A Novel Therapy for Melanoma Developed in Mice: Transformation of Melanoma into Dendritic Cells with Listeria monocytogenes
Fig 1
Listeria induced transformation of melanoma into dendritic cells.
A, Kinetic analysis of BMDC, murine (B16F10) and human (A-375 and Mel-H0) melanoma cells infected with different LM strains (LMWT, LMΔLLO). Results are expressed as CFU (mean ± SD) obtained with triplicate samples from three independent experiments (P<0.05). B, Different phagocytic parameters analysed in melanoma and BMDC: phagocytic rates after incubation with [35S]-labelled LM strains for 45 min (left plot). Radioactivity associated with cell lysates (CPM) was quantified in a β2 counter as the bacterial phagocytic rates. Results are expressed as cpm of internalized bacteria (mean ± SD) (p < 0.05). Replication indexes (RI) analysis is shown in middle plot. RIs were calculated as the ratio of the number of CFU at 16 h divided by the amount of CFU at 0 h. This parameter was considered as an indicator of bacterial growth. Results are expressed as CFU (mean ± SD) (p < 0.05). The percentages of cytosolic fractions are shown in right plot after purification of phagosomal and cytosolic fractions as in Material and Methods. Results are expressed as percentages of total internalized CFU in PNS (mean ± SD) (p < 0.05). C, Images correspond to confocal microscopy examination of melanoma and BMDC infected with GFP-LMWT. GFP-LMWT (green channel) co-localize with MHC-II molecules (red channel). Western blots correspond to the analysis in purified phagosomes for different MIIC markers: a/b stable MHC-II chains; Rab5a and LLO forms bound to MHC-class II molecules. CFU values of purified phagosomes are shown below western blots. D, BMDC and B16F10 infected with LM strains or non-infected (NI) were surface stained for the following markers: CD11c-PE, CD11b-FITC, F4/80-PE, CD40-PE, Gr-1-FITC and anti-MHC-II-APC. Samples were acquired using FACSCanto flow cytometer and percentages of positive cells for each antibody are shown. Results are expressed as the mean ± SD of triplicates (p<0.05). E, Same melanoma cells infected with different LM strains or non-infected (NI) as in D for 24 hours. Supernatants were recovered, filtered through 3 μm syringe to discard bacteria and the levels of pro-inflammatory cytokines MCP-1, TNF-alfa, IL-6, IL-10 or IL-12 were analysed using the CBA kit (Becton Dickinson) by flow cytometry. Results were expressed as cytokine concentration (pg/ml of mean ± SD, P<0,05).