Biological and molecular studies on specific immune cells treated with checkpoint inhibitors for the thera-personal approach of breast cancer patients (ex-vivo study).

Immunotherapy becomes a promising line of treatment for breast cancer (BC) however, its success rate is still limited. Methods: The study was designed to optimize the condition for producing an effective dendritic cell (DCs) based immunotherapy by using DCs and T lymphocytes together with tumor-infiltrating lymphocytes (TILs) and tumor-infiltrating DCs (TIDCs), treated with anti-PD1 and anti-CTLA4 monoclonal antibodies. This mixture of immune cells was co-cultured with autologous breast cancer cells (BCCs) isolated from 26 BC females. Results: There was a significant upregulation of CD86 and CD83 on DCs (p = 0.001 and 0.017, respectively), similarly upregulation of CD8, CD4 and CD103 on T cells (p = 0.031, 0.027, and 0.011, respectively). While there was a significant downregulation of FOXP3 and combined CD25.CD8 expression on regulatory T cells (p = 0.014 for both). Increased CD8/Foxp3 ratio (p < 0.001) was also observed. CD133, CD34 and CD44 were downregulated on BCCs (p = 0.01, 0.021, and 0.015, respectively). There was a significant increase in interferon-γ (IFN-γ, p < 0.001), lactate dehydrogenase (LDH, p = 0.02), and a significant decrease in vascular endothelial growth factor (VEGF, p < 0.001) protein levels. Gene expression of FOXP3 and Programmed cell death ligand 1 (PDL-1) were downregulated in BCCs (p < 0.001, for both), similarly cytotoxic T lymphocyte antigen-4 (CTLA4, p = 0.02), Programmed cell death 1 (PD-1, p < 0.001) and FOXP3 (p < 0.001) were significantly downregulated in T cells. Conclusion: Ex-vivo activation of immune cells (DCs, T cells, TIDCs, and TILs) with immune checkpoint inhibitors could produce a potent and effective BC immunotherapy. However, these data should be validated on an experimental animal model to be transferred to the clinical setting.

A novel potential effective strategy for enhancing the antitumor immune response in breast cancer patients using a viable cancer cell-dendritic cell-based vaccine.

Dendritic cells (DCs) have been used in a number of clinical trials for cancer immunotherapy; however, they have achieved limited success in solid tumors. Consequently the aim of the present study was to identify a novel potential immunotherapeutic target for breast cancer patients through in vitro optimization of a viable DC-based vaccine. Immature DCs were primed by viable MCF-7 breast cancer cells and the activity and maturation of DCs were assessed through measuring CD83, CD86 and major histocompatibility complex (MHC)-II expression, in addition to different T cell subpopulations, namely CD4+ T cells, CD8+ T cells, and CD4+CD25+ forkhead box protein 3 (Foxp3)+ regulatory T cells (Tregs), by flow cytometric analysis. Foxp3 level was also measured by enzyme-linked immunosorbent assay (ELISA) in addition to reverse-transcription quantitative polymerase chain reaction. The levels of interleukin-12 (IL-12) and interferon-γ (IFN-γ) were determined by ELISA. Finally, the cytotoxicity of cytotoxic T lymphocytes (CTLs) was evaluated through measuring lactate dehydrogenase (LDH) release by ELISA. The results demonstrated that CD83+, CD86+ and MHC-II+ DCs were significantly elevated (P<0.001) following priming with breast cancer cells. In addition, there was increased activation of CD4+ and CD8+ T-cells, with a significant decrease of CD4+CD25+Foxp3+ Tregs (P<0.001). Furthermore, a significant downregulation of FOXP3 gene expression (P<0.001) was identified, and a significant decrease in the level of its protein following activation (P<0.001) was demonstrated by ELISA. Additionally, significant increases in the secretion of IL-12 and IFN-γ (P=0.001) were observed. LDH release was significantly increased (P<0.001), indicating a marked cytotoxicity of CTLs against cancer cells. Therefore viable breast cancer cell-DC-based vaccines could expose an innovative avenue for a novel breast cancer immunotherapy.