In clinical laboratory diagnosis, the research focus is still on biological specimens such as blood, urine, saliva, and cerebrospinal fluid, which can be obtained by non-invasive or minimally invasive techniques (17). In general, abnormal proteins in body fluids such as CA125 and HE4 have been the first choice for ovarian cancer examination. However, detection sensitivity and specificity continue to fall short of the desired aim, and there are certain limits in early diagnosis (18). The exploration of peptide markers has attracted much attention in recent years and has potential clinical application prospects. Peptide markers can be relatively resistant to protease degradation, and small peptides may survive even if the protein is degraded by proteases in the tumor extracellular space or circulation (19). Therefore, it may provide more information than the precursor protein (Protein containing the target polypeptide sequence). Moreover, advances in technologies such as peptidomics, peptide microarray, and reaction monitoring (SRM) analysis support the detection of more low-molecular-weight, tumor-specific peptides in body fluids. In addition to high specificity, SRM analysis provides sensitivity and linearity, providing an opportunity for accurate quantification of peptides (20, 21).

The research has shown that peptides with different clinical features can be identified, quantified and compared using detection techniques, and these low molecular weight peptides are more likely to be markers for diagnosis. A prospective study about urinary micropeptides in patients with ovarian cancer has shown that they presenced in about 62.5% of patients, and were related to the pathological stage. Under 100% specificity, the detection sensitivity of CA125 was 23.2%, while that of urinary micropeptides was 62.5% (22). At the peptidome level, more peptides could be observed in serum samples compared to plasma. However, serum shows many strong artificial peptide signals that hinder the sensitive detection of endogenous peptides. Therefore, most investigators have turned their goals to plasma samples (23, 24). Wang and colleagues (20) used SRM method to detect peptide biomarkers and found that 11 (64.7%) of 17 patients with early ovarian cancer had positive scores of small peptides from peptidyl prolyl cis-trans isomerase A(PPIA), which may be a useful diagnostic marker for ovarian cancer. Lu’s group (18) isolated three promising predictive small peptides from plasma samples of 140 patients with epithelial ovarian cancer (EOC) and 158 patients with benign ovarian tumors (BOT), and their combination with CA125 increased the specificity (0.944) and AUC (0.904) of CA125 while maintaining an acceptable sensitivity of 0.804.

Traditional imaging techniques rely on non-specific imaging methods for disease examination, such as image contrast using anatomical, physiological, or metabolic abnormalities, and are often less efficient at targeting (25). In fact, molecular or biochemical alterations in disease often precede morphological and functional alterations and can more accurately characterize tumor properties or biological processes (26). Based on the observation that peptides can bind specifically to disease-associated receptors, coupling imaging molecules to peptides is a simple and accurate way to improve traditional imaging. In general, the size of the polypeptide can provide sufficient attachment sites for the chelating agent molecule, which is more suitable for linking with the chelating agent than the larger molecule, without losing the binding affinity with the receptor (27). The advantages of using radiopeptide imaging include the ease of synthesis and radiolabeling, as well as the ease of modification that enhances renal excretion and significantly improves kinetics (28). Furthermore, the small molecular weight of peptide allows for rapid blood clearance and renal excretion, which makes it among the key features of an ideal imaging probe. Many selected peptides or peptide analogues used to target OC-related molecules or receptors show high affinity and specificity for the receptors, such as RGD, NGR, etc ( Table 1 ) (37, 38).

The RGD (Arg-Gly-Asp) peptide recognizes the highly expressed α_vβ₃ integrin in ovarian cancer cells and can specifically bind α_vβ₃ with high affinity after radiolabeling. Imaging probes based on RGD peptides show good imaging effects on ovarian cancer and are one of the most promising diagnostic probes (39). Janssen and colleagues (29) synthesized radiolabeled dimeric RGD peptide E- [c (RGDfK)]₂, which showed specific targeting, good tumor uptake, and rapid renal excretion in the OVCAR-3 xenograft model. Yang’s group (30)found that ¹⁸F-RGD2 had a higher tumor/background ratio than ¹⁸F-FDG in SKOV-3 xenograft mice (6.04 ± 0.92 vs. 3.34 ± 0.79), and in addition, the anti-angiogenic effect was sensitively and reliably detected at low doses of paclitaxel. At present, imaging probes based on RGD peptides have entered preclinical or clinical trials. In a clinical study of ovarian and breast cancer, the PET/CT imaging analysis found that RGD-based imaging probes (¹⁸F-FPPRGD₂) can provide independent information with ¹⁸F-FDG, although the foci uptake and tumor/background ratio of ¹⁸F-FPPRGD₂ were lower than ¹⁸F-FDG, but 1 patient increased uptake after 1 week of anti-angiogenesis therapy. This is a potential benefit of RGD peptide-based PET tracers with the potential to predict anti-angiogenesis therapy early (31).

Aminopeptidase N (APN/CD13), a membrane protein and marker of tumor angiogenesis, is upregulated in various solid tumors, including melanoma, prostate, ovarian, lung, and breast cancers (40). NGR (Asn-Gly-Arg) peptide specifically recognizes aminopeptidase N on the surface of tumor cells. Radiolabeled NGR has been reported to have high uptake in ovarian tumors and is excreted through the kidneys (41). Meng and colleagues (32)prepared Cy5.5-labeled, NGR-conjugated iron oxide nanoparticles (Cy5.5-NGR-Fe₃O₄ NPs) as imaging nanoprobe. MR and NIRF dual-modality imaging showed that NGR targeting effectively improved tumor uptake and enhanced imaging contrast, in contrast, the control group image did not change significantly. In another work, NGR peptide-based imaging probes exhibited selective targeting and quick blood clearance of ES2 tumors, with high contrast tumor visualization occurring at 1 h and a maximal tumor/background ratio of 10.30 ± 0.26 (33).

In addition to radiolabeling, optical imaging of fluorescently labeled peptides may be advantageous because it may improve the detection of metastatic tumor deposits while reducing the risk of population radioactivity exposure (42, 43). Liu and colleagues (34) report the biological characterization of fluorescently labeled GE137 (c-Met targeting peptide) in ovarian cancer cells. The peptide demonstrated selective tumor uptake and a consistently high signal-to-noise ratio, allowing real-time observation of subcutaneous submillimeter SKOV3 tumor deposition in mice. Besides, some fluorescent probes can be used to detect ovarian cancer metastasis. In ovarian cancer mice, Liu’s group found that GnRHa-ICG was specifically localized in peritoneal tumors with high tumor-to-muscle and tumor-to-gut ratios and stable fluorescent signals compared to ICG injection alone (35). Intraoperative specific fluorescence imaging has been reported to improve tumor stage and size and improve prognosis. Wang’s team (36) designed in vivo assembly of NIR-II emitting down conversion nanoparticles (DCNPs) modified with DNA and FSHβ peptides. Imaging showed that the tumor background ratio remained at 12.5 for 20-28 hours after injection, the images could clearly distinguished the tumor margin, and HE staining demonstrated that even macroscopically invisible ultra-small tumors ≤ 1 mm could be detected and completely resected.

Paclitaxel combined with platinum-based chemotherapy is the first-line regimen for ovarian cancer. However, the low selectivity and short retention period of current chemotherapeutic agents limit their exposure time at the lesion site (47). Reasonable targeting can significantly improve the treatment effect of ovarian cancer, reduce adverse reactions and drug resistance, and enhance the bioavailability of chemotherapy drugs (48). Anticancer drugs are attached to peptide carriers via biodegradable linkers, and peptide are internalized by binding to specific receptors, which in turn deliver the drug to the target site for action ( Figure 1 ). In specific tumor-targeted therapies, the use of polypeptides as targeted ligands exhibits a variety of advantages, including low toxicity, low or no immunogenicity, and high specificity (49). Peptides are also highly permeability because they are tiny and generally hydrophilic molecules, allowing for simple and quick access into tumor locations. Polypeptides coupled to the surface of nanoparticles can mediate active targeting, as a complementary strategy to enhance permeability and retention (EPR) effects, and to facilitate targeted cell uptake of drugs (50). A variety of peptide ligands including LHRH peptides, FSHR peptides, RGD peptides, OA02 peptides have been designed and explored for OC targeted therapy ( Table 2 ), and drug delivery is achieved through various carrier systems such as liposomes, polymers, micellars, and nanoparticles.

Inhibition of tumor growth and metastasis is a strategy for ovarian cancer treatment, which is crucial for the prognosis of patients. Compared with peptides used in drug delivery systems, anticancer peptides are a rising focus in anti-tumor research (89). Anticancer peptides (ACPs) act by inducing apoptosis and disrupting cell membrane structure. Large quantities of evidence signify that cancer cells have higher mobility and more abundant microvilli compared to healthy cells. When ACPs interact with cancer cells, they cause membrane instability, cytotoxicity, and cell lysis (90). In addition, ACPs can also inhibit the proliferation of cancer cells by changing the pH around or inside the cell (91). Small peptides with anticancer effects can be discovered by phage display or chemical synthesis of peptide libraries. We summarize the small peptides that inhibit the growth and metastasis of ovarian cancer cells ( Table 3 ), some of which can directly act on ovarian cancer cells to exert anticancer effects. Zhou’s team (92) used a phage display peptide library to isolate the small peptide “SWQIGGN”, which can combine and internalize ovarian cancer cells. It not only inhibited the viability, migration, invasion and adhesion of ovarian cancer cells in vitro but also reduced the volume of ascites and controlled tumor growth and metastasis in vivo. Another research found the homologous peptide “WSGPGVWGASVK”, identified based on ovarian cancer cell-specific ligand (pc3-1), inhibits adhesion between SKOV-3 cells and HUVECs cells (93) and inhibits cancer cell invasion by down-regulating the active expression of MMP-2 and MMP-9 (94). Furthermore, several short inhibitory peptides, like as “OB3 peptide,” “Smac N7 peptide,” and “A6 peptide,” have been demonstrated to have anticancer effects by acting on ovarian cancer signaling pathways or bioprocess-related targets (95–98).

Unfortunately, due to tumor heterogeneity, drug-resistant phenotypes may emerge late in treatment, including resistance to pro-apoptotic stimuli and/or cytotoxic resistance to anticancer compounds (47). According to certain study findings, bioactive peptides may also have a function in increasing cancer cell susceptibility to chemotherapeutic medicines and reversing cancer cell resistance. Guo’s group (99)found that hexapeptide (PGPIPN), derived from bovine β-casein, significantly increased the sensitivity of DDP to human ovarian cancer cells inhibition of survival and induction of apoptosis in vitro. In an in vivo model of ovarian cancer, Alagkiozidis’s team (100)discovered that the anticancer peptide PNC-27 could target paclitaxel-resistant cells, and that the addition of PNC-27 to weekly paclitaxel injection greatly decreased tumor development. Similarly, some peptides can be designed to act on anticancer drug targets, and thus provide better inhibitory effect on drug-resistant phenotypes (107). For example, LR peptide (LSCQLYQR) is an anticancer peptide that acts on human thymidylate synthase (hTS), and active site inhibitors of hTS are commonly employed in chemotherapy (101). When LR was given to ovarian cancer cells, it could inhibit intracellular hTS expression. Also, inhibited the growth of approximately 50% of cisplatin-sensitive and resistant ovarian cancer cell lines at low micromolar concentrations (510 μM), more effectively than the same concentration of 5-FU (102).

It is worth noting that although some peptides have inhibitory effects on the growth, invasion and metastasis of ovarian cancer cells. However, due to its weak penetration capacity, it needs to be transported into cells in combination with cell penetrating peptides (CCP) or protein-specific systems to improve the bioavailability of anticancer drugs, and these delivery systems usually do not affect cell proliferation or cell cycle progression (108, 109). D3S2, an autophagy-inhibiting peptide based on the switch II region of DIRAS3, inhibition of autophagy can induce cancer cell death. D3S2 was linked to TAT (a cell-penetrating peptide) to act on OC cells to enhance penetration, and Tat-D3S2 treatment reduced the induction of autophagy in OC cell lines, as evidenced by a decrease in LC3I/LC3II conversion rate (103).EP-100 is composed of a natural ligand for GnRH linked to cleavage peptide (CLIP-71), which delivers the cleaved peptide to GnRH receptor-positive cancer cells, thereby exerting selective toxic effects (104). Kim and colleagues (105) found that EP-100 rapidly destroyed the cell membrane and increased PD-L1 levels after specific binding to ovarian cancer cells, and observed that EP-100 increased anti-tumor effector T cells and IL33 expression and induced anticancer activity in vivo when EP-100 and anti-PD-L1 antibodies were combined.

Previous reports have indicated that peptides are generally not toxic in normal host cells and thus may represent a powerful therapeutic tool. Moreover, the combination of anticancer peptides and more aggressive chemotherapeutic agents can improve efficacy, counteract chemotherapy-induced resistance, and reduce overall drug combination toxicity (110). For instance, sv6D acts as a multivalent anticancer peptide that specifically targets the C-type lectin A family 10 member (CLEC10A). In a mouse ovarian cancer model, the peptide sv6D significantly inhibited the formation of ascites when combined with the chemotherapeutic agent paclitaxel or an immunotherapeutic antibody against the receptor PD-1 (106).Combining EP-100 with PARP inhibitor (olaparib) significantly increased the number of nuclear foci of phosphorylated histone H2AX, leading to increased DNA damage in ovarian cancer cells (111). A phase II trial of paclitaxel in combination with EP-100 showed that a subset of patients with target liver lesions showed a greater overall response rate to the combination (69%) compared to paclitaxel alone (16%) (112).

Epithelial ovarian cancer is considered to be an immunogenic tumor. The presence of tumor infiltrating lymphocytes (TIL) in the tumor microenvironment has been shown to increase the 5-year survival rate and improve clinical outcomes for patients (113). As a potential immunotherapeutic route, peptide vaccines are captured by antigen-presenting cells upon entry into the body and delivered to T cells, which then recognize and initiate an immune response that promotes the production of cytotoxic T cells and enhances the body’s ability to fight tumors (114) ( Figure 2 ). Peptide vaccines generally consist of an immune adjuvant and a targeted peptide derived from a tumor-associated antigen (TAA) epitope. A retrospective analysis of vaccine treatments over 20 years in ovarian cancer found that peptide vaccines had the highest coverage rate (34.3%), which can be attributed to the fact that peptide vaccines are well tolerated and have fewer side effects (115). A number of peptide vaccines against ovarian cancer are currently in clinical trials ( Table 4 ), such as NY-ESO-1 peptide vaccine (116), folic acid receptor α peptide vaccine (120), HER-2/neu peptide vaccine (117), P53 synthetic long peptide vaccine (p53-SLP) (118), and WT1 peptide vaccine (119). Among them, the study of folate receptor alpha peptide vaccines in gynecological tumors has attracted much interest to enhance immunity to folate receptors by producing T cells. E39(EIWTHSYKV) is an HLA-A2 restricted FR peptide that induced a significant, dose-dependent in vivo immune response and decreased the risk of recurrence in a clinical phase I/IIa research (124). A recent study identified the optimal vaccination dose of E39 peptide, and the 1000 μg dose significantly improved the 2-year disease-free survival of patients (77.9% at 1000μg vs 31.2% at < 1000μg) (120). However, some researchers believe that overstimulation of E39 may lead to activation induced cell death (AICD). Berry’s group (125) developed the attenuated peptide E39’ (EIWTFSTKV) by replacing two amino acids of E39 peptide and found to elicit an effective CTL response and possibly even avoid AICD caused by repeated stimulation with E39 peptide. The safety of E39’ was subsequently demonstrated in a phase Ib trial (121). This trial also showed that sequential inoculation of 3 times E39 and 3 times E39’ resulted in greater local response and DTH response.

Most peptide vaccines are designed based on long-standing tumor-associated antigens (TAA) and have poor immunogenicity. However, peptide vaccines can generate strong immunogenic responses by increasing antigenic epitopes. Multi-epitope vaccines can carry multiple epitopes at the same time and have the ability to be recognized and bound by MHC molecules of multiple genetic backgrounds, resulting in efficient presentation (126). In a phase I clinical trial study, Kalli and colleagues (122) used five putative HLA-A2 class binding peptides (FR30, FR56, FR76, FR113 and FR238) mixed with GM-CSF to form a multi-epitope FR peptide. This experiment showed that more than 90% of ovarian cancer patients produced T-cell immune enhancement and exhibited a stable high immune memory during the observation period. It has been reported that vaccination alone in the early stage of the tumor or under minimal change disease prevents relapse. In contrast, in patients with advanced disease, combination therapy may be more effective in preventing immune escape and gaining clinical benefit (127). Combining peptide-based cancer vaccines with checkpoint medications is a prominent example. Immune checkpoint blockade can restore the body’s immune response by preventing T cell depletion and promoting activation. Zamarin and colleagues (123) evaluated the efficacy of a multi-epitope FR vaccine (TPIV200) in combination with a PD-L1 inhibitor (durvalumab) in patients with advanced platinum-resistant OC. The trial was conducted in 27 patients in 28-day cycles, receiving Durvalumab intravenously on days 1 and 15 of cycles 1-12 and three TPIV200 intradermal injections on day 1 of cycles 1-6. All patients produced a robust FRα-specific T-cell response after treatment. Despite a low overall remission rate, the median overall survival was 21 months, suggesting that combination therapy could help prolong disease control and alter patient outcomes. Likewise, other potential combination therapies such as peptide vaccines and poly ADP ribose polymerase (PARP) inhibitors, anti-angiogenic drugs should be considered.