Pancreatic adenocarcinoma (PAAD) is one of the most aggressive and lethal types of gastrointestinal cancer. Immunotherapies have revolutionized intervention strategies for various primary malignancies, but to date, it has little implementation in PAAD [1,2]. Immune evasion encompasses several complex mechanisms through which tumors evade tumoricidal immunity, escape recognition or block destructive immunoreaction, presenting a major obstacle for PAAD immunotherapy [3]. Reversion of immune evasion may therefore be conducive to the activation of anti-tumor immunity against pancreatic carcinoma, however, effective strategies remain elusive.

Expression of the major histocompatibility complex class I (MHC-I) is necessary for cytotoxic CD8+ T-cell-mediated recognition of tumors [4], [5], [6]. However, PAAD cells are characterized by heterogeneous levels of total MHC-I surface expression and very recent insights indicate the loss or reduced surface expression of MHC antigens on PAAD cells may account for tumor immune evasion [7,8]. Autophagy is an evolutionarily conserved multistep degradation mechanism in which cytoplasmic contents, such as long-lived proteins and old/damaged organelles, are wrapped by double-membraned autophagic vacuole and fused with lysosomes for degradation [9]. In PAAD, autophagy is elevated and MHC-I molecules are selectively targeted for autophagy-dependent degradation, leading to a down-regulation of expression, and even loss of MHC-I surface expression [10,11]. As such, selective inhibition of autophagy may restore surface levels of MHC-I, contribute to reversion of immune evasion, and ultimately be conducive to activating tumoricidal immunity against PAAD.

Toll-like receptors (TLR) are critical in pathogen recognition, and in response, trigger innate immune activation [12,13]. TLR9 is over-expressed on plasmacytoid dendritic cells (pDCs) and B cells [14]. Oligodeoxynucleotides (ODNs) with CpG motifs are agonists of TLR9 [15,16]. Classical research has classified CpG ODNs into three groups based on their structural features and activities in human peripheral blood mononuclear cells (PBMC), B cells and pDCs [17]. CpG-A ODNs have been observed to elicit heightened production of IFN-α by pDCs, albeit weak activation of the TLR9-dependent NF-kB signaling pathway [18]. Conversely, CpG-B ODNs have been observed to exert the opposite effect [19]. CpG-C ODNs possess a hybrid structure of Class A and B, encompassing the complete PS skeleton and CpG palindrome sequence, and have demonstrated a potent ability to induce IFN-α secretion and increase the expression of costimulatory molecules (CD80, CD86, CD40) in DCs [20], which were associated with the generation and subsequent activation of anti-tumor CD8+ T cells [21,22].

To reverse immune evasion meanwhile boosting anti-tumor T cell stimulation, we herein sought to construct a ROS-responsive DNA-based nano-orchestrator to cascade activating TLR9 and inhibiting autophagy for PAAD immunotherapy. As illustrated in Scheme 1, chloroquine phosphate (CQP) as an autophagy inhibitor [23] was firstly encapsulated in ferritin [24]. And via DNA modular self-assembly technology [25], [26], [27], [28], the generated ferritin nanocores (FNC) were then caged into ROS-responsive CpG-DNA nanoframe, subsequently obtaining FNC-laden DNA nanoframe ([email protected]). After systemic injection, [email protected] was passively enriched in tumor tissues in which the DNA nanoframe was cleaved upon the ROS stimulation [29], [30], [31]. ODNs with CpG motifs were detached and functioned as a TLR9 agonist. The liberated FNC was then endocytosed in an actively targeted manner by binding to transferrin receptor 1 (TfR1) [32]. In the lysosome, CQP was burst released from FNC due to acid-triggered destruction of ferritin [33]. Through CQP-mediated autophagy abrogation, MHC-I molecules were preserved. We demonstrated that cascade inhibiting autophagy and boosting TLR9 stimulation via our proposed DNA-based hybrid nanosystem restored MHC I at the tumor cell surface and reshape the antigen presentation of DCs, and ultimately reversed immune evasion and synergistically reinforced the function of cytotoxic T cells against PAAD cells.