الفهرس 
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Abstract
Breast cancer is a prominent cause of morbidity among women worldwide, where
epidemiological studies reported that one in 8 women will develop breast cancer in her
lifetime. In females, breast cancer is the most frequently diagnosed cancer in developed
countries and ranks the second in developing countries, and it is also a major cause of death
worldwide. In Egypt Carcinoma of the breast is the most prevalent cancer among Egyptian
women. Egypt also has a rising burden of breast cancer, where its incidence is projected to
increase by 1‑2% every year.
Radiotherapy is a principal mode of breast cancer treatment. It uses the potentiality of
ionizing radiation to cause apoptosis. One of the major treatment problems is resistance,
which result in ineffectiveness of therapy and breast cancer recurrence. Radioresistant cancer
cells do not respond to radiotherapy and the disease then becomes difficult to control.
The CXC chemokine, CXCL12, and its seven transmenmbrane G-protein coupled
receptor, CXCR4, are implicated with several types of cancer including breast cancer; where
both are highly expressed, in case of cancer, in tumor fibroblasts and cancer cells,
respectively. Their binding stimulates a signaling cascade that is associated with the behavior
of cancer cells, where it modulates cell migration, proliferation, and survival.
Thus, we aimed to evaluate the role of CXCR4 as a potential therapeutic target in
breast cancer through investigating the CXCR4 antagonist, AMD3100, effect with or without
radiotherapy in vitro. Hence, in turn we studied breast cancer cell radioresistance; which can
be referred to the tumor structure, its microenvironment and the effect of radiation itself.
These were the main axes we focused on during this study. We studied them through focusing
on the role of CXCL12 and CXCR4 and the effect of the CXCR4 antagonist AMD3100.
Monolayer cultures are the simplest and most convenient mode, but it fails to mimic
the in vivo tumors. Hence, it ignites the motivation to shift to a more complex 3D cultures;
which serves as a bridge between the simplest monolayers and the highly complex and
heterogeneous in vivo tumors. MCTSs were cultured by using the liquid overlay technique;
which is based on the ability of cells to aggregate and undergo self-assembly when grown
over a non-adhesive surface. Unlike MDA-MB-231, MCF-7 spontaneously formed
compacted and rigid MCTSs with high tolerance against mechanical forces. These MCTSs
were characterized with its multilayer cell assembly; which mimics small avascular tumors.
In breast cancer, microenvironmental CXCL12 directly stimulates paracrine growth
mechanism of CXCR4 expressing tumor cells. CXCL12 was used as a stimulator for the
MCF-7 surface receptor CXCR4 in order to mimic the impact of microenvironmental
CXCL12; to evaluate its ability to provide a protective microenvironment that improves cell
survival. CXCL12 increased the cell proliferative activity of both monolayer and MCTSs
cultured MCF-7 in a dose dependent manner along the assaying time. Also, we proved that
longer time exposure to CXCL12 increases the cell proliferation rate. Altogether draws the
attention to the role of tumor microenvironment.
We evaluated the role of CXCL12-CXCR4 as a promising therapeutic molecular
target, by studying the effect of AMD3100 on MCF-7 cells. AMD3100 is a non-peptide
specific CXCR4 antagonist. Our results revealed that the administration of AMD3100 alone
is not effective in inhibiting cell proliferative activity. Although sometimes the higher
Chapter 6: Summary
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concentration of AMD3100 exerted a significant inhibitory effect in unstimulated cells, but it
was insignificant in the stimulated ones. Hence, AMD3100 may has a dual effect. However,
the variance in the effect of AMD3100 alone on the cell, express an urge for further studying
of its role.
We evaluated the role of tumor structure in radioresistance, where MCTSs were more
resistant to radiation than cells in monolayer culture. We also evaluated the effect of tumor
microenvironment on cancer cell resistance, where CXCL12 increased the cell proliferation
rate in a dose dependent manner. However, MCTSs demonstrated more resistance to
irradiation along the increment of irradiation doses. We also indicated that longer exposure
time to CXCL12 renders the cells to be more resistant to ionizing radiation. Thus, featuring
the radioprotective role of tumor microenvironment.
We further investigated the role of CXCR4 as a possible therapeutic target by treating
the cells with CXCR4 antagonist AMD3100 and radiation. Our results demonstrated that
AMD3100 sensitizes the MCF-7 breast cancer cells to radiation in a dose-dependent manner.
It also demonstrated that AMD3100 was more effective at 72 hr post-treatment than at 48 hr;
manifesting the role of time as a factor related with AMD3100 so it could exert its inhibitory
effect. Hence, combination of AMD3100 with irradiation and targeting CXCL12-CXCR4
axis are promising treatment routes, where it could attenuate the breast cancer cells growth
and augment the effect of ionizing radiation.
MCF-7 monolayers were tested for alteration of the expression of the cell surface
receptor CXCR4, where irradiation of MCF-7 cells showed significant increase in the
expression of the surface receptor CXCR4 along with the increment in the radiation dose;
which highlights the role of CXCR4 in tumor radioresistance. Although all the groups
demonstrated dose-dependent increase in the CXCR4 receptor expression, they behaved
differently at each single dose. Out of all the unirradiated groups, only AMD3100 treated
group showed significant slight inhibition of surface receptor CXCR4.However, it was
abrogated by CXCL12. Hence, signifying the importance of tumor microenvironment and its
effect on tumor cell behavior and response to therapy.
All in all, we demonstrate in our study that breast cancer cell line MCF-7 therapy
resistance is imposed by the 3D structure of the in vivo tumor that imposes multicellular
resistance. Also the tumor microenvironment provides CXCL12 which is critical for the
signaling axis resulting in radioresistance; as our study has shown that CXCL12 protects
breast cancer cells from radiotoxicity in vitro. CXCR4 is a possible radioresistance marker as
its expression increases along the increment of the radiation dose. Hence, our results signify
that CXCR4 is a potential molecular target for therapy sensitization. Thus, our results indicate
that CXCL12-CXCR4 signaling pathway is highly implicated with breast cancer cell
resistance to radiotherapy; which in turn imposes a major clinical impediment and a real
challenge to researcher in this field.