Targeting Oncofetal Fibronectin and Neuropilin-1 in solid tumors with PL2 peptide

Targeting Oncofetal Fibronectin and Neuropilin-1 in solid tumors with PL2 peptide | 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 Targeting Oncofetal Fibronectin and Neuropilin-1 in solid tumors with PL2 peptide Prakash Lingasamy, Allan Tobi, Kaarel Kurm, Olav Tammik, Tambet Teesalu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5743295/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract To improve the precision and selectivity of anticancer therapies, affinity ligands targeting molecules of the malignancy-associated vascular signature are used. One such target is Fibronectin Extra Domain-B (Fn-EDB), an oncofetal splice variant of a major extracellular matrix protein (Fn), which is upregulated in many solid tumors as part of the angiogenic response. In this study, we conducted cell-free biopanning on recombinant Fn-EDB to identify a short peptide designated as PL2 (amino acid sequence: TSKQNSR), which specifically interacts with Fn-EDB. Notably, the C-terminal arginine of PL2 enables its interaction with neuropilin-1 (NRP-1), a receptor known to facilitate cell and tissue penetration. When administered systemically, PL2-displaying recombinant bacteriophages and iron oxide nanoworms (NWs) functionalized with PL2 peptide exhibited homing to glioblastoma and prostate tumor xenografts, followed by their extravasation and penetration into tumor parenchyma. Notably, PL2-functionalized NWs penetrated ex vivo explants of clinical ovarian carcinoma, underscoring their translational potential. These findings underscore the potential of the PL2 peptide as a promising agent for anticancer drug delivery and molecular imaging applications. One Sentence Summary: PL2 tumor penetrating peptide targeting Fibronectin Extra Domain-B (Fn-EDB) and Neuropilin-1 (NRP-1) can be used for precision delivery of payloads to the microenvironment of solid tumors Biological sciences/Biotechnology Biological sciences/Cancer Biological sciences/Drug discovery Biological sciences/Molecular biology Health sciences/Biomarkers Health sciences/Medical research Health sciences/Molecular medicine Health sciences/Oncology Physical sciences/Nanoscience and technology Fibronectin Extra Domain-B (Fn-EDB) Neuropilin-1 (NRP-1) extracellular matrix homing peptide in vivo phage display glioblastoma prostate carcinoma angiogenesis nanomedicine targeted drug delivery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Significance We developed the novel PL2 peptide via phage display, demonstrating selective dual-targeting of Fibronectin Extra-Domain-B (Fn-EDB) and Neuropilin-1 (NRP-1), key markers of the tumor microenvironment. This targeting enhances nanoparticle homing, deep-tissue penetration, and therapeutic payload delivery in solid tumors within preclinical models. Its ability to bind and penetrate clinical ovarian carcinoma tissues underscores its translational potential for precision oncology, paving the way for safer, effective cancer therapies with further optimization. Introduction Tumor blood vessels have emerged as critical targets for therapeutic intervention. Therapeutic strategies include suppressing the growth or perturbing functions of the neovessels that sustain tumor lesions 1,2 and leveraging the unique characteristics of tumor blood vessels to enhance the delivery of cytotoxic anticancer agents via specialized drug delivery systems. Therapeutic agents can be directed to malignant cells in the tumor microenvironment by using affinity ligands such as antibodies, peptides, aptamers, or small molecules that engage tumor-associated markers 3,4 . The accessibility of target receptors from the bloodstream and their adequate expression level are prerequisites for any effective affinity-targeted therapy 4 . Current affinity-targeting strategies primarily engage the cell surface biomarkers on malignant and tumor endothelial cells. Targeting the extracellular matrix (ECM) in the tumor microenvironment is less common despite its potential advantages. ECM components are estimated to be ~10-fold more abundant than cellular receptors and offer a higher capacity for drug delivery 5,6 . Compared to receptors overexpressed on the surface of malignant cells, ECM molecules provide opportunities for more stable targeting as they are predominantly deposited by genetically stable nonmalignant cells 7 . To some degree, ECM targeting peptides allow for intracellular delivery due to their ability to exploit the natural cellular uptake pathways associated with cell-matrix interactions, as shown for the PL1 peptide that uses macropinocytosis for cellular entry 7 . The ECM provides a dynamic physical and biochemical microenvironment that actively regulates essential cellular functions such as adhesion, proliferation, and migration, thereby influencing cellular developmental pathways 8,9 . Overexpression of alternatively spliced ECM isoforms is associated with cancer, and specific ECM splicing variants have been linked to stromal activation 10 . Fibronectin Extra Domain-B (Fn-EDB), an acidic 91-amino acid splice variant of fibronectin generated by type III homology repeats, is a key component of the angiogenic signature, distinguished by its overexpression in solid tumors and absence in most adult tissues, except for the female reproductive tract 11,12 . A comparative analysis of Fn-EDB expression in ~18,800 cancer samples and ~4,500 normal samples demonstrated upregulation of Fn-EDB in 15 types of cancer, including in grade I to IV malignant gliomas 13 . In addition, Fn-EDB is a specific marker for tumor neovessels 14 . To date, several classes of Fn-EDB targeting ligands, including antibodies, peptides, and aptamers, have been applied to deliver therapeutic agents, such as cytokines, cytotoxic agents, chemotherapeutic drugs, and radioisotopes, to Fn-EDB-expressing tumors 15–17 . The Fn-EDB targeting L19 antibody and its derivatives (diabodies, Single-chain variable fragment (scFv), Small immunoprotein (SIP)) have exhibited potential in preclinical and clinical studies in EDB-FN-positive cancer patients using both systemic and intratumoral administration routes 18 . Daromun, an intralesional immunocytokine combining IL-2 and TNF conjugated to the L19 antibody, has shown effectiveness in local tumor destruction and treating distant disease through an immune-mediated mechanism in early clinical studies 19–21 . In contrast to antibodies, short peptides offer easy synthesis, low immunogenicity, low cost, biocompatibility, and moderate affinity, which helps to circumvent the affinity site barrier 22–25 . Peptides, such as linear PL1 and cyclic ZD2, specifically bind to Fn-EDB and have been used to target intracranial glioblastomas and prostate cancer, respectively 26,27 . One potential limitation in the application of matrix targeting ligands for tumor delivery is the variability of accessibility in the ECM to circulating ligands. The accessibility, influenced by the degree of tumor blood vessel leakiness, generally correlates with histological grade and malignancy but varies significantly both within individual tumors and across tumor types. Several pharmacological strategies have been employed to increase tumor vascular permeability and improve tumor delivery (e.g., drugs modulating tumor blood pressure, inflammatory cytokines, and bradykinin mediators) 28,29 . We investigated the pharmacological stimulation of extravasation to enhance tumor ECM exposure to circulating targeting ligands by designing bispecific ligands that simultaneously bind the ECM molecule Tenascin-C and the cell and tissue penetration receptor neuropilin-1 (NRP-1) 30,31 . The engagement of the peptide with NRP-1 induced extravasation and tissue penetration via a mechanism involving cellular entry and vascular transcytosis through the C-end Rule (CendR) pathway 32–35 . Here, we used peptide-phage display on the recombinant Fn-EDB domain to identify a novel heptameric PL2 peptide that interacts specifically with Fn-EDB. The peptide also engages the tissue penetration receptor NRP-1 via the C-terminal arginine containing motif of the peptide, promoting cellular uptake in vitro and penetration of cell and tissue barriers in vivo . The in vivo phage playoff and systemic PL2-guided iron oxide nanoparticles showed specific accumulation in a panel of tumor xenografts implanted in mice. Our study suggests that PL2-guided agents can be used for detection, imaging, and payload delivery to solid tumors positive for the expression of Fn-EDB and NRP-1. Materials and Methods Materials Phosphate-buffered saline (PBS) was purchased from Lonza (Verviers, Belgium). K 3 [Fe(CN) 6 ], HCl, isopropanol, Triton-X, Tween-20, CHCl 3 , MeOH, Isopropyl β-D-1-thiogalactopyranoside (IPTG), and dimethylformamide (DMF) were purchased from Sigma-Aldrich (Munich, Germany). Peptides and proteins The peptides and proteins used in the study were Cys-5(6)-carboxyfluorescein (FAM)-PL2, biotin-PL2, Cys-FAM peptides, and biotin with 6-aminohexanoic acid spacer, which were purchased from TAG Copenhagen (Denmark). The plasmids pASK75-Fn7B8 and pASK75-Fn789 were kindly provided by Prof. Dr. Arne Skerra 36 . The gene fragment of Fn-EDB domain was amplified from the plasmids and cloned into a pET28a+ plasmid containing a N-terminal His 6 -tag for expression in E. coli strain BL21 Rosetta™ 2 (DE3) pLysS (Novagen, #70956). Recombinant Fn-EDB was produced as a soluble protein and purified using the HisTrap IMAC HP column (GE Healthcare, #17-0920-05) as previously described 37,38 . SDS-PAGE and mass spectrometry (MS) analyses were used to determine proteins purity, size, and sequence. The NRP-1 b1b2 domain protein was expressed and purified at the Protein Production and Analysis Facility at the Sanford Burnham Prebys Medical Discovery Institute (La Jolla, CA, US), and the NRP-1 b1 domain protein was expressed and purified in-house. Cloning, expression, purification of proteins (FN-EDB, TNC-C, NRP1, NCL and single chain antibodies FN-EDB-L19), and generation of polyclonal rabbit antibodies are described in Supplementary Information, Materials and Methods section and PL1 study protocols 27 . Cell lines and experimental animals The study utilized human glioblastoma (U87-MG, HTB-14, RRID: CVCL_0022) and prostate carcinoma (PC3, CRL1435, RRID: CVCL_0035) cells purchased from ATCC (VA, USA). Murine wild-type glioblastoma (WT-GBM) cells were kindly provided by Gabriele Bergers (UCSF, USA), and stem cell-like cancer cells P3, P13 were gifted by Rolf Bjerkvig (University of Bergen, Norway). The M21 (RRID: CVCL_D031) melanoma cells were the gift of David Cheresh (USA). The cells and tumors were prepared as described in previous studies 34,35,37–41 . Athymic nude mice (Hsd/Athymic Fox1 nu Harlan) were purchased from Harlan Sprague Dawley (HSD, Indianapolis, IN, USA) and maintained under standard housing conditions of the Animal Facility of the Institute of Biomedicine and Translational Medicine, University of Tartu (Tartu, Estonia). Inclusion and exclusion criteria for animals were based on standard experimental conditions and we selected age-matched male and female nude mice (11-15 weeks old). The orthotopic glioblastoma (GBM) tumor models were established using P13, P3 stem cell-like, and WT-GBM cells. Approximately 2–3 × 10⁵ cells suspended in 3 μL of PBS were intracranially implanted into mice brain 2 mm right and 1 mm anterior to the bregma. For the subcutaneous models, 2 - 9 × 10 6 U87-MG GBM and prostate carcinoma (PC3) cells in 100 µl of PBS were subcutaneously (s.c.) implanted in the right flank of 11 - 15-week-old male and female nude mice . No attrition was noted during the study, as all animal samples were included as per protocol. No blinding was used during the experiment and study did not include treatment groups; power calculations were not applicable. Our experimental design ensures that minimal bias (or noise) by ensuring same sex, similar animal weight at start of experiment, same animal age, same type of stabling, several animals caged together. Animal experimentation procedures were approved by the Estonian Ministry of Agriculture, Committee of Animal Experimentation, projects #42 and #48. We confirm that all methods were performed in accordance with the relevant guidelines and regulations. We confirm that the study was conducted in accordance with the ARRIVE guidelines 42 . T7 phage peptide library biopanning We employed T7-Select ® phage display system (Novagen, EMD Biosciences, MA, USA) to generate NNK-encoded X7 peptide phage libraries with a diversity of ~ 5 × 10 8 for biopanning on recombinant Fn-EDB. The first round of selection was carried out on Fn-EDB immobilized on a Costar 96-Well enzyme-linked immunosorbent assay (ELISA) plate (#3590, Corning Life Sciences, MA, USA) by coating the plate with 20 µg/ml recombinant Fn-EDB protein in 100 µl of PBS overnight at 4 °C. The plate was then blocked with 1% bovine serum albumin (BSA) in PBS overnight at 4 °C. The phage library solution (5 × 10 8 pfu in 100 µl of PBS-BSA) was incubated overnight at 4 °C, followed by 6 washes with PBS + BSA + 0.1% Tween 20 to remove non-specifically bound background phages. The bound phages were rescued and amplified in E. coli strain BLT5403 (Novagen, MA, USA) 43 . The subsequent selection rounds were performed with His 6 -tagged Fn-EDB protein (30 µg/10 µl beads) immobilized on Ni-NTA Magnetic Agarose Beads (QIAGEN, Hilden, Germany) at room temperature for 1 hour in 400 µl of PBS. The Fn-EDB immobilized beads were washed three times with PBS + BSA + 0.1% NP40, followed by incubation with the phage from the previous round (5 × 10 8 pfu in 100 µl of PBS + BSA + 0.1% NP40) for 1 hour at room temperature. The background and weakly bound phages were removed by rinsing six times with PBS + BSA + 0.1% NP40, and the bound phages were eluted with 1 ml of PBS + 50 0mM imidazole + 0.1% NP40. The recovered phages were titered and amplified for a subsequent round of selection. After 6 rounds of selection, peptide-encoding phage DNA from a randomly selected set of 48 clones from round 5 was subjected to Sanger sequencing to obtain information on the displayed peptides 43,44 . For cell-free binding studies with individually amplified phage clones, phages were incubated with Fn-EDB coated magnetic beads as described above. Furthermore, the GRPARPAR phage on NRP-1 coated beads was used as a positive control 33 , while the Nucleolin (NCL) and the C-domain of Tenascin C (TNC-C) were used as negative controls. Finally, we used phage displaying heptaglycine peptide (GGGGGGG, G7) or insertless phage clones for negative controls. In vivo playoff phage auditioning To assess the systemic homing of peptide-phages to xenograft tumor models, we employed an internally controlled competitive assay that we have termed in vivo peptide-phage playoff 45 . Briefly, the candidate Fn-EDB binding peptides, together with previously published tumor-homing peptides and control peptides, were amplified and purified by PEG-8000 precipitation, CsCl gradient ultracentrifugation, and dialysis. The equimolar pooled peptide-phages were intravenously injected into tumor-bearing mice at a concentration of 1 × 10¹⁰ pfu in 200 µL of PBS. After 2 hours of circulation, the mice were anesthetized by intraperitoneal (i.p.) injection of 350 μL containing 0.1 mg/kg dexmedetomidine and 75 mg/kg ketamine dissolved in saline or 3-4% isoflurane. Following anesthesia, the mice were perfused intracardially with Dulbecco’s Modified Eagle Medium (DMEM; Lonza Ltd, Basel, Switzerland). Tumors and organs were collected in LB + 1% NP40, and tissue homogenization was carried out to rescue peptide-phages. The lysates were then amplified in E. coli , purified through precipitation with PEG-8000, and the DNA was extracted using a DNA extraction kit (High Pure PCR Template Preparation Kit; Roche, Basel, Switzerland). To evaluate the representation of each phage in the input mixture, tumor, and control organs, we performed next-generation sequencing of phage genomic DNA using the Ion Torrent high-throughput DNA sequencing system (Thermo Fisher Scientific, Waltham, MA, USA). The FASTQ data from Ion Torrent were processed using a custom Python (RRID:SCR_024202) script that identified the barcodes and constant flanking residues, and extracted the correct length reads. Table 1 provides a detailed list of the equimolarly pooled phage peptides used in this study. Peptide Binding Assay ELISA plates (Nunc Maxisorp, Thermo Fisher Scientific Inc., MA, USA) were coated with 20 µg of PL2 peptide labeled with FAM in 100 µl of PBS and incubated at 37 °C overnight. The plate wells were blocked with 1% BSA in PBS for 1 hour at 37 °C, washed with a blocking solution (PBS containing 1% BSA and 0.1% Tween-20), and incubated with 2 µg of recombinant proteins in PBS per well for 6 hours. The wells were washed 3 times with the blocking solution, and the bound protein was detected using an anti-His-tag antibody (Cat #A2-502-100, RRID: AB_11135798, Icosagen, Tartu, Estonia) for 1 hour at 37 °C. After washing the wells 3 times with the blocking solution, a horseradish peroxidase-conjugated secondary antibody (Cat# 111-035-008, RRID: AB_2337937, Jackson Immuno Research, Cambridgeshire, UK) was added according to the manufacturer’s instructions. The wells were washed 3 times with the blocking solution, and a peroxidase reaction was initiated by adding 100 µL/well of freshly prepared solution from the TMB Peroxidase EIA Substrate Kit (Bio-Rad, Hercules, CA, USA), followed by a 5-minute incubation at 37 °C. The reaction was stopped with 1 N H 2 SO 4 , and the absorbance was measured at 450 nm using a microplate reader (Tecan Austria GmbH, Salzburg, Austria). Nanoparticle Synthesis and Functionalization The synthesis and functionalization of iron oxide nanoworms (NWs) and silver nanoparticles (AgNPs) followed previously published protocols 25,34,37,38,46–48 . For NWs, aminated NWs were PEGylated with maleimide-5K-PEG-NH 2 (JenKem Technology, TX, USA), and peptides were coupled to NWs via a thioether bond between the thiol group of a cysteine residue and the N-terminus of the peptide. The concentration of NWs was determined by constructing a calibration curve with iron oxide, and the absorbance of NWs at 400 nm was measured using a NanoDrop 2000c spectrophotometer (Thermo Scientific). For AgNPs, CF647-N-hydroxysuccinimide-dye (NHS-dye) was conjugated to the terminal amine group of PEG, and biotinylated peptides were coated on the surface of the AgNPs through NeutrAvidin (NA; Sigma-Aldrich, USA). The nanoparticles were characterized using transmission electron microscopy (TEM, Tecnai 10, Philips, Netherlands) to image, and dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern Instruments, UK) to assess the zeta potential, polydispersity, and the size, as described previously. Cell Binding and Internalization Assay The U87-MG, PPC1, and M21 cells were cultured on coverslips and treated with CF555-labeled PL2 AgNPs or non-targeted control AgNPs at 37 °C for 1 hour, as previously described 34,35,49 . After removing unbound particles with culture medium, cells were treated with an etching solution (10 mM working concentration in PBS) made by diluting 0.2 M stock solutions of Na 2 S 2 O 3 and K 3 Fe (CN) 6 in a 1: 1 ratio for 3 minutes, followed by washing with PBS. Cells were fixed with methanol at -20 °C for 1-2 minutes, and thereafter the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes) at 1 µg/mL. Finally, the coverslips were mounted on microscopy slides using Fluoromount-G medium (Electron Microscopy Sciences) for confocal imaging. Tumor-targeted delivery and biodistribution studies FAM-labeled PL2 peptide-conjugated NW or control FAM-NW (7.5 mg/kg) in PBS were administered via tail vein injection to subcutaneous U87-MG, PC3, and orthotropic WT-GBM tumor-bearing mice. Five hours after circulation, the tumors and organs were collected via cardiac perfusion of mice under deep anesthesia with 20 ml PBS/DMEM. Macroscopic images of tissues were taken using an Illuminatool Bright Light System LT-9900 (Lightools Research, Encinitas, CA, USA) before snap-freezing. The frozen tissues were then cryosectioned with Leica CM1520 (Leica Camera AG, Germany) into 8-10 µm sections and mounted on Superfrost+ slides (Thermo Fisher Scientific, MA, USA). The tissue sections were equilibrated at room temperature and fixed with 4% paraformaldehyde/-20 °C methanol. Tissue staining was performed using primary antibodies, including rabbit anti-fluorescein IgG fragment (Cat # A889, RRID: AB_221561, Thermo Fisher Scientific, MA, USA), rat anti-mouse CD31 (RRID: AB_393571, BD Biosciences, CA, USA), and in-house prepared CF647/CF546-labeled single-chain antibodies ScFV L19. Secondary antibodies used were Alexa 488 goat anti-rabbit IgG (Cat # A-11034, RRID: AB_2576217), Alexa 647 goat anti-rat IgG (Cat # A-21247, RRID: AB_141778), and Alexa 546 goat anti-mouse IgG (Cat # A-11003, RRID: AB_2534071) from Invitrogen, CA, USA. The tissue nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes) at 1 μg/ml concentration. Coverslips were mounted on glass slides with Fluoromount-G (Electron Microscopy Sciences, PA, USA) and imaged using confocal microscopy (Olympus FV1200MPE, Hamburg, Germany). The resulting images were analyzed using FV10-ASW4.2 viewer/Imaris software/Fiji ImageJ. Ex vivo clinical tumor dipping assay In compliance with the Ethics Committee of the University of Tartu, Estonia (permit #243/T27), fresh surgical ovarian carcinoma samples were collected from consenting patients undergoing surgery according to relevant guidelines and regulations accordance with the Declaration of Helsinki 50 . To perform the dipping assay, the fresh ovarian carcinoma tissues were washed with DMEM, and 1 cm 3 explants were incubated at 37 °C with PL2-NW or non-targeted control NWs (40 mg/mL Fe in DMEM supplemented with 1% BSA) for four hours. The explants were then washed with PBS, snap-frozen, cryosectioned at 10 µm, and immunostained with rabbit anti-fluorescein primary antibodies, followed by detection with the Alexa-488 anti-rabbit secondary antibody (Invitrogen, Thermo Fisher Scientific, MA, USA). Statistical Analysis The statistical analysis was conducted using Prism 6 software (GraphPad Software, Inc, RRID:SCR_002798). For comparisons between two groups, a student’s unpaired t-test was used, while an ANOVA test was applied for comparisons involving multiple groups. For Continuous data, including quantifying FAM signal in tissue sections, the fluorescence signal intensity of antibody-amplified FAM was analyzed from 12–20 confocal images using Fiji ImageJ freeware (RRID:SCR_003070) and were analyzed using the student’s unpaired t-test. Data are presented as mean values, with error bars representing ±SEM. The data are presented as mean values with error bars showing ±SEM. The significance level was set at p < 0.05, and the P-values are denoted as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. The Samples were processed in a randomized order during analysis to prevent systematic bias. Group sizes were determined based on prior studies and standard practices in the field to ensure robust data collection and reproducibility. A priori power analysis was conducted to ensure the sample size was sufficient to detect biologically relevant differences, adhering to the 3R principles 51 and guidelines such as PREPARE 52 and ARRIVE 42 . Results Identification of Fn-EDB binding peptides For biopanning, His 6 -tailed Fn-EDB domain was expressed in E. coli and purified using Ni-NTA chromatography (Supplementary Fig. S1). Subsequently, 6 rounds of selection were performed on immobilized Fn-EDB using X7 peptide T7 phage libraries. The first and fourth rounds of biopanning were performed using Fn-EDB immobilized on polystyrene multiwell plates, and other rounds were performed on Fn-EDB coated on Ni-NTA magnetic beads (Supplementary Fig. S2). We observed enrichment of Fn-EDB binding of the phages through the screening, with round 6 pool showing ~3000-fold increased binding over the naive library (Fig. 1A). Sanger sequencing of 48 random phage clones from a selection round 5 resulted in 11 unique peptide-phages that were individually tested for their interaction with Fn-EDB. Among the 11 candidates, 5 clones demonstrated the ability to bind to Fn-EDB in vitro >50 fold over control phage displaying heptaglycine (Supplementary Fig. S3). To evaluate the systemic tumor homing of the in vitro selected Fn-EDB binding peptides, in vivo phage playoff auditioning was used 45 . An equimolar mixture of candidate and control peptide-phage were intravenously injected into a panel of glioma and prostate xenograft tumor-bearing mice and, following phage circulation and removal of blood background by perfusion, representation of peptide-phages in tumors was estimated using high-throughput DNA sequencing. We observed that the phage clone displaying the heptameric TSKQNSR peptide, designated PL2, was overrepresented in tested solid tumor models (Table 1). Interestingly, the C-terminal arginine of the PL2 peptide may engage with the "C-wall" binding pocket on the b1 domain of neuropilin-1 (NRP1) 53 . Next, the binding of the PL2 peptide-displaying phage to recombinant Fn-EDB, NRP1, and control proteins (Tenascin-C C-domain, TNC-C; Nucelolin, NCL) was studied. The PL2 phages demonstrated robust binding to Fn-EDB and b1b2 fragment of NRP1 and no binding to TNC-C, a protein with size and negative surface charge similar to Fn-EDB (Fig. 1B). Interestingly, the PL2 phage exhibited ~700-fold higher binding to the recombinant NRP-1 b1b2 domain compared to the heptaglycine control phage, demonstrating a binding capacity comparable to the prototypic NRP-1 binding peptide, RPAPRPAR 33 . The binding of the PL2 displaying phage to Fn-EDB surpassed that of the previously reported Fn-EDB-binding peptide, ZD2 26 (Supplementary Fig. S4). Phage-displayed Peptides in the "Playoff" Mix Representation of the phage in tumors or in control brain tissue (fold over G7 control phage) WT GBM P3 stem cell-like P13 U87-MG PC3 Normal brain Control GGGGGGG (G7) Control 1.0 1.0 1.0 1.0 1.0 1.0 Fn-EDB-selected (round 5) TKRKGKG Clone-2 2.7 0.7 0.8 2.0 1.0 0.2 GLGGRRIKLKTS Clone-3 0.8 0.7 1.3 0.6 1.5 0.1 GRRGRVIKLKTSEPPQ Clone-4 0.8 0.5 1.9 0.8 1.3 0.3 KVKKRGA Clone-17 1.5 0.3 1.6 1.0 0.7 0.1 RESRRGRVKLAAALE Clone-33 4.0 0.6 1.0 1.2 1.3 0.2 TSKQNSR Clone- 46 11.6 32.2 4.8 19.0 4.5 0 .4 CTVRTSADC ZD2 4.5 0.7 3.1 2.1 1.7 0.2 Table 1. In vivo playoff auditioning of Fn-EDB-selected peptides in tumor xenograft models in mice . An equimolar mixture of Fn-EDB-selected phages was intravenously injected into mice bearing orthotopic WT-GBM, P3 stem cell-like, P13, and s.c. implanted U87-MG glioblastoma, or PC3 prostate carcinoma xenografts at a dose of 1 × 10 10 pfu/mouse. After 2 h of circulation, background phages were removed by perfusion, and the representation of individual phages in tumor and control tissues was evaluated by Ion-Torrent high-throughput sequencing. Phages displaying PL2 (TSKQNSR) peptide showed the highest representation across tumor models tested in tumor tissue. The data represent the mean of 3 mice for each model. The binding of alanine-substituted PL2 derivative peptide displaying phages was evaluated to identify the essential amino acids involved in the interaction with Fn-EDB. Alanine substitution of the C-terminal arginine and serine significantly reduced the binding of the PL2 phage to recombinant Fn-EDB. Surprisingly, substitution of the N-terminal threonine, lysine, and asparagine enhanced the binding of the PL2 phage to Fn-EDB (Fig. 1C). As expected, the interaction with NRP-1 was dependent on the presence of C-terminal arginine, as the phage displaying PL2 with C-terminal R >A substitution showed a marked reduction in NRP-1 binding (Fig. 1C). We then evaluated the interaction of synthetic 5(6)-carboxyfluorescein (FAM)-labeled PL2 peptide with Fn-EDB immobilized on polystyrene ELISA plates. The synthetic PL2 peptide retained its ability to bind Fn-EDB (Fig. 1D). PL2 AgNPs bind to and are internalized by tumor cells in vitro We next studied cellular internalization of the PL2-functionalized AgNPs 54 . Extracellular membrane-bound AgNPs were selectively removed by treatment with a mild, biocompatible redox-based hexacyanoferrate/thiosulfate etching solution, ensuring that only the signal from internalized AgNPs remained detectable 48,54 . CF555-labeled PL2-AgNPs were incubated with the Fn-EDB and NRP-1-expressing U87-MG glioma cells, NRP-1-positive PPC1 prostate carcinoma cells, and Fn-EDB- and NRP-1-negative M21 melanoma cells 34,35,37,38 . PL2-AgNPs exhibited robust endocytosis in U87-MG and PPC1 cells following 1-hour incubation, whereas control particles displayed negligible uptake in both U87-MG and PPC1 cells (Fig. 2A, B, Supplementary Fig. S5). In contrast, receptor-negative M21 cells did not internalize PL2-AgNPs nor control AgNPs (Fig. 2A, Supplementary Fig. S5). Post-etching, the PL2-AgNP signal showed only a modest decrease in intensity compared to non-etched controls (Fig. 2A, B), confirming that the majority of PL2-AgNPs were taken up by the cells. Systemic PL2-functionalized nanoparticles accumulate in tumor lesions Next, we investigated the potential of the PL2 peptide as a systemic tumor-targeting probe. The dextran-coated PEGylated paramagnetic iron oxide nanoworms (NWs) have been used as a theranostic nanosystem suitable for systemic affinity targeting, functioning both as a drug carrier and as a magnetic resonance imaging agent with T2 contrast properties 38,47 . FAM-labeled PL2 peptide or FAM-Cys control was conjugated to NWs with an average size of 88.9 ± 0.9 nm and Zeta Potential of -9.5 ± 0.4 mV. The conjugation of peptides to NWs did not significantly alter particle size or surface charge (Supplementary Fig. S6, A-D) 27 . In vivo homing studies were conducted in mice bearing orthotopic WT-GBM glioma, s.c. U87-MG glioma and s.c. PC3 prostate carcinoma xenograft tumor models, all of which express abundant Fn-EDB and NRP-1 34,37,38 . Mice were intravenously injected with 7.5 mg/kg of NWs. After circulation, mice were perfused to remove free NWs, and tumors along with control organs were collected, sectioned, and imaged with confocal microscopy. PL2-functionalization significantly increased NW accumulation in CD31-positive vascular structures across all tumor models (Fig. 3A-C). In some regions, PL2-NWs extravasated and accumulated within the tumor parenchyma (Fig. 3A-C, arrowheads). Following 5 h of circulation, PL2-NWs demonstrated enhanced tumor accumulation compared to control NWs, with a ~7-fold increase in PC3 tumors, ~7.5-fold increase in U87-MG tumors, and a ~2-fold increase in WT-GBM tumors (Fig. 3A-C, Supplementary Fig. S7). In contrast, the signal for PL2-functionalized and non-targeted NWs was comparable in control organs, including the liver, kidney, and lung (Fig. 3A-C, Supplementary Fig. S7). Furthermore, PL2-NWs exhibited selective accumulation in glioblastoma lesions, with minimal accumulation in nonmalignant brain in the orthotopic WT-GBM model and in the healthy brain of control mice (Supplementary Fig. S8). Macroscopic fluorescence imaging showed accumulation of the PL2-NWs, but not control NWs, in U87-MG tumors collected from mice (Fig. 4A), while no signal was detected in the control organs (liver, lung, heart, brain, kidney, and spleen) (Fig. 4A, 4D). The tumor homing pattern of the PL2-NWs was also studied by confocal imaging of U87-MG tumor tissue sections after staining with Fn-EDB- (ScFV L19) and NRP-1-specific antibodies. Fn-EDB and NRP-1 were upregulated in the tumors (Fig. 4B, 4C, 4E, 4F), and the PL2-NW signal showed extensive overlap with the receptors (Fig. 4B, 4C, arrows). PL2-NWs bind to surgical explants of human clinical cancers To evaluate the potential translational relevance of the PL2 peptide, we examined the binding and penetration capabilities of PL2-targeted NWs in fresh surgical explants of human ovarian carcinoma tissue. We first established that in these tumors, Fn-EDB were highly overexpressed (Supplementary Fig. S9). Previous studies have consistently demonstrated the overexpression of NRP-1in ovarian cancers, linking it to malignancy and poor prognosis 55,56 . Next, we performed an ex vivo tumor binding/penetration assay ("tumor dipping assay") 57,58 , and observed that PL2-NWs demonstrated a 9-fold increase of binding and penetration in tumor tissue compared to the control NWs (Fig. 5A, 5B). In certain regions, PL2-NWs penetrated a few mm into the explants, though the binding was predominantly localized to the tumor surface (Fig. 5A). In contrast, control NWs exhibited only a background fluorescence signal. These findings suggest that the PL2-NW platform holds promise as a targeted drug delivery system for ovarian carcinoma and, potentially, other solid clinical tumors. Discussion We report the development of a homing peptide named PL2, which targets the tumor-associated ECM protein Fn-EDB, a stable and abundant antigen overexpressed in many solid tumors. In addition to binding Fn-EDB, PL2 engages the cell and tissue penetration receptor NRP-1, enabling nanoparticles to internalize into cultured cells, home to solid tumors in vivo , and penetrate clinical tumor samples ex vivo . These findings suggest potential applications of PL2 in targeting solid tumors and other conditions characterized by upregulation of Fn-EDB and NRP-1, such as endometriosis 59 . ECM-reactive affinity ligands have been used to deliver anticancer payloads such as cytokines/growth factors, proapoptotic peptides, cell-permeable cytotoxic compounds, and imaging agents to tumors 4,17,60,61 . However, ECM-targeting compounds, including those targeting Fn-EDB, rely on passive delivery through the enhanced permeability and retention (EPR) effect, which is subject to significant inter- and intratumoral variability 62,63 . Additionally, within the tumor microenvironment, ECM-targeting ligands generally exhibit limited cellular uptake, necessitating the use of cleavable linkers (e.g., disulfides or hydrazones) to release drug payloads into the extracellular milieu 61,64,65 . In this study, we employed cell-free phage biopanning to identify peptides that bind Fn-EDB. Unlike other selected peptides, PL2 peptide possessed the C-terminal arginine residue forming a minimal C-end Rule (CendR) element known to mediate binding a cellular pleiotropic multiligand receptor NRP-1 53 . Although the PL2 peptide-phage ranked fifth among 11 tested phages in the cell-free Fn-EDB binding assay, it outperformed all others in an in vivo playoff assay. The functionality of the CendR element in PL2 was demonstrated in in vitro studies, where AgNPs functionalized with the PL2 peptide exhibited strong NRP-1-dependent cellular binding and internalization, consistent with our pervious finding for the PL3 peptide 30 . In vivo , functionalizing nanoparticles with the PL2 peptide significantly enhanced their tropism to prostate carcinoma and glioblastoma, with significant accumulation in the extravascular space and colocalization with both Fn-EDB and NRP-1 immunoreactivities. Finally, PL2-targeted nanoparticles effectively bound to and penetrated clinical surgical explants ex vivo , suggesting a translational potential for the targeting system. This finding is not unexpected as small homing peptides typically target evolutionarily conserved binding pockets on their target molecules and the 91-amino acid alternatively spliced EDB domain is conserved across multiple species, including mice, rats, rabbits, dogs, monkeys, and humans 27,66 . Additionally, CendR peptides consistently bind to the b1 domain of NRP-1 across these species, from mouse to human. These studies, in the context of well-established knowledge of the upregulation of Fn-EDB and NRP-1 in various primary solid tumors and metastatic lesions, highlight the potential of PL2-based nanoparticle targeting strategy for application across a range of solid tumor types 15,67 . Although our studies show that the PL2 peptide appears a promising targeting agent for nanoparticles, there is still room for improvement and opportunities to broaden the range of applications. Short homing peptides with a high degree of conformational freedom and small number of contact residues have typically moderate affinity yet are capable of high avidity interactions with their targets due to cooperative multivalent binding when displayed on nanoparticles 6,68,69 . This is illustrated by complement factor C1q-IgG interactions, where the dissociation constant (Kd) improves dramatically with increasing valency—100 μM for monomers, 1 μM for dimers, and 3 nM for tetramers 70 . Interestingly, our alanine scan revealed that at certain positions, particularly the N>A substitution at position 5, alanine substitution enhanced peptide phage binding to recombinant receptor molecules. In follow-up studies to improve the affinity of the PL2 peptide, it will be valuable to conduct secondary screens using constrained peptide libraries, created by randomizing nonessential amino acids of the PL2 peptide and/or adding additional flanking amino acids to provide stabilizing interactions. Another potential avenue for improving the PL2 peptide involves enhancing its tumor specificity. While NRP-1 is overexpressed in solid tumors, it is present at lower levels in the vascular beds of normal organs, particularly the lungs, which could result in off-target accumulation. To address this, further improvement of the PL2 peptide could include capping the C-terminal arginine with additional amino acids that are engineered or screened to be cleaved by tumor-expressed extracellular proteases. Using a similar approach, we recently reported the development of a urokinase-type plasminogen activator-dependent CendR peptide to mitigate background accumulation in nontarget tissues 31 . Such continuation studies could further facilitate the applications of PL2 as a cancer targeting agent. Many homing peptides, by binding to functionally important binding pockets on target molecules, trigger biological responses through mechanisms such as eliciting conformational changes or competing with natural ligands for receptor binding. Examples include the antitumor effects of LyP-1 71 , tumor penetration induction and immunomodulation of iRGD 72,73 , wound healing promotion by CAR 74 , and suppression of choroidal neovascularization by PL3 30,75 . Similarly, in addition to its use in affinity targeting, the PL2 peptide may possess inherent biological activity by disrupting interactions between Fn-EDB and integrins or other matrix components involved in adhesion, migration, and survival of malignant or tumor-promoting cells, potentially suppressing tumor growth, metastasis, and angiogenesis. If the biological activity of PL2 is confirmed, its improved variants, developed using the strategies outlined above, and proteolysis-resistant PL2 derivatives incorporating nonproteinogenic amino acids, will be advantageous for further applications. In conclusion, our study presents the development of the PL2 peptide, which holds potential for applications in targeted drug delivery and molecular imaging. The dual targeting of Fn-EDB and NRP-1 by the PL2 peptide offers a promising strategy for advancing targeted cancer therapies. The use of PL2 nanoparticles to tumor tissues shows promise for the development of novel cancer treatments. Further studies are needed to optimize the peptide and assess its clinical efficacy in targeting solid tumors as well as safety. Declarations Conflicts of interest The data supporting this study's findings are available from the corresponding author upon request. T.T. and P.L. hold a patent on the PL2 peptide ("Bi-Specific Extracellular Matrix Binding Peptides and Methods of Use Thereof", WO. patent no. WO 2020/161602 A1), and TT is an inventor of CendR peptides. Other authors declare that they have no competing interests. Ethics and Informed Consent Statement The fresh surgical ovarian carcinoma samples were obtained under protocols approved by the Ethics Committee of the University of Tartu, Estonia (permit #243/T27). Additionally, informed consent was obtained from all the patients, and methods for using human samples were carried out under relevant guidelines and regulations accordance with the Declaration of Helsinki. Animals and Ethics Statement Animal experimentation procedures were approved by the Estonian Ministry of Agriculture, Committee of Animal Experimentation, projects #42 and #48 (IACUC). We confirm that all methods were performed in accordance with the relevant guidelines and regulations. We confirm that the study was conducted in accordance with the ARRIVE guidelines. Data Availability Statement The datasets generated and/or analysed during the current study are included in this article and its supplementary information files. No additional data are required to be deposited in public repositories. Any additional data supporting the findings of this study are available from the corresponding author upon reasonable request. Acknowledgments T.T. was funded by the Estonian Research Council (grants PRG230 and PRG1788), EuronanomedIII projects ECM-CART and iNanoGun, and TRANSCAN3 project ReachGLIO (all coordinated by Estonian Research Council) . 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Chemotherapeutic drug delivery by tumoral extracellular matrix targeting. J Control Release 274 , 1–8 (2018). Maeda, H. Macromolecular therapeutics in cancer treatment: The EPR effect and beyond. Journal of Controlled Release vol. 164 138–144 Preprint at https://doi.org/10.1016/j.jconrel.2012.04.038 (2012). Prabhakar, U. et al. Challenges and key considerations of the enhanced permeability and retention effect for nanomedicine drug delivery in oncology. Cancer Res 73 , 2412–2417 (2013). Dal Corso, A., Cazzamalli, S., Gébleux, R., Mattarella, M. & Neri, D. Protease-Cleavable Linkers Modulate the Anticancer Activity of Noninternalizing Antibody-Drug Conjugates. Bioconjug Chem 28 , 1826–1833 (2017). Gébleux, R., Wulhfard, S., Casi, G. & Neri, D. Antibody format and drug release rate determine the therapeutic activity of non-internalizing antibody-drug conjugates. Mol Cancer Ther 14 , 2606 (2015). Zardi, L. et al. Transformed human cells produce a new fibronectin isoform by preferential alternative splicing of a previously unobserved exon. EMBO J 6 , 2337–2342 (1987). Locher, R. et al. Abundant in vitro expression of the oncofetal ED-B-containing fibronectin translates into selective pharmacodelivery of 131I-L19SIP in a prostate cancer patient. J Cancer Res Clin Oncol 140 , 35–43 (2014). Zhao, N., Qin, Y., Liu, H. & Cheng, Z. Tumor-Targeting Peptides: Ligands for Molecular Imaging and Therapy. Anticancer Agents Med Chem 18 , 74–86 (2017). Fosgerau, K. & Hoffmann, T. Peptide therapeutics: Current status and future directions. Drug Discov Today 20 , 122–128 (2015). Male, D., Champion, B. & Cooke, A. Advanced Immunology. Parasitology 96 , 643–644 (1987). Laakkonen, P. et al. Antitumor activity of a homing peptide that targets tumor lymphatics and tumor cells. Proc Natl Acad Sci U S A 101 , 9381–9386 (2004). Sugahara, K. N. et al. Tumor-penetrating iRGD peptide inhibits metastasis. Mol Cancer Ther 14 , 120–128 (2015). Sugahara, K. N. et al. Tissue-Penetrating Delivery of Compounds and Nanoparticles into Tumors. Cancer Cell 16 , 510–520 (2009). Maldonado, H. et al. Systemically administered wound-homing peptide accelerates wound healing by modulating syndecan-4 function. Nat Commun 14 , (2023). Puranen, J. et al. Intravitreal CendR peptides target laser-induced choroidal neovascularization sites in mice. Journal of Controlled Release 360 , 810–817 (2023). Additional Declarations Competing interest reported. T.T. and P.L. hold a patent on the PL2 peptide ("Bi-Specific Extracellular Matrix Binding Peptides and Methods of Use Thereof", WO. patent no. WO 2020/161602 A1), and TT is an inventor of CendR peptides. Other authors declare that they have no competing interests. Supplementary Files SupplementaryMaterials311224PL2.pdf Cite Share Download PDF Status: Published Journal Publication published 11 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 11 Jun, 2025 Reviews received at journal 09 Jun, 2025 Reviewers agreed at journal 06 Jun, 2025 Reviewers agreed at journal 05 Jun, 2025 Reviews received at journal 03 Jun, 2025 Reviewers agreed at journal 03 Jun, 2025 Reviewers invited by journal 03 Jun, 2025 Editor assigned by journal 03 Jun, 2025 Editor invited by journal 20 May, 2025 Submission checks completed at journal 20 May, 2025 First submitted to journal 31 Dec, 2024 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. 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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-5743295","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":466690002,"identity":"3f592cc8-c8ab-4b8f-bb7c-d1daed0372c9","order_by":0,"name":"Prakash Lingasamy","email":"","orcid":"","institution":"Celvia CC AS","correspondingAuthor":false,"prefix":"","firstName":"Prakash","middleName":"","lastName":"Lingasamy","suffix":""},{"id":466690003,"identity":"1fe9a77c-61ee-44a2-8167-ef801d31e159","order_by":1,"name":"Allan Tobi","email":"","orcid":"","institution":"University of Tartu","correspondingAuthor":false,"prefix":"","firstName":"Allan","middleName":"","lastName":"Tobi","suffix":""},{"id":466690004,"identity":"155275a9-03c0-41b6-8916-dc695b407323","order_by":2,"name":"Kaarel Kurm","email":"","orcid":"","institution":"University of Tartu","correspondingAuthor":false,"prefix":"","firstName":"Kaarel","middleName":"","lastName":"Kurm","suffix":""},{"id":466690005,"identity":"bfa849a3-ae20-438e-b4bd-8e7ebdebdf72","order_by":3,"name":"Olav Tammik","email":"","orcid":"","institution":"North Estonia Medical Centre","correspondingAuthor":false,"prefix":"","firstName":"Olav","middleName":"","lastName":"Tammik","suffix":""},{"id":466690006,"identity":"39d8d464-d863-4619-b5f0-c4e1562d93cc","order_by":4,"name":"Tambet Teesalu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYHACNgTzAYMNYfU8YC0JDBJgXgJDGkiENC2HCWuxZz+d9uDjD4Y6fvb2hx8S284n7mdgPvgAry08udsNZwBtkew5YyyR2HY7sYeBLdkAv8Nyt0nzALUY3MhhgGrhMZPAq4X/7TbpP0At9vefP/6R2HYOqIX/+w+8WiSAtoC8byDBYAa05QDIFjZ8Ohh4brzdbtiTJiE540yOmUXCuWTjnsNsxngdxt6fu+3BDxsbfv72449vfCizk21vb374Aa81EIBsLDMR6kfBKBgFo2AU4AcACsdEkfOJc48AAAAASUVORK5CYII=","orcid":"","institution":"University of Tartu","correspondingAuthor":true,"prefix":"","firstName":"Tambet","middleName":"","lastName":"Teesalu","suffix":""}],"badges":[],"createdAt":"2024-12-31 17:38:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5743295/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5743295/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-11299-x","type":"published","date":"2025-08-11T15:58:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84337986,"identity":"646c3ccc-14c0-4efb-943e-ca2d1b2f4e5a","added_by":"auto","created_at":"2025-06-10 18:04:26","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":67726,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification and characterization of the PL2 peptide binding to Fn-EDB and NRP-1. (A) Biopanning of T7 phage-displayed X7 peptide libraries on immobilized Fn-EDB resulted in ~3000-fold enrichment in phage binding after the sixth round of selection. (B) The selected PL2 peptide phage bound to immobilized Fn-EDB and NRP-1 b1b2 but not to control protein TNC-C, or Nucleolin. Phage binding is expressed as fold over control phage displaying heptaglycine (G7) peptide. (C) Alanine substitutions were introduced at each position of the PL2 peptide, and changes in binding were verified by measuring the percentage of changes in binding to immobilized Fn-EDB and NRP-1. The first peptide from the bottom without red in the box represents the parental PL2 peptide. (D) The interaction of synthetic FAM-PL2 peptide immobilized on an ELISA plate with recombinant His-tagged Fn-EDB was probed and detected using a rabbit anti-His-tag primary antibody and secondary goat anti-rabbit HRP antibody, followed by chromogenic peroxidase reaction. Values are expressed as mean ± standard deviation (SD) from 3 independent experiments; statistical analysis was performed using Student unpaired t-test; all statistical tests were two-sided; *p \u0026lt; 0.05; **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5743295/v1/70b3d30984598d9d91334272.jpg"},{"id":84338986,"identity":"4f996de4-083f-40dd-8b7a-1b07cc0a0968","added_by":"auto","created_at":"2025-06-10 18:12:27","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":945030,"visible":true,"origin":"","legend":"\u003cp\u003eCellular internalization of PL2 peptide-functionalized AgNPs. The PL2 AgNPs and control AgNPs labeled with CF555 fluorophore dye were incubated with PPC1 prostate carcinoma, U87-MG glioma, and M21 melanoma cells for 1 h. After washing and optional etching, the cells were processed for confocal imaging. (A) Confocal images show strong uptake of PL2-AgNPs (red) in NRP-1-positive PPC1 and U87-MG cells but not in NRP-1-negative M21 cells. Cells incubated with control particles are shown in boxes. Scale bar: 20 µm. (B) Quantification of binding and internalization of CF555-labeled particles was done using Fiji ImageJ software. Data represents 3 independent experiments. Error bars indicate mean ± SD (N = 3). Statistical significance was determined using unpaired Student's t-test (ns, p \u0026gt; 0.05; **p ˂ 0.01; ***p ≤ 0.001; ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5743295/v1/c0783783d84b7fe8c18b6e74.jpg"},{"id":84337989,"identity":"85f25689-0d06-40cc-ab6f-1955e8b44315","added_by":"auto","created_at":"2025-06-10 18:04:27","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1141823,"visible":true,"origin":"","legend":"\u003cp\u003eSystemic PL2-NWs accumulate in solid tumors. (A, B, C) PL2-NWs and control FAM-NWs were intravenously injected at 7.5 mg/kg into mice bearing (A) subcutaneous PC3 prostate carcinoma, (B) subcutaneous U87-MG glioblastoma, and (C) orthotopic WT GBM glioblastoma xenografts. After 5 h of circulation, mice were perfused with PBS/DMEM, and organs (including tumors and control tissue) were snap-frozen. Cryosections of tissues were immunostained with rabbit anti-FAM (green) and rat anti-CD31 (red) antibodies, counterstained with DAPI (blue), and analyzed using confocal microscopy. The boxes represent mice injected with control non-targeted FAM-NWs. The arrows indicate PL2-NWs (green) located along the CD31-positive tumor blood vessels, and the arrowheads indicate extravasated PL2-NWs in the tumor parenchyma. Bar charts show quantification of tissue homing of FAM signal using Fiji ImageJ software (A-C). Error bars indicate mean ± SEM (N = 3-6 mice per group). Scale bars: 100 μm. P-values were determined using Student unpaired t-test, two-tailed; ns, not significant (p \u0026gt; 0.05); *p \u0026lt; 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5743295/v1/9df41c48b365d9b8ea577f49.jpg"},{"id":84337987,"identity":"0e223b48-7e4e-4f6c-bc17-632a1d470868","added_by":"auto","created_at":"2025-06-10 18:04:27","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":928793,"visible":true,"origin":"","legend":"\u003cp\u003ePL2-NWs accumulate in glioblastoma lesions and colocalize with Fn-EDB and NRP-1. (A–C, upper row) PL2-functionalized NWs and (D–F, lower row) control NWs were injected i.v. at 7.5 mg/kg into mice bearing s.c. U87-MG glioblastoma xenografts. After 5 h of circulation, mice were perfused with PBS/DMEM, and tissues were harvested for \u003cem\u003eex vivo\u003c/em\u003e macroscopic imaging and fluorescence microscopy. (A, D) \u003cem\u003eEx vivo\u003c/em\u003emacroscopic Illumatool images of PL2- and control NWs in the green channel. The superimposed images of white light and green fluorescence channels are representative of three independent experiments. (B, C, E, F) Confocal microscopy images of NWs, Fn-EDB, and NRP-1 in tumor cryosections. PL2-NWs colocalized with Fn-EDB and NRP-1 (arrows), whereas (E, F) FAM-NWs showed only background accumulation. Tumor tissues were stained with rabbit anti-FAM (green), anti-Fn-EDB ScFv L19 (red), and rabbit anti-NRP-1 (red) antibodies. PL2-NWs (B, C) colocalize with Fn-EDB and NRP-1 (indicated by arrows), whereas control NWs (E, F) exhibit only background accumulation. Scale bar, 100 µm; the images are representative of 3 independent experiments.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5743295/v1/e5713ce55663c77474000add.jpg"},{"id":84337990,"identity":"95e53515-0c22-47ff-b687-e3a1e9a6710a","added_by":"auto","created_at":"2025-06-10 18:04:27","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":648266,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConfocal fluorescence imaging of PL2-NW binding to human ovarian carcinoma cancer explants.\u003c/strong\u003e Fresh surgical explants of human ovarian carcinoma were incubated with PL2-NWs and control NWs at 37 °C for 4 h. The tissue sections were then stained with rabbit anti-FAM primary antibodies (green) and Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. The nuclei were counterstained with DAPI (blue). The arrows indicate PL2-NW signals at the tumor rim and extravasation into the tissue. (B) Quantification of FAM fluorescence signal from the confocal images using Fiji ImageJ. Error bars represent mean ± SEM (N = 3). Statistical analysis was performed using Student's t-test; ** p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5743295/v1/783009ce16f6649fc9aa467b.jpg"},{"id":89310846,"identity":"85d15e35-45ad-4809-9037-d2684a99844b","added_by":"auto","created_at":"2025-08-18 16:10:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4752015,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5743295/v1/336afbd7-2dce-4c5d-8964-4b7b5927a427.pdf"},{"id":84337991,"identity":"bd3a7123-1490-4ed4-9fb1-2e31b944fcc9","added_by":"auto","created_at":"2025-06-10 18:04:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4997161,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials311224PL2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5743295/v1/4f7e8a5637c7ad83a0a89b53.pdf"}],"financialInterests":"Competing interest reported. T.T. and P.L. hold a patent on the PL2 peptide (\"Bi-Specific Extracellular Matrix Binding Peptides and Methods of Use Thereof\", WO. patent no. WO 2020/161602 A1), and TT is an inventor of CendR peptides. Other authors declare that they have no competing interests.","formattedTitle":"Targeting Oncofetal Fibronectin and Neuropilin-1 in solid tumors with PL2 peptide","fulltext":[{"header":"Significance","content":"\u003cp\u003eWe developed the novel PL2 peptide via phage display, demonstrating selective dual-targeting of Fibronectin Extra-Domain-B (Fn-EDB) and Neuropilin-1 (NRP-1), key markers of the tumor microenvironment. This targeting enhances nanoparticle homing, deep-tissue penetration, and therapeutic payload delivery in solid tumors within preclinical models. Its ability to bind and penetrate clinical ovarian carcinoma tissues underscores its translational potential for precision oncology, paving the way for safer, effective cancer therapies with further optimization.\u003c/p\u003e"},{"header":"Introduction ","content":"\u003cp\u003eTumor blood vessels have emerged as critical targets for therapeutic intervention. Therapeutic strategies include suppressing the growth or perturbing functions of the neovessels that sustain tumor lesions \u003csup\u003e1,2\u003c/sup\u003e and leveraging the unique characteristics of tumor blood vessels to enhance the delivery of cytotoxic anticancer agents via specialized drug delivery systems. Therapeutic agents can be directed to malignant cells in the tumor microenvironment by using affinity ligands such as antibodies, peptides, aptamers, or small molecules that engage tumor-associated markers \u003csup\u003e3,4\u003c/sup\u003e. The accessibility of target receptors from the bloodstream and their adequate expression level are prerequisites for any effective affinity-targeted therapy \u003csup\u003e4\u003c/sup\u003e. Current affinity-targeting strategies primarily engage the cell surface biomarkers on malignant and tumor endothelial cells. Targeting the extracellular matrix (ECM) in the tumor microenvironment is less common despite its potential advantages. ECM components are estimated to be ~10-fold more abundant than cellular receptors and offer a higher capacity for drug delivery \u003csup\u003e5,6\u003c/sup\u003e. Compared to receptors overexpressed on the surface of malignant cells, ECM molecules provide opportunities for more stable targeting as they are predominantly deposited by genetically stable nonmalignant cells \u003csup\u003e7\u003c/sup\u003e. To some degree, ECM targeting peptides allow for intracellular delivery due to their ability to exploit the natural cellular uptake pathways associated with cell-matrix interactions, as shown for the PL1 peptide that uses macropinocytosis for cellular entry \u003csup\u003e7\u003c/sup\u003e. The ECM provides a dynamic physical and biochemical microenvironment that actively regulates essential cellular functions such as adhesion, proliferation, and migration, thereby influencing cellular developmental pathways \u003csup\u003e8,9\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOverexpression of alternatively spliced ECM isoforms is associated with cancer, and specific ECM splicing variants have been linked to stromal activation \u003csup\u003e10\u003c/sup\u003e. Fibronectin Extra Domain-B (Fn-EDB), an acidic 91-amino acid splice variant of fibronectin generated by type III homology repeats, is a key component of the angiogenic signature, distinguished by its overexpression in solid tumors and absence in most adult tissues, except for the female reproductive tract \u003csup\u003e11,12\u003c/sup\u003e. A comparative analysis of Fn-EDB expression in ~18,800 cancer samples and ~4,500 normal samples demonstrated upregulation of Fn-EDB in 15 types of cancer, including in grade I to IV malignant gliomas \u003csup\u003e13\u003c/sup\u003e. In addition, Fn-EDB is a specific marker for tumor neovessels \u003csup\u003e14\u003c/sup\u003e. To date, several classes of Fn-EDB targeting ligands, including antibodies, peptides, and aptamers, have been applied to deliver therapeutic agents, such as cytokines, cytotoxic agents, chemotherapeutic drugs, and radioisotopes, to Fn-EDB-expressing tumors \u003csup\u003e15\u0026ndash;17\u003c/sup\u003e. The Fn-EDB targeting L19 antibody and its derivatives (diabodies, Single-chain variable fragment (scFv), Small immunoprotein (SIP)) have exhibited potential in preclinical and clinical studies in EDB-FN-positive cancer patients using both systemic and intratumoral administration routes \u003csup\u003e18\u003c/sup\u003e. Daromun, an intralesional immunocytokine combining IL-2 and TNF conjugated to the L19 antibody, has shown effectiveness in local tumor destruction and treating distant disease through an immune-mediated mechanism in early clinical studies \u003csup\u003e19\u0026ndash;21\u003c/sup\u003e. In contrast to antibodies, short peptides offer easy synthesis, low immunogenicity, low cost, biocompatibility, and moderate affinity, which helps to circumvent the affinity site barrier \u003csup\u003e22\u0026ndash;25\u003c/sup\u003e. Peptides, such as linear PL1 and cyclic ZD2, specifically bind to Fn-EDB and have been used to target intracranial glioblastomas and prostate cancer, respectively \u003csup\u003e26,27\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOne potential limitation in the application of matrix targeting ligands for tumor delivery is the variability of accessibility in the ECM to circulating ligands. The accessibility, influenced by the degree of tumor blood vessel leakiness, generally correlates with histological grade and malignancy but varies significantly both within individual tumors and across tumor types. Several pharmacological strategies have been employed to increase tumor vascular permeability and improve tumor delivery (e.g., drugs modulating tumor blood pressure, inflammatory cytokines, and bradykinin mediators) \u003csup\u003e28,29\u003c/sup\u003e. We investigated the pharmacological stimulation of extravasation to enhance tumor ECM exposure to circulating targeting ligands by designing bispecific ligands that simultaneously bind the ECM molecule Tenascin-C and the cell and tissue penetration receptor neuropilin-1 (NRP-1) \u003csup\u003e30,31\u003c/sup\u003e. The engagement of the peptide with NRP-1 induced extravasation and tissue penetration via a mechanism involving cellular entry and vascular transcytosis through the C-end Rule (CendR) pathway \u003csup\u003e32\u0026ndash;35\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHere, we used peptide-phage display on the recombinant Fn-EDB domain to identify a novel heptameric PL2 peptide that interacts specifically with Fn-EDB. The peptide also engages the tissue penetration receptor NRP-1 via the C-terminal arginine containing motif of the peptide, promoting cellular uptake \u003cem\u003ein vitro\u003c/em\u003e and penetration of cell and tissue barriers \u003cem\u003ein vivo\u003c/em\u003e. The \u003cem\u003ein vivo\u003c/em\u003e phage playoff and systemic PL2-guided iron oxide nanoparticles showed specific accumulation in a panel of tumor xenografts implanted in mice. Our study suggests that PL2-guided agents can be used for detection, imaging, and payload delivery to solid tumors positive for the expression of Fn-EDB and NRP-1.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch2\u003eMaterials\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003ePhosphate-buffered saline (PBS) was purchased from Lonza (Verviers, Belgium). K\u003csub\u003e3\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e], HCl, isopropanol, Triton-X, Tween-20, CHCl\u003csub\u003e3\u003c/sub\u003e, MeOH, Isopropyl β-D-1-thiogalactopyranoside (IPTG), and dimethylformamide (DMF) were purchased from Sigma-Aldrich (Munich, Germany).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003ePeptides and proteins\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe peptides and proteins used in the study were Cys-5(6)-carboxyfluorescein (FAM)-PL2, biotin-PL2, Cys-FAM peptides, and biotin with 6-aminohexanoic acid spacer, which were purchased from TAG Copenhagen (Denmark). The plasmids pASK75-Fn7B8 and pASK75-Fn789 were kindly provided by Prof. Dr. Arne Skerra\u0026nbsp;\u003csup\u003e36\u003c/sup\u003e. The gene fragment of Fn-EDB domain was amplified from the plasmids and cloned into a pET28a+ plasmid containing a N-terminal His\u003csub\u003e6\u003c/sub\u003e-tag for expression in \u003cem\u003eE. coli\u003c/em\u003e strain BL21 Rosetta™ 2 (DE3) pLysS (Novagen, #70956). Recombinant Fn-EDB was produced as a soluble protein and purified using the HisTrap IMAC HP column (GE Healthcare, #17-0920-05)\u0026nbsp;as previously\u0026nbsp;described\u0026nbsp;\u003csup\u003e37,38\u003c/sup\u003e. SDS-PAGE and mass spectrometry (MS) analyses were used to determine proteins purity, size, and sequence. The NRP-1 b1b2 domain protein was expressed and purified at the Protein Production and Analysis Facility at the Sanford Burnham Prebys Medical Discovery Institute (La Jolla, CA, US), and the NRP-1 b1 domain protein was expressed and purified in-house. Cloning, expression, purification of proteins (FN-EDB, TNC-C, NRP1, NCL and single chain antibodies FN-EDB-L19), and generation of polyclonal rabbit antibodies are described in Supplementary Information, Materials and Methods section and PL1 study protocols\u0026nbsp;\u003csup\u003e27\u003c/sup\u003e.\u003c/p\u003e\n\u003ch2\u003eCell lines and experimental animals\u003c/h2\u003e\n\u003cp\u003eThe study utilized human glioblastoma (U87-MG, HTB-14, RRID: CVCL_0022) and prostate carcinoma (PC3, CRL1435, RRID: CVCL_0035) cells purchased from ATCC (VA, USA). Murine wild-type glioblastoma (WT-GBM) cells were kindly provided by Gabriele Bergers (UCSF, USA), and stem cell-like cancer cells\u0026nbsp;P3, P13 were gifted by Rolf Bjerkvig (University of Bergen, Norway). The M21 (RRID: CVCL_D031) melanoma cells were the gift of David Cheresh (USA). The cells and tumors were prepared as described in previous studies\u0026nbsp;\u003csup\u003e34,35,37–41\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAthymic nude mice (Hsd/Athymic Fox1 nu Harlan) were purchased from Harlan Sprague Dawley (HSD, Indianapolis, IN, USA) and maintained under standard housing conditions of the Animal Facility of the Institute of Biomedicine and Translational Medicine, University of Tartu (Tartu, Estonia). Inclusion and exclusion criteria for animals were based on standard experimental conditions and we selected age-matched male and female nude mice (11-15 weeks old). The orthotopic glioblastoma (GBM) tumor models were established using P13, P3 stem cell-like, and WT-GBM cells. Approximately 2–3 × 10⁵ cells suspended in 3 μL of PBS were intracranially implanted into mice brain 2 mm right and 1 mm anterior to the bregma.\u0026nbsp;\u003cem\u003eFor the subcutaneous models,\u0026nbsp;\u003c/em\u003e2\u003cem\u003e-\u003c/em\u003e9 × 10\u003csup\u003e6\u003c/sup\u003e \u003cem\u003eU87-MG GBM and prostate carcinoma (PC3) cells\u0026nbsp;\u003c/em\u003ein 100 µl of PBS\u003cem\u003e\u0026nbsp;were subcutaneously (s.c.) implanted in the right flank of 11\u003c/em\u003e\u003cem\u003e-\u003c/em\u003e\u003cem\u003e15-week-old male and female nude mice\u003c/em\u003e. No attrition was noted during the study, as all animal samples were included as per protocol.\u0026nbsp;No blinding was used during the experiment and study did not include treatment groups; power calculations were not applicable. Our experimental design ensures that minimal bias (or noise) by ensuring same sex, similar animal weight at start of experiment, same animal age, same type of stabling, several animals caged together.\u003c/p\u003e\n\u003cp\u003eAnimal experimentation procedures were approved by the Estonian Ministry of Agriculture, Committee of Animal Experimentation, projects #42 and #48. We confirm that all methods were performed in accordance with the relevant guidelines and regulations. We confirm that the study was conducted in accordance with the ARRIVE guidelines\u0026nbsp;\u003csup\u003e42\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eT7 phage peptide library biopanning\u003c/h2\u003e\n\u003cp\u003eWe employed T7-Select\u003csup\u003e®\u003c/sup\u003e phage display system (Novagen, EMD Biosciences, MA, USA) to generate NNK-encoded X7 peptide phage libraries with a diversity of ~\u0026nbsp;5 × 10\u003csup\u003e8\u003c/sup\u003e for biopanning on recombinant Fn-EDB. The first round of selection was carried out on Fn-EDB immobilized on a Costar 96-Well enzyme-linked immunosorbent assay (ELISA) plate (#3590, Corning Life Sciences, MA, USA) by coating the plate with 20 µg/ml recombinant Fn-EDB protein in 100 µl of PBS overnight at 4 °C.\u0026nbsp;The plate was then blocked with 1% bovine serum albumin (BSA) in PBS overnight at 4 °C. The phage library solution (5 × 10\u003csup\u003e8\u003c/sup\u003e pfu in 100 µl of PBS-BSA) was incubated overnight at 4 °C, followed by 6 washes with PBS + BSA + 0.1% Tween 20 to remove non-specifically bound background phages. The bound phages were rescued and amplified in \u003cem\u003eE. coli\u003c/em\u003e strain BLT5403 (Novagen, MA, USA) \u003csup\u003e43\u003c/sup\u003e. The subsequent selection rounds were performed with His\u003csub\u003e6\u003c/sub\u003e-tagged Fn-EDB protein (30 µg/10 µl beads) immobilized on Ni-NTA Magnetic Agarose Beads (QIAGEN, Hilden, Germany) at room temperature for 1 hour in 400 µl of PBS. The Fn-EDB immobilized beads were washed three times with PBS + BSA + 0.1% NP40, followed by incubation with the phage from the previous round (5 × 10\u003csup\u003e8\u003c/sup\u003e pfu in 100 µl of PBS + BSA + 0.1% NP40) for 1 hour at room temperature. The background and weakly bound phages were removed by rinsing six times with PBS + BSA + 0.1% NP40, and the bound phages were eluted with 1 ml of PBS + 50 0mM imidazole + 0.1% NP40. The recovered phages were titered and amplified for a subsequent round of selection. After 6 rounds of selection, peptide-encoding phage DNA from a randomly selected set of 48 clones from round 5 was subjected to Sanger sequencing to obtain information on the displayed peptides \u003csup\u003e43,44\u003c/sup\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor cell-free binding studies with individually amplified phage clones, phages were incubated with Fn-EDB coated magnetic beads as described above. Furthermore, the GRPARPAR phage on NRP-1 coated beads was used as a positive control\u0026nbsp;\u003csup\u003e33\u003c/sup\u003e, while\u0026nbsp;the Nucleolin\u0026nbsp;(NCL) and\u0026nbsp;the C-domain of Tenascin\u0026nbsp;C (TNC-C) were used as negative controls. Finally, we used phage displaying heptaglycine peptide (GGGGGGG, G7) or insertless phage clones for negative controls. \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eIn vivo\u003c/em\u003e playoff phage auditioning\u003c/h2\u003e\n\u003cp\u003eTo assess the systemic homing of peptide-phages to xenograft tumor models, we employed an internally controlled competitive assay that we have termed \u003cem\u003ein vivo\u003c/em\u003e peptide-phage playoff \u0026nbsp;\u003csup\u003e45\u003c/sup\u003e. Briefly, the candidate Fn-EDB binding peptides, together with previously published tumor-homing peptides and control peptides, were amplified and purified by PEG-8000 precipitation, CsCl gradient ultracentrifugation, and dialysis. The equimolar pooled peptide-phages were intravenously injected into tumor-bearing mice at a concentration of 1 × 10¹⁰ pfu in 200 µL of PBS. After 2 hours of circulation, the mice were anesthetized by intraperitoneal (i.p.) injection of 350 μL containing 0.1 mg/kg dexmedetomidine and 75 mg/kg ketamine dissolved in saline or 3-4% isoflurane. Following anesthesia, the mice were perfused intracardially with Dulbecco’s Modified Eagle Medium (DMEM; Lonza Ltd, Basel, Switzerland). Tumors and organs were collected in LB + 1% NP40, and tissue homogenization was carried out to rescue peptide-phages. The lysates were then amplified in \u003cem\u003eE. coli\u003c/em\u003e, purified through precipitation with PEG-8000, and the DNA was extracted using a DNA extraction kit (High Pure PCR Template Preparation Kit; Roche, Basel, Switzerland). To evaluate the representation of each phage in the input mixture, tumor, and control organs, we performed next-generation sequencing of phage genomic DNA using the Ion Torrent high-throughput DNA sequencing system (Thermo Fisher Scientific, Waltham, MA, USA). The FASTQ data from Ion Torrent were processed using a custom Python (RRID:SCR_024202) script that identified the barcodes and constant flanking residues, and extracted the correct length reads. Table 1 provides a detailed list of the equimolarly pooled phage peptides used in this study.\u003c/p\u003e\n\u003ch2\u003ePeptide Binding Assay\u003c/h2\u003e\n\u003cp\u003eELISA plates (Nunc Maxisorp, Thermo Fisher Scientific Inc., MA, USA) were coated with 20 µg of PL2 peptide labeled with FAM in 100 µl of PBS and incubated at 37 °C overnight. The plate wells were blocked with 1% BSA in PBS for 1 hour at 37 °C, washed with a blocking solution (PBS containing 1% BSA and 0.1% Tween-20), and incubated with 2 µg of recombinant proteins in PBS per well for 6 hours. The wells were washed 3 times with the blocking solution, and the bound protein was detected using an anti-His-tag antibody (Cat #A2-502-100, RRID: AB_11135798, Icosagen, Tartu, Estonia) for 1 hour at 37 °C. After washing the wells 3 times with the blocking solution, a horseradish peroxidase-conjugated secondary antibody (Cat# 111-035-008, RRID: AB_2337937, Jackson Immuno Research, Cambridgeshire, UK) was added according to the manufacturer’s instructions. The wells were washed 3 times with the blocking solution, and a peroxidase reaction was initiated by adding 100 µL/well of freshly prepared solution from the TMB Peroxidase EIA Substrate Kit (Bio-Rad, Hercules, CA, USA), followed by a 5-minute incubation at 37 °C. The reaction was stopped with 1 N H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, and the absorbance was measured at 450 nm using a microplate reader (Tecan Austria GmbH, Salzburg, Austria).\u003c/p\u003e\n\u003ch2\u003eNanoparticle Synthesis and Functionalization\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe synthesis and functionalization of iron oxide nanoworms (NWs) and silver nanoparticles (AgNPs) followed previously published protocols \u003csup\u003e25,34,37,38,46–48\u003c/sup\u003e. For NWs, aminated NWs were PEGylated with maleimide-5K-PEG-NH\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e(JenKem Technology, TX, USA), and peptides were coupled to NWs via a thioether bond between the thiol group of a cysteine residue and the N-terminus of the peptide. The concentration of NWs was determined by constructing a calibration curve with iron oxide, and the absorbance of NWs at 400 nm was measured using a NanoDrop 2000c spectrophotometer (Thermo Scientific). For AgNPs, CF647-N-hydroxysuccinimide-dye (NHS-dye) was conjugated to the terminal amine group of PEG, and biotinylated peptides were coated on the surface of the AgNPs through NeutrAvidin (NA; Sigma-Aldrich, USA). The nanoparticles were characterized using transmission electron microscopy (TEM, Tecnai 10, Philips, Netherlands) to image, and dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern Instruments, UK) to assess the zeta potential, polydispersity, and the size, as described previously.\u003c/p\u003e\n\u003ch2\u003eCell Binding and Internalization Assay\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe U87-MG, PPC1, and M21 cells were cultured on coverslips and treated with CF555-labeled PL2 AgNPs or non-targeted control AgNPs at 37 °C for 1 hour, as previously described\u0026nbsp;\u003csup\u003e34,35,49\u003c/sup\u003e. After removing unbound particles with culture medium, cells were treated with an etching solution (10 mM working concentration in PBS) made by diluting 0.2 M stock solutions of Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e and K\u003csub\u003e3\u003c/sub\u003eFe (CN)\u003csub\u003e6\u003c/sub\u003e in a 1: 1 ratio for 3 minutes, followed by washing with PBS. Cells were fixed with methanol at\u0026nbsp;-20 °C for 1-2 minutes, and thereafter the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes) at 1 µg/mL. Finally, the coverslips were mounted on microscopy slides using Fluoromount-G medium (Electron Microscopy Sciences) for confocal imaging.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eTumor-targeted delivery and biodistribution studies\u003c/h2\u003e\n\u003cp\u003eFAM-labeled PL2 peptide-conjugated NW or control FAM-NW (7.5 mg/kg) in PBS were administered via tail vein injection to subcutaneous U87-MG, PC3, and orthotropic WT-GBM tumor-bearing mice. Five hours after circulation, the tumors and organs were collected via cardiac perfusion of mice under deep anesthesia with 20 ml PBS/DMEM. Macroscopic images of tissues were taken using an Illuminatool Bright Light System LT-9900 (Lightools Research, Encinitas, CA, USA) before snap-freezing. The frozen tissues were then cryosectioned with Leica CM1520 (Leica Camera AG, Germany) into 8-10 µm sections and mounted on Superfrost+ slides (Thermo Fisher Scientific, MA, USA). The tissue sections were equilibrated at room temperature and fixed with 4% paraformaldehyde/-20 °C methanol. Tissue staining was performed using primary antibodies, including rabbit anti-fluorescein IgG fragment (Cat # A889, RRID: AB_221561, Thermo Fisher Scientific, MA, USA), rat anti-mouse CD31 (RRID:\u003c/p\u003e\n\u003cp\u003eAB_393571, BD Biosciences, CA, USA), and in-house prepared CF647/CF546-labeled single-chain antibodies ScFV L19. Secondary antibodies used were Alexa 488 goat anti-rabbit IgG (Cat # A-11034, RRID: AB_2576217), Alexa 647 goat anti-rat IgG (Cat # A-21247, RRID: AB_141778), and Alexa 546 goat anti-mouse IgG (Cat # A-11003, RRID: AB_2534071) from Invitrogen, CA, USA. The tissue nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes) at 1 μg/ml concentration. Coverslips were mounted on glass slides with Fluoromount-G (Electron Microscopy Sciences, PA, USA) and imaged using confocal microscopy (Olympus FV1200MPE, Hamburg, Germany). The resulting images were analyzed using FV10-ASW4.2 viewer/Imaris software/Fiji ImageJ.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eEx vivo\u003c/em\u003e clinical tumor dipping assay\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eIn compliance with the Ethics Committee of the University of Tartu, Estonia (permit #243/T27), fresh surgical ovarian carcinoma samples were collected from consenting patients undergoing surgery according to relevant guidelines and regulations accordance with the Declaration of Helsinki\u0026nbsp;\u003csup\u003e50\u003c/sup\u003e. To perform the dipping assay, the fresh ovarian carcinoma tissues were washed with DMEM, and 1 cm\u003csup\u003e3\u003c/sup\u003e explants were incubated at 37 °C with PL2-NW or non-targeted control NWs (40 mg/mL Fe in DMEM supplemented with 1% BSA) for four hours. The explants were then washed with PBS, snap-frozen, cryosectioned at 10 µm, and immunostained with rabbit anti-fluorescein primary antibodies, followed by detection with the Alexa-488 anti-rabbit secondary antibody (Invitrogen, Thermo Fisher Scientific, MA, USA).\u003c/p\u003e\n\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n\u003cp\u003eThe statistical analysis was conducted using Prism 6 software (GraphPad Software, Inc, RRID:SCR_002798). For comparisons between two groups, a student’s unpaired t-test was used, while an ANOVA test was applied for comparisons involving multiple groups. For Continuous data, including quantifying FAM signal in tissue sections, the fluorescence signal intensity of antibody-amplified FAM was analyzed from 12–20 confocal images using Fiji ImageJ freeware (RRID:SCR_003070) and were analyzed using the student’s unpaired t-test. \u0026nbsp;Data are presented as mean values, with error bars representing ±SEM. The data are presented as mean values with error bars showing ±SEM. The significance level was set at p \u0026lt; 0.05, and the P-values are denoted as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. The Samples were processed in a randomized order during analysis to prevent systematic bias. Group sizes were determined based on prior studies and standard practices in the field to ensure robust data collection and reproducibility. A priori power analysis was conducted to ensure the sample size was sufficient to detect biologically relevant differences, adhering to the 3R principles\u003csup\u003e51\u003c/sup\u003e and guidelines such as PREPARE\u003csup\u003e52\u003c/sup\u003e and ARRIVE\u003csup\u003e42\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eIdentification of Fn-EDB binding peptides\u003c/h2\u003e\n\u003cp\u003eFor biopanning, His\u003csub\u003e6\u003c/sub\u003e-tailed Fn-EDB domain was expressed in \u003cem\u003eE. coli\u003c/em\u003e and purified using Ni-NTA chromatography (Supplementary Fig. S1). Subsequently, 6 rounds of selection were performed on immobilized Fn-EDB using X7 peptide T7 phage libraries. The first and fourth rounds of biopanning were performed using Fn-EDB immobilized on polystyrene multiwell plates, and other rounds were performed on Fn-EDB coated on Ni-NTA magnetic beads (Supplementary Fig. S2). We observed enrichment of Fn-EDB binding of the phages through the screening, with round 6 pool showing ~3000-fold increased binding over the naive library (Fig. 1A). Sanger sequencing of 48 random phage clones from a selection round 5 resulted in 11 unique peptide-phages that were individually tested for their interaction with Fn-EDB. Among the 11 candidates, 5 clones demonstrated the ability to bind to Fn-EDB \u003cem\u003ein vitro\u003c/em\u003e \u0026gt;50 fold over control phage displaying heptaglycine (Supplementary Fig. S3). To evaluate the systemic tumor homing of the \u003cem\u003ein vitro\u003c/em\u003e selected Fn-EDB binding peptides, \u003cem\u003ein vivo\u003c/em\u003e phage playoff auditioning was used \u003csup\u003e45\u003c/sup\u003e. An equimolar mixture of candidate and control peptide-phage were intravenously injected into a panel of glioma and prostate xenograft tumor-bearing mice and, following phage circulation and removal of blood background by perfusion, representation of peptide-phages in tumors was estimated using high-throughput DNA sequencing. We observed that the phage clone displaying the heptameric TSKQNSR peptide, designated PL2, was overrepresented in tested solid tumor models (Table 1). Interestingly, the C-terminal arginine of the PL2 peptide may engage with the \u0026quot;C-wall\u0026quot; binding pocket on the b1 domain of neuropilin-1 (NRP1) \u003csup\u003e53\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eNext, the binding of the PL2 peptide-displaying phage to recombinant Fn-EDB, NRP1, and control proteins (Tenascin-C C-domain, TNC-C; Nucelolin, NCL) was studied. The PL2 phages demonstrated robust binding to Fn-EDB and b1b2 fragment of NRP1 and no binding to TNC-C, a protein with size and negative surface charge similar to Fn-EDB (Fig. 1B). Interestingly, the PL2 phage exhibited ~700-fold higher binding to the recombinant NRP-1 b1b2 domain compared to the heptaglycine control phage, demonstrating a binding capacity comparable to the prototypic NRP-1 binding peptide, RPAPRPAR \u003csup\u003e33\u003c/sup\u003e. The binding of the PL2 displaying phage to Fn-EDB surpassed that of the previously reported Fn-EDB-binding peptide, ZD2 \u003csup\u003e26\u003c/sup\u003e (Supplementary Fig. S4). \u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" rowspan=\"2\" valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003ePhage-displayed Peptides in the \u0026quot;Playoff\u0026quot; Mix\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"6\" valign=\"top\" style=\"width: 49px;\"\u003e\n \u003cp\u003eRepresentation of the phage in tumors or in control brain tissue (fold over G7 control phage)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003eWT GBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003eP3 stem cell-like\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003eP13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003eU87-MG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003ePC3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003eNormal brain\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003eGGGGGGG (G7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003eControl\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 6px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 6px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"7\" valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003eFn-EDB-selected\u003c/p\u003e\n \u003cp\u003e(round 5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003eTKRKGKG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003eClone-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003eGLGGRRIKLKTS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003eClone-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003eGRRGRVIKLKTSEPPQ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003eClone-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eKVKKRGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003eClone-17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003eRESRRGRVKLAAALE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003eClone-33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTSKQNSR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClone-\u003c/strong\u003e\u003cstrong\u003e46\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e11.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e32.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e19.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003cstrong\u003e.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003eCTVRTSADC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003eZD2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8px;\"\u003e\n \u003cp\u003e4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1. \u0026nbsp;\u003cem\u003eIn vivo\u003c/em\u003e playoff auditioning of Fn-EDB-selected peptides in tumor xenograft models in mice\u003cem\u003e.\u003c/em\u003e An equimolar mixture of Fn-EDB-selected phages was intravenously injected into mice bearing orthotopic WT-GBM, P3 stem cell-like, P13, and s.c. implanted U87-MG glioblastoma, or PC3 prostate carcinoma xenografts at a dose of 1 \u0026times; 10\u003csup\u003e10\u003c/sup\u003e pfu/mouse. After 2 h of circulation, background phages were removed by perfusion, and the representation of individual phages in tumor and control tissues was evaluated by Ion-Torrent high-throughput sequencing. Phages displaying PL2 (TSKQNSR) peptide showed the highest representation across tumor models tested in tumor tissue. The data represent the mean of 3 mice for each model.\u003c/p\u003e\n\u003cp\u003eThe binding of alanine-substituted PL2 derivative peptide displaying phages was evaluated to identify the essential amino acids involved in the interaction with Fn-EDB. Alanine substitution of the C-terminal arginine and serine significantly reduced the binding of the PL2 phage to recombinant Fn-EDB.\u0026nbsp;Surprisingly, substitution of the N-terminal threonine, lysine, and asparagine enhanced the binding of the PL2 phage to Fn-EDB (Fig. 1C). As expected, the interaction with NRP-1 was dependent on the presence of C-terminal arginine, as the phage displaying PL2 with C-terminal R \u0026gt;A substitution showed a marked reduction in NRP-1 binding (Fig. 1C).\u003c/p\u003e\n\u003cp\u003eWe then evaluated the interaction of synthetic 5(6)-carboxyfluorescein (FAM)-labeled PL2 peptide with Fn-EDB immobilized on polystyrene ELISA plates. The synthetic PL2 peptide retained its ability to bind Fn-EDB (Fig. 1D).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003ePL2 AgNPs bind to and are internalized by tumor cells \u003cem\u003ein vitro\u003c/em\u003e\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eWe next studied cellular internalization of the PL2-functionalized AgNPs \u003csup\u003e54\u003c/sup\u003e. Extracellular membrane-bound AgNPs were selectively removed by treatment with a mild, biocompatible redox-based hexacyanoferrate/thiosulfate etching solution, ensuring that only the signal from internalized AgNPs remained detectable \u003csup\u003e48,54\u003c/sup\u003e. CF555-labeled PL2-AgNPs were incubated with the Fn-EDB and NRP-1-expressing U87-MG glioma cells, NRP-1-positive PPC1 prostate carcinoma cells, and Fn-EDB- and NRP-1-negative M21 melanoma cells \u003csup\u003e34,35,37,38\u003c/sup\u003e. PL2-AgNPs exhibited robust endocytosis in U87-MG and PPC1 cells following 1-hour incubation, whereas control particles displayed negligible uptake in both U87-MG and PPC1 cells (Fig. 2A, B, Supplementary Fig. S5). In contrast, receptor-negative M21 cells did not internalize PL2-AgNPs nor control AgNPs (Fig. 2A, Supplementary Fig. S5). Post-etching, the PL2-AgNP signal showed only a modest decrease in intensity compared to non-etched controls (Fig. 2A, B), confirming that the majority of PL2-AgNPs were taken up by the cells.\u003c/p\u003e\n\u003ch2\u003eSystemic PL2-functionalized nanoparticles\u0026nbsp;accumulate in tumor lesions\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eNext, we investigated\u0026nbsp;the potential of the PL2 peptide as a systemic tumor-targeting probe. The dextran-coated PEGylated paramagnetic iron oxide nanoworms (NWs) have been used as a theranostic nanosystem suitable for systemic affinity targeting, functioning both as a drug carrier and as a magnetic resonance imaging agent with T2 contrast properties \u003csup\u003e38,47\u003c/sup\u003e. FAM-labeled PL2 peptide or FAM-Cys control was conjugated to NWs with an average size of 88.9 \u0026plusmn; 0.9 nm and Zeta Potential of\u0026nbsp;-9.5 \u0026plusmn; 0.4 mV. The conjugation of peptides to NWs did not significantly alter particle size or surface charge (Supplementary Fig. S6, A-D) \u003csup\u003e27\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u0026nbsp;\u003c/em\u003ehoming studies were conducted in mice bearing orthotopic WT-GBM glioma, s.c. U87-MG glioma and s.c. PC3 prostate carcinoma xenograft tumor models, all of which express abundant Fn-EDB and NRP-1 \u003csup\u003e34,37,38\u003c/sup\u003e. Mice were intravenously injected with 7.5 mg/kg of NWs. After circulation, mice were perfused to remove free NWs, and tumors along with control organs were collected, sectioned, and imaged with confocal microscopy. PL2-functionalization significantly increased NW accumulation in CD31-positive vascular structures across all tumor models (Fig. 3A-C). In some regions, PL2-NWs extravasated and accumulated within the tumor parenchyma (Fig. 3A-C, arrowheads).\u003c/p\u003e\n\u003cp\u003eFollowing 5 h of circulation, PL2-NWs demonstrated enhanced tumor accumulation compared to control NWs, with a ~7-fold increase in PC3 tumors, ~7.5-fold increase in U87-MG tumors, and a ~2-fold increase in WT-GBM tumors (Fig. 3A-C, Supplementary Fig. S7). In contrast, the signal for PL2-functionalized and non-targeted NWs was comparable in control organs, including the liver, kidney, and lung (Fig. 3A-C, Supplementary Fig. S7). Furthermore, PL2-NWs exhibited selective accumulation in glioblastoma lesions, with minimal accumulation in nonmalignant brain in the orthotopic WT-GBM model and in the healthy brain of control mice (Supplementary Fig. S8).\u003c/p\u003e\n\u003cp\u003eMacroscopic fluorescence imaging showed accumulation of the PL2-NWs, but not control NWs, in U87-MG tumors collected from mice (Fig. 4A), while no signal was detected in the control organs (liver, lung, heart, brain, kidney, and spleen) (Fig. 4A, 4D). The tumor homing pattern of the PL2-NWs was also studied by confocal imaging of U87-MG tumor tissue sections after staining with Fn-EDB- (ScFV L19) and NRP-1-specific antibodies. Fn-EDB and NRP-1 were upregulated in the tumors (Fig. 4B, 4C, 4E, 4F), and the PL2-NW signal showed extensive overlap with the receptors (Fig. 4B, 4C, arrows).\u003c/p\u003e\n\u003ch2\u003ePL2-NWs bind to surgical explants of human clinical cancers\u003c/h2\u003e\n\u003cp\u003eTo evaluate the potential translational relevance of the PL2 peptide, we examined the binding and penetration capabilities of PL2-targeted NWs in fresh surgical explants of human ovarian carcinoma tissue. We first established that in these tumors, Fn-EDB were highly overexpressed (Supplementary Fig. S9). Previous studies have consistently demonstrated the overexpression of NRP-1in ovarian cancers, linking it to malignancy and poor prognosis \u003csup\u003e55,56\u003c/sup\u003e. Next, we performed an \u003cem\u003eex vivo\u003c/em\u003e tumor binding/penetration assay (\u0026quot;tumor dipping assay\u0026quot;) \u003csup\u003e57,58\u003c/sup\u003e, and observed that PL2-NWs demonstrated a 9-fold increase of binding and penetration in tumor tissue compared to the control NWs (Fig. 5A, 5B). In certain regions, PL2-NWs penetrated a few mm into the explants, though the binding was predominantly localized to the tumor surface (Fig. 5A). In contrast, control NWs exhibited only a background fluorescence signal. These findings suggest that the PL2-NW platform holds promise as a targeted drug delivery system for ovarian carcinoma and, potentially, other solid clinical tumors.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe report the development of a homing peptide named PL2, which targets the tumor-associated ECM protein Fn-EDB, a stable and abundant antigen overexpressed in many solid tumors. In addition to binding Fn-EDB, PL2 engages the cell and tissue penetration receptor NRP-1, enabling nanoparticles to internalize into cultured cells, home to solid tumors \u003cem\u003ein vivo\u003c/em\u003e, and penetrate clinical tumor samples \u003cem\u003eex vivo\u003c/em\u003e. These findings suggest potential applications of PL2 in targeting solid tumors and other conditions characterized by upregulation of Fn-EDB and NRP-1, such as endometriosis \u003csup\u003e59\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eECM-reactive affinity ligands have been used to deliver anticancer payloads such as cytokines/growth factors, proapoptotic peptides, cell-permeable cytotoxic compounds, and imaging agents to tumors \u003csup\u003e4,17,60,61\u003c/sup\u003e. However, ECM-targeting compounds, including those targeting Fn-EDB, rely on passive delivery through the enhanced permeability and retention (EPR) effect, which is subject to significant inter- and intratumoral variability \u003csup\u003e62,63\u003c/sup\u003e. Additionally, within the tumor microenvironment, ECM-targeting ligands generally exhibit limited cellular uptake, necessitating the use of cleavable linkers (e.g., disulfides or hydrazones) to release drug payloads into the extracellular milieu \u003csup\u003e61,64,65\u003c/sup\u003e. In this study, we employed cell-free phage biopanning to identify peptides that bind Fn-EDB. Unlike other selected peptides, PL2 peptide possessed the C-terminal arginine residue forming a minimal C-end Rule (CendR) element known to mediate binding a cellular pleiotropic multiligand receptor NRP-1 \u003csup\u003e53\u003c/sup\u003e. Although the PL2 peptide-phage ranked fifth among 11 tested phages in the cell-free Fn-EDB binding assay, it outperformed all others in an \u003cem\u003ein vivo\u003c/em\u003e playoff assay. The functionality of the CendR element in PL2 was demonstrated in \u003cem\u003ein vitro\u003c/em\u003e studies, where AgNPs functionalized with the PL2 peptide exhibited strong NRP-1-dependent cellular binding and internalization, consistent with our pervious finding for the PL3 peptide \u003csup\u003e30\u003c/sup\u003e . \u003cem\u003eIn vivo\u003c/em\u003e, functionalizing nanoparticles with the PL2 peptide significantly enhanced their tropism to prostate carcinoma and glioblastoma, with significant accumulation in the extravascular space and colocalization with both Fn-EDB and NRP-1 immunoreactivities. Finally, PL2-targeted nanoparticles effectively bound to and penetrated clinical surgical explants \u003cem\u003eex vivo\u003c/em\u003e, suggesting a translational potential for the targeting system. This finding is not unexpected as small homing peptides typically target evolutionarily conserved binding pockets on their target molecules and the 91-amino acid alternatively spliced EDB domain is conserved across multiple species, including mice, rats, rabbits, dogs, monkeys, and humans \u003csup\u003e27,66\u003c/sup\u003e. Additionally, CendR peptides consistently bind to the b1 domain of NRP-1 across these species, from mouse to human. These studies, in the context of well-established knowledge of the upregulation of Fn-EDB and NRP-1 in various primary solid tumors and metastatic lesions, highlight the potential of PL2-based nanoparticle targeting strategy for application across a range of solid tumor types \u003csup\u003e15,67\u003c/sup\u003e. Although our studies show that the PL2 peptide appears a promising targeting agent for nanoparticles, there is still room for improvement and opportunities to broaden the range of applications. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eShort homing peptides with a high degree of conformational freedom and small number of contact residues have typically moderate affinity yet are capable of high avidity interactions with their targets due to cooperative multivalent binding when displayed on nanoparticles \u003csup\u003e6,68,69\u003c/sup\u003e. This is illustrated by complement factor C1q-IgG interactions, where the dissociation constant (Kd) improves dramatically with increasing valency\u0026mdash;100 \u0026mu;M for monomers, 1 \u0026mu;M for dimers, and 3 nM for tetramers \u003csup\u003e70\u003c/sup\u003e. Interestingly, our alanine scan revealed that at certain positions, particularly the N\u0026gt;A substitution at position 5, alanine substitution enhanced peptide phage binding to recombinant receptor molecules. In follow-up studies to improve the affinity of the PL2 peptide, it will be valuable to conduct secondary screens using constrained peptide libraries, created by randomizing nonessential amino acids of the PL2 peptide and/or adding additional flanking amino acids to provide stabilizing interactions. Another potential avenue for improving the PL2 peptide involves enhancing its tumor specificity. While NRP-1 is overexpressed in solid tumors, it is present at lower levels in the vascular beds of normal organs, particularly the lungs, which could result in off-target accumulation. To address this, further improvement of the PL2 peptide could include capping the C-terminal arginine with additional amino acids that are engineered or screened to be cleaved by tumor-expressed extracellular proteases. Using a similar approach, we recently reported the development of a urokinase-type plasminogen activator-dependent CendR peptide to mitigate background accumulation in nontarget tissues \u003csup\u003e31\u003c/sup\u003e. Such continuation studies could further facilitate the applications of PL2 as a cancer targeting agent.\u003c/p\u003e\n\u003cp\u003eMany homing peptides, by binding to functionally important binding pockets on target molecules, trigger biological responses through mechanisms such as eliciting conformational changes or competing with natural ligands for receptor binding. Examples include the antitumor effects of LyP-1 \u003csup\u003e71\u003c/sup\u003e, \u0026nbsp; tumor penetration induction and immunomodulation of iRGD \u003csup\u003e72,73\u003c/sup\u003e, wound healing promotion by CAR \u003csup\u003e74\u003c/sup\u003e, and suppression of choroidal neovascularization by PL3 \u003csup\u003e30,75\u003c/sup\u003e. Similarly, in addition to its use in affinity targeting, the PL2 peptide may possess inherent biological activity by disrupting interactions between Fn-EDB and integrins or other matrix components involved in adhesion, migration, and survival of malignant or tumor-promoting cells, potentially suppressing tumor growth, metastasis, and angiogenesis. If the biological activity of PL2 is confirmed, its improved variants, developed using the strategies outlined above, and proteolysis-resistant PL2 derivatives incorporating nonproteinogenic amino acids, will be advantageous for further applications.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our study presents the development of the PL2 peptide, which holds potential for applications in targeted drug delivery and molecular imaging. The dual targeting of Fn-EDB and NRP-1 by the PL2 peptide offers a promising strategy for advancing targeted cancer therapies. The use of PL2 nanoparticles to tumor tissues shows promise for the development of novel cancer treatments. Further studies are needed to optimize the peptide and assess its clinical efficacy in targeting solid tumors as well as safety.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eConflicts of interest\u003c/p\u003e\n\u003cp\u003eThe data supporting this study\u0026apos;s findings are available from the corresponding author upon request. T.T. and P.L. hold a patent on the PL2 peptide (\u0026quot;Bi-Specific Extracellular Matrix Binding Peptides and Methods of Use Thereof\u0026quot;, WO. patent no. WO 2020/161602 A1), and TT is an inventor of CendR peptides. Other authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eEthics and Informed Consent Statement\u003c/p\u003e\n\u003cp\u003eThe fresh surgical ovarian carcinoma samples were obtained under protocols approved by the Ethics Committee of the University of Tartu, Estonia (permit #243/T27). Additionally, informed consent was obtained from all the patients, and methods for using human samples were carried out under relevant guidelines and regulations accordance with the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnimals and\u0026nbsp;Ethics Statement\u003c/p\u003e\n\u003cp\u003eAnimal experimentation procedures were approved by the Estonian Ministry of Agriculture, Committee of Animal Experimentation, projects #42 and #48 (IACUC). We confirm that all methods were performed in accordance with the relevant guidelines and regulations. We confirm that the study was conducted in accordance with the ARRIVE guidelines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData Availability Statement\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are included in this article and its supplementary information files. No additional data are required to be deposited in public repositories. Any additional data supporting the findings of this study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eT.T. was funded by the Estonian Research Council (grants PRG230 and PRG1788), EuronanomedIII projects ECM-CART and iNanoGun, and TRANSCAN3 project ReachGLIO\u0026nbsp;(all coordinated by Estonian Research Council)\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eP.L. \u0026ndash; conceived, coordinated the study, performed the experiments, analyzed the data, and wrote the manuscript. A.T.\u0026ndash; prepared and characterized the nanoparticles. K.K.\u0026ndash; performed ion-torrent next-generation sequencing. O.T. prepared and provided clinical samples. T.T.\u0026ndash; conceived and supervised the study, and wrote the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGuelfi, S., Hodivala-Dilke, K. \u0026amp; Bergers, G. Targeting the tumour vasculature: from vessel destruction to promotion. \u003cem\u003eNat Rev Cancer\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, (2024).\u003c/li\u003e\n\u003cli\u003eForster, J., Harriss-Phillips, W., Douglass, M. \u0026amp; Bezak, E. A review of the development of tumor vasculature and its effects on the tumor microenvironment. \u003cem\u003eHypoxia\u003c/em\u003e \u003cstrong\u003eVolume 5\u003c/strong\u003e, 21\u0026ndash;32 (2017).\u003c/li\u003e\n\u003cli\u003eRuoslahti, E., Bhatia, S. N. \u0026amp; Sailor, M. J. Targeting of drugs and nanoparticles to tumors. \u003cem\u003eJournal of Cell Biology\u003c/em\u003e \u003cstrong\u003e188\u003c/strong\u003e, 759\u0026ndash;768 (2010).\u003c/li\u003e\n\u003cli\u003eLingasamy, P. 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One such target is Fibronectin Extra Domain-B (Fn-EDB), an oncofetal splice variant of a major extracellular matrix protein (Fn), which is upregulated in many solid tumors as part of the angiogenic response. In this study, we conducted cell-free biopanning on recombinant Fn-EDB to identify a short peptide designated as PL2 (amino acid sequence: TSKQNSR), which specifically interacts with Fn-EDB. Notably, the C-terminal arginine of PL2 enables its interaction with neuropilin-1 (NRP-1), a receptor known to facilitate cell and tissue penetration. When administered systemically, PL2-displaying recombinant bacteriophages and iron oxide nanoworms (NWs) functionalized with PL2 peptide exhibited homing to glioblastoma and prostate tumor xenografts, followed by their extravasation and penetration into tumor parenchyma. Notably, PL2-functionalized NWs penetrated ex vivo explants of clinical ovarian carcinoma, underscoring their translational potential. These findings underscore the potential of the PL2 peptide as a promising agent for anticancer drug delivery and molecular imaging applications.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eOne Sentence Summary:\u003c/em\u003e PL2 tumor penetrating peptide targeting Fibronectin Extra Domain-B (Fn-EDB) and Neuropilin-1 (NRP-1) can be used for precision delivery of payloads to the microenvironment of solid tumors\u003c/p\u003e","manuscriptTitle":"Targeting Oncofetal Fibronectin and Neuropilin-1 in solid tumors with PL2 peptide","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-10 18:04:22","doi":"10.21203/rs.3.rs-5743295/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-11T14:22:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-10T00:55:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"90719681702811442325695051629620785342","date":"2025-06-06T08:49:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11832777715015992396057374170129708793","date":"2025-06-05T15:17:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-04T02:15:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"179992110568680415161897721615437360914","date":"2025-06-03T15:03:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-03T13:30:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-03T10:14:40+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-20T20:29:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-20T20:16:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-12-31T17:35:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"15744680-f351-437a-945f-824a93347c1d","owner":[],"postedDate":"June 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":49550182,"name":"Biological sciences/Biotechnology"},{"id":49550183,"name":"Biological sciences/Cancer"},{"id":49550184,"name":"Biological sciences/Drug discovery"},{"id":49550185,"name":"Biological sciences/Molecular biology"},{"id":49550186,"name":"Health sciences/Biomarkers"},{"id":49550187,"name":"Health sciences/Medical research"},{"id":49550188,"name":"Health sciences/Molecular medicine"},{"id":49550189,"name":"Health sciences/Oncology"},{"id":49550190,"name":"Physical sciences/Nanoscience and technology"}],"tags":[],"updatedAt":"2025-08-18T16:06:56+00:00","versionOfRecord":{"articleIdentity":"rs-5743295","link":"https://doi.org/10.1038/s41598-025-11299-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-08-11 15:58:05","publishedOnDateReadable":"August 11th, 2025"},"versionCreatedAt":"2025-06-10 18:04:22","video":"","vorDoi":"10.1038/s41598-025-11299-x","vorDoiUrl":"https://doi.org/10.1038/s41598-025-11299-x","workflowStages":[]},"version":"v1","identity":"rs-5743295","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5743295","identity":"rs-5743295","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}