Nanoscale Organization of TRAIL Trimers using DNA Origami to Promote Clustering of Death Receptor and Cancer Cell Apoptosis.

Through inducing death receptor (DR) clustering to activate downstream signaling, tumor necrosis factor related apoptosis inducing ligand (TRAIL) trimers trigger apoptosis of tumor cells. However, the poor agonistic activity of current TRAIL-based therapeutics limits their antitumor efficiency. The nanoscale spatial organization of TRAIL trimers at different interligand distances is still challenging, which is essential for the understanding of interaction pattern between TRAIL and DR. In this study, a flat rectangular DNA origami is employed as display scaffold, and an "engraving-printing" strategy is developed to rapidly decorate three TRAIL monomers onto its surface to form DNA-TRAIL3 trimer (DNA origami with surface decoration of three TRAIL monomers). With the spatial addressability of DNA origami, the interligand distances are precisely controlled from 15 to 60 nm. Through comparing the receptor affinity, agonistic activity and cytotoxicity of these DNA-TRAIL3 trimers, it is found that ≈40 nm is the critical interligand distance of DNA-TRAIL3 trimers to induce death receptor clustering and the resulting apoptosis.Finally, a hypothetical "active unit" model is proposed for the DR5 clustering induced by DNA-TRAIL3 trimers.

Site-Specific Modification of Virus-Like Particles for Exogenous Tumor Antigen Display and Minimizing Preexisting Immunity.

The widespread preexisting immunity against virus-like particles (VLPs) seriously limits the applications of VLPs as vaccine vectors. Enabling technology for exogenous antigen display should not only ensure the assembly ability of VLPs and site-specific modification, but also consider the effect of preexisting immunity on the behavior of VLPs in vivo. Here, combining genetic code expansion technique and synthetic biology strategy, a site-specific modification method for hepatitis B core (HBc) VLPs via incorporating azido-phenylalanine into the desired positions is described. Through modification position screening, it is found that HBc VLPs incorporated with azido-phenylalanine at the main immune region can effectively assemble and rapidly conjugate with the dibenzocycolctyne-modified tumor-associated antigens, mucin-1 (MUC1). The site-specific modification of HBc VLPs not only improves the immunogenicity of MUC1 antigens but also shields the immunogenicity of HBc VLPs themselves, thereby activating a strong and persistent anti-MUC1 immune response even in the presence of preexisting anti-HBc immunity, which results in the efficient tumor elimination in a lung metastatic mouse model. Together, these results demonstrate the site-specific modification strategy enabled HBc VLPs behave as a potent antitumor vaccine and this strategy to manipulate immunogenicity of VLPs may be suitable for other VLP-based vaccine vectors.

Antigen Capture and Immune Modulation by Bacterial Outer Membrane Vesicles as In Situ Vaccine for Cancer Immunotherapy Post-Photothermal Therapy.

Tumor antigens released from tumor cells after local photothermal therapy (PTT) can activate the tumor-specific immune responses, which are critical for eliminating the residual lesions and distant metastases. However, the limited recognition efficiency of released tumor antigens by the immune system and the immunosuppressive microenvironment lead to ineffective antitumor immunity. Here, an in situ multifunctional vaccine based on bacterial outer membrane vesicles (OMVs, 1-MT@OMV-Mal) is developed by surface conjunction of maleimide groups (Mal) and interior loading with inhibitor of indoleamine 2, 3-dioxygenase (IDO), 1-methyl-tryptophan (1-MT). 1-MT@OMV-Mal can bind to the released tumor antigens after PTT, and be efficiently recognized and taken up by dendritic cells. Furthermore, in situ injection of 1-MT@OMV-Mal simultaneously overcomes the immune inhibition of IDO on tumor-infiltrating effector T cells, leading to remarkable inhibition on both primary and distant tumors. Together, a promising in situ vaccine based on OMVs to facilitate immune-mediated tumor clearance after PTT through orchestrating antigen capture and immune modulation is presented.

A novel adoptive synthetic TCR and antigen receptor (STAR) T-Cell therapy for B-Cell acute lymphoblastic leukemia.

We developed a T-cell-receptor (TCR) complex-based chimeric antigen receptor (CAR) named Synthetic TCR and Antigen Receptor (STAR). Here, we report pre-clinical and phase I clinical trial data (NCT03953599) of this T-cell therapy for refractory and relapsed (R/R) B-cell acute lymphoblastic leukemia (B-ALL) patients. STAR consists of two protein modules each containing an antibody light or heavy chain variable region and TCR α or ß chain constant region fused to the co-stimulatory domain of OX40. T-cells were transduced with a STAR-OX40 lentiviral vector. A leukemia xenograft mouse model was used to assess the STAR/STAR-OX40 T cell antitumor activity. Eighteen patients with R/R B-ALL were enrolled into the clinical trial. In a xenograft mouse model, STAR-T-cells exhibited superior tumor-specific cytotoxicity compared with conventional CAR-T cells. Incorporating OX40 into STAR further improved the proliferation and persistence of tumor-targeting T-cells. In our clinical trial, 100% of patients achieved complete remission 4 weeks post-STAR-OX40 T-cell infusion and 16/18 (88.9%) patients pursued consolidative allogeneic hematopoietic stem cell transplantation (allo-HSCT). Twelve of 16 patients (75%) remained leukemia-free after a median follow-up of 545 (433-665) days. The two patients without consolidative allo-HSCT relapsed on Day 58 and Day 186. Mild cytokine release syndrome occurred in 10/18 (55.6%) patients, and 2 patients experienced grade III neurotoxicity. Our preclinical studies demonstrate super anti-tumor potency of STAR-OX40 T-cells compared with conventional CAR-T cells. The first-in-human clinical trial shows that STAR-OX40 T-cells are tolerable and an effective therapeutic platform for treating R/R B-ALL.

Efficacy of pp65-specific TCR-T cell therapy in treating cytomegalovirus infection after hematopoietic stem cell transplantation.

Cytomegalovirus (CMV) infection remains a major cause of mortality after hematopoietic stem cell transplantation (HSCT). Current treatments, including antiviral drugs and adoptive cell therapy with CMV-specific cytotoxic T lymphocytes (CTLs), only show limited benefits in patients. T-cell receptor (TCR)-T cell therapy offers a promising option to treat CMV infections. Here, using tetramer-based screening and single-cell TCR cloning technologies, we identified various CMV antigen-specific TCRs from healthy donors, and generated TCR-T cells targeting multiple pp65 epitopes corresponding to three major HLA-A alleles. The TCR-T cells showed efficient cytotoxicity toward epitope-expressing target cells in vitro. After transfer into immune-deficient mice bearing pp65+ HLA+ tumor cells, TCR-T cells induced dramatic tumor regression and exhibited long-term persistence. In a phase I clinical trial (NCT04153279), CMV TCR-T cells were applied to treat patients with CMV reactivation after HSCT. Except one patient who withdrew at early treatment stage, all other six patients were well-tolerated and achieved complete response (CR), no more than grade 2 cytokine release syndrome (CRS) and other adverse events were observed. CMV TCR-T cells persisted up to 3 months. Among them, two patients have survived for more than 1 year. This study demonstrates the great potential in the treatment and prevention of CMV infection following HSCT or other organ transplantation.

CXCR2-modified CAR-T cells have enhanced trafficking ability that improves treatment of hepatocellular carcinoma.

Unlike hematological malignancies, solid tumors have proved to be less susceptible to chimeric antigen receptor (CAR)-T cell therapy, which is partially caused by reduced accumulation of therapeutic T cells in tumor site. Since efficient trafficking is the precondition and pivotal step for infused CAR-T cells to exhibit their anti-tumor function, strategies are highly needed to improve the trafficking ability of CAR-T cells for solid tumor treatment. Here, based on natural lymphocyte chemotaxis theory and characteristics of solid tumor microenvironments, we explored the possibility of enhancing CAR-T cell trafficking by using chemokine receptors. Our study found that compared with other chemokines, several CXCR2 ligands showed relatively high expression level in human hepatocellular carcinoma tumor tissues and cell lines. However, both human peripheral T cells and hepatocellular carcinoma tumor infiltrating T cells lacked expression of CXCR2. CXCR2-expressing CAR-T cells exhibited identical cytotoxicity but displayed significantly increased migration ability in vitro. In a xenograft tumor model, we found that expressing CXCR2 in CAR-T cells could significantly accelerate in vivo trafficking and tumor-specific accumulation, and improve anti-tumor effect of these cells.

RNF31 Regulates Skin Homeostasis by Protecting Epidermal Keratinocytes from Cell Death.

Linear ubiquitin chain assembly complex plays an important role in regulating TNF-α signaling activation by modifying target proteins with linear (M1-linked) ubiquitin chains. In this study, we report that the epidermis-specific knockout (KO) of RNF31, the catalytic subunit of linear ubiquitin chain assembly complex, results in an early postnatal lethality in mice due to severe skin inflammation. The inflammation was mainly triggered by TNF-α-induced apoptosis in RNF31 KO keratinocytes. Mechanistically, the deficiency of RNF31 not only impaired TNF-α-induced NF-κB activation, but also significantly increased apoptosis. Consistently, deleting TNF receptor 1 could rescue the lethality of RNF31 epidermis-specific KO mice and also the skin inflammation. Collectively, our study provides an in vivo insight that linear ubiquitination is critical for maintaining the homeostasis of keratinocytes, which will shed light on designing therapeutic compounds to treat skin inflammation.

Linear ubiquitination of cFLIP induced by LUBAC contributes to TNFα-induced apoptosis.

The linear ubiquitin chain assembly complex (LUBAC) regulates NF-κB activation by modifying proteins with linear (M1-linked) ubiquitination chains. Although LUBAC also regulates the apoptosis pathway, the precise mechanism by which LUBAC regulates apoptosis remains not fully defined. Here, we report that LUBAC-mediated M1-linked ubiquitination of cellular FLICE-like inhibitory protein (cFLIP), an anti-apoptotic molecule, contributes to tumor necrosis factor (TNF) α-induced apoptosis. We found that deficiency of RNF31, the catalytic subunit of the LUBAC complex, promoted cFLIP degradation in a proteasome-dependent manner. Moreover, we observed RNF31 directly interact with cFLIP, and LUBAC further conjugated M1-linked ubiquitination chains at Lys-351 and Lys-353 of cFLIP to stabilize cFLIP, thereby protecting cells from TNFα-induced apoptosis. Together, our study identifies a new substrate of LUBAC and reveals a new molecular mechanism through which LUBAC regulates TNFα-induced apoptosis via M1-linked ubiquitination.

Bacteria-derived nanovesicles enhance tumour vaccination by trained immunity.

Trained immunity enhances the responsiveness of immune cells to subsequent infections or vaccinations. Here we demonstrate that pre-vaccination with bacteria-derived outer-membrane vesicles, which contain large amounts of pathogen-associated molecular patterns, can be used to potentiate, and enhance, tumour vaccination by trained immunity. Intraperitoneal administration of these outer-membrane vesicles to mice activates inflammasome signalling pathways and induces interleukin-1ß secretion. The elevated interleukin-1ß increases the generation of antigen-presenting cell progenitors. This results in increased immune response when tumour antigens are delivered, and increases tumour-antigen-specific T-cell activation. This trained immunity increased protection from tumour challenge in two distinct cancer models.

Intelligent Responsive Nanoparticles with Multilevel Triggered Drug Penetration for Tumor Photochemotherapy.

Nanomedicines have contradictory size requirements to overcome systemic barriers and penetrate the tumor extracellular matrix (ECM). Larger-sized nanoparticles (50-200 nm) exhibit prolonged blood circulation half-life and improved tumor enrichment, while small-sized nanoparticles (4-20 nm) easily penetrate deep tumor tissues. Therefore, the development of intelligent responsive nanomedicine systems can not only increase nanodrug tumor accumulation but also improve their penetration into the ECM. Herein, we propose an intelligent responsive nanoparticle triggered by near-infrared light (NIR). The nanoparticle was constructed by a temperature-sensitive liposome (TSL) encapsulating ultrasmall melanin nanoparticles (MNPs) loaded with doxorubicin (MNP/doxorubicin (DOX)@TSL). When exposed to NIR irradiation, the tailor-made nanoparticles not only effectively ablated the tumor cells around blood vessels but also destroyed the structural integrity and released loaded ultrasmall MNP/DOX (<10 nm) to promote deep tumor penetration and enhance interior tumor cell killing. This NIR-triggered intelligent nanoparticle successfully integrated photothermal therapy (PTT) for perivascular tumor cells and chemotherapy for deep tumor cell inhibition. The in vivo results showed remarkable tumor regression in 4T1 breast tumor-bearing mice by 74.2%. This controllable size switchable nanosystem with efficient tumor accumulation and penetration has shown great potential in improving synergistic antitumor effects of photochemotherapy.

Probiotic-Inspired Nanomedicine Restores Intestinal Homeostasis in Colitis by Regulating Redox Balance, Immune Responses, and the Gut Microbiome.

Microbiota-based therapeutics offer innovative strategies to treat inflammatory bowel diseases (IBDs). However, the poor clinical outcome so far and the limited flexibility of the bacterial approach call for improvement. Inspired by the health benefits of probiotics in alleviating symptoms of bowel diseases, bioartificial probiotics are designed to restore the intestinal microenvironment in colitis by regulating redox balance, immune responses, and the gut microbiome. The bioartificial probiotic comprises two components: an E. coli Nissle 1917-derived membrane (EM) as the surface and the biodegradable diselenide-bridged mesoporous silica nanoparticles (SeM) as the core. When orally administered, the probiotic-inspired nanomedicine (SeM@EM) adheres strongly to the mucus layer and restored intestinal redox balance and immune regulation homeostasis in a murine model of acute colitis induced by dextran sodium sulfate. In addition, the respective properties of the EM and SeM synergistically alter the gut microbiome to a favorable state by increasing the bacterial diversity and shifting the microbiome profile to an anti-inflammatory phenotype. This work suggests a safe and effective nanomedicine that can restore intestinal homeostasis for IBDs therapy.

Lipid-Peptide-mRNA Nanoparticles Augment Radioiodine Uptake in Anaplastic Thyroid Cancer.

Restoring sodium iodide symporter (NIS) expression and function remains a major challenge for radioiodine therapy in anaplastic thyroid cancer (ATC). For more efficient delivery of messenger RNA (mRNA) to manipulate protein expression, a lipid-peptide-mRNA (LPm) nanoparticle (NP) is developed. The LPm NP is prepared by using amphiphilic peptides to assemble a peptide core and which is then coated with cationic lipids. An amphiphilic chimeric peptide, consisting of nine arginine and hydrophobic segments (6 histidine, C18 or cholesterol), is synthesized for adsorption of mRNA encoding NIS in RNase-free conditions. In vitro studies show that LP(R9H6) m NP is most efficient at delivering mRNA and can increase NIS expression in ATC cells by more than 10-fold. After intratumoral injection of NIS mRNA formulated in optimized LPm NP, NIS expression in subcutaneous ATC tumor tissue increases significantly in nude mice, resulting in more iodine 131 (131 I) accumulation in the tumor, thereby significantly inhibiting tumor growth. Overall, this work designs three arginine-rich peptide nanoparticles, contributing to the choice of liposome cores for gene delivery. LPm NP can serve as a promising adjunctive therapy for patients with ATC by restoring iodine affinity and enhancing the therapeutic efficacy of radioactive iodine.

Normalizing the Immune Macroenvironment via Debulking Surgery to Strengthen Tumor Nanovaccine Efficacy and Eliminate Metastasis.

In tumor nanovaccines, nanocarriers enhance the delivery of tumor antigens to antigen-presenting cells (APCs), thereby ensuring the robust activation of tumor antigen-specific effector T-cells to kill tumor cells. Through employment of their high immunogenicity and nanosize, we have developed a "Plug-and-Display" delivery platform on the basis of bacterial outer membrane vesicles (OMVs) for tumor nanovaccines (NanoVac), which can rapidly display different tumor antigens and efficiently eliminate lung metastases of melanoma. In this study, we first upgraded the NanoVac to increase their antigen display efficiency. However, we found that the presence of a subcutaneous xenograft seriously hampered the efficiency of NanoVac to eliminate lung metastases, with the subcutaneous xenograft mimicking the primary tumor burden in clinical practice. The primary tumor secreted significant amounts of granulocyte colony-stimulating factor (G-CSF) and altered the epigenetic features of granulocyte monocyte precursor cells (GMPs) in the bone marrow, thus disrupting systemic immunity, particularly the function of APCs, and ultimately resulting in NanoVac failure to affect metastases. These changes in the systemic immune macroenvironment were plastic, and debulking surgery of primary tumor resection reversed the dysfunction of APCs and failure of NanoVac. These results demonstrate that, in addition to the formulation design of the tumor nanovaccines themselves, the systemic immune macroenvironment incapacitated by tumor development is another key factor that cannot be ignored to affect the efficiency of tumor nanovaccines, and the combination of primary tumor resection with NanoVac is a promising radical treatment for widely metastatic tumors.

Modular-designed engineered bacteria for precision tumor immunotherapy via spatiotemporal manipulation by magnetic field.

Micro-nano biorobots based on bacteria have demonstrated great potential for tumor diagnosis and treatment. The bacterial gene expression and drug release should be spatiotemporally controlled to avoid drug release in healthy tissues and undesired toxicity. Herein, we describe an alternating magnetic field-manipulated tumor-homing bacteria developed by genetically modifying engineered Escherichia coli with Fe3O4@lipid nanocomposites. After accumulating in orthotopic colon tumors in female mice, the paramagnetic Fe3O4 nanoparticles enable the engineered bacteria to receive and convert magnetic signals into heat, thereby initiating expression of lysis proteins under the control of a heat-sensitive promoter. The engineered bacteria then lyse, releasing its anti-CD47 nanobody cargo, that is pre-expressed and within the bacteria. The robust immunogenicity of bacterial lysate cooperates with anti-CD47 nanobody to activate both innate and adaptive immune responses, generating robust antitumor effects against not only orthotopic colon tumors but also distal tumors in female mice. The magnetically engineered bacteria also enable the constant magnetic field-controlled motion for enhanced tumor targeting and increased therapeutic efficacy. Thus, the gene expression and drug release behavior of tumor-homing bacteria can be spatiotemporally manipulated in vivo by a magnetic field, achieving tumor-specific CD47 blockage and precision tumor immunotherapy.

Engineered Bacterial Outer Membrane Vesicles as Controllable Two-Way Adaptors to Activate Macrophage Phagocytosis for Improved Tumor Immunotherapy.

The most immune cells infiltrating tumor microenvironment (TME), tumor-associated macrophages (TAMs) closely resemble immunosuppressive M2-polarized macrophages. Moreover, tumor cells exhibit high expression of CD47 "don't eat me" signal, which obstructs macrophage phagocytosis. The precise and efficient activation of TAMs is a promising approach to tumor immunotherapy; however, re-education of macrophages remains a challenge. Bacteria-derived outer membrane vesicles (OMVs) are highly immunogenic nanovesicles that can robustly stimulate macrophages. Here, an OMV-based controllable two-way adaptor is reported, in which a CD47 nanobody (CD47nb) is fused onto OMV surface (OMV-CD47nb), with the outer surface coated with a polyethylene glycol (PEG) layer containing diselenide bonds (PEG/Se) to form PEG/Se@OMV-CD47nb. The PEG/Se layer modification not only mitigates the immunogenicity of OMV-CD47nb, thereby remarkedly increasing the dose that can be administered safely through intravenous injection, but also equips the formulation with radiation-triggered controlled release of OMV-CD47nb. Application of radiation to tumors in mice injected with the nanoformulation results in remodeling of TME. As two-way adaptors, OMV-CD47nb activates TAM phagocytosis of tumor cells via multiple pathways, including induction of M1 polarization and blockade of "don't eat me" signal. Moreover, this activation of TAMs results in the stimulation of T cell-mediated antitumor immunity through effective antigen presentation.

Personalized cancer vaccines from bacteria-derived outer membrane vesicles with antibody-mediated persistent uptake by dendritic cells.

Nanocarriers with intrinsic immune adjuvant properties can activate the innate immune system while delivering tumor antigen, thus efficiently facilitating antitumor adaptive immunity. Bacteria-derived outer membrane vesicles (OMVs) are an excellent candidate due to their abundance of pathogen associated molecular patterns. However, during the uptake of OMVs by dendritic cells (DCs), the interaction between lipopolysaccharide and toll-like receptor 4 induces rapid DC maturation and uptake blockage, a phenomenon we refer to as "maturation-induced uptake obstruction" (MUO). Herein we decorated OMV with the DC-targeting αDEC205 antibody (OMV-DEC), which endowed the nanovaccine with an uptake mechanism termed as "not restricted to maturation via antibody modifying" (Normandy), thereby overcoming the MUO phenomenon. We also proved the applicability of this nanovaccine in identifying the human tumor neoantigens through rapid antigen display. In summary, this engineered OMV represents a powerful nanocarrier for personalized cancer vaccines, and this antibody modification strategy provides a reference to remodel the DC uptake pattern in nanocarrier design.

Nanotechnology-empowered vaccine delivery for enhancing CD8+ T cells-mediated cellular immunity.

After centuries of development, using vaccination to stimulate immunity has become an effective method for prevention and treatment of a variety of diseases including infective diseases and cancers. However, the tailor-made efficient delivery system for specific antigens is still urgently needed due to the low immunogenicity and stability of antigens, especially for vaccines to induce CD8+ T cells-mediated cellular immunity. Unlike B cells-mediated humoral immunity, CD8+ T cells-mediated cellular immunity mainly aims at the intracellular antigens from microorganism in virus-infected cells or genetic mutations in tumor cells. Therefore, the vaccines for stimulating CD8+ T cells-mediated cellular immunity should deliver the antigens efficiently into the cytoplasm of antigen presenting cells (APCs) to form major histocompatibility complex I (MHCI)-antigen complex through cross-presentation, followed by activating CD8+ T cells for immune protection and clearance. Importantly, nanotechnology has been emerged as a powerful tool to facilitate these multiple processes specifically, allowing not only enhanced antigen immunogenicity and stability but also APCs-targeted delivery and elevated cross-presentation. This review summarizes the process of CD8+ T cells-mediated cellular immunity induced by vaccines and the technical advantages of nanotechnology implementation in general, then provides an overview of the whole spectrum of nanocarriers studied so far and the recent development of delivery nanotechnology in vaccines against infectious diseases and cancer. Finally, we look forward to the future development of nanotechnology for the next generation of vaccines to induce CD8+ T cells-mediated cellular immunity.

Enhancing CAR-T cell efficacy in solid tumors by targeting the tumor microenvironment.

Chimeric antigen receptor (CAR)-T cell therapy has achieved successful outcomes against hematological malignancies and provided a new impetus for treating solid tumors. However, the efficacy of CAR-T cells for solid tumors remains unsatisfactory. The tumor microenvironment has an important role in interfering with and inhibiting the effector function of immune cells, among which upregulated inhibitory checkpoint receptors, soluble suppressive cytokines, altered chemokine expression profiles, aberrant vasculature, complicated stromal composition, hypoxia and abnormal tumor metabolism are major immunosuppressive mechanisms. In this review, we summarize the inhibitory factors that affect the function of CAR-T cells in tumor microenvironment and discuss approaches to improve CAR-T cell efficacy for solid tumor treatment by targeting those barriers.

Chimeric STAR receptors using TCR machinery mediate robust responses against solid tumors.

Chimeric antigen receptor T (CAR-T) cell therapies have demonstrated high response rate and durable disease control for the treatment of B cell malignancies. However, in the case of solid tumors, CAR-T cells have shown limited efficacy, which is partially attributed to intrinsic defects in CAR signaling. Here, we construct a double-chain chimeric receptor, termed as synthetic T cell receptor (TCR) and antigen receptor (STAR), which incorporates antigen-recognition domain of antibody and constant regions of TCR that engage endogenous CD3 signaling machinery. Under antigen-free conditions, STAR does not trigger tonic signaling, which has been reported to cause exhaustion of traditional CAR-T cells. Upon antigen stimulation, STAR mediates strong and sensitive TCR-like signaling, and STAR-T cells exhibit less susceptibility to dysfunction and better proliferation than traditional 28zCAR-T cells. In addition, STAR-T cells show higher antigen sensitivity than CAR-T cells, which holds potential to reduce the risk of antigen loss-induced tumor relapse in clinical use. In multiple solid tumor models, STAR-T cells prominently outperformed BBzCAR-T cells and generated better or equipotent antitumor effects to 28zCAR-T cells without causing notable toxicity. With these favorable features endowed by native TCR-like signaling, STAR-T cells may provide clinical benefit in treating refractory solid tumors.

A Bioinspired Nanoprobe with Multilevel Responsive T1 -Weighted MR Signal-Amplification Illuminates Ultrasmall Metastases.

Metastasis remains the major cause of death in cancer patients. Thus, there is a need to sensitively detect tumor metastasis, especially ultrasmall metastasis, for early diagnosis and precise treatment of cancer. Herein, an ultrasensitive T1 -weighted magnetic resonance imaging (MRI) contrast agent, UMFNP-CREKA is reported. By conjugating the ultrasmall manganese ferrite nanoparticles (UMFNPs) with a tumor-targeting penta-peptide CREKA (Cys-Arg-Glu-Lys-Ala), ultrasmall breast cancer metastases are accurately detected. With a behavior similar to neutrophils' immunosurveillance process for eliminating foreign pathogens, UMFNP-CREKA exhibits a chemotactic "targeting-activation" capacity. UMFNP-CREKA is recruited to the margin of tumor metastases by the binding of CREKA with fibrin-fibronectin complexes, which are abundant around tumors, and then release of manganese ions (Mn2+ ) to the metastasis in response to pathological parameters (mild acidity and elevated H2 O2 ). The localized release of Mn2+ and its interaction with proteins affects a marked amplification of T1 -weighted magnetic resonance (MR) signals. In vivo T1 -weighted MRI experiments reveal that UMFNP-CREKA can detect metastases at an unprecedented minimum detection limit of 0.39 mm, which has significantly extended the detection limit of previously reported MRI probe.