Precision tumor discrimination and eradication is a prerequisite for successful cancer therapies. Immune cells, as key players in the host defense system, have evolved the remarkable capability to traffic through the body, recognize and kill tumor cells. Harnessing these intrinsic properties has led to the development of cell-based immunotherapy, a promising therapeutic modality. For example, genetically engineered T cells, NK cells, and macrophages, equipped with chimeric antigen receptors (CARs), enhance cytotoxic activity toward cancer cells. Cell surface engineering techniques displaying natural and artificial receptors further boost tumor cell-targeting. Despite great potential, engineered immune cell strategies have limitations. The need to engineer cells with different antigen receptors for various tumors is time and resource-intensive and unsuitable as broad-spectrum therapy. More importantly, few antigens are absolutely tumor-specific; most targeted antigens are also present in normal tissues, causing life-threatening adverse side effects. Therefore, developing alternative tactics independent of antigen recognition is highly desirable. Neutrophils, the most prevalent innate immunity effector cells, are particularly interesting due to their capacity for inflammation targeting and pathogen elimination. Recent work has shown that neutrophil-released neutrophil elastase (ELANE) or its homolog (porcine pancreatic elastase, PPE) can kill many cancer cell types while preserving non-cancer cells and trigger an abscopal effect mediated by cytotoxic T lymphocytes to attenuate primary and distal tumor growth. The crucial factor driving this broad specificity is the selective expression of histone H1 isoforms in numerous tumor types. Mechanistically, ELANE or PPE enters the cell, proteolytically liberates the CD95 death domain (DD), induces DNA damage, provoking histone H1 isoform nucleo-cytoplasmic translocation to bind CD95 DD and co-localize with mitochondria, initiating apoptosis. ELANE or PPE's cancer-selective property depends on elevated histone H1 isoforms, not tumor-restricted antigens, suggesting their potential as powerful tumor-agnostic and mutation-agnostic targeted agents. Onchilles Pharma is exploiting ELANE and PPE therapies clinically. Adoptive transfer of engineered neutrophils to release ELANE appears effective, but neutrophil-based therapies face challenges such as uncertain phenotypes, low delivery efficacy (cells trapped in the lungs), and complexity of manipulation and storage. The cell-free biomimetic strategy bridges the gap between cells and nanomaterials, offering potential to circumvent problems encountered by cell-based therapies. Integrating the tumor homing properties of immune cells and the defined functional responses of nanomaterials enables a synthetic system to effectively transport therapeutic payloads to tumor sites and achieve spatiotemporal control and on-demand intracellular release. However, unsatisfactory cancer cell killing and inadequate immune activation of ELANE/PPE alone limit its application. Amplifying tumor cell killing activity while retaining precise targeting is needed. Considering the crucial role of histone H1 nucleo-cytoplasmic translocation in the precision tumor discrimination and killing effect of ELANE/PPE, the authors hypothesized that accelerating histone H1 translocation would maximize therapeutic effect. Histone H1 binds to linker DNA, protecting it from DNA-damaging agents. DSBs cause histone H1 release, increasing DNA sensitivity to DSB inducers. ROS, especially singlet oxygen (¹O₂), can cause DSBs. Introducing a ¹O₂ generator into the therapeutic system could enhance selective cancer cell killing.

The introduction extensively reviews existing literature on cancer therapies, focusing on the use of engineered immune cells and their limitations. It highlights the challenges associated with antigen recognition-based therapies, emphasizing the need for alternative approaches. The review then introduces neutrophils and the promising results obtained with neutrophil elastase (ELANE) and its homolog porcine pancreatic elastase (PPE) in selectively targeting cancer cells. The mechanism of action of PPE is discussed along with the challenges associated with neutrophil-based therapies. The authors transition to the cell-free biomimetic approach and its limitations, preparing the reader for their proposed solution.

The authors describe the fabrication and characterization of their biomimetic nanodevice (FKPN). The Fe-porphyrin MOF was synthesized using a solvent-thermal method, characterized by SEM, TEM, UV-vis absorption spectra, and FTIR. NLS peptides were covalently grafted to the porphyrin ligands using EDC/NHS chemistry. PPE was adsorbed onto the positively charged FK through electrostatic interactions, confirmed by UV-vis analysis, zeta potential measurements, and nitrogen adsorption studies. Neutrophil membrane (NM) fragments were obtained from purified and activated neutrophils and coated onto FKP using sonication and extrusion to create FKPN. The size, morphology, and composition of FKPN were characterized using various techniques, including TEM, HAADF-STEM, and elemental mapping. The stability of NM coating was assessed over time. The presence of representative adhesion proteins (L-selectin, CXCR4, β1 integrin) on FKPN was confirmed by Western blot. The GSH-triggered unlocking of FKPN was validated using XPS, which showed a change in the Fe 2p_3/2 band from Fe³⁺ to Fe²⁺ after GSH treatment. The morphology change and GSH consumption were also quantified. The GSH-responsive release of porphyrin and PPE was determined using fluorescence spectroscopy and ICP-MS. The catalytic activity of released PPE was measured using PNA substrate. Singlet oxygen (¹O₂) generation was monitored using SOSG. Cellular uptake of FKPN by cancer and non-cancer cells was assessed using fluorescence microscopy and flow cytometry. The internalization mechanism was investigated using various inhibitors and low temperature. Intracellular ¹O₂ levels were measured using DCFH-DA. DNA damage was evaluated by Western blotting (γH2AX) and comet assays. Immunofluorescence was used to assess cytoplasmic histone H1.0 localization, CD95 DD-H1.0 interaction, and mitochondrial trafficking. Co-immunoprecipitation confirmed CD95 DD binding to H1.0. Cell viability was determined using CCK-8 and live/dead staining assays. Apoptosis was analyzed by flow cytometry. ICD markers (HMGB1, ATP) were quantified by ELISA and ATP assay kits. In vivo biocompatibility was evaluated using hematological analysis and H&E staining of major organs. Plasma pharmacokinetics was determined using ICP-MS. In vivo biodistribution was assessed using in vivo and ex vivo fluorescence imaging. In vivo immune activation and antitumor studies were performed using a 4T1 orthotopic breast cancer mouse model, including analysis of DC maturation, CD4⁺ and CD8⁺ T cell populations. Tumor growth, survival, and histopathological analyses were conducted. The abscopal effect was evaluated using a bilateral 4T1 tumor model and immunofluorescence staining. DC and T-cell blocking experiments were performed to assess the role of these immune cells in the abscopal effect.

The study successfully synthesized and characterized a novel biomimetic nanodevice (FKPN) that mimics neutrophils' function. FKPN demonstrates high selectivity towards cancer cells due to the GSH-triggered unlocking mechanism and neutrophil membrane camouflage. In vitro studies show that FKPN effectively induces cancer cell death through a mechanism involving PPE-mediated CD95 DD liberation and ¹O₂-induced DSBs, leading to amplified histone H1 nucleo-cytoplasmic translocation. This process results in CD95 DD-H1.0 interactions and subsequent mitochondrial apoptosis. Moreover, FKPN induces immunogenic cell death (ICD) in cancer cells, releasing HMGB1 and ATP, which activate the immune system. In vivo experiments confirmed the superior tumor-targeting ability of FKPN. Treatment with FKPN combined with laser irradiation significantly inhibited primary tumor growth and induced a robust immune response characterized by increased DC maturation and CD4⁺ and CD8⁺ T cell infiltration into tumor tissues. Notably, the treatment demonstrated an abscopal effect, effectively suppressing the growth of distal, untreated tumors. The abscopal effect was shown to be dependent on DCs and both CD4⁺ and CD8⁺ T cells. The study demonstrated that FKPN exhibits good biocompatibility and biosecurity in vivo.

The findings of this study demonstrate the successful design and development of a novel biomimetic nanodevice (FKPN) for precise cancer therapy. The unique combination of neutrophil membrane camouflage, GSH-triggered drug release, PPE activity, and in situ ¹O₂ generation within the nucleus creates a highly selective and effective anticancer agent. The mechanism of action, involving amplified histone H1 translocation, is distinct from traditional antigen-recognition-based therapies, offering a potential solution to address the limitations of current immunotherapies. The induction of ICD further enhances the therapeutic effect by activating the adaptive immune response and mediating an abscopal effect. The in vivo studies confirm the translational potential of FKPN as a promising therapeutic strategy for solid tumors. The observed abscopal effect highlights the potential for systemic anti-tumor immunity elicited by the nanodevice. Further research should investigate different cancer types and explore the possibility of combining FKPN with other therapeutic modalities for enhanced efficacy.

This research successfully developed a novel neutrophil-mimicking nanodevice (FKPN) for precise cancer therapy. FKPN leverages the unique properties of neutrophils and nanomaterials to selectively target and kill cancer cells while minimizing harm to healthy tissues. The in vitro and in vivo data strongly suggest FKPN's potential as a highly effective and safe cancer treatment, especially considering the abscopal effect and the current clinical development of PPE. Future studies should focus on clinical translation and exploring the nanodevice's efficacy across diverse cancer types.

While the study demonstrates promising results, several limitations should be considered. The study primarily focused on breast cancer cell lines and a mouse model. Further research is necessary to confirm FKPN's efficacy and safety in other cancer types and larger animal models. The use of laser irradiation might limit the clinical applicability; non-invasive methods for ¹O₂ generation should be explored. The exact mechanisms of histone H1 translocation and immune activation might require further investigation. The long-term effects of FKPN treatment require further study.