IFNα gene/cell therapy curbs colorectal cancer colonization of the liver by acting on the hepatic microenvironment (2015)
Figure 2

Figure 2. Tie2-IFNα mice display reduced tumor burden and prolonged survival.

A. Contrast-enhanced magnetic resonance imaging (MRI) of the liver from representative Tie2-GFP mice (red frame) or Tie2-IFNα (blue frame) that were intrasplenically injected with 5x103 CT26. Red arrows identify CRC liver metastases of representative z-sections. Tumors were characterized as hypointense and slightly-hyperintense regions in T1 and T2 weighted sequences, respectively. Each panel refers to a single mouse analyzed at different time points; n.a.=not assessed, refers to a mouse euthanized before the specified time point; scale bars=5mm.

B. Percentage of mice bearing at least one CRC liver metastasis estimated by MRI analysis. Mice were treated as described in A. Mock/Tie2-GFP n=11, Tie2-IFNα n=9; the oblique black line pattern within the red columns depicts the percentage of mice euthanized or that died before the indicated time point; p-values were calculated by Fisher’s exact test.

C. Tumor volume quantification measured by MRI analysis of the same mice described in B. Each symbol corresponds to the same mouse analyzed at different time points; horizontal bars=mean values; note that Mock/Tie2-GFP mice were euthanized or died before day 54; p-values were calculated by Mann-Whitney test.

D, E. Quantitative real-time PCR analyses of the relative expression levels of the interferon inducible genes Oas1 (D) and Irf7 (E) within brain (n=4, n=2), kidney (n=4, n=3) and metastatic liver (n=4, n=7) of Tie2-GFP or Tie2-IFNα mice euthanized 25 days post-intrasplenic injection of 5x103 CT26. The basal expression of Oas1 and Irf7 estimated in brain of control mice (i.e. Tie2-GFP injected with saline), was set to 1 and utilized to calculate the fold increase values depicted. Mean values are shown; error bars=S.E.M.; p-value was calculated by Mann-Whitney test.

F. Eighty-four or 54 days post-NaCl or 5x103 CT26 intrasplenic injection, Sham/CTRL (n=5) or Tie2-IFNα mice (n=7) were infected with LCMV (Armstrong strain, 200 pfu intraperitoneally injected). Eight days post-infection white blood cells were isolated and analyzed for LCMV specific CD8+ T cell response. Left panel: percentage of total CD8+ T cells; middle panel: percentage of CD8+/NP118+ T cells (NP118=recombinant dimeric H-2d/Ig fusion protein complexed with the immune-dominant H-2d-restricted LCMV NP118-126 peptide); right panel: percentage of CD8+/IFNγ+ T cells after in vitro stimulation with virus-specific H-2d-restricted peptide (NP118-126); data pooled from two independent experiments; mean values are shown; error bars=S.E.M.; differences were not statistically significant (by unpaired Student’s t-Test).

G. Kaplan-Meier survival curves of the indicated groups of mice described in A. Sham (n=3), Mock(wt)/Tie2-GFP (n=11); Tie2-IFNα (n=9); data pooled from three independent experiments; p=0.001 by log-rank/Mantel-Cox test. The inset images show representative macroscopic photographs of the metastatic progression in the liver of a control animal (red frame: Mock/Tie2-GFP, day 35 post-CT26 injection) opposed to the lack of lesions in Tie2-IFNα treated mice at later time points (blue frame: day 450 post-CT26 injection).