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. 2025 Jul 29;16:1437. doi: 10.1007/s12672-025-03149-z

Different inhibitory effect of a neuropilin-1 monoclonal antibody on different types of hepatocellular carcinoma

Yafei Zhang 1,2, Xiaofeng Dou 2, Jianghua Yan 3, Rongshui Yang 2,✉, Xinhui Su 1,2,✉
PMCID: PMC12307266  PMID: 40731174

Abstract

As a co-receptor for vascular endothelial growth factor, neuropilin receptor type-1 (NRP-1) plays a crucial role in tumor angiogenesis, growth, and metastasis, and is regarded as a promising target for cancer molecular imaging and therapy. However, few data on inhibitory effect of an anti-NRP-1 monoclonal antibody on HCC with different NRP-1 expression levels have been reported. This study aimed to investigate inhibitory effect of an anti-NRP-1 monoclonal antibody (A6-11-26) on different types of HCC, with a view to further understanding the role of A6-11-26 in HCC. Three different types of HCC cell lines (Bel-7402, SMMC-7721 and HepG2) were conducted in this study. MTT, colony formation test, cell morphology, and flow cytometry were used to assess the inhibitory effect of A6-11-26 on three HCC cell lines. The in vivo growth inhibitory effect of A6-11-26 was evaluated in mice xenograft models bearing three HCC cell lines respectively. Immunohistochemistry analyses was performed to characterize the expressions of vascular endothelial growth factor receptor (VEGFR) and NRP-1 in HCC tissues after A6-11-26 administration. A6-11-26 displayed to inhibit the proliferation, migration and apoptosis of HCC cells in vitro and tumor growth in vivo. The inhibitory effect of A6-11-26 was dose-dependent and dependent on the level of NRP-1 expression. The decreasing expressions of VEGFR and NRP-1 in HCC tissues after A6-11-26 treatment had also dose-dependent and NRP-1 expression characteristics. Taken together, an anti-NRP-1 monoclonal antibody (A6-11-26) can inhibit HCC growth through decreasing NRP-1 and VEGFR expression accordingly with NRP-1 expression characteristics, suggesting that A6-11-26 may be a potential targeted medicine for HCC with high NRP-1 expression.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12672-025-03149-z.

Keywords: Neuropilin-1, Monoclonal antibody, Hepatocellular carcinoma, Inhibitory effect

Introduction

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and the third leading cause of cancer mortality in the world [1], and the second in China [2]. HCC primarily develops from chronic infection with hepatitis B or C virus, alcohol abuse, and metabolic syndrome related to diabetes and obesity [3–5]. The management of HCC is complex, as HCC is a heterogeneous disease which usually affects a chronically inflamed liver and often requires a team of clinicians from different areas [1]. Although significant improvement in diagnosis of HCC has been achieved in the last two decades, HCC prognosis remains poor. The overall 5-year survival rate of HCC patients has been reported to be only 16% [6], since the majority of patients with HCC are already in an intermediate or advanced stage when diagnosed and no longer have the chance to undergo surgery, and then receive systemic therapies [2]. HCC is a chemotherapy-refractory tumor and thus difficult to treat cancer. In recent years, a strategy of systemic therapy based on combinations of targeted therapy with immunotherapy has clinical benefits, but the improvements in patient outcomes have been modest and incremental [6, 7]. Thus, studies that focus on the development of novel molecular targeted agents for HCC are urgently needed and of great importance. Neuropilin-1 (NRP-1) was initially regarded as a co-receptor of semaphorin that mediates neuronal growth and signaling. Subsequent studies have revealed that NRP-1 is abundantly expressed in endothelial cells and participates in VEGF-induced angiogenesis by acting as a co-receptor for vascular endothelial growth factor receptor (VEGFR) [8, 9]. It is reported that NRP-1 is upregulated in various tumors such as glioma, lung cancer, breast cancer, gastric cancer, pancreatic cancer, bile duct cancer, and colon cancer [10–12]. Elevated expression of NRP-1 is significantly associated with poor outcomes in breast, colon, lung and liver cancer patients [11, 13]. As reported by Bergé M et al. [14], inhibition of NRP-1 function which can be blocked by N peptide, leads to tumor growth inhibition in HCC mice. These findings indicated that NRP-1 could serve as a molecular imaging and therapeutic target for cancer. In previous studies we have shown that a monoclonal antibody of NRP-1 (A6-11-26) prepared in our lab [15] exhibited the ability to inhibit glioma growth and breast cancer cell adhesion [11, 16]. Moreover, SPECT (single photon emission computed tomography) imaging with 131I-labeled A6-11-26 could detect tumors with NRP-1 overexpression [17]. However, to our best knowledge, few study on inhibitory effect of an anti-NRP-1 monoclonal antibody on HCC with different NRP-1 expression levels has not been reported. Herein, in this study, we aimed to investigate the inhibitory effect of A6-11-26 on different types of HCC in mice, with a view to further understanding the role of A6-11-26 in HCC.

Materials and methods

Materials

Anti-NRP-1 monoclonal antibodies (A6-11-26, designed by Dr. J. Yan) were prepared by our lab [12]. Goat anti-mouse IgG antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Three human HCC cell lines (Bel-7402, SMMC-7721, and HepG2) were provided by Cancer Research Center, Medical College of Xiamen University (Xiamen, China) after being purchased from Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). Female nude mice (aged, 6–8 weeks) were purchased from the Experimental Animal Center of Xiamen University (Xiamen, China).

Cell culture

According to routine protocols, three cell lines including Bel-7402, SMMC-7721 and HepG2 were cultured in DMEM supplemented with 10% FBS (Gibco, Thermo Fisher Scientific) at 37 °C with 5% CO2 in a humidified atmosphere.

Cell growth inhibition test

In vitro cell growth inhibition was performed by MTT test. Three HCC cell lines, Bel-7402, SMMC-7721 and HepG2 (3.0 × 104 cells/well) were seeded in 96 well plates, respectively. After 24 h, cells were incubated with various concentrations (0, 25, 50, 100, 200, 400 µg/mL) of A6-11-26 at 37 °C for 24, 48 and 72 h, respectively. Subsequently, 20 µL of 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT, Sigma- Aldrich, St Louis, Missouri, USA) solution at a concentration of 5 mg/mL was added to each well and incubated additionally for another 4 hours. Absorbance was measured at 492 nm using a scientific microplate reader (Multiskan Spectrum; Thermo Fisher, USA). The percentage of inhibition (%) was determined using the formula:

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Observation of morphological changes by light microscopy

The three HCC cell lines, Bel-7402, SMMC-7721 and HepG2 (3.0 × 104 cells/well) were seeded in 96 well plates for 24 h, respectively, and then were treated with various concentrations (0, 25, 50, 100, 200, 400 µg/mL) of A6-11-26 for 24 h, 48 h and 72 h. After then, the morphological changes of these cells were observed by phase contrast microscopy (Leica, Nusslich, Germany).

Colony inhibition test

The three HCC cell lines, Bel-7402, SMMC-7721 and HepG2 (1.0 × 103 cells/well) were seeded in 6 well plates for 24 h, respectively, and then were treated with various concentrations (0, 25, 50, 100, 200, 400 µg/mL) of A6-11-26 for 2 weeks. Next, these cells were fixed by methanol and stained with 0.5% crystal violet. The number of colonies was counted manually under a microscope.

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Cell apoptosis assay

Bel-7402, SMMC-7721 and HepG2 cells (5.0 × 105 cells/well) were cultured with various concentrations (0, 25, 50, 100, 200, 400 µg/mL) of A6-11-26 at 37 °C for 48 h, stained with 10 µL Propidium Iodide (PI) and 5 µL Annexin V-FITC (Becton, Dickinson and Company) and then incubated for 15 min in the dark. Cell apoptosis was analyzed by flow cytometry (Partec GmbH).

Preparation of HCC animal models and therapy

The animal study protocol has been approved by the Institutional Animal Care and Use Committee of Zhongshan Hospital Xiamen University and was conducted in accordance with the ARRIVE guidelines. The three different HCC cell lines, Bel-7402, SMMC-7721 and HepG2 (1.0 × 106 cells) were implanted subcutaneously in right lower limb of nude mice, respectively. Tumors were allowed to grow to 0.8–1.0 cm in diameter and then the tumor-bearing mice were randomly divided into 3 groups (n = 3 in each group): PBS group, low dose (1 mg/kg) group and high dose (5 mg/kg) group. A6-11-26 was given by tail vein injection every 2 d for 5 times. The body weight and tumor size were observed and recorded every 2 days. After 28 days of observation, the mice were sacrificed, and tumors were removed and weighed. The tumor volume was calculated as follows: The tumor volume = length × width × height × π/6. The growth curves of the xenograft tumors of HepG2, SMMC-7721 and Bel-7402 were documented. The tumor volume change rate (TVR%) were calculated according to the formula, TVR% = (tumor volume treated - first tumor volume)/(first tumor volume) × 100%. The tumor growth index ratio (TGI%) were calculated according to the formula:

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Immunohistochemical analysis

The tumor tissues were harvested and immediately frozen in dry ice. After that, thawed tumor tissues were sliced into 5 μm pieces of tumor slices. After being blocked with 2% BSA (Bovine serum albumin) in phosphate-buffered saline (PBS, pH = 7.4), these slides were incubated with 1:100 diluted anti-NRP-1mAb (A6-11-26) and anti-VEGF mAb (Sigma), respectively, for 12 h at 4℃, After being washed, the slides were then incubated for 2 h at room temperature with secondary antibodies goat anti-rat IgG antibody (1:1000; Sigma). These slides were then examined and photographed using an inverted microscope (Moticam 2005, Motic). The results were analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Inc.).

Western blot

The tumor tissues were sonicated and homogenized with modified Davidson Fluid (mDF) (0.5 mL) containing 1% Triton X-100 (Thermo Fisher Scientific). After centrifugation, the supernatant portions (40 µg) were subjected to SDS-PAGE gel and transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, Massachusetts, USA). To avoid interference between different antibodies, the membrane was cut to sizes that cover the internal reference and target bands respectively for transfer, followed by antibody incubation. Membranes were blocked with 2% BSA for at least 1 h, followed by overnight incubation with A6-11-26 (1:100). After extensive washing, blots were then incubated with goat anti-mouse IgG HRP secondary antibody (1: 1000; Sigma) and visualized by the ECL detection reagent (Roth, Karlsruhe, Germany) and data were analyzed with LAS 3000 digital software (Fujifilm Life Science, Santa Clara, California, USA). GAPDH blotting was used as an internal control.

Quantitative real-time PCR (qRT-PCR)

Total RNA isolated from tumor tissues was extracted with TRIZOL reagent according to the manufacturer’s instructions. The cDNA was synthesized from 1 µg of total RNA from each sample using MMLV transcriptase (ToYoBo, Shanghai, China) with random primers. Semi-Quantitative RT-PCR was performed with 2 × Phanta Max Master Mix (Vazyme, Nanjing, China). The qRT-PCR procedures were as follows: Briefly, the template cDNA was first denatured at 95 °C for 3 min. The amplification step included 25 cycles of denaturation for 15 s at 95 °C, 15 s of annealing at 60 °C, and elongation at 72 °C for 30 s.

Primers used in this study were:

NRP-1-RT-F (Homo sapiens): 5′-AAATGCGAATGGCTGATTCAG-3′;

NRP-1-RT-R (Homo sapiens): 5′-CTCCATCGAAGACTTCCACGTAGT-3′;

β-actin-RT-F (Homo sapiens): 5′-CAAGAGATGGCCACGGCTGCT-3′;

β-actin-RT-R (Homo sapiens): 5′-TCCTTCTGCATCTGTCGGCA-3′.

Statistical analysis

All the statistical analyses were performed using the SPSS 18.0 (SPSS Company, Chicago, USA). Statistical data were shown as mean ± standard deviation. For data where variables fail to meet the normality assumption based on the Shapiro-Wilk test, non-parametric tests were used for analysis. Non-parametric tests were used to compare the differences between groups of different HCC cell lines, as well as to compare the different dosages or time points within the same cell line. In vivo transplanted tumor model, non-parametric tests were used to analyze the differences in growth parameters between groups. GraphPad Prism 9.0 (GraphPad Software California, USA) was utilized to draw statistical charts. A P value < 0.05 was defined as statistically significant.

Results

Different inhibitory effect of A6-11-26 on proliferation of different HCC cells

The MTT assay was used to detect the inhibitory effect of A6-11-26 on the proliferation of three human HCC cells. As shown in Fig. 1, the growth of these cells was significantly inhibited by A6-11-26 with dose-dependent, among which the strongest inhibitory effect of A6-11-26 was found in 400 µg/mL dose group (P < 0.05). Importantly, the inhibition rates in the three HCC cells were obviously different, and were lower in HepG2 (22.75 ± 0.04%) than those of SMMC-7721 (42.09 ± 0.06%) and Bel-7402 (47.56 ± 0.10%) (P < 0.05) as 400 µg/mL dose group at 72 h. The inhibitory effect of A6-11-26 progressively increased from HepG2 and SMMC-7721, and then to Bel-7402. Furthermore, it was also found that morphological changes of the three HCC cells were obviously observed at 24 h after administration, including lower cell, thinner cell, rough surface and intracellular particle, demonstrating cell growth inhibition (Fig. S1).

Fig. 1.

Fig. 1

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Inhibitory effect of A6-11-26 on the proliferation of three different human HCC cells (A HepG2, B SMMC-7721 and C Bel-7402). *P < 0.05, **P < 0.01

In order to further investigate the anti-proliferative activity of A6-11-26, the colony formation assay was carried out in the three HCC cells. As presented in Fig. 2, the number and size of the three HCC colonies were significantly reduced on day 7 with increasing doses of A6-11-26 compared to the control group. The number and size of colony decreased successively from HepG2 and SMMC-7721, and then to Bel-7402 (P < 0.05). Overall, these results showed that anti-proliferative activity of A6-11-26 correlated with the malignant potential of HCC cell lines, suggesting that A6-11-26 might be a potential anti HCC agent.

Fig. 2.

Fig. 2

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Inhibitory effect of A6-11-26 on colony formation of three different human HCC cells (HepG2, SMMC-7721 and Bel-7402). *P < 0.05, **P < 0.01

Different inhibitory effect A6-11-26 on apoptosis of different HCC cells

Apoptosis is a form of cell death; thus, this study examined the occurrence of apoptosis after A6-11-26 treatment. In the three HCC cells, when treated with different concentrations of A6-11-26 for 24 h, the apoptotic rates in HepG2, SMMC-7721 and Bel-7402 increased with the increase of the doses of A6-11-26, which was dose-dependent (P < 0.05) as shown in Fig. 3 and Table S1. The apoptotic rates progressively increased from HepG2 to SMMC-7721 and then to Bel-7402 (P < 0.05).

Fig. 3.

Fig. 3

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Inhibitory effect of A6-11-26 on apoptosis of three different human HCC cells (HepG2, SMMC-7721 and Bel-7402)

In vivo different inhibitory effect of A6-11-26 on tumor growth

We evaluated the in vivo inhibitory effect of A6-11-26 on the human HCC tumor xenografts in nude mice. The growth curves of the xenograft tumors of HepG2, SMMC-7721 and Bel-7402 were documented, which demonstrated that the volume of tumors in the control group was larger than that in the A6-11-26 administration groups (Fig. 4). Notably, the high-dose regimen had significantly reduced tumor volume compared to the low-dose intervention (P < 0.05). When nude mice bearing HepG2, SMMC-7721 and Bel-7402 tumors were treated with various concentrations of A6-11-26, there was a significant difference in the TGI between the high dose and low dose groups of A6-11-26 for HepG2, SMMC-7721, and Bel-7402 xenograft tumors (all P < 0.05). At the last observation on the 27th day, the TGI rates of the low dose and high dose groups for HepG2, SMMC-7721, and Bel-7402 xenograft tumors were 19.54% vs. 44.51%, 21.77% vs. 51.05%, and 42.00% vs. 58.43% respectively. A6-11-26 significantly reduced TVR at all 3 utilized doses on day 9 post-treatment for tumors bearing HepG2, SMMC-7721 and Bel-7402 cells, respectively, with dose-dependent (P < 0.001, Fig. S2). Furthermore, the reduction of TVR of high dose group progressively decreased from HepG2 (84.45 ± 4.01%) and SMMC-7721 (77.97 ± 5.21), to Bel-7402 (38.17 ± 1.41) (P < 0.05).

Fig. 4.

Fig. 4

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Inhibitory effect of A6-11-26 on xenograft tumor volume in vivo (A HepG2, B SMMC-7721 and C Bel-7402), control group: PBS, low dose: 1 mg/kg, high dose: 5 mg/kg

In vivo different inhibitory effect of A6-11-26 on the expression of NRP-1 and VEGFR

NRP-1 is considered a co-receptor of VEGF which promotes tumor angiogenesis, growth, and metastasis. To investigate whether the anti-neoplastic action of A6-11-26 is associated with the modulation of VEGF, we investigated the effects of A6-11-26 on the expression of VEGFR and NRP-1 in tumors bearing these three different human HCC cell lines. As shown in Figs. 5, 6 and 7, A6-11-26 concentration dependently attenuated the expressions of NRP-1(Figs. 5 and 6) and VEGFR (Fig. 7). The different was statistical significance among the three groups, including control group, low dose and high dose group. Moreover, the expression of VEGF and NRP-1 in hepatocellular carcinoma tissues decreased successively from HepG2, SMMC-7721 to Bel-7402 (P < 0.05).

Fig. 5.

Fig. 5

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Inhibitory effect of A6-11-26 on the expression of NRP-1 in xenograft tumor from mice bearing three different human HCC cells (HepG2, SMMC-7721 and Bel-7402). A Western blot, B Semi-quantitative analysis of western blot, C PCR, D Semi-quantitative analysis of PCR. Graphics have been cropped and the original blots/gels are presented in Supplementary Figure S3

Fig. 6.

Fig. 6

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Inhibitory effect of A6-11-26 on the expression of NRP-1 in xenograft tumor from mice bearing three different human HCC cells (HepG2, SMMC-7721 and Bel-7402) (×400). A Immunohistochemical analysis. B Semi-quantitative analysis of immunohistochemistry

Fig. 7.

Fig. 7

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Inhibitory effect of A6-11-26 on the expression of VEGFR in xenograft tumor from mice bearing three different human HCC cells (HepG2, SMMC-7721 and Bel-7402). A Immunohistochemical analysis. B Semi-quantitative analysis of immunohistochemistry

Discussion

HCC is primarily characterized by hypervascularity and remarkable vascular abnormalities with above 20 mm arterialization and sinusoidal capillarization [18]. The increase of tumor vascularity may be caused by pro-angiogenic factors leading to angiogenic sprouting or existing vessels into tumor [19]. Additionally, a recent study has demonstrated that tumor blood supply influences the prognosis of HCC [20].Thus, angiogenesis is involved in the development and pathogenesis of HCC [18]. Among pro-angiogenic factors, vascular endothelial growth factor (VEGF) plays a crucial role in the promotion of angiogenesis in HCC tumors. Blocking any step in this pathway can effectively inhibit the tumor-associated angiogenesis and thereby inhibit the growth and metastasis of HCC tumor. In recent years, a variety of anti-tumor angiogenesis drugs targeting VEGF/VEGF receptor (VEGFR) signaling pathway have been approved treatments for advanced HCC, including bevacizumab, Axitinib, Sorafenib and Sunitinib [21]. However, anti-tumor angiogenic drugs may not be sufficient to eradicate tumors since a great many of processes and factors can result ineffectiveness and resistance to angiogenesis inhibitors, particularly those associated with the tumor endothelium [21]. Strategies for improving treatment effect of angiogenesis inhibitors, targeting other elements of angiogenic pathways with other novel therapies are under investigation [18].

NRP-1 is expressed on endothelial cells and acts as a co-receptor for VEGF165, thereby participating in the formation of new blood vessels [22]. Tumor growth can be inhibited by blocking the binding of NRP-1 to VEGF, which can specifically target and inhibit tumor neovascularization [22, 23]. Meanwhile, it has been found that inhibition of NRP-1 expression ameliorates tumor drug-resistance, with better effects than anti-VEGF strategy [24]. Furthermore, over-expression of NRP-1 was found in several tumor cells, such as glioma U87 cells, breast cancer MCF7 cells, HCC Bel-7402 cells [11, 13, 16]. Our previous studies have shown that blocking NRP-1 expression by an anti-NRP-1 monoclonal antibody (A6-11-26) inhibited tumor cell growth and reduce tumor cell migration [11, 16, 22]. However, there are few report on whether A6-11-26 is of different inhibitory effect on different cell types in a homogeneous tumor or not due to tumor heterogeneity. Based on our previous demonstrations that the expression level of NRP-1 was obviously different in three HCC cell lines, and progressively increased from HepG2, SMMC7721 to Bel-7402 [13], we further investigate the inhibitory effect of A6-11-26 on the HCC in the present study. Our results showed that both in vitro and in vivo, A6-11-26 could inhibit the growth of HCC. The inhibition rates increased as the increase of the doses of A6-11-26 and from HepG2, SMMC7721 to Bel-7402, indicating the inhibitory responses to A6-11-26 were of dose-dependent and NRP-1 expression-dependent.

In this study, we performed the MTT assay, colony inhibition test, and cell apoptosis to determine inhibitory effect of A6-11-26 on three HCC cell lines, respectively. These tests showed A6-11-26 could not only decrease the tumor cell growth, change tumor cell morphology, reduce the number and size of tumor cell colonies, but also promote tumor cell apoptosis. The changes of cell morphology in these three cell lines included lower cell, thinner cell, rough surface and intracellular particles, consistently with the previous literature by Chen [16], suggesting cell growth inhibition. Furthermore, the inhibitory response to A6-11-26 increased as the increase of the doses of A6-11-26 and level of NPR-1 expression (from HepG2, SMMC7721 to Bel-7402), indicating the inhibitory effects of A6-11-26 were of dose-dependent and NRP-1 expression- relationship. The possible mechanism may be that A6-11-26 can block NRP-1 interactions with VEGF, thereby influencing VEGF signaling. Studies have confirmed that NRP-1 contributes to tumor angiogenesis by mediating VEGF-A-induced VEGFR trafficking and specifically regulating the ERK signaling pathway [25]. In current study, inhibition of NRP-1 by A6-11-26 suppressed its activity and may have downregulated these downstream signaling pathways. Furthermore, the blockade of NRP-1 by A6-11-26 likely induces cell cycle arrest through upregulation of p27 and downregulation of cyclin E and cyclin-dependent kinase 2 (CDK2), thereby inhibiting cellular proliferation [26]. Interestingly, the results demonstrate that the NRP-1 antibody exhibits stronger anti-proliferative effects on Bel-7402 cells than on SMMC-7721 cells at 48 h, while showing greater pro-apoptotic activity in SMMC-7721 cells compared to Bel-7402 cells. This differential response may be attributed to the complexity of molecular regulatory networks, along with the independence or crosstalk of signaling pathways [27–29].

In vivo studies, mice bearing three human HCC cells xenografts were conducted to assess the effects of A6-11-26 administrated by different doses. The results showed that A6-11-26 takes inhibitory effects on growth capacity of HCC with obvious dose-effect relationship and NRP-1 expression-dependent. Moreover, immunohistochemical analysis showed that A6-11-26 concentration dependently significantly attenuated the expressions of NRP-1 and VEGFR. This effect may benefit from the multiple inhibitory effects of the A6-11-26 antibody on NRP-1 and may be associated with multiple mechanisms. For example, it could be due to the inhibition of c-Met [30] and the reduction of p130Cas phosphorylation [31]. It may also be attributed to the fact that NRP-1 activates the tumor microenvironment, thereby promoting tumor growth, while the neutralizing antibody can slow down tumor growth [32]. Alternatively, in endothelial cells, VEGF concurrently binds to both NRP-1 and VEGFR to form a ternary complex that facilitates receptor internalization and intracellular trafficking, which is critical for activating downstream intracellular signaling pathways [33]. A6-11-26 may inhibit the formation of this ternary complex, thereby suppressing tumor progression. In addition, these results may indicate that the NRP-1 antibody is associated with the inhibition of vascular endothelial cell growth and the reduction of VEGFR expression. The downregulation of VEGFR expression in the A6-11-26 treatment group may be associated with known pathways such as PI3K/AKT/mTOR or RAF/MEK/ERK [34, 35]. Our previous studies have demonstrated iodine-131-labeled A6-11-26 (131I-A6-11-26) can specifically accumulated in the U87MG tumor at 24 h and then showed a gradual increase of uptake from 48 to 120 h [17]. These data suggested that the over-expression of NRP-1 in cancer cells and the neovascularizations of tumor leaded to the enrichment of A6-11-26 in tumor tissues. Briefly, A6-11-26 may act as a promising drug targeting NRP-1. However, the widespread expression of NRP-1 was found in normal tissues, including liver, lung and kidney have moderate NRP-1 expression, and it may affect A6-11-26 distribution to tumor tissue, resulting in decrease therapy effect of A6-11-26 on tumor. Subsequently, it is necessary for an appropriate A6-11-26 dose to saturate normal tissue antigenic sinks to achieve better therapy affect. Our previous study demonstrated that the distribution of A6-11-26 in tumors is overwhelmingly higher than in normal tissues as described above, and indicated that a dose between 10 and 20 mg·kg-1 of A6-11-26 may be the optimal dose that maximized tumor exposure [36].

However, this study has certain limitations. Firstly, we did not conduct further research on whether A6-11-26 inhibits HCC strictly through the NRP-1 target. Potential off-target effects or indirect modulation of NRP-1-associated pathways cannot be fully excluded. However, A6-11-26 was explicitly designed as an NRP-1-targeting agent, and its observed efficacy aligns with the differential NRP-1 expression profiles across HCC models [13], supporting a target-mediated mechanism. The current results are generally reasonable. In future studies, NRP-1 knockout or overexpression experiments will be conducted to validate the specific inhibitory effect of A6-11-26 on HCC via the NRP-1 pathway. Moreover, we will also design a radioactive derivative of A6-11-26 to further improve the validation of its target specificity. Secondly, due to some constraints, we did not further elaborate on the molecular mechanism of A6-11-26 in inhibiting HCC. Nevertheless, the study of the molecular mechanism plays an important role in explaining the inhibitory effect of A6-11-26 on HCC. We will improve this in subsequent studies by including CRISPR screening to identify target networks and proteomics to track activation states of key signaling pathways [37]. These approaches will clarify its on-target specificity and mechanistic landscape, bridging the current knowledge gap.

In conclusion, an anti-NRP-1 monoclonal antibody (A6-11-26) can inhibit HCC growth through decreasing NRP-1 and VEGFR expression with NRP-1 expression characteristics, suggesting that A6-11-26 may be a potential targeted medicine for HCC with high NRP-1 expression.

Electronic supplementary material

Supplementary Material 1 (12.4MB, docx)

Author contributions

YZ, XS and JY conceived and designed experiments and interpreted data. YZ and XD performed experiments. RY, YZ and XS wrote the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (NSFC) (82071965).

Data availability

The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The animal study protocol has been approved by the Institutional Animal Care and Use Committee of Zhongshan Hospital Xiamen University and was conducted in accordance with the ARRIVE guidelines.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Rongshui Yang, Email: horseyrs@163.com.

Xinhui Su, Email: suxinhui@zju.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (12.4MB, docx)

Data Availability Statement

The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.

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