Secretion of extracellular vesicles (EVs) is a ubiquitous mechanism of intercellular communication based on the exchange of effector molecules, such as growth factors, cytokines, and nucleic acids. Recent studies identified tumor-derived EVs as central players in tumor progression and the establishment of the tumor microenvironment (TME). However, studies on EVs from classical Hodgkin lymphoma (cHL) are limited. The growth of malignant Hodgkin and Reed–Sternberg (HRS) cells depends on the TME, which is actively shaped by a complex interaction of HRS cells and stromal cells, such as fibroblasts and immune cells. HRS cells secrete cytokines and angiogenic factors thus recruiting and inducing the proliferation of surrounding cells to finally deploy an immunosuppressive TME. In this study, we aimed to investigate the role of tumor cell-derived EVs within this complex scenario. We observed that EVs collected from Hodgkin lymphoma (HL) cells were internalized by fibroblasts and triggered their migration capacity. EV-treated fibroblasts were characterized by an inflammatory phenotype and an upregulation of alpha-smooth muscle actin (α-SMA), a marker of cancer-associated fibroblasts. Analysis of the secretome of EV-treated fibroblast revealed an enhanced release of pro-inflammatory cytokines (e.g., IL-1α, IL-6, and TNF-α), growth factors (G-CSF and GM-CSF), and pro-angiogenic factors such as VEGF. These soluble factors are known to promote HL progression. In line, ingenuity pathway analysis identified inflammatory pathways, including TNF-α/NF-κB-signaling, as key factors directing the EV-dependent phenotype changes in fibroblasts. Confirming the in vitro data, we demonstrated that EVs promote α-SMA expression in fibroblasts and the expression of proangiogenic factors using a xenograft HL model. Collectively, we demonstrate that HL EVs alter the phenotype of fibroblasts to support tumor growth, and thus shed light on the role of EVs for the establishment of the tumor-promoting TME in HL.
The most common intra-ocular malignancy is uveal melanoma (UM). Metastatic disease occurs in approximately 50% of UM patients and presents most frequently in the liver. Novel therapies that prevent the development of metastases are needed and immunotherapy is a potential option. Because of its origin in the immune-privileged eye, UM may be particularly responsive to T cell-based immunotherapy. MelanA is frequently over-expressed by human cutaneous melanomas. MelanA expression has also been detected in UM primary and metastatic tumor-tissue sections and could serve as a target antigen for immunotherapy of UM. Here we report that primary and metastatic UM cell lines express MelanA and that this protein can be used as a target antigen for transfection of mature dendritic cells (mDC). MelanA expression was confirmed in primary and metastatic UM cell lines using intracellular flow cytometry and quantitative PCR (qPCR). In addition, sequencing of UM-derived MelanA demonstrated that MelanA in primary and metastatic UM cells has an identical sequence as compared to the published MelanA consensus sequence. As an initial step towards activating UM-specific T cells by UM-derived mRNA transfected DC we are generating MelanA mRNA transfected mDC. To facilitate both MHC class I and class II presentation of MelanA epitopes after mRNA electroporation into mDC, we included the endosomal targeting sequence DCLamp linked to the MelanA protein. Monocytes were isolated from leukapheresis products of healthy donors by density centrifugation and plastic adherence. Monocytes were differentiated to monocyte-derived DC by culturing with IL-4 and GM-CSF for 6 days to obtain immature DC (iDC). iDCs were matured with a cytokine mixture containing IL-4, GM-CSF, IL-1α, IL-6, TNFα, and PGE2 for 48 hours. The resulting mDC expressed cell surface maturation markers CD25, CD83, and CD86 as confirmed by flow cytometry. mDC were transfected with either MelanA-mRNA of MelanA-DCLamp-mRNA, using an established electroporation protocol. To assess transfection efficiency and monitor MelanA expression in transfected mDC, MelanA-mRNA and MelanA-DCLamp-mRNA expression was demonstrated by qPCR and MelanA-protein expression was shown by intracellular flow cytometry at different time points post-transfection using variable mRNA concentrations. The detected amount of MelanA-mRNA and protein was dependent on the amount of transfected mRNA and presented transiently. Subsequently, total RNA was isolated from MelanA+ primary and metastatic UM cell lines, then reversely transcribed into cDNA, amplified by PCR and in vitro transcribed to mRNA. As confirmed by qPCR, MelanA-mRNA was present in the pool of UM-derived mRNA and can be used for transfection of mDC. Collectively, these data demonstrate that mDC can be transfected with MelanA-mRNA and suggest MelanA could serve as a target for immunotherapy of UM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1585. doi:1538-7445.AM2012-1585
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