细胞因子、PM21颗粒刺激与TGF-beta预处理的联合应用可产生细胞毒性增强并能浸润实体瘤的自然杀伤细胞

In this study, feeder cell-free PM21 particle activation of Natural Killer (NK) cells was combined with cytokine activation and TGF-beta conditioning to produce highly proliferative and cytotoxic NK cells that can infiltrate solid tumors. Development of NK cell therapeutics is a promising anti-cancer therapy. For clinical applications, NK cells are typically either ex vivo activated with cytokines or expanded with feeder cells or by feeder cell-free methods. One such method for feeder cell-free expansion of highly cytotoxic NK cells uses plasma membrane particles containing surface IL-21 and 41BBL (PM21). Additionally, NK cells expanded with IL-12, IL-15, and IL-18 in combination with PM21 stimulation (CAP-NK cells) exhibit robust proliferation, potent cytotoxicity, and memory-like features. To further improve their activity against solid tumors-a setting often resistant to NK cells-we introduced TGF-beta conditioning. Previous studies have shown that ex vivo TGF-beta conditioning induces IFNgamma hypersecretion. To assess whether this could enhance anti-tumor function, PM21- and CAP-NK cells were expanded in the presence of TGF-beta. The CAP-based expansion method still resulted in enhanced NK-cell expansion, even with TGF-beta conditioning. CAP-based expansion remained highly effective even with TGF-beta conditioning, achieving an average 4000±700-fold expansion by day 14 compared to 1800±60-fold for PM21-NK cells. TGF-beta conditioned NK cells retained high viability after cryopreservation (>80% immediately and at 16 h post-thaw) and 99±1% expressed CD25 by day 7. These cells produced significantly more IFNgamma upon stimulation than unconditioned CAP-NK cells. Moreover, TGF-beta conditioned CAP-NK cells acquired a tissue-resident-like phenotype (CD103+, CD49a+, CD300a−) not observed in PM21- or CAP-NK cells, which would be expected to enhance their ability to infiltrate tumors. To test this, labeled TGF-beta conditioned CAP-NK and PM21-NK cells were co-cultured with large lung tumor spheroids and monitored via live-cell imaging. TGF-beta conditioned NK cells penetrated tumors faster and deeper than PM21-NK cells, which remained mostly at the periphery, resulting in greater spheroid killing-even in tumors overexpressing TGF-beta.TGF-beta conditioning also enhanced cytotoxicity against multiple solid tumor cell line spheroids, including lung, pancreatic, and neuroblastoma. Overall, these findings support TGF-beta conditioned CAP-NK cells as a potent cellular therapy candidate for solid tumors.