In the TME, FN-EDB as a key ECM protein, through a regulation of multiple signaling pathways, activated the series of signaling pathways closely related to tumor cell behavior (Fig. 2), including integrin signaling pathways, vascular formation signaling pathways, signaling pathways, immune regulatory signaling pathways, tumor stem cell signaling pathway, inflammatory signaling pathways and epithelial-stromal transformation (EMT) signaling pathway promote the occurrence and development of tumor and provides the theoretical basis for regulating targeted immunotherapy of tumor under various pathological conditions.

The interaction between integrins αvβ3 and αvβ5 with FN-EDB is a finely regulated process involving multiple layers of interaction. First, structural complementarity forms the basis of this interaction, with FN-EDB's specific three-dimensional structure complementing the binding sites of integrins, promoting tight molecular interactions (36). Secondly, the αv subunit of integrins contains specific domains responsible for recognizing FN-EDB, while the β3 or β5 subunits contribute to the overall structural stability of the integrin. FN-EDB can also interact with other integrin subtypes, albeit with differing binding characteristics (37). α5β1 integrin binds to the RGD sequence in fibronectin and participates in cell adhesion, while α8β1 integrin primarily interacts with other domains of fibronectin but can also interact with FN-EDB (38). αvβ1 integrin has the ability to bind to various ECM proteins, including FN-EDB (39) and αvβ6 and αvβ8 integrin subtypes, especially their interactions with epithelial and tumor cells, suggest their potential binding with FN-EDB under specific cell types and pathological conditions (40). Additionally, α4β1 (VLA-4) integrin, primarily interacting with VCAM-1 and the CS-1 region of fibronectin, may also interact with FN-EDB. α9β1 integrin, although less studied in its direct interaction with FN-EDB, can also bind to some domains of fibronectin (41).

The diversity of integrin subtypes and their interactions with FN-EDB highlight the complexity and importance of the ECM in cell function and pathology. The dynamic nature of the ECM, including its composition and physical state, such as rigidity or disassembly, can also regulate integrin activity and subsequently influence their interactions with FN-EDB (42). This intricate network of interactions between FN-EDB and integrins underscores the multifaceted role of the ECM in cellular behavior and disease progression.

Integrins are a bidirectional signaling molecule (Fig. 3) and integrins are transmembrane receptor that play a key role in cell-ECM interactions. On one hand, they transduce information from the extracellular environment to modulate cellular responses, a process often referred to as ‘inside-out’ signaling. On the other hand, the active state of integrins can also be modulated by intracellular signaling molecules that, when integrins are bound to their ligands, can trigger an intracellular signaling pathway, a process known as ‘outside-in’ signaling (33). Integrins mediate inside-in and inside-out signaling, required for various cellular processes such as cell adhesion, migration, survival, proliferation and gene expression (36).

In outside-in signaling inactive integrins are described as their basal state, waiting for the activation of ECM ligands, such as fibronectin, which contains the RGD sequence. Upon ligand binding, integrins undergo a conformational change to become active integrins, initiating a cascade of intracellular signaling events. FAK and Src homology 2 (SH2) domain-containing proteins (Src) are key components of the signaling complex, which assemble following integrin activation, leading to the recruitment of various signaling molecules and the subsequent activation of downstream signaling pathways, including the FAK/PI3K/AKT and MAPK/ERK pathways (35); for example, the monomeric form of talin protein, which then regulates actin polymerization and affects cell morphology and motility.

The inside-out signaling pathway involves the regulation of intracellular signaling molecules on the activation of integrins, thereby regulating the affinity of integrins for their ligands (43). Talin is a cytoskeletal protein considered a key regulator of integrin activation and is critical for integrin activation and the formation of focal adhesions that are essential for the transmission of mechanical and biochemical signals between the ECM and the cell interior (44). By interacting with integrin and its enhanced ability to bind to ECM, it participates in the transmission of signals, ultimately realizing the directed movement of cells and the remodeling of cellular structure (45). In addition, talin activation in turn affects the actin cytoskeleton assembly and the formation of focal adhesions. Actin polymerization is thought to be a critical process downstream of integrin activation, which contributes to cell polarity, survival, migration and the assembly of the ECM (46).

FN-EDB promotes cell adhesion to the ECM by binding to specific integrin isoforms such as α v β 3 and α v β 5. This adhesion is fundamental to cell stability and is critical for cellular sensing and response to external signals (47). The interaction of FN-EDB with integrins activates a series of downstream signaling events that triggers the activation of integrin signaling pathways, including cellular signaling cascade in FAK, PI3K, AKT and MAPK/ERK (48).

The reorganization of the cytoskeleton underlies cell migration and morphological changes; FN-EDB positively regulates this process through a signaling pathway activated by integrins. This is extremely critical for the physiological and pathological processes such as maintaining cell adhesion, cytoskeletal structure reorganization, gene expression, tumor cell invasion and metastasis (49).

FN-EDB plays a pivotal role in regulating classical integrin signaling pathways within the TME. This process is initiated by the binding of integrins to ECM components, particularly FN-EDB, leading to the activation of FAK at Tyr397, resulting in its autophosphorylation [phosphorylated (p-)FAK-Y397] (50). This event marks the initiation of intracellular signaling events crucial for tumor progression.

Abnormal activation of FAK signaling can be achieved through various mechanisms, including integrin-mediated autophosphorylation and interactions with kinase families such as Src and Grb2. FAK promotes activation of Ras, which in turn activates RAF, MEK and ultimately ERK1/2, initiating the ERK/MAPK signaling pathway (48,51). This phosphorylation ultimately leads to the translocation of ERK1/2 to the nucleus, where it regulates the expression of genes involved in cell proliferation, differentiation and survival. The activation state of ERK1/2 is closely associated with the invasiveness and metastatic potential of tumor cells, with its high expression often correlating with poor prognosis in various tumors (52).

Parallel to the ERK/MAPK pathway, FAK also participates in the PI3K-Akt pathway, a key regulator of cell survival, proliferation and metabolism. FAK activation of PI3K leads to the phosphorylation and activation of Akt, which then phosphorylates multiple downstream targets, influencing cellular metabolism and survival signals. The sustained activation of Akt is also associated with tumor invasiveness and chemoresistance, making it a significant target for cancer therapy (51–53).

In clinical research, the activation status of FAK, ERK1/2 and Akt signaling pathways has become an important biomarker for assessing tumor progression and prognosis. Targeted therapeutic strategies against these signaling pathways, such as the use of FAK inhibitors, MEK inhibitors and PI3K/Akt inhibitors, have shown potential therapeutic value in clinical trials for various types of cancer (54).

FN-EDB not only regulates classical integrin signaling pathways but also modulates atypical β1-integrin signaling pathways, which are critical for tumor cell plasticity and metastatic potential. Unlike the classical FAK-ERK/MAPK or PI3K-Akt signaling axes, the atypical β1-integrin signaling mechanism involves integrin-mediated endocytosis, leading to the internalization of integrin-ECM complexes into endosomes (55). This process allows for the continued signaling within the cell through the formation of endosomal vesicles.

In endosomes, activated integrins co-localize with phosphorylated FAK (p-FAK-Y397), indicating the sustained transmission of integrin signals within the cell (56). The presence of integrin ligands like fibronectin and integrin-activating proteins such as talin in endosomes further confirms the connection between functional receptor-ligand coupling and active FAK pools associated with endosomes (57).

In endosomal lumen, integrin signaling continues through the activation of FAK, which can occur independently of its typical activation at the plasma membrane. This atypical activation of FAK in endosomes is associated with the regulation of cytoskeletal dynamics and cell migration, processes essential for tumor invasion (58). Key regulators such as early endosome antigen 1 and Ras-related protein Rab21 play crucial roles in the endocytosis and signaling of integrins, potentially promoting the assembly of specific signal complexes on endosomes (59).

Inhibition of endocytosis, for example through dynamin inhibitors, significantly reduces the activation of FAK and ERK1/2, highlighting the central role of endocytosis in integrin-ECM signaling (60). Additionally, endocytosis is crucial for maintaining cell resistance to anoikis, revealing its protective role in cell survival signaling (61).

Integrin signaling on endosomes may involve different molecular mechanisms and regulatory proteins than those at focal adhesions on the cell membrane, such as direct interaction with integrins and the assembly of unique signaling complexes. Furthermore, FN-EDB-mediated integrin activation can activate Rho GTPases signaling pathways, leading to the activation of RhoA and its downstream effector ROCK, which are crucial for regulating cell contraction, migration and invasion (62).

Endosomal integrin signaling also intersects with the JNK signaling pathway, potentially affecting cell stress responses and apoptosis (63). Non-classical integrin signaling through FN-EDB can activate NF-κB, which is associated with inflammation and cell survival regulation, further highlighting the multifaceted role of FN-EDB in tumor biology (64).

In clinical research, atypical β1-integrin signaling mechanisms are related to the development and progression of various types of tumor. For example, in breast cancer, atypical β1-integrin signaling enhances tumor invasiveness and metastatic potential by promoting EMT and angiogenesis (65). In non-small cell lung cancer, atypical integrin signaling is associated with tumor cell proliferation, drug resistance and metastasis. Integrin-mediated signaling can enhance tumor cell invasiveness and promote immune evasion within the TME (66). In glioblastoma (67), atypical β1-integrin signaling is linked to the maintenance of tumor stem cell properties, which may contribute to tumor recurrence and treatment resistance.

In summary, the regulation of both classical and atypical integrin signaling pathways by FN-EDB underlines its multifaceted role in tumor progression. Understanding the complex interactions between FN-EDB, integrins and their downstream signaling molecules is key for deciphering the intricate regulatory networks that control tumor angiogenesis, invasion and metastasis. Future research aimed at dissecting these pathways may reveal new therapeutic targets for cancer treatment, providing strategies to disrupt the complex signaling networks regulated by FN-EDB in the TME.

The activation of ERK1/2 and Akt not only regulates immediate cellular responses but also extends to the regulation of gene expression. Specifically, these kinases promote the nuclear translocation of transcription factors such as NF-κB and hypoxia-inducible factor-1alpha (HIF-1α). In the hypoxic environment within tumor masses, the HIF-1α dimeric protein complex remains stable and activates the expression of numerous genes involved in angiogenic processes (70). HIF-1-induced proteins include VEGF and basic fibroblast growth factor (bFGF), which promotes vascular permeability, while the latter promotes endothelial cell growth (71). Other secreted factors such as platelet-derived growth factor (PDGF), angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2) promote chemotaxis, while hepatocellular signaling controls migration and cell-cell adhesion, thereby guiding the formation and stabilization of newly formed blood vessels (72). Other HIF-1-induced gene products include MMP, where MMP resolves the ECM, promoting endothelial cell migration and releasing associated growth factors. Once activated, these factors bind to the promoter regions of genes encoding angiogenic factors, including VEGF, to amplify the angiogenic response (73).

Endothelial cells and pericytes are fundamental cellular components of new blood vessels and their interactions regulate angiogenesis (74). FN-EDB has been shown to disrupt the interaction between pericytes and endothelial cells, leading to pericyte detachment and vascular system instability, resulting in further angiogenesis and remodeling of the tumor vasculature (75). FN-EDB-induced signaling pathways enhance endothelial cell proliferation, migration and invasion, which are crucial for vascular sprout formation (76). Pericytes provide support by covering the basal surface of endothelial cells and regulate vascular contraction and relaxation under normal physiological conditions. Newly formed vessels often lack pericytes, but endothelial cells recruit these pericytes to provide additional structural support, enhancing tumor vascularization (77).

FN-EDB promotes formation of tubular structures by promoting the proliferation and organization of endothelial cells. It achieves this by activating FAK and subsequent downstream signaling molecules, including ERK/MAPK and PI3K-Akt pathways that enhance cell adhesion and cytoskeletal rearrangements, required for tube morphogenesis (78).

In the TME, in addition to endothelial cells, many other cell types contribute to angiogenesis. FN-EDB interacts with tumor-associated macrophages (TAMs) and other immune cells, promoting ECM remodeling and creating favorable physical and chemical conditions for angiogenesis (79). Factors secreted into the TME activate TAMs, producing VEGF and MMP, further promoting angiogenesis. Neutrophils, a significant component of immune cell infiltration, promote tumor angiogenesis through various mechanisms, including the release of MMP into the TME, triggering the release of VEGF and other angiogenic factors (80). Other immune cell types (such as B and T cells) secrete VEGF-A, bFGF, MMP9, interferon γ (IFNγ) and interleukin (IL)-17, indirectly affecting angiogenesis (81). Adipocytes release a variety of cytokines, chemokines and hormones (collectively referred to as adipokines), many of which are pro-angiogenic factors (82).

FN-EDB expression and activity can be further enhanced by tumor-secreted cytokines, forming a positive feedback loop that drives tumor angiogenesis. This positive feedback mechanism amplifies the impact of FN-EDB on tumor angiogenesis (83). VEGF and other growth factors respond to the upregulation of FN-EDB signaling, creating a feedforward mechanism that strengthens the angiogenic process. This autocrine and paracrine signaling enhances the recruitment of endothelial cells, pericytes and smooth muscle cells, promoting the stability and maturation of newly formed blood vessels (84).

ZD2, a small peptide aptamer, selectively targets the TME-specific fibronectin extra domain B (FN-EDB), a protein overexpressed in various types of cancer. Its tumor-targeting capability positions ZD2 as a promising agent for diagnostic imaging and therapeutic applications (88,89). Recent advances include the development of a Tc-99m-labeled aptamer (EDB) as an effective tumor imaging agent, demonstrating high specificity for EDB-positive cell lines in vitro and favorable pharmacokinetics in vivo, with rapid blood clearance and renal excretion. The potential of this agent to reflect tumor metastasis status offers a valuable tool for early cancer diagnosis and therapeutic response monitoring (90,91).

FN-EDB, a splice variant of fibronectin, has emerged as a promising target for the design of clinically translatable magnetic resonance imaging (MRI) and positron emission tomography (PET) contrast agents. A study investigated the MRI dose-response effect of ZD2-N3-Gd (HP-DO3A) (MT218), a targeted contrast agent, in a rat prostate cancer xenograft model and its detection potential in mouse lung and pancreatic cancer models (92). MT218 exhibited high relaxivities (r1 and r2), favorable physicochemical properties, pharmacokinetics and safety, leading to effective tumor enhancement at reduced contrast agent doses. In the realm of PET imaging, a ZD2-(⁶⁸Ga-NOTA) conjugate was developed for hepatocellular carcinoma (HCC) imaging, demonstrating high affinity for FN-EDB in a marmoset model, rapid tumor accumulation and a stable state within 5 min post-injection (93). Additionally, a ⁶⁴Cu-DOTA conjugated to the ZD2 peptide was created for sensitive detection and risk stratification of prostate cancer (94). These findings validate the feasibility of using ZD2 peptide-based radiopharmaceuticals for PET imaging, offering new perspectives for clinical management of patients with HCC and prostate cancer.

ZD2-Gd (HP-DO3A), a molecular MRI contrast agent targeting FN-EDB, has demonstrated significant contrast enhancement in primary and metastatic triple-negative breast cancer tumors, outperforming the nonspecific clinical agent Gd (HP-DO3A) (95–97). The high expression of this agent in the tumor ECM positions it as a promising tool for precise cancer diagnosis. Furthermore, Cy5-EDBp, (¹⁸F)-EDBp and (¹⁷⁷Lu)-EDBp have emerged as potential candidates for surgical navigation, radionuclide imaging and targeted radionuclide therapy, respectively (98).

The development of novel targeted contrast agents, such as the EDB-FN-targeted imaging probe (ZD2-Gd-DOTA-Cy7), has shown good performance in dual-modality MRI and near-infrared fluorescence imaging for pancreatic cancer, as well as in the precise assessment of chemotherapy efficacy (99). This probe requires only half the dose of conventional clinical gadolinium contrast agents to achieve accurate cancer imaging and evaluation of therapeutic responses. By conjugating ZD2 with commonly used clinical MRI gadolinium contrast agents (Gd-DOTA) and the near-infrared fluorescence dye Cy7, researchers have created an EDB-FN-targeted imaging probe. This probe, combined with dual-modality imaging techniques, has been used to assess the efficacy of chemotherapy regimens, offering a new approach for personalized treatment of pancreatic cancer patients (100).

In summary, ZD2 peptide and its derived targeted probes have shown considerable potential and application prospects in the field of tumor diagnosis and therapy targeting FN-EDB. With continuing technological advances and clinical trials, ZD2 peptide and its associated targeted probes are expected to play an increasingly vital role in future cancer therapy.

APTEDB Peptide, a molecule designed based on structural features of FN-EDB, ensures high affinity and selective binding to the TME by recognition of key amino acid residues (101). Ranjbar et al (102) achieved specific binding and high tumor uptake in colorectal cancer models by Tc-APTEDB probe, demonstrating its potential as a diagnostic imaging probe. The high binding specificity of the APTEDB peptide, which selectively targets the fibronectin extra domain B (FN-EDB) overexpressed in the tumor microenvironment, enables the development of novel tumor imaging modalities. For example, APTEDB has been conjugated to imaging agents, such as fluorescent dyes, quantum dots, and radionuclides, to create targeted probes that enhance the detection and visualization of tumors with high contrast and resolution (103). These targeted imaging modalities have shown promise in preclinical models, providing a non-invasive approach to cancer detection and monitoring. In the field of optical imaging, the coupling of APTEDB with fluorescent labels or quantum dots enables the visualization of tumor regions with high contrast and resolution (104). In PET and MRI, the coupling of APTEDB to a radionuclide or contrast agent, respectively, enhances the detection of tumors in preclinical models (105). Using FN-EDB-specific peptide [APT (EDB)] Sun et al (106) conjugated thermal cross crosslinked superparamagnetic iron oxide nanoparticles [APT (EDB)-TCL-SPIONs] in a breast cancer stem-cell-like cell xenograft mouse model. The results of these studies confirm the broad application of APTEDB peptides in tumor-targeted therapy and imaging (107).

Recent advances in nanotechnology have facilitated the development of FN-EDB-targeting nanoparticles for enhanced diagnostic and therapeutic capabilities in oncology (108). Surface modification of these nanoparticles has been instrumental in augmenting their accumulation and internalization within the TME. Jailkhani et al (109) identified a high-affinity nanobody, alpaca-derived libraries of nanobodies (NJB2), which selectively bind to FN-EDB expressed in the ECM of tumors and metastases, offering a non-invasive diagnostic tool for cancer detection and a targeted approach to fibrotic diseases. Using immunoPET/CT, ⁶⁴Cu-NJB2 nanobodies demonstrate the ability to detect primary and metastatic tumors across various cancer models, highlighting their potential as robust tools for oncological imaging (110). In parallel, Rossin et al (111) successfully radiolabeled an anti-ED-B fibronectin human antibody derivative, L19-SIP, using an enzymatic radiobromination method. In vivo studies in animal models revealed rapid and specific tumor targeting by (76) Br-L19-SIP, with subsequent validation of its potential in tumor imaging through small animal PET. Berndorff et al (112) developed 99 mtc-tagged L19 derivatives to target ED-B fibronectin for the angiogenesis imaging in tumors. Compounds 99 mTc-AP39,99 mTc-L19-His and 99 mTc-L19-Hi20, were synthesized and evaluated in vivo. 99mTc-AP39 shows high tumor uptake, rapid blood clearance and excellent imaging properties, providing a promising tool for visualizing tumor-expressing ED-B.

These studies underline the value of targeted molecular imaging with radiolabeled antibodies in enhancing the detection and monitoring of cancer, paving the way for more precise and personalized therapeutic strategies.

At present, antibodies (L19) are often targeted with cytokines in FN-EDB for tumor treatment and diagnosis. These other antitumor drugs are composed of mAbs and cytokines (such as IL-12, IL-15 and IL-21), TNF-α, IFN-γ and tissue factor to deliver cytokines directly to the TME to activate the immune response (117).

L19-IL2, a novel fusion protein consisting of the single-chain variable fragment of the humanized monoclonal antibody L19 targeting FN-EDB and IL-2 represents a promising advancement in cancer therapeutics (118). This targeted approach enhances the delivery of IL-2 directly to the TME, thereby mitigating the systemic side effects associated with conventional IL-2 therapy.

The L19 antibody, which selectively binds to FN-EDB overexpressed in various malignancies, has been effectively fused with IL-2 to create darleukin. This fusion protein not only exhibits high tumor specificity but also activates cytotoxic T lymphocytes and natural killer (NK) cells, bolstering the immune response against cancer cells (119). Moreover, L19-IL2 may reshape the immunosuppressive TME, facilitating the activation and proliferation of tumor-specific T cells and the maturation and antigen presentation of dendritic cells.

Preclinical studies have demonstrated the efficacy of L19-IL2 in suppressing the growth of FN-EDB-positive tumors and extending survival in animal models, including nude and SCID mice (120). The transition of L19-IL2 into clinical trials marks a significant step, with ongoing research aimed at assessing its safety, tolerability and efficacy in human subjects. Initial clinical investigations are focused on determining the maximum tolerated dose, recommended phase II dose and characterizing the pharmacokinetic and pharmacodynamic profiles of L19-IL2 (121).

The potential for L19-IL2 to be combined with other therapeutic modalities, such as immune checkpoint inhibitors, chemotherapy and radiotherapy, is currently being explored in clinical trials. For example, in a randomized phase II clinical trial, one group of patients received dacarbazine (DTIC; 1,000 mg/m² on day 1 of a 21-day cycle) as a single agent, while in the other two groups, L19IL2 was increased (22.5 million international units of IL 2 equivalents) depending on two different dosing regimens. Analysis of efficacy results showed that patients receiving L19IL2 combined with DTIC had statistically significant results for overall response rate and median progression-free survival compared with DTIC monotherapy (122). The synergy between L19-IL2 and stereotactic ablation body radiotherapy (SABR) is of particular interest, as evidenced by a phase II clinical trial investigating their combined use in patients with oligometastatic non-small cell lung cancer (NSCLC) (123). This multicenter, randomized controlled trial, involving 126 patients, is designed to evaluate the 1.5-year progression-free survival as the primary endpoint, highlighting the potential of integrating L19-IL2 with SABR to enhance local tumor control and systemic immune responses (124).

IL-12 was covalently linked with a monoclonal antibody to produce an IL-12 antibody drug conjugate (ADC) to deliver IL-12 to tumor cells enriched at the target antigen level. This IL-12 delivery targeting a specific antigen minimizes IL-12 exposure in normal tissues, allowing it to show lower toxicity and an improved therapeutic index (125). The development of ADC therapy underwent several stages. The first generation ADC has limited therapeutic efficacy due to problems such as linker instability and insufficient payload toxicity. The second generation ADC improved efficacy by improving the stability of the linker and using more payloads. The third generation of ADC further optimized the selectivity of antibodies, the design of linker and the toxicity and bystander effects of payload, so that ADC therapy has achieved significant therapeutic effects in some hematological tumors and solid tumors (126,127).

AS1409 is formed by covalently linking the humanized anti-FN-EDB antibody BC-1 with IL-12, aiming to deliver IL-12 to the tumor-associated vasculature (128). Ongaro et al (129) developed a tandem-dimer form of a full-human fusion protein, L19-L19-IL12, created by fusing human IL-12 as a tandem dimer with the L19 antibody using protein engineering techniques. This variant, equipped with an optimized linker, demonstrated enhanced tumor targeting, prolonged retention at the tumor site and rapid clearance from blood and normal organs, suggesting its potential as a promising alternative to L19-IL12 and warranting further clinical investigation.

Clinical trials are currently underway to evaluate ADCs targeting FN-EDB across different types of tumor for safety, pharmacokinetics and antitumor effects. PYX-201, an ADC comprising a monoclonal antibody against FN-EDB, a cleavable linker and an auristatin payload, is being assessed in Phase I clinical trials for various solid tumors, including squamous cell carcinoma of the head and neck, non-small cell lung cancer, ovarian cancer, soft tissue sarcoma and pancreatic ductal adenocarcinoma (130). The ADC's internalization and endosomal-lysosomal pathway release the payload, achieving precise cytotoxicity against tumor cells.

EDB-ADCs have demonstrated antitumor activity and enhanced immune cell infiltration in multiple cancer models, with improved efficacy when combined with immune checkpoint blockade and have shown good safety profiles in non-human primates (131). These findings underscore the potential of FN-EDB-targeting ADCs as a valuable tool in the oncology therapeutic arsenal, providing a rationale for their further development and clinical application.

L19 can also be coupled to other cytokines, including IL-10 (132), IL-15 (133) and granulocyte-macrophage colony-stimulating factor (granulocyte-macrophage colony stimulating factor; GM-CSF) (133), IFN-γ (134–136), and TNF-α (137–140), Most of these cytokines are produced by immune cells and act on the tumor.

Trachsel et al (132) demonstrated that L19-IL-10 fusion protein, when delivered to rheumatoid arthritis models, exhibits superior efficacy in alleviating arthritis symptoms and inhibiting disease progression compared with non-targeted IL-10. This targeted approach suggests a potential therapeutic benefit in chronic inflammatory conditions. Kaspar et al (133) investigated the antitumor properties of L19-IL-15 and L19-GM-CSF, revealing potent antitumor activities in both subcutaneous and metastatic cancer models. These immunocytokines, by selectively binding to tumor vasculature, effectively inhibited tumor growth and metastasis, underscoring the potential of L19-based targeted therapy in oncology (133). IFNγ, a critical immunomodulatory cytokine, has been explored in the context of L19 fusion proteins to enhance tumor immunogenicity and promote immune cell infiltration. Ruan et al (134) developed an L19-IFNγ variant fusion protein with reduced affinity for its cognate receptor, thereby minimizing off-target organ capture and enhancing tumor-specific localization. Di Nitto et al (135) designed an L19-IFNγ KRG fusion protein that demonstrated improved tumor homing and pharmacokinetic properties in preclinical models. Niu et al (136) show that the combination of L19-IFNγ KRG with immune checkpoint inhibitors, such as anti-PD-1 antibodies, induce tumor growth delay and increase intra-tumoral concentrations of T cells and NK cells, highlighting the synergistic potential of this combination therapy.

TNF-α is known for its potent antitumor effects. L19-TNF is an immune cytokine fusion protein targeting tumor vasculature resulting from the fusion of the human monoclonal antibody L19 to TNF. L19 antibody can specifically bind FN-EDB on the surface of tumor vascular endothelial cells and accurately deliver TNF to tumor tissue, which has also been studied in the form of L19-mTNF fusion protein (140). Spitaleri et al (139) reported on the efficacy and safety of L19-TNF monotherapy in patients with advanced solid tumors in a phase I/II clinical trial (139). A total of 34 patients were enrolled into six dose groups, with L19-TNF doses increasing from 1.3–13 µg/kg, every 3 weeks, administered on days 1, 3 and 5. The aforementioned study showed that in terms of safety, the patients mainly experienced mild chills, nausea and vomiting but no hematology or unexpected serious toxicity. In terms of efficacy, no objective tumor response was observed, but out of 31 evaluable patients, 19 had transient stable disease-states (137). Therefore, its potential for combination chemotherapy to play a greater role in the treatment of advanced solid tumors can be further explored. In an additional exploratory clinical trial, the investigators evaluated the safety and clinical activity of L19-TNF in combination with melphalam-isolated limb perfusion (ILP) containing melphalam in patients with locally advanced limb melanoma (138). A total of 17 patients were included, of which 7 received 325 µg L19-TNF and 10 received 650 µg L19-TNF. The results showed that the non-hematologic toxicity of L19-TNF ILP was low, but four patients developed severe myelosuppression. Although the TNF equivalent dose of L19-TNF was only 3.13 and 6.25% of the approved TNF (Beromun^®) dose of 4 mg, the clinical efficacy was marked. In the 650 µg dose group, 89% of patients had objective responses, five achieved complete response (CR) and four patients had CR status until 12 months; no CR was observed in the 325 µg group. This suggests that L19-TNF ILP has promising clinical activity and potential application in the treatment of locally advanced limb melanoma (138). Danielli et al (137) fused two immune cytokines, IL-2 and TNF-α, to a combination of monoclonal L19 antibody that binds to FN-EDB and this selectively targeting L19 antibody could allow the drug to accumulate in the injected lesion, potentially producing a more durable immunoreactive. Melanoma is an aggressive skin malignancy (141), especially in the local advanced stage (stage IIIB/C). Traditional surgical resection may not achieve ideal results, and patients face a high risk of recurrence and metastasis. New treatments are needed. The Neo-DREAM trial is a phase III study to evaluate the efficacy of neoadjuvant intratumoral injection of Darleukin/Fibromun (L19IL2 + L19TNF) in patients with clinical stage IIIB/C melanoma. The study randomizes patients into two groups: Darleukin/Fibromun, followed by surgery and adjuvant therapy and surgery and adjuvant therapy alone (142). Darleukin/Fibromun precisely targets FN-EDB in the tumor extracellular matrix (ECM) by using immune cytokines IL-2 and TNF-α with the monoclonal antibody L19 to stimulate the local immune response. According to the June 2022 issue of Ann Surg Oncol, the study observed significant patient objective response rates (ORR) and excellent complete response rates (CR) and partial response rates (PR) (143). Notably, the incidence of distant effect (abscopal effect) in non-injected lesions was ≤53.8% (7/13), indicating that Darleukin/Fibromun can not only effectively control tumors at the injection site, but also produce immune killing in distant uninjected lesions. In terms of safety, the side effects recorded in the study were relatively few and controllable and Darleukin/Fibromun showed improved safety compared with traditional treatments.

These studies collectively illustrate the versatility and potential of L19-based immunocytokines in modulating the TME and enhancing antitumor immune responses. The ongoing clinical development of L19-IL2 and other L19-based fusion proteins signifies a promising direction in cancer immunotherapy, with the potential to improve patient outcomes through targeted delivery of therapeutic agents to the tumor site. The synergistic effects observed with combination therapies further emphasize the importance of exploring multimodal treatment strategies to maximize clinical benefits.

Tumor-specific PDCs) represent a burgeoning class of therapeutics in cancer immunotherapy, harnessing the targeting capabilities of tumor-specific peptides to deliver cytotoxic agents directly to cancer cells (144). This strategy aims to enhance therapeutic efficacy while minimizing damage to healthy tissues, offering a promising approach to improve cancer treatment.

The design of PDCs necessitates careful selection of a linker that ensures drug stability and is cleavable within the TME to release the active drug. Equally critical is the choice of toxin, which must possess potent antitumor activity and retain its pharmacological properties post-conjugation. Researchers have developed a bispecific peptide, HyPEP (EDB-VEGF), capable of binding to cancer cells overexpressing EDB and VEGF (145). In animal models, this conjugate has demonstrated the ability to inhibit tumor growth, with enhanced effects when combined with anti-PD-1 antibodies, suggesting its potential in immunotherapy (146). The PL1 peptide, identified for its ability to bind to FN-EDB and Tenascin C subtypes in the tumor ECM, triggers cellular uptake primarily through electrostatic interactions.

This finding provides a novel perspective for developing targeted cancer therapeutics. By conjugating carboplatin (CBT) with a cyclic cell-penetrating peptide, a new drug delivery system has been developed. This conjugate selectively binds to cancer cells overexpressing integrins and EDB-FN and releases CBT in the acidic tumor environment, exhibiting high selectivity and low toxicity for cancer cells (147). In the treatment of prostate cancer, PDCs targeting EDB-FN have shown therapeutic effects in animal models by providing selective cytotoxicity (148). To overcome the short circulation half-life of PDCs, a strategy involving the formation of hybrid complexes with anti-hapten antibodies has been developed, significantly extending the PDC's circulation half-life, enhancing tumor accumulation and penetration, thereby inhibiting tumor growth (149). The expression of VEGF, closely associated with the development of various tumors, has been targeted by cetuximab (Erbitux), a monoclonal antibody that inhibits tumor angiogenesis by blocking the binding of VEGF to its receptors, thus exerting antitumor effects (150).

Current research on PDCs in cancer therapy has made significant progress. As clinical trials continue to assess the safety and efficacy of PDCs, their potential to revolutionize cancer treatment strategies continues to grow.

In the medical research domain, innovative nanosystems have been developed to enhance drug targeting and bioavailability while minimizing systemic side effects (104,151,152). Gold nanoparticles (NPs) with distinct surface modifications have demonstrated the ability to selectively bind various structural domains of fibronectin (FN), with interaction strengths influenced by the NPs' physicochemical properties. Particularly, cationic NPs (NP-NH3+) interact with acidic domain-rich regions of FN, potentially impairing its function (153).

For instance, a system encapsulating bevacizumab within immunoglobulin-1 functionalized PLGA nanoparticles has been investigated for the treatment of atherosclerosis, showing improved drug targeting and bioavailability with reduced systemic adverse effects (151). Noh et al (104) developed APTEDB-cyNP @ CpG nanomedicine, which coupled APTEDB to PEG and DSPE to build cyanine nanoparticles coated with CpG adjuvant for photothermal therapy and immunotherapy of cancer. In addition, APTEDB-DSPE-docetaxel (DTX) nanoparticles formed by combining APTEDB peptide and DTX show excellent targeting and anti-tumor effects in glioma treatment (154). These nanodrugs have exhibited significant therapeutic effects in tumor models, inducing immunogenic cell death and enhancing T-cell antitumor activity.

To enhance the therapeutic efficiency for malignant glioblastoma multiforme (152), aptamer-like peptide-modified liposomal nanoplatforms for systemic small interfering RNA (siRNA) delivery have been developed. These nanoplatforms specifically target tumor cells and suppress tumor growth by silencing the expression of key genes, such as cyclophilin A. Furthermore, FN-EDB expression in atherosclerotic plaques has been explored, leading to the design of FN-EDB-specific nanoparticles for enhanced plaque detection and localized drug delivery, demonstrating favorable targeting and drug delivery outcomes in animal models (155).

These studies collectively highlight the potential of nanotechnology in improving drug delivery efficiency and tumor therapy, offering new directions for future clinical applications.

Small molecule inhibitors targeting FN-EDB disrupt FN-EDB interactions with integrin receptors to impede tumor cell adhesion, migration and invasion. These inhibitors mitigate tumor growth and metastasis by attenuating interactions between cancer cells and the ECM, modulating signaling pathways within the TME that influence angiogenesis and immune cell infiltration (156).

BRAF/MEK inhibitors exemplify cell growth suppressants that induce immunogenic cell death, activating antitumor T cell responses, particularly in melanomas harboring BRAF (V600E) mutations (157). VEGF-VEGFR inhibitors, such as bevacizumab, disrupt the VEGF pathway, curtailing recruitment of immunosuppressive cells and enhancing T cell tumor infiltration, demonstrating significant efficacy in combination with atezolizumab for non-small cell lung cancer (158).

CSF1R inhibitors, by modulating immune cell activity in the TME, reduce the number of TAMs, bolstering the antitumor immune response (159). Metabolic pathway inhibitors, including glutaminase and arginase inhibitors, are under investigation to regulate the metabolism of both tumor and immune cells, further potentiating immune responses (160).

The advent of small molecule immune checkpoint inhibitors offers a novel avenue for directly targeting the PD-1/PD-L1 axis, promising similar therapeutic effects to antibody drugs with improved pharmacokinetics and oral bioavailability. Emerging small molecule immunotherapeutic candidates, such as ABBV-CLS-484, target both tumor and immune cells, enhancing immune cell activity and sensitizing tumors to immune attack, showing significant antitumor effects in animal models (161).

These advances highlight the key role of small molecule inhibitors in activating and augmenting the immune system against cancer, offering new therapeutic prospects for precision oncology.

CAR-T cell therapy has revolutionized cancer treatment by genetically engineering the T cells of patients to recognize and attack specific tumor cells. This approach has demonstrated marked efficacy in hematological malignancies, leading to the approval of several CAR-T products. The evolution of CAR-T therapy, from the first generation with a single-chain variable fragment to the fourth generation incorporating multiple signaling pathways, has been driven by the pursuit of enhanced efficacy and safety.

Despite the success in blood cancers, CAR-T cell therapy faces challenges in solid tumors due to the suppressive TME and the lack of specific tumor-associated antigens (162). To address this, researchers have turned to FN-EDB, a cancer-fetal antigen highly expressed in the tumor vasculature and cancer cells, making it a potential target for CAR-T cell therapy in solid tumors (163).

Investigators have developed recombinant T cells expressing T cell receptors (rTCRs) fused with CARs (rTCR-CAR T cells) that target FN-EDB (164). These rTCR-CAR T cells have shown the ability to bypass TME suppression by combining the single-chain variable fragment with CD3ε. Preclinical studies have shown that rTCR-CAR T cells exert antitumor effects on various solid tumors, including thyroid cancer and breast cancer, by inhibiting the effects of FN-EDB in cell adhesion, migration and angiogenesis (165,166). These rTCR-CAR T cells exhibited cytotoxicity against EDB-positive tumor cells in vitro and effectively inhibited tumor growth and reduced tumor vascular density in vivo without significant toxicity.

Further research has constructed a BBz CAR targeting EDB-FN, using lentiviral transduction to express CAR molecules on T cells (167). In vitro and in vivo experiments confirm the ability of CAR T cells to activate and lyse EDB-positive cells, inhibiting tumor growth and vascular density in U-87 MG xenograft models.

The innovative rTCR-CAR T cell therapy is currently being evaluated in multiple clinical trials for its safety and efficacy in cancer treatment, particularly in targeting FN-EDB within the TME (168,169). Combination therapy strategies under exploration include the use of CD70 CAR-T cells with PARP inhibitors, aiming to enhance efficacy by optimizing CAR-T cell structures and constructing bispecific CAR-T cells (170).

Additionally, the combination of CD70 CAR-T cells with oncolytic viruses (OVs) exploits the selective replication and destructive capacity of OVs within tumor cells, promoting an inflammatory TME to improve treatment outcomes for solid tumors (171). Epigenetic modulators, such as decitabine and givinostat, have shown potential in enhancing the efficacy of CD70 CAR-T cells by increasing CD70 expression levels (172).

The combination of CAR-T cells with immune checkpoint inhibitors, such as anti-PD-1 and anti-PD-L1 antibodies, restores the function of tumor-infiltrating lymphocytes, enhancing their ability to attack cancer cells and improving the persistence and efficacy of CAR-T cells within the TME (173). Furthermore, the co-expression of cytokines such as IL-12, IL-15 and IL-18 with CAR-T cells has demonstrated advantages in boosting their activity and durability (174).

Moreover, the combination of CAR-T cells with small molecule drugs, such as the BTK inhibitor ibrutinib, has potential to improve patient responses in clinical trials (175). The scope of combination therapies for CAR-T cells extends beyond the aforementioned strategies to include radiotherapy, chemotherapy, hematopoietic stem cell transplantation and targeted therapies, all aiming to enhance treatment efficacy.

In summary, CAR-T cell therapy targeting FN-EDB holds significant potential in the treatment of solid tumors, with ongoing clinical trials exploring its effectiveness. The future of cancer treatment may benefit from more effective and personalized options offered by CAR-T cell therapies. As research progresses, optimizing CAR-T cell design, enhancing treatment efficacy, minimizing side effects and exploring combination applications with other therapeutic modalities will remain a focal point in this field.

The present study focused on the involvement of FN-EDB and FN-EDB and its diagnostic role as a biomarker in tumors and FN-EDB inhibition as a strategy for immunotherapy. FN-EDB has emerged as a pivotal player in tumorigenesis, with its elevated expression levels in malignant tissues correlating with aggressive tumor behavior, including invasion, lymph node metastasis and poor prognosis. This association positions FN-EDB as a potential biomarker for cancer diagnosis and a therapeutic target. The utility of FN-EDB as a biomarker has been harnessed in the development of various imaging techniques, such as MRI contrast agents using FN-EDB-specific peptides like ZD2, enhancing the specificity and sensitivity of tumor detection. Significant advances have been made in targeting FN-EDB for cancer immunotherapy, with approaches including ADCs, CAR-T cell therapy and immune checkpoint blockade. Clinical trials for several therapeutic and diagnostic modalities targeting FN-EDB are underway, with some, like the phase III trial of daromun for neoadjuvant treatment, showing promising results (184,185).

Despite these developments, challenges remain in translating FN-EDB-targeted therapies into clinical practice. The precise mechanisms by which FN-EDB contributes to the TME are not fully understood and its complex interactions with cancer cell signaling pathways require further investigation. The differential expression of FN-EDB across tumor types hints at diverse roles in oncogenesis, necessitating a deeper exploration of the underlying biological mechanisms. While preclinical studies have shown promise for FN-EDB-targeted conjugate therapies, the transition from animal models to clinical settings demands rigorous clinical trials to validate safety and efficacy in humans. Future research should focus on elucidating the role of FN-EDB in tumor biology, dissecting its signaling pathways in oncogenesis and understanding the basis for its variable expression across different tumors.

A thorough understanding of FN-EDB's function will pave the way for the development of more effective and safer immunotherapies targeting FN-EDB. Concurrently, the pursuit of safer and more effective conjugate drugs, along with the development of non-invasive and precise diagnostic strategies for tumor grading, will be crucial for enhancing cancer treatment and diagnosis outcomes. Although challenges persist, ongoing research into FN-EDB holds the promise of yielding more potent immunotherapeutic agents and clinical strategies, offering renewed hope for patients with cancer.