Enzyme-Instructed Peptide Self-Assembly as A Cell Membrane Lichen Activating Macrophage-Mediated Cancer Immunotherapy

Enzyme-Instructed Peptide Self-Assembly as A Cell Membrane Lichen Activating Macrophage-Mediated Cancer Immunotherapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Enzyme-Instructed Peptide Self-Assembly as A Cell Membrane Lichen Activating Macrophage-Mediated Cancer Immunotherapy Yanbin Cai This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3314213/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Targeted immunomodulation for reactivating innate cells, especially macrophages, holds great promise to complement current adaptive immunotherapy. Nevertheless, there is still a lack of high-performance inhibitors for blocking macrophage phagocytosis checkpoints in immune quiescent solid tumors so far. Herein, a peptide-antibody combo-supramolecular in situ assembled CD47 and CD24 bi-target inhibitor (PAC-SABI) is described, which undergoes biomimetic surface propagation like lichens on cancer cell membranes through ligand-receptor binding and enzyme-triggered reactions. Primarily, the PAC-SABIs demonstrate specific avidity for the overexpressed CD24 on the cancer cell surface with anti-CD24 monoclonal antibody (mAb). Subsequently, they exhibit alkaline phosphatase-catalyzed rapid dephosphorylation of phosphopeptides, constructing a three-dimensional nanofiber network and reinstating blockade of CD47 signaling. By concurrent inhibition of CD47 and CD24 signaling, PAC-SABIs stimulate macrophage phagocytosis and initiate T cell antitumor response. Remarkably, compared with anti-CD24 mAb, PAC-SABIs enhance the phagocytic ability of macrophages by 3–4 times in vitro and in vivo while facilitating infiltration of CD8 + T cells into 4T1 tumors. Moreover, combining PAC-SABIs with anti-PD-1 therapy effectively suppressed 4T1 tumor growth in murine models, surmounting other treatment groups with a 60-day survival rate of 57%. The in vivo construction of PAC-SABI-based nanoarchitectonics provides an efficient platform for bridging innate and adaptive immunity to maximize therapeutic potency. Biological sciences/Biotechnology/Biomaterials/Biomedical materials Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy Immunotherapy innate immune checkpoint macrophage phagocytosis peptide self-assembly alkaline phosphatase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Adaptive immune checkpoint (IC) inhibitors targeting programmed cell death protein 1 (PD-1) or its ligand programmed death-ligand 1 (PD-L1) have shown efficacy in treating some cancer types by disrupting inhibitory T cell pathways 1 – 3 . Unfortunately, their effectiveness remains limited against immune quiescent tumors like breast and pancreatic cancers (BCs and PCs), underscoring the importance of innate immune cells and enhanced antigen processing to overcome this challenge 4 – 6 . As highly prevalent non-malignant cells in the tumor microenvironment (TME), macrophages play a crucial innate immune role via phagocytosis, antigen presentation, and inflammatory cytokine production, linking innate and adaptive immunity 5 , 7 . Yet cancer cells can evade macrophage clearance by upregulating anti-phagocytic “don’t eat me” membrane proteins 8 – 11 . Thus, inhibiting these anti-phagocytic signals or receptors represents a promising immunotherapeutic approach that could synergize with cancer cell elimination, activate CD8 + T cells, and initiate an anti-cancer immune response. The CD47-signal regulatory protein alpha (SIRPα) axis, discovered in the late 2000s, was the first identified checkpoint inhibiting cancer phagocytosis through innate immunity 12 – 14 . Despite ongoing clinical trials testing the inhibitors and antibodies targeting this axis for cancer therapy, two primary challenges have hindered successful clinical application 15 . One is the ubiquitous CD47 expression on normal cells (“antigen sink”), especially red blood cells (RBCs) and platelets. High maintenance dosing required to saturate peripheral antigens causes severe toxicity like anemia and thrombocytopenia 16 . The other challenge is the limited treatment response in solid tumors 17 , 18 , likely due to additional immunomodulatory receptors beyond CD47 and the inefficiency of solely blocking anti-phagocytic membrane proteins given the tumor microenvironment (TME) complexity. Consequently, interest has grown in identifying other macrophage-associated immune checkpoints (ICs) to potentially expand innate immunotherapy benefits to more patients 19 . Recently, CD24 was identified as a novel cancer cell “don’t eat me” signal facilitating immune evasion via binding the macrophage sialic acid-binding Ig-like lectin 10 (Siglec-10) 20 . Both the CD24-Siglec10 and CD47-SIRPα pathways enable immunoreceptor tyrosine-based inhibitory motif phosphorylation, inhibiting macrophage phagocytosis 15 , 20 . As innate cancer immunology understanding increases, developing inhibitors to simultaneously block CD47 and CD24 signaling is critical to foster optimal anti-tumor immunity. Current inhibitor designs against membrane protein targets in solid tumors are primarily based on the intrinsic structures and properties of the proteins themselves, without adequately accounting for the native curved topological structure of cell membranes 21 – 23 . Owing to the heightened molecular requisites for concurrent and synergistic engagement of two distinct proteinaceous targets, this phenomenon may greatly impede binding kinetics and attenuate the pharmacological potency of the bi-target inhibitor 24 . By incorporating more information on membrane contexts into rational structure-based design, inhibitors are expected to achieve improved binding affinity and selectivity for membrane protein targets. Recent studies have demonstrated that alkaline phosphatase (ALP)-responsive peptide self-assembly enables interaction with proteins or protein assemblies on cell membranes, making it a crucial tool for influencing cell-cell interactions and deciding the destiny of cells in multicellular systems 25 – 27 . The enzyme-instructed self-assembly (EISA) effectively modulates the phosphorylated precursors, enabling precise control over their distinctive spatiotemporal functions within the tumor microenvironment (TME) while concurrently reducing toxicity in the blood circulatory system 28 , 29 . By incorporating specific protein targeting motifs, these precursors can be directed towards the formation of self-assembly units with well-organized structures at the subcellular level within the cell or pericellular space 30 – 33 . Furthermore, the plasma membrane’s topological structure tends to facilitate the formation of subsequent peptide assemblies through the coordination of ligand binding with target proteins, which is advantageous in achieving maximum inhibitory effects on multiprotein signaling 34 – 36 . Motivated by the aforementioned context, we propose an in situ self-assembly strategy involving functional peptide assemblies to activate the interaction between macrophages and cancer cells. Herein, we developed a peptide-antibody combo-supramolecular in situ assembled CD47 and CD24 bi-target inhibitor (PAC-SABI) to stimulate macrophage phagocytic activity against malignant cells, thus eliciting potent anti-tumor immunity. As illustrated in Scheme 1 , PAC-SABI undergoes biomimetic, ligand-directed surface propagation like lichens on cancer cell membranes, with conformational maturation mediated by target binding and enzyme-responsive morphological transformation to achieve in situ self-assembly and presentation of bioactive motifs. This precision targeting underpins PAC-SABI’s potent inhibition of the “don’t eat me” signal transmission driven by cancerous overproduction of CD47 and CD24. Our rationally designed modular system demonstrates that responsive nanoarchitectonics enables meticulous molecular manipulation to construct versatile peptide-antibody therapeutics with enhanced performance. Looking ahead, this proof-of-concept establishes supramolecular peptide engineering as a facile yet powerful approach to merge multitargeting, programmability, and spatial control within a singular nanoscale platform for more efficacious next-generation immunotherapies. 2. Results 2.1 Molecular Design and Self-Assembly Behavior To test the feasibility of our concepts and investigate the assembly behavior, we synthesized the peptide molecules as depicted in the Scheme 1 A. The multifunctional peptide-based inhibitor employed a modular design. In the molecular structure, the fluorophores nitrobenzoxadiazole (NBD) and cyanine 5.5 (Cy5.5) were incorporated as capping groups at the ends of the peptides, and enabled real-time tracking of molecular assemblies in vitro and in vivo through fluorescence imaging. The typical self-assembled peptide, Lys-Leu-Val-Phe-Phe, with a sequence derived from Alzheimer’s-disease-associated beta-amyloid, was the peptide backbone that accomplishes molecular assembly. The hydrophilic PEG2000 was introduced and implemented to modify the small molecular prodrug assemblies for prolonging circulation in blood and increasing accumulation in the tumor. Moreover, Pep-20 (Ala-Trp-Ser-Ala-Thr-Trp-Ser-Asn-Tyr-Trp-Arg-His) incorporated in our molecular design was identified to specifically bind to both human and murine CD47 for blockage of CD47/SIRPα interaction 37 . The peptide monomers were assembled into micelles in the aqueous solution, which were subsequently connected to the anti-CD24 mAb. The linkage allowed for active targeting of CD24 overexpressing cancer cells, thereby exerting a synergistic macrophage immunomodulation (Scheme 1 B). The tyrosine of Pep-20 enabled the synthesis of phosphorylated analogs (pTyr) to promote in situ rearrangement and growth of molecular assemblies leading to the formation of PAC-SABI nanofibers upon dephosphorylation in the presence of ALP (Scheme 1 C and 1 D). Expanding upon our supramolecular platform, we engineered additional supramolecular assembled CD47 mono-target inhibitor (SAMIs) to validate the feasibility and efficacy of PAC-SABIs directed by ligand-receptor interaction and enzyme catalysis. Solid-phase peptide synthesis and liquid synthesis were employed, and the detailed procedures were described in the Schemes S1 and S2 (Supporting Information). The molecular identification of the intermediates and final products were confirmed in Figure S1 -8 (Supporting Information). The critical assembly concentration (CAC) of the peptide molecules (Pep) and conjugates (Pep-PEG) was measured using a hydrophobic environmental responsive fluorescent probe, 1-phenylnaphthalene-8-sulfonate (ANS). In PBS (pH 7.4), the CACs of Pep and Pep-PEG were determined to be 42.6 and 26.6 10 − 6 M, respectively (Fig. 1 A). The lower CAC of Pep-PEG could be attributed to the presence of hydrophilic PEG chains, which imparted the conjugate with amphiphilic character and enhanced its self-assembly capability in aqueous medium. The fluorescence associated with NBD may potentially serve as a reliable indicator for monitoring the self-assembly of Pep and Pep-PEG. In comparison to concentrations below the CAC, we discerned a noticeable rise in detectable NBD fluorescence at CAC concentrations, which was further amplified under ALP conditions (Fig. 1 B and 1 C). This heightened fluorescence quantum yields implied that the dephosphorylation process mediated by ALP facilitated the formation of self-assembled aggregates of Pep and Pep-PEG, wherein NBD residues were encapsulated within a highly hydrophobic environment. After the PEG modification, the zeta potential of the peptide molecules in PBS (pH 7.4) shifted from − 21.1 ± 1.1 mV (Pep) to -9.1 ± 1.2 mV (Pep-PEG) due to the surface charge screening effect (Fig. 1 D). The increasing conjugation between Pep-PEG and negatively charged anti-CD24 mAb resulted in a gradual decrease in the zeta potential. When the ratio of mAb to Pep-PEG reached 1: 10, the conjugate molecules (PAC-SABIs) exhibited a potential of -11.7 ± 0.8 mV, and remained stable despite the escalation of Pep-PEG (Table S1 , Supporting Information). Therefore, the ratio of 1: 10 was considered to be the saturation point for the following study. In order to accurately monitor the secondary structure transformation of the peptide molecule assemblies, we conducted circular dichroism (CD) spectroscopy and Fourier transform infrared spectroscopy (FTIR) analysis. In the presence of ALP, the CD spectrum of Pep exhibited a prominent negative peak at 217 nm and a strong positive peak near 195 nm, the characteristic features of a spectrum generated by β-sheet (Fig. 1 E and 1 F). The PAC-SABIs exposed to ALP were found to undergo conformational transformation within the assembly environment, leading to an elevation in the proportion of α-helical content, as evidenced by the emergence of positive peaks at 193nm and negative peaks near 222nm (Fig. 1 E and 1 F). The interaction of ALP with Pep-PEG or PAC-SABIs was further corroborated through characterization results of FTIR. The spectra presented a maximum absorption at 1654 cm − 1 indicating formation of α- helix structure (1650–1658 cm − 1 ) in the amide-Ⅰ band (Fig. 1 G). Then, high-performance liquid chromatograph (HPLC) analysis was carried out to monitor the kinetics of EISA. Although the PEG and mAb modification might produce steric hindrance for molecular recognition and ALP reaction, Pep, Pep-PEG, and PAC-SABIs all displayed high conversion efficiency, with a conversion rate of approximately 90% at 60 min (Fig. 1 H). In addition, we observed the morphology transformation of Pep and PAC-SABIs by transmission electron microscopy (TEM) in a time-dependent manner. Pep exhibited a quasi-circular aggregate, wherein these peptide assemblies subsequently aggregated and manifested a spherical structure after a 10-min incubation period with ALP. Then, the advent of a short fiber structure was observed at 30 min. By the 60-minute mark, the majority of the spherical structure was dissipated, giving way to a substantial presence of fibrous structures. At the 120-minute interval, the entire assembly system was predominantly occupied by characteristic nanofibers with a diameter of 6.2 ± 0.7 nm (Fig. 1 I). However, the PAC-SABIs were observed as irregular aggregates. During the initial 30 min of enzyme catalyzed reactions, these aggregates rapidly coalesced into entangled nanofibers. Subsequently, these nanofibers underwent further extension and bundling, resulting in the formation of a three-dimensional (3D) network after 60 min. At 120 min, nearly all precursors transformed into nanofibers with a diameter of 13.2 ± 2.9 nm (Fig. 1 I). These results demonstrated that ALP mediated enzymatic reactions acted as a trigger for initiating the rearrangement of peptide molecules, promoting the rapid formation of ordered superstructures by influencing all the microscopic events in the assembly process. 2.2 Expression of Target Innate ICs in BC and PC In order to assess the impact of CD47 and CD24 signaling on the regulation of macrophage-mediated immune response against cancer, we investigated the expression of CD47 and CD24 in different tumor types. Analysis of RNA sequencing data obtained from The Cancer Genome Atlas (TCGA) revealed a significant overexpression of CD47 and CD24 in nearly all examined tumors (Fig. 2 A). Furthermore, when compared to the well-established adaptive immune checkpoint PD-L1, CD47 and CD24 exhibited a consistent up-regulation in BC and PC (Fig. 2 A). The levels of CD47 and CD24 expression in PC and BC were notably elevated compared to their corresponding normal tissues, respectively, while no significant variation was detected in PD-L1 expression (Fig. 2 B-D). Stratification of patients based on their CD47 and CD24 expression levels demonstrated a significant correlation with improved overall survival in individuals with lower CD47 expression among PC patients and lower CD24 expression among BC patients (Fig. 2 E and 2 F). Single-cell RNA sequencing conducted on a primary BC sample, focusing on examining the expression patterns of CD47 and CD24 at the individual cancer cell level, revealed a prominent expression of CD47 and CD24 in cancer cells, whereas other cell clusters exhibited relatively low expression levels (Fig. 2 G and 2 H). These results suggested that CD47 and CD24 may serve as potential markers specific to cancer cells. Moreover, immunofluorescence (IF) analysis of two cancer nodule sections obtained from patient 1 with BC indicated the presence of CD47 and CD24, which had a broad distribution within the cytoplasm and membrane of malignant cells (Fig. 2 I and 2 J). IF results acquired from consecutive sections of BC tissue in patient 2 showed robust CD47 and CD24 protein expression by cancer cells, indicating redundancy of these two membrane-bound “don’t-eat-me” signals (Fig. 2 K and 2 L). As illustrated in the Fig. 2 M, CD47 and CD24 function as innate ICs, diminishing the phagocytic action of macrophages on cancer cells in a synergistic manner. By utilizing the interface receptor ligand interaction and ALP catalysis, PAC-SABIs can precisely identify the cancer cell membrane and establish stable assemblies. This process efficiently inhibits CD47 and CD24 signals, regulating TAM phagocytic activity and, eventually, boosting anti-tumor immune responses. 2.3 In Situ Self-assembly on Cancer Cell Membranes Having determined the effective assembly of PAC-SABIs under static external conditions, we proceeded to investigate the self-assembly behavior of PAC-SABIs within BC and PC cellular environments using the fluorescence of NBD. Firstly, the widespread expression of ALP in tissue sections of BC and PC cell lines (4T1 and PAN02), as well as on the surface of clinical samples of BC and PC, was verified using the BCIP/NBT color development kit (Figure S9, Supporting Information). Then, the 4T1 and PAN02 cells were co-incubated with NBD-labeled PAC-SABIs at 37 ℃, and changes in NBD fluorescence were directly monitored using confocal laser scanning microscopy (CLSM) imaging at different time points (Fig. 3A and 3B). As anticipated, the treated 4T1 and PAN02 cells exhibited a time-dependent augmentation in NBD fluorescence emission on the cellular surface. Following a 30-min co-incubation period, the fluorescence appeared as a diffuse signal surrounding the cancer cells as PAC-SABIs bound to the membrane’s outer surface. Then, a prompt aggregation of fluorescent clusters on the membrane was observed at the 60-min mark. By the 120-min interval, fluorescence became uniform and encompassed the cell surface, signifying the in situ self-assembly of PAC-SABIs on cancer cell membrane (Fig. 3A and 3B). This observation is further supported by the findings from differential interference contrast (DIC) and fluorescence microscopy imaging (Fig. 3C). As shown in Fig. 3D-F, the green fluorescence signal from NBD exhibited a strong co-localization with the red fluorescence signal from the Dil dye after co-incubation for 120 min, suggesting that a majority of PAC-SABI molecules were assembled and localized on the 4T1 cell membrane. Under identical conditions, 4T1 cells treated with SAMIs exhibited robust intracellular green fluorescence, reflecting the internalization of SAMIs into the cells, a phenomenon commonly observed when other nanostructures are incubated with live cells (Figure S10, Supporting Information). Blocking experiments were conducted with anti-CD24 antibodies, and the surface fluorescence signals of pretreated 4T1 cells were significantly reduced during dynamic incubation with PAC-SABI, indicating that the specificity of the interaction between CD24 and PAC-SABI promotes the membrane in situ self-assembly process (Figure S11, Supporting Information). Additionally, as shown in Figure S12 (Supporting Information), transmission electron microscopy (TEM) revealed the morphologies of PAC-SABIs assembled on the top surface of cell membranes. We also observed the formation of superstructure networks over time on the 4T1 and PAN02 cell membranes treated with PAC-SABIs through scanning electron microscopy (SEM). During the co-incubation process of 30 to 120 min, PAC-SABIs, like the proliferation process of lichens, first attached to the cell’s adherent edge, then migrated along the cell surface, and finally formed a nanoscale fiber network assembly covering the membrane (Fig. 3G and 3H). To further emulate the TME and 3D spatial architecture of tumors in vivo, we constructed 4T1 spheroid models. After incubating Cy5.5-labeled PAC-SABIs with the 3D cellular spheres for 120 min, fluorescence monitoring by CLSM revealed Cy5.5 envelopment of the outermost Calcein-AM stained cell layer (Figs. 3I and 3J). Scanning multiple confocal planes along the z-axis showed Cy5.5 fluorescent binding on the surfaces of sequential cross-sections, corroborating PAC-SABI assembly on the membrane exteriors. Consistent with 2D culture results, pretreatment with anti-CD24 mAb markedly reduced external fluorescence of the layered spheroids (Fig. 3J). These results demonstrated the impressive capacity of PAC-SABIs for in situ self-assembly within dynamic physiological milieus, potentially enabling productive immune checkpoint binding and multiprotein signal inhibition. Figure 3. In situ self-assembly of PAC-SABIs on BC and PC cell membranes. A) Time-dependent CLSM images of 4T1 cells treated with NBD-labeled PAC-SABIs. Scale bar: 20 µm. B) Time-dependent CLSM images of PAN02 cells treated with NBD-labeled PAC-SABIs. Scale bar: 20 µm. C) Merged DIC and fluorescent images of 4T1 cell treated with NBD-labeled PAC-SABIs for 120 min. Scale bar: 20 µm. D) CLSM images of 4T1 cell treated with Dil dye (red) and NBD-labeled PAC-SABIs for 120 min. Scale bar: 20 µm. E, F) The fluorescence distribution of NBD-labeled PAC-SABIs on 4T1 cell. G) Time-dependent SEM images of 4T1 cells treated with PAC-SABIs. The red arrows point to the PAC-SABI nanofibers on cell membrane. Scale bar: 1 µm. H) Time-dependent SEM images of PAN02 cells treated with PAC-SABIs. The red arrows point to the PAC-SABI nanofibers on cell membrane. Scale bar: 1 µm. I) CLSM images of 3D 4T1 spheroids treated with Cy5.5-labeled PAC-SABIs, Calcein AM, and Hoechst 33342. Scale bar: 50 µm. J) CLSM images of 3D 4T1 spheroids along the z-axis position. Scale bar: 50 µm. Due to the ability of PAC-SABI to form nanofiber networks on the surface of cancer cells, we conducted a series of in vitro experiments including wound healing, cell invasion, and clone formation assays to verify whether PAC-SABIs affect the physiological activity of BC and PC cells. The results of the wound healing assay on the inhibitory ability of SAMI and PAC-SABI on cancer cell movement are shown in Figure S13A and S14A (Supporting Information). The control group, consisting of highly metastatic 4T1 and PAN02 cells, exhibited robust migration and healing capabilities, as evidenced by a wound healing rate of 100% within 24 h post-scratching. However, treatment with PAC-SABI significantly decreased the wound healing rates of 4T1 and PAN02 cells to 45.9% and 20.1%, respectively, which were notably lower than the rates of 72.7% and 69.8% observed in the SAMI treatment group (Figure S13D and S14D, Supporting Information). The cell invasion assay was conducted by using Transwell chambers with pre-coating Matrigel, which aimed to simulate allowing the cancer cells to degrade the extracellular matrix barrier (ECM) and migrate through the vessels (Figure S13B and S14B, Supporting Information). Based on the findings from the control group, it can be concluded that the highly metastatic 4T1 and PAN02 cells possess the ability to break through and dissolve the ECM barrier, facilitating their migration from the primary tumor site and subsequent establishment of new metastatic sites. The PAC-SABIs yielded a substantial inhibitory effect on the invasion of 4T1 and PAN02 cells, with the inhibition rates of 83.4% and 91.1%, respectively (Figure S13E and S14E, Supporting Information). Furthermore, we conducted a plate colony formation assay to examine the impact of PAC-SABI on the proliferative capacity of tumorigenic 4T1 and PAN02 cells (Figure S13C and S14C, Supporting Information). The results showed that the number of tumorigenic cells significantly decreased, indicating that PAC-SABI can significantly inhibit the colony formation ability of 4T1 and PAN02 cells, with inhibition rates of 79.1% and 80.3%, respectively, which were significantly superior to the inhibitory effect of SAMI (Figure S13F and S14F, Supporting Information). 2.4 Promotion of Phagocytic Clearance of Cancer Cells via PAC-SABIs Treatment In Vitro To evaluate the phagocytic elimination of cancer cells by macrophages, 4T1 and PAN02 cells labeled with the pH-sensitive dye pHrodo Red were co-cultured with RAW264.7 cells pre-exposed to tumor conditioned media (TCM). Over 2 h, PAC-SABI-treated 4T1 and PAN02 cells exhibited considerably enhanced susceptibility to engulfment and degradation within acidic phagolysosomes compared to IgG controls (Figs. 4 A and 4 B; Figure S15, Supporting Information). Investigating the therapeutic potential of these findings, we assessed whether PAC-SABIs could amplify the phagocytosis of cancer cells by diverse macrophage populations beyond direct anti-CD24 mAb or SAMI blockade. 3D reconstructions of confocal image stacks showed increased vulnerability of 4T1-pHRodo-Red + cells to phagolysosomal uptake when treated with anti-CD24 mAb or SAMIs. Dual CD47 and CD24 inhibition by PAC-SABIs further enhanced engulfment of 4T1 cells by TCM-exposed bone marrow-derived macrophages (BMDMs) and RAW264.7 cells (Fig. 4 C). Fluorescence activated cell sorting (FACS) quantitation revealed robustly amplified phagocytosis with PAC-SABI addition, with approximately 4- and 2-fold greater uptake by BMDMs and RAW264.7 cells respectively compared to anti-CD24 mAb alone (Figs. 4 D-G; Figure S16, Supporting Information). In agreement with the results in murine macrophages, human THP-1 monocyte-derived macrophages stimulated by TCM exhibited enhanced clearance of human breast cancer MDA-MB-231 cells treated with PAC-SABIs compared to other groups (Figure S17, Supporting Information). Primary human donor-derived macrophages (HDDMs) were also generated by the methodology previously outlined by Barkal et al 20 . to assess PAC-SABI efficacy (Fig. 5 A). HDDMs were subsequently conditioned with tumor-derived media from MDA-MB-231 to generate TAMs. Immunofluorescence (IF) and FACS evidenced upregulation of the M2 marker CD206 in TAMs versus control HDDMs (Figs. 5 B and 5 C). 2D and 3D confocal microscopy of living cells provided further proof that PAC-SABIs effectively inhibited “don't eat me” signals, highlighting their potential as a valuable innate anti-tumor immunotherapeutic tool (Figs. 5 D-F). 2.5 Biodistribution and Tumor Targeting In Vivo To monitor and quantify the biodistribution up to 120 h after intravenous injection of Cy5.5 labeled PAC-SABI, the in vivo imaging system (IVIS) spectroscopy was employed, and we constructed subcutaneous xenograft models of BC and PC in mice to mitigate systematic errors and individual variances. As shown in Figs. 6 A and 6 C, there were notable differences in the distribution of fluorescence among free Cy5.5, SAMI, and PAC-SABI in mice. Cy5.5, serving as the representative small molecule probe, exhibits swift distribution and elimination throughout the mice’s bodies, without displaying any discernible specific targeting effects on BC and PC tissues. Due to the absence of active targeting and ALP triggered self-assembly properties, the SAMIs exhibited limited accumulation of Cy5.5 fluorescence signal in the tumor regions of BC and PC. However, when PAC-SABIs were administered intravenously in subcutaneous tumor mice, PAC-SABI conjugated with anti-CD24 mAb demonstrated enhanced fluorescence signal in the tumor regions of BC and PC, reaching its maximum intensity at the 48-h time point. The PAC-SABIs, utilizing the EISA and PEG effects, effectively improved its biodistribution by accumulating more signals in the tumor regions, thereby prolonging its retention time in the tumor for up to 120 h. Simultaneously, the distribution of signals in non-tumor tissues was reduced, and the elimination time was shortened (Figs. 6 A and 6 C). The area under the curve (AUC), which is a crucial parameter for the enrichment of PAC-SABI and SAMI in tumor tissue, was determined through quantitative analysis of fluorescence signals. Following the quantitative calculation of fluorescence intensity in the tumor region, excluding background signal, a time-dependent curve was plotted for PAC-SABI and SAMI. In the mouse models of BC and PC tumors, the AUC (0-120 h) of PAC-SABI was observed to be approximately 3 times greater than that of SAMI. Furthermore, our investigation revealed that the elimination of PAC-SABI molecules in BC and PC exhibited a remarkably slow rate, with only a modest decrease of 16.2% and 25.6% between 48 and 120 h, respectively (Figs. 6 B and 6 D). After the intravenous administration of free Cy5.5, SAMIs, and PAC-SABIs, the mice were euthanized using carbon dioxide inhalation at the 48-h mark, and the major organs (heart, liver, spleen, lung, and kidney) as well as tumors were harvested for ex vivo imaging (Fig. 6 E and 6 F). Similar to the in vivo results, free Cy5.5 cleared rapidly from the body with negligible accumulation in the tumor and major organs. IVIS imaging showed notable differences in tissue biodistribution between SAMI and PAC-SABI. The distribution of PAC-SABI exhibited distinct selectivity within tumor tissues of BC and PC, with partial retention observed in metabolic organs such as the liver and kidney. By comparison, no significant variance was observed in the biological distribution of SAMI within the lungs, kidneys, and tumors, with the majority of molecules predominantly localized in the liver. The discernible dissimilarity between these two molecules can be attributed to the presence of targeted ligands and EISA, which augment the precise recognition of PAC-SABI molecules towards cancer cells and facilitate its proficient molecular assembly within tumors, while SAMI accumulated non-specifically in the liver during the metabolic process. To visually observe the distribution of the PAC-SABIs in the tumor tissue of BC and PC, we analyzed the Cy5.5 fluorescence in the frozen tumor sections. Interestingly, a notable accumulation of Cy5.5 fluorescence was observed within the cancer cell regions of the 4T1 tumor, whereas the fluorescence intensity was diminished in the fibroblast-rich areas (Fig. 6 G). This phenomenon was similarly observed within the interior of PAN02 tumors, where PAC-SABI exhibited a concentration within the cancer cell area rather than the paracancerous or fibroid regions (Fig. 6 G). These results indicated that the rapid formation of the PAC-SABI superstructure network leads to its effective accumulation and retention in tumors, which may further provide persistent blockade of innate immune checkpoints in cancer cells, thereby inducing effective and sustained macrophage-mediated immune responses. 2.6 Antitumor Efficacy and Immune Response In Vivo Generally, the toxicity of inhibitors, especially the blood toxicity associated with CD47 blockade therapy, is a key criterion affecting clinical transformation. To evaluate the systemic toxicity of PAC-SABIs, complete blood count was obtained from healthy BALB/c mice on the 1st, 7th, 14th, and 21st days after 4 times of intravenous injection of PAC-SABIs every other day. The main parameters evaluated included red blood cell, hemoglobin, hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, mean platelet volume and platelets. All of these parameters were shown to be within reference ranges for healthy BALB/c mice (Figure S18A, Supporting Information). Subsequently, following approximately one month of treatment, we collected the main organs of mice in each group, including heart, liver, spleen, lung, and kidney, and sliced them for H&E staining. No notable histological alterations or pathological lesions were observed (Figure S18B, Supporting Information). In summary, PAC-SABIs exhibited high biocompatibility characteristics in mice without significant systemic toxicity, supporting its potential application in clinical practice. Encouraged by the aforementioned experimental results, we hypothesized that PAC-SABI therapy has the potential to instruct macrophages in eliciting proficient in vivo anti-tumor phagocytic reactions, consequently leading to the inhibition of tumor growth. To mimic the natural progression of human BC and PC, the orthotopic BC model utilizing luciferase-labeled 4T1 (4T1-Luc) tumor-bearing BALB/c mice and the orthotopic PC model utilizing luciferase-labeled PAN02 (PAN02-Luc) tumor-bearing C57BL/6 mice were constructed for in vivo therapeutic efficacy evaluation. According to the depicted illustration (Fig. 7 A), after the establishment of in situ BC and PC in mice, control IgG, anti-CD24, SAMI and PAC-SABI were administered every other day for a total of 4 doses. The evaluation of tumor regression/progression was conducted through the periodic monitoring of tumor bioluminescence (BLI) at 7-day intervals. Over time, on the 22nd day post-treatment, the PAC-SABI group exhibited significantly reduced tumor BLI signal intensities in comparison to the other groups, suggesting a notable deceleration in tumor growth among the BC and PC mice subjected to PAC-SABI treatment (Fig. 7 B, 7 C, 7 F, and 7 G). Conversely, mice treated with Control IgG and anti-CD24 mAb exhibited limited therapeutic efficacy, as the majority of BC and PC mice succumbed within 40 and 50 days, respectively (Fig. 7 D and 7 H). The overall survival rate of BC mice treated with PAC-SABI on the 50th day and that of PC mice on the 60th day were 60% and 40%, respectively, showing a significant survival benefit of PAC-SABIs (Fig. 7 D and 7 H). In order to examine the regulatory effects of PAC-SABIs in vivo, the changes that occurred in the 4T1 TME were analyzed after intravenous administration. Hematoxylin and eosin (H&E) staining of xenograft sections in both the IgG control and anti-CD24 mAb groups revealed that the cancer cells were densely arranged in bands and clusters, with disordered arrangement (Fig. 8 A). In the PAC-SABI group, a notable presence of inflammatory cells and tissue necrosis was observed within the tumor interstitium, showing a greater extent of inflammatory cell infiltration and tissue necrosis compared to the SAMI group (Fig. 8 A). Then, IF staining was conducted on the typical immunosuppressive 4T1 tumor tissue. In the PAC-SABI group, we observed a noteworthy manifestation of macrophages engaging in phagocytic and cytotoxic activities against cancer cells, characterized by extensive macrophage infiltration and internalization of cancer cell components (Fig. 8 A). This could potentially be attributed to the heightened phagocytic capacity resulting from the suppression of combined innate ICs. The nuclear density within this particular region was lower than that in adjacent regions, indicating a reduction in cancer cell density. Conversely, this tumoricidal phenomenon was less frequently observed in the remaining treatment groups. In addition, the IF results showed that CD8 + T cells in the IgG control group were mainly distributed at the edge of the tumor, while the infiltration of CD8 + T cells within the tumor tissue in the PAC-SABI group was significantly enhanced (Fig. 8 A). FACS analyses were performed on GFP + 4T1 tumors to further explore how PAC-SABI inhibition altered the tumor immune microenvironment. Consistent with the IF results, we found that the highest levels of in vivo phagocytosis by infiltrating TAMs were achieved in the PAC-SABI group, which was 3-fold higher than that in the IgG control group (Fig. 8 B and 8 C). As shown in Fig. 8 D and 8 E, anti-CD24 mAb, SAMI, and PAC-SABI treatments all potentiated the activation state of TAMs towards a pro-inflammatory antitumoral phenotype, indicating another potential mechanism of innate IC blockade strategy. Moreover, FACS analysis of T cells in tumor tissues treated with PAC-SABIs demonstrated a significant increase in the percentage of tumor-infiltrating CD8 + and CD4 + T cells (Fig. 8 B and 8 F). We speculated that the phagocytosis of activated macrophages might increase the cross-presentation of tumor antigen to T cells, which could have enhanced the effectiveness of adaptive anti-tumor immune response. As the main immunosuppressive cells, the number of FOXP3 regulatory T cells (Tregs) within the tumor following PAC-SABI treatment was found to be significantly decreased (Fig. 8 B and 8 G). This reduction may have also potentially contributed to the transition of the macrophage population from a suppressive M2 phenotype to an M1 phenotype. Previous studies have shown that reprogramming the TME can impact the cytokine secretion of immune cells, resulting in heightened levels of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and interleukin-6 (IL-6), and reduced levels of the anti-inflammatory cytokine transforming growth factor beta (TGF-β). As shown in the Fig. 8 H-K, compared with the IgG control group, the level of TGF-β in the PAC-SABI group was significantly decreased, while the levels of TNF-α, IFN-γ, and IL-6 were increased by 3.3-, 10.6-, and 4.3-fold, respectively. These findings strongly indicated that PAC-SABIs have the ability to counteract the anti-inflammatory properties of the tumor immune microenvironment. 2.8 Inhibition on BC and PC Liver Metastasis In Vivo To investigate if the ability of PAC-SABIs to inhibit cancer cell migration and invasion in vitro translates to decreased metastasis in vivo, we used an experimental liver metastasis model of BC and PC. 4T1-luc and PAN02-luc cells were injected into the spleens of BALB/c and C57BL/6 mice, respectively, which enabled monitoring and quantification of metastasized cancer cells in the liver with BLI. Starting from the day before injection of cancer cells into the spleen, mice were given intravenous injections of IgG control antibody, anti-CD24 mAb, SAMIs, and PAC-SABIs every other day for a total of 4 doses. The BLI intensity started to increase soon after injection of cancer cells in the IgG control and anti-CD24 mAb groups, and patchy metastatic lesions formed in the liver region (Figure S19A and S19E, Supporting Information). However, a significant decrease in BLI intensity was observed in the PAC-SABI group compared to the IgG control group after two weeks of injection (Figure S19B and S19F, Supporting Information). Then, the mice were euthanized, and reductions in the number and size of liver metastases were confirmed macroscopically in the PAC-SABI group (Figure S19C and S19G, Supporting Information). Additionally, H&E staining of the liver further demonstrated that PAC-SABIs facilitated construction of nanofibrous barriers on the cancer cell membrane, inhibiting early metastasis and effectively stopping the growth of previously formed metastasis in mouse liver metastasis models (Figure S19D and S19H, Supporting Information). 2.9 Enhanced Anti-PD-1 Therapy After Phagocytosis Modulation Based on the aforementioned in vivo experimental findings, regulating the phagocytosis of cancer cells by macrophages exhibited great potential in adaptive anti-tumor immune induction. Therefore, it is postulated that the combination of PAC-SABI and PD-1 pathway blockade could potentially result in an augmented anti-tumor immune response in vivo, consequently yielding optimal therapeutic outcomes. To test this, we inoculated 2 × 10 5 GFP + 4T1 cells into the third mammary fat pad of female BALB/c mice. Mice were then administered intravenously with PAC-SABIs or PBS every 2 days for a total of 4 doses, followed by anti-PD-1 or IgG control intraperitoneal injection on days 7, 10, 13, and 16. The 4T1 tumor growth and survival rate were measured over time to assess the immunotherapeutic efficacy of different treatment regimens (Fig. 9 A). The untreated group and the anti-PD-1 group showed rapid tumor growth and poor median survival. Compared with these two groups, the PAC-SABI group showed modest tumor growth inhibition and improved survival (Fig. 9 B-D). Remarkably, anti-PD-1 therapy after PAC-SABI activation of macrophages produced the greatest tumor suppression effect and achieved a 60-day optimal survival rate of 57% (Fig. 9 D). At the experimental end points, the lungs were dissected for analysis. Histological examination of representative lung sections using H&E staining showed a lower incidence of pulmonary metastatic nodules in the SAMI group compared to untreated and anti-PD-1 groups (Fig. 9 E). More importantly, the combination of PAC-SABI and anti-PD-1 therapy effectively impeded lung metastasis development, with few observable nodules. Given the promising results, FACS was used to investigate macrophage phagocytic activity towards 4T1 cells. The combination treatment led to a substantial increase in phagocytosing CD45 + CD11b + F4/80 + macrophages compared to other groups (Fig. 9 F). Furthermore, CD8 + T cell infiltration was directly observed to be improved with combination therapy through IF staining (Fig. 9 G). These findings suggested that the combination elicited robust innate and adaptive immune responses, leading to tumor growth inhibition and metastasis rejection. 3. Discussion In conclusion, we have crafted a peptide-antibody conjugate that functions as a supramolecular, in situ assembled CD47 and CD24 bi-target inhibitor, explicitly devised to modulate the phagocytic activity of macrophages and enhance the immune response against cancer. The modularly constructed PAC-SABI not only accumulated effectively in BC and PC tissues but also rapidly self-organized into well-structured interface nanofiber networks, a process facilitated by both an active targeting mechanism and EISA-induced conformational transitions. Governed by rigorous spatiotemporal oversight, PAC-SABI demonstrates prolonged presence within the TME, thereby attenuating the hematological repercussions commonly linked to CD47-focused therapies. In its distinctive lichen-mimetic growth phase on the membrane facade, PAC-SABI obstructs the collaborative engagement of CD47 and CD24 with SIRPα and Siglec10. This strategic intervention curtails the broad dissemination of the “don’t eat me” signal emanating from cancer cells. When introduced in vivo, PAC-SABI acts as an intermediary conduit, merging the innate and adaptive immune responses. In synergy with anti-PD-1 regimens, it markedly inhibits tumor proliferation. The architectural blueprint of PAC-SABI serves as a harbinger, potentially guiding the schematic outlines for multifaceted peptide therapeutics. These novel designs aspire to manipulate membrane proteins via in situ self-assembly methodologies, striving for immunotherapeutic modalities of heightened safety and potency. 4. Methods 4.1 Synthesis and Characterization of PAC-SABIs: The intermediates including Pep and Pep-PEG were acquired by solid phase peptide synthesis, and PAC-SABIs were obtained by liquid phase synthesis. The detailed synthesis procedures and mass spectrum analysis are presented in the Supporting Information. 4.2 Fluorescence Assays: The CACs of Pep and Pep-PEG were determined using the ANS fluorescence assay. ANS was dissolved in dimethylformamide (DMF) to a concentration of 1 mM. Subsequently, 1 µL of ANS was added to 100 µL of varying concentrations of Pep and Pep-PEG. The resulting solution was transferred to a quartz cuvette and analyzed using a Perkin Elmer LS-55 fluorescence spectrophotometer (Perkin Elmer, USA). The CAC was determined from the matched curve, which was obtained by the ANS fluorescent intensities at 475 nm plotted against different concentrations of Pep and Pep-PEG. The self-assembly process of Pep and Pep-PEG was monitored using NBD fluorescence. The fluorescence intensities of NBD-labeled Pep and Pep-PEG were measured at CAC and half of the CAC concentrations via a fluorescence spectrometer (excitation wavelength: 467 nm). To evaluate the ALP-triggered dephosphorylation and in situ self-assembly process, the NBD fluorescence intensity was detected after incubating Pep and Pep-PEG at CAC concentration with ALP (1 U / mL) at 37 °C for 1 h. 4.3 CD Spectroscopy: All the CD spectra analyses were recorded on a CD spectropolarimeter (Applied Photophysics Ltd, UK) in quartz cuvettes with an optical path length of 0.1 cm. Data were collected from 190 to 260 nm with a scanning speed of 100 nm/min at room temperature. 4.4 FTIR Spectroscopy: All the FTIR analyses were carried out by a Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific, USA). A 100 µL solution of different formulations was spun down at 10000 g for 1 h, and the pellet was blown dry with nitrogen. The dry samples were mixed with KBr and pressed into pellets for further analysis. 4.5 TEM Characterization: Pep and PAC-SABI solutions (100 μM) with or without ALP (1 U / mL) incubation were prepared and placed at room temperature for morphology observation at different time points. Copper grids with a porous carbon mesh were used for sample preparation. 10 µL of the Pep and PAC-SABI solution was placed onto a copper grid with a carbon membrane and left for 3 min, followed by removal of excess solutions with filter papers. Then, a small drop of uranyl acetate solution (2% weight/volume in water) was added to the copper grid for negative staining of samples, and the grid was blotted with filter paper after 1 minute. Finally, the samples were left on filter paper overnight to facilitate further analysis. 4.6 Cell Culture, Animal studies and human specimens: Murine BC 4T1 cell line, murine PC PAN02 cell line, human BC MDA-MB-231 cell line, murine macrophage cell line RAW264.7, and human macrophage cell line THP-1 were purchased from the American Type Culture Collection (ATCC) and cultured according to the supplier’s recommendations, supplemented with 10% fetal bovine serum (FBS) and antibiotics. The cell culture supernatant of BC cell lines 4T1 and MDA-M-231 was collected as TCM, and was used to induce the transformation of RAW264.7 macrophages and phorbol myristate acetate (PMA; 100 ng/ml)-treated THP-1 macrophages into TAMs, respectively. C57BL/6 mice (male, 6 weeks old) were purchased from BesTest Bio-Tech Co., Ltd (Zhuhai, China), and BALB/c mice (female, 6 weeks old) were purchased from Guangdong Nanmo Biotechnology Co., Ltd (Zhongshan, China). All animal experiments were carried out in compliance with NIH guidelines and all animal-experimental protocol were approved by the Animal Experimentation Ethics Committee of Zhujiang Hospital of Southern Medical University (LAEC-2021-040 and LAEC-2022-073). After obtaining approval from the institutional review board of Nanfang Hospital and Guangdong Provincial People’s Hospital of Southern Medical University (NFEC-2023-309, KY2023-179-01), human peripheral blood, BC and PC specimens were obtained, and the appropriate informed consent was obtained for all sample donors. 4.7 BMDMs and HDDMs Generation and Stimulation: BMDM isolation was performed according to a previously published protocol with minor modification. Briefly, after sacrificing C57BL/6 mice and disinfecting the skin with 75% alcohol, the hind legs were cut off and placed in a sterile petri dish containing sterile and ice-cold PBS. The bone marrow was flushed with PBS using a syringe with a 25-gauge needle. The supernatant bone marrow cells were collected and then washed with PBS and resuspended with complete conditioned media for BMDM differentiation (100 mL complete medium consisted of 74 mL Dulbecco’s modified Eagle’s medium (DMEM) + 15 mL macrophage colony-stimulating factor (M-CSF, 25 ng/mL) + 10 mL FBS + 1 mL penicillin-streptomycin solution (PS)), seeded on tissue culture plates, and incubated at 37 °C with 5% CO 2 . Primary HDDMs were generated from venous blood of healthy volunteers, diluted with 2 × PBS (pH 7.4) and separated with Ficoll density gradient as described previously. Monocytes were then differentiated into macrophages by culture in Iscove’s modified Dulbecco’s medium (IMDM) + 10% AB human serum (Life Technologies) for 7 to 10 days. To stimulate macrophages with TCM, we cultured BMDMs and HDDMs with complete medium containing 50% TCM from 4T1 and MDA-MB-231 cells, respectively. 4.8 Characterization of the Self-Assembly Process of PAC-SABI in 2D and 3D Cell Culture Environments: 4T1 or PAN02 cells were inoculated and incubated overnight at a density of 1 × 10 5 cells / dish in laser confocal petri dishes. NBD-labeled PAC-SABIs (100 μM) were co-incubated with 4T1 or PAN02 cells for 30, 60, and 120 min, followed by washing with PBS three times. Then, the NBD fluorescence at different time points was observed by CLSM imaging. To reveal the self-assembly process of PAC-SABI on cancer cell membranes, 4T1 (1 × 10 5 cells/dish) cells were cultured with NBD-labeled PAC-SABI (100 μM) for 120 min. After 3 rounds of washing with PBS, the membrane dye CellTracker CM-Dil (1 mM; Invitrogen, USA) was added and incubated for 5 min at 37 °C, and then for an additional 15 min at 4 °C. CLSM imaging was used to detect the spatial distribution of NBD and Dil fluorescence. 3D spheroids of 4T1 cells were constructed according to the previously reported method. Cy5.5-labeled PAC-SABI (100 μM) was co-incubated with 4T1 3D spheroids for 90 min, followed by washing with PBS three times. Then, the outermost cells and nuclei of the spheroids were stained with Calcein-AM and Hoechst 33342, respectively. Finally, CLSM imaging was used to detect spatial distributions of fluorescence signals with Cy5.5, FITC and DAPI channels in spheroids and different focal plane along the z axis. 4.9 Detection of Cell Surface Distribution Using Bio-TEM: The 4T1 and PAN02 cells were seeded on cell culture dishes with a diameter of 10 mm at a density of 1 × 10 5 cells / dish and cultured overnight, followed by replacement with fresh culture medium containing PAC-SABIs at a concentration of 100 μM for 120 min. Then, the PAC-SABI-containing medium was removed and the cells were washed three times with PBS. The cells attached to the bottom of the dishes were gently harvested using a cell scraper and collected in a centrifuge tube. After centrifugation at 1500–3000 rpm for 5 min, the supernatant was carefully discarded, and then a 4 °C precooled fixative was slowly added along the tube wall and then placed in a 4 °C refrigerator overnight. The samples were then fixed with a 1% osmium acid solution for 1 h followed by dehydration using a gradient concentration of ethanol. The samples were embedded and sliced with a LEICA EM UC7 ultra-thin microtome to obtain 70–90 nm slices. The sections were stained with lead citrate solution, uranyl acetate, and 50% ethanol saturated solution for 5-10 min, respectively, and then observed under TEM imaging. 4.10 Detection of Cell Surface Distribution Using SEM: 4T1 and PAN02 cells were seeded into 12-well plates containing cover glass slips at a density of 1 × 10 5 cells / well and cultured overnight. PAC-SABIs were added into each well at the final concentration of 100 μM and cultured for 120 min. Then, PAC-SABI-treated 4T1 and PAN02 cells were dehydrated for the time course of 24 h, processing from 30% to 100% ethanol. The samples were then sputter coated with gold (CRC-150 Sputter Coater, USA) and imaged using a Zeiss Ultra 55 SEM (Carl Zeiss, Germany). 4.11 Phagocytosis Assay Using Flow Cytometry: The in vitro phagocytosis assays described in this study were performed by co-culture GFP + 4T1 cells and macrophages at a ratio of 100,000 target cells to 50,000 macrophages for 120 min in a humidified, 5% CO 2 incubator at 37 °C in ultra-low-attachment 96-well U-bottom plates (Corning, USA) in serum-free IMDM. 4T1 cells with endogenous fluorescence were harvested from plates using TrypLE Express (Life Technologies, Poland) and treated with PAC-SABIs for 120 min prior to co-culture. After co-culture, phagocytosis assays were stopped by placing plates on ice, centrifuged at 400 g for 5 min at 4 °C and stained with anti-CD11b (Biolegend, cat. no. 101209, clone M1/70) to identify macrophages. Assays were analyzed by flow cytometry on a Sony SA3800 Flow Cytometer (Sony Biotechnology, Japan) or a CytoFLEX (Beckman, USA). Phagocytosis was measured as the number of CD11b + , GFP + macrophages, quantified as a percentage of the total CD11b + macrophages. 4.12 Phagocytosis Assay Using Live-cell Microscopy: Non-fluorescently labeled 4T1, PAN02, or MDA-MB-231 cells were harvested and labeled with pHrodo Red, SE (Thermo Fisher Scientific, USA) as per manufacturer instructions at a concentration of 1:30,000 in PBS for 1 h at 37°C, followed by two washes with DMEM + 10% FBS + 100 U/mL PS. 50,000 macrophages were added to a transparent 96 well plate and allowed to adhere at 37°C. After macrophage adherence, 100,000 pHrodo-Red-labeled 4T1, PAN02, or MDA-MB-231 cells pretreated with PAC-SABIs for 120 minutes were added in serum-free IMDM. The plate was centrifuged gently at 50 g for 2 min in order to promote the timely settlement of 4T1, PAN02, or MDA-MB-231 cells into the same plane as adherent macrophages. Phagocytosis events were calculated as the number of pHrodo red + events per visual field and the fluorescent signals were captured by a Nikon AX confocal laser microscope (Nikon, Japan). 4.13 In Vivo and Ex Vivo Fluorescence Imaging: To obtain breast and pancreatic subcutaneous xenografts, 1 × 10 6 4T1 or PAN02 cells were implanted into the right hind limb of BALB/c and C57BL/6 mice, respectively. When the tumor size reached ≈ 50 mm 3 mice were randomly sorted into free Cy5.5, Cy5.5 SAMI, and Cy5.5 PAC-SABI groups. The 4T1 and PAN02 tumor-bearing mice were correspondingly intravenously injected with free Cy5.5, Cy5.5-labled SAMI, and Cy5.5-labled PAC-SABI (the dose of Cy5.5 was 1 mg / kg) for in vivo fluorescence imaging with IVIS system (Perkin Elmer, USA). Then, the mice were sacrificed to collect the major organs (heart, liver, spleen, lung, and kidney) and tumors for ex vivo fluorescence imaging. Finally, the 4T1 and PAN02 tumors were embedded in Tissue-Tek OCT compound (Sakura, Japan), and cryosections of 8 µm thickness were prepared. The Cy5.5 signal was detected using a fluorescence microscope (Nikon, Japan). 4.14 Blood Examination and Histology: To record the complete blood count data, healthy BALB/c mice were intravenously injected with PAC-SABIs at a dose of 15 mg / kg every other day for a total of 4 administrations. The mice were sacrificed before blood collection (0.5 mL), and complete blood count evaluations at 1, 7, 14 and 21 d postinjection of PAC-SABIs were carried out at the Nanfang Hospital of Southern Medical University. After about 1 month following treatment with PAC-SABIs, the mice were sacrificed and the major organs (heart, liver, spleen, lung and kidney) were collected. The organs were immersed in a 4% paraformaldehyde solution for an overnight fixation period, followed by dehydration in a 25% sucrose solution. Subsequently, the fixed tissues were sliced into sections with a thickness of 8 μm, and the sections were stained with H&E (Beyotime Biotech, China) as per the manufacturer’s instructions. Finally, a microscope was employed to examine the samples for any histological alterations. 4.15 Anticancer Treatment Studies: The anticancer efficacy of PAC-SABIs was evaluated in the breast and pancreatic orthotopic xenograft tumor models. 4T1-luc (1 × 10 6 ) suspended in a 25 µL PBS and Matrigel (Corning, USA) mixture (1:1, v/v) was injected into the right third mammary fat pad of BALB/c mice. PAN02-luc (1 × 10 6 ) suspended in a 25 µL PBS and Matrigel (Corning, USA) mixture (1:1, v/v) were injected into the tail region of the pancreatic parenchyma of C57BL/6 mice. The PAC-SABI treatment began 5 days after the tumor implantation, and the BLIs of orthotopic 4T1-luc and PAN02-luc tumor-bearing mice were detected every 7 days to monitor the tumor growth in each treatment group. For BLI, mice were given D-luciferin potassium salt (150 mg/kg) intraperitoneally and imaged 10 min later in an IVIS system (Perkin Elmer, USA). Survival (in days) of mice in the different treatment groups were monitored throughout the period of study. In order to study the amplified anticancer immune therapy, 4T1-luc (1 × 10 6 ) suspended in a 25 µL PBS and Matrigel (Corning, USA) mixture (1:1, v/v) was injected into the right third mammary fat pad of BALB/c mice. On the 5th day, 4T1 tumor-bearing mice were randomly divided into 4 groups, and were immunized with PAC-SABIs (15 mg / kg) or PBS on day 5, 7, 9, and 11 through intravenous injection. Then, intraperitoneal injection with or without anti-PD-1 antibody (10 mg / kg) was performed on days 12, 15, 18, and 21. Mouse body weights and tumor sizes (length and width measured by calipers) were measured every other day. The tumor volume was calculated with following the formula: V = (L × W 2 )/2, where V is the volume (mm 3 ), L is the biggest diameter (mm), and W is the smallest diameter (mm). To study the role of PVA-CD40 in prevention of lung metastasis, the histological examination of representative lung sections was conducted using H&E staining at the end points of different treatment procedures. The mice were monitored regularly for death throughout the whole 60-day survival period. 4.16 Analysis of Macrophages and T Cells in Tumor: The 4T1 tumor-bearing mice were euthanized, and tumor tissues were collected and frozen in optimal cutting temperature medium on dry ice. Tumor sections were cut using a cryotome, mounted on slides and stained with different primary antibodies: Anti-F4/80 antibody (Abcam, cat. no. ab6640), Anti-CK19 antibody (Abcam, cat. no. ab52625), Anti-CD8 antibody (Invitrogen, cat. no. MA5-29682) overnight at 4 °C according to the manufacturer’s instructions. Following the addition of fluorescently labelled goat anti-rat IgG H&L (Abcam, cat. no. ab150167) and goat anti-rabbit IgG H&L (Abcam, cat. no. ab150077), the slides were analyzed with a confocal microscope (Nikon, Japan) For flow cytometry analysis, 4T1 tumors were cut into small pieces and homogenized in cold staining buffer to form single cell suspensions in the presence of digestive enzyme. Cells were stained with fluorescence-labelled antibodies: anti-mouse CD45 antibody (Elabscience, cat. no. E-AB-F1136J, clone 30-F11), anti-mouse CD11b antibody (Biolegend, cat. no. 101209, clone M1/70), anti-mouse F4/80 antibody (Biolegend, cat. no. 123116, clone BM8), anti-mouse CD206 antibody (Biolegend, cat. no. 141703, clone C068C2), anti-mouse CD80 antibody (Biolegend, cat. no. 104705, clone 16-10A1), anti-mouse CD3 antibody (BDbioscience, cat. no. 555274, clone 17A2), anti-mouse CD4 antibody (BDbioscience, cat. no. 553051, clone RM4-5), anti-mouse CD8 antibody (BDbioscience, cat. no. 553033, clone 53-6.7), and anti-mouse Foxp3 antibody (BDbioscience, cat. no. 563101, clone R16-715) following the manufacturer’s instructions. The stained cells were measured on a Sony SA3800 Flow Cytometer (Sony Biotechnology, Japan) or a CytoFLEX (Beckman, USA). The numbers presented in the flow cytometry analysis images are percentage based. 4.17 Statistical analysis: Data are presented as mean ± standard error of the mean (S.E.M.). Student’s t-test was used for two-group comparisons, and Kaplan–Meier curves were used to analyze the survival study. Statistical significance was defined as * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analysis was performed using GraphPad Prism 5 and SPSS version 22.0. References Offringa, R., Kötzner, L., Huck, B. & Urbahns, K. The expanding role for small molecules in immuno-oncology. Nat. Rev. Drug Discov. 21, 821–840 (2022). Kraehenbuehl, L., Weng, C.-H., Eghbali, S., Wolchok, J. D. & Merghoub, T. Enhancing immunotherapy in cancer by targeting emerging immunomodulatory pathways. Nat. Rev. Clin. Oncol. 19, 37–50 (2022). Topalian, S. L., Taube, J. M. & Pardoll, D. M. Neoadjuvant checkpoint blockade for cancer immunotherapy. Science 367, eaax0182 (2020). Zhang, X. et al. Reprogramming tumour-associated macrophages to outcompete cancer cells. Nature 619, 616–623 (2023). Nalio Ramos, R. et al. Tissue-resident FOLR2 + macrophages associate with CD8 + T cell infiltration in human breast cancer. Cell 185, 1189–1207.e25 (2022). Kubli, S. P., Berger, T., Araujo, D. V., Siu, L. L. & Mak, T. W. Beyond immune checkpoint blockade: emerging immunological strategies. Nat. Rev. Drug Discov. 20, 899–919 (2021). Su, S. et al. Immune Checkpoint Inhibition Overcomes ADCP-Induced Immunosuppression by Macrophages. Cell 175, 442–457.e23 (2018). Morrissey, M. A., Kern, N. & Vale, R. D. CD47 Ligation Repositions the Inhibitory Receptor SIRPA to Suppress Integrin Activation and Phagocytosis. Immunity 53, 290–302.e6 (2020). CD24 Is a “Don’t Eat Me” Signal That Promotes Tumor Immune Escape. Cancer Discov. 9, 1156–1156 (2019). Yu, J. et al. SIRPα-Fc fusion protein IMM01 exhibits dual anti-tumor activities by targeting CD47/SIRPα signal pathway via blocking the “don’t eat me” signal and activating the “eat me” signal. J. Hematol. Oncol. 15, 167 (2022). Li, D. et al. SLAMF3 and SLAMF4 are immune checkpoints that constrain macrophage phagocytosis of hematopoietic tumors. Sci. Immunol. 7, eabj5501 (2022). Jaiswal, S., Chao, M. P., Majeti, R. & Weissman, I. L. Macrophages as mediators of tumor immunosurveillance. Trends Immunol. 31, 212–219 (2010). Chao, M. P. et al. Calreticulin Is the Dominant Pro-Phagocytic Signal on Multiple Human Cancers and Is Counterbalanced by CD47. Sci. Transl. Med. 2, (2010). Eladl, E. et al. Role of CD47 in Hematological Malignancies. J. Hematol. Oncol. 13, 96 (2020). Liu, Y. et al. Emerging phagocytosis checkpoints in cancer immunotherapy. Signal Transduct. Target. Ther. 8, 104 (2023). Sikic, B. I. et al. First-in-Human, First-in-Class Phase I Trial of the Anti-CD47 Antibody Hu5F9-G4 in Patients With Advanced Cancers. J. Clin. Oncol. 37, 946–953 (2019). Liu, X., Kwon, H., Li, Z. & Fu, Y. Is CD47 an innate immune checkpoint for tumor evasion? J. Hematol. Oncol. 10, 12 (2017). Logtenberg, M. E. W., Scheeren, F. A. & Schumacher, T. N. The CD47-SIRPα Immune Checkpoint. Immunity 52, 742–752 (2020). Cassetta, L. & Pollard, J. W. A timeline of tumour-associated macrophage biology. Nat. Rev. Cancer 23, 238–257 (2023). Barkal, A. A. et al. CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy. Nature 572, 392–396 (2019). Setiawan, D., Brender, J. & Zhang, Y. Recent advances in automated protein design and its future challenges. Expert Opin. Drug Discov. 13, 587–604 (2018). Rawson, S., McPhillie, M. J., Johnson, R. M., Fishwick, C. W. G. & Muench, S. P. The potential use of single-particle electron microscopy as a tool for structure-based inhibitor design. Acta Crystallogr. Sect. Struct. Biol. 73, 534–540 (2017). Zmuda, F. et al. Identification of Novel Trypanosoma cruzi Proteasome Inhibitors Using a Luminescence-Based High-Throughput Screening Assay. Antimicrob. Agents Chemother. 63, e00309-19 (2019). Brown, D. P. et al. Novel multifunctional pharmacology of lobinaline, the major alkaloid from Lobelia cardinalis. Fitoterapia 111, 109–123 (2016). Ding, Y. et al. Enzyme-Instructed Peptide Assembly Favored by Preorganization for Cancer Cell Membrane Engineering. J. Am. Chem. Soc. 145, 4366–4371 (2023). Feng, Z., Han, X., Wang, H., Tang, T. & Xu, B. Enzyme-Instructed Peptide Assemblies Selectively Inhibit Bone Tumors. Chem 5, 2442–2449 (2019). Wu, C. et al. Lysosome-Targeted and Fluorescence‐Turned “On” Cytotoxicity Induced by Alkaline Phosphatase‐Triggered Self‐Assembly. Adv. Healthc. Mater. 11, 2101346 (2022). Wang, H., Feng, Z. & Xu, B. Assemblies of Peptides in a Complex Environment and their Applications. Angew. Chem. Int. Ed Engl. 58, 10423–10432 (2019). Shy, A. N., Kim, B. J. & Xu, B. Enzymatic Noncovalent Synthesis of Supramolecular Soft Matter for Biomedical Applications. Matter 1, 1127–1147 (2019). Cai, Y. et al. Supramolecular ‘Trojan Horse’ for Nuclear Delivery of Dual Anticancer Drugs. J. Am. Chem. Soc. 139, 2876–2879 (2017). Zhan, J., Cai, Y., He, S., Wang, L. & Yang, Z. Tandem Molecular Self-Assembly in Liver Cancer Cells. Angew. Chem. Int. Ed Engl. 57, 1813–1816 (2018). Zhan, J. et al. Organelle-inspired supramolecular nanomedicine to precisely abolish liver tumor growth and metastasis. Bioact. Mater. 9, 120–133 (2022). Zhang, W. et al. Parallelism to Conquer: Binary Supramolecular Peptide Amphiphiles Launch Synergistic Stromal Reprogramming to Remove the Baffle in Pancreatic Cancer Therapy. Adv. Funct. Mater. 32, 2203767 (2022). Mamuti, M. et al. A Polyvalent Peptide CD40 Nanoagonist for Targeted Modulation of Dendritic Cells and Amplified Cancer Immunotherapy. Adv. Mater. Deerfield Beach Fla 34, e2109432 (2022). Zhang, K. et al. A Monotargeting Peptidic Network Antibody Inhibits More Receptors for Anti-Angiogenesis. ACS Nano 15, 13065–13076 (2021). Ren, H. et al. A bioactivated in vivo assembly nanotechnology fabricated NIR probe for small pancreatic tumor intraoperative imaging. Nat. Commun. 13, 418 (2022). Wang, H. et al. CD47/SIRPα blocking peptide identification and synergistic effect with irradiation for cancer immunotherapy. J. Immunother. Cancer 8, e000905 (2020). Tang, Z., Kang, B., Li, C., Chen, T. & Zhang, Z. GEPIA2: an enhanced web server for large-scale expression profiling and interactive analysis. Nucleic Acids Res. 47, W556–W560 (2019). Declarations Conflict of Interest All authors declare no conflict of interest. Acknowledgments We acknowledge the financial support from National Natural Science Foundation of China (31900952, 51973090, 32271372, 32101058, 82273256), Guangdong Basic and Applied Basic Research Foundation (2023A1515012734). Science and Technology Projects of Guangzhou (SL2024A04J01735, SL2024A04J02587). Schemes Scheme 1 is available in the supplementary files section. Additional Declarations There is NO Competing Interest. Supplementary Files SupportingInformation.docx floatimage1.jpeg Scheme 1. Design and proposed mechanism of PAC-SABIs. A) The structure of the designed peptide molecules of Pep-PEG. B) Schematic illustration of nanofibers of PAC-SABIs formation process including peptide self-assembling, mAb modification, and ALP catalysis. C) Schematic illustration of immune quiescent microenvironment and in vivo construction of peptide-antibody combo-supramolecular in situ assembled CD47 and CD24 bi-target inhibitor. D) The proposed mechanism of PAC-SABI-mediated activation of macrophage phagocytosis against cancer cell. Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3314213","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":233918350,"identity":"97608ab7-c417-4b66-b97a-01485b3d6dd2","order_by":0,"name":"Yanbin Cai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYDACHhBhYIPEIU5LQRrJWj4cJkGLfM8Zw8cVBuftdWckMD5428Ygb05Ii8HZHmPDMwa3E7fdSGA2nNvGYLizgZAWfh4zyQaD2wlmNxLYpHnbGBIMDhByWD+P+c8Gg3P2QC3sv4nSwnC2x4yxweAAI9BhbMxEaTE4c6wY6LDkxG1nHjZLzjknYbiBoMN6kjd+bPhjZ292PPnghzdlNvKEHcbAYQBlMDYACQmC6oGA/QExqkbBKBgFo2AkAwB9rj7xcjdEjgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-2139-4750","institution":"Southern Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yanbin","middleName":"","lastName":"Cai","suffix":""}],"badges":[],"createdAt":"2023-08-31 15:35:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3314213/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3314213/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":43379701,"identity":"11b7aa79-6454-43e4-9072-6f3df3154993","added_by":"auto","created_at":"2023-09-19 16:45:07","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1542900,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of PAC-SABIs. A) Determination of the CACs of the Pep and Pep-PEG. B) Fluorescence spectrum of NBD-labeled Pep (pH = 7.4). Insert: fluorescence emission image of NBD-labeled Pep and NBD-labeled Pep + ALP (1 U / mL). C) Fluorescence spectrum of NBD-labeled Pep-PEG (pH = 7.4). Insert: fluorescence emission image of NBD-labeled Pep-PEG and NBD-labeled Pep-PEG + ALP (1 U / mL). D) Zeta potentials of Pep, Pep-PEG and PAC-SABIs detected by dynamic light scattering. E) The CD spectrum of Pep, Pep-PEG and PAC-SABIs in the presence or absence of ALP (1 U / mL). F) Secondary structure calculation of Pep, Pep-PEG and PAC-SABIs in the presence or absence of ALP (1 U / mL). G) The FTIR spectra of Pep, Pep-PEG and PAC-SABIs in the presence or absence of ALP (1 U / mL). H) Conversion rates of Pep, Pep-PEG and PAC-SABIs incubated with ALP (1 U / mL) over time. I) Time-dependent TEM images of Pep and PAC-SABIs. The red arrows point to the formation of nanofibers. Scale bar: 200 nm.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3314213/v1/0c5c734d1d05a4e64582a5c5.jpeg"},{"id":43379697,"identity":"b170cc28-7308-4388-ac97-0526539c6784","added_by":"auto","created_at":"2023-09-19 16:45:06","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1479824,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of CD47 and CD24 in BC and PC. A) Heatmap showing the normalized expression of CD47, CD24, and PD-L1 in a pan-cancer cohort. B-D) The expression of CD47, CD24 and PD-L1 in BC and PC compared to matched normal tissue by analyzing GEPIA\u003csup\u003e38\u003c/sup\u003e. E) Overall survival for PC patients (n = 178) with high versus low CD47 expression as defined by median. F) Overall survival for BC patients (n = 1070) with high versus low CD24 expression as defined by median. G) UMAP dimension 1 and 2 plots displaying cells from a primary sample of BC, and cells colored by cluster identity (n = 20000 single cells). H) CD44, CD47, CD24 and PD-L1 expression overlaid onto UMAP space. I, J) IF analysis of CD47 and CD24 in Patient 1. K, L) IF analysis of CD47 and CD24 in Patient 2. M) Schematic illustration of immune quiescent microenvironment in BC, in situ assembly of PAC-SABIs on the BC cell surface, and blockage of CD47 and CD24 phagocytic checkpoints mediated by PAC-SABIs.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3314213/v1/dc10b64b2e68cf2e3eccee41.jpeg"},{"id":43379703,"identity":"5646ed0d-c341-47df-959f-f4c0a578e6ea","added_by":"auto","created_at":"2023-09-19 16:45:07","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1249009,"visible":true,"origin":"","legend":"\u003cp\u003eIn situ self-assembly of PAC-SABIs on BC and PC cell membranes. A) Time-dependent CLSM images of 4T1 cells treated with NBD-labeled PAC-SABIs. Scale bar: 20 μm. B) Time-dependent CLSM images of PAN02 cells treated with NBD-labeled PAC-SABIs. Scale bar: 20 μm. C) Merged DIC and fluorescent images of 4T1 cell treated with NBD-labeled PAC-SABIs for 120 min. Scale bar: 20 μm. D) CLSM images of 4T1 cell treated with Dil dye (red) and NBD-labeled PAC-SABIs for 120 min. Scale bar: 20 μm. E, F) The fluorescence distribution of NBD-labeled PAC-SABIs on 4T1 cell. G) Time-dependent SEM images of 4T1 cells treated with PAC-SABIs. The red arrows point to the PAC-SABI nanofibers on cell membrane. Scale bar: 1 μm. H) Time-dependent SEM images of PAN02 cells treated with PAC-SABIs. The red arrows point to the PAC-SABI nanofibers on cell membrane. Scale bar: 1 μm. I) CLSM images of 3D 4T1 spheroids treated with Cy5.5-labeled PAC-SABIs, Calcein AM, and Hoechst 33342. Scale bar: 50 μm. J) CLSM images of 3D 4T1 spheroids along the z-axis position. Scale bar: 50 μm.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3314213/v1/abce833a6fb5385aa624c0ae.jpeg"},{"id":43379696,"identity":"c69becae-75f0-41ea-b0fd-21502036f123","added_by":"auto","created_at":"2023-09-19 16:45:06","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1501777,"visible":true,"origin":"","legend":"\u003cp\u003ePromotion of phagocytic clearance of cancer cells via PAC-SABIs treatment in vitro. A) Phagocytosis images of pHrodo-red\u003csup\u003e+\u003c/sup\u003e over time (hours). Scale bar: 50 μm. B) Phagocytosis of 4T1 cells, in the presence of IgG control or PAC-SABIs. The error bars represent the mean ± S.E.M. (n = 3 independent experiments). C) Representative 3D reconstruction of CLSM images of in vitro phagocytosis of 4T1 cells (pHrodo-red\u003csup\u003e+\u003c/sup\u003e, red) by BMDMs or RAW264.7 cells (Calcein, AM; green) in the presence of IgG control, anti-CD24 mAb, SAMIs and PAC-SABIs after 120 min of co-culture. D) Representative flow cytometry plots depicting BMDM phagocytosis of 4T1 cells treated with IgG control, anti-CD24 mAb, SAMIs and PAC-SABIs. E) Quantitative analysis of BMDM flow cytometry results. The error bars represent the mean ± S.E.M. (n = 3 independent experiments; \u003csup\u003e**\u003c/sup\u003eP = 0.01, \u003csup\u003e***\u003c/sup\u003eP = 0.001). F) Representative flow cytometry plots depicting RAW264.7 cell phagocytosis of 4T1 cells treated with IgG control, anti-CD24 mAb, SAMIs and PAC-SABIs. G) Quantitative analysis of RAW264.7 cell flow cytometry results. The error bars represent the mean ± S.E.M. (n = 3 independent experiments; \u003csup\u003e**\u003c/sup\u003eP = 0.01).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3314213/v1/27439e979b348ed5888c882b.jpeg"},{"id":43379702,"identity":"8612cccb-5370-493f-8370-a021ee691b21","added_by":"auto","created_at":"2023-09-19 16:45:07","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":886306,"visible":true,"origin":"","legend":"\u003cp\u003ePAC-SABIs promote phagocytic clearance of cancer cells by HDDMs. A) Schematic illustration of HDDMs generation and stimulation (Created with BioRender.com). B) IF staining of CD11b and CD206 in HDDMs after stimulation with TCM for 48 h. Scale bar: 50 μm. C) FACS-based measurement of CD206 expression by HDDMs stimulated with TCM (blue) vs. cell medium control (red). D) Normalized phagocytosis of MDA-MB-231 cells, in the presence of IgG control, anti-CD24 mAb, SAMIs and PAC-SABIs. The error bars represent the mean ± S.E.M. (n = 3 donors; \u003csup\u003e*\u003c/sup\u003eP = 0.05, \u003csup\u003e**\u003c/sup\u003eP = 0.01). E) Representative CLSM images of in vitro phagocytosis of MDA-MB-231 cells (pHrodo-red\u003csup\u003e+\u003c/sup\u003e, red) by HDDMs (Calcein, AM; green) in the presence of IgG control, anti-CD24 mAb, SAMIs and PAC-SABIs after 120 min of co-culture. Scale bar: 50 μm. F) Representative 3D reconstruction of CLSM images of in vitro phagocytosis of MDA-MB-231 cells (pHrodo-red\u003csup\u003e+\u003c/sup\u003e, red) by HDDMs (Calcein, AM; green) in the presence of IgG control, anti-CD24 mAb, SAMIs and PAC-SABIs after 120 min of co-culture.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3314213/v1/e6f6db002a17aa923221bf2e.jpeg"},{"id":43379699,"identity":"7adeb7f6-4953-42f6-9e53-a443f7117a60","added_by":"auto","created_at":"2023-09-19 16:45:06","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2196504,"visible":true,"origin":"","legend":"\u003cp\u003eBiodistribution and tumor targeting of \u003csup\u003eCy5.5\u003c/sup\u003ePAC-SABIs in vivo. A) Representative near infrared (NIR) fluorescence images of free Cy5.5, \u003csup\u003eCy5.5\u003c/sup\u003eSAMIs and \u003csup\u003eCy5.5\u003c/sup\u003ePAC-SABIs on 4T1 subcutaneous tumor-bearing mice after intravenous injection. Images were acquired at 0, 2, 6, 12, 24, 48, 72, 96 and 120 h post injection. B) Time-dependent quantitative calculation of the average fluorescence intensity in 4T1 tumor area and the AUC of \u003csup\u003eCy5.5\u003c/sup\u003eSAMIs and \u003csup\u003eCy5.5\u003c/sup\u003ePAC-SABIs. The error bars represent the mean ± S.E.M. (n = 3 independent experiments). C) Representative near infrared (NIR) fluorescence images of free Cy5.5, \u003csup\u003eCy5.5\u003c/sup\u003eSAMIs and \u003csup\u003eCy5.5\u003c/sup\u003ePAC-SABIs on PAN02 subcutaneous tumor-bearing mice after intravenous injection. Images were acquired at 0, 2, 6, 12, 24, 48, 72, 96 and 120 h post injection. D) Time-dependent quantitative calculation of the average fluorescence intensity in PAN02 tumor area and the AUC of \u003csup\u003eCy5.5\u003c/sup\u003eSAMIs and \u003csup\u003eCy5.5\u003c/sup\u003ePAC-SABIs. The error bars represent the mean ± S.E.M. (n = 3 independent experiments). E) Ex vivo NIR fluorescence images of 4T1 tumor and major organs (heart, liver, spleen, lung, and kidney) collected post 48 h injection. F) Ex vivo NIR fluorescence images of PAN02 tumor and major organs (heart, liver, spleen, lung, and kidney) collected post 48 h injection. G) Fluorescence images of 4T1 and PAN02 subcutaneous tumor sections at 48 h post-injection of \u003csup\u003eCy5.5\u003c/sup\u003ePAC-SABIs. A refers to the area of fibroids; B refers to the area of cancer cells; C refers to the paracancerous area. Scale bar: 100 μm.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3314213/v1/e5dd21bb46741ea71c630e53.jpeg"},{"id":43380493,"identity":"f9652d4f-5737-4c24-97ba-89c10b3c25da","added_by":"auto","created_at":"2023-09-19 16:53:07","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":651330,"visible":true,"origin":"","legend":"\u003cp\u003eAntitumor efficacy of PAC-SABIs in vivo. A) Scheme of the PAC-SABIs therapeutic strategy for 4T1 orthotopic tumor (Created with BioRender.com). B) In vivo BLIs of 4T1 orthotopic tumor-bearing mice on days 7, 14, 21, and 28. C) Quantification analysis of the in vivo BLI signal of 4T1 orthotopic tumor in each treatment group. The error bars represent the mean ± S.E.M. (n = 5; \u003csup\u003e**\u003c/sup\u003eP = 0.01, \u003csup\u003e***\u003c/sup\u003eP = 0.001). D) Kaplan–Meier survival curves of 4T1 orthotopic tumor-bearing mice treated with the indicated formulation (n= 5; \u003csup\u003e**\u003c/sup\u003eP = 0.01). E) Scheme of the PAC-SABIs therapeutic strategy for PAN02 orthotopic tumor (Created with BioRender.com). F) In vivo BLIs of PAN02 orthotopic tumor-bearing mice on days 7, 14, 21, and 28. G) Quantification analysis of the in vivo BLI signal of PAN02 orthotopic tumor in each treatment group. The error bars represent the mean ± S.E.M. (n = 5; \u003csup\u003e*\u003c/sup\u003eP = 0.05, \u003csup\u003e***\u003c/sup\u003eP = 0.001). H) Kaplan–Meier survival curves of PAN02 orthotopic tumor-bearing mice treated with the indicated formulation (n= 5; \u003csup\u003e*\u003c/sup\u003eP = 0.05).\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3314213/v1/043d0f2fd8af561f76aa0b56.jpeg"},{"id":43379704,"identity":"6e114b6f-5ad7-40b7-a6af-b562a78242d8","added_by":"auto","created_at":"2023-09-19 16:45:07","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1415666,"visible":true,"origin":"","legend":"\u003cp\u003eImmune response in vivo. A) Representative H\u0026amp;E and IF staining of cytokeratin 19 (CK19), F4/80\u003csup\u003e+ \u003c/sup\u003emacrophages and CD8\u003csup\u003e+\u003c/sup\u003e T cells for the corresponding 4T1 tumor tissues after different treatments. Scale bars are marked in the figures. B) Representative flow cytometry plots depicting CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+ \u003c/sup\u003emacrophages phagocytosis, CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003ehi\u003c/sup\u003e macrophages, CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003ehi\u003c/sup\u003e macrophages, CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+ \u003c/sup\u003eT cells, and CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+ \u003c/sup\u003eT cells in 4T1 tumors after different treatments. C-G) Quantification analysis of CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+ \u003c/sup\u003emacrophages phagocytosis, CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003ehi\u003c/sup\u003e macrophages, CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003ehi\u003c/sup\u003e macrophages, CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+ \u003c/sup\u003eT cells, and CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+ \u003c/sup\u003eT cells in 4T1 tumors after different treatments. The error bars represent the mean ± S.E.M. (n = 4 independent experiments;\u003csup\u003e *\u003c/sup\u003eP = 0.05, \u003csup\u003e**\u003c/sup\u003eP = 0.01, \u003csup\u003e***\u003c/sup\u003eP = 0.001). H-K) Cytokine levels (TNF-α, IFN-γ, IL-6, and TGF-β) in the plasma of mice after different treatments determined using ELISA. The error bars represent the mean ± S.E.M. (n = 4 independent experiments;\u003csup\u003e *\u003c/sup\u003eP = 0.05, \u003csup\u003e**\u003c/sup\u003eP = 0.01, \u003csup\u003e***\u003c/sup\u003eP = 0.001).\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3314213/v1/8ed25bbf82edab1e95cc0001.jpeg"},{"id":43381082,"identity":"79eb950e-a9b3-4e62-a2fe-6e8f7f6e956a","added_by":"auto","created_at":"2023-09-19 17:01:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2781402,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3314213/v1/efec86b8-63ea-46f8-8fe6-1f842a24dcdf.pdf"},{"id":43379707,"identity":"90411922-3831-449c-8305-6ab7ee00759e","added_by":"auto","created_at":"2023-09-19 16:45:10","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":47112551,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-3314213/v1/ef33121092bdc0111b6e2d3e.docx"},{"id":43380491,"identity":"47282499-b143-47f1-80b9-437ee9c31f59","added_by":"auto","created_at":"2023-09-19 16:53:06","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1186671,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. \u003c/strong\u003eDesign and proposed mechanism of PAC-SABIs. A) The structure of the designed peptide molecules of Pep-PEG. B) Schematic illustration of nanofibers of PAC-SABIs formation process including peptide self-assembling, mAb modification, and ALP catalysis. C) Schematic illustration of immune quiescent microenvironment and in vivo construction of peptide-antibody combo-supramolecular in situ assembled CD47 and CD24 bi-target inhibitor. D) The proposed mechanism of PAC-SABI-mediated activation of macrophage phagocytosis against cancer cell.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3314213/v1/abff63c8bef8171d3c0996c4.jpeg"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Enzyme-Instructed Peptide Self-Assembly as A Cell Membrane Lichen Activating Macrophage-Mediated Cancer Immunotherapy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAdaptive immune checkpoint (IC) inhibitors targeting programmed cell death protein 1 (PD-1) or its ligand programmed death-ligand 1 (PD-L1) have shown efficacy in treating some cancer types by disrupting inhibitory T cell pathways\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Unfortunately, their effectiveness remains limited against immune quiescent tumors like breast and pancreatic cancers (BCs and PCs), underscoring the importance of innate immune cells and enhanced antigen processing to overcome this challenge\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. As highly prevalent non-malignant cells in the tumor microenvironment (TME), macrophages play a crucial innate immune role via phagocytosis, antigen presentation, and inflammatory cytokine production, linking innate and adaptive immunity\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Yet cancer cells can evade macrophage clearance by upregulating anti-phagocytic \u0026ldquo;don\u0026rsquo;t eat me\u0026rdquo; membrane proteins\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Thus, inhibiting these anti-phagocytic signals or receptors represents a promising immunotherapeutic approach that could synergize with cancer cell elimination, activate CD8\u003csup\u003e+\u003c/sup\u003e T cells, and initiate an anti-cancer immune response.\u003c/p\u003e \u003cp\u003eThe CD47-signal regulatory protein alpha (SIRPα) axis, discovered in the late 2000s, was the first identified checkpoint inhibiting cancer phagocytosis through innate immunity\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Despite ongoing clinical trials testing the inhibitors and antibodies targeting this axis for cancer therapy, two primary challenges have hindered successful clinical application\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. One is the ubiquitous CD47 expression on normal cells (\u0026ldquo;antigen sink\u0026rdquo;), especially red blood cells (RBCs) and platelets. High maintenance dosing required to saturate peripheral antigens causes severe toxicity like anemia and thrombocytopenia\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The other challenge is the limited treatment response in solid tumors\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, likely due to additional immunomodulatory receptors beyond CD47 and the inefficiency of solely blocking anti-phagocytic membrane proteins given the tumor microenvironment (TME) complexity. Consequently, interest has grown in identifying other macrophage-associated immune checkpoints (ICs) to potentially expand innate immunotherapy benefits to more patients\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Recently, CD24 was identified as a novel cancer cell \u0026ldquo;don\u0026rsquo;t eat me\u0026rdquo; signal facilitating immune evasion via binding the macrophage sialic acid-binding Ig-like lectin 10 (Siglec-10)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Both the CD24-Siglec10 and CD47-SIRPα pathways enable immunoreceptor tyrosine-based inhibitory motif phosphorylation, inhibiting macrophage phagocytosis\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. As innate cancer immunology understanding increases, developing inhibitors to simultaneously block CD47 and CD24 signaling is critical to foster optimal anti-tumor immunity.\u003c/p\u003e \u003cp\u003eCurrent inhibitor designs against membrane protein targets in solid tumors are primarily based on the intrinsic structures and properties of the proteins themselves, without adequately accounting for the native curved topological structure of cell membranes\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Owing to the heightened molecular requisites for concurrent and synergistic engagement of two distinct proteinaceous targets, this phenomenon may greatly impede binding kinetics and attenuate the pharmacological potency of the bi-target inhibitor\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. By incorporating more information on membrane contexts into rational structure-based design, inhibitors are expected to achieve improved binding affinity and selectivity for membrane protein targets. Recent studies have demonstrated that alkaline phosphatase (ALP)-responsive peptide self-assembly enables interaction with proteins or protein assemblies on cell membranes, making it a crucial tool for influencing cell-cell interactions and deciding the destiny of cells in multicellular systems\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The enzyme-instructed self-assembly (EISA) effectively modulates the phosphorylated precursors, enabling precise control over their distinctive spatiotemporal functions within the tumor microenvironment (TME) while concurrently reducing toxicity in the blood circulatory system\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. By incorporating specific protein targeting motifs, these precursors can be directed towards the formation of self-assembly units with well-organized structures at the subcellular level within the cell or pericellular space\u003csup\u003e\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Furthermore, the plasma membrane\u0026rsquo;s topological structure tends to facilitate the formation of subsequent peptide assemblies through the coordination of ligand binding with target proteins, which is advantageous in achieving maximum inhibitory effects on multiprotein signaling\u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Motivated by the aforementioned context, we propose an in situ self-assembly strategy involving functional peptide assemblies to activate the interaction between macrophages and cancer cells.\u003c/p\u003e \u003cp\u003eHerein, we developed a peptide-antibody combo-supramolecular in situ assembled CD47 and CD24 bi-target inhibitor (PAC-SABI) to stimulate macrophage phagocytic activity against malignant cells, thus eliciting potent anti-tumor immunity. As illustrated in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, PAC-SABI undergoes biomimetic, ligand-directed surface propagation like lichens on cancer cell membranes, with conformational maturation mediated by target binding and enzyme-responsive morphological transformation to achieve in situ self-assembly and presentation of bioactive motifs. This precision targeting underpins PAC-SABI\u0026rsquo;s potent inhibition of the \u0026ldquo;don\u0026rsquo;t eat me\u0026rdquo; signal transmission driven by cancerous overproduction of CD47 and CD24. Our rationally designed modular system demonstrates that responsive nanoarchitectonics enables meticulous molecular manipulation to construct versatile peptide-antibody therapeutics with enhanced performance. Looking ahead, this proof-of-concept establishes supramolecular peptide engineering as a facile yet powerful approach to merge multitargeting, programmability, and spatial control within a singular nanoscale platform for more efficacious next-generation immunotherapies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Molecular Design and Self-Assembly Behavior\u003c/h2\u003e \u003cp\u003eTo test the feasibility of our concepts and investigate the assembly behavior, we synthesized the peptide molecules as depicted in the Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. The multifunctional peptide-based inhibitor employed a modular design. In the molecular structure, the fluorophores nitrobenzoxadiazole (NBD) and cyanine 5.5 (Cy5.5) were incorporated as capping groups at the ends of the peptides, and enabled real-time tracking of molecular assemblies in vitro and in vivo through fluorescence imaging. The typical self-assembled peptide, Lys-Leu-Val-Phe-Phe, with a sequence derived from Alzheimer\u0026rsquo;s-disease-associated beta-amyloid, was the peptide backbone that accomplishes molecular assembly. The hydrophilic PEG2000 was introduced and implemented to modify the small molecular prodrug assemblies for prolonging circulation in blood and increasing accumulation in the tumor. Moreover, Pep-20 (Ala-Trp-Ser-Ala-Thr-Trp-Ser-Asn-Tyr-Trp-Arg-His) incorporated in our molecular design was identified to specifically bind to both human and murine CD47 for blockage of CD47/SIRPα interaction\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The peptide monomers were assembled into micelles in the aqueous solution, which were subsequently connected to the anti-CD24 mAb. The linkage allowed for active targeting of CD24 overexpressing cancer cells, thereby exerting a synergistic macrophage immunomodulation (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The tyrosine of Pep-20 enabled the synthesis of phosphorylated analogs (pTyr) to promote in situ rearrangement and growth of molecular assemblies leading to the formation of PAC-SABI nanofibers upon dephosphorylation in the presence of ALP (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Expanding upon our supramolecular platform, we engineered additional supramolecular assembled CD47 mono-target inhibitor (SAMIs) to validate the feasibility and efficacy of PAC-SABIs directed by ligand-receptor interaction and enzyme catalysis. Solid-phase peptide synthesis and liquid synthesis were employed, and the detailed procedures were described in the Schemes S1 and S2 (Supporting Information). The molecular identification of the intermediates and final products were confirmed in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-8 (Supporting Information).\u003c/p\u003e \u003cp\u003eThe critical assembly concentration (CAC) of the peptide molecules (Pep) and conjugates (Pep-PEG) was measured using a hydrophobic environmental responsive fluorescent probe, 1-phenylnaphthalene-8-sulfonate (ANS). In PBS (pH 7.4), the CACs of Pep and Pep-PEG were determined to be 42.6 and 26.6 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e M, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The lower CAC of Pep-PEG could be attributed to the presence of hydrophilic PEG chains, which imparted the conjugate with amphiphilic character and enhanced its self-assembly capability in aqueous medium. The fluorescence associated with NBD may potentially serve as a reliable indicator for monitoring the self-assembly of Pep and Pep-PEG. In comparison to concentrations below the CAC, we discerned a noticeable rise in detectable NBD fluorescence at CAC concentrations, which was further amplified under ALP conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). This heightened fluorescence quantum yields implied that the dephosphorylation process mediated by ALP facilitated the formation of self-assembled aggregates of Pep and Pep-PEG, wherein NBD residues were encapsulated within a highly hydrophobic environment. After the PEG modification, the zeta potential of the peptide molecules in PBS (pH 7.4) shifted from \u0026minus;\u0026thinsp;21.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 mV (Pep) to -9.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 mV (Pep-PEG) due to the surface charge screening effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The increasing conjugation between Pep-PEG and negatively charged anti-CD24 mAb resulted in a gradual decrease in the zeta potential. When the ratio of mAb to Pep-PEG reached 1: 10, the conjugate molecules (PAC-SABIs) exhibited a potential of -11.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 mV, and remained stable despite the escalation of Pep-PEG (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Supporting Information). Therefore, the ratio of 1: 10 was considered to be the saturation point for the following study.\u003c/p\u003e \u003cp\u003eIn order to accurately monitor the secondary structure transformation of the peptide molecule assemblies, we conducted circular dichroism (CD) spectroscopy and Fourier transform infrared spectroscopy (FTIR) analysis. In the presence of ALP, the CD spectrum of Pep exhibited a prominent negative peak at 217 nm and a strong positive peak near 195 nm, the characteristic features of a spectrum generated by β-sheet (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). The PAC-SABIs exposed to ALP were found to undergo conformational transformation within the assembly environment, leading to an elevation in the proportion of α-helical content, as evidenced by the emergence of positive peaks at 193nm and negative peaks near 222nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). The interaction of ALP with Pep-PEG or PAC-SABIs was further corroborated through characterization results of FTIR. The spectra presented a maximum absorption at 1654 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicating formation of α- helix structure (1650\u0026ndash;1658 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in the amide-Ⅰ band (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Then, high-performance liquid chromatograph (HPLC) analysis was carried out to monitor the kinetics of EISA. Although the PEG and mAb modification might produce steric hindrance for molecular recognition and ALP reaction, Pep, Pep-PEG, and PAC-SABIs all displayed high conversion efficiency, with a conversion rate of approximately 90% at 60 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). In addition, we observed the morphology transformation of Pep and PAC-SABIs by transmission electron microscopy (TEM) in a time-dependent manner. Pep exhibited a quasi-circular aggregate, wherein these peptide assemblies subsequently aggregated and manifested a spherical structure after a 10-min incubation period with ALP. Then, the advent of a short fiber structure was observed at 30 min. By the 60-minute mark, the majority of the spherical structure was dissipated, giving way to a substantial presence of fibrous structures. At the 120-minute interval, the entire assembly system was predominantly occupied by characteristic nanofibers with a diameter of 6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). However, the PAC-SABIs were observed as irregular aggregates. During the initial 30 min of enzyme catalyzed reactions, these aggregates rapidly coalesced into entangled nanofibers. Subsequently, these nanofibers underwent further extension and bundling, resulting in the formation of a three-dimensional (3D) network after 60 min. At 120 min, nearly all precursors transformed into nanofibers with a diameter of 13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). These results demonstrated that ALP mediated enzymatic reactions acted as a trigger for initiating the rearrangement of peptide molecules, promoting the rapid formation of ordered superstructures by influencing all the microscopic events in the assembly process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Expression of Target Innate ICs in BC and PC\u003c/h2\u003e \u003cp\u003eIn order to assess the impact of CD47 and CD24 signaling on the regulation of macrophage-mediated immune response against cancer, we investigated the expression of CD47 and CD24 in different tumor types. Analysis of RNA sequencing data obtained from The Cancer Genome Atlas (TCGA) revealed a significant overexpression of CD47 and CD24 in nearly all examined tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Furthermore, when compared to the well-established adaptive immune checkpoint PD-L1, CD47 and CD24 exhibited a consistent up-regulation in BC and PC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The levels of CD47 and CD24 expression in PC and BC were notably elevated compared to their corresponding normal tissues, respectively, while no significant variation was detected in PD-L1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D). Stratification of patients based on their CD47 and CD24 expression levels demonstrated a significant correlation with improved overall survival in individuals with lower CD47 expression among PC patients and lower CD24 expression among BC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Single-cell RNA sequencing conducted on a primary BC sample, focusing on examining the expression patterns of CD47 and CD24 at the individual cancer cell level, revealed a prominent expression of CD47 and CD24 in cancer cells, whereas other cell clusters exhibited relatively low expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). These results suggested that CD47 and CD24 may serve as potential markers specific to cancer cells. Moreover, immunofluorescence (IF) analysis of two cancer nodule sections obtained from patient 1 with BC indicated the presence of CD47 and CD24, which had a broad distribution within the cytoplasm and membrane of malignant cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). IF results acquired from consecutive sections of BC tissue in patient 2 showed robust CD47 and CD24 protein expression by cancer cells, indicating redundancy of these two membrane-bound \u0026ldquo;don\u0026rsquo;t-eat-me\u0026rdquo; signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL). As illustrated in the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM, CD47 and CD24 function as innate ICs, diminishing the phagocytic action of macrophages on cancer cells in a synergistic manner. By utilizing the interface receptor ligand interaction and ALP catalysis, PAC-SABIs can precisely identify the cancer cell membrane and establish stable assemblies. This process efficiently inhibits CD47 and CD24 signals, regulating TAM phagocytic activity and, eventually, boosting anti-tumor immune responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 In Situ Self-assembly on Cancer Cell Membranes\u003c/h2\u003e \u003cp\u003eHaving determined the effective assembly of PAC-SABIs under static external conditions, we proceeded to investigate the self-assembly behavior of PAC-SABIs within BC and PC cellular environments using the fluorescence of NBD. Firstly, the widespread expression of ALP in tissue sections of BC and PC cell lines (4T1 and PAN02), as well as on the surface of clinical samples of BC and PC, was verified using the BCIP/NBT color development kit (Figure S9, Supporting Information). Then, the 4T1 and PAN02 cells were co-incubated with NBD-labeled PAC-SABIs at 37 ℃, and changes in NBD fluorescence were directly monitored using confocal laser scanning microscopy (CLSM) imaging at different time points (Fig.\u0026nbsp;3A and 3B). As anticipated, the treated 4T1 and PAN02 cells exhibited a time-dependent augmentation in NBD fluorescence emission on the cellular surface. Following a 30-min co-incubation period, the fluorescence appeared as a diffuse signal surrounding the cancer cells as PAC-SABIs bound to the membrane\u0026rsquo;s outer surface. Then, a prompt aggregation of fluorescent clusters on the membrane was observed at the 60-min mark. By the 120-min interval, fluorescence became uniform and encompassed the cell surface, signifying the in situ self-assembly of PAC-SABIs on cancer cell membrane (Fig.\u0026nbsp;3A and 3B). This observation is further supported by the findings from differential interference contrast (DIC) and fluorescence microscopy imaging (Fig.\u0026nbsp;3C). As shown in Fig.\u0026nbsp;3D-F, the green fluorescence signal from NBD exhibited a strong co-localization with the red fluorescence signal from the Dil dye after co-incubation for 120 min, suggesting that a majority of PAC-SABI molecules were assembled and localized on the 4T1 cell membrane. Under identical conditions, 4T1 cells treated with SAMIs exhibited robust intracellular green fluorescence, reflecting the internalization of SAMIs into the cells, a phenomenon commonly observed when other nanostructures are incubated with live cells (Figure S10, Supporting Information). Blocking experiments were conducted with anti-CD24 antibodies, and the surface fluorescence signals of pretreated 4T1 cells were significantly reduced during dynamic incubation with PAC-SABI, indicating that the specificity of the interaction between CD24 and PAC-SABI promotes the membrane in situ self-assembly process (Figure S11, Supporting Information). Additionally, as shown in Figure S12 (Supporting Information), transmission electron microscopy (TEM) revealed the morphologies of PAC-SABIs assembled on the top surface of cell membranes. We also observed the formation of superstructure networks over time on the 4T1 and PAN02 cell membranes treated with PAC-SABIs through scanning electron microscopy (SEM). During the co-incubation process of 30 to 120 min, PAC-SABIs, like the proliferation process of lichens, first attached to the cell\u0026rsquo;s adherent edge, then migrated along the cell surface, and finally formed a nanoscale fiber network assembly covering the membrane (Fig.\u0026nbsp;3G and 3H).\u003c/p\u003e \u003cp\u003eTo further emulate the TME and 3D spatial architecture of tumors in vivo, we constructed 4T1 spheroid models. After incubating Cy5.5-labeled PAC-SABIs with the 3D cellular spheres for 120 min, fluorescence monitoring by CLSM revealed Cy5.5 envelopment of the outermost Calcein-AM stained cell layer (Figs.\u0026nbsp;3I and 3J). Scanning multiple confocal planes along the z-axis showed Cy5.5 fluorescent binding on the surfaces of sequential cross-sections, corroborating PAC-SABI assembly on the membrane exteriors. Consistent with 2D culture results, pretreatment with anti-CD24 mAb markedly reduced external fluorescence of the layered spheroids (Fig.\u0026nbsp;3J). These results demonstrated the impressive capacity of PAC-SABIs for in situ self-assembly within dynamic physiological milieus, potentially enabling productive immune checkpoint binding and multiprotein signal inhibition.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 3.\u003c/b\u003e In situ self-assembly of PAC-SABIs on BC and PC cell membranes. A) Time-dependent CLSM images of 4T1 cells treated with NBD-labeled PAC-SABIs. Scale bar: 20 \u0026micro;m. B) Time-dependent CLSM images of PAN02 cells treated with NBD-labeled PAC-SABIs. Scale bar: 20 \u0026micro;m. C) Merged DIC and fluorescent images of 4T1 cell treated with NBD-labeled PAC-SABIs for 120 min. Scale bar: 20 \u0026micro;m. D) CLSM images of 4T1 cell treated with Dil dye (red) and NBD-labeled PAC-SABIs for 120 min. Scale bar: 20 \u0026micro;m. E, F) The fluorescence distribution of NBD-labeled PAC-SABIs on 4T1 cell. G) Time-dependent SEM images of 4T1 cells treated with PAC-SABIs. The red arrows point to the PAC-SABI nanofibers on cell membrane. Scale bar: 1 \u0026micro;m. H) Time-dependent SEM images of PAN02 cells treated with PAC-SABIs. The red arrows point to the PAC-SABI nanofibers on cell membrane. Scale bar: 1 \u0026micro;m. I) CLSM images of 3D 4T1 spheroids treated with Cy5.5-labeled PAC-SABIs, Calcein AM, and Hoechst 33342. Scale bar: 50 \u0026micro;m. J) CLSM images of 3D 4T1 spheroids along the z-axis position. Scale bar: 50 \u0026micro;m.\u003c/p\u003e \u003cp\u003eDue to the ability of PAC-SABI to form nanofiber networks on the surface of cancer cells, we conducted a series of in vitro experiments including wound healing, cell invasion, and clone formation assays to verify whether PAC-SABIs affect the physiological activity of BC and PC cells. The results of the wound healing assay on the inhibitory ability of SAMI and PAC-SABI on cancer cell movement are shown in Figure S13A and S14A (Supporting Information). The control group, consisting of highly metastatic 4T1 and PAN02 cells, exhibited robust migration and healing capabilities, as evidenced by a wound healing rate of 100% within 24 h post-scratching. However, treatment with PAC-SABI significantly decreased the wound healing rates of 4T1 and PAN02 cells to 45.9% and 20.1%, respectively, which were notably lower than the rates of 72.7% and 69.8% observed in the SAMI treatment group (Figure S13D and S14D, Supporting Information). The cell invasion assay was conducted by using Transwell chambers with pre-coating Matrigel, which aimed to simulate allowing the cancer cells to degrade the extracellular matrix barrier (ECM) and migrate through the vessels (Figure S13B and S14B, Supporting Information). Based on the findings from the control group, it can be concluded that the highly metastatic 4T1 and PAN02 cells possess the ability to break through and dissolve the ECM barrier, facilitating their migration from the primary tumor site and subsequent establishment of new metastatic sites. The PAC-SABIs yielded a substantial inhibitory effect on the invasion of 4T1 and PAN02 cells, with the inhibition rates of 83.4% and 91.1%, respectively (Figure S13E and S14E, Supporting Information). Furthermore, we conducted a plate colony formation assay to examine the impact of PAC-SABI on the proliferative capacity of tumorigenic 4T1 and PAN02 cells (Figure S13C and S14C, Supporting Information). The results showed that the number of tumorigenic cells significantly decreased, indicating that PAC-SABI can significantly inhibit the colony formation ability of 4T1 and PAN02 cells, with inhibition rates of 79.1% and 80.3%, respectively, which were significantly superior to the inhibitory effect of SAMI (Figure S13F and S14F, Supporting Information).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Promotion of Phagocytic Clearance of Cancer Cells via PAC-SABIs Treatment In Vitro\u003c/h2\u003e \u003cp\u003eTo evaluate the phagocytic elimination of cancer cells by macrophages, 4T1 and PAN02 cells labeled with the pH-sensitive dye pHrodo Red were co-cultured with RAW264.7 cells pre-exposed to tumor conditioned media (TCM). Over 2 h, PAC-SABI-treated 4T1 and PAN02 cells exhibited considerably enhanced susceptibility to engulfment and degradation within acidic phagolysosomes compared to IgG controls (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; Figure S15, Supporting Information). Investigating the therapeutic potential of these findings, we assessed whether PAC-SABIs could amplify the phagocytosis of cancer cells by diverse macrophage populations beyond direct anti-CD24 mAb or SAMI blockade. 3D reconstructions of confocal image stacks showed increased vulnerability of 4T1-pHRodo-Red\u0026thinsp;+\u0026thinsp;cells to phagolysosomal uptake when treated with anti-CD24 mAb or SAMIs. Dual CD47 and CD24 inhibition by PAC-SABIs further enhanced engulfment of 4T1 cells by TCM-exposed bone marrow-derived macrophages (BMDMs) and RAW264.7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Fluorescence activated cell sorting (FACS) quantitation revealed robustly amplified phagocytosis with PAC-SABI addition, with approximately 4- and 2-fold greater uptake by BMDMs and RAW264.7 cells respectively compared to anti-CD24 mAb alone (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-G; Figure S16, Supporting Information).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn agreement with the results in murine macrophages, human THP-1 monocyte-derived macrophages stimulated by TCM exhibited enhanced clearance of human breast cancer MDA-MB-231 cells treated with PAC-SABIs compared to other groups (Figure S17, Supporting Information). Primary human donor-derived macrophages (HDDMs) were also generated by the methodology previously outlined by Barkal et al\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. to assess PAC-SABI efficacy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). HDDMs were subsequently conditioned with tumor-derived media from MDA-MB-231 to generate TAMs. Immunofluorescence (IF) and FACS evidenced upregulation of the M2 marker CD206 in TAMs versus control HDDMs (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). 2D and 3D confocal microscopy of living cells provided further proof that PAC-SABIs effectively inhibited \u0026ldquo;don't eat me\u0026rdquo; signals, highlighting their potential as a valuable innate anti-tumor immunotherapeutic tool (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Biodistribution and Tumor Targeting In Vivo\u003c/h2\u003e \u003cp\u003eTo monitor and quantify the biodistribution up to 120 h after intravenous injection of Cy5.5 labeled PAC-SABI, the in vivo imaging system (IVIS) spectroscopy was employed, and we constructed subcutaneous xenograft models of BC and PC in mice to mitigate systematic errors and individual variances. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, there were notable differences in the distribution of fluorescence among free Cy5.5, SAMI, and PAC-SABI in mice. Cy5.5, serving as the representative small molecule probe, exhibits swift distribution and elimination throughout the mice\u0026rsquo;s bodies, without displaying any discernible specific targeting effects on BC and PC tissues. Due to the absence of active targeting and ALP triggered self-assembly properties, the SAMIs exhibited limited accumulation of Cy5.5 fluorescence signal in the tumor regions of BC and PC. However, when PAC-SABIs were administered intravenously in subcutaneous tumor mice, PAC-SABI conjugated with anti-CD24 mAb demonstrated enhanced fluorescence signal in the tumor regions of BC and PC, reaching its maximum intensity at the 48-h time point. The PAC-SABIs, utilizing the EISA and PEG effects, effectively improved its biodistribution by accumulating more signals in the tumor regions, thereby prolonging its retention time in the tumor for up to 120 h. Simultaneously, the distribution of signals in non-tumor tissues was reduced, and the elimination time was shortened (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The area under the curve (AUC), which is a crucial parameter for the enrichment of PAC-SABI and SAMI in tumor tissue, was determined through quantitative analysis of fluorescence signals. Following the quantitative calculation of fluorescence intensity in the tumor region, excluding background signal, a time-dependent curve was plotted for PAC-SABI and SAMI. In the mouse models of BC and PC tumors, the AUC (0-120 h) of PAC-SABI was observed to be approximately 3 times greater than that of SAMI. Furthermore, our investigation revealed that the elimination of PAC-SABI molecules in BC and PC exhibited a remarkably slow rate, with only a modest decrease of 16.2% and 25.6% between 48 and 120 h, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eAfter the intravenous administration of free Cy5.5, SAMIs, and PAC-SABIs, the mice were euthanized using carbon dioxide inhalation at the 48-h mark, and the major organs (heart, liver, spleen, lung, and kidney) as well as tumors were harvested for ex vivo imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Similar to the in vivo results, free Cy5.5 cleared rapidly from the body with negligible accumulation in the tumor and major organs. IVIS imaging showed notable differences in tissue biodistribution between SAMI and PAC-SABI. The distribution of PAC-SABI exhibited distinct selectivity within tumor tissues of BC and PC, with partial retention observed in metabolic organs such as the liver and kidney. By comparison, no significant variance was observed in the biological distribution of SAMI within the lungs, kidneys, and tumors, with the majority of molecules predominantly localized in the liver. The discernible dissimilarity between these two molecules can be attributed to the presence of targeted ligands and EISA, which augment the precise recognition of PAC-SABI molecules towards cancer cells and facilitate its proficient molecular assembly within tumors, while SAMI accumulated non-specifically in the liver during the metabolic process. To visually observe the distribution of the PAC-SABIs in the tumor tissue of BC and PC, we analyzed the Cy5.5 fluorescence in the frozen tumor sections. Interestingly, a notable accumulation of Cy5.5 fluorescence was observed within the cancer cell regions of the 4T1 tumor, whereas the fluorescence intensity was diminished in the fibroblast-rich areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). This phenomenon was similarly observed within the interior of PAN02 tumors, where PAC-SABI exhibited a concentration within the cancer cell area rather than the paracancerous or fibroid regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). These results indicated that the rapid formation of the PAC-SABI superstructure network leads to its effective accumulation and retention in tumors, which may further provide persistent blockade of innate immune checkpoints in cancer cells, thereby inducing effective and sustained macrophage-mediated immune responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Antitumor Efficacy and Immune Response In Vivo\u003c/h2\u003e \u003cp\u003eGenerally, the toxicity of inhibitors, especially the blood toxicity associated with CD47 blockade therapy, is a key criterion affecting clinical transformation. To evaluate the systemic toxicity of PAC-SABIs, complete blood count was obtained from healthy BALB/c mice on the 1st, 7th, 14th, and 21st days after 4 times of intravenous injection of PAC-SABIs every other day. The main parameters evaluated included red blood cell, hemoglobin, hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, mean platelet volume and platelets. All of these parameters were shown to be within reference ranges for healthy BALB/c mice (Figure S18A, Supporting Information). Subsequently, following approximately one month of treatment, we collected the main organs of mice in each group, including heart, liver, spleen, lung, and kidney, and sliced them for H\u0026amp;E staining. No notable histological alterations or pathological lesions were observed (Figure S18B, Supporting Information). In summary, PAC-SABIs exhibited high biocompatibility characteristics in mice without significant systemic toxicity, supporting its potential application in clinical practice.\u003c/p\u003e \u003cp\u003eEncouraged by the aforementioned experimental results, we hypothesized that PAC-SABI therapy has the potential to instruct macrophages in eliciting proficient in vivo anti-tumor phagocytic reactions, consequently leading to the inhibition of tumor growth. To mimic the natural progression of human BC and PC, the orthotopic BC model utilizing luciferase-labeled 4T1 (4T1-Luc) tumor-bearing BALB/c mice and the orthotopic PC model utilizing luciferase-labeled PAN02 (PAN02-Luc) tumor-bearing C57BL/6 mice were constructed for in vivo therapeutic efficacy evaluation. According to the depicted illustration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), after the establishment of in situ BC and PC in mice, control IgG, anti-CD24, SAMI and PAC-SABI were administered every other day for a total of 4 doses. The evaluation of tumor regression/progression was conducted through the periodic monitoring of tumor bioluminescence (BLI) at 7-day intervals. Over time, on the 22nd day post-treatment, the PAC-SABI group exhibited significantly reduced tumor BLI signal intensities in comparison to the other groups, suggesting a notable deceleration in tumor growth among the BC and PC mice subjected to PAC-SABI treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). Conversely, mice treated with Control IgG and anti-CD24 mAb exhibited limited therapeutic efficacy, as the majority of BC and PC mice succumbed within 40 and 50 days, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eD and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). The overall survival rate of BC mice treated with PAC-SABI on the 50th day and that of PC mice on the 60th day were 60% and 40%, respectively, showing a significant survival benefit of PAC-SABIs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eD and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to examine the regulatory effects of PAC-SABIs in vivo, the changes that occurred in the 4T1 TME were analyzed after intravenous administration. Hematoxylin and eosin (H\u0026amp;E) staining of xenograft sections in both the IgG control and anti-CD24 mAb groups revealed that the cancer cells were densely arranged in bands and clusters, with disordered arrangement (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). In the PAC-SABI group, a notable presence of inflammatory cells and tissue necrosis was observed within the tumor interstitium, showing a greater extent of inflammatory cell infiltration and tissue necrosis compared to the SAMI group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Then, IF staining was conducted on the typical immunosuppressive 4T1 tumor tissue. In the PAC-SABI group, we observed a noteworthy manifestation of macrophages engaging in phagocytic and cytotoxic activities against cancer cells, characterized by extensive macrophage infiltration and internalization of cancer cell components (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). This could potentially be attributed to the heightened phagocytic capacity resulting from the suppression of combined innate ICs. The nuclear density within this particular region was lower than that in adjacent regions, indicating a reduction in cancer cell density. Conversely, this tumoricidal phenomenon was less frequently observed in the remaining treatment groups. In addition, the IF results showed that CD8\u003csup\u003e+\u003c/sup\u003e T cells in the IgG control group were mainly distributed at the edge of the tumor, while the infiltration of CD8\u003csup\u003e+\u003c/sup\u003e T cells within the tumor tissue in the PAC-SABI group was significantly enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eFACS analyses were performed on GFP\u003csup\u003e+\u003c/sup\u003e 4T1 tumors to further explore how PAC-SABI inhibition altered the tumor immune microenvironment. Consistent with the IF results, we found that the highest levels of in vivo phagocytosis by infiltrating TAMs were achieved in the PAC-SABI group, which was 3-fold higher than that in the IgG control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eB and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eD and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eE, anti-CD24 mAb, SAMI, and PAC-SABI treatments all potentiated the activation state of TAMs towards a pro-inflammatory antitumoral phenotype, indicating another potential mechanism of innate IC blockade strategy. Moreover, FACS analysis of T cells in tumor tissues treated with PAC-SABIs demonstrated a significant increase in the percentage of tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eB and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). We speculated that the phagocytosis of activated macrophages might increase the cross-presentation of tumor antigen to T cells, which could have enhanced the effectiveness of adaptive anti-tumor immune response. As the main immunosuppressive cells, the number of FOXP3 regulatory T cells (Tregs) within the tumor following PAC-SABI treatment was found to be significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eB and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eG). This reduction may have also potentially contributed to the transition of the macrophage population from a suppressive M2 phenotype to an M1 phenotype. Previous studies have shown that reprogramming the TME can impact the cytokine secretion of immune cells, resulting in heightened levels of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and interleukin-6 (IL-6), and reduced levels of the anti-inflammatory cytokine transforming growth factor beta (TGF-β). As shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eH-K, compared with the IgG control group, the level of TGF-β in the PAC-SABI group was significantly decreased, while the levels of TNF-α, IFN-γ, and IL-6 were increased by 3.3-, 10.6-, and 4.3-fold, respectively. These findings strongly indicated that PAC-SABIs have the ability to counteract the anti-inflammatory properties of the tumor immune microenvironment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Inhibition on BC and PC Liver Metastasis In Vivo\u003c/h2\u003e \u003cp\u003eTo investigate if the ability of PAC-SABIs to inhibit cancer cell migration and invasion in vitro translates to decreased metastasis in vivo, we used an experimental liver metastasis model of BC and PC. 4T1-luc and PAN02-luc cells were injected into the spleens of BALB/c and C57BL/6 mice, respectively, which enabled monitoring and quantification of metastasized cancer cells in the liver with BLI. Starting from the day before injection of cancer cells into the spleen, mice were given intravenous injections of IgG control antibody, anti-CD24 mAb, SAMIs, and PAC-SABIs every other day for a total of 4 doses. The BLI intensity started to increase soon after injection of cancer cells in the IgG control and anti-CD24 mAb groups, and patchy metastatic lesions formed in the liver region (Figure S19A and S19E, Supporting Information). However, a significant decrease in BLI intensity was observed in the PAC-SABI group compared to the IgG control group after two weeks of injection (Figure S19B and S19F, Supporting Information). Then, the mice were euthanized, and reductions in the number and size of liver metastases were confirmed macroscopically in the PAC-SABI group (Figure S19C and S19G, Supporting Information). Additionally, H\u0026amp;E staining of the liver further demonstrated that PAC-SABIs facilitated construction of nanofibrous barriers on the cancer cell membrane, inhibiting early metastasis and effectively stopping the growth of previously formed metastasis in mouse liver metastasis models (Figure S19D and S19H, Supporting Information).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Enhanced Anti-PD-1 Therapy After Phagocytosis Modulation\u003c/h2\u003e \u003cp\u003eBased on the aforementioned in vivo experimental findings, regulating the phagocytosis of cancer cells by macrophages exhibited great potential in adaptive anti-tumor immune induction. Therefore, it is postulated that the combination of PAC-SABI and PD-1 pathway blockade could potentially result in an augmented anti-tumor immune response in vivo, consequently yielding optimal therapeutic outcomes. To test this, we inoculated 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e GFP\u003csup\u003e+\u003c/sup\u003e 4T1 cells into the third mammary fat pad of female BALB/c mice. Mice were then administered intravenously with PAC-SABIs or PBS every 2 days for a total of 4 doses, followed by anti-PD-1 or IgG control intraperitoneal injection on days 7, 10, 13, and 16. The 4T1 tumor growth and survival rate were measured over time to assess the immunotherapeutic efficacy of different treatment regimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). The untreated group and the anti-PD-1 group showed rapid tumor growth and poor median survival. Compared with these two groups, the PAC-SABI group showed modest tumor growth inhibition and improved survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eB-D). Remarkably, anti-PD-1 therapy after PAC-SABI activation of macrophages produced the greatest tumor suppression effect and achieved a 60-day optimal survival rate of 57% (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eD). At the experimental end points, the lungs were dissected for analysis. Histological examination of representative lung sections using H\u0026amp;E staining showed a lower incidence of pulmonary metastatic nodules in the SAMI group compared to untreated and anti-PD-1 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eE). More importantly, the combination of PAC-SABI and anti-PD-1 therapy effectively impeded lung metastasis development, with few observable nodules. Given the promising results, FACS was used to investigate macrophage phagocytic activity towards 4T1 cells. The combination treatment led to a substantial increase in phagocytosing CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e macrophages compared to other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eF). Furthermore, CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration was directly observed to be improved with combination therapy through IF staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eG). These findings suggested that the combination elicited robust innate and adaptive immune responses, leading to tumor growth inhibition and metastasis rejection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eIn conclusion, we have crafted a peptide-antibody conjugate that functions as a supramolecular, in situ assembled CD47 and CD24 bi-target inhibitor, explicitly devised to modulate the phagocytic activity of macrophages and enhance the immune response against cancer. The modularly constructed PAC-SABI not only accumulated effectively in BC and PC tissues but also rapidly self-organized into well-structured interface nanofiber networks, a process facilitated by both an active targeting mechanism and EISA-induced conformational transitions. Governed by rigorous spatiotemporal oversight, PAC-SABI demonstrates prolonged presence within the TME, thereby attenuating the hematological repercussions commonly linked to CD47-focused therapies. In its distinctive lichen-mimetic growth phase on the membrane facade, PAC-SABI obstructs the collaborative engagement of CD47 and CD24 with SIRPα and Siglec10. This strategic intervention curtails the broad dissemination of the \u0026ldquo;don\u0026rsquo;t eat me\u0026rdquo; signal emanating from cancer cells. When introduced in vivo, PAC-SABI acts as an intermediary conduit, merging the innate and adaptive immune responses. In synergy with anti-PD-1 regimens, it markedly inhibits tumor proliferation. The architectural blueprint of PAC-SABI serves as a harbinger, potentially guiding the schematic outlines for multifaceted peptide therapeutics. These novel designs aspire to manipulate membrane proteins via in situ self-assembly methodologies, striving for immunotherapeutic modalities of heightened safety and potency.\u003c/p\u003e"},{"header":"4. Methods","content":"\u003cp\u003e\u003cstrong\u003e4.1 Synthesis and Characterization of PAC-SABIs:\u0026nbsp;\u003c/strong\u003eThe intermediates including Pep and Pep-PEG were acquired by solid phase peptide synthesis, and PAC-SABIs were obtained by liquid phase synthesis. The detailed synthesis procedures and mass spectrum analysis are presented in the Supporting Information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2 Fluorescence Assays:\u003c/strong\u003e The CACs of Pep and Pep-PEG were determined using the ANS fluorescence assay. ANS was dissolved in dimethylformamide (DMF) to a concentration of 1 mM. Subsequently, 1 \u0026micro;L of ANS was added to 100 \u0026micro;L of varying concentrations of Pep and Pep-PEG. The resulting solution was transferred to a quartz cuvette and analyzed using a Perkin Elmer LS-55 fluorescence spectrophotometer (Perkin Elmer, USA). The CAC was determined from the matched curve, which was obtained by the ANS fluorescent intensities at 475 nm plotted against different concentrations of Pep and Pep-PEG.\u003c/p\u003e\n\u003cp\u003eThe self-assembly process of Pep and Pep-PEG was monitored using NBD fluorescence. The fluorescence intensities of NBD-labeled Pep and Pep-PEG were measured at CAC and half of the CAC concentrations via a fluorescence spectrometer (excitation wavelength: 467 nm). To evaluate the ALP-triggered dephosphorylation and in situ self-assembly process,\u0026nbsp;the NBD fluorescence intensity was detected after incubating Pep and Pep-PEG at CAC concentration with ALP (1 U / mL) at 37 \u0026deg;C for 1 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3 CD Spectroscopy:\u003c/strong\u003e All the CD spectra analyses were recorded on a CD spectropolarimeter (Applied Photophysics Ltd, UK) in quartz cuvettes with an optical path length of 0.1 cm. Data were collected from 190 to 260 nm with a scanning speed of 100\u0026nbsp;nm/min at room temperature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4 FTIR Spectroscopy:\u003c/strong\u003e All the FTIR analyses were carried out\u0026nbsp;by a Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific, USA). A 100\u0026nbsp;\u0026micro;L solution of different formulations was spun down at 10000 g for 1 h, and the pellet was blown dry with nitrogen. The dry samples were mixed with KBr and pressed into pellets for further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5 TEM Characterization:\u003c/strong\u003e Pep and PAC-SABI solutions (100 \u0026mu;M) with or without ALP (1 U / mL) incubation were prepared and placed at room temperature for morphology observation at different time points. Copper grids with a porous carbon mesh were used for sample preparation. 10 \u0026micro;L of the Pep and PAC-SABI solution was placed onto a copper grid with a carbon membrane and left for 3 min, followed by removal of excess solutions with filter papers. Then, a small drop of uranyl acetate solution (2% weight/volume in water) was added to the copper grid for negative staining of samples, and the grid was blotted with filter paper after 1 minute. Finally, the samples were left on filter paper overnight to facilitate further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.6 Cell Culture, Animal studies and human specimens:\u0026nbsp;\u003c/strong\u003eMurine BC 4T1 cell\u0026nbsp;line, murine PC PAN02 cell line, human BC MDA-MB-231 cell line, murine macrophage cell line RAW264.7, and human macrophage cell line THP-1 were purchased from the American Type Culture Collection (ATCC)\u0026nbsp;and cultured according to the supplier\u0026rsquo;s recommendations, supplemented with 10% fetal bovine serum (FBS) and antibiotics. The cell culture supernatant of BC cell lines 4T1 and MDA-M-231 was collected as TCM, and was used to induce the transformation of RAW264.7 macrophages and phorbol myristate acetate (PMA; 100 ng/ml)-treated THP-1 macrophages into TAMs, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC57BL/6 mice (male, 6 weeks old) were purchased from BesTest Bio-Tech Co., Ltd (Zhuhai, China), and BALB/c mice (female, 6 weeks old) were purchased from Guangdong\u0026nbsp;Nanmo Biotechnology Co., Ltd (Zhongshan, China). All animal experiments were carried out in compliance with NIH guidelines and all animal-experimental protocol were approved by the Animal Experimentation Ethics Committee of Zhujiang Hospital of Southern Medical University (LAEC-2021-040 and LAEC-2022-073).\u003c/p\u003e\n\u003cp\u003eAfter obtaining approval from the institutional review board of Nanfang Hospital and\u0026nbsp;Guangdong Provincial People\u0026rsquo;s Hospital\u0026nbsp;of Southern Medical University (NFEC-2023-309, KY2023-179-01), human peripheral blood, BC and PC specimens were obtained, and the appropriate informed consent was obtained for all sample donors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.7 BMDMs and HDDMs Generation and Stimulation:\u0026nbsp;\u003c/strong\u003eBMDM isolation was performed according to a previously published protocol with minor modification. Briefly, after sacrificing C57BL/6 mice and disinfecting the skin with 75% alcohol, the hind legs were cut off and placed in a sterile petri dish containing sterile and ice-cold PBS.\u0026nbsp;The bone marrow was flushed with PBS using a syringe with a 25-gauge needle. The supernatant bone marrow cells were collected and then washed with PBS and resuspended with complete conditioned media for BMDM differentiation (100 mL complete medium consisted of 74 mL Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) + 15 mL macrophage colony-stimulating factor (M-CSF, 25 ng/mL) + 10 mL FBS + 1 mL penicillin-streptomycin solution (PS)), seeded on tissue culture plates, and incubated at 37 \u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u0026nbsp;Primary HDDMs\u0026nbsp;were generated from venous blood of healthy volunteers, diluted with 2 \u0026times; PBS (pH 7.4) and separated with Ficoll density gradient\u0026nbsp;as described previously. Monocytes were then differentiated into macrophages by culture in Iscove\u0026rsquo;s modified Dulbecco\u0026rsquo;s medium (IMDM) + 10% AB human serum (Life Technologies)\u0026nbsp;for 7 to 10 days. To stimulate macrophages with TCM, we cultured BMDMs and HDDMs with complete medium containing 50% TCM from 4T1 and MDA-MB-231 cells, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.8 Characterization of the Self-Assembly Process of PAC-SABI in 2D and 3D Cell Culture Environments:\u0026nbsp;\u003c/strong\u003e4T1 or PAN02 cells were inoculated and incubated overnight at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells / dish in laser confocal petri dishes. NBD-labeled PAC-SABIs (100 \u0026mu;M) were co-incubated with 4T1 or PAN02 cells for 30, 60, and 120 min, followed by washing with PBS three times. Then, the NBD fluorescence at different time points was observed by CLSM imaging.\u0026nbsp;To reveal the self-assembly process of PAC-SABI on cancer cell membranes, 4T1 (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/dish) cells were cultured with NBD-labeled PAC-SABI (100 \u0026mu;M) for 120 min. After 3 rounds of washing with PBS, the membrane dye CellTracker CM-Dil (1 mM; Invitrogen, USA) was added and incubated for 5 min at 37 \u0026deg;C, and then for an additional 15 min at 4 \u0026deg;C.\u0026nbsp;CLSM imaging was used to detect the spatial distribution of NBD and Dil fluorescence.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3D spheroids of 4T1 cells were constructed according to the previously reported method. Cy5.5-labeled PAC-SABI (100 \u0026mu;M) was co-incubated with 4T1 3D spheroids for 90 min, followed by washing with PBS three times. Then, the outermost cells and nuclei of the spheroids were stained with Calcein-AM and Hoechst 33342, respectively.\u0026nbsp;Finally, CLSM imaging was used to detect spatial distributions of fluorescence signals with Cy5.5, FITC and DAPI\u0026nbsp;channels in spheroids and different focal plane along the z axis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.9 Detection of Cell Surface Distribution Using Bio-TEM:\u0026nbsp;\u003c/strong\u003eThe 4T1 and PAN02 cells were seeded on cell culture dishes with a diameter of 10\u0026nbsp;mm at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells / dish and cultured overnight, followed by replacement with fresh culture medium containing PAC-SABIs at a concentration of 100 \u0026mu;M for 120 min. Then, the PAC-SABI-containing medium was removed and the cells were washed three times with PBS. The cells attached to the bottom of the dishes were gently harvested using a cell scraper and collected in a centrifuge tube. After centrifugation at 1500\u0026ndash;3000 rpm for 5 min, the supernatant was carefully discarded, and then a 4 \u0026deg;C precooled fixative was slowly added along the tube wall and then placed in a 4 \u0026deg;C refrigerator overnight. The samples were then fixed with a 1% osmium acid solution for 1 h followed by dehydration using a gradient concentration of ethanol. The samples were embedded and sliced with a LEICA EM UC7 ultra-thin microtome to obtain 70\u0026ndash;90 nm slices. The sections were stained with lead citrate solution, uranyl acetate, and 50% ethanol saturated solution for 5-10 min, respectively, and then observed under TEM imaging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.10 Detection of Cell Surface Distribution Using SEM:\u0026nbsp;\u003c/strong\u003e4T1 and PAN02 cells were seeded into 12-well plates containing cover glass slips at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells / well and cultured overnight. PAC-SABIs were added into each well at the final concentration of 100 \u0026mu;M and cultured for 120 min. Then, PAC-SABI-treated 4T1 and PAN02 cells were dehydrated for the time course of 24 h, processing from 30% to 100% ethanol. The samples were then sputter coated with gold (CRC-150 Sputter Coater, USA) and imaged using a Zeiss Ultra 55 SEM (Carl Zeiss, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.11 Phagocytosis Assay Using Flow Cytometry:\u003c/strong\u003e The in vitro phagocytosis assays described in this study were performed by co-culture GFP\u003csup\u003e+\u003c/sup\u003e 4T1 cells and macrophages at a ratio of 100,000 target cells to 50,000 macrophages for 120 min in a humidified, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37 \u0026deg;C in ultra-low-attachment 96-well U-bottom plates (Corning, USA) in serum-free IMDM. 4T1 cells with endogenous fluorescence were harvested from plates using TrypLE Express (Life Technologies, Poland) and treated with PAC-SABIs for 120 min prior to co-culture.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAfter co-culture, phagocytosis assays were stopped by placing plates on ice, centrifuged at 400 g for 5 min at 4 \u0026deg;C and stained with anti-CD11b (Biolegend, cat. no. 101209, clone M1/70) to identify macrophages. Assays were analyzed by flow cytometry on a Sony SA3800 Flow Cytometer (Sony Biotechnology, Japan) or a CytoFLEX (Beckman, USA). Phagocytosis was measured as the number of CD11b\u003csup\u003e+\u003c/sup\u003e, GFP\u003csup\u003e+\u003c/sup\u003e macrophages, quantified as a percentage of the total CD11b\u003csup\u003e+\u003c/sup\u003e macrophages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.12 Phagocytosis Assay Using Live-cell Microscopy:\u003c/strong\u003e Non-fluorescently labeled 4T1, PAN02, or MDA-MB-231 cells were harvested and labeled with pHrodo Red, SE (Thermo Fisher Scientific, USA) as per manufacturer instructions at a concentration of 1:30,000 in PBS for 1 h at 37\u0026deg;C, followed by two washes with DMEM + 10% FBS + 100 U/mL PS. 50,000 macrophages were added to a transparent 96 well plate and allowed to adhere at 37\u0026deg;C. After macrophage adherence, 100,000 pHrodo-Red-labeled 4T1, PAN02, or MDA-MB-231 cells pretreated with PAC-SABIs for 120 minutes were added in serum-free IMDM. The plate was centrifuged gently at 50 g for 2 min in order to promote the timely settlement of 4T1, PAN02, or MDA-MB-231 cells into the same plane as adherent macrophages.\u0026nbsp;Phagocytosis events were calculated as the number of pHrodo red\u003csup\u003e+\u003c/sup\u003e events per visual field and the fluorescent signals were captured by a Nikon AX confocal laser microscope (Nikon, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.13 In Vivo and Ex Vivo Fluorescence Imaging:\u003c/strong\u003e To obtain breast and pancreatic subcutaneous xenografts, 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e 4T1 or PAN02 cells were implanted into the right hind limb of BALB/c and C57BL/6 mice, respectively. When the tumor size reached \u0026asymp; 50 mm\u003csup\u003e3\u003c/sup\u003e mice were randomly sorted into free Cy5.5, \u003csup\u003eCy5.5\u003c/sup\u003eSAMI, and \u003csup\u003eCy5.5\u003c/sup\u003ePAC-SABI groups. The 4T1 and PAN02 tumor-bearing mice were correspondingly intravenously injected with free Cy5.5, Cy5.5-labled SAMI, and Cy5.5-labled PAC-SABI (the dose of Cy5.5 was 1 mg / kg) for in vivo fluorescence imaging with IVIS system (Perkin Elmer, USA). Then, the mice were sacrificed to collect the major organs (heart, liver, spleen, lung, and kidney) and tumors for ex vivo fluorescence imaging. Finally, the 4T1 and PAN02 tumors were embedded in Tissue-Tek OCT compound (Sakura, Japan), and cryosections of 8 \u0026micro;m thickness were prepared. The Cy5.5 signal was detected using a fluorescence microscope (Nikon, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.14 Blood Examination and Histology:\u0026nbsp;\u003c/strong\u003eTo record the complete blood count data, healthy BALB/c mice were intravenously injected with PAC-SABIs at a dose of 15 mg / kg\u0026nbsp;every other day for a total of 4 administrations. The mice were sacrificed before blood collection (0.5 mL), and complete blood count evaluations at 1, 7, 14 and 21 d postinjection of PAC-SABIs were carried out at the Nanfang Hospital of Southern Medical University. After about 1 month following treatment with PAC-SABIs, the mice were sacrificed and the major organs (heart, liver, spleen, lung and kidney) were collected. The organs were immersed in a 4% paraformaldehyde solution for an overnight fixation period, followed by dehydration in a 25% sucrose solution. Subsequently, the fixed tissues were sliced into sections with a thickness of 8 \u0026mu;m, and the sections were stained with H\u0026amp;E (Beyotime Biotech, China) as per the manufacturer\u0026rsquo;s instructions. Finally, a microscope was employed to examine the samples for any histological alterations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.15 Anticancer Treatment Studies:\u003c/strong\u003e The anticancer efficacy of PAC-SABIs was evaluated in the breast and pancreatic orthotopic xenograft tumor models. 4T1-luc (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) suspended in a 25 \u0026micro;L PBS and Matrigel (Corning, USA) mixture (1:1, v/v) was injected into the right third mammary fat pad of BALB/c mice. PAN02-luc (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) suspended in a 25 \u0026micro;L PBS and Matrigel (Corning, USA) mixture (1:1, v/v) were injected into the tail region of the pancreatic parenchyma of C57BL/6 mice. The PAC-SABI treatment began 5 days after the tumor implantation, and the BLIs of orthotopic 4T1-luc and PAN02-luc tumor-bearing mice were detected every 7 days to monitor the tumor growth in each treatment group. For BLI, mice were given D-luciferin potassium salt (150 mg/kg) intraperitoneally and imaged 10 min later in an IVIS system (Perkin Elmer, USA). Survival (in days) of mice in the different treatment groups were monitored throughout the period of study.\u003c/p\u003e\n\u003cp\u003eIn order to study the amplified anticancer immune therapy, 4T1-luc (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) suspended in a 25 \u0026micro;L PBS and Matrigel (Corning, USA) mixture (1:1, v/v) was injected into the right third mammary fat pad of BALB/c mice.\u0026nbsp;On the 5th day, 4T1 tumor-bearing mice were randomly divided into 4 groups, and were immunized with PAC-SABIs (15 mg / kg) or PBS on day 5, 7, 9, and 11 through intravenous injection. Then, intraperitoneal injection with or without anti-PD-1 antibody (10 mg / kg) was performed on days 12, 15, 18, and 21. Mouse body weights and tumor sizes (length and width measured by calipers) were measured every other day. The tumor volume was calculated with following the formula: V = (L \u0026times; W\u003csup\u003e2\u003c/sup\u003e)/2, where V is the volume (mm\u003csup\u003e3\u003c/sup\u003e), L is the biggest diameter (mm), and W is the smallest diameter (mm). To study the role of PVA-CD40 in prevention of lung metastasis, the histological examination of representative lung sections was conducted using H\u0026amp;E staining at the end points of different treatment procedures.\u0026nbsp;The mice were monitored regularly for death throughout the whole 60-day survival period.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.16 Analysis of Macrophages and T Cells in Tumor:\u0026nbsp;\u003c/strong\u003eThe\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e4T1 tumor-bearing mice were euthanized, and tumor tissues were collected and frozen in optimal cutting temperature medium on dry ice.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eTumor sections were cut using a cryotome, mounted on slides and stained with different primary antibodies: Anti-F4/80 antibody (Abcam,\u0026nbsp;cat. no. ab6640), Anti-CK19 antibody (Abcam,\u0026nbsp;cat. no. ab52625), Anti-CD8 antibody (Invitrogen, cat. no. MA5-29682) overnight at 4 \u0026deg;C according to the manufacturer\u0026rsquo;s instructions. Following the addition of fluorescently labelled goat anti-rat IgG H\u0026amp;L (Abcam, cat. no. ab150167) and goat anti-rabbit IgG H\u0026amp;L (Abcam, cat. no. ab150077), the slides were analyzed with a confocal microscope (Nikon, Japan)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor flow cytometry analysis, 4T1 tumors were cut into small pieces and homogenized in cold staining buffer to form single cell suspensions in the presence of digestive enzyme. Cells were stained with fluorescence-labelled antibodies: anti-mouse CD45 antibody (Elabscience, cat. no. E-AB-F1136J, clone 30-F11), anti-mouse CD11b antibody (Biolegend, cat. no. 101209, clone M1/70), anti-mouse F4/80 antibody (Biolegend, cat. no. 123116, clone BM8), anti-mouse CD206 antibody (Biolegend, cat. no. 141703, clone C068C2), anti-mouse CD80 antibody (Biolegend, cat. no. 104705, clone 16-10A1), anti-mouse CD3 antibody (BDbioscience, cat. no.\u0026nbsp;555274, clone 17A2), anti-mouse CD4 antibody (BDbioscience, cat. no. 553051, clone RM4-5), anti-mouse CD8 antibody (BDbioscience, cat. no. 553033, clone 53-6.7), and anti-mouse Foxp3 antibody (BDbioscience, cat. no. 563101, clone R16-715) following the manufacturer\u0026rsquo;s instructions. The stained cells were measured on a Sony SA3800 Flow Cytometer (Sony Biotechnology, Japan) or a CytoFLEX (Beckman, USA). The numbers presented in the flow cytometry analysis images are percentage based.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.17 Statistical analysis:\u0026nbsp;\u003c/strong\u003eData are presented as mean \u0026plusmn; standard error of the mean (S.E.M.). Student\u0026rsquo;s t-test was used for two-group comparisons, and Kaplan\u0026ndash;Meier curves were used to analyze the survival study. Statistical significance was defined as \u003csup\u003e*\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003ep \u0026lt; 0.01 and \u003csup\u003e***\u003c/sup\u003ep \u0026lt; 0.001. Statistical analysis was performed using GraphPad Prism 5 and SPSS version 22.0.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOffringa, R., K\u0026ouml;tzner, L., Huck, B. \u0026amp; Urbahns, K. The expanding role for small molecules in immuno-oncology. Nat. Rev. Drug Discov. 21, 821\u0026ndash;840 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKraehenbuehl, L., Weng, C.-H., Eghbali, S., Wolchok, J. D. \u0026amp; Merghoub, T. Enhancing immunotherapy in cancer by targeting emerging immunomodulatory pathways. Nat. Rev. Clin. Oncol. 19, 37\u0026ndash;50 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTopalian, S. L., Taube, J. M. \u0026amp; Pardoll, D. M. Neoadjuvant checkpoint blockade for cancer immunotherapy. Science 367, eaax0182 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, X. \u003cem\u003eet al.\u003c/em\u003e Reprogramming tumour-associated macrophages to outcompete cancer cells. Nature 619, 616\u0026ndash;623 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNalio Ramos, R. \u003cem\u003eet al.\u003c/em\u003e Tissue-resident FOLR2 + macrophages associate with CD8 + T cell infiltration in human breast cancer. Cell 185, 1189\u0026ndash;1207.e25 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKubli, S. P., Berger, T., Araujo, D. V., Siu, L. L. \u0026amp; Mak, T. W. Beyond immune checkpoint blockade: emerging immunological strategies. Nat. Rev. Drug Discov. 20, 899\u0026ndash;919 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu, S. \u003cem\u003eet al.\u003c/em\u003e Immune Checkpoint Inhibition Overcomes ADCP-Induced Immunosuppression by Macrophages. Cell 175, 442\u0026ndash;457.e23 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorrissey, M. A., Kern, N. \u0026amp; Vale, R. D. CD47 Ligation Repositions the Inhibitory Receptor SIRPA to Suppress Integrin Activation and Phagocytosis. Immunity 53, 290\u0026ndash;302.e6 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCD24 Is a \u0026ldquo;Don\u0026rsquo;t Eat Me\u0026rdquo; Signal That Promotes Tumor Immune Escape. Cancer Discov. 9, 1156\u0026ndash;1156 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu, J. \u003cem\u003eet al.\u003c/em\u003e SIRPα-Fc fusion protein IMM01 exhibits dual anti-tumor activities by targeting CD47/SIRPα signal pathway via blocking the \u0026ldquo;don\u0026rsquo;t eat me\u0026rdquo; signal and activating the \u0026ldquo;eat me\u0026rdquo; signal. J. Hematol. Oncol. 15, 167 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, D. \u003cem\u003eet al.\u003c/em\u003e SLAMF3 and SLAMF4 are immune checkpoints that constrain macrophage phagocytosis of hematopoietic tumors. Sci. Immunol. 7, eabj5501 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJaiswal, S., Chao, M. P., Majeti, R. \u0026amp; Weissman, I. L. Macrophages as mediators of tumor immunosurveillance. Trends Immunol. 31, 212\u0026ndash;219 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChao, M. P. \u003cem\u003eet al.\u003c/em\u003e Calreticulin Is the Dominant Pro-Phagocytic Signal on Multiple Human Cancers and Is Counterbalanced by CD47. Sci. Transl. Med. 2, (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEladl, E. \u003cem\u003eet al.\u003c/em\u003e Role of CD47 in Hematological Malignancies. J. Hematol. Oncol. 13, 96 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, Y. \u003cem\u003eet al.\u003c/em\u003e Emerging phagocytosis checkpoints in cancer immunotherapy. Signal Transduct. Target. Ther. 8, 104 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSikic, B. I. \u003cem\u003eet al.\u003c/em\u003e First-in-Human, First-in-Class Phase I Trial of the Anti-CD47 Antibody Hu5F9-G4 in Patients With Advanced Cancers. J. Clin. Oncol. 37, 946\u0026ndash;953 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, X., Kwon, H., Li, Z. \u0026amp; Fu, Y. Is CD47 an innate immune checkpoint for tumor evasion? J. Hematol. Oncol. 10, 12 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLogtenberg, M. E. W., Scheeren, F. A. \u0026amp; Schumacher, T. N. The CD47-SIRPα Immune Checkpoint. Immunity 52, 742\u0026ndash;752 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCassetta, L. \u0026amp; Pollard, J. W. A timeline of tumour-associated macrophage biology. Nat. Rev. Cancer 23, 238\u0026ndash;257 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarkal, A. A. \u003cem\u003eet al.\u003c/em\u003e CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy. Nature 572, 392\u0026ndash;396 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSetiawan, D., Brender, J. \u0026amp; Zhang, Y. Recent advances in automated protein design and its future challenges. Expert Opin. Drug Discov. 13, 587\u0026ndash;604 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRawson, S., McPhillie, M. J., Johnson, R. M., Fishwick, C. W. G. \u0026amp; Muench, S. P. The potential use of single-particle electron microscopy as a tool for structure-based inhibitor design. Acta Crystallogr. Sect. Struct. Biol. 73, 534\u0026ndash;540 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZmuda, F. \u003cem\u003eet al.\u003c/em\u003e Identification of Novel Trypanosoma cruzi Proteasome Inhibitors Using a Luminescence-Based High-Throughput Screening Assay. Antimicrob. Agents Chemother. 63, e00309-19 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrown, D. P. \u003cem\u003eet al.\u003c/em\u003e Novel multifunctional pharmacology of lobinaline, the major alkaloid from Lobelia cardinalis. Fitoterapia 111, 109\u0026ndash;123 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing, Y. \u003cem\u003eet al.\u003c/em\u003e Enzyme-Instructed Peptide Assembly Favored by Preorganization for Cancer Cell Membrane Engineering. J. Am. Chem. Soc. 145, 4366\u0026ndash;4371 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng, Z., Han, X., Wang, H., Tang, T. \u0026amp; Xu, B. Enzyme-Instructed Peptide Assemblies Selectively Inhibit Bone Tumors. Chem 5, 2442\u0026ndash;2449 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, C. \u003cem\u003eet al.\u003c/em\u003e Lysosome-Targeted and Fluorescence‐Turned \u0026ldquo;On\u0026rdquo; Cytotoxicity Induced by Alkaline Phosphatase‐Triggered Self‐Assembly. Adv. Healthc. Mater. 11, 2101346 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, H., Feng, Z. \u0026amp; Xu, B. Assemblies of Peptides in a Complex Environment and their Applications. Angew. Chem. Int. Ed Engl. 58, 10423\u0026ndash;10432 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShy, A. N., Kim, B. J. \u0026amp; Xu, B. Enzymatic Noncovalent Synthesis of Supramolecular Soft Matter for Biomedical Applications. Matter 1, 1127\u0026ndash;1147 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai, Y. \u003cem\u003eet al.\u003c/em\u003e Supramolecular \u0026lsquo;Trojan Horse\u0026rsquo; for Nuclear Delivery of Dual Anticancer Drugs. J. Am. Chem. Soc. 139, 2876\u0026ndash;2879 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhan, J., Cai, Y., He, S., Wang, L. \u0026amp; Yang, Z. Tandem Molecular Self-Assembly in Liver Cancer Cells. Angew. Chem. Int. Ed Engl. 57, 1813\u0026ndash;1816 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhan, J. \u003cem\u003eet al.\u003c/em\u003e Organelle-inspired supramolecular nanomedicine to precisely abolish liver tumor growth and metastasis. Bioact. Mater. 9, 120\u0026ndash;133 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, W. \u003cem\u003eet al.\u003c/em\u003e Parallelism to Conquer: Binary Supramolecular Peptide Amphiphiles Launch Synergistic Stromal Reprogramming to Remove the Baffle in Pancreatic Cancer Therapy. Adv. Funct. Mater. 32, 2203767 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMamuti, M. \u003cem\u003eet al.\u003c/em\u003e A Polyvalent Peptide CD40 Nanoagonist for Targeted Modulation of Dendritic Cells and Amplified Cancer Immunotherapy. Adv. Mater. Deerfield Beach Fla 34, e2109432 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, K. \u003cem\u003eet al.\u003c/em\u003e A Monotargeting Peptidic Network Antibody Inhibits More Receptors for Anti-Angiogenesis. ACS Nano 15, 13065\u0026ndash;13076 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen, H. \u003cem\u003eet al.\u003c/em\u003e A bioactivated in vivo assembly nanotechnology fabricated NIR probe for small pancreatic tumor intraoperative imaging. Nat. Commun. 13, 418 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, H. \u003cem\u003eet al.\u003c/em\u003e CD47/SIRPα blocking peptide identification and synergistic effect with irradiation for cancer immunotherapy. J. Immunother. Cancer 8, e000905 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang, Z., Kang, B., Li, C., Chen, T. \u0026amp; Zhang, Z. GEPIA2: an enhanced web server for large-scale expression profiling and interactive analysis. Nucleic Acids Res. 47, W556\u0026ndash;W560 (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eAll authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe acknowledge the financial support from National Natural Science Foundation of China (31900952, 51973090, 32271372, 32101058, 82273256), Guangdong Basic and Applied Basic Research Foundation (2023A1515012734). Science and Technology Projects of Guangzhou (SL2024A04J01735, SL2024A04J02587).\u003c/p\u003e"},{"header":"Schemes","content":"Scheme 1 is available in the supplementary files section."}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Immunotherapy, innate immune checkpoint, macrophage phagocytosis, peptide self-assembly, alkaline phosphatase","lastPublishedDoi":"10.21203/rs.3.rs-3314213/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3314213/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTargeted immunomodulation for reactivating innate cells, especially macrophages, holds great promise to complement current adaptive immunotherapy. Nevertheless, there is still a lack of high-performance inhibitors for blocking macrophage phagocytosis checkpoints in immune quiescent solid tumors so far. Herein, a peptide-antibody combo-supramolecular in situ assembled CD47 and CD24 bi-target inhibitor (PAC-SABI) is described, which undergoes biomimetic surface propagation like lichens on cancer cell membranes through ligand-receptor binding and enzyme-triggered reactions. Primarily, the PAC-SABIs demonstrate specific avidity for the overexpressed CD24 on the cancer cell surface with anti-CD24 monoclonal antibody (mAb). Subsequently, they exhibit alkaline phosphatase-catalyzed rapid dephosphorylation of phosphopeptides, constructing a three-dimensional nanofiber network and reinstating blockade of CD47 signaling. By concurrent inhibition of CD47 and CD24 signaling, PAC-SABIs stimulate macrophage phagocytosis and initiate T cell antitumor response. Remarkably, compared with anti-CD24 mAb, PAC-SABIs enhance the phagocytic ability of macrophages by 3\u0026ndash;4 times in vitro and in vivo while facilitating infiltration of CD8\u003csup\u003e+\u003c/sup\u003e T cells into 4T1 tumors. Moreover, combining PAC-SABIs with anti-PD-1 therapy effectively suppressed 4T1 tumor growth in murine models, surmounting other treatment groups with a 60-day survival rate of 57%. The in vivo construction of PAC-SABI-based nanoarchitectonics provides an efficient platform for bridging innate and adaptive immunity to maximize therapeutic potency.\u003c/p\u003e","manuscriptTitle":"Enzyme-Instructed Peptide Self-Assembly as A Cell Membrane Lichen Activating Macrophage-Mediated Cancer Immunotherapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-19 16:45:01","doi":"10.21203/rs.3.rs-3314213/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"feedf130-a54a-40d6-907f-568e9e146d3b","owner":[],"postedDate":"September 19th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":24776329,"name":"Biological sciences/Biotechnology/Biomaterials/Biomedical materials"},{"id":24776330,"name":"Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy"}],"tags":[],"updatedAt":"2024-06-20T14:25:12+00:00","versionOfRecord":[],"versionCreatedAt":"2023-09-19 16:45:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3314213","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3314213","identity":"rs-3314213","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}