Figure 6.

1Gy x 4 fractions (1Gy x 4F) or 4Gy x 1 fraction (4Gy x 1F) combined with anti-GCC CAR-T cell therapy exhibited significantly better tumor growth control and survival than CAR-T therapy alone in a bilateral tumor model. (A) Primary tumor growth comparison shows a significant difference (p<0.02) between mice receiving 1Gy x 4 fractions vs. mice receiving CAR-T cells only. Four mice from the CAR-T group were euthanized by day 30 due to tumor burden and only one mouse survived to the end of the experiment. Also, mice receiving 1Gy x 4 fractions combined with CAR-T cells showed significantly better control in the primary tumor (p<0.009) than 4Gy x 1 fraction + CAR-T cell therapy. Although no significant differences were observed in the control of the secondary tumor (B), the dual therapy 1Gy x 4 fractions combined with CAR-T cell therapy showed enhanced control of the primary tumor and a significant survival rate ((C), p<0.0001) than mice receiving CAR-T cell therapy or radiation therapy alone. (D) We also analyzed the CAR-T phenotype on cells collected at the time of their death (n=3). With exception to the UTD control, all groups had representative populations in the primary tumors including CD4⁺PD-1⁺ (C23), terminally differentiated CD4⁺ T cells (CD4⁺CD69⁺, CD45RA⁺, CCR7^-; C28), and activated CD8⁺cells, (CD69⁺, PD-1⁺; C26). Also, in the secondary tumors, except for the UTD group, we did not observe changes in the expression of CD4 (C28) or CD8 (C34, C36) between the groups. E) The red clusters in both primary and secondary tumors, represent the distribution of the CD3⁺ populations. (n= 5 mice per group). The t-SNE maps were generated using the R-based software interface Cytofkit (https://www.bioconductor.org/packages/3.5/bioc/html/cytofkit.html).