Tumor-Derived Autophagosomes Coated With Nanodots as Future Personalized Cancer Vaccines

Abstract

In a recent study published in Nature Nanotechnology, You et al. describe how coating tumor autophagosomes with nanodots in situ offers a promising strategy for personalizing cancer vaccines in the treatment of tumors [1]. Here, we explore how titanium nitride-based MXene (Ti₂NX) nanodots help tumor autophagosomes escape fusion with lysosomes, allowing drainage to lymph nodes (LNs), and priming of T cells. This Research Highlight also summarizes the therapeutic effects of Ti₂NX nanodot-coated autophagosomes in different murine tumor models.

Autophagy is an essential intracellular process involving the formation of autophagosomes that degrade and recycle cellular components to maintain cellular homeostasis. Autophagosomes, double-membrane vesicles that engulf and transport intracellular material to lysosomes for degradation [2], are being exploited as vaccines in cancer immunotherapy based on their capacity to carry tumor antigens that can be taken up and cross-presented by antigen-presenting cells (APCs), such as dendritic cells (DCs). Autophagosomes are conventionally prepared intracellularly by increasing lysosomal pH, with compounds like Bafilomycin A1, chloroquine, hydroxychloroquine, or ammonium chloride [2], to prevent fusion with lysosomes and allow autophagosome isolation through cell disruption and gradient centrifugation. However, this conventional approach may reduce autophagosome immunogenicity as increasing lysosomal pH can promote the formation of multivesicular bodies (MVBs) that fuse with autophagosomes. You et al. developed 3 nm Ti₂NX nanodots that coat tumor-derived autophagosomes and inhibit autophagy by preventing autophagosome fusion with lysosomes or MVBs [1]. Unlike conventional autophagy inhibitors that affect autophagy in cancer and immune cells, Ti₂NX nanodots selectively target cancer cells and preserve immune cell function.

The fate of Ti₂NX nanodot-coated autophagosomes (NCAPs) follows a sophisticated and well-coordinated process (Figure 1) that begins with nanodots shielding phosphatidylinositol-4-phosphate (PI4P), expressed on the autophagosome surface, through molecular interactions (e.g., hydrogen bonding) with the phosphate groups of PI4P. This shielding prevents the recruitment of functional proteins, such as SNARE syntaxin 17, that mediate fusion of autophagosomes with lysosomes or MVBs. Subsequent accumulation of NCAPs within tumor cells induces intracellular stress, leading to inflammasome-associated pyroptosis and release of NCAPs from tumor cells for recognition and transport by migratory DCs to the LNs. The size range of NCAPs (200–700 nm) and presence of C-type lectin domains containing 9 A (CLEC9A) ligands on the surface of NCAPs enhance recognition by DCs.

Once in the LNs, NCAPs are processed by LN-resident and migratory DCs for cross-presentation. Tumor antigens carried by NCAPs are then fragmented and presented by DCs to activate T cells. In addition to tumor antigens, NCAPs carry other immunostimulating molecules, such as damage-associated molecular patterns, that may facilitate APC maturation and enhance T-cell priming. The nanodot itself also promotes APC maturation and drives macrophage polarization into antitumor phenotype M1. Finally, activated T cells migrate to the tumor tissues to eliminate cancer cells. Research by Skwarczynski et al. and Zhao et al. involving polypeptides also confirmed that nanoaggregates (300 nm) of small nanoparticles (10–30 nm) composed of antigen–adjuvant conjugates can effectively modulate APC maturation and elicit robust humoral responses against bacteria following subcutaneous administration [3, 4].

To help recognize the translational potential of NCAPs, You et al. initially prepared and isolated NCAPs from allogenic cells to produce Allo-NCAPs in vitro before in vivo studies in a murine breast cancer model. After subcutaneous administration, Allo-NCAPs largely accumulated in LNs, inducing immune infiltration, activating local DCs, CD4 T cells, and CD8 T cells, and promoting the production of inflammatory cytokines, such as tumor necrosis factor-α, interleukin-6 and interleukin-1β, by bone marrow-derived DCs. Notably, the therapeutic effect of Allo-NCAPs surpassed conventionally prepared autophagosomes (Con-APs) following two subcutaneous immunizations with Allo-NCAPs significantly decreasing 4T1 tumor growth in 71% of the mice, while only 14% of Con-AP treated mice showed tumor regression. Higher granzyme B expression and lower CD206 expression were also observed in tumors of mice receiving Allo-NCAPs, compared to mice receiving Con-APs, suggesting more immune activation and less immunosuppression by Allo-NCAPs.

To expand the applications of NCAPs as personalized cancer vaccines, You et al. injected Ti₂NX nanodots intratumorally to endogenously produce Self-NCAPs within mice self-tumors. Three tumor models, including breast cancer (4T1), melanoma (B16-F10), and colon cancer (CT26), were used to explore the antitumoral efficiency of Self-NCAPs. Findings showed that Allo-NCAPs and Self-NCAPs eliminated B16-F10 tumors in mice, demonstrating the potential of Self-NCAPs as a promising cancer vaccine. In the bilateral 4T1 model, doxorubicin, cisplatin, NLRP3 inducer BMS-986299, and Self-NCAPs were delivered only to the right-sided tumor in mice, and among these treatments, only Self-NCAPs eliminated the tumor on the right side. More importantly, Self-NCAPs induced the strongest abscopal effect by targeting and almost eliminating the left 4T1 tumor, while other treatments failed to eliminate either the right or left tumor. This significant outcome was accompanied by stronger pro-inflammatory tumor microenvironments observed in mice treated with Self-NCAPs, characterized by higher numbers of mature DCs in LNs, increased circulation and accumulation of IFN-γ⁺ CD8 T cells in LNs, and greater CD8 T-cell infiltration in right and left tumors.

Intratumoral injections of Ti₂NX nanodots also eliminated established tumors in the colon cancer model, with no relapse observed even after rechallenging of CT26. Again, this outcome was accompanied by a highly pro-inflammatory tumor microenvironment in mice treated with Self-NCAPs. High numbers of CD4 and CD8 T cells also infiltrated tumor tissues in mice treated with Self-NCAPs, with a large proportion of CD8 T cells secreting IFN-γ, and significantly less immunosuppressive Tregs were present in tumors of mice treated with Self-NCAPs compared to those receiving phosphate-buffered saline. The long-lasting antitumor protection provided by Self-NCAPs was associated with a high proportion of effector memory CD8 T cells circulating in peripheral blood. In addition, You et al. demonstrated that the robust antitumor responses correlated with recognition of CT26-specific antigen Slc20a1, as a higher IFN-γ ELISpot response was generated in Self-NCAP-treated mice-derived splenocytes re-stimulated with Slc20a1, compared to restimulation with tumor lysates. These findings indicated that intratumoral administration of Self-NCAPs induced tumor-specific immune responses by priming T cells with tumor-specific antigens. However, it is worth noting that intravenous delivery of Ti₂NX nanodots in mice had the potential to eliminate 4T1 tumor with similar efficiency to intratumoral delivery. Importantly, the safety of systemic nanodot administration was validated by You et al., highlighting it as an alternative approach when intratumoral administration is not feasible. This finding broadens the application of NCAPs in personalized cancer therapies.

Over the past decade, neoantigen-based vaccines have dominated research into personalized cancer vaccines; however, extensive identification of patient-specific neoantigens, which can be recognized by highly antitumoral T cells, is required for the development of personalized neoantigen immunotherapies. The persistence of such rare but highly functional T cells can also be limited by high expression of exhaustion markers, such as PD-1, CTLA-4, LAG-3, and TIM-3 [5]. Hence, combination therapies of checkpoint inhibition and neoantigen-based immunotherapies have proven improved effectiveness. The research conducted by You et al. may help overcome the challenges of developing personalized cancer vaccines by eliminating the need for tumor antigen identification. Tumor-derived autophagosomes generated in situ provide all necessary components for immune activation, including tumor-associated antigens, neoantigens, and immunostimulatory molecules, such as damage-associated molecular patterns. This approach preserves immune function, boosts antigen presentation, and induces durable T-cell responses. Its efficacy in multiple murine tumor models and systemic safety highlight its potential for scalable, next-generation cancer immunotherapy.

Lantian Lu: visualization (lead), writing – original draft (lead). Mariusz Skwarczynski: writing – review and editing (lead). Both authors have read and approved the final manuscript.

The authors have nothing to report.

The authors declare no conflicts of interest.