Additional file 2: of Reprogramming the murine colon cancer microenvironment using lentivectors encoding shRNA against IL-10 as a component of a potent DC-based chemoimmunotherapy

Figure S2. The influence of the shIL10–3-based therapy on lymphocyte and myeloid cell subpopulations infiltrating MC38 tumors. The figure presents changes in proportions of myeloid cells (A) and lymphoid cells (B) after therapy. To calculate the mean ± SD at least 5 mice per group were analyzed. The differences between the groups were estimated using nonparametric Kruskal-Wallis test followed by Dunn’s multi comparison test (* p < 0.05, ** p < 0.01, *** p < 0.001). (TIF 343 kb

Data - Polymyxin B modulates the activity of Lipooligosaccharide E. coli B; Kicielińska and all

Data - Polymyxin B modulates the activity of Lipooligosaccharide E. coli B; Kicielińska and al

Cytokine concentration in serum obtained from control or treated tumor bearing mice.

<p>The concentration of IL-6, IL-1β and TNF-α in murine serum was evaluated by ELISA kits (eBioscience). The presence of the IFN-γ and IL-10 were not observed. To calculate the mean 9–15 mice of each presented group were tested. Cytokine concentration in serum obtained from healthy mice: IL-1β – 5.46 pg/ml; IL-6–0 pg/ml; TNF-α—0 pg/ml.</p

Cytokine production by spleen cells after ConA (A) or LOS (B) stimulation.

<p>The concentration of IFN-γ and IL-10 was measured in supernatants collected from above splenocytes (2x10⁶ cells/ml) incubated for 48h with ConA (0.5 μg/ml) or LOS (1 μg/ml), using commercially available ELISA kits (BD Bioscience or eBioscience). To calculate the mean, six mice of each group were analyzed. The production of IL-4 after ConA stimulation was not observed. Cells from healthy mice stimulated with ConA produced 3.53 ng/ml of IFN-γ and 0.89 ng/ml of IL-10. Cells from healthy mice stimulated with LOS produced 0.06 ng/ml of IFN-γ and 3.51 ng/ml IL-10.</p

Phenotypic characteristics of splenocytes.

<p>A. Phenotypic characteristics of myeloid (A) and lymphoid cells (B) in spleen. Spleen cells from mice treated with LOS and/or PmB were incubated with CD11b-PerCP-Cy5.5, B220-APC, Ly6G-APC-Cy7, Ly6C-PE, MHCII-FITC or anti-CD4-APC, anti- CD8-PE-Cy7, anti-CD49b-PE and anti-CD19-FITC. Phenotype analysis was carried out using the Becton Dickinson FACSCalibur apparatus with Cell Quest Software. To calculate the mean, six mice of each group were tested. The percentages of myeloid cells in healthy mice were: CD11b⁺—0.93%, monocytic cells among CD11b⁺ cells—13%, granulocytic cells among CD11b⁺ cells—28%. The percentages of lymphoid cells in healthy mice were: CD4⁺—13.45%, CD8⁺ 5.95%- CD19⁺—70.6%, CD49b⁺—6.11%.</p

Phenotypic characteristics of myeloid (A) and lymphoid cells (B) in spleen.

<p>Spleen cells from mice treated with LOS and/or PmB were incubated with CD11b-PerCP-Cy5.5, B220-APC, Ly6G-APC-Cy7, Ly6C-PE, MHCII-FITC or anti-CD4-APC, anti- CD8-PE-Cy7, anti-CD49b-PE and anti-CD19-FITC. Phenotype analysis was carried out using the Becton Dickinson FACSCalibur apparatus with Cell Quest Software. To calculate the mean, three mice of each group were tested. The percentages of myeloid cells in healthy mice were: CD11b⁺—3.50%, monocytic cells among CD11b⁺ cells—30.50%, granulocytic cells among CD11b⁺ cells—30.90%. The percentages of lymphoid cells in healthy mice were: CD4⁺—17.43%, CD8⁺ 9.26%- CD19⁺—61.67%, CD49b⁺—8.00%. Cytokine production by spleen cells after ConA (C) or LOS (D) stimulation. The concentration of IL-6, IFN-γ and IL-10 was measured in supernatants collected from above splenocytes (2x10⁶ cells/ml) incubated for 48h with ConA (0.5 μg/ml) or LOS (1 μg/ml), using commercially available ELISA kits (BD Bioscience or eBioscience). To calculate the mean, three mice of each group were analyzed. Cells from healthy mice stimulated with ConA produced 1.07 ng/ml of IFN-γ, 0.10 ng/ml IL-6 and 0,23 ng/ml of IL-10. Cells from healthy mice stimulated with LOS produced 0.01 ng/ml of IFN-γ, 0.33 ng/ml IL-6 and 3.52 ng/ml IL-10.</p

Table_1_Inhibition of MC38 colon cancer growth by multicomponent chemoimmunotherapy with anti-IL-10R antibodies, HES-MTX nanoconjugate, depends on application of IL-12, IL-15 or IL-18 secreting dendritic cell vaccines.docx

BackgroundThe tumor microenvironment (TME) provides a conducive environment for the growth and survival of tumors. Negative factors present in TME, such as IL-10, may limit the effectiveness of cellular vaccines based on dendritic cells, therefore, it is important to control its effect. The influence of IL-10 on immune cells can be abolished e.g., by using antibodies against the receptor for this cytokine - anti-IL-10R. Furthermore, the anticancer activity of cellular vaccines can be enhanced by modifying them to produce proinflammatory cytokines, such as IL-12, IL-15 or IL-18. Additionally, an immunomodulatory dose of methotrexate and hydroxyethyl starch (HES-MTX) nanoconjugate may stimulate effector immune cells and eliminate regulatory T cells, which should enhance the antitumor action of immunotherapy based on DC vaccines. The main aim of our study was to determine whether the HES-MTX administered before immunotherapy with anti-IL-10R antibodies would change the effect of vaccines based on dendritic cells overproducing IL-12, IL-15, or IL-18.MethodsThe activity of modified DCs was checked in two therapeutic protocols - immunotherapy with the addition of anti-IL10R antibodies and chemoimmunotherapy with HES-MTX and anti-IL10R antibodies. The inhibition of tumor growth and the effectiveness of the therapy in inducing a specific antitumor response were determined by analyzing lymphoid and myeloid cell populations in tumor nodules, and the activity of restimulated splenocytes.Results and conclusionsUsing the HES-MTX nanoconjugate before immunotherapy based on multiple administrations of anti-IL-10R antibodies and cellular vaccines capable of overproducing proinflammatory cytokines IL-12, IL-15 or IL-18 created optimal conditions for the effective action of these vaccines in murine colon carcinoma MC38 model. The applied chemoimmunotherapy caused the highest inhibition of tumor growth in the group receiving DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg at the level of 72.4%. The use of cellular vaccines resulted in cytotoxic activity increase in both immuno- or chemoimmunotherapy. However, the greatest potential was observed both in tumor tissue and splenocytes obtained from mice receiving two- or three-component vaccines in the course of combined application. Thus, the designed treatment schedule may be promising in anticancer therapy.</p

Cytokine production by BM-DCs stimulated with T4 phage.

<p>The concentrations of (A) IL-6, (B) TNF-a, (C) IL-12 and (D) IL-10 were determined by ELISA assay in supernatants collected after 24 h stimulation of 1×10⁶ DCs/ml. The data present the means of 3 independent experiments. K – control, alb – albumin.</p

Mouse Cytokine Antibody Array map.

<p>Mouse Cytokine Antibody Array map.</p

Human Cytokine Antibody Array map.

<p>Human Cytokine Antibody Array map.</p