Loss of function in tumor suppressors like p53 drives tumorigenesis and therapeutic resistance. P53 regulates various cellular pathways and is frequently mutated in cancers, including hepatocellular carcinoma (HCC). Beyond its tumor-suppressive roles, p53 also modulates the immune tumor microenvironment (TME) by influencing interactions between tumor and immune cells. It can induce antitumor immune responses by regulating genes encoding cytokines, chemokines, and pathogen recognition receptors. Genetic restoration of p53 activates immune cells, promoting tumor antigen-specific adaptive immunity and upregulating NKG2D ligands for NK cell activity. It also suppresses pro-tumorigenic tumor-associated macrophage (TAM) polarization. Recent studies suggest that immunogenic cancer cell death enhances p53 pathway efficacy. However, developing therapeutic agents targeting p53 function and overcoming immunotherapy resistance in HCC remains challenging. HCC is a prevalent and deadly liver cancer with a unique immune microenvironment. Immune checkpoint blockade (ICB) therapies, while effective in some cancers, show limited success in HCC due to insufficient tumor immunogenicity and an immunosuppressive TME. Combining ICB with other therapies shows promise but needs further improvement. This study addresses the need for combination therapy to potentiate ICB response in HCC by using a targeted mRNA nanoparticle (NP) platform to induce p53 expression and reprogram the TME, testing it in combination with ICB in p53-null murine HCC models.

The literature extensively documents the role of p53 in tumor suppression and its impact on the immune system. Studies have shown p53's ability to regulate the expression of various immune-related molecules, leading to the modulation of anti-tumor immune responses. The immunosuppressive nature of the tumor microenvironment in HCC has also been well-established, highlighting the challenges in achieving effective immunotherapy. Existing strategies for combining ICB with other therapies show some success, yet the need for more effective combinatorial approaches remains. The use of mRNA-based therapies for cancer treatment is a growing area, with research showing the potential for targeted delivery and enhanced therapeutic efficacy. This study builds on these prior findings by investigating a novel combination therapy targeting p53 restoration in HCC.

This study developed and optimized a CXCR4-targeted mRNA nanoparticle (NP) platform for delivering p53 mRNA to HCC cells. The NPs were engineered using a lipid-polymer hybrid NP self-assembly strategy. The platform incorporated a CXCR4-targeting peptide (CTCE) for selective HCC targeting, conjugated to DSPE-PEG-Mal via a thiol-maleimide Michael addition reaction. A scrambled peptide (SCP) served as a non-targeted control. The researchers optimized the CTCE peptide surface density to maximize cellular uptake in RIL-175 murine HCC cells, confirmed by flow cytometry and confocal microscopy. To optimize mRNA encapsulation and translation, they synthesized a series of Go-CN compounds and selected Go-CN for its high mRNA retention ability, confirmed by luciferase-RNA NP transfection assays. The resulting CTCE-p53 NPs were approximately 110 nm in size, with a spherical and uniform structure verified by TEM. In vitro studies demonstrated that the CTCE-targeted NPs significantly enhanced p53 protein expression and reduced HCC cell viability compared to non-targeted NPs, confirmed by Western blotting and immunofluorescence. Pharmacokinetics (PK) and biodistribution (BioD) studies in healthy and HCC-bearing mice showed prolonged mRNA circulation and enhanced tumor accumulation of CTCE-targeted NPs compared to non-targeted NPs and free mRNA. In vivo studies used orthotopic and ectopic p53-null murine HCC models. Mice were treated with CTCE-p53 NPs, anti-PD-1 (aPD1), or a combination thereof. Tumor growth was monitored by high-frequency ultrasound imaging. Flow cytometry assessed immune cell infiltration and activation in tumor tissues. Multiplexed analysis measured immune cytokine levels. In vivo safety was assessed by monitoring body weight, organ weights, blood chemistry, and histopathology. The study also investigated in vivo safety via weight monitoring, organ weight analysis, blood chemistry, and histopathological examination.

The CXCR4-targeted p53 mRNA NPs effectively delivered p53 mRNA to HCC cells, restoring p53 expression. The combination of CTCE-p53 NPs and anti-PD-1 therapy significantly inhibited tumor growth in both orthotopic and ectopic p53-null murine HCC models, exceeding the efficacy of either treatment alone. Combination therapy significantly increased the number of infiltrating CD8+ T cells and the fraction of activated IFN-γ+TNF-α+ CD8+ T cells. It also increased the fraction of M1-like tumor-associated macrophages (TAMs) while decreasing M2-like TAMs. This combination also led to increased levels of TNF-α and IL-1β cytokines. In the orthotopic model, combination therapy significantly prolonged survival, reduced ascites and pleural effusions, and reduced lung metastases. In vivo safety studies showed no significant adverse effects from the combination treatment.

This study successfully demonstrates that restoring p53 expression using a CXCR4-targeted mRNA NP platform, in combination with anti-PD-1 therapy, significantly enhances antitumor efficacy in p53-deficient HCC. The results strongly suggest that the combination therapy globally reprograms the immune TME, shifting it from an immunosuppressive state to one that supports antitumor immunity. The observed increase in CD8+ T cells, activated CD8+ T cells, M1 TAMs, and pro-inflammatory cytokines highlights the multifaceted impact of the combination therapy on the immune microenvironment. The enhanced survival and reduced metastasis further underscore the clinical potential of this therapeutic strategy. These findings warrant further investigation into the precise mechanisms by which p53 restoration influences immune cell function within the tumor microenvironment. Additional research should explore the effects of different p53 mutations and the potential for combining this therapy with other immunotherapies or targeted therapies to further improve its efficacy.

This study provides compelling preclinical evidence supporting the use of a novel combination therapy of CXCR4-targeted p53 mRNA nanoparticles and immune checkpoint blockade for the treatment of p53-deficient HCC. The significant improvement in tumor growth inhibition, immune response, survival, and reduced metastasis in murine models suggests substantial clinical potential. Future research should focus on translating these findings into clinical trials to evaluate the safety and efficacy in human patients. Investigating the long-term effects and potential for combination with other therapies are also crucial next steps.

The study was conducted using murine models, and the results may not perfectly translate to human HCC. The relatively small sample size in some of the in vivo experiments limits the statistical power. Further research is needed to fully elucidate the mechanisms underlying the observed therapeutic effects and to assess the potential for resistance development.