Panel A: Flow cytometry histograms show TNFR1 expression in MC38 wild-type (WT), 1A3 clone, 1C1 clone, and 1E10 clone cells. Line graphs depict individual tumor growth over time for MC38 WT, TNFR1KO 1A3 clone, TNFR1KO 1C1 clone, TNFR1KO 1E10 clone, and Polyclonal TNFR1KO. The rightmost graph summarizes the mean tumor size over time for these groups. Panel B: Flow cytometry histograms show TNFR1 expression in EO771 WT, 1D2 clone, 2B9 clone, and 2H8 clone cells. Line graphs depict individual tumor growth over time for EO771 WT, TNFR1KO 1D2 clone, TNFR1KO 2B9 clone, TNFR1KO 2H8 clone, and Polyclonal TNFR1KO. The rightmost graph summarizes the mean tumor size over time for these groups. Panel C: Flow cytometry histograms show TNFR1 expression in CT26 WT, 1B5 clone, 1D8 clone, and 2E9 clone cells. Line graphs depict individual tumor growth over time for CT26 WT, TNFR1KO 1B5 clone, TNFR1KO 1D8 clone, TNFR1KO 2E9 clone, and Polyclonal TNFR1KO. The rightmost graph summarizes the mean tumor size over time for these groups. Panel D: Flow cytometry histograms show TNFR1 expression in B16OVA WT, B5 clone, G1 clone, and G9 clone cells. Line graphs depict individual tumor growth over time for B16OVA WT, TNFR1KO B5 clone, TNFR1KO G1 clone, and TNFR1KO G9 clone. The rightmost graph summarizes the mean tumor size over time for these groups. Panel E: Flow cytometry histograms show TNFR1 expression in EO771 WT, EO771 TNFR1KO clone 2H8, and EO771 TNFR1 addback from clone 2H8 cells. Panel F: Three line graphs depict individual tumor growth over time for EO771 WT, EO771 TNFR1KO clone 2H8, and EO771 TNFR1 addback from clone 2H8. Panel G: Two sets of bioluminescence images show tumor generation and individual follow-up in mice with multifocal HCC induced by hydrodynamic gene co-transfer of plasmids to express cMyc, GFP, and luciferase, with or without a CRISPR/Cas9 silencing system to knockdown TNFR1. Panel H: A table shows the fraction of tumor-free mice at different time points and the results of Fisher's exact test statistical comparisons.
TNFR1KO variants are rejected or slowly progress in transplantable models and in a genetically induced multifocal HCC model. (A–D) TNFR1KO clonal variants of the indicated transplantable cell lines were subcutaneously engrafted in immunocompetent syngeneic mice in comparison to their WT cell counterparts. Flow cytometry histograms showing the expression or silencing of TNFR1 (on the left). Individual tumor follow-up and lethal tumor engraftment (left panels) are presented alongside summaries of data (mean ± SEM in the right panels, also providing two-way ANOVA statistical comparisons). Fractions in the top left corner of each graph show the number of mice that experienced complete tumor rejection (n = 5–6). (E) FACS histograms showing TNFR1 expression in EO771 WT, EO771 TNFR1KO (clone 2H8), and its lentivirally transduced back with TNFR1 to regain expression. (F) Tumor engraftment experiments in immunocompetent syngeneic mice of the WT and TNFR1KO variant of EO771 in comparison with the KO variant lentivirally transduced to regain expression of TNFR1 (unpaired t test statistical comparisons) (n = 6). (G) Tumor generation and individual follow-up by sequential bioluminescence imaging of multifocal HCC induced by hydrodynamic gene co-transfer of plasmids to express cMyc, GFP, and luciferase with a CRISPR/cas9 silencing system to knock down p53. As indicated in the figure, in a group of mice, the TNFR1 CRISPR guide was added to the hydrodynamic mixture and controlled by an empty vector in the other group. (H) Fraction of mice without evidence of tumors at the indicated time points and Fisher’s exact test statistical comparisons (n = 11). Experiments are representative of at least two similarly performed. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****).