Evaluation of findings

In both epidemiological and experimental studies of tumour biology there have been a concrete
evidence that alcohol increased the mammary CSC population both in vitro and in vivo models.
CSCs are small cell populations that exists within tumours. The identification and classification
of CSCs population within breast cancer is done by the expression of distinctive cell surface and
intracellular markers and can be differentially and separated from non-CSCs. All of the
significant features of malignancies, such as tumour initiation, heterogeneity, therapeutic
resistance, recurrence, and metastasis, are triggered by CSCs.
Several experiments were done to evaluate the effect of long-term alcohol use on breast cancer
development/metastasis. The results of the experiment show that Chronic alcohol intake
increased the number of breast cancer stem cell- CSCs.

Figure A, shows that the ALDEFLUOR test was used to detect CSCs in human breast cancer cell
lines MCF7 or MCF7 overexpressing ErbB2 cells (MCF7-ErbB2). The cell lines were treated to
alcohol (0 or 100 mg/dl) for 10 days. Figure A, denotes a large difference between the control
groups (Control) and alcohol (EtOH) treated MCF7 cells. There number of positive cancer cells
increases in both MCF7 and MCF7-ErbB2 after alcohol was added to the cells. However, alcohol
increased the number of CSCs substantially more in MCF7-ErbB2 cells than in MCF7 that
expresses low level of ErbB2.
Figure B, illustrates that MCF7-ErbB2 cells were exposed to alcohol (0, 100 mg/dl) for 10 days
and then mammosphere formation was evaluated under the microscope. Figure A, shows that
the mammospheres grew in the MCF7-EtbB2 cells that were exposed to alcohol, there is an
increase in the formation of mammosphere in MCF7-ErbB2+EtOH.

Figure C,illustrates that for 10 days, alcohol (0, 100 mg/dl) was exposed to MCF7, MCF7-ErbB2
or BT474 cells. The number of mammospheres was determined by using relative
mammosphere ratio. There is a huge difference between the respective control groups
(Control) and alcohol treated (EtOH). In all three types of cells the relative mammosphere ratio
increased. However, alcohol increases significantly more the relative mammosphere ratio in
MCF7-ErbB2 and BT474 cells than in MCF7.

Figure D, shows that FVB MMTV Neu mice were fed with a liquid diet containing ethanol (0 or
6.7%). CD44+ is used as a cells surface marker to identify and qualify CSC population within
breast cancer cells. After alcohol exposure, the mammary tumour tissues were assessed for the
expression of CD44.Figure D, Shows that chronic alcohol (12 months) exposure increased CD44
positive cells in the mammary tumours.