الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
Breast cancer (BC) is the greatest frequently diagnosed cancer and the leading cause of cancer death among females worldwide including Egypt.
The tumor microenvironment (TME) and its associated molecules, infiltrating immune cells, soluble factors and altered extracellular matrix, are involved in promoting tumor growth and metastasis. Autophagy and cancer stem cells (CSCs) are two main players within the TME.
Autophagy is a form of programmed cell death that is activated under stress conditions, e.g. in the absence of adequate amounts of nutrients or as a result of damage caused by cell toxins (Borys et al 2017). On the other hand, cancer stem cells (CSCs) are immortal tumor-initiating cells that can self-renew and have pluripotent capacity which is liable for tumor genesis, growth of tumor, relapse, and drug resistance of numerous cancers including breast cancer (Chen et al 2013).
Increased serum level of the proinflammatory cytokine IL-8 in patients with BC has an independent prognostic significance for post relapse survival. Secretion of the multifunctional IL-8 by tumor cells promotes vascularization via enhancing endothelial cell proliferation, survival and matrix metalloproteinase production (A. Li et al., 2003). It has been suggested that targeting IL-8 signaling within the TME may stop disease progression and help in preparing tumors to chemotherapeutic and biological agents (Waugh & Wilson, 2008).
The aim of the current study was to investigate the autophagic activities and cancer stem cells in breast cancer tumor microenvironment supplemented with anti-IL-8 monoclonal antibodies.
To achieve this aim, the present study was conducted on 15 breast cancer patients enrolled for radical mastectomy at Department of Experimental and Clinical Surgery, Medical Research Institute, Alexandria University. Fresh sterile samples of primary breast tumor tissue as well as breast normal tissue were obtained immediately after surgical resection. These samples were used for construction of tumor tissue culture systems either enriched with or without anti-IL-8 mAbs. Autophagic activities and CSCs’ maintenance were evaluated within these tissue culture systems by immunofluorescence technique using LC3B and cell surface markers CD44/CD24 respectively.
The results of the current study revealed that the spontaneous autophagic activity in the breast tumor tissue culture system as measured by LC3B fluorescence intensity was significantly greater than that in corresponding normal ones (P=0.005). Neutralizing IL-8 activities within the breast tumor tissue culture system by its specific mAbs significantly decreased the tumor confined autophagic activities (P=0.002).
The CSCs maintenance represented by cell surface marker CD44 fluorescence intensity levels in untreated breast tumor tissue cultures was insignificantly higher than that in corresponding normal ones. (P=0.191). These levels of CD44 were significantly lowered in both tumor and normal tissue culture systems when they were supplemented with anti- IL-8 mAbs. (P= 0.012 , P=0.023 respectively ).
Summary, Conclusion and Recommendations
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The expression levels of cell surface marker CD24 in both tumor tissue culture systems either supplemented with or without anti-IL-8 mAbs were found to be higher than those detected in their corresponding normal ones. Supplementing either breast tumor or normal tissues with anti- IL-8 mAb resulted in decreased CD24 expression compared to their corresponding un-supplemented ones.
Our results also revealed that autophagy is positively associated with CD44 expression and negatively associated with CD24 expression within the breast tumor tissue culture system treated with anti-IL8 mAbs.
Most of the statistical correlations between levels of either autophagy or CSCs in different tissue culture systems created in the current study and the clinicopathological parameters of the studied patients were found to be insignificant.
Accordingly, we can conclude that in our own designed breast tumor/ normal tissue culture systems:
The spontaneous autophagic activity in tumor tissues is significantly greater than that in the corresponding normal ones. This autophagic activity is significantly decreased via neutralizing IL-8 effects by its specific monoclonal antibodies.
The CSCs maintenance as represented by CD44 in tumor tissues is statistically insignificantly higher than that in the corresponding normal ones. These levels of CD44 have been lowered in both tumor and normal tissues when they were supplemented with IL-8 mAbs.
The significant decreased level of CD44 expression in the breast normal tissue culture system supplemented with anti-IL8 mAb suggested that this tissue may contain CD44 expressing cancer initiating cells.
The CD24 expression levels in both tumor tissue culture systems either supplemented with or without IL-8 mAbs are higher than those detected in their corresponding normal tissue cultures.
Supplementing either breast tumor or normal tissues with IL-8 mAb leads to decreased CD24 expression compared to their corresponding non-supplemented ones.
Autophagy is positively associated with CD44 expression and negatively associated with CD24 expression within the breast tumor tissue culture system treated with anti-IL8 mAbs.
The ability of anti-IL-8 mAb to reduce both autophagic activities and stemness of the tumor cells within the breast TME indicates the effectiveness of neutralizing IL-8 activities by its specific monoclonal antibodies (anti-IL-8 mAb) that could be suggested as a novel approach for breast cancer immunotherapy.
Thus, we recommend developing a new immunotherapeutic strategy for BC based on the ability of anti-IL-8 mAb to suppress the TME local autophagic activities and CSCs maintenance. Achievement of consequent studies using large samples of BC patients of different stages of the disease is also recommended to identify the proper targeted patient populations of the proposed strategy. In addition, proceeding successive studies to determine the optimum anti-tumor effect of anti-IL-8 mAbs and performing early phase clinical trials to test the safety and efficacy of the proposed immunotherapeutic strategy are highly recommended.