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 and has high heterogeneity and further can be classified into 6 subtypes. It is characterized by its aggressive behavior that is related to large tumor size, high histological grade, lymph node involvement, poor prognosis, low survival rate and lack of targeted therapy.
On the hand, exosomes are nano-sized vesicles that carry cargoes of bioactive molecules, like 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 supposed to be a key role in the regulation of cell proliferation, differentiation, migration, cell-cell communication, apoptosis, and angiogenesis. Many studies have reported the role STAT3 in TNBC where it was found to be constitutively activated in these cells. Additionally, Notch signaling regulates the discrimination of breast epithelial cells during normal development and plays a critical role in breast cancer development via the abnormal expression of the Notch up-and down-stream effectors. Thus, the main objective of the present study is to evaluate the 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 Notch signaling pathway and cell death mechanisms. To approach the objective of the present study two types of breast cancer cell lines were utilized; TNBC, MDA-MB231 (mesenchymal stem-like) and HCC1806 (basal-like), and NTNBC, MCF7 (Estrogen-receptor-positive). Accordingly, each cell line was divided into the following groups:
Un-Treated gp:
Untreated cells
EXO gp:
Cells were treated with 20μl of exosomes (EXO) (equivalent to 20μg/protein); depending on the exosomes up-take test.
DOXO gp:
Cells were treated with IC50 of doxorubicin (DOXO)
EXO-DOXO gp:
Cells were treated with 20μl of EXO and IC50 of DOXO
InST gp:
Cells were treated with IC50 of AG490, STAT3 pathway inhibitor.
InST-EXO gp:
InST cells were treated cells with 20μl of EXO.
InST-DOXO gp:
InST cells were treated with IC50 of DOXO.
InST-EXO-DOXO gp:
InST cells were treated with 20μl of EXO and IC50 of DOXO.
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 and pan-STAT3 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).
Summary and Conclusion
57
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.
Regarding cell cycle analysis of TNBC and NTNBC cells, treated with EXO revealed an increase in cell population arrested at the S-phase. Meanwhile, the apoptotic assay revealed an increase in necrotic cells. These observations may suggest tumor progression and aggressiveness thus throwing more lights on the roles of exosomes in strengthen tumorigenic behavior of NTNBC or TNBC. Cell cycle analysis of DOXO treated TNBC cells revealed an increase in cell population arrested at Go/G1 denoting its pro-apoptotic effect. Also, apoptosis assay showed an increase in the percentage of late apoptotic and necrotic cells in MDA-MB231, whereas in HCC1806 cells only necrosis was observed. However, treatment of NTNBC cells with DOXO led to the increase in cells arrested at Sub G1 revealing its pro-apoptotic effect. The results of cell cycle analysis of TNBC cells treated with EXO-DOXO may suggest cell cycle progression. Moreover, the results and observations may support more the role of exosomes in enhancing the tumorigenic behavior of recipient BC cells especially in induction of chemoresistance of BC cells to DOXO treatment.
Summary and Conclusion
58
However, EXO treatment of both InST-TNBC and InST-NTNBC cells revealed an increase in apoptotic cells accompanied by a decrease in proliferative cells compared to EXO treated cells. Notably, this observation is compatible with that of proliferative assay. On the contrary, cell cycle analysis of InST-TNBC cells showed an increase in cell population arrested at G2/M phase associated with the decrease in G0/G1 compared to EXO treated cells. These observations may point out to the enhanced tumorigenic role of exosomes in TNBC cells than NTNBC owing either to the origin of exosomes or to the type of recipient cells. Meanwhile, treatment of InST-TNBC and InST-NTNBC cells with DOXO showed the prevalence of apoptotic cells which may suggest that inhibition of STAT3 signaling pathway increased breast cancer cell response to the proapoptotic effect of DOXO (with different extents). In addition, apoptotic assay of InST-TNBC and InST-NTNBC cells with EXO-DOXO showed that the majority of cells were in apoptotic phase compared to EXO-DOXO treated cells without inhibition which may point out to the anti-cancer effect of AG490. In addition, the present study aimed to investigate the influence of exosomes on TNBC tumorigenic behavior through Notch signaling pathway. The genes expression of Notch signaling pathway markers, NOTCH1, NOTCH2, JAG1, HES1 and DLL4 in MDA-MB231, HCC1806 and MCF7 after different treatments were assessed via qRT-PCR. The results of gene expression of Notch pathway markers may point out to the crucial and important role of exosomes in potentiating the tumorigenic behavior of BC especially TNBC including tumor progression, metastasis and chemoresistance.
In conclusion, the present study is carried out to evaluate the role of human TNBC-derived exosomes in potentiating the aggressiveness of different sub-types TNBC as well as NTNBC tumors. The results revealed that exosomes may participate in tumor progression, invasion, and migration in addition to metastasis and chemoresistance. Additionally, the results showed that exosomes may have a significant role in manipulating several signaling pathways implicated in cell cycle progression and tumorigenesis such as Notch signaling pathway. Thus, the present research study may provide more evidences on the tumorigenic role of exosomes in the aggressiveness of TNBC. In this aspect, it could be suggested that the mechanisms involved in the proposed tumorigenic role of exosomes may include Notch signaling pathway mediated through STAT3 signaling.