Single-cell sequencing-guided design of synergistic chemo-immunotherapy nanodrugs for cGAS-STING activation in prostate cancer therapy
Yu Jiang, Yaowu Zhang, Jingqi Hou, Heng Liu, Xianyu Dai, Yuchuan Hou
TL;DR
This paper introduces a new nanodrug that uses single-cell sequencing to activate the immune system in prostate cancer by targeting VSIG4 and triggering the cGAS-STING pathway.
Contribution
A novel nanodrug design is proposed, combining chemo-immunotherapy to convert 'cold' prostate tumors into 'hot' ones through VSIG4 downregulation and cGAS-STING activation.
Findings
VSIG4 is identified as a key regulator of macrophage fate in prostate tumors.
Mn/Shik@CpG nanodrugs synergistically activate cGAS-STING and induce immunogenic cell death.
The treatment remodels the tumor immune microenvironment and elicits durable immune memory.
Abstract
Characterizing the tumor immune microenvironment (TIME) to explore potential therapeutic targets is fundamental to advancing precision tumor immunotherapy. However, the immunosuppressive nature of “cold” tumors, notably prostate cancer, poses a significant barrier to immunotherapy, demanding new approaches to simultaneously reinvigorate anti-tumor immunity and modulate the molecular drivers of immune evasion. Here, we identified VSIG4 as a key regulator of prostate tumor-resident macrophage fate through single-cell sequencing analysis. Meanwhile, a shikonin (Shik)-mediated downregulation of VSIG4 in macrophages is verified, potentially attenuating its immunosuppressive effects. Building on these findings, cytosine guanine dinucleotide (CpG) oligodeoxynucleotide (ODN)-modified manganese (Mn)-Shik metal-polyphenol network nanodrugs (Mn/Shik@CpG NDs) are designed to reverse the “cold”…
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Figure 8- —https://doi.org/10.13039/501100001809National Natural Science Foundation of China
- —https://doi.org/10.13039/100015800Jilin Province Development and Reform Commission
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Taxonomy
Topicsinterferon and immune responses · Ferroptosis and cancer prognosis · Cancer Immunotherapy and Biomarkers
Introduction
Precision medicine has revolutionized the landscape of tumor immunotherapy [1], and the heterogeneous immune cells within the tumor microenvironment (TME) represent a critical avenue for identifying key therapeutic targets. However, despite the identification of numerous immune-related targets that influence tumor progression, achieving effective drug delivery to the tumor region and ensuring therapeutic efficacy remain formidable challenges for conventional drugs. Notably, nanodrugs (NDs) represent a promising platform that surpasses the limitations of traditional drug delivery, with distinct advantages in enhancing tumor-targeting and modulating adaptive immune responses [2]. The rational design of NDs tailored to specific therapeutic targets within the TME may help overcome existing limitations in tumor immunotherapy.
As a classic immunologically “cold” tumor of the genitourinary system, prostate cancer serves as a key model for studying antitumor immunotherapy [3]. Its TME is characterized by a significant enrichment of immunosuppressive cell populations, such as tumor-associated macrophages (TAMs), regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs), which further curtails the inherently sparse infiltration of effector T cells [4]. It is well-established that macrophages exhibit considerable plasticity, allowing them to dynamically alter their functional state to meet the pathological demands of various tissues and diseases [5, 6]. Within the prostate cancer environment, TAMs play a pivotal role in tumor progression and immune regulation, a complexity likely tied to their intricate differentiation pathways [7]. However, the key mechanisms governing their fate remain largely elusive.
Single-cell sequencing has revolutionized the study of tumor heterogeneity, allowing us to parse complex cellular subpopulations and their functional trajectories in ways that bulk analysis cannot. This technique enables the precise identification of the key regulators responsible for the immunosuppressive functions of TAMs. Therefore, targeting such molecular targets presents a promising strategy to dismantle the suppressive barrier that TAMs impose on antitumor T cell immunity. While reprogramming immunosuppressive macrophages is a vital first step, achieving a durable antitumor response ultimately hinges on robust T cell activation. However, T cell effector function is not autonomous. Their function is fundamentally governed by an intricate network of signals from the innate immune system. The dependency underscores the need for a therapeutic strategy that can effectively bridge the innate and adaptive arms of immunity to truly dismantle the immunosuppressive network in prostate cancer.
The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway is a key mediator between innate and adaptive immunity and an important regulator of the tumor microenvironment. It orchestrates the initial innate activation required to prime adaptive responses, influencing the interplay between tumor cells and their surrounding immune and stromal components. It triggers interferon production in response to various DNA sources, including cytosolic dsDNA from tumor cells. The subsequent secretion of interferons promotes dendritic cell (DC) maturation, thereby amplifying the cytotoxic function of CD8^+^ T cells [5]. Divalent manganese ions (Mn^2+^) have recently emerged as powerful sensitizers of the cGAS-STING pathway [8], capable of promoting its activation under physiological conditions [9]. This provides a rationale for coupling STING sensitization with agents that release endogenous ligands for the pathway. For instance, agents that induce immunogenic cell death pathways such as necroptosis are known to release a host of damage-associated molecular patterns (DAMPs), including cytosolic dsDNA [10]. This accumulation of cytosolic dsDNA then acts as a primary trigger for the innate immune response, an effect that is strongly amplified by Mn^2+^ to effectively prime the adaptive immune system.
Immunoadjuvants are agents that enhance the magnitude, breadth, and durability of an immune response [11]. Cytosine-guanine dinucleotide (CpG) oligodeoxynucleotides (ODNs) are particularly promising immunoadjuvants that stimulate innate and adaptive immunity by activating Toll-like receptor 9 (TLR9), leading to DC maturation, T Helper 1 (Th1) cell differentiation, and pro-inflammatory cytokine production [12]. As synthetic single-stranded DNA, CpG ODNs possess a strong negative charge at physiological pH, making them ideal for straightforward incorporation onto ND surfaces via electrostatic adsorption [13]. By combining DAMP release resulting from immunogenic cell death (ICD), cGAS-STING pathway activation, and the immunostimulatory properties of CpG ODNs, a powerful and synergistic antitumor immune effect can be elicited.
Herein, we present a chemo-immunotherapeutic strategy built upon a synergistic framework of immunogenic cell death (ICD), STING pathway sensitization, and potent immunoadjuvant activity to reverse the immunosuppressive TME of prostate cancer. Specifically, we employed metal-polyphenol self-assembly and electrostatic adsorption to construct CpG-coated manganese-Shik nanodrugs (Mn/Shik@CpG NDs) as our final therapeutic agent. While manganese-coordinated polyphenol nanoplatforms show promise [14–16], their efficacy is often curtailed by the marked heterogeneity of the tumor immune microenvironment (TIME). We therefore engineered the NDs tailored to specific molecular features of prostate cancer, seeking to surpass the therapeutic limits of generalized formulations via rational, context-dependent engineering. Within the acidic and glutathione (GSH)-rich TME, the NDs are designed to disassemble, releasing Mn^2+^, Shik, and CpG ODN. Shik-induced necroptosis and reactive oxygen species (ROS) production generates damage-associated dsDNA, which in turn initiates the cGAS-STING pathway. This signaling is then amplified by Mn^2+^, a cGAS-STING pathway sensitizer. Concurrently, as an immunoadjuvant, CpG ODN promotes DC maturation and forges a critical link between the innate and adaptive immune systems. And our initial finding that Shik influences a key gene governing TAM fate proved to be an important driver of success in remodeling the “cold” TME. By harnessing the synergistic antitumor immune effects of Shik, Mn^2+^, and CpG ODN, our Mn/Shik@CpG NDs demonstrated potent therapeutic efficacy in prostate cancer.
Results and discussion
Shik affects the key macrophage gene VSIG4 in the prostate TME
Variations in single-cell processing can lead to differential cell capture rates. To dissect the role of macrophages in prostate cancer, we utilized a publicly available single-cell sequencing dataset that was generated with a protocol optimized for the preservation of immune cells [17] (Fig. 1a). Following the original cell annotations, we applied the scANVI method [18] for batch correction (Supplementary Fig. 1). The credibility of our major cell clusters was validated by the concordance between MetaTiME automatic annotation [19] (Supplementary Fig. 2) and manual annotation using canonical marker genes (Supplementary Fig. 3). A comprehensive list of marker genes used for annotation is provided in Table S1. Since monocytes, macrophages, and DCs all arise from a common monocyte-macrophage DC progenitor (MDP), we grouped these related lineages for a unified sub-cluster analysis [20].
Fig. 1. Single-cell analysis of the prostate cancer immune microenvironment. (a) Minimum-distortion embedding (MDE) dimensionality reduction plot visualizing major cell types in the integrated dataset. (b) MDE plot showing subtypes of MDP-derived cells. (c) Stacked bar plot illustrating the proportion of MDP-derived cell subtypes across different sample types. (d) Uniform manifold approximation and projection (UMAP) plot visualizing cell subtypes after metacell calculation on MDP-derived cells using the SEACells algorithm. (e) Pseudotime trajectory of metacells, with the red color gradient indicating later pseudotime points. (f–g) Scatter plots showing the correlation between original pseudotime and predicted pseudotime from ridge regression models trained on all genes (f) or a curated set of 11 genes (g). (h) Heatmap showing the expression dynamics of 7 key genes along the pseudotime trajectory (blue: low expression, red: high expression). (i) Representative western blot bands for VSIG4 expression in BMDMs at M0-like state, after M2 polarization, and after Shik treatment (2 µg/ml Shik for 24 h) of M2-polarized cells. (j) Densitometric quantification of VSIG4 protein levels. Data are presented as mean±SD (n = 3). **p < 0.01
To delineate the fate trajectories of macrophages within the prostate cancer microenvironment, we moved beyond the classic M1/M2 dichotomy, which is often insufficient to capture their true functional heterogeneity. Instead, our unbiased, data-driven clustering revealed four distinct macrophage populations: antigen-presenting macrophages (APMΦ) (CD74^high^), inflammatory macrophages (IMΦ) (IL1B^high^, CCL20^+^, IL23A^+^), repair-associated macrophages (RMΦ) (CD25^+^, LYVE1^+^), and M0-like macrophages, enriched for genes involved in metabolism, redox regulation, and mitochondrial function (Supplementary Fig. 4). Monocytes were classified as classical monocytes (CMo) (S100A9^+^, FCGR3A^−^) or non-classical monocytes (NCMo) (S100A9^+^, FCGR3A^+^). Consistent with the original study, a population exhibiting hybrid monocyte-macrophage characteristics was annotated as mono-macrophages (MoMΦ)^17^ (Supplementary Fig. 5). The monocyte-macrophage DC progenitor (MDP)-derived cells were further classified as shown in Fig. 1b.
An analysis of cell proportions highlighted a distinct immunological profile for each tissue type (Fig. 1c). Healthy tissues were characterized by high frequencies of CMo and APMΦ, whereas adjacent normal tissues had a greater abundance of DCs. In contrast, the tumor microenvironment itself was defined by a clear predominance of repair-associated macrophages (RMΦ). Effective antitumor immunity is heavily dependent on the functional state of immune cells within the TME [21]. While the proportion of IMΦ was comparable across all sample types, the predominance of RMΦ in tumor samples likely suppresses the activity of other pro-inflammatory and antigen-presenting cells. This phenotype aligns with the behavior of M2-like macrophages [22] and is consistent with the immunologically cold nature of prostate cancer.
Since macrophages at various differentiation stages play distinct roles in the TME [23], investigating their developmental pathways is crucial to understanding the prostate cancer immune landscape. We next performed a pseudotime analysis on the macrophage lineage. Despite a shared monocytic origin for some DCs, they are widely recognized as a distinct hematopoietic lineage from macrophages [24]. Therefore, DCs were excluded from this trajectory analysis. To mitigate the impact of noise inherent in single-cell data while preserving cellular heterogeneity, we employed SEACells to compute metacells [25] prior to performing trajectory inference (Fig. 1d and e). The analysis positioned RMΦ at the terminal end of the pseudotime trajectory, with M0-like MΦ and CMo serving as progenitor populations. The apparent developmental trajectory towards the RMΦ phenotype directly accounts for their dominance within the prostate TME and highlights their important role in driving tumor progression.
Using adaptive threshold regression, a model constructed from just 11 genes achieved an excellent r^2^ value of 0.96 (Fig. 1g), nearly identical to a model trained on all genes (Fig. 1f), indicating that this compact gene set effectively captures the cellular states along the trajectory. Further statistical validation with the Kendall’s tau test identified a core set of six macrophage differentiation fate-determining genes (MDFGs) with the highest significance: FOLR2, RASD1, VSIG4, NMB, DNAB4, and GEM. Analysis of their expression along the pseudotime axis showed that, with the exception of EREG, all MDFGs were upregulated as cells progressed along the trajectory (Fig. 1h). Given the immunosuppressive role of the terminally differentiated RMΦ, we examined differential gene expression between RMΦ and other cell types along the trajectory (Supplementary Fig. 6). Notably, VSIG4 consistently emerged as a top differential gene distinguishing RMΦ from all other populations in the monocyte-macrophage lineage within the tumor tissue. This finding, combined with its strong positive correlation with pseudotime, suggests that VSIG4 expression in tissue-resident macrophages is intimately linked to prostate cancer progression.
VSIG4, also known as CRIg, is exclusively expressed on a subset of tissue-resident macrophages [26]. It plays a pivotal role in maintaining immune homeostasis by inhibiting complement pathway activation and suppressing early T cell activation, while also promoting the differentiation of induced regulatory T cells (iTreg) [27, 28]. While beneficial in controlling inflammatory diseases, these functions contribute to an immunosuppressive TME that hampers the efficacy of cancer immunotherapy [29]. Despite observations of elevated VSIG4 expression in elderly prostate cancer patients [30], its functional role in tumor progression remains poorly defined. Research by Liao et al. has positively correlated VSIG4 expression with M2 macrophage differentiation [31]. This aligns with our single-cell data, where RMΦ exhibit an M2-like, immunosuppressive gene signature. Therefore, targeting VSIG4 expression on these macrophages presents a rational strategy for remodeling the prostate cancer TME. During macrophage differentiation, the MEK/ERK pathway promotes PPARγ signaling, which in turn drives M2 polarization [32]. Shik has been shown to directly bind the ligand-binding domain of PPARγ, disrupting its interaction with coactivators, and to epigenetically repress PPARγ target gene expression by modulating histone modifications [33]. These findings prompted us to investigate whether Shik, likely through its known inhibition of PPARγ, could modulate VSIG4 expression. Western blot analysis substantiated this hypothesis, revealing a significant downregulation of VSIG4 expression in M2-polarized mouse bone marrow-derived macrophages (BMDMs) following treatment with 2 µg/mL Shik for 24 h (Fig. 1i and j). Given the established role of VSIG4 in suppressing early T cell activation, this result lends further support to the findings of Yuan et al., which demonstrated that Shik not only downregulates PD-L1 expression on macrophages but also increases T cell proportions [34]. This preliminary finding cemented the potential of Shik as a VSIG4-modulating agent. To translate this activity into a more robust therapeutic, we then rationally designed a multi-component ND, Mn/Shik@CpG, that integrates Shik with Mn^2+^ and the immunoadjuvant CpG to not only alleviate immunosuppression but also to actively ignite a potent anti-tumor immune response.
Synthesis and characterization of Mn/Shik@CpG NDs
Extending our previous research in metal-polyphenol supramolecular chemistry [35], we synthesized Mn/Shik NDs and subsequently coated them with CpG ODN via electrostatic adsorption. Transmission electron microscopy (TEM) confirmed that both the core Mn/Shik NDs and the final Mn/Shik@CpG NDs were uniformly spherical and monodisperse. Complementary elemental mapping highlighted a homogeneous distribution of manganese ions within the nanostructure. Moreover, unlike the Mn/Shik counterparts (Supplementary Fig. 7), the Mn/Shik@CpG NDs exhibited a distinct and even nitrogen signal, thereby providing visual confirmation of the successful CpG ODN surface coating (Fig. 2a). Dynamic light scattering (DLS) analysis showed that the hydrodynamic diameter of Mn/Shik@CpG NDs (95.1 ± 52.3 nm) was slightly larger than that of the uncoated Mn/Shik NDs (90.0 ± 60.8 nm), consistent with the addition of a surface CpG layer. Both formulations exhibited a low polydispersity index (PDI < 0.3), indicating a narrow and monodisperse size distribution (Fig. 2b). This size range is optimal for leveraging the enhanced permeability and retention (EPR) effect in solid tumors [36]. To assess colloidal stability under physiological conditions, we monitored the hydrodynamic diameter variations of the NDs over 7 days. In H_2_O, the NDs maintained a consistent size (~100 nm) and low PDI (< 0.2), confirming structural integrity. In serum-containing medium, a slight increase in hydrodynamic size (~150 nm) and PDI (< 0.4) was observed (Supplementary Fig. 8), which is attributable to the formation of a protein corona, a characteristic phenomenon in biological fluids. Surface charge of the NDs was evaluated by measuring their ζ-potential. Uncoated Mn/Shik NDs displayed a ζ-potential of −39.2 ± 5.5 mV, which ensures colloidal stability and prevents aggregation in vivo. The addition of the polyanionic CpG ODN further increased the negative surface charge to −46.7 ± 7.0 mV for Mn/Shik@CpG NDs (Fig. 2c), enhancing their dispersibility and stability, which is beneficial for systemic circulation and biological function [37].
Fig. 2. Synthesis and characterization of Mn/Shik NDs and Mn/Shik@CpG NDs. (a) Representative TEM image with elemental mapping of Mn/Shik@CpG NDs. (b–c) Hydrodynamic diameter (b) and zeta potential (c) of the NDs. (d–f) High-r esolution XPS spectra of the O 1s (d), C 1s (e), and Mn 2p (f) regions for Mn/Shik NDs. (g–k) High-resolution XPS spectra of the P 2p (g), O 1s (h), C 1s (i), Mn 2p (j), and N 1s (k) regions for Mn/Shik@CpG NDs. (l) UV-vis absorption spectra of different formulations. (m–n) TME-responsive disassembly of Mn/Shik NDs (m) and Mn/Shik@CpG NDs (n) in different environments. (o–p) Representative live/dead fluorescence staining images (o) and quantification of viable RM-1 cells (p) after treatment with different formulations (equivalent to 12 μg/mL Mn/Shik@CpG NDs) for 24 h
X-ray photoelectron spectroscopy (XPS) was used to analyze the elemental composition and chemical states. The C 1 s spectrum of Mn/Shik NDs was deconvoluted into peaks corresponding to C-C/C-H (284.8 eV), C-O (286.4 eV), and C = O (287.8 eV) bonds, confirming the successful incorporation of Shik (Fig. 2d). The O 1 s spectrum showed peaks for C-O (532.7 eV), C = O (531.0 eV), and Mn-O (532.0 eV), the last of which confirms coordination (Fig. 2e). The Mn 2p spectrum showed the Mn 2p_3/2_ and Mn 2p_1/2_ doublet (641.1 eV and 652.8 eV) and a satellite peak (~645.0 eV), which is characteristic of Mn^2+^ (Fig. 2f). For Mn/Shik@CpG NDs, XPS analysis provided definitive evidence of CpG conjugation. The binding energy of the primary P 2p_3/2_ peak (133.8 eV) is typical for the phosphate/thiophosphate backbone, confirming not only the presence of phosphorus but also its correct chemical state, thus verifying successful surface modification (Fig. 2g). The C 1 s spectrum showed a new peak at 286.2 eV, attributed to C-N bonds from the CpG bases (Fig. 2h). Concurrently, analysis of the O 1 s spectrum revealed a notable increase in the relative intensity of the Mn-O component peak (Fig. 2i). This finding, along with a markedly improved signal-to-noise ratio in the Mn 2p spectrum (Fig. 2j), suggests that the phosphate groups on the CpG backbone likely served as additional coordination sites. This would stabilize the ND framework, facilitating a more robust incorporation of manganese ions. Lastly, the N 1 s spectrum further confirmed the presence of CpG with peaks for amine/amide (-NH_2_/-NH-) and imine (-N = C-) nitrogen at 399.6 eV and 401.5 eV, respectively (Fig. 2k).
UV-visible (UV-vis) absorption spectra provided further evidence of ND formation (Fig. 2l). Free Shik exhibits characteristic absorption peaks at ~275 nm and ~520 nm. In the Mn/Shik NDs spectrum, these peaks were retained but the visible peak showed a red shift of 10–15 nm, which is characteristic of metal-catechol coordination that alters the molecular conjugated system. Additionally, a hypochromic effect (overall decrease in absorbance) was observed, suggesting π-π stacking between the closely packed Shik molecules within the ND core. The spectrum of Mn/Shik@CpG NDs displayed a superposition of these features with an additional shoulder peak at ~260 nm, perfectly corresponding to the characteristic absorbance of CpG nucleic acid bases.
The stability of the NDs was tested under conditions mimicking the TME (acidic pH 6.4 and high GSH 10 mM) [38, 39]. UV-vis spectroscopy further showed that in both acidic and GSH-rich environments, the characteristic absorption peak of Shik underwent a blue-shift, indicating the disassembly of the NDs and release of the drug [40] (Fig. 2m-n). Of note, to maintain drug stability and ensure that the subsequent Mn^2+^ could effectively sensitize the cGAS-STING pathway [8], we used MnCl_2_ as the raw material. Nevertheless, our results still show the disassembly of both NDs in a GSH-rich environment. Mechanistically, the sulfur atom in the thiol group of GSH has a higher metal coordination affinity than oxygen, allowing it to engage in competitive coordination with Shik. This process disrupts the key linkages maintaining ND stability, leading to structural disintegration and drug release. Quantitative release kinetics (Supplementary Fig. 9 and Supplementary Fig. 10) revealed that both NDs maintained structural integrity under simulated physiological conditions, exhibiting negligible disassembly. Conversely, the acidic and GSH-rich environments triggered a responsive, time-dependent disassembly, which increased progressively to reach a plateau at approximately 24 h. Building on these chemical findings, we next investigated whether the drug release triggered by acidic conditions, coupled with the concurrent depletion of GSH, would manifest as potent cytotoxicity against cancer cells in vitro. To this end, we treated RM-1 cells with the key ND components and the two final ND formulations, and subsequently assessed cell viability using a live/dead staining assay (Fig. 2p-q). As visualized in the fluorescence microscopy images, RM-1 cells treated with either Mn/Shik NDs or Mn/Shik@CpG NDs exhibited widespread cell death at a level comparable to that induced by the free Shik, indirectly showing the efficient and responsive release of the therapeutic cargo within a simulated TME.
To probe the chemical transformations during ND formation, we first examined the Fourier-transform infrared (FTIR) spectrum of Shik, which was defined by two key features: a broad -OH stretching band near 3350 cm^− 1^ indication of extensive hydrogen bonding, and the sharp C = O stretching vibration of its quinone structure at ~1620 cm^− 1^ (Supplementary Fig. 11). The MnCl_2_ mainly showed peaks related to adsorbed water (~3400 cm^− 1^ and ~1630 cm^− 1^). In the spectrum of the Mn/Shik NDs, a significant red-shift of the C = O peak and a more diffuse -OH band confirmed that both carbonyl and hydroxyl groups participated in coordination with manganese ions. Finally, the spectrum of Mn/Shik@CpG NDs presented two new, strong absorption peaks completely absent in the uncoated version: a peak at approximately 1245 cm^− 1^ clearly attributed to the P = O stretching vibration of the CpG thiophosphate backbone, and another broad, strong band appearing at approximately 1080 cm^− 1^, is assigned to the overlapping stretching vibrations of the P-O-C linkages and the numerous C-O bonds within the sugar rings of the CpG backbone, which dominate this spectral region after surface modification. Furthermore, the sharp -OH signal observed on the Mn/Shik NDs spectrum reverted to a single broad band in the MSC spectrum. This spectral change occurs because the abundant hydrogen-bonding sites on the CpG chains engage the surface -OH groups of Shik, forming a new and more complex intermolecular network that disrupts the previously ordered surface environment.
In vitro antitumor performance and innate immune activation
To assess the selective cytotoxicity of our formulations, we next performed a CCK8 assay comparing their effects on the murine prostate cancer cell line RM-1 against a normal murine fibroblast cell line L929 (Supplementary Fig. 12). Free Shik was highly toxic to both cell lines. Free Mn^2+^ was largely inert, showing negligible cytotoxicity, likely due to its chelation by components in the culture medium. In comparison, both ND formulations displayed a cancer-selective killing effect, exerting dose-dependent cytotoxicity against RM-1 cells while showing significantly less impact on the normal L929 cells. For instance, at 20 µg/mL, Mn/Shik@CpG NDs reduced RM-1 viability to 5.5% while leaving 59.0% of L929 cells viable. The calculated IC_50_ values for Mn/Shik NDs and Mn/Shik@CpG NDs on RM-1 cells were 9.6 µg/mL and 12.1 µg/mL, respectively (Supplementary Fig. 13).
To further assess the impact of our NDs on key hallmarks of cancer beyond short-term viability, we next evaluated their effects on long-term proliferative capacity and cell migration using colony formation and scratch wound healing assays, respectively. Consistent with the viability data, free Mn^2+^ had a negligible impact on either the clonogenic or migratory potential of RM-1 cells. In contrast, both Mn/Shik NDs and Mn/Shik@CpG NDs markedly suppressed the ability of cells to form colonies, resulting in significantly fewer and smaller colonies compared to controls (Supplementary Fig. 14). Additionally, both NDs strongly inhibited cell migration, as evidenced by delayed closure of the scratch wound (Fig. 3l and Supplementary Fig. 15). It is worth noting that the acute cytotoxicity of free Shik precluded the formation of macroscopic colonies. Analogously, in the scratch assay, the extensive cell mortality rendered the quantification of the effective wound area unfeasible. These observations mandated the use of a relatively attenuated dosage for the free Shik group in subsequent mechanistic investigations to ensure sufficient cell viability for analysis.
Fig. 3. In vitro antitumor and cGAS-STING activating effects of the NDs. Treatments: (I) PBS, (II) MnCl_2_, (III) Shik, (IV) Mn/Shik NDs, and (V) Mn/Shik@CpG NDs. RM-1 cells were treated at a concentration equivalent to 12 µg/mL Mn/Shik@CpG NDs (3.5 µg/mL for free Shik) for 24 h. (a–b) Representative fluorescence images (a) and quantification (b) of intracellular ROS generation in RM-1 cells using a DCFH-DA probe. (c) Immunofluorescence staining showing subcellular localization of HMGB1. (d–e) ELISA-based quantification of HMGB1 (d) and ATP (e) released into the culture supernatant. (f–g) Representative flow cytometry histograms (f) and quantification of median fluorescence intensity (MFI) (g) for CRT exposure on the surface of non-permeabilized RM-1 cells. (h–i) Representative flow cytometry plots (h) and quantification (i) of Annexin V^+^/PI^+^ RM-1 cells. (j–k) Representative western blot bands (j) and heatmap of relative expression levels (k) for key proteins in the cGAS-STING pathway. (l) Quantification of wound healing rate in the scratch assay. (m) ELISA-based quantification of IFN-β concentration in the culture supernatant. Data are presented as mean±SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001
Shik and manganese are known to induce ROS production [41–44]. We confirmed this using a DCFH-DA probe, which showed that both Mn/Shik NDs and Mn/Shik@CpG NDs significantly increased intracellular ROS levels in RM-1 cells (Fig. 3a). The NDs induced higher ROS levels than free Shik, whose acute cytotoxicity likely limited the duration of ROS production (Fig. 3b). Necroptosis induced by Shik is a form of immunogenic cell death (ICD), which is characterized by the release of DAMPs [45]. Accordingly, we next assessed the key hallmarks of ICD after treatment. Immunofluorescence (IF) staining (Fig. 3c) and ELISA (Fig. 3d) confirmed, respectively, the release of high-mobility group box 1 protein (HMGB1) from the nucleus into the cytoplasm and extracellular space. Simultaneously, flow cytometry analysis revealed a significant increase in cell-surface exposure of calreticulin (CRT), another key ICD marker (Fig. 3f). The median CRT exposure was higher in the ND-treated groups (Fig. 3g), possibly due to a more sustained drug release profile compared to free Shik [46]. Finally, ATP secretion into the culture medium was also significantly elevated in these groups (Fig. 3e). To quantify the extent and nature of cell death, we performed flow cytometry using Annexin V and propidium iodide (PI) staining. The analysis revealed that treatment with Shik, as well as with both ND formulations, induced a substantial increase in the population of Annexin V-positive and PI-positive (Annexin V^+^/PI^+^) cells, which is primarily attributable to necroptosis (Fig. 3h-i). In the context of other positive findings for ICD, this result indicates that the TME-responsive release of Shik from the NDs effectively promotes necroptosis of the tumor cells. To confirm that cell death resulted from Shik-mediated necroptosis, we evaluated the phosphorylation status of hallmark mediators via WB (Supplementary Fig. 16). As anticipated, both NDs elicited a marked elevation in the phosphorylation levels of RIPK1, RIPK3, and MLKL, which paralleled that induced by free Shik, indicating the biological activity of Shik was fully preserved.
The ROS-induced DNA damage and release of DAMPs provide the necessary ligands to activate the cGAS-STING pathway [8, 47, 48]. WB analysis confirmed the activation of this pathway, showing a significant increase in the phosphorylation of TBK1 and IRF3, hallmarks of cGAS-STING pathway activation, in cells treated with Mn/Shik NDs and Mn/Shik@CpG NDs (Fig. 3j-k), while this activation was negligible with free Mn^2+^ (likely due to poor cellular uptake) and absent with free Shik, suggesting that Shik-induced DAMPs alone were insufficient to trigger robust STING signaling without Mn^2+^ sensitization. Correspondingly, ELISA results showed a significant secretion of IFN-β, a key type I interferon [49], only in the ND-treated groups (Fig. 3m). Our NDs thus create a highly immunostimulatory milieu, where the synergistic action of DAMPs and type I interferons shatters local immune tolerance and ignites a powerful innate response, thereby setting the stage for effective adaptive immunity.
Given that DCs and macrophages are essential for initiating adaptive immunity, we investigated the functional sequelae of induced cell death by co-culturing drug-treated RM-1 cells with BMDMs or BMDCs (Supplementary Fig. 17 and Supplementary Fig. 18). Consistently, free Mn^2+^ elicited no appreciable phenotypic shifts, a result attributable to restricted cellular uptake. In the free Shik group, the induction of ICD released DAMPs which precluded the “silent” clearance typically associated with necroptosis, triggering an inflammatory response that drove discernable BMDC maturation and M1 macrophage polarization. This effect was markedly amplified in the Mn/Shik NDs group, where the intracellular delivery of Mn^2+^, potentially acting in concert with residual cGAMP from the dying tumor cells, sensitized and activated the cGAS-STING pathway. The resulting surge in type I interferons acted as a potent driver for both DC maturation and macrophage reprogramming. Finally, consistent with the established role of CpG ODN as a TLR9 agonist in promoting innate immune activation [50], the Mn/Shik@CpG NDs group exhibited the most pronounced phenotype, achieving maximal levels of DC maturation (31.3 ± 1.4%) and M1/M2 ratio (2.6 ± 0.2%). Collectively, these in vitro findings imply a strong potential for our NDs to reverse the immunosuppressive landscape. However, given the intricate immune network of the TME, this efficacy warrants rigorous validation in vivo.
In vivo safety and biodistribution
The in vivo safety of the formulations was evaluated in healthy C57BL/6 mice. Mice were administered PBS, MnCl_2_, Shik, Mn/Shik NDs, or Mn/Shik@CpG NDs at an equivalent dosage of 10 mg/kg Mn/Shik@CpG NDs. A hemolysis assay confirmed the biocompatibility of the NDs, as they induced negligible changes in osmotic pressure when incubated with murine venous blood (Fig. 4b). To evaluate systemic safety, the body weight of the mice in each group was monitored over a two-week period. All groups exhibited a steady and gradual increase in body weight, with no significant differences observed between them, indicating that all formulations were well-tolerated at the administered doses (Fig. 4c). Furthermore, hematoxylin and eosin (H&E) staining of major organs (hearts, livers, spleens, lungs, and kidneys) revealed no discernible histopathological abnormalities or signs of toxicity in any of the treatment groups (Supplementary Fig. 19), confirming their good biocompatibility. Together, no significant abnormalities were observed in blood biochemistry panels for liver and kidney function (Fig. 4e), establishing the systemic safety of the NDs at the therapeutic dose.
Fig. 4. In vivo antitumor efficacy of Mn/Shik NDs and Mn/Shik@CpG NDs. Treatments: (I) PBS, (II) MnCl_2_, (III) Shik, (IV) Mn/Shik NDs, and (V) Mn/Shik@CpG NDs. Mice received intravenous injections on days 0 and 5 at a dose equivalent to 10 mg/kg Mn/Shik@CpG NDs. (a) Schematic of the experimental design for evaluating the therapeutic efficacy and immune activation in a primary subcutaneous tumor model. (b) Hemolysis assay of the NDs and the quantification after 10 h incubation. (c–d) Mouse body weight (c) and tumor growth curves (d) over time. (e) Ex vivo fluorescence imaging of major organs and tumors after tail vein injection of ICG or ^ICG^Mn/Shik@CpG NDs. (f) Serum biochemistry analysis for key indicators of liver and kidney function 24 h post-injection. (g–h) Representative images of H&E-stained (g) and Ki67-immunostained (h) tumor sections harvested on day 14. Data are presented as mean±SD (n = 5)
Having confirmed their systemic safety, we next sought to track their in vivo fate by labeling the Mn/Shik@CpG NDs with the near-infrared dye ICG. In vivo imaging at 12 h post-injection revealed significant ^ICG^Mn/Shik@CpG ND accumulation in the tumor region (Supplementary Fig. 20). Conversely, tumors in the free ICG group exhibited only a faint fluorescence signal. Subsequent ex vivo imaging of major organs and tumors corroborated the superior tumor-targeting capability of the NDs, revealing high tumor accumulation with minimal off-target distribution, except for expected uptake by the mononuclear phagocyte system (MPS) in the liver and spleen (Fig. 4e). Unlike the NDs, free ICG was primarily sequestered in the liver and spleen. Collectively, these results confirm that the Mn/Shik@CpG NDs possess a favorable biodistribution profile, which is likely attributable to the hydrophilic CpG coating acting as a “stealth” layer to prolong systemic circulation [51, 52].
In vivo antitumor efficacy
The antitumor efficacy of Mn/Shik@CpG NDs was evaluated in a subcutaneous RM-1 prostate cancer model (Fig. 4a). As shown by the tumor growth curves (Fig. 4d), the PBS and Mn^2+^ groups exhibited rapid tumor progression with no significant difference between them, confirming that Mn^2+^ alone has few antitumor effects in vivo. Treatment with free Shik moderately slowed tumor growth, likely due to its anti-angiogenic effects rather than direct, sustained cytotoxicity [53]. Moreover, both the Mn/Shik and Mn/Shik@CpG groups demonstrated significant tumor growth inhibition. Interestingly, the addition of the CpG adjuvant did not confer a statistically significant benefit in retarding primary tumor growth when compared to the uncoated NDs. This can be attributed to two factors. Firstly, the combination of Mn^2+^ and Shik already exerts a highly potent local cytotoxic effect, leaving little room for further improvement from an adjuvant that does not directly kill tumor cells. Secondly, the primary role of CpG is to stimulate and shape the adaptive immune response, the full benefits of which are more apparent in long-term outcomes like metastasis and recurrence, rather than in the initial reduction of the primary tumor mass. Images of the excised tumors at the study endpoint corroborated the growth curve data (Supplementary Fig. 21). H&E staining confirmed extensive necrosis, consistent with the known necroptosis-inducing activity of Shik, only in the ND groups (Fig. 4g). This widespread cell death was complemented by a strong anti-proliferative effect, as evidenced by a marked reduction in the proliferation marker Ki67 in tumors from these same groups (Fig. 4h).
In vivo immune activation efficacy
To understand the immunological basis for the potent antitumor effect, we next characterized the composition and activation state of the tumor immune microenvironment (TIME) in treated mice. Mirroring the in vitro mechanistic sequence, we first evaluated the activation status of necroptosis, the upstream initiator of the signaling cascade. WB profiling of tumor lysates revealed distinct phosphorylation of core necroptotic mediators in the ND-treated cohorts (Supplementary Fig. 22). Conversely, and diverging from in vitro observations, the free Shik group failed to induce appreciable necroptosis, a deficit attributable to its transient intratumoral retention. These biochemical findings align with the histological necrosis patterns observed in H&E sections. Building on this foundation, RT-qPCR analysis revealed a marked transcriptional upregulation of Ifnb1 in the ND-treated groups (Supplementary Fig. 23), which translated directly into pervasive IFN-β protein infiltration throughout the tumor parenchyma, as visualized by IF (Fig. 5a) and IHC (Supplementary Fig. 24) profiling. To delineate the upstream signaling cascade driving this response, we performed WB analysis on tumor lysates. The results confirmed substantial cGAS-STING pathway engagement, characterized by distinct elevations in p-TBK1 and p-IRF3 levels (Supplementary Fig. 25). While the Mn/Shik@CpG NDs elicited the most potent activation, free Shik, diverging from in vitro results, failed to induce appreciable pathway engagement, likely due to its transient intratumoral retention. These biochemical findings were spatially corroborated by IHC, which displayed intensified p-TBK1 and p-IRF3 staining within the ND-treated tumors, peaking in the CpG-coated cohort (Supplementary Fig. 26). To ascertain whether this cytokine surge translated into functional antigen presentation, we analyzed the maturation status of tumor-infiltrating DCs (CD11c^+^CD80^+^CD86^+^) via flow cytometry, showing an increase in DC maturation in the ND-treated groups (Fig. 5c). The effect was most pronounced in the Mn/Shik@CpG NDs group, where the proportion of mature DCs reached 30.3%±2.3%, highlighting the synergistic effect of the CpG adjuvant (Fig. 5d).
Fig. 5. In vivo immune activation by Mn/Shik NDs and Mn/Shik@CpG NDs. Treatments: (I) PBS, (II) MnCl_2_, (III) Shik, (IV) Mn/Shik NDs, and (V) Mn/Shik@CpG NDs. Tumors were harvested on day 14 following treatments on days 0 and 5 (10 mg/kg Mn/Shik@CpG NDs equivalent). (a–b) Representative immunofluorescence images of IFN-β (a) and VSIG4 (b) in paraffin-embedded tumor sections. (c–d) Representative flow cytometry density plots (c) and quantification (d) of mature DCs (CD11c^+^CD80^+^CD86^+^). (a–f) Density plots (e) and quantification (f) of NK cells (CD45^+^CD3^−^NK1.1^+^). (g–h) Density plots (g) and quantification (h) of CD8^+^ T cells (CD45^+^CD3^+^CD8^+^). (i–j) Density plots (i) and quantification (j) of Treg cells (CD45^+^CD3^+^CD4^+^Foxp3^+^). (k–l) Density plots (k) of M1 (CD45^+^CD11b^+^CD86^+^) and M2 (CD45^+^CD11b^+^CD206^+^) macrophages and quantification of the M1/M2 ratio (l) within tumors. Data are presented as mean±SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001
We next assessed the infiltration of cytotoxic innate lymphocytes. In line with the ability of Shik to boost NK cell activity [54] and the crosstalk between mature DCs and NK cells [55], we observed a significant increase in tumor-infiltrating NK cells (CD45^+^CD3^−^NK1.1^+^) in the Shik and ND groups (Fig. 5e), with the Mn/Shik@CpG NDs group showing the highest infiltration (2.44-fold increase over control) (Fig. 5f). This wave of innate immune activation establishes an immediate first line of defense while simultaneously creating the inflammatory milieu required to initiate a durable adaptive response.
The innate immune activation contributes to an adaptive T cell response [56]. Flow cytometry revealed a profound shift in the T cell landscape within the tumors (Fig. 5g). Both ND formulations led to a significant increase in the infiltration of CD8^+^ T cells (from 11.5 ± 0.6% in the control group to 23.0 ± 2.9% in the Mn/Shik@CpG NDs group) (Fig. 5h). Simultaneously, a notable decrease in the percentage of immunosuppressive Treg cells (CD3^+^CD4^+^Foxp3^+^) was detected (Fig. 5i-j), indicating reversal of the suppressive TME nature [57]. Immunofluorescence staining confirmed that the infiltrating CD8^+^ T cells were distributed throughout the tumor parenchyma, poised to execute their effector functions (Supplementary Fig. 27).
Our in vivo analysis culminated with an examination of the macrophage population, the very cell type that inspired this study, where flow cytometry revealed a stark repolarization of TAMs. Both ND groups showed a significant increase in the proportion of pro-inflammatory M1 macrophages (CD11b^+^CD86^+^) and a decrease in immunosuppressive M2 macrophages (CD11b^+^CD206^+^) (Fig. 5k). This resulted in a 2.26-fold and 5.14-fold increase in the M1/M2 ratio for the Mn/Shik and Mn/Shik@CpG groups, respectively, suggesting a shift towards an anti-tumor phenotype (Fig. 5l). Finally, to bridge our initial single-cell discoveries with our in vivo findings, we stained tumor sections for VSIG4. Transcriptional analysis via RT-qPCR confirmed a marked suppression of Vsig4 mRNA in ND-treated tumors (Supplementary Fig. 28), a trend recapitulated at the translational level by WB (Supplementary Fig. 29). Spatially, both IHC (Supplementary Fig. 30) and IF (Fig. 5b) staining visualized a sparse distribution of VSIG4^+^ cells within the treated TME, contrasting sharply with the dense accumulation in controls, providing a direct molecular correlation for the observed reduction in M2 macrophage infiltration.
Long-Term antitumor efficacy and immune memory
The ultimate benchmark of a successful immunotherapy is its ability to establish a durable, systemic antitumor response capable of preventing both tumor recurrence and metastatic spread. We evaluated this using two challenging models. The lung is a preferential site for prostate cancer metastasis [58]. We therefore evaluated the ND efficacy in preventing metastatic colonization using a lung metastasis model established by intravenous injection of RM-1 cells. Representative photographs of excised lungs revealed that pretreatment with Mn/Shik@CpG NDs almost completely prevented the formation of pulmonary nodules (Fig. 6b and f). Strikingly, the Mn/Shik NDs group exhibited only a modest reduction in metastatic burden, while the other control groups (PBS, MnCl_2_, and Shik) displayed extensive metastasis, with lungs nearly filled with tumor nodules. Similarly, H&E staining of lung sections revealed that, in stark contrast to the Mn/Shik@CpG NDs group which retained its normal lung architecture, deep lung tissues in all other groups were almost entirely effaced by invasive tumor cells (Fig. 6e). Consequently, the high mortality rate associated with extensive pulmonary metastasis in this model prompted a survival analysis, which revealed that pretreatment with Mn/Shik@CpG NDs significantly prolonged the survival of the mice (Fig. 6d).
Fig. 6. Long-term antitumor efficacy and immune memory induction by Mn/Shik@CpG NDs. Treatments: (I) PBS, (II) MnCl_2_, (III) Shik, (IV) Mn/Shik NDs, and (V) Mn/Shik@CpG NDs. Mice received intravenous injections on days 0 and 5 at a dose equivalent to 10 mg/kg Mn/Shik@CpG NDs. (a) Schematic of experimental design for evaluating the prevention of metastasis and protection against tumor rechallenge. (b) Representative photographs of excised lungs from the lung metastasis model on day 10 post-metastatic challenge. (c) Bar plot of terminal tumor volumes from the primary challenge and subsequent rechallenge on day 14. (d) Kaplan-Meier survival curves of mice in the lung metastasis model. (e–f) Representative H&E-stained lung sections (e) and quantification of metastatic nodules (f). (g) Representative immunofluorescence images of CD8^+^ T cell infiltration in rechallenged tumors. (h) Quantification of CD8^+^ T cell proportions in rechallenged tumors. (i–j) Representative flow cytometry density plots (I) and quantification (j) of effector memory CD8^+^ T cells (CD3^+^CD8^+^CD44^+^CD62L^−^) in the spleen on day 14. Data are presented as mean±SD (n = 3). ***p < 0.001, and ****p < 0.0001
Subsequentially, we assessed protection against tumor recurrence in a rechallenge model, where cured mice were inoculated with a second tumor on the contralateral flank. In this stringent test of immune memory, a clear difference was observed between the two ND formulations (Supplementary Fig. 31). While both were effective against the primary tumor, the growth of the secondary tumor was significantly slower only in the Mn/Shik@CpG NDs group (Fig. 6c). The failure of the core Mn/Shik NDs to protect against tumor rechallenge indicates the essential role of the CpG adjuvant in generating a lasting memory response. Flow cytometry of the secondary tumors confirmed this, showing a 1.9-fold increase of CD8^+^ T cells infiltration in the Mn/Shik@CpG NDs group compared with the PBS group (17.8 ± 2.5% vs. 9.4 ± 1.0%) (Fig. 6h and Supplementary Fig. 32), another finding supported by immunofluorescence staining (Fig. 6g).
To definitively demonstrate the establishment of immune memory, we analyzed memory T cell populations in the spleens (Fig. 6i). Flow cytometry revealed that mice treated with Mn/Shik@CpG NDs had a significantly expanded population of both central memory (Tcm, CD44^+^CD62L^+^) and, most notably, effector memory (Tem, CD44^+^CD62L^−^) CD8^+^ T cells (Fig. 6j). The increase in Tem cells was particularly striking compared to the Mn/Shik group, providing clear evidence of CpG-driven memory formation. Collectively, these findings suggest that Mn/Shik@CpG NDs induce a durable systemic immunity capable of preventing tumor metastasis and establishing an antigen-specific immune memory that protects against subsequent tumor recurrence.
Transcriptomic analysis of rechallenged tumors confirms immune activation
While our Mn/Shik@CpG NDs could suppress tumor recurrence in the rechallenge model, it was essential to determine whether this effect was mainly attributable to the direct cytotoxicity of residual NDs or a bona fide, memory-driven immune response. To this end, we performed bulk RNA-sequencing on tumor masses harvested from the rechallenged mice in both the PBS and Mn/Shik@CpG NDs groups. Bulk RNA-sequencing revealed a transcriptomic reprogramming in the rechallenged tumors following ND treatment, evidenced by the identification of 469 differentially expressed genes (DEGs) between the two groups (Supplementary Fig. 33). A Z-score heatmap of the top 50 DEGs (ranked by adjusted p-value) revealed that the ND treatment induced pronounced transcriptomic reprogramming of the rechallenged tumor, characterized by a coordinated upregulation of most of these genes in the Mn/Shik@CpG NDs group (Fig. 7a).
Fig. 7. Bulk RNA-sequencing analysis of rechallenged tumors from mice treated with either PBS or Mn/Shik@CpG NDs. (a Z-score heatmap of the top 50 differentially expressed genes (DEGs). (b–c) Gene Ontology (GO) (b) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (c) enrichment analyses of the significant DEGs. (d–i) Gene Set Enrichment Analysis (GSEA) plots for representative immune-related pathways, including immune response (d), adaptive immune response (e), allograft rejection (f), T cell activation (g), antigen processing and presentation (h), and natural killer cell mediated cytotoxicity (i)
The set of significant DEGs was then further analyzed for functional enrichment using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. The GO enrichment analysis strongly pointed to significant alterations in the TIME across all three domains (Fig. 7b). Specifically, within the Biological Process (BP) domain, top-ranking terms were almost exclusively related to leukocyte cell-cell adhesion and activation, suggesting a highly active state of immune cell engagement. The Cellular Component (CC) domain depicted a near-complete immunological process, from the engulfment of tumor-associated antigens by APCs to their presentation via MHC molecules to activate memory T cells at the immune synapse. The Molecular Function (MF) domain not only highlighted a similar antigen presentation process via MHC molecules but also indicated a TME rich in active cytokine signaling. Similarly, KEGG enrichment analysis revealed pathways related to antigen processing and presentation, as well as cytokine-cytokine receptor interactions (Fig. 7c). Complementing the GO results, the concurrent enrichment of pathways for Th1, Th2, and Th17 cell differentiation suggests the induction of a mature, multi-lineage T helper cell response. Furthermore, the enrichment of the “natural killer cell mediated cytotoxicity” pathway indicates the involvement of the innate immune system in this memory response. Tellingly, the enrichment of pathways like “graft-versus-host disease” and “allograft rejection” offered a transcriptomic parallel to the macroscopic tumor control we observed.
To further explore functional trends, we conducted Gene Set Enrichment Analysis (GSEA) on pathways of interest (Fig. 7d-i). The results showed a significant positive enrichment for both overarching immune processes, such as the general immune response, adaptive immune response, and allograft rejection, as well as for more specific cellular and molecular pathways, including “T cell activation”, “natural killer cell mediated cytotoxicity”, and “antigen processing and presentation via MHC class II”. Collectively, the transcriptomic data support our research logic, suggesting that the NDs activate a durable immune response to control tumor recurrence.
While our findings demonstrate the efficacy of the single-cell sequencing-guided design pipeline, several limitations warrant discussion. Although we identified the downregulation of VSIG4 by Shik, the precise biophysical interaction between Shik and the VSIG4 promoter region or protein structure remains to be fully elucidated in future structural biology studies. In addition, while the RM-1 model provides a robust platform for mechanistic investigation, future clinical translation will require validating this strategy across a broader spectrum of prostate cancer subtypes to account for patient heterogeneity.
Conclusion
Our study establishes a comprehensive therapeutic paradigm that integrates the data-driven discovery of an immunotherapy target with the synergistic NDs. Through interrogation of the clinical TME, we identified VSIG4 as a pivotal modulator of TAM fate. Guided by this insight, the resulting Mn/Shik@CpG NDs effectively induce tumor cell death while remodeling the TIME in vivo, fostering long-term immunological memory capable of protecting against both recurrence and metastasis. Critically, by validating the translation of a bioinformatics-identified target into a rationally assembled nanoplatform, our study exemplifies a cohesive “discovery-to-design” workflow. This strategy serves as a conceptual blueprint for bridging high-dimensional omics data with therapeutic engineering, advancing the development of precision nanomedicines tailored to specific disease microenvironments.
Methods
Materials
MnCl_2_, Shik, and ICG were purchased from Aladdin Industrial Corporation (Shanghai, China). Polyvinylidene difluoride (PVDF) membranes were obtained from Sigma-Aldrich. SDS-polyacrylamide gels were sourced from Changzhou Boyi Biotech Co., Ltd. (Changzhou, China). The enhanced chemiluminescence (ECL) reagent was purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). PBS, Dulbecco’s Modified Eagle Medium (DMEM), and RPMI 1640 medium were obtained from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Tianhang Biotechnology Co., Ltd. (Zhejiang, China). Penicillin/streptomycin solution was supplied by Biosharp (Hefei, China). CoraLite^®^ Plus 555-conjugated anti-CRT antibody, anti-Foxp3-APC antibody, anti-p-TBK1 antibody, anti-TBK1 antibody and anti-IRF3 antibody were all purchased from Proteintech (Wuhan, China). Anti-HMGB1 primary antibody was from Abcam (Cambridge, UK). Anti-VSIG4 primary antibody was purchased from Abmart Shanghai Co.,Ltd. (Shanghai, China). Anti-p-IRF3 and anti-GAPDH antibodies were from Bioss (Beijing, China). Goat anti-mouse AF488 secondary antibody and HRP-conjugated goat anti-rabbit IgG secondary antibody were purchased from Abcam (Cambridge, UK). For flow cytometry, the following antibodies were purchased from Biolegend (San Diego, CA, USA): anti-CD45-FITC antibody, anti-CD11c-APC antibody, anti-CD86-PE antibody, anti-CD80-PerCP/Cyanine5.5 antibody, anti-CD3-PerCP/Cyanine5.5 antibody, anti-CD4-PE antibody, anti-CD8a-APC antibody, anti-CD11b-PerCP/Cyanine5.5 antibody, anti-CD206-PE antibody, and anti-NK1.1-APC antibody. CpG ODN and the LIVE/DEAD^™^ fixable dead cell stain kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The cell counting kit-8 (CCK-8), ROS assay kit, antifade mounting medium with DAPI, ATP assay kit, BCA protein assay kit, and RIPA lysis buffer were all sourced from Beyotime Biotechnology (Shanghai, China). The calcein-AM/PI double staining kit, normal goat serum, and the H&E staining kit were purchased from Wuhan Servicebio Technology Co., Ltd. (Wuhan, China). The mouse HMGB1 ELISA kit and the mouse IFN-beta ELISA kit were from Bioss. The alanine aminotransferase (ALT) assay kit, aspartate aminotransferase (AST) assay kit, urea assay kit, and creatinine (Cr) assay kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
Cell culture and animals
The murine prostate cancer cell line RM-1 was obtained from Beina Chuanglian Biotech Institute (Beijing, China). The L929 cell line was from the China-Japan Friendship Hospital of Jilin University. RM-1 cells were maintained in RPMI 1640 medium, while L929 cells were kept in high-glucose DMEM. To generate BMDMs, bone marrow was harvested from the tibias, femurs, and iliac crests of C57BL/6J mice, following established protocols [59]. These cells were then differentiated for 7 days in DMEM containing 20% FBS and 20 ng/mL of macrophage colony-stimulating factor (M-CSF). For M2 polarization, the resulting BMDMs were subsequently treated with IL-4 (20 ng/mL) and IL-13 (20 ng/mL) for 24 h. All culture media were supplemented with 10% FBS and 1% penicillin-streptomycin, and all cell lines were cultured at 37 °C in a humidified 5% CO₂ atmosphere. Male C57BL/6J mice (6 − 8 weeks old) were sourced from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed in a specific pathogen-free (SPF), temperature-controlled facility at the Institute of Translational Medicine, The First Hospital of Jilin University (Changchun, China). All animal procedures were performed in accordance with institutional guidelines and received ethical approval from the Animal Ethics Committee of the First Hospital of Jilin University (Changchun, China; Approval No. JDYY20250648).
Re-analysis of Single-Cell RNA sequencing data
Raw single-cell RNA sequencing data and associated annotations for the prostate cancer dataset GSE181294 were downloaded from the Gene Expression Omnibus (GEO). To mitigate batch effects, we focused our analysis on high-quality samples with over 3000 estimated cells. Using high-quality samples allowed us to optimize data quality without substantially reducing the total cell count. Most of the computational work was carried out using the OmicVerse framework [60]. For quality control, we filtered out cells with fewer than 250 detected genes, under 600 total UMIs, or over 20% mitochondrial gene expression. The Scrublet al.gorithm [61] was used to remove potential doublets, yielding a final dataset of over 100,000 high-quality cells. Subsequent analyses were based on the top 4000 highly variable genes. We performed batch correction using the scANVI method [18], referencing the original study’s annotations. Cell clustering was achieved with the Leiden algorithm. While major cell populations were annotated using canonical marker genes (Table S1), finer subtypes were manually assigned based on a combination of known markers and differentially expressed genes (DEGs), which were identified using the Wilcoxon signed-rank test in Scanpy. We computed metacells using the SEACells module [25], and then performed pseudotime analysis on these metacells with pyVIA. To pinpoint key genes correlated with pseudotime, we first used ridge regression to model gene expression against pseudotime. An adaptive threshold regression model was then built by iteratively adding genes with the highest coefficients until the change in r^2^ per iteration fell below 0.01. The final gene set was further refined by assessing the Kendall’s Tau correlation between each gene and the calculated pseudotime.
Synthesis of Mn/Shik NDs and Mn/Shik@CpG NDs
The Mn/Shik NDs were prepared based on our previously described method for self-delivering supramolecular NDs [35]. In brief, methanolic solutions of Shik (5 mg/mL) and MnCl_2_ (1 mg/mL) were rapidly co-injected into a Tris buffer solution (10 mM, pH 8.5) under vigorous vortexing, followed by gentle stirring for 1 h at room temperature. The resulting Mn/Shik NDs were collected by centrifugation (13,500 rpm, 10 min) and washed twice with deionized water. For the final product, the purified Mn/Shik NDs (1 mg/ml) were resuspended in a CpG ODN solution (300 µg/mL) and stirred overnight at room temperature. The resulting Mn/Shik@CpG NDs were then purified by centrifugation (13,000 rpm, 10 min) to remove any unbound CpG. All NDs were stored at 4 °C until use.
Characterization of NDs
ND morphology was examined using a JEM-2100 F transmission electron microscope (TEM; JEOL, Japan). A Malvern Zetasizer Nano ZS was used to determine the hydrodynamic diameter and zeta potential by dynamic light scattering (DLS). Absorbance spectra were recorded on a Shimadzu 2600 UV-vis-NIR spectrophotometer. To visualize in vitro responsiveness, the NDs were incubated for 4 h in either purified water, PBS at pH 7.4 or 6.4, or a GSH-rich solution (10 mM), after which disassembly was assessed by monitoring changes in the UV-vis absorption spectra. To evaluate in vitro disassembly kinetics, the ND dispersion was sealed in a dialysis bag and immersed in the indicated release media under constant shaking. At scheduled intervals, 3 mL supernatant was withdrawn and immediately replaced with an equal volume of fresh buffer to maintain sink conditions. To quantify Shik release, the absorbance of the collected supernatant was measured at 517 nm. The released amount of Shik was subsequently calculated by referencing a standard calibration curve. A Bruker IFS80V spectrometer was used to record Fourier transform infrared (FTIR) spectra. X-ray photoelectron spectroscopy (XPS) was performed on a VG ESCALAB MKII spectrometer. The manganese content was precisely quantified using an inductively coupled plasma atomic emission spectrometer (ICP-AES; Optima 3300DV, PerkinElmer). To quantify the CpG ODN loading, the absorbance at 260 nm of the solution was measured before and after the coating reaction using a CLARIOSTAR microplate reader (BMG LABTECH, Germany). The concentration of CpG ODN in the supernatant was determined via a standard calibration curve, from which the mass of the coated CpG ODN was calculated by subtracting the supernatant content from the total input.
Treatment regimen and dosage standardization
Treatment dosages were standardized based on the mass composition of the NDs (Table S2). For assays evaluating direct cytotoxicity, specifically CCK-8, scratch wound healing, colony formation, and live/dead staining, all formulations were administered at a concentration equivalent to 12 µg/mL of Mn/Shik@CpG NDs. Conversely, for all other in vitro mechanistic studies, the dosage of free Shik was adjusted to 3.5 µg/mL (approximating its IC50 against RM-1 cells) to mitigate excessive acute toxicity and ensure sufficient cellular viability for analysis. Notably, considering the higher sensitivity of primary cells compared to immortalized cancer lines, the dosage of free Shik was reduced to 2 µg/mL for BMDMs to avoid non-specific cell death. For in vivo experiments, the administration was standardized to a single dose equivalent to 10 mg/kg of Mn/Shik@CpG NDs, calculated according to the body weight of each mouse.
In vitro cytotoxicity assays
The cytotoxic profile of each formulation was determined against both RM-1 cancer cells and L929 fibroblasts using a standard CCK-8 assay. Cells were seeded in 96-well plates for 24 h before being exposed to various drug concentrations for another 24 h. Following the addition of 10 µL of CCK-8 solution to each well, the optical density (OD) at 450 nm was measured. Cell viability was normalized to untreated controls. IC_50_ values were calculated by fitting the dose-response data to a four-parameter logistic (4PL) model via non-linear regression, with the IC_50_ defined as the concentration required to inhibit viability by 50%. Cell death was visualized with a Calcein-AM/PI double staining kit. RM-1 cells seeded in confocal dishes were treated with the standardized dosage of formulations (equivalent to 12 µg/mL Mn/Shik@CpG NDs) for 24 h, and images were captured on an Olympus IX73 inverted fluorescence microscope.
Colony formation assay
RM-1 cells were seeded in 6-well plates at a density of 1,000 cells per well and allowed to adhere overnight. Cells were then treated with the indicated formulations (equivalent to 12 µg/mL Mn/Shik@CpG NDs) for 24 h. The drug-containing medium was then replaced with fresh complete medium, and the cells were cultured for an additional 14 days to allow colony formation. Finally, colonies were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and then photographed.
Scratch wound healing assay
RM-1 cells were cultured in 6-well plates until they reached approximately 80% confluence, a density optimized to accommodate the rapid proliferation rate of this cell line. A linear wound was created by scratching the monolayer with a sterile 200 µL pipette tip. After washing with PBS to remove detached cells, the monolayer was incubated in serum-free medium containing the indicated treatments (equivalent to 12 µg/mL Mn/Shik@CpG NDs). Images of the wound area were captured at 0 h and 24 h using an inverted microscope, and the wound closure rate was quantified using ImageJ software.
Intracellular ROS detection
RM-1 cells, cultured in 6-well plates, were treated for 24 h with the indicated formulations. After washing with PBS, cells were incubated with a DCFH-DA ROS probe for 30 min. Fluorescence was immediately visualized using an inverted fluorescence microscope (Olympus IX73), with all images captured under identical exposure settings. The average fluorescence intensity per cell was quantified in ImageJ.
In vitro ICD assays
ICD induction was evaluated by measuring CRT exposure, HMGB1 release, and ATP secretion after treating RM-1 cells with the indicated formulations for 24 h. For CRT exposure, treated RM-1 cells were harvested and stained with an AF488-conjugated anti-mouse CRT antibody for 30 min at 4 °C prior to analysis on a BD LSRFortessa^™^ flow cytometer. For HMGB1 localization, treated cells grown in confocal dishes were fixed, permeabilized, blocked, and incubated with a primary antibody against mouse HMGB1, followed by an AF488-conjugated secondary antibody and DAPI counterstaining. Images were acquired with a confocal laser scanning microscope (Olympus FluoView FV1000). The amounts of HMGB1 and ATP released into the culture supernatant were quantified using commercial ELISA kits.
Apoptosis and necroptosis analysis
RM-1 cells were treated with the indicated formulations (equivalent to 12 µg/mL Mn/Shik@CpG NDs) for 24 h. Subsequently, cells were harvested, washed with cold PBS, and resuspended in binding buffer. They were then stained with Annexin V-FITC and PI for 15 min at room temperature in the dark, followed by immediate analysis using a BD LSRFortessaTM flow cytometer.
Western blot analysis
Harvested cells or homogenized tissue fragments were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. Equal protein amounts were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked and incubated overnight at 4 °C with primary antibodies against the targets of interest. After incubation with an HRP-conjugated secondary antibody, protein bands were visualized using ECL reagents.
Direct co-culture assays
RM-1 cells were first seeded and exposed to the indicated formulations for 24 h. Subsequently, the culture medium was discarded, and the tumor cells were washed carefully with PBS to remove any residual free therapeutics. Subsequently, BMDMs or BMDCs were seeded into the same wells containing the pre-treated tumor cells at a 2:1 tumor cells/immune cells ratio. The co-culture was maintained for another 24 h. Afterward, the mixed cell populations were harvested, stained with fluorophore-conjugated antibodies against immune cell markers (BMDCs: CD45, CD80, CD86; BMDMs: CD45, CD86, CD206), and analyzed by flow cytometry.
RT-qPCR
Total RNA was extracted from RM-1 tumor tissues harvested on day 14 using Trizol (Invitrogen, CA, USA). Subsequently, cDNA was synthesized and real-time PCR was performed using one-step reverse transcription PCR kit (Tiangen, Beijing, China). Gapdh was served as internal control for quantification of mRNAs. The mRNA relative expression was calculated with the 2^−ΔΔCt^ method. Primer sequences can be seen in Table S3.
In Vivo safety assessment
For the hemolysis assay, venous blood was collected from C57BL/6J mice into anticoagulant tubes. After dilution with an equal volume of PBS, 100 µL of the diluted blood was added to 1 mL of either pure water (positive control), PBS (negative control), Mn/Shik NDs, or Mn/Shik@CpG NDs (at an equivalent dosage of 10 mg/kg Mn/Shik@CpG NDs). Samples were incubated at 37 ℃ for 10 h and then centrifuged at 5,000 rpm for 5 min. The absorbance of the supernatant at 545 nm was measured using a CLARIOstar microplate reader. The hemolysis rate was calculated as: (OD_sample_ - OD_PBS_)/(OD_water_ - OD_PBS_) × 100%. For systemic toxicity assessment, mice were intravenously injected with the formulations at an equivalent dosage of 10 mg/kg Mn/Shik@CpG. After 24 h, blood was collected to prepare serum, and liver/kidney function indexes were measured using commercial assay kits. Simultaneously, major organs were harvested, fixed in 4% formaldehyde, and processed for H&E staining. The standard procedure included paraffin embedding, dehydration, sectioning, and deparaffinization, followed by staining with an H&E kit. Stained slides were scanned using an Olympus VS200 slide scanner.
Biodistribution
RM-1 tumors were established on the right flank of male C57BL/6J mice. Mice were then intravenously injected with free ICG or ^ICG^Mn/Shik@CpG NDs. 12 h post-injection, in vivo fluorescence imaging was performed. Subsequently, mice were euthanized, and major organs and tumors were excised for ex vivo imaging. All fluorescence imaging was conducted using an IVIS Lumina XRMS Series III system (PerkinElmer), with an excitation wavelength of 675 nm and an emission wavelength of 695 nm.
In Vivo antitumor efficacy
RM-1 cells (1 × 10^6^) were injected subcutaneously into the right flank of C57BL/6J mice. When the tumor volume reached approximately 30 mm^3^, mice were randomly assigned to five groups. On days 0 and 5, mice received a 200 µL tail vein injection of PBS, MnCl_2_, Shik, Mn/Shik NDs, or Mn/Shik@CpG NDs (at an equivalent dosage of 10 mg/kg Mn/Shik@CpG NDs). Tumor volumes and body weights were recorded every other day. Mice were euthanized when the tumor volume approached 1,500 mm^3^ as per ethical guidelines. Tumor volume was calculated as (W^2^×L)/2. At the experimental endpoint, mice were euthanized, and tumors and major organs were harvested for histological analysis. H&E, IF and IHC staining of paraffin-embedded tumor sections were performed and scanned by Servicebio Technology Co. (Wuhan, China).
Analysis of antitumor immune effects
On day 14 post-treatment, tumors were harvested and dissociated into single-cell suspensions. After Fc receptor blocking, cells were stained with fluorophore-conjugated antibodies against various immune cell markers (DCs: CD45, CD11c, CD86, CD80; CD8^+^ T cells: CD45, CD3, CD4, CD8; Tregs: CD45, LIVE/DEAD stain, CD4, Foxp3; TAMs: CD45, CD11b, CD206, CD86; NK cells: CD45, CD3, NK1.1) and analyzed by flow cytometry.
Metastasis inhibition study
Primary tumors were established and treated as described above. Two days after the final treatment, 1 × 10^6^ RM-1 cells were injected intravenously to initiate lung metastasis. To prevent primary tumors from reaching the ethical endpoint, subcutaneous tumors in all mice were surgically resected under isoflurane anesthesia on day 3 after the intravenous cell injection. The survival time of each mouse was recorded to generate a Kaplan-Meier survival curve. On day 10 after the intravenous injection, lung tissues were isolated for photography and H&E staining.
Immune memory study
Primary tumors were established and treated as described previously. A rechallenge was performed on day 7 by injecting 1 × 10^6^ RM-1 cells into the contralateral flank, and all mice were euthanized on day 14. To analyze memory T cell populations, splenocytes were harvested and stained with antibodies against CD3, CD8, CD62L, and CD44. Tumor-infiltrating T cells from the rechallenged tumors were also analyzed as described above.
Bulk RNA-sequencing and analysis
For bulk RNA-sequencing, secondary tumors from the rechallenge model were excised at the study endpoint. Total RNA extraction, library construction, and sequencing were performed by Servicebio Technology Co. (Wuhan, China). For the analysis, raw read counts were filtered to retain genes with a total count ≥ 10. Differential expression analysis was performed using the R package DESeq2. Genes were considered significant DEGs at an adjusted *p-*value < 0.05 and an absolute log₂ fold change > 1.0. A Z-score heatmap was created to visualize the expression patterns of the top 50 DEGs, ranked by adjusted *p-*value. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the list of significant DEGs using the R package clusterProfiler. Significant enrichment was defined by a Benjamini-Hochberg (BH) adjusted p-value < 0.05 and a q-value < 0.2. The top 5 GO terms from each category and the top 15 KEGG pathways, ranked by adjusted p-value, were plotted. Finally, the ranked gene list was used as input for Gene Set Enrichment Analysis (GSEA) using the gseGO and gseKEGG functions from the clusterProfiler R package. Gene sets with a p-value < 0.05 were considered significantly enriched.
Statistical analysis
All data are presented as the mean ± standard deviation (SD). Statistical significance between two groups was determined using a two-tailed Student’s t-test. For multiple group comparisons, one-way analysis of variance (ANOVA) was used. P-values were considered statistically significant as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Supplementary Information
Supplementary Material 1
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