Open Access

Neuropilin‑1 expression in primary and metastatic prostate cancer: Expression patterns and clinicopathological correlations

  • Authors:
    • Kamil Kowalczyk
    • Maciej Kaczorowski
    • Aleksandra Piotrowska
    • Paweł Kiełb
    • Krzysztof Dudek
    • Adam Gurwin
    • Jakub Karwacki
    • Dariusz Kowalczyk
    • Wojciech Krajewski
    • Tomasz Szydełko
    • Agnieszka Hałoń
    • Piotr Dzięgiel
    • Bartosz Małkiewicz
  • View Affiliations

  • Published online on: August 8, 2025     https://doi.org/10.3892/mco.2025.2887
  • Article Number: 92
  • Copyright: © Kowalczyk et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Despite significant advancements in prostate cancer (PCa) diagnostics, it remains a challenge for accurate diagnosis and effective treatment. The aging global population and the established correlation between PCa incidence and advancing age suggest an anticipated rise in cases. Traditional clinicopathological parameters, such as prostate‑specific antigen (PSA) levels, Gleason Grade Group, and pT stage, highlight the need for novel biomarkers to improve prognostic accuracy and risk assessment. The present study investigated the role of neuropilin‑1 (NRP‑1) in PCa progression, with a focus on lymph node metastases. Findings reveal that higher NRP‑1 expression is associated with a lower percentage of metastatic lymph nodes (9.5 vs. 15.0%; P=0.027) and significantly lower postoperative PSA levels (0.02 vs. 0.21 ng/ml, P=0.039), both considered favorable prognostic factors. These observations align with prior hypotheses suggesting that NRP‑1's function may depend on its ligand, semaphoring 3A (SEMA3A) or vascular endothelial growth factor (VEGF), with SEMA3A exhibiting anti‑tumoral properties in hormone‑sensitive PCa. However, NRP‑1 expression showed no correlation with key clinicopathological parameters, such as pT stage or Gleason score, nor did it influence 5‑year survival rates. These results suggest that while NRP‑1 has potential as a biomarker, its prognostic utility as a standalone factor remains limited. Further studies are warranted to validate these findings in larger cohorts and to explore the molecular mechanisms underlying NRP‑1's role in PCa.

Introduction

Prostate cancer (PCa) is the second most common malignant tumor in men worldwide, with 1,466,680 new cases diagnosed in 2022, accounting for ~397,000 fatalities (1). Although its mortality rate is comparatively low, it continues to be the primary cause of cancer-related death in numerous nations across the Caribbean, sub-Saharan Africa, Central and South America, as well as Sweden in Europe. Increasing incidence of PCa was noted in China, Baltic countries and Eastern Europe, which might be a result of widely adopted prostate-specific antigen (PSA) testing and more frequent transurethral resections (2-4).

Given the global trend of increasing life expectancy and the established correlation between PCa incidence and age, the number of men newly diagnosed with this type of cancer will likely rise in the foreseeable future (5). Prediction of patient prognosis following prostatectomy is currently based on classic clinicopathological parameters, however incorporating data related to specific tissue biomarkers might be a valuable addition in this context, especially for patients with aggressive disease prone to early recurrence.

Neuropilin-1 (NRP-1), a member of neuropilin family of receptor proteins, was initially identified as modulator of nervous system development, functioning as semaphorin coreceptor alongside plexin. Later, it was found to be receptor of vascular endothelial growth factor (VEGF) family (6). NRP-1, together with NRP-2, transfers signals for angiogenesis, cellular communication and migration (7). NRP-1has emerged as a focal point in tumor biology research due to its participation in vascular and lymphatic development, as well as its role in tumor cells (6,8,9).

Elevated expression of NRP-1 was demonstrated not only in PCa, but also in breast, colon, lung and bladder cancers as well as in neuroblastoma (10-14). NRP-1 is involved in tumor cell proliferation, migration, survival and therapy resistance (6,14,15).

As a transmembrane co-receptor NRP-1, alongside with VEGFR2 (VEGF tyrosine kinase receptor), activates VEGF-A-mediated vascular development (16,17). Targeted disruption of a single allele of VEGF gene in murine models is sufficient to induce vascular abnormalities and results in embryonic lethality, highlighting that the in vivo level of VEGF is crucial for proper vascular development (18,19). Furthermore, by interacting with VEGF165 NRP-1 activates c-MET receptor tyrosine kinase pathway, which leads to the subsequent activation of downstream effectors such as Src kinase and signal transducer and activator of transcription 3 (STAT3), culminating in the upregulation of anti-apoptotic proteins like myeloid cell leukemia-1 (Mcl-1). Mcl-1 is an established survival factor in PCa cells, which is upregulated in patients with PCa with bone metastasis (20). b1/b2 binding site of NRP-1 also interacts with extracellular domain of epidermal growth factor receptor (EGFR) leading to its phosphorylation and subsequent activation of the downstream PI3K/AKT/mTOR signaling pathway. This activation promotes proliferation and migration of PCa cells. Additionally, hypoxic conditions within the tumor microenvironment (TME) induce the accumulation of hypoxia-inducible factor 1-alpha (HIF1-α), which transcriptionally upregulates NRP-1 expression, further amplifying these oncogenic signaling cascades (21,22).

Proangiogenic function of NRP-1 is dependent on its b1/b2 binding site, whereas a1/a2 binding sites respond for semaphorin3A (SEMA3A) recognition (23). In addition to binding VEGF, NRP-1 is a co-receptor for SEMA3A, a protein involved in the regulation of sympathetic ganglion development and axon pathfinding (24). SEMA3A also acts as a tumor suppressor by promoting apoptosis, suppressing cell migration and angiogenesis, at least partly in NRP-1-dependent manner. Binding to NRP-1 initiates signaling cascades that inhibit endothelial cell lamellipodia formation, adhesion, migration and survival, thereby effectively suppressing angiogenesis. VEGF165 competes with SEMA3A for NRP-1 binding on endothelial cells and promotes the internalization of cell-surface NRP-1. Interestingly, SEMA3A requires a lower concentration to achieve similar effects, suggesting a higher affinity for NRP-1 (25,26). Notably, both VEGF165 and SEMA3A induce vascular permeability through interaction with their shared receptor (27). Hence, different NRP-1 functions in PCa may result from different downstream pathways activated by individual ligands (24,28-32).

Additionally, recent research indicates that NRP-1 expression is dynamically regulated during the development of castration-resistant PCa (CRPC), particularly in response to androgen-targeted therapies (ATTs) and suggests its upregulation in metastatic compared with localized PCa. PCa cells with NRP-1 knockdown exhibited a less invasive morphology, characterized by reduced vimentin and enhanced E-cadherin expression, which was associated with decreased metastatic dissemination in a zebrafish xenograft model. Yet, the exact signaling mechanism between the androgen receptor (AR) and NRP-1 remains unclear (33).

Previous studies on potential prognostic and predictive utility of NRP-1 in PCa are encouraging, however, immunohistochemistry (IHC)-based data are limited and inconclusive (20,21,32-34). Moreover, the significance of NRP-1 levels in lymph node metastases of PCa has not been investigated. The present study systematically analyzed clinicopathological correlations of NRP-1 immunoreactivity in primary PCa and matched lymph node metastases.

Materials and methods

Patients and clinicopathological data

Patients included in the present study were those with histologically confirmed prostate adenocarcinoma (PRAD) who underwent radical prostatectomy with extended pelvic lymph node dissection between 2012 and 2018 at the University Urology Centre in Wrocław, Poland. Additional inclusion criteria were the availability of adequate formalin-fixed, paraffin-embedded (FFPE) tissue samples from both primary tumors and metastatic lymph nodes, as well as complete clinical documentation. Patients who had received any form of antitumor therapy prior to surgery, including androgen deprivation therapy, chemotherapy, or radiotherapy, were excluded from the analysis. Further exclusion criteria included incomplete clinical data, insufficient quality or quantity of tissue material for immunohistochemical evaluation, or a history of another malignancy. Out of an initial group of 95 patients, 73 men aged 42 to 79 years with PCa and lymph node metastases were included in the final analysis. A total of 22 patients were excluded due to incomplete clinical data and/or technical issues encountered during the construction and IHC staining of tissue microarrays (TMAs).

The tissue samples used in the present study were obtained from the archives of the Department of Clinical and Experimental Pathology (Wroclaw Medical University). For the purposes of the present study, archived FFPE tissue blocks were retrieved and accessed between January 2022 and March 2023, during the process of constructing the TMAs and performing immunohistochemical analyses.

Tumor stage and grade were compliant with the 8th edition of American Joint Committee on Cancer staging and 2019 International Society of Urological Pathology prostatic carcinoma grading consensus, respectively (35,36). The study protocol was approved (approval no. KB-545/2020) by the Ethics Committee of Wrocław Medical University (Wrocław, Poland). All procedures were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from each recruited patient prior to sample collection.

Histopathological assessment was independently conducted by two experienced uropathologists. In cases of discrepancy, the final diagnosis was established by consensus during a joint review. Risk groups for biochemical recurrence of localized and locally advanced PCa were stratified according to European Association of Urology (EAU) (37). The radicality of prostatectomy was assessed by histopathological evaluation of surgical margins and by clinical measurements of PSA levels, with a threshold of <0.2 ng/ml at the first post-surgical measurement, typically done 6 weeks after the procedure.

Under hematoxylin and eosin (H&E) staining, normal prostate tissue is composed of well-formed glandular units lined by a dual layer: a basal cell layer and an inner luminal epithelial layer. The glands are typically round to oval, well-spaced, and separated by fibromuscular stroma. By contrast, PRAD, particularly acinar adenocarcinoma (the most common type), is characterized by infiltrative, irregularly shaped small glands that lack basal cells. These glands are often crowded, angulated, and may display cribriform, fused, or solid growth patterns in higher Gleason grades. Nuclei are typically enlarged with prominent nucleoli. These features are especially evident at the tumor invasive front, which defines the boundary between neoplastic and benign tissue. Surgical margin status was determined during standard histopathological evaluation of radical prostatectomy specimens. Margins were considered positive if cancer cells were present at the inked resection surfaces (capsular, apical, or bladder neck), and negative if no tumor cells were observed at these margins. The appearance of tumor glands at the margin is histologically similar to the rest of the carcinoma, but their location at or near the inked edge determines their clinical significance. Importantly, no unique architectural or cytological feature distinguishes tumor cells at the margin from those elsewhere in the tumor; the defining factor is their proximity to the resection surface. Overall survival (OS) was assessed from the date of radical prostatectomy, irrespective of the cause of death. The follow-up period ranged from 0 to 96 months. Background clinical and histopathological data were retrieved from archival hospital records.

IHC

Archival FFPE tissue samples were used for NRP-1 IHC. Matched samples from the primary tumor and lymph node metastasis were analyzed for each patient using bright-field light microscope. TMAs with the tumor tissues were assembled as previously described (38). IHC reactions were performed on 4-µm TMA paraffin sections using Autostainer Link48 (Agilent Technologies, Inc.). Slides were deparaffined and epitope retrieval was carried out by treating the slides with EnVision FLEX Target Retrieval Solution (Agilent Technologies, Inc.) (97˚C, 20 min; pH 9) using PTLink (Agilent Technologies, Inc.). In order to block endogenous peroxidase activity slides were incubated for 5 min at room temperature with EnVision FLEX Peroxidase-Blocking Reagent (Agilent Technologies, Inc.). Afterwards, rabbit monoclonal primary antibody against NRP-1 (1:300; cat. no. ab81321; Abcam) was applied for 20 min at room temperature. Then slides were incubated with secondary antibodies conjugated with horseradish peroxidase-EnVision FLEX/HRP (20 min at room temperature). The reactions were visualized using substrate for horseradish peroxidase (3,3'-diaminobenzidine) with incubation for 10 min. Additionally, all sections were counterstained 5 min with EnVision FLEX Hematoxylin (Agilent Technologies, Inc.). Finally, slides were dehydrated in ethanol (70, 96%, absolute) and xylene. All slides were closed with coverslips in Dako Mounting Medium (Agilent Technologies, Inc.). The primary antibody was diluted in EnVision FLEX Antibody Diluent (Agilent Technologies, Inc.).

For IHC scoring, all slides were digitized using Pannoramic MIDI scanner (3DHISTECH, Ltd.). To assess NRP-1 expression, six TMA cores were evaluated for each patient (three from the primary prostate tumor and three from the metastatic lymph node). H-score was used for semi-quantitative analysis of IHC reactions. In brief, points for staining intensity (0: negative, 1: weak, 2: moderate, 3: strong) were multiplied by the percentage of positive cancer cells. Sum of products for each intensity gave the final H-score ranging from 0 to 300. Tumors were categorized into low (H-score ≤100), moderate (H-score 101-200) and high (H-score >200) expression groups.

The immunohistochemical evaluation of NRP-1 expression was performed independently by two experienced genitourinary pathologists. In cases of scoring discrepancies, the final H-score was determined by consensus.

Statistical analysis

The mean, standard deviation, minimum, maximum, median (Me), lower quartile (Q1), and upper quartile (Q3) for quantitative variables were calculated. The empirical distribution of quantitative variables was examined to fit a normal distribution using the Kolmogorov-Smirnov and Shapiro-Wilk tests. Spearman's rank correlation coefficient was calculated to assess the association between monotonic relationships between variables. Differences in NRP-1 expression levels between sample types were assessed using Student's t-test (for normally distributed data) or the Mann-Whitney U test (for non-normally distributed data). For subgroup comparisons, expression levels were categorized as low or moderate (≤200), or high (>200) based on predefined H-score thresholds. The Mann-Whitney U test was used to assess differences in quantitative parameters between two independent groups with varying NRP-1 marker expression levels. For qualitative factors, Pearson's Chi-square test or Fisher's exact test was employed to evaluate independence. Kaplan-Meier survival analysis was performed to assess the impact of NRP-1 expression on 5-year OS, with comparisons between groups conducted using the log-rank test. A significance level of P<0.05 was applied for all statistical tests. All statistical analyses were performed using Statistica v.13.3 (TIBCO Software Inc.).

Results

Positive NRP-1 staining was detected in all specimens-primary and metastatic tumors. NRP-1 expression was significantly higher in lymph node metastases (NRP1-N) compared with primary prostate tumors (NRP1-P), with Me H-scores of 215 vs. 170, respectively (P<0.001). High NRP-1 expression (>200 H-score) was more frequent in lymph node metastases than in primary prostate tumors (57.5% vs. 27.4%; P<0.001), whereas moderate expression was predominant in primary tumors (65.8%). The proportion of low expression was small in both groups as seen in Table I. The general characteristics of the patients are presented in Table II.

Table I

NRP1 expression in primary prostate cancers and lymph node metastases from 73 patients after radical prostatectomy.

Table I

NRP1 expression in primary prostate cancers and lymph node metastases from 73 patients after radical prostatectomy.

 Prostate NRP1-PLymph nodes NRP1-NTest result
Marker expression  Z=3.527
     Mean (SD)173(59)207(62)P<0.001
     Median [Q1; Q3]170 [130; 215]215 [160; 250] 
     Minimum-Maximum30-28080-300 
Level of expression, n (%)   χ2=13.8
     Low (≤100 H-score)5 (6.8%)2 (2.8%)df=2
     Moderate (100-200 H-score)48 (65.8%)29 (39.7%)P=0.001
     High (>200 H-score)20 (27.4%)42 (57.5%) 

[i] NRP1-P, neuropilin-1 expression in prostate cancer; NRP1-N, neuropilin-1 expression in lymph node metastases; SD, standard deviation; Q1, lower quartile; Q3, upper quartile.

Table II

Clinicopathological characteristics stratified according to NRP1-P expression.

Table II

Clinicopathological characteristics stratified according to NRP1-P expression.

 NRP-1 expression 
VariableHigh >200 H-score N=20Low or moderate ≤200 H-score N=53P-value
Body weight (kg), Me [Q1; Q3]80 [74;95]85 [80;94]0.390a
Height (cm), Me [Q1; Q3]175 [170;180]176 [170;180]0.508a
Body mass index (kg/m2), mean (SD)27.6 (4.4)28.3 (3.5)0.516b
Anesthesiologists Classification, n (%)  0.658c
     ASA I5 (19.2)8 (12.3) 
     ASA II19 (73.1)47 (72.3) 
     ASA III2 (7.7)9 (13.9) 
     ASA IV0 (0.0)1 (1.5) 
Karnofsky (score), Me [Q1; Q3]100 [100; 100]100 [90; 100]0.268a
ECOG, n (%)  0.344c
     019 (73.1)38 (58.4) 
     17 (26.9)25 (38.5) 
     20 (0.0)2 (3.1) 
Preoperative PSA (ng/ml), Me [Q1; Q3]14.2 [8.4; 26.7]21.4 [14.0; 36.1]0.104a
EAU risk group, n (%):  0.835c
     10 (0.0)1 (1.5) 
     23 (11.5)5 (7.7) 
     313 (50.0)36 (55.4) 
     410 (38.5)23 (35.4) 
Age (years), mean (SD)64.2 (4.7)65.3 (5.9)0.456b
Hospitalization (days), Me [Q1; Q3]7 [6; 10]7 [6; 8]0.354a
pT, n (%)  0.313c
     pT2a0 (0.0)1 (1.5) 
     pT2b0 (0.0)0 (0.0) 
     pT2c2 (7.7)8 (12.3) 
     pT3a8 (30.8)8 (12.3) 
     pT3b16 (61.5)48 (73.9) 
Gleason postoperative, n (%):  0.569c
     3+31 (3.8)0 (0.0) 
     3+44 (15.4)9 (13.8) 
     4+39 (34.6)13 (20.0) 
     3+51 (3.8)3 (4.6) 
     4+41 (3.8)2 (3.1) 
     5+30 (0.0)2 (3.1) 
     4+57 (26.9)28 (43.1) 
     5+43 (11.5)7 (10.8) 
     5+50 (0.0)1 (1.5) 
Postoperative ISUP GGG, n (%):  0.202c
     11 (5.0)0 (0.0) 
     24 (20.0)5 (9.4) 
     36 (30.0)11 (20.8) 
     42 (10.0)7 (13.2) 
     57 (35.0)30 (56.6) 
Comorbidities, n (%)   
     Hypertension12 (60.0)26 (49.1)0.442d
     Type 2 diabetes mellitus2 (10.0)7 (13.2)1.000d
     Ischemic heart disease2 (10.0)4 (7.6)0.663d
     Hypercholesterolemia1 (5.0)4 (7.6)1.000d
     COPD2 (10.0)1 (1.9)0.180d
     Varicose veins of the lower limbs1 (5.0)2 (3.8)1.000d
ECE, n (%)18 (90.0)46 (86.8)1.000d
Surgical margin, n (%)15 (75.0)36 (67.9)0.776d
Involved lymph nodes (%)9.5 [5.1; 21.0]15.0 [9.1; 30.0]0.027d
Extranodal extension, n (%)4 (20.0)15 (28.3)0.561d
NRP-1-N (H-score), Me [Q1; Q3]240 [198; 285]210 [130; 240]0.010a

[i] aMann-Whitney U test;

[ii] bt-test for independent sample;

[iii] cchi-square test;

[iv] dFisher's exact test. NRP1-P, neuropilin-1 expression in prostate cancer; Me, median; Q1, lower quartile; Q3, upper quartile; ECOG, Eastern Cooperative Oncology Group; PSA, prostate-specific antigen; EAU, European Association of Urology; ISUP GGG, International Society of Urological Pathology Gleason Grade Group; COPD, chronic obstructive pulmonary disease; ECE, extracapsular extension; NRP1-N, neuropilin-1 expression in lymph node metastases.

The expression of NRP-1 in lymph node metastases was significantly higher than in primary tumors (median H-score: 215 vs. 170; P<0.001, Fig. 1). High expression (>200 H-score) was more frequently observed in metastatic than primary tumors (57.5% vs. 27.4%; P<0.001). A total of 14 patients (19.2%) had high NRP-1 expression both primary and metastatic tumors.

Patients with high NRP-1 expression in primary tumors presented significantly lower percentage of affected lymph nodes (9.5% vs. 15.0%, P=0.027, Fig. 2) and significantly lower postoperative PSA level (0.02 vs. 0.21 ng/ml, P=0.039, Fig. 3). A comparison of NRP-1 expression levels in the PCa and metastatic lymph nodes is demonstrated in Fig. 4. However, no association was observed between NRP-1 expression and any of the analyzed parameters-postoperative International Society of Urological Pathology Gleason Grade Group (ISUP GGG), pT stage, extranodal extension, surgical margin, neurovascular invasion, lymph-vascular invasion, EAU risk group, Body mass intex, patient's age. Also, 5-year survival was not related to NRP-1 expression (Fig. 5).

Discussion

Despite significant advancements in PCa diagnostics in recent years, it continues to pose a considerable challenge for urologists and uropathologists with regard to accurate diagnosis and effective treatment strategies. Considering the global increase in life expectancy and the well-documented link between PCa incidence and aging, it is projected that the incidence of PCa will increase in the near future (5). The current reliance on traditional clinicopathological parameters, such as PSA levels, GGG, and pT stage, underscores the need to identify novel, specific biomarkers that could improve the accuracy of survival prognosis and the assessment of cancer dissemination risk in patients following prostatectomy and to incorporate them into current urological guidelines for PCa management.

The present study provided new insights into the role of NRP-1in PCa progression, particularly in the context of lymph node metastases. None of previous studies on NRP-1 in PCa investigated its expression in lymph node metastases, highlighting the novelty of the present study (20,21,33,39-42).

Although the literature on the clinical implications of NRP-1 expression in PCa is limited, several studies reported a positive association between NRP-1 levels in PCa tissue and poor prognosis. In a study by Hu et al (42) NRP-1 expression correlated with OS; however, only in Caucasian-American patients and not in Chinese and African American men. In another study, IHC analysis of TMAs demonstrated a positive correlation between elevated NRP-1 expression and higher Gleason grade, pathological T stage, lymph node involvement and primary treatment failure. Moreover, multivariate analyses identified NRP-1 expression at the time of radical prostatectomy as an independent prognostic factor for biochemical recurrence following radiotherapy, as well as for metastatic progression and cancer-specific mortality. Interestingly, NRP-1 was identified as a gene repressed by androgen signaling, with its expression upregulated during the adaptive response to ATTs, ultimately contributing to the progression toward metastatic CRPC (33).

Notably, roles of NRP-1 observed in animal and in vitro models of PCa, may not reflect clinical conditions (20,21,32,34). Moreover, other studies reporting high biomarker expression in PCa also identified certain methodological inconsistencies related to selection bias and result assessment methods (20,21).

In one study, NRP-1 overexpression was positively associated with PCa progression and bone metastasis. However, a bias in patient selection was noted, as only two patients with a Gleason score of 6 were included in the analysis, compared with 43 cases with Gleason scores of 8-10(20).

Another study reported significantly higher NRP-1 levels in PCa specimens from patients with poor prognosis (higher Gleason score, high PSA, advanced T stage lymph node metastasis). Further, NRP-1 expression was associated with worse disease-free survival, shorter OS and cancer-specific survival (21). Nonetheless, IHC staining in that study was evaluated using the immunoreactivity score, which has certain limitations and lacks the broader a dynamic range provided by the H-score used in the analysis of the present study. Although the H-score is more time-consuming and requires a higher level of expertise from the pathologist, it offers a more detailed assessment of the material, accounting for intra-tumoral heterogeneity in antigen expression and potentially improving accuracy (43,44).

In addition, some studies failed to corroborate earlier findings or yielded unclear results (39-41). Talagas et al (39) did not find any association between NRP-1 and unfavorable clinicopathological parameters in patients with clinically localized PCa after radical prostatectomy. In turn, elevated endothelial expression of NRP-1 was an independent predictor for the absence of distant relapse in PCa.

Another study reported a 10-fold increase in NRP-1 mRNA levels in tumor specimens compared with benign tissue samples. Interestingly, a comparison between benign prostate hyperplasia (BPH) and malignant tissues revealed that NRP-1 IHC staining was elevated in PCa specimens; however, it was confined to the stroma and absent in malignant epithelial cells, which contradicts previous studies. In addition, the stroma exhibited positive staining in all samples, with no significant variation in intensity across different tumor specimens. Consequently, staining intensity could not be associated with Gleason score or recurrence risk (40).

Finally, Yacoub et al (41) hypothesized in their study, that the function of NRP-1 in PCa varies depending on the specific ligand to which it is attached (SEMA3A/VEGF). In contrast to VEGF, SEMA3A presented lower protein expression in hormone-refractory PCa (HRPC) compared with localized cancer. In localized cancer, SEMA3A was linked to lower pT stage and showed a close correlation with NRP-1 expression.

No significant relationship was observed between NRP-1 and VEGF in localized disease, and no significant correlation was found between NRP-1 and SEMA3A in HRPC. The expression of both SEMA3A and NRP-1 was linked to predictors of favorable prognosis, such as lower preoperative PSA levels, a low Gleason score for NRP-1, and a lower pT stage for SEMA3A, while VEGF expression correlated with elevated PSA levels.

Consequently, the function of NRP-1 in PCa may vary depending on VEGF/SEMA3A ratio. When SEMA3A is the primary ligand, as observed in clinically localized PCa, NRP-1 expression could be indicative of a favorable prognosis due to its anti-tumoral effects. Conversely, in cases of metastatic HRPC where VEGF is the predominant ligand, NRP-1 expression might be associated with a more aggressive disease profile due to its pro-tumoral nature (16,17).

The analysis of the present study revealed that patients with high NRP-1 expression in PCa samples exhibited a significantly lower percentage of affected lymph nodes compared with those with lower expression (9.5% vs. 15.0%, P=0.027). The percentage of metastatic lymph nodes, commonly referred to as lymph node density, is widely recognized as a reliable predictor of clinical recurrence. Its prognostic value has been demonstrated in multiple studies, emphasizing its importance in guiding postoperative management and risk stratification in patients with PCa (45,46).

Additionally, the group with higher NRP-1 staining exhibited a significantly lower postoperative PSA level (0.02 vs. 0.21 ng/ml, P=0.039), which is also considered a positive prognostic factor. Postoperative PSA levels are strongly correlated with an increased risk of biochemical recurrence and overall mortality in patients following prostatectomy (47-50). These observations could be explained by the hypothesis proposed by Yacoub et al (41), suggesting that the function of NRP-1may depend on the ligand to which it is bound (SEMA3A or VEGF). SEMA3A may mediate anti-angiogenic and pro-apoptotic effects through NRP-1, potentially counteracting the pro-tumoral influence of VEGF, as documented in several studies (26,30,31).

Notably, all patients in the present cohort study had lymph node involvement; however, their disease was clinically localized and had been treated with curative intent. Consequently, these cases represent hormone-sensitive PCa. These findings highlight the importance of further exploring NRP-1expression as a prognostic biomarker in this specific patient subgroup.

Despite a thorough outcome analysis, no statistically significant associations were identified between high NRP-1 expression and key clinicopathological parameters, including pT stage, Gleason score, surgical margin status, or extracapsular extension. Moreover, NRP-1 expression was not found to influence 5-year survival rates, a result consistent with earlier studies (39,41). These findings suggest that, while NRP-1 may play a role in specific TME interactions, its utility as a standalone prognostic marker for long-term outcomes in PCa remains limited.

While the present study highlighted the potential of NRP-1 as a biomarker, several important limitations must be acknowledged. Most notably, the retrospective design and single-center setting represent significant constraints that may limit the generalizability and external validity of the present study's findings. Although these factors are common in exploratory studies, the results should be interpreted with caution and the findings underscore the need for prospective validation, preferably focusing on hormone-sensitive patients with PCa and lymph node involvement. Secondly, the lack of data on specific VEGF/SEMA3A ratios in patient samples is another limitation-studies incorporating quantitative analysis of VEGF and SEMA3A expression will help clarify the ligand-dependent role of NRP-1 in tumor biology. The patient groups in the present study were relatively small-future research should focus on validating these findings in larger, multicenter cohorts and exploring the molecular mechanisms underlying NRP-1's role in PCa. Additionally, investigating its interaction with other pathways critical to PCa progression, such as AR signaling, could also yield valuable information. TMAs were selected for the present study to enable efficient, standardized immunohistochemical analysis across a large number of patient samples. TMAs allow simultaneous staining and evaluation of multiple cases under uniform conditions, minimizing inter-assay variability and conserving valuable tissue. This approach is particularly advantageous when assessing biomarker expression semi-quantitatively across matched tumor regions. However, it is important to note that TMAs represent only selected areas of the tumor and do not include its full architecture, such as surgical margins, which limits their use for margin assessment. Finally, a comparative analysis of NRP-1 staining between cancerous and non-cancerous tissues (such as BPH tissue) could further enhance the study's robustness. In conclusion the present study evaluated the role of NRP-1in PCa, particularly in lymph node metastases-a novel area of focus. Positive NRP-1 immunohistochemical staining was detected in both PCa and metastatic lymph nodes. Patients with high NRP-1 expression demonstrated significantly lower percentage of affected lymph nodes and lower postoperative PSA level. NRP-1 expression was not associated with other clinicopathological parameters or survival rates. These findings highlight a complex ligand-dependent role of NRP-1, where SEMA3A may mediate anti-tumoral effects in clinically localized PCa, counteracting VEGF's pro-tumoral influence in more advanced cancers. While the results of the present study suggested that NRP-1 holds promise as a prognostic biomarker in clinically localized, hormone-sensitive PCa, its clinical utility remains to be established. Additional studies are necessary to validate these observations in larger patient cohorts and to explore the molecular mechanisms that regulate NRP-1 function in PCa. In particular, assessing NRP-1 expression in the context of VEGF/SEMA3A balance and AR signaling pathways may provide critical insights for its future use in establishing prognosis and therapy.

Acknowledgements

Not applicable.

Funding

Funding: The present study was supported by (grant no. SUBZ.C090.23.080) the Wroclaw Medical University.

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

KK, MK and BM confirm the authenticity of all the raw data. KK, MK, BM and PK conceived and designed the study. KK, AG, DK, MK, JK, TS, PD and AH acquired data (acquired and managed patients and provided facilities). AP, KD, MK, KK, WK and AH analyzed and interpreted the data (such as statistical, biostatistics and computational analyses). KK and MK wrote and/or revised the manuscript, and provided administrative, technical or material support (such as reporting or organizing data and constructing databases). BM, TS, AH, WK, PD and MK supervised the study. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

The study protocol was approved (approval no. KB-545/2020) by the Ethics Committee of Wrocław Medical University (Wrocław, Poland). All procedures were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from each recruited patient prior to sample collection.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Volume 23 Issue 4

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Spandidos Publications style
Kowalczyk K, Kaczorowski M, Piotrowska A, Kiełb P, Dudek K, Gurwin A, Karwacki J, Kowalczyk D, Krajewski W, Szydełko T, Szydełko T, et al: Neuropilin‑1 expression in primary and metastatic prostate cancer: Expression patterns and clinicopathological correlations. Mol Clin Oncol 23: 92, 2025.
APA
Kowalczyk, K., Kaczorowski, M., Piotrowska, A., Kiełb, P., Dudek, K., Gurwin, A. ... Małkiewicz, B. (2025). Neuropilin‑1 expression in primary and metastatic prostate cancer: Expression patterns and clinicopathological correlations. Molecular and Clinical Oncology, 23, 92. https://doi.org/10.3892/mco.2025.2887
MLA
Kowalczyk, K., Kaczorowski, M., Piotrowska, A., Kiełb, P., Dudek, K., Gurwin, A., Karwacki, J., Kowalczyk, D., Krajewski, W., Szydełko, T., Hałoń, A., Dzięgiel, P., Małkiewicz, B."Neuropilin‑1 expression in primary and metastatic prostate cancer: Expression patterns and clinicopathological correlations". Molecular and Clinical Oncology 23.4 (2025): 92.
Chicago
Kowalczyk, K., Kaczorowski, M., Piotrowska, A., Kiełb, P., Dudek, K., Gurwin, A., Karwacki, J., Kowalczyk, D., Krajewski, W., Szydełko, T., Hałoń, A., Dzięgiel, P., Małkiewicz, B."Neuropilin‑1 expression in primary and metastatic prostate cancer: Expression patterns and clinicopathological correlations". Molecular and Clinical Oncology 23, no. 4 (2025): 92. https://doi.org/10.3892/mco.2025.2887