Anti-CD20 CAR exPBNK significantly inhibit growth of Raji cells in xenografted mice. Anti-CD20 CAR exPBNK significantly inhibit growth of Raji cells in xenografted mice. A, Live-imaging demonstrating the extent of Raji-Luc progression. Raji-Luc cells (5 × 105) were i.p. injected into NSG mice on day 0. Three doses of (5 × 106 cells/dose) of either anti-CD20 CAR exPBNK (with anti-CD20 CAR mRNA electroporation) or mock exPBNK (without anti-CD20 CAR mRNA electroporation) cells were injected i.p. on days 9, 16, and 23, respectively. Mice treated with medium and mice without tumor inoculation served as controls. Whole mouse luciferase activity was measured once weekly at various time points. B, photons emitted from luciferase-expressing cells were measured in regions of interest that encompassed the entire body and quantified using Living Image software. Signal intensities (total Flux) are shown at the time points indicated in untreated (medium), anti-CD20 CAR− exPBNK (MOCK)-, and anti-CD20 CAR+ exPBNK (CAR)-treated mice and plotted as mean ± SEM. C, mice were followed until death or sacrificed when tumor size reached 2 cm3. The Kaplan–Meier survival curves for all groups were generated following therapy initiation using animal sacrifice as the terminal event. Comparison of survival for the four groups showed a statistically significant difference. The CAR exPBNK-treated Raji-Luc (n = 6) mice had significantly extended survival time compared with the untreated mice (n = 5; P < 0.001) and the mock exPBNK-treated mice (n = 8; P < 0.001). There was no statistically significant difference in survival between the untreated Raji mice and the mock exPBNK-treated Raji mice. Five NSG mice without tumor inoculation were used as controls. D, peripheral blood was collected from Raji-Luc xenografted mice 7 days after the last NK injection. Circulating CD20+ tumor cells (left) and NK cells (right) from the medium-only control group (n = 2), mock exPBNK group (n = 6), and CAR exPBNK group (n = 5), were quantified using the TruCOUNT method by flow cytometry analysis with anti-CD20-PE and anti-CD56-FITC antibodies. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not statistically significant. E, tumor masses were dissected from 3 mice in each group, washed with D-PBS, and soaked in D-PBS with d-luciferin. Bioluminescent images were acquired and analyzed with Living Image software (Xenogen; top). Then the tumor masses were embedded in optimal cutting temperature (OCT) compound and frozen at −80°C. Of note, 10 μm sections were cut in a cryostat microtome, fixed with cold acetone, and stained with H&E (bottom; original magnification, ×200). F, frozen tumor sections from 3 mice of each group were stained with rabbit anti-mouse CD56 and then with goat anti-rat Alexa fluor 488 secondary antibodies. Rabbit serum and tumor sections without NK treatment were used as negative controls. Nuclei were counterstained with DAPI (original magnification, ×100). Data shown are representative of three independent assays. Yaya Chu et al. Cancer Immunol Res 2015;3:333-344 ©2015 by American Association for Cancer Research