preparation
extracted from the Engelbreth-Holm-Swarm (EHS) mouse
sarcoma, a tumor rich in ECM proteins such as laminin, collagen
IV, entactin, and heparin sulfate proteoglycans. It is ideal for the
promotion and maintenance of differentiated phenotypes in a
variety of cell cultures including primary epithelial cells,
endothelial cells, and human induced pluripotent stem cells
(iPSC). One of the challenges of using Matrigel in the 2.5D
migration assay is that it is hard to handle under room
temperature conditions. Sometimes it thickens the media too
much making the solution heterogenous rather than
homogenous as wanted.
Fetal Bovine Serum (FBS): A by-product of the beef industry
produced from fetal blood collected at commercial
slaughterhouses. It is the most widely used serum-supplement
for cell cultures because it contains low levels of antibodies and
high levels of growth and survival enhancing factors to cells in
vitro. The rich variety of proteins within the serum provides cells
with an environment in which they can survive, grow, and
multiply. One of the challenges of using FBS in the 2.5D
migration assay is the possibility of it altering the cells migration
and differentiation patterns. This may occur due to the hormones
contained within the serum.
Based on our results for the proneural PBT003 cell line, we can confirm our second hypothesis. TNF-alpha did prove to promote
greater cellular adhesion and dissemination among the differentiated PBT003 tumor cells. We are still in the process of testing this
hypothesis for the two mesenchymal cell lines (PBT017 and PBT030) and the other proneural cell line (PBT008). Unfortunately, based on
time restraints, we were only able to analyze our data for the PBT003 cell line. Thus, we have yet to completely test our primary
hypothesis. In the future, we hope to analyze our results for the other cell lines so that we can better understand if and why cells from the
mesenchymal subtype migrate farther and proliferate more frequently under the dot migration assay conditions than cells of the proneural
subtype.
C.B. D. E.
1. Baghdadchi, Negin, "Cytokine Control of Glioma Adhesion and Migration"
(2014). Electronic Theses, Projects, and Dissertations. Paper 93.
2. Brown, Christine E., Charles D. Warden, Renate Starr, Xutao Deng, Behnam Badie, Yate-Ching Yuan, Stephen J.
Forman,
and Michael E. Barish. 2013. “Glioma IL13Rᶐ2 Is Associated with Mesenchymal Signature Gene Expression and
Poor Patient Prognosis.” Edited by Ilya Ulasov. PLoS ONE 8 (10): e77769. doi:10.1371/journal.pone.0077769.
3. Das, Asha M, Alexander M M Eggermont, and Timo L M ten Hagen. 2015. “A Ring Barrier–based Migration Assay to
Assess Cell Migration in Vitro.” Nature Protocols 10 (6): 904–15. doi:10.1038/nprot.2015.056.
4. Juntilla, Melissa R., and Frederic J. De Sauvage. "Influence of Tumour Micro-environment
Heterogeneity on Therapeutic Response."Nature. Nature, 19 Sept. 2013. Web. 21 July 2015.
5. Lee, C.-W., Lin, W.-N., Lin, C.-C., Luo, S.-F., Wang, J.-S., Pouyssegur, J. and Yang, C.-M. (2006),
Transcriptional regulation of VCAM-1 expression by tumor necrosis factor-α in human tracheal smooth muscle
cells: Involvement of MAPKs, NF-κB, p300, and histone acetylation. J. Cell. Physiol., 207: 174–186. doi: 10.1002
/jcp.20549
6. Meacham, Corbin E., and Sean J. Morrison. 2013. “Tumour Heterogeneity and Cancer Cell Plasticity.” Nature 501 (7467):
328–37. doi:10.1038/nature12624.
7. Phillips, H. et al. Molecular subclasses of high-grade gliomas predict prognosis, delineate a pattern of
disease progression, and resemble stages in neurogenesis.Cancer Cell 9, 157–173 (2006)
8. Verhaak, R.G., Hoadley, K.A., Purdom, E., Wang, V., Qi, Y., Wilkerson, M.D., Miller, C.R., Ding, L.,
Golub, T., Mesirov, J.P., Alexe, G., et al. (2010) Integrated genomic analysis identifies clinically relevant subtypes
of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell. 17(1):98-110.
ACKNOWLEDGEMENTS
Barish Lab, Department of Neuroscience
Dr. Michael E. Barish - Primary Investigator
Patrick Le - Mentor
Dr. Ying Wang - Mentor
Blake Bruwster - Mentor
Christine Brown - Collaborator
Alicia Mizes - Summer student
Raina Siegal - Summer studentFigure 9. A schematic overview of a ring barrier-based 2D migration assay: protocol published in Nature America, Inc. 21 May 2015
B.
A.
Primary Antibodies Stained For:
Hoechst (Counterstain) → DAPI 350nm
KI-67 → FITC 488nm
VCAM-1→ TRITC 555nm
A. B.
Figure 8.
A. A visual representation of Ki-67 expression within a PBT003 dot
migration seated at 2.5 x 106
cells per mL with and without TNF-
alpha.
B. A visual representation of VCAM-1 expression within a PBT003 dot
migration seated at 2.5 x 106
cells per mL with and without TNF-
alpha
(Antibody expression analyzed using CellProfiler software.)
Graph 1.
A. A comparison of Ki-67 luminance (proportion of 16-bit scale) and
the proportion of the total cells within the control and TNF-alpha
condition.
B. A comparison of VCAM-1 luminance (proportion of 16-bit scale)
and the proportion of the total cells within the control and TNF-
alpha condition.
The serum-differentiated PBT003 cell slides were scanned using a Hamamatsu NanoZoomer. After the slides were scanned, we went
through 80 images to find two that best represented the control and TNF-alpha conditions (Figure 7A,B). These two images were from dot
migrations seeded at 2.5 x 106
cells per mL (the second highest cell concentration used). As is evident, Figure 7A contains far fewer cells than
Figure 7B. It is also clear, looking at the primary antibodies key, that Figure B contains more TRITC (bright orange/yellow-fluorescence)-
marked cells expressing VCAM-1, and FITC (green-fluorescence)-marked Ki-67 cells. Both of these findings seem reasonable as TNF-alpha
has been shown in previous literature to promote increased expression of VCAM-1 in stem-like PBT003 cells.1
Furthermore, previous
literature has shown that TNF-alpha induces phenotypes similar to those of mesenchymal subtypes.2
As we know, PBT003 cells are of the
proneural subtype and not the mesenchymal.
After scanning, we further analyzed the same two images in Figure 7 with CellProfiler software (Figure 8A,B) to obtain more quantitative
results. In both Figure 8A and B it appears that there are fewer cells in the control condition as compared to the TNF-alpha condition. It also
appears that for both Figure 8 A and B there is a greater staining by the primary antibodies Ki-67 and VCAM-1 for the TNF-alpha condition
than for the control condition. This is visible as cells depicted in colors towards the white end of the spectrum display higher levels of antibody
binding then cells depicted in the blue end of the spectrum. Both Graph 1A and B show similar conclusions. In Graph 1A the control condition
had a larger proportion of total cells expressing Ki-67 at a lower luminance than the TNF-alpha condition which had a higher proportion of
total cells expressing Ki-67 at a higher luminance. In Graph 1B the control condition had a larger proportion of total cells expressing VCAM-1
at a lower luminance than the TNF-alpha condition which had a higher proportion of total cells expressing VCAM-1 at a higher luminance.
Something important to note is that in Figure 8A there appears to be fewer cells and less staining by the Ki-67 antibody for both the control
and TNF-alpha conditions than there is in Figure 8B for the VCAM-1 antibody. This makes sense as Ki-67 is a proliferation marker expressed
only in the nucleus of the cell while VCAM-1 is an adhesion marker expressed in the entire cell body.
Figure 7.
A. An overhead view of a PBT003 dot migration seated at 2.5 x 106
cells per
mL without TNF-alpha.
B. An overhead view of a PBT003 dot migration seated at 2.5 x 106
cells per
mL with TNF-alpha.
(Images taken with Hamamatsu NanoZoomer Digital Pathology.)
cells per mL