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Supplementary Figures 7–11: Safety levels of systemic IL-12 induced by cDNA expression as a cancer therapeutic

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posted on 16.11.2021, 11:34 by Constanza Savid-Frontera, Maria Estefania Viano, Natalia S. Baez, Della Reynolds, Mariana Matellon, Howard A. Young, Maria Cecilia Rodriguez-Galan

Suppl. Fig. 7- Systemic IL-12 expression generates the appearance and disappearance of different cell clusters in SLO. WT C57BL/6 and Foxp3-EGFP C57BL/6 mice were subcutaneously inoculated with 1 x 106 B16 cells. When solid tumors were visible mice were hydrodynamically injected with 1g of an empty vector (control) or IL-12 cDNA. Seven days after hydrodynamic injection spleen and both inguinal lymph nodes were harvested. Single cell suspensions were obtained for each organ and stained with Zombie Acqua Dye, CD4, CD8, B220, CD11b and Gr-1 antibodies for flow cytometry analysis. Foxp3 expression was analyzed in the CD4 population based on GFP detection. Samples were acquired in a BD LSR Fortessa cytometer. The graphs represent: (A) a tSNE dimensionality reduction showing flow cytometry data of SLO cells from control or IL-12 treated mice, analyzed with phonograph clustering and (B) heat-map showing the distribution of the different parameters analyzed on the tSNE map. Graphs in (C) represent the respective tSNE map corresponding to each SLO from control or IL-12 treated mice. Plots are obtained from concatenated files of 5000 cells of each mouse from 4-5 mice per group.

Suppl. Fig. 8- Flow cytometry dimensional reduction analysis of splenocytes from control and IL-12 cDNA-treated mice shows different cell clusters. WT C57BL/6 and Foxp3-EGFP C57BL/6 mice were subcutaneously inoculated with 1 x 106 B16 cells. When solid tumors were visible mice were hydrodynamically injected with 1g of an empty vector (control) or IL-12 cDNA. Seven days after hydrodynamic injection spleens and both inguinal lymph nodes were harvested and cells were stained with Zombie Acqua Dye, CD4, CD8, B220, CD11b, CD44, F4/80 and Gr-1 antibodies for flow cytometry analysis. Samples were acquired in a BD LSR Fortessa cytometer. The graphs represent: (A) a tSNE dimensionality reduction showing flow cytometry data of SLO cells from control or IL-12 treated mice, analyzed with phonograph clustering and (B) the distribution of 3 of the different non-T non-B subpopulations analyzed on the tSNE map. Histograms in (C) represent the differential expression for each subpopulation (yellow, red, and orange) of the different parameters analyzed. Plots are obtained from concatenated files of 5000 cells of each mouse from 4-5 mice per group.

Suppl. Fig. 9- Flow cytometry dimensional reduction analysis of tumor-draining and non-draining inguinal LN from control and IL-12 cDNA-treated mice shows different cell clusters. WT C57BL/6 and Foxp3-EGFP C57BL/6 mice were subcutaneously inoculated with 1 x 106 B16 cells. When solid tumors were visible mice were hydrodynamically injected with 1g of an empty vector (control) or IL-12 cDNA. Seven days after hydrodynamic injection spleens and both inguinal lymph nodes were harvested and cells were stained with Zombie Acqua Dye, CD4, CD8, B220, CD11b, CD44, F4/80 and Gr-1 antibodies for flow cytometry analysis. Samples were acquired in a BD LSR Fortessa cytometer. The graphs represent: (A) a tSNE dimensionality reduction showing flow cytometry data of tumor cells from control or IL-12 treated mice, analyzed with phonograph clustering. Graphs in (B) represent the respective tSNE map corresponding to ndLN or dLN from control or IL-12 treated mice. Histograms in (C) show the expression of the indicated parameters for the indicated clusters present in the CD8+ T cells subpopulation ((1) activated/memory phenotype, orange circles and (2) naïve phenotype, red circles). Histograms in (D) and (E) show the expression of different markers among a subpopulation of CD4+ T cells ((3) yellow circles). In (F), the expression of the indicated surface markers among the selected (in black circles (4) and (5)) non-T non-B subpopulations is show. Plots are obtained from concatenated files of 5000 cells of each mouse from 4-5 mice per group.

Suppl. Fig. 10- Systemic IL-12 cDNA expression generates changes in leukocytes distribution within tumors. WT C57BL/6 and Foxp3-EGFP C57BL/6 mice were subcutaneously inoculated with 1 x 106 B16 cells. When solid tumors were visible mice were hydrodynamically injected with 1g of an empty vector (control) or IL-12 cDNA. Seven days after hydrodynamic injection tumors were harvested, cell suspensions were obtained and stained with Zombie Acqua Dye, CD45, CD4, CD8, B220, CD11b, CD44, F4/80 and Gr-1 antibodies for flow cytometry analysis. Samples were acquired in a BD LSR Fortessa cytometer. The graphs represent: (A) a tSNE dimensionality reduction showing flow cytometry data of the tumors from control or IL-12 treated mice, analyzed with phonograph clustering. (B) heat-map showing the distribution of B cells and CD4+ and CD8+ T cells analyzed on the tSNE map. Graphs in (C) represent the tSNE map corresponding to tumors from control or IL-12cDNA-treated mice. In (D), the distribution in tSNE map and expression of different surface markers from two subpopulations of tumor-infiltrating leukocytes are shown. Plots are obtained from concatenated files of 3000 cells of each mouse from 4-5 mice per group.

Suppl. Fig. 11- IL-12 systemic expression increases the percentage of both CD8+ and IFN+ CD8+ T in EL4 tumors. WT C57BL/6 mice were subcutaneously inoculated with 1 x 106 EL4 cells. When solid tumors were visible mice were hydrodynamically injected with 1g of an empty vector (control) or IL-12 cDNA. Seven days after hydrodynamic injection EL4 tumors were removed, mechanically disrupted, and resuspended in 0.2g of tumor/5mL of Supplemented media. (A) Representative FSC vs SSC plots from EL4 cells alone (left) of EL4 tumors obtained from control or IL-12 cDNA treated mice (right). Leukocyte were gated based on FSC vs SSC features since they can be easily discriminated from EL4 cells as shown in the dotplots. Six hundred microliters of the tumor suspensions were either (B) surface stained with an anti-CD8 antibody or (C) seeded into a 96-well plate and cultured in Supplemented media for 5h in the presence of PMA/Ionomycin, Breferdin A and Monensin. Cells were then surface stained with an anti-CD8 antibody, washed, fixed, permeabilized and stained with an anti-IFN antibody (or isotype control). Data are expressed as mean + SEM of a pool of 2 independent experiments with 3-4 mice per group. Statistical analysis was performed using Student t test, *p<0.05.

Funding

Fundación para el Progreso de la Medicina GC N°1, Secretaria de Ciencia y Tecnología de la UNC (SeCyT-UNC).

the Intramural Research Program of the Center for Cancer Research, National Cancer Institute (NCI): ZIA BC 009283.

History