TUSC2 Immunogene Therapy Synergizes with Anti–PD-1 through Enhanced Proliferation and Infiltration of Natural Killer Cells in Syngeneic Kras-Mutant Mouse Lung Cancer Models

Combined TUSC2 and anti–PD-1 upregulates NK and cytotoxic T cells and downregulates regulatory cells. A, Effect of TUSC2 on peripheral NK, T cells, and B cells in tumor-free mice. Pooled samples of n = 3 mice/group. In vivo uptake of TUSC2 nanovesicles determined based on exogenous TUSC2 expression by RT-PCR in sorted T, B, NK, and lineage-negative cells. B, Effect of treatments on NK, T, and B cells at week 2 of tumor implantation. C, TUSC2 treatment altered MDSC status. Gating strategy for MDSC; CD45⁺>CD3^–>MHCII^low>CD11b⁺>Gr-1⁺. Granulocytic MDSC: CD11b⁺Gr-1^high and monocytic MDSC: CD11b⁺Gr-1^low. D, Effect of treatment on Tregs (CD4⁺CD25⁺). E, Surface expression of PD-1, CTLA-4, and Tim-3 on T cells. F, Effect on ratios of NK/MDSC and CD8⁺ T cells/Tregs. Data shown as mean ± SD; n = 5; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

TUSC2 combination increased infiltration of NK and CD8 T cells and impeded MDSCs and Tregs. A, Immunohistochemistry images for formalin fixed tumor. Vectra automated imaging system was used to take high-resolution images (20×) with imaging of 25% of the tumor area. n = 5 tumor sections/groups were imaged. Approximately 100 images per treatment group were analyzed by InForm software for H-Scoring. *, P < 0.05; **, P < 0.01; ***, P < 0.001. B, Chemokine gene expression analysis in tumor samples (n = 3/treatment) shown and determined by NanoString technology. C, Concentration of CCL4 and CCL5 chemokines in serum induced by TUSC2 treatment are shown. Mean ± SD; n = 3; ***, P < 0.001.

Dependence of the antitumor activity of TUSC2 on NK cells. Antitumor activity of TUSC2 + anti–PD-1 treatment was affected by depletion of (A) NK cells and (B) CD8⁺ T cells, shown in tumor intensity graphs (n = 5 mice/group). C, Ratio of IFNγ (Th1) and IL4 (Th2) serum cytokines determined by Luminex assay. D, Concentration of IL18 and IL15 cytokine in NK cell–depleted and nondepleted mice. Mean ± SD, n = 3. E, Fold expression of IL15Ra and IL18R1 in sorted NK cells from mice treated with TUSC2. Mean ± SD, n = 3. F, NanoString analysis of mRNA expression of IL15Ra and IL18R1 in tumors treated with TUSC2. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

TUSC2 + anti–PD-1 treatment significantly improved survival in a Kras-mutant lung metastasis model. A, PD-L1 expression determined by flow cytometry in 344SQ-luc and CMT167-luc cells. B, Sequential treatment shown schematically. C, Survival is shown in Kaplan–Meier curves after treatment (n = 10 mice/group). D, Bioluminescence showed lung specific colonization of tumor cells. A representative of 3 independent experiments shown. E, Dissected lung images show tumor nodule status 2 weeks after tumor implantation. (F) NK, (G) Treg, (H) MDSC, and (I) PD-L1⁺ and PD-L2⁺ leukocyte infiltration determined by flow cytometry of single cells prepared from metastasized lungs. Data were normalized based on per gram of tumor tissue (23). PD + CT indicate anti–PD-1 + anti–CTLA-4 treatment. Mean ± SD; n = 5. *, P < 0.05; **, P < 0.01; ***, P < 0.001.