Figure 7.

Intravital multiphoton confocal microscopy imaging of multifocal HCC-bearing livers in mice undergoing immunotherapy

CD2-RFP transgenic mice were subjected to multifocal HCC induction by the five-plasmid hydrodynamic gene transfer and subjected to intravital microscopy of surgically exposed livers following the treatment with RatIgG (control) or the triple immunomodulatory combination (anti-PD-1 + anti-CTLA-4 + IL-2) started on day 35.

(A) Representative microphotographs of non-tumor and tumor liver on day 43 showing endogenous CD2-RFP T cells and the corresponding quantifications according to experimental treatments.

(B) Quantification of the density of the red fluorescent cells corresponding to transferred RFP⁺ pmel-1 T cells and images from the indicated experimental treatments groups.

(C) Experiments as in (B) in non-transgenic C57Bl/6 mice that had undergone hydrodynamic HCC induction and that were transferred with resting RFP⁺ pmel-1 CD8⁺ T cells on day 32. Mice were given control or triple immunomodulatory treatment on day 35, and intravital microscopy images were acquired on day 43. Representative images are shown as well as quantifications pertaining to motility, migration speed, and meandering index from four imaged cases are represented. These images correspond to Video S2. See also Figure S6.