Research
in the Cancer Stem Cell Lab
Overall Goals
Protocols
Overall
Goals
The long-term research
objective of my laboratory is to investigate the fundamental questions
in cancer stem cell biology. We would like to establish the role of
microenvironment in maintaining the balance between self-renewal and
differentiation of cancer stem cells. A nswering this question will
provide information on how to keep the cancer stem cell from initiating
tumors and their re-growth. We also want to identify the
characteristics
of cancer stem cells that make them different from the normal tissue
stem cells. Identifying the unique properties of cancer stem cells can
be exploited therapeutically to target tumor-initiating cells. Lastly,
we would like to determine whether all cancer stem cells are created
equal, i.e. whether a drug designed to kill the multiple myeloma cancer
stem cell could also
kill a breast cancer stem cell. If the hypothesis that cancer
stem
cells from different tissues share similar characteristics is
validated, it will revolutionize the current thinking about cancer,
implying that generative compartments of different cancers may share
common characteristics, raising the possibility that one drug or group
of drugs can be used to treat multiple malignancies.
 |
| The
working
hypothesis is that cancer stem cells are found in a specialized
microenvironment niche which keeps the cells in a non-proliferative
state. Altering the conditions in favor of differentiation and
proliferation leads to tumor re-growth. |
Multiple
Myeloma
Multiple
myeloma (MM) is an incurable malignancy of the bone marrow (BM) plasma
cells (PM) responsible for 1% of hematopoietic cancers and 19% of
deaths from these malignancies. MM is characterized by the
overproduction of monoclonal immunoglobulin leading to secondary
amyloidosis causing renal failure, suppression of hematopoiesis leading
to anemia and succeptibility to infections, and bone lesions caused by
the increase in bone resorption and decrease in bone formation due to
the overstimulation of osteoclasts, bone resorbing cells, and a
decrease in osteoblasts, bone forming cells, causing bone thinning,
pain
and fractures. Most MM are thought to
originate from a pre-malignant condition termed monoclonal gammopathy
of undetermined significance (MGUS), with 10% of patients with MGUS
progressing to MM.
With the emergence
of
novel biologically based therapeutics such as bortezomib and
lenalidomide, considerable progress has been made in treatment of
MM.
While these drugs show remarkable improvement over conventional
therapies, such as melphalan and VAD, in treating MM by reducing the
tumor burden and alleviating the symptoms of MM, even patients in
complete remission inevitably succumb to a relapse. This suggests
that
a progenitor cell responsible for tumor regrowth is drug resistant and
escapes therapeutic targeting by even the most advanced drugs.
Because
clonal expansion
of primary MM cells outside their BM microenvironment has been
unsuccessful, most pre-clinical studies have utilized MM cell lines
derived from leukemic phase cells that have escaped BM
dependence. It seems likely that BM niches maintain
MM cancer stem
cells (MM-CSC) in a quiescent, drug-resistant state. To date
pre-clinical models do not take into account adhesion-mediated
drug-resistance and none allow testing of drug efficacy on MM-CSC
populations as only
PCs are evaluated in pre-clinical
studies to determine
the impact of new drugs. Therefore, we designed a 3-D tissue
culture model which takes into account the BM microenvironment allowing
us to culture the BM cells under physiological conditions.
Bone
Marrow 3-D Culture
As
a site of hematopoiesis, BM has
a complex organization with multiple cell types occupying distinct
niches. Discrete extracellular matrix microenvironments within
the BM help to separate endosteum, an interface between bone and BM,
from the central marrow.
BM
extracellular matrix, a proteinacious matrix of mainly fibronectin,
laminin, and
collagens is responsible for maintaining the BM architecture by
providing a scaffold for the cellular compartments occupying the
BM.
Cell
culture systems involving growth of cells on the surface of tissue
culture plastic do not accurately represent tissue architecture or the
complex interactions between cells and their microenvironment. To
adequately study B cell development, pathogenesis, and neoplasia, a
culture system that places BM cells within their physiological
environment is required. Because
the BM microenvironment can influence the therapeutic efficacy by
conferring drug resistance, more
effective culture systems designed to study BM-localized malignancies
must incorporate all compartments of the malignant clone, including
cancer stem and progenitor cells, to identify their therapeutic
vulnerabilities.
We designed a 3-D tissue culture model where we reconstruct the
endosteum and BM in vitro,
providing access to all of the BM compartments and allowing us to
manipulate the system to understand the role of micronevironment in the
initiation, promotion, progression, and relapse of MM.
The
Original Model
In the 3-D culture, endosteal
(rEnd) and
BM (rBM) matrices have been reconstructed to mimic the in vivo composition of BM.
rEnd was reconstructed by surface coating of the tissue culture plastic
with collagen I/fibronectin mixture and overlaid BM mononuclear cells
suspended in fibronectin/Matrigel mixture, which is representative of
the in vivo central marrow
composed of fibronectin, laminin, and collagen IV. 3-D cultures
maintain the cellular composition of in
vivo BM. In 3-D cultures, at day 0, BM cells are randomly
distributed throughout the 3-D matrix.
As early as day 1 of culture, cells begin to migrate within the ECM,
marked by the appearance of leading edge protrusions. By day 4,
both
diffuse and tight colonies are seen. By day 14, the architecture of the
rBM closely resembled that of the in vivo BM. Stromal cells
appear at the rEnd after a week in culture and grow to cover the
surface of the culture vessel.
Physical stratification of the BM includes distinct niches with dormant
hematopoietic progenitor cells (HPC) found at the bone surface in close
association with the endosteum, and more differentiated hematopoietic
cells such as B cells and PCs found in the central marrow, with the
most differentiated cells localized furthest away from the rEnd.
The interaction between HPC, endosteal osteoblasts and
osteoclasts, bone forming and resorbing cells respectively, is
essential for the maintenance of the HPC niche and mobilization of stem
cells. In 3-D culture, after completion of redistribution and
proliferation, rBM is stratified into approximately top, middle and
rEnd layers within the 1mm thcik 3-D culture. Similar to the
architecture of the in vivo
BM, the endosteal niche of rBM is composed
of fibroblastic stromal cells, adipocytes, and osteoclastic and
osteoblastic cells capable of mineralizing calcium. Exclusion of
the
rEnd coating from the culture greatly reduces the stromal
compartment. Obtaining the entire stromal compartment in 3-D is a
marked improvement over standard 2-D methods where only a single stomal
cell type differentiates from BM, depending on the culture media.
Mimicking their localization in intact BM, CD34+ HPC are found at rEnd
in contact with stromal cells, CD19+ B cells localize mainly to the
middle layer, and CD138+ PCs mainly to the top layer of the rBM.
Characterization
of the myeloma cancer stem cell
As essentially all MM patients
ultimately
relapse, drug-resistant cancer stem cells must escape current
therapies. Similarly to BM mediated quiescence of hematopoietic
progenitors, it seems likely that microenvironment plays a central role
in maintaining MM cancer stem cells in a quiescent state, perhaps
delaying relapse or alternatively preserving the malignant clone
throughout therapy. Therefore, we have a number of ongoing
projects aimed at establishing the identity of the MM cancer stem cell,
determining its tumorigenic capacity, and defining its microenvironment
niche.
Breast Cancer
The morphology of
normal
mammary glands is characterized by an inner layer of polarized
epithelial cells with secretory surfaces facing a central lumen and
basal surfaces surrounded by an outer layer of myoepithelial
cells. When normal mammary epithelial cells are cultured ex vivo
in Matrigel, a source of extracellular matrix, they form
alveolar
structures resembling their in vivo morphology. Upon contact with
either Matrigel or basement membrane proteins such as laminin and
collagen IV, normal mammary epithelial cells become polarized, form
spherical acini or branched tubules, and in the presence of prolactin
initiate β-casein secretion. In contrast to normal cells, tumor
mammary epithelial cells form large, non-polarized, undifferentiated
colonies without lumena when grown in Matrigel.
|
|
Acini formed by cancerous
MCF7 cells are not polarized and lack central lumena present in normal
mammary acini. Red - mitochondrial marker.
|
Polarized acinus with a
central lumen formed by MCF7 cells transfected with CEACAM1, a
cell-cell
adhesion molecule. Transfection of MCF7 cells with CEACAM1-4S,
CEACAM1
with a short cytoplasmic domain, reverts MCF7 acini to a normal
morphogenic phenotype. Green - luminal marker; red -
mitochondrial marker.
|
Characterization of the breast cancer stem cell
niche
The lab is starting a number of
project aimed at defining the microenvironment niche maintaining the
proliferative quiescence of the breast cancer stem cell.
Last modified:
March 12, 2009
