Predicting the response of immunotherapy in two different breast cancer models. WT C57BL/6 mice were inoculated with one million breast cancer cells (mesenchymal PB3 cells in A or epithelial PB2 cells in B). 2 wk p.i., mice were imaged by PET/CT using anti-CD8 ⁸⁹Zr-PEG20-VHH X118. (A) PET-CT images of the mesenchymal tumor-bearing mouse (n = 3); (left) PET/CT maximum intensity projection; (middle and right) coronal CT (middle) and PET-CT (right) images taken through a cross section of the tumor. The box outlines the tumor. (B) PET-CT images of the epithelial PB2 tumor-bearing mice (n = 3). (left) PET-CT maximum intensity projection. (middle and right) Coronal CT (middle) and PET-CT (right) images taken through a cross section of the tumor. (C and D) PET images of the tumors are shown. The PET signals in the tumor are rendered as a heat map. Below each image is the corresponding 3D graph, in which the z axis represents the strength of the PET signal (arbitrary units). The CD8 T cell signal was more homogenously distributed in epithelial tumors, whereas mesenchymal tumors showed a more heterogeneous signal distribution. Where relevant, areas with lower PET signals are indicated by arrows. On the right are graphs that show three randomly chosen transects (arrows) across each of the tumors, plotting the intensity of the PET signal along that transect. The first derivative of this function is shown below each graph to record the presence of local maxima. (E and F) Mean tumor growth with or without receiving therapy. Mice were injected subcutaneously with one million cells (mesenchymal cells in E or epithelial cells in F), followed by 200 µg anti-CTLA4 therapy (clone 9H10) three times per week for 20 d. The epithelial tumors showed a strong response, whereas the mesenchymal tumors did not (n = 5 for each cohort). Error bars represent standard deviation.