Mentored Professional Enrichment Experience
Applicant: Megan Logsdon
Name of Project/Experience: Bone Marrow-Derived Multipotent Mesenchymal Stem
Cells (BMSC) in Cell-Based Therapies for Epithelial Ovarian Cancer
Location where Project/Experience will take place:
911 N. Rutledge, Room 329, Springfield, IL
Mentor Name and Contact Information:
Mary McAsey, Ph.D., mmcasey@siumed.edu
913 N. Rutledge, Room 1325, Springfield, IL
Phone: Office: 217-545-4692, Clinic: 217-545-3128, Laboratory: 217-545-2173
RATIONALE
As the deadliest gynecological cancer, epithelial ovarian cancer is one of the main causes
of cancer mortalities in women1. While improvements in chemotherapies (intravenous
and intraperitoneal) have been considerable, the survival rate of advanced stage ovarian
cancer after 5 years has effectively stagnated over the past 30 years2. A significant factor
in the severity of epithelial ovarian cancer progression is the hypoxic environment of the
peritoneal cavity, which induces cancer cell expression of a transcription factor (hypoxiainducible factor-1, or HIF-1)3,4. HIF-1 expression advances the development and
metastasis of tumors, through gene transcription events, which result in metastatic
implantation, vasculogenesis, growth, and survival5,6. Furthermore, it is difficult to reach
the locations of these ovarian tumors with therapeutic treatments, due to a lack of
functioning vessels in the region. Bone marrow-derived multipotent mesenchymal
stromal cells (BMSC), however, may be selectively attracted to hypoxic regions.
BMSC may be engineered to express the gene cytosine deaminase (CD), which acts to
convert 5 fluorocytosine (5-FC, a non-toxic pro-drug) to 5-fluorouricil (a cytotoxic
chemotherapeutic drug), which can enter cancer cells through non-facilitated diffusion.
In this research, BMSC may also be engineered to express the reporter genes green
fluorescent protein (GFP) and firefly luciferase (Luc), which are important for imaging of
BMSC in vitro and in vivo. This engineering of BMSC is accomplished through
1
Parkin DM, Bray F, Ferlay J, Pisani P. CA Cancer J Clin. 2005;55:74-108.
Jemal A, Sieger R, Ward E., et al. CA Cancer J Clin.2006;56:106-30
3
Naora H, Montell DJ. Nat Rev Cancer. 2005;5:355-66.
4
Mutch DG, Williams S. Clin Obstet Gynecol 1994;37:406-22.
5
Ratcliffe N, Terhune PG, Longnecker DS. Arch Pathol Lab Med 1996;120:1111-5.
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Stadlmann S, Amberger A, Pollheimer J, et al. Gynecol Oncol 2005;97:784-9.
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lentiviral vectors, which are based on HIV-17. Lentiviral vectors introduce the GFP and
Luc genes, as well as coordinate their expression by BMSC. Lentiviral vectors will also
introduce the CD gene in BMSC.
Previous studies have shown that human bone marrow cells are able to respond to soluble
factors from ovarian cancer cell lines (HEY and SKOV3) by invading the extracellular
matrix. Additionally, these human bone marrow cells migrate to ovarian cancer cells in
vitro, as well as to ovarian cancer multicellular tumor spheroids in vitro. Multicellular
tumor spheroids are a better representation of solid tumors than the monolayer cultures,
and the BMSC have been shown to move towards and adhere to the spheroids (derived
from HEY), and NOT to fibroblast spheroids. Studies have also shown that lentiviral
vectors used in this research have the ability to alter BMSC in order to cause expression
of green fluorescent protein (GFP) and firefly luciferase (Luc). GFP and Luc expression
aids in assessment of successful transduction of BMSC, which will be determined using
flow cytometry and fluorescence and bioluminescence imaging. Finally, ovarian cancer
cell lines have been engineered to stably express red fluorescent protein (AsRed) and
renilla luciferase. Consistent expression of AsRed and Renilla in cancer cell lines allows
for distinguishing between ovarian cancer cells and BMSC in vitro and in vivo, through
fluorescence and bioluminescence imaging.
GOALS
This project aims to accomplish two goals:
1) To evaluate cytosine deaminase-transduced BMSC on their ability to invade and
migrate to ovarian cancer cells (using HEY, SKOV3, and SKOV3ip1 cells). The
BMSC will be transduced to express green fluorescent protein, firefly luciferase,
and cytosine deaminase genes. HEY, SKOV3, and SKOV3ip1 lines will be
engineered to stably express AsRed and Renilla.
2) To determine the cytotoxic effect of transduced BMSC; specifically, this project
intends to assess the most advantageous number of stem cells for the maximum
cytotoxic effects of cytosine deaminase-transduced BMSC. The BMSC will be
transduced to express green fluorescent protein and cytosine deaminase genes.
The HEY cancer cell line will be used, and will be engineered to stably express
AsRed and Renilla.
These two goals work towards a larger goal of the research on transduced BMSC to
determine the maintenance of the stem cells’ character, multipotent potential, and
migratory response.
METHODS
To accomplish the first goal,transduced BMSC will be plated within cloning rings on
glass cover slips. Engineered HEY, SKOV3, and SKOV3ip1 cells will be seeded on the
slips external to the cloning rings. Several controls will be employed: 1) no cells plated
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Hanawa, H., P.F. Kelly, A.C. Nathwani, et al. Mol. Ther. 2002. 5: 242-251.
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onto cover slips external to cloning rings; 2) unlabeled BMSC (with no GFP or Luc
expression) plated onto cover slips external to cloning rings; and 3) immortalized, nontumorigenic ovarian surface epithelial (IOSE) cell lines will be plated external to the
cloning rings.
To accomplish the second goal, CD-transduced BMSC will be cultured with HEY cell
lines engineered to express AsRed and Renilla in vitro. Differing concentrations of the
transduced BMSC and engineered HEY cells will be cultured, and increasing amounts of
5 fluorocytosine will be added to the cultures, in order to establish the half maximal
inhibitory concentration (IC50) for 5 fluorocytosine in these conditions. Several controls
will be employed: 1) BMSC transduced to express GFP and Luc will be incubated with
HEY cells engineered to express AsRed and Renilla; 2) HEY cells will be treated with
increasing amounts of 5 fluorocytosine; 3) BMSC transduced to express GFP and Luc
will be treated with increasing amounts of 5-FC; and 4) BMSC transduced to express
GFP and CD will be treated with increasing amounts of 5-FC.
ANALYSIS
For the first goal of assessing invasion and migration of transduced BMSC, the data will
be analyzed in several stages. After waiting four hours from plating, the cloning rings
will be removed and the cells will be imaged using fluorescence microscopy. For four
days following, the cells will be monitored and photographed daily, under direct
fluorescent microscopy of GFP-labeled BMSC and AsRed-labeled cancer cells. We
anticipate that the transduced BMSC will migrate to ovarian cancer cells in vitro, as the
unmanipulated BMSC have already been shown to do. Migrating cells will be counted
and analysis of variance will be used to assess migration difference among the test and
control groups
In accomplishing the second goal of determining the cytotoxic effect of BMSC, the data
will also be analyzed in several stages. After 24, 48, and 72 hours from initial culturing,
cells will be rinsed twice, using phosphate buffered saline (PBS) and 40 l Thiazolyl
Blue Tetrazolium Bromide (MTT) reagent incubated for two hours at 37C and at 5%
CO2. At the conclusion, 100 l dimethyl sulfoxide (DMSO) will be added to cells in
order to stop the reaction. Relative numbers of viable cells will be determined by an
optical density (OD) reading of the cells at 570nm with reference to 630nm. We hope to
identify a correct half maximal inhibitory concentration for the 5-FC in the cultured cell
conditions.
SUPPORT
1. Do you request support funds? Yes
2. Would you be able to participate if a scholarship is not available? Yes
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