الفهرس 
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Abstract
Breast cancer (BC) is a heterogeneous disorder with different pathological and histological characteristics. The tumor microenvironment (TME) of breast cancer is a critical contributing factor to immune evasion and immunotherapeutic resistance. The majority of tumor infiltrating lymphocytes (TILs) within the TME consists of CD4+ helper cells, CD4+ regulatory T-cells and CD8+ cytotoxic T lymphocytes (CTLs).
Regulatory T cells (Tregs), have been extensively characterized in the peripheral blood and immune infiltrates of different cancers including breast cancer, contributing to the development of an immunosuppressive TME.
Interleukin- 2 (IL-2) is a multifunctional cytokine that is produced mainly by CD4+ T cells and, to a lesser extent by CD8+ T cells, NK cells and NKT cells. IL-2 involves in the activation and differentiation of T and B cells, enhances the cytolytic functions of NK cells as well as activation and proliferation of several non-lymphoid cells. The IL-2 administration is reported to induce apparently curative and durable regressions in cancer patients such as in metastatic melanoma and renal cell cancer.
The current study aimed at investigating the effect of supplementing breast cancer TME with interleukin-2 on the level of tumor infiltrating T regulatory cells.
To achieve this aim, freshly excised breast tumor/normal tissue samples were collected from twenty Egyptian females who were scheduled for modified radical mastectomy for histologically proved breast cancer. Levels of cell surface marker CD4 and CD4/CD25 expressing cells have been detected in our own designed breast tumor/ normal tissue culture systems supplemented with /without exogenous IL-2 using immunofluorescence technique.
In the current study, nineteen patients (95%) showed invasive ductal carcinoma (IDC). According to TNM classification, 50% of patients presented as stage II, 35% of patients were stage III while 15% of patients were stage I.
Regarding the tumor size, 60% of patients were having tumor of size T2 followed by T3 constituting 35% of patients and 5% of patients with T1. No metastasis was reported among studied patients.
Results of our study revealed that the detected levels of the cell surface molecule CD4 in either breast tumor or normal tissues cultured without exogenous IL-2 are significantly higher than those detected in corresponding tissues cultured with exogenous IL-2 ( p <0.001).
Results also revealed that breast tumor tissue cultured without exogenous IL-2 showed higher CD4 level (p=0.118) than that of corresponding normal ones, while CD4 level has been found to be significantly lower (p=0.001) in breast tumor tissue cultured with exogenous IL-2 than in corresponding breast normal tissue.
These results highlights the spontaneous situations of both tissue culture systems which represent the actual cellular and molecular components of intact TME and breast normal tissue of each breast cancer patient under the study. In addition, they reflect the magnitude of tumor infiltrating CD4+ cell population. IL-2 sensitive CD4+ cells are abundant in TME resulting in increased IL-2 inducing apoptosis of these cells than in breast normal cells that is represented as decreased number of CD4+ cells in IL-2 treated tumor tissue than that of corresponding normal one.
Our study also showed significant lower level of detected CD4+CD25+ cells in breast tumor tissue cultured with exogenous IL-2 than without exogenous IL-2 (p <0.001). This is due to the consumption of IL‐2 by high affinity IL‐2 receptor CD25 that in turn, cause shortage of unbounded IL-2 in the TME. This insufficient amount of free IL-2 could not activate T cell proliferation including CD4+CD25+ cells themselves.
Also, level of detected CD4+ CD25+ cells in breast normal tissue cultured with exogenous IL-2 is significantly higher than that of corresponding breast normal tissues cultured without exogenous IL-2 (where p=0.049). This result highlights the fact that IL-2 activates CD4+cells in the steady state and stimulates their differentiation into different CD4+ subsets including CD4+ CD25+Tregs.
However, level of detected CD4+CD25+ cells in breast tumor tissue cultured with exogenous IL-2 was found to be significantly lower than that of corresponding breast normal tissues cultured with exogenous IL-2 (p<0.001). This result underlines the different structural component between breast TME with more IL-2 sensitive CD4+ CD25+ cells and the steady state breast normal tissue. Therefore, consumption of IL‐2 by TME high affinity IL‐2 receptor CD25 is increased by the exogenous IL-2 that in turn cause decreased free IL-2 within the TME.
Our results also revealed that level of detected CD4+CD25+ cells in breast tumor tissue cultured without exogenous IL-2 was found to be significantly higher than that of corresponding breast normal tissues cultured without exogenous IL-2 (p=0.249). This result represents the spontaneous proliferation of CD4+CD25+ cells within their ex vivo tissue culture systems. Breast tumor tissue culture system symbolize the actual TME where the level of tumor infiltrating Treg cells are much more higher than those in breast normal one.
Results of the current study also revealed a significant positive correlation between the levels of CD4+T cells and CD4+ CD25+ T regulatory cells in either breast tumor or normal tissue cultured without exogenous IL-2 (p= 0.003, p<0.001). This also represents the natural relationship between the two cell populations regarding CD4+ CD25+ T cells as a subset of CD4+ T cells.
In the current study, we also correlated the levels of CD4+ CD25+ T reg cells and CD4+ cells with the clinical and pathological outcomes of the patients. Level of CD4+ cells was significantly higher in breast tumor tissue cultured with IL-2 of patients with tumor sizes less than 2cm (T1) and in stage I breast tumor than later stages. This result can be explained regarding CD4+ cells like many other TILs of early stage breast cancer patients still retain their intact signal transduction activities (IL-2/IL-2R signaling pathway)than later stage.
In the current study, the mean percentage of CD4+ CD25+ Tregs were significantly higher in breast tumor tissue cultured with IL-2 of patients with tumor sizes more than 2cm (p=0.039) than tumor sizes less than 2cm and higher in later stages (p=0.026) than in early breast cancer stages. This result follows the fact that tumor size is a time-d ependent prognostic factor where larger tumor size is associated with later cancer stage which is characterized by immune suppressive TME and high level CD4+ CD25+ Tregs.
Hence, we can propose a novel breast cancer IL-2 mediated immunotherapeutic strategy through eliminating the magnitude of immune suppressive CD4+ CD25+ T reg cells in the TME that is in turn transformed into immune supportive one.
6.2. Conclusion
According to the current study findings, it can be concluded that:
1. High affinity IL‐2 receptor CD25 expressed on CD4+CD25+ Treg cells in breast cancer tumor tissue results in consumption of exogenous IL-2 that is leading to decreased the magnitude of both CD4+ and CD4+CD25+ levels in TME .
2. The IL-2 suppressive effect on tumor infiltrating CD4+ CD25+ T regulatory cells detected in the current study demonstrates a novel mechanism of a significant value as an anti-tumor approach of breast cancer.
3. Both CD4+ and CD4+CD25+ T cell population in TME of early stage breast cancer patients are effectively responded to exogenous IL-2 than those in the TME of later stage patients.
4. Early stage breast cancer patients may represent the proper group of patient targeted by the current proposed IL-2 mediated therapeutic strategy.
6.3. Recommendations
from our findings, we recommend the following:
1. IL-2 as a promising immunotherapeutic approach of breast cancer.
2. Using breast tumor tissue samples from large patient population of different stages of breast cancer to identify the proper targeted patient population.
3. Using different concentrations of the IL-2 to determine the optimum concentration that gives the optimum anti-tumor activities in the breast tumor tissue culture system.
4. Proceeding early phase clinical trials to test the safety and efficacy of the proposed IL-2 anti- cancer immunotherapeutic strategy.