(108d) The Direct Osteolytic Potential of MCF-7 and MDA-MB-231 Breast Cancer Cells in An in Vitro Bone Environment

The direct osteolytic potential of MCF-7 and
MDA-MB-231 breast cancer cells in an in
vitro bone environment

JE Dumas, CJ Ihenacho, and MO Platt

Department of Biomedical
Engineering, Georgia Institute of Technology, Atlanta, GA

Introduction. Breast cancer affects
one in eight women, and its propensity to metastasize to bone causes further
health complications.  Cathepsins and other proteases have been reported to be upregulated in breast cancer tissues, facilitating the
invasion and metastasis of breast cancer. 
It is generally established that cancer cells influence osteoclast
activity by releasing factors such as parathyroid hormone that stimulate the
production of RANKL, which increases osteoclastogenesis
and increased bone resorption (i.e., bone degradation).  Growth factors released from the
degraded matrix then bind to cancer cells stimulating proliferation.  This vicious cycle causes osteolytic lesions and considerable patient morbidity.  The direct resorptive
capacity of breast cancer cells is not well understood and could contribute to
this osteolytic cycle.      Therefore, it is
important to fully elicit putative mechanisms of direct resorption
of bone by metastasized breast cancer cells to optimize bone metastasis
treatment strategies. To do so, engineered bone biocomposites,
are useful to determine individual mechanisms behind these pathologies in a
controlled environment.  In this
study, a bone biocomposite platform is used to study
the induction of bone resorptive activity by two breast
cancer cell lines noted for differences in their aggressive/metastatic
abilities MDA-MB-231, and MCF-7.

Materials
and Methods.  Bone biocomposites were synthesized using
polyurethane and bovine mineralized bone particles.  The bone biocomposites
were molded in cylindrical rods, and were subsequently cut into discs (500 µm
thickness).  After sterilization,
the discs were placed in a 12 well plate and 5.0 x 10⁴ MDA-MB-231and
MCF-7 cells (transfected with RFP and GFP, respectively) were seeded on the
composite surface.  Media was
collected every two days, and cells were lysed after seven days.  Protein concentration was determined and
lysates were prepared for Western blot. 
Media was analyzed for calcium using an Arsenazo
III assay. Collagen fragment (CTX) concentrations
in cell media were measured using ELISA.   

Results
and Discussion.  After nine days of culture, MDA-MB-231 exhibited a
2-fold increase in calcium released into the medium as compared to MCF-7s and
all above the background no cell control.  Increased
calcium released in the culture media was an indicator that breast cancer
cells, in the absence of active osteoclasts, may be capable of resorbing bone,
and that the bone biocomposites we developed may be a
useful tissue engineered platform to study these mechanisms.  Further, culture on the bone biocomposites but not on tissue culture plastic, exhibited
increased expression of cathepsin K, the major enzyme
that degrades type I collagen (the major structural component of bone), which
is coupled with bone de-mineralization. 
Increased cathepsin K expression coincides
with increased CTX concentrations in the culture media.   

Conclusions. Bone biocomposites
synthesized in this study demonstrated that breast cancer cells are capable of
mineral dissolution and collagen cleavage that may lead to osteolytic
lesions in metastasized cancer. 
This observation emphasizes the importance of early intervention of bone
metastasis as the breast cancer cells may directly begin resorption
of bone, prior to activation of the neighboring osteoclasts and may present a
mechanism that improves their homing and colonization of the foreign
environment.

Figure  SEQ Figure \* ARABIC 1. (A) Fluorescent image of MCF-7 cells attached on the surface of a bone biocomposite at day 4. (B) Western blot indicating an increase
of cathepsin K expression of MDA-MB-231 cells seeded
on bone biocomposite as compared to cells seeded on
tissue culture plastic.  (C) Calcium
accumulated in culture media from bone biocomposite
degradation over a 9 day period of both MDA-MB-231 and
MCF-7 cells. (D) CTX collagen fragments accumulated in culture media between
days 7 and 9.