Background: The broader application of adoptive cell therapy (ACT) in cancer immunotherapies (particularly for solid tumors) has always been limited by the immunosuppressive tumor microenvironment (TME) and the insufficient targetability of effector T cells, resulting in unsatisfied therapeutic outcome. Here, we designed a new strategy by using aptamer-based immunoliposomes to modify PD-1-silencing T cells, which were activated by dendritic cell (DC)/tumor fusion cells (FCs) to improve the antitumor potency of cytotoxic T lymphocytes (CTLs/CD8⁺ T cells). Methods: PD-1 gene was knocked out from CD8⁺ T cells using CRISPR/Cas9 system to liberate T cell activity from immunosuppression. The PD-1⁻ T cells were stimulated with DC/tumor FCs, followed by further functional modification of tumor-specific nanoliposomes (hEnd-Apt/CD3-Lipo) to generate FC/PD-1⁻ CTLs. The activation and proliferation and specificity of the modified FC/PD-1⁻ CTLs were measured. The antitumor activity of these CTLs against HepG2-tumors was evaluated in xenograft NOD/SCID mice, and the antitumor mechanism was investigated based on tissue immunohistochemistry and serum ELISA. Results: Our results indicated that the modification of hEnd-Apt/CD3-Lipo nanocomposites on the FC/PD-1⁻ CTLs had a more substantial synergetic effect in inhibiting tumor growth and prolonging animal survival, rather than other control liposomes. Furthermore, the hEnd-Apt/CD3-Lipo-modified FC/PD-1⁻ CTLs showed a stronger antitumor outcome in the tumor-bearing mouse model, through the mechanisms of suppressing tumor cell proliferation, promoting tumor apoptosis, reducing angiogenesis but increasing the infiltration of the FC/PD-1⁻ CTLs in the tumor tissue, as well as upregulating the systemic levels of IFN-γ, IL-2, TNF-α and IL-6 cytokines, by comparison of the control settings. Conclusion: In sum, our investigation suggests an enhancement of antitumor effect by the surface modification of endoglin-targeting nanoliposomes upon DC/tumor FC-activated PD-1⁻ CTLs, therefore, provides a new tumoral endoglin-targeted approach as a promising strategy to reduce immunosuppression of tumor microenvironment and improve the immunotherapeutic outcome of anticancer ACT.

Luteolin, a natural flavonoid compound, has demonstrated anti-inflammatory, antioxidant, and broad anti-tumor properties. Recent studies suggest that its anti-tumor effects are linked to enhanced CTL function—including proliferation, survival, and cytotoxicity-via inhibition of the YAP/Wnt signaling pathway in tumor cells. Consequently, luteolin has potential as an adjuvant in combination therapies with adoptive immunotherapy. This study first assessed luteolin’s tumor-inhibitory effects in vitro and in vivo using cytotoxicity assays, Transwell invasion tests, wound healing assays, and analyses of post-treatment tumor growth and survival time. Additionally, we explored whether luteolin combined with a DC/tumor fusion vaccine could synergistically enhance overall antitumor efficacy by boosting activation, proliferation, cytokines secretion, and cytotoxicity of effector T cells. Our findings indicate that luteolin, as a standalone agent, can inhibit the proliferation and invasion of colon and lung cancer cells both in vitro and in vivo to a certain extent. When combined with activated CTLs, it upregulated the expression of CD25 and CD69 in effector cells and resulted in higher levels of IL-2, TNF-α, and IFN-γ secretion in vitro. In vivo, this combination significantly curtailed subcutaneous tumor growth and extended the mean survival time of tumor-bearing mice (HCT116, A549), outperforming luteolin monotherapy. Furthermore, the efficacy of this combination therapy may be attributable to enhanced apoptosis in tumor cells, reduced proliferation, and decreased YAP expression. The combination of luteolin and DC/tumor fusion vaccine-activated CTLs presents a novel approach for cancer treatment. As an adjuvant, luteolin downregulates YAP expression in tumor cells, enhancing CTL proliferation, cytotoxicity, and survival, thus improving tumor recognition and selective targeting. This strategy is promising for safe and effective tumor treatment.