Cancer stem cells (CSCs) underpin the resistance of breast cancer (BC) cells to therapy. Dendritic cell (DC)-based treatment is efficacious and safe, but the efficiency of this technique for targeting CSCs in BC treatment requires further investigation. The present study aimed to investigate the ability of DCs pulsed with breast CSC antigens to activate effector lymphocytes for killing BC cells. CD44 + /CD24-CSCs were isolated from BCA55-121, an in-house patient-derived BC cell line, and acquisition of stemness properties was confirmed by upregulated expression of OCT4A and a superior proliferative capacity in colony formation assays compared with whole population of BCA55-121 (BCA55-121-WP). DCs were differentiated from monocytes from peripheral blood of healthy donors and pulsed with CSC total RNA. Maturation of the CSC RNA-pulsed DCs was confirmed by increased expression of CD11c, CD40, CD83, CD86 and HLA-DR, as well as reduced CD14 expression compared with monocytes. Total lymphocytes co-cultured with CSC RNA-pulsed DCs were analyzed by flow cytometry for markers including CD3, CD4, CD8, CD16 and CD56. The results revealed that the co-cultures contained mostly cytotoxic CD8 + T lymphocytes followed by CD4 + T lymphocytes and smaller populations of natural killer (NK) and NKT cells. ELISA was used to measure IFN-γ production, and it was revealed that activated CD4 + and CD8 + lymphocytes produced more IFN-γ compared with naïve T cells, suggesting that CD8 + T cells were effector T cells. CSC RNA was a more efficient antigen source compared with RNA from mixed BC cells for activating tumor antigen-specific killing by T cells. These CSC-specific effector T cells significantly induced BC cell apoptosis at a 20:1 effector T cell:tumor cell ratio. Of note, the breast CSCs cultures demonstrated resistance to effector T cell killing, which was in part due to increased expression of programmed death ligand 1 in the CSC population. The present study highlights the potential use of CSC RNA for priming DCs in modulating an anticancer immune response against BC.
Dendritic cell (DC)–based T-cell activation is an alternative immunotherapy in breast cancer. The anti-programmed death ligand 1 (PD-L1) can enhance T-cell function. Nucleolin (NCL) is overexpressed in triple-negative breast cancer (TNBC). The regulation of PD-L1 expression through autophagy and the anti–PD-L1 peptide to help sensitize T cells for NCL-positive TNBC cell killing has not been evaluated. Results showed the worst clinical outcome in patients with high NCL and PD-L1. Self-differentiated myeloid-derived antigen-presenting cells reactive against tumors presenting NCL or SmartDCs-NCL producing GM-CSF and IL-4, could activate NCL-specific T cells. SmartDCs-NCL plus recombinant human ribosomal protein substrate 3 (RPS3) successfully induced maturation and activation of DCs characterized by the reduction of CD14 and the induction of CD11c, CD40, CD80, CD83, CD86, and HLA-DR. Interestingly, SmartDCs-NCL plus RPS3 in combination with anti–PD-L1 peptide revealed significant killing activity of the effector NCL-specific T cells against NCLHigh/PD-L1High MDA-MB-231 and NCLHigh/PD-L1High HCC70 TNBC cells at the effector: a target ratio of 5:1 in 2-D and 10:1 in the 3-D culture system; and increments of IFNγ by the ELISpot assay. No killing effect was revealed in MCF-10A normal mammary cells. Mechanistically, NCL-specific T-cell–mediated TNBC cell killing was through both apoptotic and autophagic pathways. Induction of autophagy by curcumin, an autophagic stimulator, inhibited the expression of PD-L1 and enhanced cytolytic activity of NCL-specific T cells. These findings provide the potential clinical approaches targeting NCLHigh/PD-L1High TNBC cells with NCL-specific T cells in combination with a PD-L1 inhibitor or autophagic stimulator.
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