ABSTRACT
Protein-coated microbeads provide a consistent approach for activating and expanding populations of T cells for immunotherapy but do not fully capture the properties of antigen presenting cells. In this report, we enhance T cell expansion by replacing the conventional, rigid bead with a mechanically soft elastomer. Polydimethylsiloxane (PDMS) was prepared in a microbead format and modified with activating antibodies to CD3 and CD28. A total of three different formulations of PDMS provided an extended proliferative phase in both CD4+-only and mixed CD4+-CD8+ T cell preparations. CD8+ T cells retained cytotoxic function, as measured by a set of biomarkers (perforin production, LAMP2 mobilization, and IFN-γ secretion) and an in vivo assay of targeted cell killing. Notably, PDMS beads presented a nanoscale polymer structure and higher rigidity than that associated with conventional bulk material. These data suggest T cells respond to this higher rigidity, indicating an unexpected effect of curing conditions. Together, these studies demonstrate that adopting mechanobiology ideas into the bead platform can provide new tools for T cell based immunotherapy.
Subject(s)
Dimethylpolysiloxanes/chemistry , Microspheres , T-Lymphocytes/cytology , Antibodies/chemistry , CD28 Antigens/immunology , CD3 Complex/immunology , Cell Proliferation , Cell Survival , Emulsions , Humans , Immunotherapy , Particle Size , Surface Properties , T-Lymphocytes/physiology , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/physiologyABSTRACT
BACKGROUND: Non-small cell lung cancer (NSCLC) is a highly metastatic cancer with limited therapeutic options, so development of novel therapies that target NSCLC is needed. During the early stage of metastasis, the cancer cells undergo an epithelial-mesenchymal transition (EMT), a phase in which Wnt/ß-catenin signaling is known to be involved. Simultaneously, AEG-1 has been demonstrated to activate Wnt-mediated signaling in some malignant tumors. METHODS: Human NSCLC cell lines and xenograft of NSCLC cells in nude mice were used to investigate the effects of AEG-1 on EMT. EMT or Wnt/ß-catenin pathway-related proteins were characterized by western blot, immunofluorescence and immunohistochemistry. RESULTS: In the present study, we demonstrated that astrocyte elevated gene-1(AEG-1) ectopic overexpression promoted EMT, which resulted from the down-regulation of E-cadherin and up-regulation of Vimentin in lung cancer cell lines and clinical lung cancer specimens. Using an orthotopic xenograft-mouse model, we also observed that AEG-1 overexpression in human carcinoma cells led to the development of multiple lymph node metastases and elevated mesenchymal markers such as Vimentin, which is a characteristic of cells in EMT. Furthermore, AEG-1 functioned as a critical protein in the regulation of EMT by directly targeting multiple positive regulators of the Wnt/ß-catenin signaling cascade, including GSK-3ß and CKIδ. Notably, overexpression of AEG-1 in metastatic cancer tissues was closely associated with poor survival of NSCLC patients. CONCLUSIONS: These results reveal the critical role of AEG-1 in EMT and suggest that AEG-1 may be a prognostic biomarker and its targeted inhibition may be utilized as a novel therapy for NSCLC.
