A Novel Therapy for Melanoma Developed in Mice: Transformation of Melanoma into Dendritic Cells with Listeria monocytogenes

Listeria monocytogenes is a gram-positive bacteria and human pathogen widely used in cancer immunotherapy because of its capacity to induce a specific cytotoxic T cell response in tumours. This bacterial pathogen strongly induces innate and specific immunity with the potential to overcome tumour induced tolerance and weak immunogenicity. Here, we propose a Listeria based vaccination for melanoma based in its tropism for these tumour cells and its ability to transform in vitro and in vivo melanoma cells into matured and activated dendritic cells with competent microbicidal and antigen processing abilities. This Listeria based vaccination using low doses of the pathogen caused melanoma regression by apoptosis as well as bacterial clearance. Vaccination efficacy is LLO dependent and implies the reduction of LLO-specific CD4+ T cell responses, strong stimulation of innate pro-inflammatory immune cells and a prevalence of LLO-specific CD8+ T cells involved in tumour regression and Listeria elimination. These results support the use of low doses of pathogenic Listeria as safe melanoma therapeutic vaccines that do not require antibiotics for bacterial removal

<i>Listeria</i> infection of melanoma induces nitric oxide and iNOS expression.

<p>^aB16F10 murine melanoma or BMDC were infected with LM^WT (B16F10-LM^WT, BMDC-LM^WT) or non-infected (B16F10-NI, BMDC-NI) for 24 hours.</p><p>^bNO produced was measured in cell supernatants. Results are expressed as nmol of NO produced by 10⁵ cells (mean ± SD, <i>P</i><0.005) obtained with triplicate samples.</p><p>^cCell surfaces markers of B16F10 and BMDC infected or not with LM^WT were analysed by FACS using the following antibodies: CD11c-PE, iNOS-FITC, CD86-V450 and MHC-II-APC. Samples were acquired using FACSCanto flow cytometer. Results are expressed as the percentages of positive cell (mean ± SD, <i>P</i><0.005).</p><p><i>Listeria</i> infection of melanoma induces nitric oxide and iNOS expression.</p

<i>Listeria</i> vaccination of melanoma shows a dual action, tumour regression by apoptosis and bacterial clearance.

<p><b><i>A</i></b>, C57BL/6 female were inoculated <i>i</i>.<i>p</i>. with 5 x 10⁵ B16F10/mice (n = 5) for 7 (7-D) or 15 days (15-D). Mice were bled, sacrificed and treated for histological analysis as described in <i>Material and Methods</i>. Images correspond to sections of peritoneum infiltrates or liver metastases. <b><i>B</i></b>, C57BL/6 female were inoculated <i>i</i>.<i>p</i>. with 5 x 10⁵ B16F10/mice (n = 5) as in <b><i>A</i></b> for none (NT), 7 (7-D) or 15 days (15-D). Spleens from sacrificed mice (n = 5) were stained for histological analysis using different antibodies as described in <i>Material and Methods</i> and images correspond to sections. Results are expressed as percentages of positive cells (mean ± SD) (<i>P</i> < 0.05). <b><i>C</i></b>, C57BL/6 female were inoculated <i>i</i>.<i>p</i>. with 5 x 10⁵ B16F10/mice (n = 5) for 7 days and next injected <i>i</i>.<i>p</i>. or not (NT) with 5 x 10³ bc/mice of different LM strains (LM^WT or LM^ΔLLO) for 5 additional days. Mice were sacrificed, bled to collect sera and photographed before collecting melanoma and lungs. Images correspond to the peritoneum of mice and the recovered melanoma. Plots correspond to measurements of diameters of collected melanoma. Results are expressed as the mean ± SD (<i>P</i> < 0,05). <b><i>D</i></b>, Melanoma recovered from LM^WT or LM^ΔLLO vaccinated mice or from non-vaccinated mice (NV) as in <b><i>C</i></b> were analysed for early and late apoptosis by FACS according to <i>Materials and Methods</i> after double staining with 7-AAD (IP labelled) and annexin V (anexina labelled). Results are expressed as the percentages of late apoptotic cells, necrotic death, (Q2 area corresponding to double positive for 7-AAD and annexin V cells) and the percentages of early apoptotic cells, programmed cell death (Q4 area corresponding to annexin V positive while 7-AAD negative cells) (mean ± SD) (<i>p</i> < 0.05).</p

Efficiency of <i>Listeria</i> vaccination of melanoma is mediated by activation of LLO_rec specific CD8⁺ T cells and inhibition of LLO_rec specific CD4⁺ T cells.

<p>C57BL/6 female were inoculated <i>i</i>.<i>p</i>. with 5 x 10⁵ B16F10/mice (n = 5) for 7 days and next injected <i>i</i>.<i>p</i>. or not (NT) with 5 x 10³ bc/mice of GFP-LM^WT strain for 3 additional days. Mice were bled and sacrificed. <b><i>A</i></b>, Immune cells plot (left) corresponds with spleens were homogenized and cell populations were analysed by FACS. Results were expressed as the mean of the percentages of positive cells ± SD. LM growth plot (right) corresponds with spleen homogenates examined for CFU in blood-agar plates. Results are expressed as CFU (mean ± SD) obtained with triplicate samples from three independent experiments (<i>P</i>< 0,05). <b><i>B</i></b>, Levels of pro-inflammatory cytokines (MCP-1, TNF-alfa, IFN-gamma, IL-6, IL-10, IL-12) were analysed in sera of mice using the CBA kit (Becton Dickinson) by flow cytometry. Results were expressed as cytokine concentration (pg/ml of mean ± SD, <i>P</i><0,05). <b><i>C</i></b>, Spleen cells obtained from homogenates after inoculation with melanoma B16F10 (5 x 10⁵ cells/mice) for 7 days and vaccination with LM^WT for 5 days (LM^WT-MEL). Cells were stimulated 5 h with recombinant LLO (0.1 μg/ml) in the presence of brefeldin A for intracellular cytokine staining. LLO-stimulated spleen cell surface was stained for CD4 or CD8 and fixed and permeabilized using cytofix/cytoperm kit. Stimulated cells were surface stained for CD4 or CD8 using anti-CD4⁺FITC-labeled or anti-CD8⁺APC-labelled and data gated to include histograms show the percentages of LLO-CD4⁺ and IFN-gamma producers (lower left) and LLO-CD8⁺ and IFN-gamma producers (lower right) (R2 and R3 gates). Experiments were performed in triplicate and results are expressed as the mean ± SD (<i>p</i> < 0.05). <b><i>D</i></b>, Spleen cells obtained from homogenates after inoculation with melanoma B16F10 pre-infected with LM^WT (5 x 10⁵ cells/mice) for 7 days. Cells were stimulated 5 h with recombinant LLO (0.1 μg/ml) in the presence of brefeldin A for intracellular cytokine staining. Procedures were performed as in <b><i>C</i></b> and results expressed as the mean ± SD (<i>p</i> < 0.05).</p

Frequencies of LLO_296–304 specific CD8⁺ T cells in spleens of B16F10 treated mice or non-treated after LM^WT therapy.

<p>^aC57BL/6 mice non-treated with murine melanoma were inoculated with saline <i>i</i>.<i>p</i> for 7 days and next injected <i>i</i>.<i>p</i> with LM^WT for 3 days as described in <i>Materials and Methods</i>. Splenocytes from mice treated with LM^WT were incubated with recombinant dimeric H-2K^b: Ig fusion protein (BD Biosciences) loaded with LLO_296–304 peptide. The staining cocktail contained the dimeric fusion protein loaded with the peptides, CD8 and anti-IFN-gamma antibodies. CD8⁺ cells were gated for anti-IFN-gamma staining (% Gated dimer-CD8) to calculate the frequencies of CD8⁺-LLO_296–304. Results are expressed as percentages of triplicate samples ± SD. <i>P</i><0.05.</p><p>^bB16F10 murine melanoma were inoculated <i>i</i>.<i>p</i> into C57BL/6 mice for 7 days and next injected <i>i</i>.<i>p</i> with LM^WT for 3 additional days as described in <i>Materials and Methods</i>. Splenocytes from mice were incubated with recombinant dimeric H-2Kb: Ig fusion protein as in a. <i>P</i><0.05.</p><p>^cB16F10 murine melanoma pre-infected with LM^WT was inoculated into C57BL/6 mice for 7 days as described in Materials and Methods. Splenocytes from mice were incubated with recombinant dimeric H-2Kb: Ig fusion protein as in a. <i>P</i><0.05.</p><p>Frequencies of LLO_296–304 specific CD8⁺ T cells in spleens of B16F10 treated mice or non-treated after LM^WT therapy.</p

<i>Listeria</i> induced transformation of melanoma into dendritic cells.

<p><b><i>A</i></b>, Kinetic analysis of BMDC, murine (B16F10) and human (A-375 and Mel-H0) melanoma cells infected with different LM strains (LM^WT, LM^ΔLLO). Results are expressed as CFU (mean ± SD) obtained with triplicate samples from three independent experiments (<i>P</i><0.05). <b><i>B</i></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) (<i>p</i> < 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) (<i>p</i> < 0.05). The percentages of cytosolic fractions are shown in right plot after purification of phagosomal and cytosolic fractions as in <i>Material and Methods</i>. Results are expressed as percentages of total internalized CFU in PNS (mean ± SD) (<i>p</i> < 0.05). <b><i>C</i></b>, Images correspond to confocal microscopy examination of melanoma and BMDC infected with GFP-LM^WT. GFP-LM^WT (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. <b><i>D</i></b>, 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 (<i>p</i><0.05). <b><i>E</i></b>, 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, <i>P</i><0,05).</p