Abstract:

Background and purpose: Gallic acid (GA) induces tumor cells apoptosis and inhibits angiogenesis. Beyond directly attacking tumor cells, another crucial aspect of GA is its ability to modulate and enhance immune system function. For example, it can improve T cell metabolism, alleviate T cell exhaustion, and promote the formation of memory T cell phenotypes. Although several chimeric antigen receptor T (CAR-T) cells products have gained market approval, the technology still faces significant challenges. These limitations include off-target effects, a predisposition to T cell exhaustion and so on. Moreover, similar to exhaustion, cellular senescence is a major hindrance that impairs T cell function. This study aimed to investigate the effects of GA on the anti-tumor function of CAR-T cells both in vitro and in vivo. We further evaluated the impact of GA on CAR-T cells senescence and memory phenotypes, as well as the impact of GA and CAR-T cells on immune cell infiltration within the tumor microenvironment (TME). Methods: Second-generation CAR targeting mouse glypican 3 (GPC3) and human epidermal growth factor receptor 2 (HER2) were constructed to generate CAR-T cells. CAR-T cells were co-cultured with GA at a concentration of 5 μg/mL, and flow cytometry was used to assess the senescence status and memory phenotype of CAR-T cells and their killing ability against tumor cells at different effector-to-target ratios. Senescence markers included p53, p21, γ-H2AX and senescence-associated β-galactosidase (SA-β-gal), while CCR7 served as the memory phenotype marker. A subcutaneous tumor model was established to explore the effects of GA on the anti-tumor function of CAR-T cells and immune cell infiltration within the TME. Results: We successfully generated human HER2 and murine GPC3 CAR-T cells, achieving a purity of 30%-50%. GA enhanced the in vitro killing ability of CAR-T cells targeting mouse GPC3 and human HER2 (P<0.001) at different E:T ratios, delayed the senescence of mouse GPC3 CAR-T cells (p53, p21, γ-H2AX, P<0.05; SA-β-gal, P<0.001; CCR7, P<0.001). And GA promoted the differentiation of CAR-T cells toward a memory phenotype (P<0.001). Additionally, GPC3 CAR-T cells inhibited tumor cell growth (P<0.05), prolonged mouse survival (P<0.001), and enhanced the infiltration capacity of CAR-cells (P<0.001) and endogenous immune cells [CD4⁺ T cells, P<0.05; CD8⁺ T cells, P<0.01; natural killer (NK) cells, P<0.01]. Conclusion: GA can enhance the cytotoxic activity of CAR-T cells in vitro, and delay the senescence of CAR-T cells. Furthermore, by modulating TME, GA improved immune cell infiltration, thereby augmenting the overall anti-tumor efficacy of CAR-T cells.

Key words: Gallic acid, Chimeric antigen receptor T cells, Anti-solid tumor, Senescence, Tumor microenvironment