Generation of ENO1 Single Chain Variable Fragment and Its Inhibitory effect on cervical cancer cells

Generation of ENO1 Single Chain Variable Fragment and Its Inhibitory effect on cervical cancer cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Generation of ENO1 Single Chain Variable Fragment and Its Inhibitory effect on cervical cancer cells Rui Yang, Yuanyuan Chen, Xiaofeng Liu, Pengyu Dai, Tingting Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6854783/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective Enolase l (a-enolase, ENO1) is highly expressed in a variety of tumor cells, including cervical cancer. The eno1 monoclonal antibody could inhibit the invasion and migration of cervical cancer cells;however, it could not enter into cells. To overcome the limination, this study prepared ENO1 Single-Chain Variable Fragment (anti-ENO1 scFv) and validated its in vitro anti-cervical cancer cell effect to address the issue of antibody entry into cells. Methods Firstly, the nucleotide sequence of anti-ENO1 scFv was inserted into pET-30a prokaryotic expression vector by restriction enzyme digest sites (Nde I and HindIII); Then,the expression was induced by isopropyl β-D- thiogalactoside(IPTG) in E.coli BL21(DE3) cells. Second,Ni–NTA chromatography was used for the purification, and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (WB). Thirdly, the anti-cervical cancer effect of anti-ENO1 scFv in vitro was studied through cell proliferation assay, colony formation assay, wound healing assay, and Transwell assay. Results The recombinant plasmid pET30a-ENO1 scFv was successfully constructed. SDS-PAGE analysis showed that the expression of anti-ENO1 scFv could be located in the periplasmic space and extracellular space of BL21 and about 180 ug/mL purified anti-ENO1 scFv was obtained. Cell experiments showed that anti-ENO1 scFv could inhibit the activity of ENO1, significantly reduce the content of pyruvate and lactic acid, inhibit cell proliferation, invasion and migration, and clone formation of cervical cancer cells ( p <0.05). Conclusion The present study demonstrated that anti-ENO1 scFv can specifically block the expression of ENO1 on the cell membrane and inhibit the activity of ENO1 glycolytic enzyme in tumor cells, and it is expected to become a potential anti-tumor drug for cervical cancer. Biological sciences/Biological techniques Biological sciences/Cancer Biological sciences/Immunology ENO1 Single-Chain Variable Fragment prokaryotic expression system cervical cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 What does this study add to the clinical work The results of this study demonstrate that we have successfully prepared a single-chain antibody against ENO1. It can inhibit the migration, invasion and proliferation of cervical cancer cells. This study provides a new biological candidate drug for precise treatment of cervical cancer based on metabolic targets. Introduction Cervical cancer is one of the three major cancers affecting women under 45 In developed countries, the five-year survival rate for cervical cancer patients ranges from 60–70%[1], while in developing countries, this value is obviously reduced to one-third of that in developed countries[2]. Early-stage cervical cancer can be cured with surgery,chemoradiation or a combination of treatment modalities, advanced cervical cancer is often incurable and once recurrent,relatively refractory to treatment.When cervical cancer relapse after radiotherapy, systemic treatment is usually performed[3].Some patients can be saved through local treatments such as surgical resection and radiation therapy[4]. Late stage patients with stage IIB and above should be treated with radiotherapy and chemotherapy .In the United States, survival has not significantly improved for cervical cancer patients since the 1970s[5], demonstrating the urgent need to improve on current treatment approaches for cervical cancer. Metabolic reprogramming represents a hallmark of cancer cell physiology, with the distinctive tendency of these cells to favor relatively energy-efficient glycolytic pathways over oxidative phosphorylation garnering considerable interest in the field of oncology[6]. During the process of glycolysis, numerous pivotal metabolic enzymes, notably Enolase 1 (alpha-enolase, ENO1), have been identified as being overexpressed or hyperactivated in cancerous cells. ENO1 functions as a multifunctional oncoprotein, serving several critical roles. Primarily, it functions as a key enzyme within the glycolytic pathway, playing an essential role in regulating cellular proliferation and tolerance to hypoxic conditions[7]. Additionally, ENO1 serves as a plasminogen receptor located on the cell membrane, thereby facilitating cancer cell proliferation, invasion, and migration[8]. Moreover, ENO1 is also found in the nucleus, where it modulates the expression of genes closely linked to tumorigenesis[9]. The resultant overproduction of lactic acid further contributes to the establishment of an acidic microenvironment, which is detrimental to the survival and functionality of adjacent immune cells[10]. The degradation of the extracellular matrix (ECM) is a pivotal process in tumor metastasis, necessitating the action of various proteolytic enzymes, including plasmin and matrix metalloproteinases (MMPs)[11]. Fibrinogen levels have been shown to correlate positively with tumor aggressiveness[12], and fibrinogen-binding proteins are integral in allowing tumor cells to evade innate immune responses[13]. Among the fibrinogen-binding proteins, ENO1 has emerged as a promising candidate for applications in tumor diagnosis, therapeutic intervention, and prognostic prediction[14]. In our research laboratory, we previously generated an ENO1 monoclonal antibody utilizing hybridoma technology. Our findings indicate that this particular antibody possesses the capability to impede the proliferation, clonogenicity, invasion, and metastatic potential of cervical cancer cells. Nonetheless, the clinical application of monoclonal antibodies is fraught with challenges[14]. The foremost issue arises from their substantial molecular weight, which restricts their ability to traverse the cell membrane and penetrate the cytoplasm[15].And ENO1 involved in glycolysis is mainly located in the cytoplasm, so solving the problem of its entry into the cell is imminent. In recent years, molecular biology and genetic engineering technology have made it possible to construct antibody fragments, such as Single Chain Antibody Fragment (scFv,~30kda) and single domain antibody (sdAd)[16].The scFv is a recombinant protein formed by the combination of the variable regions of the heavy chain (VH) and light chain (VL) of antibodies, linked by a peptide linker[17].With the advantages of low molecular weight, good tissue permeability and weak immunogenicity, scFv has been widely promoted in the diagnosis and treatment of many clinical diseases[18].ScFv targeted therapy in tumors refers to coupling scFv with other effector molecules, such as toxins and viruses, and delivering them to the corresponding tumor cells, so as to enhance the tumor-killing ability of the effector molecules[16].Currently, it has been actively used in the research and treatment of breast cancer, prostate cancer and other diseases[19, 20]. In this study, specific anti-ENO1 scFv was prepared on the basis of previous experiments. Preliminary cellular experiments showed that anti-ENO1 scFv inhibited glycolysis, proliferation, invasion, migration and cell clone formation of HeLa cells, suggesting that scFv-ENO1 has the ability to specifically block ENO1, and providing data support for further in vivo experiments. Materials Hela cells,E. coli strainDH5a, E. coli strain BL21 (DE3), The pFastBacTMDual-EN01 single chain antibody strain is stored in the Institute of Pathogen Biology, School of Basic Medical Sciences, Lanzhou University. Expi293FTM cells were purchased from Thermo Fisher and kept in our laboratory. ExpiFectamine™ 293 (A14527CN) transfection kit, DMEM medium were purchased from Thermo Fisher Scientific (USA). Lactic acid kit and pyruvic acid kit were purchased from Nanjing Jiancheng Biological Co., Ltd (Nanjing,China),2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonatobenzene)-2H-tetrazolium monosodium salt (CCK-8) kit, cell culture flasks and Transwell were purchased from Gansu Weibo Xin Bio-technology Co.,Ltd(Lanzhou, China). bicinchoninic acid(BCA) protein assay kit was from Beijing Solar bio Technology Co., Ltd (Beijing, China). Cell culture plates were obtained from Wuhan Xavier Biotechnology Co., Ltd (Wuhan, China). Matrigengel was purchased from Shanghai Nova Pharmaceutical Technology Co., Ltd (Shanghai, China). Methods 1. Construction of anti-ENO1 scFv E. coli expression vector Using pFastBacTM-Dual-ENO1 recombinant plasmid containing anti-ENO1 scFv gene as template, using F as forward primer and R as reverse primer, PCR reaction was carried out, and the specific conditions were as follows:The template was denatured at 94 C for 5 minutes, then 30 cycles of amplification were performed, each cycle was held at 94 C for 30 seconds, at 55°C for 1 minute, and finally extended at 72°C for 10 minutes (Table. 1).The amplified DNA fragment was purified, digested with restriction enzymes Nde I and HindIII, and then inserted into the pET-30a expression vector which had been digested with the same restriction enzymes.The final plasmid was named pET-30a/ENO1.The recombinant expression vector pET-30a/ENO1 was transformed into E. coli DH5α cells. Subsequently, it was stored in glycerol at -80℃ for later use. The clone picked up from a solid Luria–Bertani (LB) medium plate containing kanamycin (50mg/ml) was confirmed by DNA sequencing. Table 1 Forward primers (F)and reverse primer (R) harboring restriction enzymes Nde I/ HindIII sequence were used for cloning harboring anti-ENO1cscFv sequence to pET-30a expression vector by 3-step PCR. Primer Primer sequence (5’–3’) F 5’-GGAATTCCATATGATGAGAGTGCTGATTCTTTTGTGGC-3’ Nde I R 5’-CAAGCTTCTAGTGGTGGTGGTGGTGGTGTTTTATTTCCAACTTTGTC-3’ HindIII 2. Expression of anti-ENO1 scFv in E. coli ENO1 is one of the key enzymes in the glycolysis pathway, considering that it may affect the metabolism of E. coli, we expressed anti-ENO1 scFv using normal LB medium and LB medium with glucose added to observe whether there is any difference in the expression yield. 2.1.37℃, 0.5M IPTG induct for 4h Add 100ug/ml kanamycin to LB liquid medium, and add E. coli BL21 cells carrying recombinant plasmids into LB liquid medium. Cultivate at 37°C for 4 hours. When the optical density (OD450) reaches 0.4–0.6, add isopropyl β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM to the culture medium to induce the expression of recombinant proteins. Subsequently, the culture was shaken at 180 rpm at 37°C for 4 hours.Harvest cells by centrifugation (8000 rpm, 20 minutes, 4°C) and then lysed by sonication. The cell lysate was then centrifugated at 4°C, 8000rpm for 30 min. 2.2.16℃, 0.5M IPTG induct for 15 hours The preliminary steps are as described above. When the optical density (OD450) reaches 0.4–0.6, add isopropyl β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM to the culture medium to induce the expression of recombinant proteins. Subsequently, the culture was shaken at 180 rpm at 16°C for 15 hours.The subsequent operation is the same as the previous steps. Analyze the expression of anti-ENO1 single chain antibody (scFv) using SDS-PAGE. SDS–PAGE was performed using a 5% stacking gel and a 12% separating gel in 1%Tris–glycine buffer.Subsequently, stain with 250 µ L Coomassie Brilliant Blue. 3. Purification of anti-ENO1 scFv Subsequently, the supernatant was collected and loaded into a Ni–NTA column which had been previously equilibrated with the binding buffer (0.5M Na2HPO4, 0.5M NaH2PO4 and 0.5M NaCl). After thorough washing with binding buffer, the anti-ENO1 scFv was eluted using elution buffer (0.5M Na2HPO4, 0.5M NaH2PO4, 0.5M NaCl, and 10-500mM imidazole). The elution fractions containing anti-ENO1scFv were pooled and concentrated.The anti-ENO1 scFv obtained was validated using SDS-PAGE analysis.Finally, the product was desalted by dialysis against PBS buffer (0.01 M, pH 7.4) at 4°C for 16 h and stored at 80°C until use. 4. The inhibitory effect of anti-ENO1 scFv on the proliferation of cervical cancer cells HeLa cells in the logarithmic growth phase were seeded in 96-well plates at a density of 5 × 10⁴ cells/100 µL per well and incubated overnight in a culture incubator. HeLa cells were treated with graded concentrations of anti-ENO1 scFv and 3-BrPA, followed by 16-hour incubation. A negative control group was simultaneously established for comparative analysis.The original culture medium was replaced with DMEM supplemented with CCK-8 solution, followed by 2-hour incubation of the 96-well plate in a CO₂ incubator. Subsequently, optical density (OD) at 450 nm was measured using a microplate reader, and cell viability was calculated accordingly. 5. Determination of glucose metabolites Cells were seeded in 6-well plates at a density of 5 × 10⁵ cells/well and incubated overnight under 5% CO₂ at 37°C. Dilute anti-ENO1 scFv (IC50 concentration) and 3-BrPA with DMEM without phenol red,and add appropriate concentrations of anti-ENO1 scFv and 3-BrPA to 6-well plates, respectively. Afterwards, place the culture plate in the incubator and continue to cultivate for 20 hours.Set up a blank group and a control group separately. Culture supernatants were collected post-incubation for quantification of pyruvate and lactate levels using commercial assay kits. 6. Experiment of plate cloning formation HeLa cells were seeded in 6-well plates at graded densities (400, 600, and 1000 cells/well) to determine the optimal seeding density, followed by overnight incubation at 37°C with 5% CO₂. Cells were treated with anti-ENO1 scFv and 3-BrPA, with PBS-treated groups serving as negative controls. Continuous culture was maintained for 2–3 weeks under standard conditions (37°C, 5% CO₂).The assay was concluded when macroscopically visible colonies (> 50 cells/colony) formed. Culture medium was aspirated. cells were Fixed with 4% paraformaldehyde (30 min, RT),Stained with 0.1% crystal violet (15 min, light-protected).Gently rinsed under running water to remove residual dye .Air-dried plates were imaged using a documentation system, and colony quantification was performed with ImageJ software. 7. Wound healing test Cervical cancer HeLa cells were seeded in 6-well plates at 1×10⁶ cells/well and incubated overnight at 37°C/5% CO₂ until reaching ~ 100% confluency. A standardized wound was created using a sterile 200uL pipette tip, followed by PBS washing to remove dislodged cells.Next, dilute anti-ENO1 scFv and 3-BrPA with serum-free DMEM and add them to a 6-well plate. Incubate cells at 37°C and 5% CO2 for another 24 hours. Wound closure dynamics were documented at 0h(baseline), 12h, and 24h intervals using an inverted phase-contrast microscope (20× objective). Wound areas were measured using ImageJ.And analyze the average migration rate. 8. Transwell assay Cell Migration Assay: Add HeLa cells (5 × 104) to the upper chamber of the Transwell chamber, and add 600ul of DMEM medium containing 15% FBS to the lower chamber of the Transwell chamber. Add homemade ENO1 monoclonal antibody (ENO1mAb), anti-ENO1 scFv, and 3-BrPA (15ug/ml) to the upper chamber, respectively. And incubating at 37℃ and 5% CO2 for 48 hours. Then, migrated cells were fixed with 4% paraformaldehyde (600 µL, 30 min) ,stained with 0.1% crystal violet (600 µL, 20 min),and washed with PBS to remove non migrating cells. Finally, five random fields per insert were imaged under a Nikon inverted microscope (100× magnification) and count the number of migrating cells. Cell Invasion Assay: Thaw Matrigel™ at 4°C overnight,dilute 1:8 (v/v) with serum-free DMEM,take 60ul Diluted Matrigel was added to the upper chamber of Transwell chamber (37°C, 5% CO2,3 h polymerization).Add HeLa cells (5 × 104) to the upper chamber of the Transwell chamber, and add 600ul of DMEM medium containing 15% FBS to the lower chamber of the Transwell chamber. The Subsequent steps are the same as the cell migration experiment.The subsequent steps are the same as the cell migration experiment, including cell fixation, staining, and image acquisition. Results 1. Construction of expression vector pET30a-ENO1 scFv The anti-ENO1 scFv gene was amplified using sequence-specific primers, yielding a 903 bp product (Fig. 1 A). Construction and identification results of anti-ENO1 scFv prokaryotic vector(Fig. 2 ).Positive clones were selected from LB agar plates containing 50 mg/L kanamycin and subjected to Sanger sequencing. Sequencing analysis confirmed successful construction of the recombinant pET-30a/ENO1 expression vector. The full-length ORF of the anti-ENO1 scFv was 903bp and it encoded a 298 amino acid peptide with a deduced molecular weight of 32 KDa(Fig. 1 B).6xHis-tag at C-terminus of the recombinant protein was used for purification. 2. Preparation of anti-ENO1 scFv 2.1. Expression of anti-ENO1 scFv in E. coli Induction parameters critically determine protein expression efficiency in prokaryotic systems[21].Compared with LB medium without glucose, the expression level of anti-ENO1 scFv was significantly increased in LB medium containing 2g/L glucose, and a specific protein band appeared at a molecular weight of about 32 kDa(Fig. 2 A). Induced at 37 ℃ for 4 hours, SDS-PAGE analysis showed that anti-ENO1 scFv was more expressed in bacterial precipitation(Fig. 2 B). 16 ℃ induction for 15 hours resulted in a higher expression of single chain antibodies in the bacterial supernatant(Fig. 2 C). This result indicates that under the same concentration of IPTG induction, Low-temperature induction significantly facilitated soluble scFv secretion (>80% soluble fraction).Supernatant-derived antibodies maintained native conformation, enabling tag-based purification without denaturation. 2.2. Anti-ENO1 scFv Purification & Validation The culture supernatant containing anti-ENO1 scFv was purified via Ni- NTA chromatography. Stepwise elution with imidazole gradients (50–500 mM) revealed optimal purity at 500 mM imidazole (Fig. 2 D). Target fractions dialyzed against PBS (1:30 v/v, 4°C, 24h),Final concentration: 180 µg/mL (BCA protein assay). Western Blot:Primary antibody: Mouse anti-His tag mAb (1:2000),Secondary antibody: HRP-conjugated goat anti-mouse IgG (1:5000),Specific band detected at 25–35 kDa (Fig. 2 E). 3. Anti ENO1 scFv Affects the growth of cervical cancer cells 3.1. Anti-ENO1 scFv inhibits the proliferation of HeLa cells In this experiment, 3-BrPA served as the positive control for evaluating the inhibitory effect of anti-ENO1 scFv on HeLa cell proliferation, owing to its well-documented cytotoxic activity against most cancer cells[22].After co incubation of anti-ENO1 scFv and 3-BrPA at different concentrations (25–150) ul with Hela cells for 16 hours, the inhibitory effect of anti-ENO1 scFv on Hela cell proliferation was detected using CCK-8. Compared with the blank control group, the survival rate of HeLa cells in the anti ENO1 single chain antibody group was significantly reduced in a dose-dependent manner. At a concentration of 150ug/ml, the proliferation rate of Hela cells was 64.33 ± 7.506, which was significantly different from the blank control group (100%) ( p < 0.01), while there was no significant statistical difference compared to the proliferation rate of 67 ± 12.12 in the 3-BrPA group (Fig. 3 A, p < 0.05). This indicates that anti ENO1 scFv has a significant inhibitory effect on the growth of cervical cancer HeLa cells, and its effect is not significantly different from 3-BrPA at the same dose. 3.2. Plate cloning experiment To verify whether inhibiting tumor cell glycolysis inhibits tumor cell proliferation and clone formation ability, we used plate clone formation experiments to detect the effect of anti-ENO1 single chain antibodies on Hela cell clone formation ability(Fig. 3 B).The clone formation rates of the anti-ENO1 scFv and 3-BrPA groups were 36.95 ± 0.085% and 30.05 ± 5.445%, respectively, significantly lower than those of the PBS group (48.55 ± 0.045%) (Fig. 3 C, P < 0.01).The results indicate that anti-ENO1 scFv has a certain inhibitory effect on the cloning ability of cervical cancer cells. 4. Determination of glucose metabolites The small molecular weight of single-chain antibodies enables direct cellular internalization. Upon entering cervical cancer HeLa cells, anti-ENO1 scFv is hypothesized to inhibit cytoplasmic ENO1 activity. We assessed its impact on glycolysis using a glycolysis assay kit.3-BrPA and anti-ENO1 scFv were incubated with HeLa cells for 24 hours, respectively, and the pyruvate content in the anti-ENO1 scFv group and 3-BrPA group was 0.099 ± 0.0002, respectively µ Mol/mL, 0.089 ± 0.0004 µ Mol/mL, compared to the control group (0.164 ± 0.0002) µ Mol/L (Fig. 4 A, P < 0.01). The lactate content in the anti-ENO1 scFv group and 3-BrPA group was 13.89 ± 2.02 mmol/gprot and 7.79 ± 0.54 mmol/gprot, respectively, significantly lower than the control group (18.61 ± 0.82 mmol/gprot) (Fig. 4 B, P < 0.001), and the lactate content in the 3-BrPA group was significantly lower than that in the ScFV-ENO1 group ( P < 0.001). These results confirm that anti-ENO1 scFv suppresses glycolysis in HeLa cells, though its efficacy is inferior to 3-BrPA at equivalent doses(Fig. 4 B, P < 0.001). 5. Anti-Migratory and Anti-Invasive Effects Wound healing assay: To investigate the effect of anti ENO1 single chain antibody on Hela cell migration ability, as shown in Fig. 5 A. The analysis results are shown in Fig. 5 B. The free ENO1mAb, anti ENO1 scFv, and 3-BrPA groups all inhibited the migration of cervical cancer Hela cells within 24 hours, with average migration rates of 8.24 ± 3.1%、12.13 ± 2.1%、12.66 ± 1.7%, respectively. There was a statistical difference compared to the PBS group (21.59 ± 3.5%) ( P 0.05) between the free ENO1 monoclonal antibody group, the anti ENO1 single chain antibody group, and the 3-BrPA group. Transwell assay: Further detection of the effect of anti-ENO1 scFv on tumor migration and invasion using the Transwell assay. The results showed that when HeLa cells were treated with ENO1mAb, anti-ENO1 scFv, and 3-BrPA, the number of migrating cells was 177 ± 31, 283 ± 28, and 268 ± 18, respectively, and the difference was significant compared to the control group (514 ± 44) ( P < 0.01) (Fig. 5 C). This result indicates that anti-ENO1 scFv can significantly inhibit HeLa cell migration. Similarly, when HeLa cells were treated with ENO1mAb, anti-ENO1 scFv, and 3-BrPA, the number of invading cells was 264 ± 32, 481 ± 44, and 444 ± 30, respectively, and the difference was significant ( P < 0.01) compared to the control group (972 ± 40) (Fig. 5 D). These results confirmed that anti-ENO1 scFv can significantly inhibit HeLa cell invasion. Discussion Studies have found that ubiquitination down-regulation of ENO1 could significantly reduce the proliferation and invasive metastatic ability of cancer cells[23].During the initial phase of this laboratory's investigations, the ENO1 protein was expressed by baculovirus expression system , and the hybridoma technology was used to successfully prepare high potency monoclonal antibody. Subsequently, PLGA/FA-SS-PLGA nanoparticle mediated ENO1mAb was prepared, which can enter Hela cells through folate receptor-mediated endocytosis and inhibit cervical cancer cell invasion, proliferation, and clone formation[14]. The repeatability of the above experiment is poor in the later stage.To overcome the challenge of ENO1 antibody internalization into tumor cells, this study focused on the preparation of anti-ENO1 scFv. The selection of a suitable expression system for single-chain variable fragments (scFv) significantly influences their expression levels. In contrast to mammalian cell, Escherichia coli proliferates at a much faster rate, which considerably reduces the time required for the purification, analysis, and application of the expressed proteins[24].Consequently,E. coli is extensively employed for the production of exogenous proteins with single structural domains, non-glycosylated, molecular weight <50 KDa, soluble folded proteins or insoluble proteins[25].In the present investigation, anti-ENO1 scFv was expressed using an E. coli expression system, and SDS-PAGE analysis showed that anti-ENO1 scFv was soluble in expression.Our findings indicated that the yield was lower when expressing scFv using regular LB medium, which may be due to the inhibition of ENO1 enzyme activity by anti-ENO1 scFv, thereby affecting E. coli metabolism. Furthermore, extended induction periods with IPTG could result in protein degradation within the culture medium, contributing to a reduction in yields. Thus, replacing the regular LB medium and adding glucose to the medium to improve the energy metabolism efficiency of Escherichia coli, it was found that the expression yield was increased. High purity anti-ENO1 scFv was obtained using Ni–NTA chromatography. After co incubation of anti-ENO1 scFv with cervical cancer HeLa cells, the kit detection shown that the contents of lactic acid and pyruvate of glycolysis products decreased significantly. This demonstrates that anti-ENO1 scFv can directly inhibit the activity of the glycolytic enzyme ENO1 and exert an inhibitory effect on glycolysis. It has been found that overexpression of ENO1 can promote the proliferation, invasion and metastasis of tumor cells by regulating the FAK/PI3K/AKT signaling pathway and up-regulating the expression of the glycolysis-related gene LDHA, thus promoting glycolysis[26].It is suggested that the use of anti-ENO1 scFv may block ENO1 enzyme activity and thus exert anti-tumor effects. Research has indicated that a majority of tumor stem cells, including those associated with colon and breast cancers, exhibit an augmented Warburg effect, which facilitates their growth and sustains their stemness[27-29].Glucose uptake, lactate production, glycolytic enzyme expression, and ATP content were reported to be significantly increased in tumor stem cells, whereas inhibition of glycolysis or glucose deprivation led to a decrease in the cancer stem cell (CSC) population[30]. Regulation of glycolysis is critical for cancer stem cell differentiation and cancer biology[31]. Recently, many central metabolic enzymes, including glycolytic enzymes, have been identified to bind RNA in different cell types and organisms, One of these RNA-binding metabolic enzymes is ENO1, the catalytic activity of the ENO1 is directly regulated by RNAs leading to metabolic rewiring in mouse embryonic stem cells (mESCs)[32].Our study showed that anti-ENO1 scFv significantly inhibited the clone formation ability of HeLa cells, leading to speculation that anti-ENO1 scFv could inhibit cervical cancer stem cell population formation. Therefore, we speculate that anti-ENO1scFv inhibits ENO1 enzyme activity, leading to reduced RNA regulation and disrupting glycolysis and cancer stem cell differentiation.ENO1 has been reported to function as an oncogene in bladder cancer by regulating cell cycle and apoptosis[33].It has also been found that silencing of ENO1 induces apoptosis and inhibits proliferation and clone formation in breast cancer cells and lung adenocarcinoma cells[34, 35].In our study, we observed that higher concentrations of anti-ENO1 scFv significantly inhibited the proliferation of Hela cells, suggesting that anti-ENO1 may inhibit the proliferation of cervical cancer cells by inducing apoptosis. Research has demonstrated that, alongside its presence in the cytoplasm and nucleus, ENO1 is also localized on the surface of the cell membrane, functioning as a receptor for plasminogen[9], which subsequently binds to plasminogen.This interaction is activated through the action of urokinase-type plasminogen activator, facilitating the plasmin-mediated breakdown of extracellular matrix barriers and thereby aiding in cell adhesion and migration[36].In addition, epithelial mesenchymal transition (EMT) is closely related to tumor cell migration, invasion and metastasis. Several studies have shown that knockdown of ENO1 leads to restoration of E-calmodulin expression, which inhibits EMT and promotes tumor cell migration and invasion[37-39].The wound healing assay and Transwell assay in this study showed that anti-ENO1 scFv significantly inhibited the migration of cervical cancer cells, suggesting that anti-ENO1 scFv can inhibit the migration of tumor cells by blocking the activation of fibrinolytic enzymes through binding to the ENO1 receptor on the cell membrane, which may inhibit the invasive ability of Hela cells by inhibiting EMT. In summary, we prepared an effective anti-ENO1 scFv, which blocked the fibrinogen receptor highly expressed on the membrane of cervical cancer cells and inhibited the migration and invasion of tumor cells. Notably, the scFv was able to penetrate the cytoplasm directly, leading to the inhibition of glycolysis, and consequently, a decrease in the proliferation and colony-forming ability of cervical cancer cells. TThis study provides a novel biologic candidate for metabolic target-based precision treatment of cervical cancer. Declarations Competing Interests Patent:Human-Mouse Chimeric Antibodies against ENO1 and Single-Chain Variable Fragment targeting ENO1 and their applicationspatent applicant：Liu Huiling, Yang Rui, Chen Yuanyuan,Zhu Bingdong,Li Wen,Zhang Tingting, Dai Pengyu, Ma Xinyun,Nan Yang and Liu Xiaofeng Patent inventor： Gansu Provincial HospitalPatent application number：CN202311301662.8status of application: Invention publicspecific aspect of manuscript covered in patent application：The patent mainly relates to the preparation of anti-ENO1 scFv and some cell experiments. Author Contribution Rui Yang and Yuanyuan Chen.wrote the main manuscript text.Rui Yang,Bingdong Zhu,Yuanyuan Chen and Huiling Liu.Conceptualization.Rui Yang,Yuanyuan Chen and Xiaofeng Liu.Methodology.Pengyu Dai and Xinyun Ma.Date curation.Rui Yang and Tingting Zhang. prepared figures 1-5.Bingdong Zhu and Huiling Liu.Writing-review & editing.All authors critically reviewed the manuscript and agreed to submit for publication. Acknowledgement The author would like to thank Professor Bingdong Zhu from the Institute of Pathogen Biology, School of Basic Medical Sciences, and Lanzhou University for providing an experimental platform. Data Availability "GenBank accession number for nucleotide and protein sequences of anti-ENO1 scFv：BankIt2970929 Seq1 PV793678 and are available from the corresponding author on reasonable request."It should be emphasized that when storing nucleotide sequence information in the Genbank database, delayed disclosure is chosen, and currently only GenBank accession number are provided. References Weiderpass, A. M. et al. Estimates of incidence and mortality of cervical cancer in 2018: a worldwide analysis. Lancet Glob Health . 8 , e191–e203. https://doi.org/10.1016/s2214-109x(19)30482-6 (2020). Lin, F. L., Roden, K. Y., Hung, R. B. S., Wu, C. F. & TC Cervical Cancer Immunotherapy: Facts and Hopes. Clin. Cancer Res. 27 , 4953–4973. https://doi.org/10.1158/1078-0432.Ccr-20-2833 (2021). Du, K. G., Zeng, W., Wu, S. & X Advances in systemic treatment for recurrent metastatic cervical cancer. Br. J. Hosp. Med. (Lond) . 85 , 1–10. https://doi.org/10.12968/hmed.2024.0279 (2024). Chang, K. H. J., Koom, J. S., Lee, W. S., Kim, K. C., Kim, G. E. & YB Radiotherapy is a safe and effective salvage treatment for recurrent cervical cancer. Gynecol. Oncol. 151 , 208–214. https://doi.org/10.1016/j.ygyno.2018.08.029 (2018). Laversanne, B. F. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 74 , 229–263. https://doi.org/10.3322/caac.21834 (2024). Sui, L. B. & L Metabolic reprogramming in cervical cancer and metabolomics perspectives. Nutr. Metab. (Lond) . 18 , 93. https://doi.org/10.1186/s12986-021-00615-7 (2021). Ferri-Borgogno, C. M., Cappello, S., Novelli, P. & F α-Enolase: a promising therapeutic and diagnostic tumor target. Febs j. 278 , 1064–1074. https://doi.org/10.1111/j.1742-4658.2011.08025.x (2011). Cao, H. J. Y. et al. Upregulation of α enolase (ENO1) crotonylation in colorectal cancer and its promoting effect on cancer cell metastasis. Biochem. Biophys. Res. Commun. 578 , 77–83. https://doi.org/10.1016/j.bbrc.2021.09.027 (2021). Sanchez, A. F. A., Ortiz-Hernandez, T. W., Casiano, G. L. & CA Alpha-Enolase: Emerging Tumor-Associated Antigen, Cancer Biomarker, and Oncotherapeutic Target. Front. Genet. 11 , 614726. https://doi.org/10.3389/fgene.2020.614726 (2020). Ghosh, P. S., Kumar, S. & S Tumor glycolysis, an essential sweet tooth of tumor cells. Semin Cancer Biol. 86 , 1216–1230. https://doi.org/10.1016/j.semcancer.2022.09.007 (2022). Bos, N. D. X., Massagué, P. D. & J Metastasis: from dissemination to organ-specific colonization. Nat. Rev. Cancer . 9 , 274–284. https://doi.org/10.1038/nrc2622 (2009). Egelund, A. P. A., Petersen, R. & HH The plasminogen activation system in tumor growth, invasion, and metastasis. Cell. Mol. Life Sci. 57 , 25–40. https://doi.org/10.1007/s000180050497 (2000). Fragoso, A. N. D. A., Bobes, G., Laclette, R. J. & JP Plasminogen-binding proteins as an evasion mechanism of the host's innate immunity in infectious diseases. Biosci. Rep. 38 . https://doi.org/10.1042/bsr20180705 (2018). Li, G. Y. et al. ENO1 monoclonal antibody inhibits invasion, proliferation and clone formation of cervical cancer cells. Am. J. Cancer Res. 11 , 1946–1961 (2021). in LiverTox: Clinical and Research Information on Drug-Induced Liver Injury . National Institute of Diabetes and Digestive and Kidney Diseases: Bethesda (MD). (2012). Cao, L. H. & X Antibody variable region engineering for improving cancer immunotherapy. Cancer Commun. (Lond) . 42 , 804–827. https://doi.org/10.1002/cac2.12330 (2022). Prinslow, B. L. E. et al. Stapling scFv for multispecific biotherapeutics of superior properties. MAbs 15 , 2195517. https://doi.org/10.1080/19420862.2023.2195517 (2023). Song, B. J. & Y Engineering a cell-penetrating hyperstable antibody scFv(Ras) - An extraordinary approach to cancer therapeutics. Synth. Syst. Biotechnol. 6 , 343–350. https://doi.org/10.1016/j.synbio.2021.10.002 (2021). Hervé-Aubert, A. C. et al. Targeting HER2-breast tumors with scFv-decorated bimodal nanoprobes. J. Nanobiotechnol. 16 , 18. https://doi.org/10.1186/s12951-018-0341-6 (2018). Pardo, K. C. et al. Novel PSCA targeting scFv-fusion proteins for diagnosis and immunotherapy of prostate cancer. J. Cancer Res. Clin. Oncol. 143 , 2025–2038. https://doi.org/10.1007/s00432-017-2472-9 (2017). Srivastava, J. J. & P Strong synthetic stationary phase promoter-based gene expression system for Escherichia coli. Plasmid 109 , 102491. https://doi.org/10.1016/j.plasmid.2020.102491 (2020). Matyjaszczyk, C. M. et al. The Anticancer Drug 3-Bromopyruvate Induces DNA Damage Potentially Through Reactive Oxygen Species in Yeast and in Human Cancer Cells. Cells 9. https://doi.org/10.3390/cells9051161 (2020). Shen, D. T. et al. CCDC65 as a new potential tumor suppressor induced by metformin inhibits activation of AKT1 via ubiquitination of ENO1 in gastric cancer. Theranostics 11 , 8112–8128. https://doi.org/10.7150/thno.54961 (2021). Morales, R. G. L., Ceccarelli, E. S. & EA New tools for recombinant protein production in Escherichia coli: A 5-year update. Protein Sci. 28 , 1412–1422. https://doi.org/10.1002/pro.3668 (2019). Sivaccumar, S. A., Ruvo, J. P. & M Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments. Int. J. Mol. Sci. 21 . https://doi.org/10.3390/ijms21176324 (2020). Liu, F. Q. F. et al. Alpha-enolase promotes cell glycolysis, growth, migration, and invasion in non-small cell lung cancer through FAK-mediated PI3K/AKT pathway. J. Hematol. Oncol. 8 , 22. https://doi.org/10.1186/s13045-015-0117-5 (2015). Kim, C. Y. C. & JH Cancer stem cell metabolism: target for cancer therapy. BMB Rep. 51 , 319–326. https://doi.org/10.5483/bmbrep.2018.51.7.112 (2018). Deshpande, D. A., Arfuso, K., Newsholme, F., Dharmarajan, P. & A Cancer stem cell metabolism: a potential target for cancer therapy. Mol. Cancer . 15 , 69. https://doi.org/10.1186/s12943-016-0555-x (2016). Liu, Y. M., Huang, P. & P Cancer stem cells, metabolism, and therapeutic significance. Tumour Biol. 37 , 5735–5742. https://doi.org/10.1007/s13277-016-4945-x (2016). Merino, M. et al. The role of signaling pathways in cervical cancer and molecular therapeutic targets. Arch. Med. Res. 45 , 525–539. https://doi.org/10.1016/j.arcmed.2014.10.008 (2014). Perez-Perri, H. I. et al. Riboregulation of Enolase 1 activity controls glycolysis and embryonic stem cell differentiation. Mol. Cell. 82 , 2666–2680e11. https://doi.org/10.1016/j.molcel.2022.05.019 (2022). Castello, H. M. W., Schwarzl, A., Preiss, T. & T A brave new world of RNA-binding proteins. Nat. Rev. Mol. Cell. Biol. 19 , 327–341. https://doi.org/10.1038/nrm.2017.130 (2018). Correction: Up-regulated ENO1 promotes the bladder cancer cell growth and proliferation via regulating β-catenin. Biosci. Rep. 41 . https://doi.org/10.1042/bsr-2019-0503_cor (2021). Li, Z. J., Miao, H., Ding, L. & J Silencing of ENO1 inhibits the proliferation, migration and invasion of human breast cancer cells. J. buon . 25 , 696–701 (2020). Zhang, Z. J., Chen, S., He, Z., Xu, Z., Li, Y. & Z CircRNA-ENO1 promoted glycolysis and tumor progression in lung adenocarcinoma through upregulating its host gene ENO1. Cell. Death Dis. 10 , 885. https://doi.org/10.1038/s41419-019-2127-7 (2019). Molloy, S. R. G. et al. Proteomic identification of lynchpin urokinase plasminogen activator receptor protein interactions associated with epithelial cancer malignancy. J. Proteome Res. 6 , 1016–1028. https://doi.org/10.1021/pr060518n (2007). Jiang, M. Q. et al. The moonlighting function of glycolytic enzyme enolase-1 promotes choline phospholipid metabolism and tumor cell proliferation. Proc. Natl. Acad. Sci. U S A . 120 , e2209435120. https://doi.org/10.1073/pnas.2209435120 (2023). Borgoni, P. M. et al. Alpha-enolase (ENO1) controls alpha v/beta 3 integrin expression and regulates pancreatic cancer adhesion, invasion, and metastasis. J. Hematol. Oncol. 10 , 16. https://doi.org/10.1186/s13045-016-0385-8 (2017). Sun, H. C. K., Lv, Y., Ping, L. & Y ENO1 and Cancer. Mol. Ther. Oncolytics . 24 , 288–298. https://doi.org/10.1016/j.omto.2021.12.026 (2022). Additional Declarations Competing interest reported. Patent:Human-Mouse Chimeric Antibodies against ENO1 and Single-Chain Variable Fragment targeting ENO1 and their applications patent applicant：Liu Huiling, Yang Rui, Chen Yuanyuan,Zhu Bingdong,Li Wen,Zhang Tingting, Dai Pengyu, Ma Xinyun,Nan Yang and Liu Xiaofeng Patent inventor： Gansu Provincial Hospital Patent application number：CN202311301662.8 status of application: Invention public specific aspect of manuscript covered in patent application：The patent mainly relates to the preparation of anti-ENO1 scFv and some cell experiments. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6854783","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":479683115,"identity":"52e92456-a7ea-47ac-9fc3-42eb9973a1bd","order_by":0,"name":"Rui Yang","email":"","orcid":"","institution":"University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Yang","suffix":""},{"id":479683116,"identity":"288ff09a-b194-4539-b344-37dc454ff0ac","order_by":1,"name":"Yuanyuan Chen","email":"","orcid":"","institution":"University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Chen","suffix":""},{"id":479683117,"identity":"f7e04349-f365-4c9f-bbc0-c9ae1db38d4c","order_by":2,"name":"Xiaofeng Liu","email":"","orcid":"","institution":"University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiaofeng","middleName":"","lastName":"Liu","suffix":""},{"id":479683118,"identity":"5fedaef2-dfd8-4005-ad81-7d17c130c501","order_by":3,"name":"Pengyu Dai","email":"","orcid":"","institution":"University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Pengyu","middleName":"","lastName":"Dai","suffix":""},{"id":479683119,"identity":"582e03d6-084f-465b-b1f5-1566a4703816","order_by":4,"name":"Tingting Zhang","email":"","orcid":"","institution":"University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Zhang","suffix":""},{"id":479683120,"identity":"2d8979f6-6def-45d1-89b0-b772985803c4","order_by":5,"name":"Xinyun Ma","email":"","orcid":"","institution":"University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xinyun","middleName":"","lastName":"Ma","suffix":""},{"id":479683121,"identity":"e2be1758-0c0b-4ec3-b1d3-2d29a6460dba","order_by":6,"name":"Bingdong Zhu","email":"","orcid":"","institution":"Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Bingdong","middleName":"","lastName":"Zhu","suffix":""},{"id":479683122,"identity":"ed450c2e-3365-49c3-80c6-baf80f56bb58","order_by":7,"name":"Huiling Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYBACA2YGNiBpwcPAwP/9wwcDGztitUgAtTCYMc4oSEsmrIUBpIVBAsQ2Y+b5cIixgaAWdh6zxzwFEjLm/AvSHtsYHGBmYD98dAN+h/GYG/MAHWY548Fx4xyDO3wMPGlpNwhoMZMGaTG4cbBBOsfgGTODBI8ZsVoOM0hbGBxmbCBey/k2NmkG4rSwlUnOAdvCw2zYY5CWzEbIL/b9h7dJvPljY29w/gzjgx9/bOz42Q8fw6sFASQSIDQbccpBgP8A8WpHwSgYBaNgZAEArbo9Amwwr34AAAAASUVORK5CYII=","orcid":"","institution":"University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Huiling","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-06-09 13:08:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6854783/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6854783/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85993741,"identity":"418f84b7-368e-4e63-b0d0-b79674c04671","added_by":"auto","created_at":"2025-07-04 05:42:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":998086,"visible":true,"origin":"","legend":"\u003cp\u003eA. The schematic structure of anti-ENO1 scFv gene; B. Construction and identification results of anti-ENO1 scFv prokaryotic vector, a.PCR amplification product results, b. PCR product and plasmid double restriction result, c.PCR validation result of positive clone bacteria, d.Positive clone plasmid small extraction double enzyme digestion results, e.Positive bacterial liquid sequencing.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6854783/v1/b9a94b97c7cdc15ade77d0ef.png"},{"id":85993745,"identity":"8706dcc8-4bb2-4134-a52f-ce022eb59aba","added_by":"auto","created_at":"2025-07-04 05:42:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":167019,"visible":true,"origin":"","legend":"\u003cp\u003eInduced expression results under different conditions, A. shows the expression of glucose in LB medium with and without glucose,① empty bacteria ② LB supernatant ③ LB precipitation ④ LB+GS supernatant ⑤ LB+GS precipitation.B.16 ℃ and 0.5M IPTG induct 15 hours.C.16 ℃ and 0.5M IPTG induct 15 hours, and the arrow indicates a single chain antibody band. ① Empty bacteria ② whole bacteria ③ supernatant ④precipitation ⑤ Marker.D.SDS-PAGE analysis of anti-ENO1 scFv, where①⑦supernatant②Heteroprotein ③125mM④150mM⑤⑥Marker⑧-⑪are all 500mM imidazole gradient elution bands.E.WB dentification result of anti-ENO1 scFv.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6854783/v1/7002d2cbfc606ac17cd5ebbf.png"},{"id":85994009,"identity":"5b4abb5a-3bbb-459f-bff1-31033bf5ea97","added_by":"auto","created_at":"2025-07-04 05:50:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1124995,"visible":true,"origin":"","legend":"\u003cp\u003eA.The inhibitory effect of anti-ENO1 scFv on Hela cell proliferation. Different concentrations of anti-ENO1 scFv were co incubated with Hela cells at 37 °C for 16 hours, and the inhibitory effect of ScFV-ENO1 on Hela cell proliferation was evaluated by CCK-8 assay. The average soil SEM, n=3, ns \u003cem\u003eP\u003c/em\u003e\u0026gt;0.05, * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, * *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01;B and C.Observing the effect of anti-ENO1 scFv on Hela cell clone formation ability through plate cloning experiments. The average soil SEM, n=3, * *\u003cem\u003e P\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6854783/v1/38a42d4bd9891b4bce08a197.png"},{"id":85994007,"identity":"1cba6797-3898-40f1-8b74-9d4aacbc8cc7","added_by":"auto","created_at":"2025-07-04 05:50:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":132121,"visible":true,"origin":"","legend":"\u003cp\u003eAnti-ENO1 scFv inhibits glycolysis in Hela cells. Incubate anti-ENO1 scFv and 3-BrPA with Hela cells at 37 ° C for 24 hours,and determine the content of pyruvate and lactate using a glycolysis kit. A. Acetate content. B. Lactic acid content. Average soil SEM, n=5, * * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6854783/v1/ac3b1e96661b32c9d73b2bab.png"},{"id":85993749,"identity":"e9227deb-0910-42ac-b4ff-b4cd6ce3a613","added_by":"auto","created_at":"2025-07-04 05:42:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1871444,"visible":true,"origin":"","legend":"\u003cp\u003eInhibitory effect of anti-ENO1 scFv on the migration and invasion of cervical cancer HeLa cells. Wound healing experiments and Transwell assay were used to evaluate the effect of anti-ENO1 scFv on the migration and invasion of HeLa cells. The results of Wound healing experiments indicate that anti-ENO1 scFv can inhibit cell migration at both 12h and 24h. Average soil SEM, n=3, * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.5, * * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01(Fig. 5A,B).The results of Transwell assay indicate that anti-ENO1 scFv can significantly inhibit the migration and invasion ability of cells. Average soil SEM, n=5, *\u003cem\u003e P\u003c/em\u003e\u0026lt;0.01, * *\u003cem\u003e P\u003c/em\u003e\u0026lt;0.01, * * * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, * * *\u003cem\u003e P\u003c/em\u003e\u0026lt;0.0001(Fig. 5C,D)\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6854783/v1/c069f80d61acd35295d4a00b.png"},{"id":93650995,"identity":"b9e92233-c9cb-47c6-931b-42c715826cea","added_by":"auto","created_at":"2025-10-16 05:47:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5114505,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6854783/v1/0742b480-a8be-47ef-a04f-1a3a2d9277bf.pdf"}],"financialInterests":"Competing interest reported. Patent:Human-Mouse Chimeric Antibodies against ENO1 and Single-Chain Variable Fragment targeting ENO1 and their applications\npatent applicant：Liu Huiling, Yang Rui, Chen Yuanyuan,Zhu Bingdong,Li Wen,Zhang Tingting, Dai Pengyu, Ma Xinyun,Nan Yang and Liu Xiaofeng \nPatent inventor： Gansu Provincial Hospital\nPatent application number：CN202311301662.8\nstatus of application: Invention public\nspecific aspect of manuscript covered in patent application：The patent mainly relates to the preparation of anti-ENO1 scFv and some cell experiments.","formattedTitle":"Generation of ENO1 Single Chain Variable Fragment and Its Inhibitory effect on cervical cancer cells","fulltext":[{"header":"What does this study add to the clinical work","content":"\u003cp\u003eThe results of this study demonstrate that we have successfully prepared a single-chain antibody against ENO1. It can inhibit the migration, invasion and proliferation of cervical cancer cells. This study provides a new biological candidate drug for precise treatment of cervical cancer based on metabolic targets.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eCervical cancer is one of the three major cancers affecting women under 45 In developed countries, the five-year survival rate for cervical cancer patients ranges from 60\u0026ndash;70%[1], while in developing countries, this value is obviously reduced to one-third of that in developed countries[2]. Early-stage cervical cancer can be cured with surgery,chemoradiation or a combination of treatment modalities, advanced cervical cancer is often incurable and once recurrent,relatively refractory to treatment.When cervical cancer relapse after radiotherapy, systemic treatment is usually performed[3].Some patients can be saved through local treatments such as surgical resection and radiation therapy[4]. Late stage patients with stage IIB and above should be treated with radiotherapy and chemotherapy .In the United States, survival has not significantly improved for cervical cancer patients since the 1970s[5], demonstrating the urgent need to improve on current treatment approaches for cervical cancer.\u003c/p\u003e \u003cp\u003eMetabolic reprogramming represents a hallmark of cancer cell physiology, with the distinctive tendency of these cells to favor relatively energy-efficient glycolytic pathways over oxidative phosphorylation garnering considerable interest in the field of oncology[6]. During the process of glycolysis, numerous pivotal metabolic enzymes, notably Enolase 1 (alpha-enolase, ENO1), have been identified as being overexpressed or hyperactivated in cancerous cells. ENO1 functions as a multifunctional oncoprotein, serving several critical roles. Primarily, it functions as a key enzyme within the glycolytic pathway, playing an essential role in regulating cellular proliferation and tolerance to hypoxic conditions[7]. Additionally, ENO1 serves as a plasminogen receptor located on the cell membrane, thereby facilitating cancer cell proliferation, invasion, and migration[8]. Moreover, ENO1 is also found in the nucleus, where it modulates the expression of genes closely linked to tumorigenesis[9]. The resultant overproduction of lactic acid further contributes to the establishment of an acidic microenvironment, which is detrimental to the survival and functionality of adjacent immune cells[10]. The degradation of the extracellular matrix (ECM) is a pivotal process in tumor metastasis, necessitating the action of various proteolytic enzymes, including plasmin and matrix metalloproteinases (MMPs)[11]. Fibrinogen levels have been shown to correlate positively with tumor aggressiveness[12], and fibrinogen-binding proteins are integral in allowing tumor cells to evade innate immune responses[13]. Among the fibrinogen-binding proteins, ENO1 has emerged as a promising candidate for applications in tumor diagnosis, therapeutic intervention, and prognostic prediction[14].\u003c/p\u003e \u003cp\u003eIn our research laboratory, we previously generated an ENO1 monoclonal antibody utilizing hybridoma technology. Our findings indicate that this particular antibody possesses the capability to impede the proliferation, clonogenicity, invasion, and metastatic potential of cervical cancer cells. Nonetheless, the clinical application of monoclonal antibodies is fraught with challenges[14]. The foremost issue arises from their substantial molecular weight, which restricts their ability to traverse the cell membrane and penetrate the cytoplasm[15].And ENO1 involved in glycolysis is mainly located in the cytoplasm, so solving the problem of its entry into the cell is imminent.\u003c/p\u003e \u003cp\u003eIn recent years, molecular biology and genetic engineering technology have made it possible to construct antibody fragments, such as Single Chain Antibody Fragment (scFv,~30kda) and single domain antibody (sdAd)[16].The scFv is a recombinant protein formed by the combination of the variable regions of the heavy chain (VH) and light chain (VL) of antibodies, linked by a peptide linker[17].With the advantages of low molecular weight, good tissue permeability and weak immunogenicity, scFv has been widely promoted in the diagnosis and treatment of many clinical diseases[18].ScFv targeted therapy in tumors refers to coupling scFv with other effector molecules, such as toxins and viruses, and delivering them to the corresponding tumor cells, so as to enhance the tumor-killing ability of the effector molecules[16].Currently, it has been actively used in the research and treatment of breast cancer, prostate cancer and other diseases[19, 20].\u003c/p\u003e \u003cp\u003eIn this study, specific anti-ENO1 scFv was prepared on the basis of previous experiments. Preliminary cellular experiments showed that anti-ENO1 scFv inhibited glycolysis, proliferation, invasion, migration and cell clone formation of HeLa cells, suggesting that scFv-ENO1 has the ability to specifically block ENO1, and providing data support for further in vivo experiments.\u003c/p\u003e"},{"header":"Materials","content":"\u003cp\u003eHela cells,E. coli strainDH5a, E. coli strain BL21 (DE3), The pFastBacTMDual-EN01 single chain antibody strain is stored in the Institute of Pathogen Biology, School of Basic Medical Sciences, Lanzhou University. Expi293FTM cells were purchased from Thermo Fisher and kept in our laboratory. ExpiFectamine\u0026trade; 293 (A14527CN) transfection kit, DMEM medium were purchased from Thermo Fisher Scientific (USA). Lactic acid kit and pyruvic acid kit were purchased from Nanjing Jiancheng Biological Co., Ltd (Nanjing,China),2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonatobenzene)-2H-tetrazolium monosodium salt (CCK-8) kit, cell culture flasks and Transwell were purchased from Gansu Weibo Xin Bio-technology Co.,Ltd(Lanzhou, China). bicinchoninic acid(BCA) protein assay kit was from Beijing Solar bio Technology Co., Ltd (Beijing, China). Cell culture plates were obtained from Wuhan Xavier Biotechnology Co., Ltd (Wuhan, China). Matrigengel was purchased from Shanghai Nova Pharmaceutical Technology Co., Ltd (Shanghai, China).\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eMethods\u003c/h2\u003e\n \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\n \u003ch2\u003e1. Construction of anti-ENO1 scFv E. coli expression vector\u003c/h2\u003e\n \u003cp\u003eUsing pFastBacTM-Dual-ENO1 recombinant plasmid containing anti-ENO1 scFv gene as template, using F as forward primer and R as reverse primer, PCR reaction was carried out, and the specific conditions were as follows:The template was denatured at 94 C for 5 minutes, then 30 cycles of amplification were performed, each cycle was held at 94 C for 30 seconds, at 55\u0026deg;C for 1 minute, and finally extended at 72\u0026deg;C for 10 minutes (Table. 1).The amplified DNA fragment was purified, digested with restriction enzymes Nde I and HindIII, and then inserted into the pET-30a expression vector which had been digested with the same restriction enzymes.The final plasmid was named pET-30a/ENO1.The recombinant expression vector pET-30a/ENO1 was transformed into E. coli DH5\u0026alpha; cells. Subsequently, it was stored in glycerol at -80℃ for later use. The clone picked up from a solid Luria\u0026ndash;Bertani (LB) medium plate containing kanamycin (50mg/ml) was confirmed by DNA sequencing.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eForward primers (F)and reverse primer (R) harboring restriction enzymes Nde I/ HindIII sequence were used for cloning harboring anti-ENO1cscFv sequence to pET-30a expression vector by 3-step PCR.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"2\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePrimer\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePrimer sequence (5\u0026rsquo;\u0026ndash;3\u0026rsquo;)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026rsquo;-GGAATTCCATATGATGAGAGTGCTGATTCTTTTGTGGC-3\u0026rsquo; Nde I\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026rsquo;-CAAGCTTCTAGTGGTGGTGGTGGTGGTGTTTTATTTCCAACTTTGTC-3\u0026rsquo; HindIII\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003e2. Expression of anti-ENO1 scFv in E. coli\u003c/h3\u003e\n\u003cp\u003eENO1 is one of the key enzymes in the glycolysis pathway, considering that it may affect the metabolism of E. coli, we expressed anti-ENO1 scFv using normal LB medium and LB medium with glucose added to observe whether there is any difference in the expression yield.\u003c/p\u003e\n\u003ch3\u003e2.1.37℃, 0.5M IPTG induct for 4h\u003c/h3\u003e\n\u003cp\u003eAdd 100ug/ml kanamycin to LB liquid medium, and add E. coli BL21 cells carrying recombinant plasmids into LB liquid medium. Cultivate at 37\u0026deg;C for 4 hours. When the optical density (OD450) reaches 0.4\u0026ndash;0.6, add isopropyl \u0026beta;-D-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM to the culture medium to induce the expression of recombinant proteins. Subsequently, the culture was shaken at 180 rpm at 37\u0026deg;C for 4 hours.Harvest cells by centrifugation (8000 rpm, 20 minutes, 4\u0026deg;C) and then lysed by sonication. The cell lysate was then centrifugated at 4\u0026deg;C, 8000rpm for 30 min.\u003c/p\u003e\n\u003ch3\u003e2.2.16℃, 0.5M IPTG induct for 15 hours\u003c/h3\u003e\n\u003cp\u003eThe preliminary steps are as described above. When the optical density (OD450) reaches 0.4\u0026ndash;0.6, add isopropyl \u0026beta;-D-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM to the culture medium to induce the expression of recombinant proteins. Subsequently, the culture was shaken at 180 rpm at 16\u0026deg;C for 15 hours.The subsequent operation is the same as the previous steps. Analyze the expression of anti-ENO1 single chain antibody (scFv) using SDS-PAGE. SDS\u0026ndash;PAGE was performed using a 5% stacking gel and a 12% separating gel in 1%Tris\u0026ndash;glycine buffer.Subsequently, stain with 250 \u0026micro; L Coomassie Brilliant Blue.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e3. Purification of anti-ENO1 scFv\u003c/h2\u003e\n \u003cp\u003eSubsequently, the supernatant was collected and loaded into a Ni\u0026ndash;NTA column which had been previously equilibrated with the binding buffer (0.5M Na2HPO4, 0.5M NaH2PO4 and 0.5M NaCl). After thorough washing with binding buffer, the anti-ENO1 scFv was eluted using elution buffer (0.5M Na2HPO4, 0.5M NaH2PO4, 0.5M NaCl, and 10-500mM imidazole). The elution fractions containing anti-ENO1scFv were pooled and concentrated.The anti-ENO1 scFv obtained was validated using SDS-PAGE analysis.Finally, the product was desalted by dialysis against PBS buffer (0.01 M, pH 7.4) at 4\u0026deg;C for 16 h and stored at 80\u0026deg;C until use.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e4. The inhibitory effect of anti-ENO1 scFv on the proliferation of cervical cancer cells\u003c/h3\u003e\n\u003cp\u003eHeLa cells in the logarithmic growth phase were seeded in 96-well plates at a density of 5 \u0026times; 10⁴ cells/100 \u0026micro;L per well and incubated overnight in a culture incubator. HeLa cells were treated with graded concentrations of anti-ENO1 scFv and 3-BrPA, followed by 16-hour incubation. A negative control group was simultaneously established for comparative analysis.The original culture medium was replaced with DMEM supplemented with CCK-8 solution, followed by 2-hour incubation of the 96-well plate in a CO₂ incubator. Subsequently, optical density (OD) at 450 nm was measured using a microplate reader, and cell viability was calculated accordingly.\u003c/p\u003e\n\u003ch3\u003e5. Determination of glucose metabolites\u003c/h3\u003e\n\u003cp\u003eCells were seeded in 6-well plates at a density of 5 \u0026times; 10⁵ cells/well and incubated overnight under 5% CO₂ at 37\u0026deg;C. Dilute anti-ENO1 scFv (IC50 concentration) and 3-BrPA with DMEM without phenol red,and add appropriate concentrations of anti-ENO1 scFv and 3-BrPA to 6-well plates, respectively. Afterwards, place the culture plate in the incubator and continue to cultivate for 20 hours.Set up a blank group and a control group separately. Culture supernatants were collected post-incubation for quantification of pyruvate and lactate levels using commercial assay kits.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e6. Experiment of plate cloning formation\u003c/h2\u003e\n \u003cp\u003eHeLa cells were seeded in 6-well plates at graded densities (400, 600, and 1000 cells/well) to determine the optimal seeding density, followed by overnight incubation at 37\u0026deg;C with 5% CO₂. Cells were treated with anti-ENO1 scFv and 3-BrPA, with PBS-treated groups serving as negative controls. Continuous culture was maintained for 2\u0026ndash;3 weeks under standard conditions (37\u0026deg;C, 5% CO₂).The assay was concluded when macroscopically visible colonies (\u0026gt;\u0026thinsp;50 cells/colony) formed. Culture medium was aspirated. cells were Fixed with 4% paraformaldehyde (30 min, RT),Stained with 0.1% crystal violet (15 min, light-protected).Gently rinsed under running water to remove residual dye .Air-dried plates were imaged using a documentation system, and colony quantification was performed with ImageJ software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e7. Wound healing test\u003c/h2\u003e\n \u003cp\u003eCervical cancer HeLa cells were seeded in 6-well plates at 1\u0026times;10⁶ cells/well and incubated overnight at 37\u0026deg;C/5% CO₂ until reaching\u0026thinsp;~\u0026thinsp;100% confluency. A standardized wound was created using a sterile 200uL pipette tip, followed by PBS washing to remove dislodged cells.Next, dilute anti-ENO1 scFv and 3-BrPA with serum-free DMEM and add them to a 6-well plate. Incubate cells at 37\u0026deg;C and 5% CO2 for another 24 hours. Wound closure dynamics were documented at 0h(baseline), 12h, and 24h intervals using an inverted phase-contrast microscope (20\u0026times; objective). Wound areas were measured using ImageJ.And analyze the average migration rate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e8. Transwell assay\u003c/h2\u003e\n \u003cp\u003eCell Migration Assay: Add HeLa cells (5 \u0026times; 104) to the upper chamber of the Transwell chamber, and add 600ul of DMEM medium containing 15% FBS to the lower chamber of the Transwell chamber. Add homemade ENO1 monoclonal antibody (ENO1mAb), anti-ENO1 scFv, and 3-BrPA (15ug/ml) to the upper chamber, respectively. And incubating at 37℃ and 5% CO2 for 48 hours. Then, migrated cells were fixed with 4% paraformaldehyde (600 \u0026micro;L, 30 min) ,stained with 0.1% crystal violet (600 \u0026micro;L, 20 min),and washed with PBS to remove non migrating cells. Finally, five random fields per insert were imaged under a Nikon inverted microscope (100\u0026times; magnification) and count the number of migrating cells.\u003c/p\u003e\n \u003cp\u003eCell Invasion Assay: Thaw Matrigel\u0026trade; at 4\u0026deg;C overnight,dilute 1:8 (v/v) with serum-free DMEM,take 60ul Diluted Matrigel was added to the upper chamber of Transwell chamber (37\u0026deg;C, 5% CO2,3 h polymerization).Add HeLa cells (5 \u0026times; 104) to the upper chamber of the Transwell chamber, and add 600ul of DMEM medium containing 15% FBS to the lower chamber of the Transwell chamber. The Subsequent steps are the same as the cell migration experiment.The subsequent steps are the same as the cell migration experiment, including cell fixation, staining, and image acquisition.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e1. Construction of expression vector pET30a-ENO1 scFv\u003c/h2\u003e \u003cp\u003eThe anti-ENO1 scFv gene was amplified using sequence-specific primers, yielding a 903 bp product (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Construction and identification results of anti-ENO1 scFv prokaryotic vector(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).Positive clones were selected from LB agar plates containing 50 mg/L kanamycin and subjected to Sanger sequencing. Sequencing analysis confirmed successful construction of the recombinant pET-30a/ENO1 expression vector. The full-length ORF of the anti-ENO1 scFv was 903bp and it encoded a 298 amino acid peptide with a deduced molecular weight of 32 KDa(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).6xHis-tag at C-terminus of the recombinant protein was used for purification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2. Preparation of anti-ENO1 scFv\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.1. Expression of anti-ENO1 scFv in E. coli\u003c/h2\u003e \u003cp\u003eInduction parameters critically determine protein expression efficiency in prokaryotic systems[21].Compared with LB medium without glucose, the expression level of anti-ENO1 scFv was significantly increased in LB medium containing 2g/L glucose, and a specific protein band appeared at a molecular weight of about 32 kDa(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Induced at 37 ℃ for 4 hours, SDS-PAGE analysis showed that anti-ENO1 scFv was more expressed in bacterial precipitation(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). 16 ℃ induction for 15 hours resulted in a higher expression of single chain antibodies in the bacterial supernatant(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This result indicates that under the same concentration of IPTG induction, Low-temperature induction significantly facilitated soluble scFv secretion (\u0026gt;80% soluble fraction).Supernatant-derived antibodies maintained native conformation, enabling tag-based purification without denaturation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Anti-ENO1 scFv Purification \u0026amp; Validation\u003c/h2\u003e \u003cp\u003eThe culture supernatant containing anti-ENO1 scFv was purified via Ni- NTA chromatography. Stepwise elution with imidazole gradients (50\u0026ndash;500 mM) revealed optimal purity at 500 mM imidazole (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Target fractions dialyzed against PBS (1:30 v/v, 4\u0026deg;C, 24h),Final concentration: 180 \u0026micro;g/mL (BCA protein assay). Western Blot:Primary antibody: Mouse anti-His tag mAb (1:2000),Secondary antibody: HRP-conjugated goat anti-mouse IgG (1:5000),Specific band detected at 25\u0026ndash;35 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3. Anti ENO1 scFv Affects the growth of cervical cancer cells\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.1. Anti-ENO1 scFv inhibits the proliferation of HeLa cells\u003c/h2\u003e \u003cp\u003eIn this experiment, 3-BrPA served as the positive control for evaluating the inhibitory effect of anti-ENO1 scFv on HeLa cell proliferation, owing to its well-documented cytotoxic activity against most cancer cells[22].After co incubation of anti-ENO1 scFv and 3-BrPA at different concentrations (25\u0026ndash;150) ul with Hela cells for 16 hours, the inhibitory effect of anti-ENO1 scFv on Hela cell proliferation was detected using CCK-8. Compared with the blank control group, the survival rate of HeLa cells in the anti ENO1 single chain antibody group was significantly reduced in a dose-dependent manner. At a concentration of 150ug/ml, the proliferation rate of Hela cells was 64.33\u0026thinsp;\u0026plusmn;\u0026thinsp;7.506, which was significantly different from the blank control group (100%) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while there was no significant statistical difference compared to the proliferation rate of 67\u0026thinsp;\u0026plusmn;\u0026thinsp;12.12 in the 3-BrPA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This indicates that anti ENO1 scFv has a significant inhibitory effect on the growth of cervical cancer HeLa cells, and its effect is not significantly different from 3-BrPA at the same dose.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Plate cloning experiment\u003c/h2\u003e \u003cp\u003eTo verify whether inhibiting tumor cell glycolysis inhibits tumor cell proliferation and clone formation ability, we used plate clone formation experiments to detect the effect of anti-ENO1 single chain antibodies on Hela cell clone formation ability(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).The clone formation rates of the anti-ENO1 scFv and 3-BrPA groups were 36.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.085% and 30.05\u0026thinsp;\u0026plusmn;\u0026thinsp;5.445%, respectively, significantly lower than those of the PBS group (48.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.045%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).The results indicate that anti-ENO1 scFv has a certain inhibitory effect on the cloning ability of cervical cancer cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4. Determination of glucose metabolites\u003c/h2\u003e \u003cp\u003eThe small molecular weight of single-chain antibodies enables direct cellular internalization. Upon entering cervical cancer HeLa cells, anti-ENO1 scFv is hypothesized to inhibit cytoplasmic ENO1 activity. We assessed its impact on glycolysis using a glycolysis assay kit.3-BrPA and anti-ENO1 scFv were incubated with HeLa cells for 24 hours, respectively, and the pyruvate content in the anti-ENO1 scFv group and 3-BrPA group was 0.099\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0002, respectively \u0026micro; Mol/mL, 0.089\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0004 \u0026micro; Mol/mL, compared to the control group (0.164\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0002) \u0026micro; Mol/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The lactate content in the anti-ENO1 scFv group and 3-BrPA group was 13.89\u0026thinsp;\u0026plusmn;\u0026thinsp;2.02 mmol/gprot and 7.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54 mmol/gprot, respectively, significantly lower than the control group (18.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82 mmol/gprot) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and the lactate content in the 3-BrPA group was significantly lower than that in the ScFV-ENO1 group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These results confirm that anti-ENO1 scFv suppresses glycolysis in HeLa cells, though its efficacy is inferior to 3-BrPA at equivalent doses(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e5. Anti-Migratory and Anti-Invasive Effects\u003c/h2\u003e \u003cp\u003eWound healing assay: To investigate the effect of anti ENO1 single chain antibody on Hela cell migration ability, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. The analysis results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB. The free ENO1mAb, anti ENO1 scFv, and 3-BrPA groups all inhibited the migration of cervical cancer Hela cells within 24 hours, with average migration rates of 8.24\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1%、12.13\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1%、12.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7%, respectively. There was a statistical difference compared to the PBS group (21.59\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5%) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was no statistically significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) between the free ENO1 monoclonal antibody group, the anti ENO1 single chain antibody group, and the 3-BrPA group.\u003c/p\u003e \u003cp\u003eTranswell assay: Further detection of the effect of anti-ENO1 scFv on tumor migration and invasion using the Transwell assay. The results showed that when HeLa cells were treated with ENO1mAb, anti-ENO1 scFv, and 3-BrPA, the number of migrating cells was 177\u0026thinsp;\u0026plusmn;\u0026thinsp;31, 283\u0026thinsp;\u0026plusmn;\u0026thinsp;28, and 268\u0026thinsp;\u0026plusmn;\u0026thinsp;18, respectively, and the difference was significant compared to the control group (514\u0026thinsp;\u0026plusmn;\u0026thinsp;44) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). This result indicates that anti-ENO1 scFv can significantly inhibit HeLa cell migration. Similarly, when HeLa cells were treated with ENO1mAb, anti-ENO1 scFv, and 3-BrPA, the number of invading cells was 264\u0026thinsp;\u0026plusmn;\u0026thinsp;32, 481\u0026thinsp;\u0026plusmn;\u0026thinsp;44, and 444\u0026thinsp;\u0026plusmn;\u0026thinsp;30, respectively, and the difference was significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to the control group (972\u0026thinsp;\u0026plusmn;\u0026thinsp;40) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These results confirmed that anti-ENO1 scFv can significantly inhibit HeLa cell invasion.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eStudies have found that ubiquitination down-regulation of ENO1 could significantly reduce the proliferation and invasive metastatic ability of cancer cells[23].During the initial phase of this laboratory\u0026apos;s investigations, the ENO1 protein was expressed by baculovirus expression system , and the hybridoma technology was used to successfully prepare high potency monoclonal antibody. Subsequently, PLGA/FA-SS-PLGA nanoparticle mediated ENO1mAb was prepared, which can enter Hela cells through folate receptor-mediated endocytosis and inhibit cervical cancer cell invasion, proliferation, and clone formation[14]. The repeatability of the above experiment is poor in the later stage.To overcome the challenge of ENO1 antibody internalization into tumor cells, this study focused on the preparation of anti-ENO1 scFv.\u003c/p\u003e\n\u003cp\u003eThe selection of a suitable expression system for single-chain variable fragments (scFv) significantly influences their expression levels. In contrast to mammalian cell, Escherichia coli proliferates at a much faster rate, which considerably reduces the time required for the purification, analysis, and application of the expressed proteins[24].Consequently,E. coli is extensively employed for the production of exogenous proteins with single structural domains, non-glycosylated, molecular weight \u0026lt;50 KDa, soluble folded proteins or insoluble proteins[25].In the present investigation, anti-ENO1 scFv was expressed using an E. coli expression system, and SDS-PAGE analysis showed that anti-ENO1 scFv was soluble in expression.Our findings indicated that the yield was lower when expressing scFv using regular LB medium, which may be due to the inhibition of ENO1 enzyme activity by anti-ENO1 scFv, thereby affecting E. coli metabolism. Furthermore, extended induction periods with IPTG could result in protein degradation within the culture medium, contributing to a reduction in yields. Thus, replacing the regular LB medium and adding glucose to the medium to improve the energy metabolism efficiency of Escherichia coli, it was found that the expression yield was increased.\u003c/p\u003e\n\u003cp\u003eHigh purity anti-ENO1 scFv was obtained using Ni\u0026ndash;NTA chromatography. After co incubation of anti-ENO1 scFv with cervical cancer HeLa cells, the kit detection shown that the contents of lactic acid and pyruvate of glycolysis products decreased significantly. This demonstrates that anti-ENO1 scFv can directly inhibit the activity of the glycolytic enzyme ENO1 and exert an inhibitory effect on glycolysis. It has been found that overexpression of ENO1 can promote the proliferation, invasion and metastasis of tumor cells by regulating the FAK/PI3K/AKT signaling pathway and up-regulating the expression of the glycolysis-related gene LDHA, thus promoting glycolysis[26].It is suggested that the use of anti-ENO1 scFv may block ENO1 enzyme activity and thus exert anti-tumor effects.\u003c/p\u003e\n\u003cp\u003eResearch has indicated that a majority of tumor stem cells, including those associated with colon and breast cancers, exhibit an augmented Warburg effect, which facilitates their growth and sustains their stemness[27-29].Glucose uptake, lactate production, glycolytic enzyme expression, and ATP content were reported to be significantly increased in tumor stem cells, whereas inhibition of glycolysis or glucose deprivation led to a decrease in the cancer stem cell (CSC) population[30]. Regulation of glycolysis is critical for cancer stem cell differentiation and cancer biology[31]. Recently, many central metabolic enzymes, including glycolytic enzymes, have been identified to bind RNA in different cell types and organisms, One of these RNA-binding metabolic enzymes is ENO1, the catalytic activity of the ENO1 is directly regulated by RNAs leading to metabolic rewiring in mouse embryonic stem cells (mESCs)[32].Our study showed that anti-ENO1 scFv significantly inhibited the clone formation ability of HeLa cells, leading to speculation that anti-ENO1 scFv could inhibit cervical cancer stem cell population formation. Therefore, we speculate that anti-ENO1scFv inhibits ENO1 enzyme activity, leading to reduced RNA regulation and disrupting glycolysis and cancer stem cell differentiation.ENO1 has been reported to function as an oncogene in bladder cancer by regulating cell cycle and apoptosis[33].It has also been found that silencing of ENO1 induces apoptosis and inhibits proliferation and clone formation in breast cancer cells and lung adenocarcinoma cells[34, 35].In our study, we observed that higher concentrations of anti-ENO1 scFv significantly inhibited the proliferation of Hela cells, suggesting that anti-ENO1 may inhibit the proliferation of cervical cancer cells by inducing apoptosis.\u003c/p\u003e\n\u003cp\u003eResearch has demonstrated that, alongside its presence in the cytoplasm and nucleus, ENO1 is also localized on the surface of the cell membrane, functioning as a receptor for plasminogen[9], which subsequently binds to plasminogen.This interaction is activated through the action of urokinase-type plasminogen activator, facilitating the plasmin-mediated breakdown of extracellular matrix barriers and thereby aiding in cell adhesion and migration[36].In addition, epithelial mesenchymal transition (EMT) is closely related to tumor cell migration, invasion and metastasis. Several studies have shown that knockdown of ENO1 leads to restoration of E-calmodulin expression, which inhibits EMT and promotes tumor cell migration and invasion[37-39].The wound healing assay and Transwell assay in this study showed that anti-ENO1 scFv significantly inhibited the migration of cervical cancer cells, suggesting that anti-ENO1 scFv can inhibit the migration of tumor cells by blocking the activation of fibrinolytic enzymes through binding to the ENO1 receptor on the cell membrane, which may inhibit the invasive ability of Hela cells by inhibiting EMT.\u003c/p\u003e\n\u003cp\u003eIn summary, we prepared an effective anti-ENO1 scFv, which blocked the fibrinogen receptor highly expressed on the membrane of cervical cancer cells and inhibited the migration and invasion of tumor cells. Notably, the scFv was able to penetrate the cytoplasm directly, leading to the inhibition of glycolysis, and consequently, a decrease in the proliferation and colony-forming ability of cervical cancer cells. TThis study provides a novel biologic candidate for metabolic target-based precision treatment of cervical cancer. \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003ePatent:Human-Mouse Chimeric Antibodies against ENO1 and Single-Chain Variable Fragment targeting ENO1 and their applicationspatent applicant：Liu Huiling, Yang Rui, Chen Yuanyuan,Zhu Bingdong,Li Wen,Zhang Tingting, Dai Pengyu, Ma Xinyun,Nan Yang and Liu Xiaofeng Patent inventor： Gansu Provincial HospitalPatent application number：CN202311301662.8status of application: Invention publicspecific aspect of manuscript covered in patent application：The patent mainly relates to the preparation of anti-ENO1 scFv and some cell experiments.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eRui Yang and Yuanyuan Chen.wrote the main manuscript text.Rui Yang,Bingdong Zhu,Yuanyuan Chen and Huiling Liu.Conceptualization.Rui Yang,Yuanyuan Chen and Xiaofeng Liu.Methodology.Pengyu Dai and Xinyun Ma.Date curation.Rui Yang and Tingting Zhang. prepared figures 1-5.Bingdong Zhu and Huiling Liu.Writing-review \u0026amp; editing.All authors critically reviewed the manuscript and agreed to submit for publication.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe author would like to thank Professor Bingdong Zhu from the Institute of Pathogen Biology, School of Basic Medical Sciences, and Lanzhou University for providing an experimental platform.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003e\"GenBank accession number for nucleotide and protein sequences of anti-ENO1 scFv：BankIt2970929 Seq1 PV793678 and are available from the corresponding author on reasonable request.\"It should be emphasized that when storing nucleotide sequence information in the Genbank database, delayed disclosure is chosen, and currently only GenBank accession number are provided.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWeiderpass, A. 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K., Lv, Y., Ping, L. \u0026amp; Y ENO1 and Cancer. \u003cem\u003eMol. Ther. Oncolytics\u003c/em\u003e. \u003cb\u003e24\u003c/b\u003e, 288\u0026ndash;298. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.omto.2021.12.026\u003c/span\u003e\u003cspan address=\"10.1016/j.omto.2021.12.026\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ENO1, Single-Chain Variable Fragment, prokaryotic expression system, cervical cancer","lastPublishedDoi":"10.21203/rs.3.rs-6854783/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6854783/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnolase l (a-enolase, ENO1) is highly expressed in a variety of tumor cells, including cervical cancer. The eno1 monoclonal antibody could inhibit the invasion and migration of cervical cancer cells;however, it could not enter into cells. To overcome the limination, this study prepared ENO1 Single-Chain Variable Fragment (anti-ENO1 scFv) and validated its in vitro anti-cervical cancer cell effect to address the issue of antibody entry into cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirstly, the nucleotide sequence of anti-ENO1 scFv was inserted into pET-30a prokaryotic expression vector by restriction enzyme digest sites (Nde I and HindIII); Then,the expression was induced by isopropyl β-D- thiogalactoside(IPTG) in E.coli BL21(DE3) cells. Second,Ni–NTA chromatography was used for the purification, and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (WB). Thirdly, the anti-cervical cancer effect of anti-ENO1 scFv in vitro was studied through cell proliferation assay, colony formation assay, wound healing assay, and Transwell assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe recombinant plasmid pET30a-ENO1 scFv was successfully constructed. SDS-PAGE analysis showed that the expression of anti-ENO1 scFv could be located in the periplasmic space and extracellular space of BL21 and about 180 ug/mL purified anti-ENO1 scFv was obtained. Cell experiments showed that anti-ENO1 scFv could inhibit the activity of ENO1, significantly reduce the content of pyruvate and lactic acid, inhibit cell proliferation, invasion and migration, and clone formation of cervical cancer cells (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study demonstrated that anti-ENO1 scFv can specifically block the expression of ENO1 on the cell membrane and inhibit the activity of ENO1 glycolytic enzyme in tumor cells, and it is expected to become a potential anti-tumor drug for cervical cancer.\u003c/p\u003e","manuscriptTitle":"Generation of ENO1 Single Chain Variable Fragment and Its Inhibitory effect on cervical cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-04 05:42:24","doi":"10.21203/rs.3.rs-6854783/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cd35472c-1d26-497f-b7e6-633abff3bdc8","owner":[],"postedDate":"July 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50937356,"name":"Biological sciences/Biological techniques"},{"id":50937357,"name":"Biological sciences/Cancer"},{"id":50937358,"name":"Biological sciences/Immunology"}],"tags":[],"updatedAt":"2025-12-22T05:38:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-04 05:42:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6854783","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6854783","identity":"rs-6854783","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}