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Supplementary Figures 1–6: 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. 1- FACS analysis of Target YAC-1 cells and effector cells used for Killing assay. Details of the assays are described in Material and Methods section. (A) Representative SSC vs FSC dotplot or (B) representative histogram of CFSE expression of the co-cultures of target plus effector cells. R1 was used to gate target YAC-1 cells. C) Representative dotplots of SSC vs FSC (left) or Annexin V vs 7ADD (right) from YAC-1 target cells cultured alone.

Suppl. Fig. 2- Systemic effects after IL-12 alone or IL-12 plus IL18 cDNA hydrodynamic injections. WT C57BL/6 mice were subcutaneously inoculated with 1 x 106 B16 cells. When solid tumors were visible mice were hydrodynamically injected with: control (10g), IL-12 (5g) or IL-12 (5g) plus IL-18 (10g) cDNAs. Seven days after hydrodynamic injection mice were euthanized and (A) IL-12 levels were determined by ELISA in the sera of control or IL-12 B16 tumor-bearing mice 24 h post-hydrodynamic injection of empty or IL-12 cDNAs (1g) and p values were calculated by Student t test. (B) Statistical lineal correlation test (r2) was performed for IL-12 sera levels vs tumor fold change in IL-12 B16 tumor-bearing mice 7 days after hydrodynamic injection of 1g of IL-12 cDNA. Data represent the pool of 2-3 independent experiments with 3-5 mice per group.

Suppl. Fig. 3- Differential expression of leukocyte and T lymphocyte markers between PBMC and EL4 cells. PBMC cells from a control mouse and EL4 cells were obtained and stained for different leukocyte lineage markers or T cells, gating on living cells. Expression of CD45, CD45.1, CD45.2, CD4 and CD8 of both type of cells has been plotted in the same graph for comparison purposes (red, PBMC and light blue, EL4).

Suppl. Fig. 4- Systemic IL-12 expression generates a significant increase in total leukocyte number in spleen. 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 were harvested and cells were stained with Zombie Acqua Dye, CD4, CD8, B220, CD11b and Gr-1 antibodies for flow cytometry analysis. Samples were acquired in a BD LSR Fortessa cytometer. Foxp3 expression was analyzed in the CD4 population based on GFP detection. Total cell numbers were calculated and plotted in each control vs IL-12 cDNA-treated mice. Data is the result of 3 independent experiments with 3-5 mice per group. Statistical analysis was performed using Student t test. *p<0.05, ***p<0.001.

Suppl. Fig. 5- Systemic IL-12 expression generates different total cell numbers of leucocytes and cell subsets between tumor draining (dLN) and non-draining lymph nodes (ndLN). 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 both inguinal lymph nodes were taken, harvested, counted, and stained with Zombie Acqua Dye, CD3, 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. Total cell numbers were calculated and plotted in each control vs IL-12 cDNA-treated mice. Data represent the pool of 3 independent experiments with 3-5 mice per group. Statistical analysis was performed using student t test. *p<0.05, ***p<0.001 represents the statistical difference between control and IL-12 mice for each tissue analyzed.

Suppl. Fig. 6- Systemic IL-12 expression induces a positive effector versus regulatory ratio in OLS. 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 tumor-draining and non-draining inguinal lymph nodes were taken, harvested, counted, and stained with Zombie Acqua Dye, CD3, CD4 and CD8 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. Graphs show the ratio of total cell number of CD8+ T cells to total CD4+ T cells and CD8+ T cells to Tregs (CD3+CD4+Foxp3+) cells respectively. Data represent the pool of 2 independent experiments with 3-5 mice per group. Statistical analysis was performed using student t test. *p<0.05, ***p<0.001, ****p<0.0001 represents the statistical difference between control and IL-12 mice for each tissue analyzed.


Funding

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

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

History