The relationship between the Glasgow Microenvironment Score and markers of epithelial-mesenchymal transition in TNM II-III colorectal cancer.

Recently published work on the Glasgow Microenvironment Score (GMS) demonstrated its relevance as a biomarker in TNM II-III colorectal cancer (CRC). Epithelial-mesenchymal transition (EMT) markers in CRC have also shown promise as prognostic biomarkers. This study aimed to assess the relationship between GMS and markers of EMT in stage II-III CRC. A previously constructed tissue microarray of CRC tumors resected between 2000 and 2007 from the Western Infirmary, Stobhill, and Gartnavel General Hospitals in Glasgow was used. Immunohistochemistry was performed for 5 markers of EMT: E-cadherin, ß-catenin, Fascin, Snail, and Zeb1. Two-hundred and thirty-eight TNM II-III CRC with valid scores for all EMT markers and GMS were assessed. The prognostic significance of markers of EMT in this cohort and relationships between GMS and markers of EMT were determined. High cytoplasmic and nuclear ß-catenin and membrane Zeb-1 were significant for worse cancer-specific survival (hazard ratio [HR] 1.67, 95% confidence interval [CI] 1.01-2.76, P < .05; HR 2.22, 95% CI 1.24-3.97, P < .01; and HR 2.00, 95% CI 1.07-3.77, P = .03, respectively). GMS 0 was associated with low membrane Fascin (P = .03), whereas membrane and cytoplasmic Fascin were observed to be highest in GMS 1, but lower in GMS 2. Nuclear ß-catenin was lowest in GMS 0, but highest in GMS 2 (P = .03), in keeping with its role in facilitating EMT. Novel associations were demonstrated between GMS categories and markers of EMT, particularly ß-catenin and Fascin, which require further investigation in independent cohorts.

Mesothelin­specific T cell cytotoxicity against triple negative breast cancer is enhanced by 40s ribosomal protein subunit 3­treated self­differentiated dendritic cells.

Triple negative breast cancer (TNBC) lacks targeted treatment resulting in poor prognosis. Targeting overexpressing mesothelin (MSLN) using MSLN­specific T cells is an attractive treatment approach and the aim of the present study. The expression of MSLN in human TNBC paraffin sections was analyzed by immunohistochemistry. Lentiviral vector harbored granulocyte­macrophage colony stimulating factor (GM­CSF), interleukin­4 (IL­4) and MSLN cDNAs was constructed to generate self­differentiated myeloid­derived antigen­presenting­cells reactive against tumor expressing MSLN dendritic cell (MSLN­SmartDC) for MSLN­specific T cell activation. The results showed high MSLN in 32.8% of all breast cancer subtypes and 57% in TNBC. High MSLN was significantly associated with TNBC subtype and the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. MSLN­SmartDC exhibited comparable phenotype to DC generated by exogenous cytokine treatment and an addition of 40s ribosomal protein subunit 3 (RPS3), a toll­like receptor 4 ligand, enhanced DC maturation and function by upregulation of CD40, CD80 and CD83 expressions and IL­12p70 secretion. MSLN­specific CD8+CD69+ IFN­Î³+ T cells were detected in T cells activated by both MSLN­SmartDC and RPS3­MSLN­SmartDC. MSLN­specific T cells activated by these DCs showed more specific killing capability against naturally expressed MSLN­HCC70 and artificially MSLN­overexpressing MDA­MB­231 compared with parental MDA­MB­231 in both two dimensional (2D)­ and 3D­culture systems. In conclusion, the results demonstrated the efficacy of MSLN­SmartDC to promote MSLN­specific T cells response against TNBC and RPS3 can enhance the cytolytic activity of these T cells providing an alternative treatment approach for patients with TNBC.

Cytotoxic T Cells Activated by Self-differentiated Monocyte-derived Dendritic Cells Against Multiple Myeloma Cells.

BACKGROUND/AIM: B cell maturation antigen (BCMA) is an ideal target for adoptive T cell therapy of multiple myeloma (MM). In this study, we evaluated self-differentiated monocyte-derived dendritic cells expressing BCMA (SD-DC-BCMA) to activate T cells for killing MM cells. MATERIALS AND METHODS: Lentivirus-modified SD-DC-BCMA harboring tri-cistronic cDNAs encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and BCMA was generated. Cytotoxicity of T cells activated by SD-DC-BCMA against MM cells was evaluated. RESULTS: T cells activated by SD-DC-BCMA exhibited a dose-dependent cytotoxicity against BCMA-expressing MM cells and produced high IFN-γ levels, compared to inactivated T cells or control T cells. A significantly higher killing ability of T cells activated by SD-DC-BCMA was further demonstrated in BCMA-overexpressing cells when compared with BCMA-negative cells. CONCLUSION: The potency of SD-DC-BCMA to activate T cells for antigen-specific cancer killing provides a framework for therapeutic application of adoptive T cell therapy in MM.

Adoptive Transfer of Anti-Nucleolin T Cells Combined with PD-L1 Inhibition against Triple-Negative Breast Cancer.

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.

Breast cancer stem cell RNA-pulsed dendritic cells enhance tumor cell killing by effector T cells.

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.