الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Triple Negative Breast Cancer (TNBC) is a subgroup of breast cancer that accounts for approximately 15-20 % among breast cancer. TNBC have high heterogeneity and further can be classified into 6 subtypes. It is characterized by its aggressive behavior that is associated with large tumor size, high histological grade, lymph node involvement, poor prognosis, low survival rateand lack of targeted therapy.
On the hand, exosomes are nano-sized vesicles that carry cargoes of bioactive molecules, such as nucleic acids, proteins, lipids, and metabolites.They are involved in cell-cell communication by transferring molecular cargoes to recipient cells.Accordingly, the tumorigenic role of exosomes in enhancing the aggressive of TNBC has been suggested. In this aspect, JAK/STAT is a fundamental intracellular signaling pathway that assumes a key role in the regulation of cell proliferation, differentiation, migration, cell-cell communication, apoptosis, and angiogenesis. Several studies have reported the role STAT3 in TNBC where it was found to be constitutively activated in these cells. Additionally, Wnt/β-catenin signaling pathway is considered to be a critical pathway as it is central regulator of several cellular tumorigenic processes. Deregulation of Wnt/β-catenin signaling pathway in TNBCis closely implicated in disease progression and failure of the treatment methods through enhancing aggressiveness of the disease and resistance to chemotherapy. Meanwhile, autophagy is a catabolic lysosomal degradation pathway of defective cytoplasmic materials, misfolded proteins and damaged organelles that maintain cellular homeostasis and survival. It acts as a cell survival mechanism in response to different stress conditions such as hypoxia, starvation and reactive oxygen species (ROS).There is growing evidence that autophagy plays an important role in TNBC progression.
Thus, the main objective of the present study is to evaluatethe of exosomes in potentiating the tumorigenic behavior of TNBC cells. The proposed role of exosomes was evaluated in the view of the influences of down-regulation of STAT pathway in TNBC on Wnt signaling pathway and cell death mechanisms; apoptosis and autophagy. To approach the objective of the present study two types of breast cancer cell lines were utilized TNBC; MDA-MB231 (masenchymal stem-like) and HCC1806 (basal-like), and NTNBC; MCF7 (Estrogen-receptor-positive).
Accordingly, each cell line was divided into the following groups:
1. UN-Treated gp:
Untreated control cells
2. EXO gp:
Cells were treated with 20μl of exosomes (EXO) (equivalent to 20μg/protein); depending on the exosomes up-take test.
3. DOXO gp:
Cells were treated with IC50 of doxorubicin (DOXO)
4. EXO-DOXO gp:
Cells were treated with IC50 of DOXO and 20μl of EXO.
5. InSTgp:
Cells were treated with IC50 of AG490, STAT3 pathway inhibitor.
6. InST-EXO gp:
InST cells were treated cells with 20μl of EXO.
7. InST-DOXO gp:
InST cells were treated with IC50 of DOXO.
8. InST-EXO-DOXO gp:
InST cells were treated with IC50 of EXO and DOXO.
Summary and Conclusion
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In the present study, blood samples were collected from TNBC patients and exosomes were extracted characterized by scanning electron microscope (SEM) and detecting of protein biomarkers by western blotting (W.B). SEM analysis of exosomes revealed the presence of exosomes with their characteristic biconcave shape and different sizes (30 – 300 nm), as well as the large size subclass was predominant. Meanwhile, characterization of exosomal protein biomarkers by W.B revealed the presence of CD63, PCD6IP (Alix) and TSG101, indicating the presence of exosomal surface-marker proteins, where CD63 is the most abundant relative to TSG101 and PDCDIP.Additionally,W.B technique for detection of p-STAT3 (phosphorylated) and pan-STAT3 (un-phosphorylated) in each cell line before and AG490 treatment was carried out. The uptake of PKH67 labeled exosomes by MDA-MB231, HCC1806 and MCF7 cell lines was detected under fluorescent microscope (FM).The FM images showed that the exosomal uptake efficiency was higher in TNBC cell lines (slightly higher in MDA-MB231 cells than HCC1806 cells) than in NTNBC cell line (MCF7 cells). Morphologically, the treatment of TNBC cells with exosomes (EXO) lead to the elongation of the spindle shape structure of epithelial cell However, the treatment of NTNBC with exosomes resulted in cellular aggregates. Moreover, EXO treatment of both to TNBC and NTNBC cells resulted in an increase in cell proliferation, migration and colonization cells. All of which may suggest the ability of TNBC derived exosomes of significantly increasing recipient cells’ invasion potential via transferring functional cargo molecules to those cells promoting cell proliferation,colonization and migration. Morphological changes associated with DOXO treatment markedly affected NTNBC cells; MCF7 than TNBC cells;
MDA-MB231 and HCC1806, respectively. The observed alterations are in consistent with the changes in tumorigenic behavior of BC cells as reflected by proliferation, migration and colonization especially in MDA-MB231. However, EXO-DOXO treatment TNBC cells with still showed elongated spindle shape structures compared to DOXO treated cells. In the same context, treatment of TNBC and NTNBC cells with EXO-DOXO increased cell proliferation, migration and colonization of BC cells. All of these may add more evidence and support for the implication of exosomes in chemoresistance of BC cells to DOXO treatment. Interestingly, EXO treatment of InST-TNBC and InST-NTNBC cells revealed different responses compared to EXO treated cells. In InST-MDA-MB231, EXO treatment showed a decrease in the frequency of spindle shaped cells, while in InST-MCF7 cells, the cell aggregations and clumps were disappeared. In contrast, addition of EXO to InST-HCC1806 cells revealed the presence of cells mixture among them small circular shaped cells resemble hematopoietic stem cell like with increase in the cell viability as well as dead cells. The proliferation and migration of InST-TNBC and InST-NTNBC treated with EXO were less pronounced compared to EXO treated cells. Regarding colonization, InST-TNBC treated with EXO showed a reduction in the colony formation ability while, in InST-NTNBC cells treated with EXO, the ability for colony formation was prohibited. Thus, this may point out to the enhanced anti-tumorigenic effects of AG490 through STAT3 inhibition in both subtypes of BC. Meanwhile, it may provide more evidences that the tumorigenic influences of exosome may be mediated by the activity of STAT3 signaling. Moreover, the inhibition of STAT3 pathway enhanced the anti-tumor effects of DOXO treatment in BC cells. Moreover, treatment of InST-TNBC cells with EXO-DOXO caused a decrease in the proliferation, migration and colonization of cells compared to that in cells treated with EXO-DOXO only. While, in InST-NTNBC cells, the proliferation and migration were reduced whereas, colonization was completely diminished. These results may point out to the anti-tumorigenic effect of AG490.
Summary and Conclusion