IntroductionTraditional anticancer therapies have long viewed tumor cell death as the primary biological and therapeutic endpoint, with chemotherapy serving as a cornerstone for treating unresectable malignancies1,2. While the direct tumoricidal effects of chemotherapeutic agents are well established, the resulting cell death triggers a cascade of events that profoundly reshape the tumor microenvironment (TME), ultimately influencing therapeutic outcomes.One potential immunological benefit of chemotherapy is the induction of immunogenic cell death (ICD)3,4,5, a regulated form of cell death characterized by the release of damage-associated molecular patterns (DAMPs)6,7,8. These signals promote dendritic cell (DC) maturation and antigen presentation, linking cytotoxic therapy to adaptive antitumor immunity. However, in many solid tumors, this pro-immunogenic potential is rarely realized6,9. A major contributing factor is the concurrent induction of cyclooxygenase-2 (COX2) expression and subsequent production of prostaglandin E2 (PGE2) by several chemotherapeutic agents9,10. PGE2 is a potent immunosuppressive mediator that impairs DC function11, inhibits T-cell activity12,13, and has been proposed to act as an inhibitory DAMP (iDAMP), counteracting ICD-associated immune activation14. Thus, combining chemotherapy with COX2 or PGE2 inhibition has been explored as a strategy to resolve this signaling conflict. However, such combinations have shown limited and inconsistent benefits in both preclinical and clinical studies15,16,17. The reasons for this failure remain incompletely understood, representing a critical barrier to the rational design of chemoimmunotherapeutic strategies. Notably, most combination approaches have focused on the presence or absence of immunosuppressive signaling while largely neglecting the temporal coordination between immune suppression relief and ICD induction.Emerging evidence highlights that the temporal sequence of combination therapies is a key determinant of their immunomodulatory outcomes and overall efficacy18,19. For example, the CheckMate 064 study in melanoma demonstrated that initiating treatment with nivolumab followed by ipilimumab resulted in superior outcomes, whereas the reverse sequence induced immunosuppressive T-cell phenotypes, leading to resistance to subsequent nivolumab20,21. Consequently, the efficacy of immunotherapy may be influenced by the preexisting immunological landscape22. In the TME, elevated PGE2 fosters an immunosuppressive state, which is further amplified by chemotherapy-induced PGE2 signaling, impairing the immune system’s response to chemotherapy-induced DAMPs. In such a TME, dominated by immunosuppressive mediators such as PGE2, ICD-associated signals may be functionally silenced, rendering the cytotoxic event immunologically ineffective23,24,25. These findings support a fundamental immunological premise: the immunogenic potential of chemotherapy-induced ICD depends on a permissive TME in which PGE2-mediated immunosuppression has been alleviated.This issue is particularly pronounced in triple-negative breast cancer (TNBC), an aggressive subtype where cytotoxic chemotherapy remains the primary treatment modality26,27. The TME of TNBC is heavily reliant on PGE2 signaling, contributing to severe immune suppression and resistance to immunotherapy28,29. The disappointing outcomes of combining chemotherapy with celecoxib in TNBC models illustrate the limitations of simple coadministration strategies in this context9,30. These failures suggest that concurrent drug exposure alone is insufficient to resolve the underlying immunosignal conflict. Beyond the immunological barrier, the dense and fibrotic stroma of TNBC further hinders the synchronized delivery of free drugs, which is essential for achieving precise temporal control31. The immune signal conflict, combined with these physical barriers, highlights the urgent need for an advanced delivery platform capable of implementing a precisely coordinated, sequential therapeutic approach.To address these limitations, a spatiotemporally programmed nanomedicine, R-Gem@Cel-PV, was designed to resolve the inherent NOT-AND immunosignal conflict that undermines conventional chemoimmunotherapy (Fig. 1). This system is based on a phospholipase A2 (PLA2)-responsive gemcitabine prodrug (GPC), further functionalized with c(RGDfk) peptide to enhance spatial control. The design first exploits the overexpressed PLA2 in the TME to trigger the initial release of celecoxib, which resolves the NOT condition by preemptively blocking PGE2 synthesis. Subsequently, gemcitabine is activated by intracellular esterases, fulfilling the AND condition required for immune priming. By enforcing this spatiotemporal decoupling—unachievable with free drug combinations—this strategy aligns immune suppression relief with subsequent ICD signaling. This work establishes temporal control of immunosuppression relief as a critical determinant of chemotherapy-induced immune engagement and offers a generalizable framework for engineering nanomedicines capable of reprogramming immune-refractory tumors.Fig. 1The alternative text for this image may have been generated using AI.Full size imageSpatiotemporally programmed nanomedicine enables sequentially regulated chemoimmunotherapy. a Schematic of the enzyme-responsive R-Gem@Cel-PV nanomedicine, engineered for the sequential release of celecoxib and a gemcitabine prodrug (GPC) to achieve spatiotemporal control. b The underlying immunosignal conflict model governing the immune response to gemcitabine-induced tumor cell death, highlighting that the concurrent release of immunostimulatory DAMPs and inhibitory PGE2 nullifies the antitumor immune response. c The R-Gem@Cel-PV nanomedicine enforces a spatiotemporal decoupling of these opposing immunosignals. Release of celecoxib suppresses PGE2 and primes the microenvironment. The subsequent activation of gemcitabine then releases DAMPs into this nonsuppressive milieu, allowing them to function as potent immunostimulants. This programmed sequence fundamentally shifts the tumor immune microenvironment (TIME) from suppressive to immunostimulatory, orchestrating robust DC maturation and T-cell activation to drive potent immunity against both primary and metastatic tumors. Figure created with Adobe Photoshop and Adobe IllustratorResultsGemcitabine induces a PGE2-mediated immunosuppressive barrier in TNBCTo establish a therapeutic benchmark and examine the limitations of conventional chemo-COX2 inhibition strategies, this study evaluated a physical combination of gemcitabine (Gem) and the COX2 inhibitor celecoxib (Cel). This analysis aimed to determine whether gemcitabine elicits a dominant immunosuppressive barrier that restricts the immunogenic potential of chemotherapy in TNBC.Our initial validation confirmed the clinical relevance of the COX2/PGE2 axis in TNBC. Gemcitabine consistently upregulated Ptgs2 expression and PGE2 production across a broad range of preclinical models, including patient-derived xenografts (PDXs) and tumor spheres (Supplementary Fig. 1). Moreover, elevated expression of PGE2 biosynthesis genes correlated with poor patient survival (Supplementary Fig. 2). Despite this strong rationale, the conventional combination in vivo revealed a critical therapeutic dilemma: it achieved only a modest improvement in tumor growth control (Supplementary Figs. 3a and 4) but induced significant systemic toxicity, as evidenced by weight loss (Supplementary Fig. 4d) and clear cardiotoxicity (Supplementary Fig. 5). This outcome highlights the failure of physical coadministration and points to a more complex, unresolved biological issue.It is hypothesized that this failure arose from an underlying spatiotemporal immunosignal conflict, wherein gemcitabine simultaneously triggers opposing immunosignals. On the one hand, gemcitabine induced the release of immunostimulatory DAMPs, including surface calreticulin (CRT), high-mobility group box 1 (HMGB1), and adenosine triphosphate (ATP), effects that were not impaired by celecoxib (Supplementary Figs. 6 and 7). On the other hand, gemcitabine potentiated a counteracting immunosuppressive response, as evidenced by the upregulation of intratumoral COX2 and systemic PGE2 (Supplementary Fig. 8a–d). This was further confirmed by RNA sequencing (RNA-seq) analysis, which revealed the concurrent upregulation of both immunostimulatory and immunosuppressive factors, resulting in a conflicting gene signature with enrichment of immune activation and suppression genes, creating a paradoxical state (Supplementary Fig. 8e). This observation was further supported by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, which highlighted a significant enrichment of immune-related signatures (Supplementary Fig. 8f, g). Consequently, gemcitabine monotherapy impaired DC maturation both in vivo and in vitro (Supplementary Figs. 9 and 10a–c). Although celecoxib addition partially restored DC maturation and shifted intratumoral gene expression from a tolerogenic to an immunogenic profile (Supplementary Fig. 11), the therapeutic benefit of this combination was largely dependent on CD8+ T cells (Supplementary Fig. 12), emphasizing the importance of a robust T-cell response.To provide definitive genetic evidence of the core mechanism behind this conflict, this study further demonstrated that exogenous PGE2 alone was sufficient to impair DC maturation and T-cell-mediated cytotoxicity in vitro (Supplementary Fig. 10d–h). Notably, using Ptgs2 (COX2) knockout 4T1 cells, this study showed that genetic ablation of Ptgs2 completely abolished gemcitabine-induced PGE2 production and restored the ability of gemcitabine-treated cells to promote DC maturation both in vitro and in vivo (Supplementary Fig. 13).These results indicate that while overcoming gemcitabine-potentiated PGE2 signaling is essential for an effective antitumor immune response, it cannot be safely or effectively achieved through the physical combination of free drugs.Design of a spatiotemporally programmed nanomedicineTo overcome the intrinsic limitations of free drug combinations, a spatiotemporally programmed nanomedicine, R-Gem@Cel-PV, was engineered to enforce a predefined temporal order between PGE2 blockade and ICD induction.PLA2 expression is typically low in normal tissues but significantly upregulated in cancerous tissues, highlighting its potential role in tumor progression32,33. Leveraging this differential expression, a PLA2-responsive gemcitabine prodrug (GPC) was developed. PLA2 overexpression in murine orthotopic TNBC tumors, compared to normal mammary glands, was first confirmed (Supplementary Fig. 14), validating the premise for spatial control. Based on PLA2’s catalytic mechanism34, which requires substrate flexibility (Supplementary Fig. 15), a C5-linker (glutaric acid) was selected to bridge gemcitabine and the lipid backbone (1-palmitoyl-sn-glycero-3-phosphocholine, LPC), resulting in the creation of GPC. As illustrated in Fig. 2a, GPC was designed for dual responsiveness: PLA2 first hydrolyzes the sn-2 ester bond, releasing glutaryl-anchored gemcitabine (Gem-C5). Subsequently, low-specific esterases abundant in TNBC tumor cells (Supplementary Fig. 16) hydrolyze the ester bond of Gem-C5, further activating the drug.Fig. 2The alternative text for this image may have been generated using AI.Full size imageDesign and characterization of a spatiotemporally programmed nanomedicine for sequential drug delivery. a Chemical structure of GPC, indicating cleavage sites for PLA2 and intracellular esterase. b HRMS analysis of GPC hydrolysis by PLA2 (5 U/mL), showing spectra for GPC and its product, Gem-C5. c HRMS monitoring of the sequential enzymatic conversion of GPC. GPC was incubated with PLA2 (5 U/mL) for 4 h, followed by the addition of PLE (30 U/mL) at t = 0. Spectra were acquired at the indicated time points. An untreated GPC sample is shown as a control. d Size distribution of R-Gem@Cel-PV determined by dynamic light scattering (DLS) in the presence or absence of enzyme. e Cumulative release of gemcitabine and celecoxib from R-Gem@Cel-PV over 72 h in buffer containing PLA2 (2 U/mL) and PLE (7.5 U/mL). f–h AFADESI-MSI analysis of intracellular drug levels in 4T1 cells after a 4-h treatment. The treatment groups are defined as: 1, free drug mixture of GPC and celecoxib (G + C); 2, Gem@Cel-PV; and 3, R-Gem@Cel-PV. All treatments were administered at a gemcitabine-equivalent dose of 64 μg/mL. f Workflow schematic of AFADESI-MSI analysis for molecular imaging of GPC, celecoxib, and gemcitabine in MRM mode, with their respective mass-to-charge ratios (m/z) listed. Representative ion intensity maps (g) and semi-quantitative analysis (h) of intracellular drug signals. i–l In vivo tumor targeting and biodistribution. Mice bearing 4T1 tumors were intravenously injected with the following formulations: Ctrl, untreated cells; G1, free Cy5 dye; G2, Cy5-labeled Gem@Cel-PV (Gem@Cy5-PV); G3, c(RGDfk)-modified Gem@Cy5-PV (R-Gem@Cy5-PV). i Representative whole-body fluorescence imaging at the indicated time points. j Ex vivo fluorescence imaging of tumors and major organs harvested 12 h post-treatment, with corresponding quantification of fluorescence intensity. k Representative fluorescence images of tumor sections 12 h post-injection, with nuclei stained by DAPI. Scale bar: 50 μm. l Representative CLSM images of tumor sections 12 h after treatment, stained for nuclei (DAPI) and vasculature (CD31). Scale bar: 20 μm. Data are presented as means ± SEM. b–e n = 3 independent experiments; h n = 4 independent experiments; i–l n = 3 biological replicates. Statistical analysis was performed using two-tailed Student’s t-test (for e) and one-way ANOVA with Tukey’s multiple comparisons test (for h, j). *p