To generate polyclonal antibodies against gicerin, 10 female BALB/c mice (6–8 weeks old) were immunized with recombinant gicerin protein. The primary immunization was performed subcutaneously with 50 µg of recombinant rat gicerin protein (aa 1–545; accession number Q9EPF2-1) (29), which exhibits ~70% peptide-sequence homology to the human gicerin ortholog, emulsified in an equal volume of Complete Freund's Adjuvant (MilliporeSigma). Booster injections were administered on days 14 and 28 using an identical dose of antigen emulsified in Incomplete Freund's Adjuvant (MilliporeSigma). On day 42, mice were deeply anesthetized using isoflurane (induction at 3–5% and maintenance at 2–3% in oxygen) and the absence of reflex responses (pedal withdrawal and palpebral reflexes) was confirmed before proceeding. For terminal blood collection, cardiac puncture was performed under deep anesthesia using a 1 ml syringe with a 23-gauge needle. Blood (~0.5–1.0 ml) was withdrawn from the left ventricle. Following the procedure, sacrifice of the mice was performed by cervical dislocation while under deep anesthesia, or alternatively by thoracotomy to confirm the cessation of cardiac activity. Finally, mortality was verified by the absence of a heartbeat and fixed, dilated pupils. All animal procedures were conducted in accordance with institutional guidelines and were approved by the Animal Care and Use Committee of Kyoto Prefecture University (approval no. KPU240401). A total of 55 mice, including those reserved as backups, were used for antibody production and tumor cell transplantation in the present study.

Collected blood samples were allowed to clot at room temperature for 1 h prior to centrifugation at 1,500 × g for 10 min at 4°C to isolate serum. The sera from all mice were pooled and total IgG was purified using a Protein G Sepharose column (GE Healthcare) following the manufacturer's protocol. Bound IgG was eluted with 0.1 M glycine-HCl buffer (pH 2.7) and immediately neutralized with 1 M Tris-HCl (pH 9.0). The eluates were subsequently dialyzed against phosphate-buffered saline (PBS) (pH 7.4) and the IgG concentrations were determined spectrophotometrically. Similarly, serum was collected from non-immunized normal mice and IgG was purified using protein G affinity chromatography. The resulting antibodies were used as pre-immune IgG.

For the isolation of gicerin-specific antibodies, affinity purification was performed using a custom-made column prepared by covalently coupling recombinant gicerin protein to CNBr-activated Sepharose 4B beads (Cytiva). The purified IgG was subsequently loaded onto the affinity column equilibrated with PBS. After extensive washing, bound antibodies were eluted with 0.1 M glycine-HCl buffer (pH 2.7) and immediately neutralized, as aforementioned. The eluted antibodies were then dialyzed against PBS and protein concentrations were quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.). The final purified anti-gicerin antibodies were subsequently adjusted to a concentration of 1 mg/ml in PBS and stored at −80°C until use. The overview of antibody production and purification is shown in Fig. 1A.

Recombinant gicerin protein and mouse antibodies were separated by SDS-PAGE on 8% gels. Protein samples were mixed with Laemmli sample buffer, heated at 95°C for 5 min and loaded onto an 8% polyacrylamide gel. Electrophoresis was carried out under reducing conditions using a Mini-PROTEAN Tetra System (Bio-Rad Laboratories, Inc.) at a constant voltage of 100 V. After electrophoresis, the gel was briefly rinsed with distilled water and stained using Coomassie Brilliant Blue R-250 staining solution (Bio-Rad Laboratories, Inc.) for 1 h at room temperature. The gel was subsequently destained with a solution containing 40% methanol and 10% acetic acid until protein bands became clearly visible.

Each well of the polystyrene ELISA plates (Sumitomo Bakelite Co., Ltd.) was coated with 0.2 µg of recombinant gicerin proteins in PBS and the plate was incubated over night at 4°C. Each of the following incubation steps were preceded by washing the wells twice with PBS containing 0.05% Tween 20. The wells were blocked for nonspecific binding by the addition of a commercial blocking buffer (DS Pharma Biomedical Co., Ltd.) and were incubated at 37°C for 2 h. The serial dilutions of pre-immune IgG and anti-gicerin IgG were added vertically to the wells and kept for incubation at 37°C for 1 h. The HRP-conjugated rabbit IgG diluted (1:5,000) in PBS was dispensed into each well. The plate was incubated for 1 h at 37°C. Later, a substrate buffer containing TMB (Sumitomo Bakelite Co., Ltd.) was added to each well and kept for incubation at 37°C for 15 min. The reaction was terminated by the addition of a stopping reagent (1.25M sulfuric acid). The absorbance was recorded at 450 nm using the ELISA plate reader (DS Pharma Biomedical Co., Ltd.). The ELISA titer was defined as the highest dilution factor of anti-gicerin IgG that yielded an absorbance value at least three times higher than that obtained with pre-immune IgG.

Reaggregation assay and determination of IC₅₀ for anti-gicerin antibody: A reaggregation assay was performed to evaluate the inhibitory effect of anti-gicerin mouse IgG on cell-cell adhesion in gicerin transfected L929 cells (RIKEN BioResource Research Center; cat. no. RCB2619) (30). The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; high glucose) supplemented with L-glutamine, sodium pyruvate, 10% fetal bovine serum (FBS) and antibiotics (Nacalai Tesque, Inc.) and maintained at 37°C in a humidified atmosphere containing 5% CO₂. Then, the cells were detached using 0.02% EDTA in calcium- and magnesium-free PBS to obtain a single-cell suspension. The cells were then washed twice with calcium- and magnesium-containing Hank's Balanced Salt Solution (HBSS) and resuspended at a concentration of 1×10⁶ cells/ml. Serial dilutions of purified anti-gicerin mouse IgG (0, 0.01, 0.1, 1, 10 and 100 ng/ml) or control non-immune mouse IgG were prepared in HBSS. Equal volumes of antibody solution and cell suspension were mixed and transferred to 96-well U-bottom low-adhesion plates (100 µl/well). The mixtures were incubated at 37°C for 30 min to allow antibody binding. Subsequently, the plate was gently agitated on an orbital shaker (50 rpm) and incubated for an additional 45 min at 37°C to promote cell-cell reaggregation. Following incubation, the degree of cell aggregation in each well was assessed by phase-contrast microscopy. A cell aggregate composed of at least four cells was counted as a single unit for analysis (21). Images of representative fields (five per well) were captured at ×200 magnification. Aggregation was quantified by calculating the inhibition of cell aggregation (%), defined as follows: Inhibition of cell aggregation (%)=100 × (1-number of single cells/total number of cells).

NUGC-4 cells (JCRB Cell Bank; cat no. JCRB0834), HUVEC cells [JCRB Cell Bank; cat. no. IFO50271; F10 (passage 10)] and murine melanoma B16 cells (RIKEN BioResource Research Center; cat. no. RCB1283) were cultured in DMEM (high glucose) supplemented with L-glutamine, sodium pyruvate, 10% FBS and antibiotics (Nacalai Tesque, Inc.) and maintained at 37°C in a humidified atmosphere containing 5% CO₂.

Monolayers of NUGC-4 and HUVEC cells were fixed using Zamboni's fixative and incubated with a murine polyclonal anti-gicerin antibody (1:500; generated as aforementioned) at 37°C, followed by an FITC-conjugated goat anti-mouse IgG (1:100; MilliporeSigma; cat. no. AP124F) at 37°C. In addition, tumor tissues harvested from mice were fixed in Zamboni's solution and frozen sections (20 µm thickness) were subjected to immunofluorescence staining using the anti-gicerin primary antibody followed by the FITC-conjugated secondary antibody as aforementioned. Fluorescence microscopy revealed the distinct membrane-localized expression of gicerin on cells. A commercially available mounting medium containing DAPI was employed following the manufacturer's instructions. While nuclear staining was achieved, the combined display of phase-contrast, FITC (gicerin) and DAPI slightly reduced the clarity of cell contours in our setup; therefore, the final images are presented without DAPI to best illustrate the relationship between cell morphology and gicerin expression in the same field of view.

Cultured NUGC-4, HUVEC and B16 cells were rinsed twice with calcium- and magnesium-free Hanks' balanced salt solution and collected using a cell scraper. The harvested monolayer cells were subsequently homogenized in PBS using a Polytron homogenizer. The homogenates were centrifuged at 1,000 × g for 10 min at 4°C to remove cellular debris, followed by centrifugation of the supernatants at 18,000 × g for 30 min at 4°C. The resulting pellets were resuspended in 10 mM Tris-acetate buffer (pH 8.0) containing 1 mM EDTA and 0.5% Nonidet P-40 and incubated at 4°C for 1 h with continuous rotation. The suspensions were subsequently centrifuged at 40,000 × g for 90 min at 4°C and the final supernatants were collected for western blot analysis, as described by Taniura et al (31). Protein concentrations were quantified using BCA Protein Assay Reagent (Pierce; Thermo Fisher Scientific, Inc.) at an absorbance of 562 nm, with bovine serum albumin as a standard. Equal amounts of total protein (20 µg) extracted from HUVEC, NUGC-4 and B16 cells, along with 10 ng recombinant gicerin protein and mouse IgG, were separated by SDS-PAGE using 7.5% polyacrylamide gels and electrotransferred on to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Inc.). The membranes were blocked with Bullet Blocking One reagent (Nacalai Tesque, Inc.; cat. no. 1379) and subsequently incubated for 1 h at room temperature with primary anti-gicerin antibodies which were generated as aforementioned) diluted 1:1,000 in 2% skimmed milk in Tris-buffered saline (TBS) buffer (150 mM NaCl/10 mM Tris-HCl, pH 8.0). After six washes with TBST buffer (TBS buffer containing 0.05% Tween-20), the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG (Nacalai Tesque, Inc.; cat. no. 21860-61l) diluted 1:1,000 in TBS buffer containing 2% skimmed milk. Following four washes with TBST and two final washes with TBS, immunoreactive bands were visualized using 0.05% diaminobenzidine (MilliporeSigma) in 50 mM Tris-HCl (pH 7.6) containing 0.03% hydrogen peroxide for 5 min at room temperature. The reaction was stopped by rinsing in distilled water. Images of the membranes were captured using a ChemiDoc imaging system (Bio-Rad Laboratories, Inc.) as previously described (19,20).

Subcutaneous implantation of tumor cells into the cervical region of nude mice offers several advantages (32). The cervical area provides a soft and accessible site that facilitates precise and reproducible injection of the cell suspension, minimizing procedural variability. Tumors formed in this region tend to grow outward beneath the skin, allowing for easy visual assessment and accurate measurement of tumor size using calipers. Additionally, the relatively stable blood supply in the cervical skin reduces the risk of ulceration, thereby enabling long-term observation and reliable evaluation of therapeutic efficacy. Since the tumor remains localized in the cervical area, vital organs such as those of the gastrointestinal and urinary systems are less likely to be affected, contributing to the maintenance of the general condition of the animals and allowing for consistent assessment of drug effects and toxicity. Moreover, because cervical tumors rarely interfere with the mobility or feeding behavior of the animals, this model supports the ethical conduct of experiments while minimizing distress to the animals. After harvesting the NUGC-4 cells with trypsin-EDTA (Nacalai Tesque, Inc.), the cells were split into two groups. One group was incubated with anti-gicerin IgG (10 µg/ml), whereas the other was incubated with pre-immune IgG (10 µg/ml) for 1 h at 37°C. Cells were washed and resuspended in serum-free DMEM (3×10⁷ cells/ml) and 0.2 ml cell suspension was injected subcutaneously into the dorsal cervical region of nude mice (9-week-old female BALB/c Slc-nu/nu mice; n=5 per group). Tumor volume was calculated every other day starting at 1-week post-inoculation using the formula (AxB²)/2, where A and B represent the largest and smallest tumor diameters, respectively.

After the final measurement, mice were deeply anesthetized using isoflurane (induction at 3–5% and maintenance at 2–3% in oxygen) and the absence of reflex responses (pedal withdrawal and palpebral reflexes) was confirmed before proceeding with the experiment. Sacrifice was performed by cervical dislocation while the mice were in a state of deep anesthesia, or alternatively by thoracotomy to confirm the cessation of cardiac activity. Mortality was verified by the absence of a heartbeat and fixed, dilated pupils. In accordance with institutional animal care guidelines, the maximum allowable tumor volume was limited to 1.5 cm³. Mice were sacrificed immediately upon reaching this limit or upon exhibiting signs of pain, ulceration or impaired mobility; however, no animals exhibited such conditions during the course of the experiment.

Pathologically, the tumors were excised, 10% buffered-formalin-fixed for 48 h at room temperature, paraffin-embedded and stained with hematoxylin for 5 min at room temperature (22–25°C), followed by eosin staining for 3 min at room temperature [hematoxylin and eosin (H&E) staining] for a histopathological assessment of local invasion. For immunofluorescence staining, tumor tissues were fixed in Zamboni's fixative, followed by cryoprotection in 20% sucrose solution for 24 h at 4°C. The samples were subsequently frozen at −30°C and 20-µm frozen sections were prepared using a cryostat. The frozen sections were blocked with 1% non-fat dry milk in PBS containing 0.1% Tween-20 for 1 h at room temperature, and were subsequently incubated with a murine polyclonal anti-gicerin antibody (1:500; generated as aforementioned) at 37°C, followed by an FITC-conjugated goat anti-mouse IgG (1:100; MilliporeSigma; cat. no. AP124F) at 37°C as aforementioned. Fluorescence microscopy revealed the distinct membrane-localized expression of gicerin on cells.

NUGC-4 cells (2.5×10⁶ cells/ml) were pretreated with anti-gicerin antibodies or pre-immune IgG under the same conditions. Each mouse (n=5 per group) received a 0.1 ml tail-vein injection of either of the treated cell suspensions. After 1 week, mice were sacrificed and lungs were collected, formalin-fixed, paraffin-embedded and stained with H&E, as aforementioned. The number of metastatic foci was counted in all lung lobes and expressed as nodules per cm². In these experiments, mice were deeply anesthetized using isoflurane (induction at 3–5% and maintenance at 2–3% in oxygen) and the absence of reflex responses (pedal withdrawal and palpebral reflexes) was confirmed prior to the performance of any procedure. Sacrifice was performed as aforementioned and mortality was verified by the absence of a heartbeat and the presence of fixed, dilated pupils. In cases where mice exhibited clinical signs such as respiratory distress or lethargy, sacrifice was to have been performed immediately to minimize suffering; however, no animals exhibited such symptoms during the course of the experiment.

In vitro cell adhesion of NUGC-4 cells to gicerin proteins: Cell adhesion experiments were carried out as reported previously (21). Aliquots (1 ml) of PBS containing 10ng/ml of recombinant gicerin proteins were spotted on a 35 mm culture dish and incubated for 1 h in a humidified 5% CO₂ in incubator at 37°C. Then the dishes were washed with DMEM three times and preincubated with DMEM containing 10% FBS for 1 h to block nonspecific binding. Aliquots (2 ml) of the cell suspension (1×10⁶ cells/ml) were plated on the test dishes. To assess the inhibition of adhesion activity by anti-gicerin antibody, the cells were preincubated for 30 min at 37°C with the pre-immune IgG or anti-gicerin IgG at a final concentration of 100 ng/ml before plating on the dishes. The test dishes were incubated for 1 h at 37°C, washed twice with DMEM and the adhesion cells were observed by phase-contrast microscopy.

Endogenous gicerin-positive HUVEC cells, a vascular endothelial cell line, were cultured on dishes with DMEM containing 10% FBS at 37°C and used as feeder layers. The NUGC-4 cell suspensions were preincubated with anti-gicerin polyclonal antibodies or pre-immune IgG for 1 h at 37°C. After five washing in PBS and centrifugation at 800 × g for 10 min at 4°C, the NUGC-4 cells were resuspended in DMEM and seeded on to the HUVEC monolayers. Following a 30 min incubation at 37°C, the cultures were gently washed with DMEM and removed and the NUGC-4 cells adhering to the feeder layers were observed under a light microscope (Nikon Corporation). The cell number per area was determined for five areas and the average scores and standard deviation (SD) were calculated.

NUGC-4 cells were seeded into 6-well tissue culture plates at a density of 2×10⁴ cells per well in complete DMEM supplemented with 10% FBS. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂. After 4 h to allow for cell attachment, 10 µl of phosphate-buffered saline (PBS), 10 µl of pre-immune IgG, or 10 µl (10 ng/ml) of anti-gicerin IgG (10 ng/ml) was added to each well, respectively. For each treatment group, a total of four independent plates (n=4) were prepared and analyzed. At 0, 24, 48 and 72 h after seeding, one well from each plate was harvested. Cells were gently washed with PBS, detached using 0.25% trypsin-EDTA and resuspended in culture medium. Cell viability was assessed using trypan blue exclusion and viable cells were counted with a hemocytometer. Only unstained (viable) cells were included in the count.

Doubling time (T_d) was calculated using the following formula: Td=t⋅log(2)log(Nt/N0)

where t is the elapsed time (in h), N₀ is the number of viable cells at time 0 and Nt is the number of viable cells at time t. The mean doubling time were calculated for each treatment group. Statistical comparison was performed between the pre-immune IgG group and anti-gicerin IgG using Student's t-test.

NUGC-4 cells on culture dishes coated with gicerin protein (as aforementioned) were cultured in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere containing 5% CO₂ until they reached confluence. A straight scratch was made across the center of each well using a sterile yellow 200-µl micropipette tip to detach cells along the line. The medium was carefully aspirated to remove the detached cells and replaced with fresh serum-free DMEM. The cells were then treated with either pre-immune IgG (100 ng/ml) or anti-gicerin IgG (100 ng/ml) and incubated under the same conditions. Cell migration into the scratched area was monitored daily under a light microscope for up to 96 h.

NUGC-4 cells were cultured in DMEM supplemented with 10% FBS and seeded into 24-well culture plates (Corning, Inc.). Cells were subsequently incubated at 37°C in a humidified atmosphere containing 5% CO₂ until they reached semi-confluence. After removal of the culture medium, cells were gently rinsed with PBS. Subsequently, serum-free DMEM containing either pre-immune IgG or anti-gicerin polyclonal antibody was added to each well. The final antibody concentration was adjusted to 10 µg/ml. Cells were incubated at 37°C for 1 h. Following antibody treatment, cells were washed gently two or three times with PBS to remove any unbound antibodies. Fresh mouse serum (complement-active) or heat-inactivated mouse serum (treated at 56°C for 30 min) was subsequently diluted in serum-free DMEM to a final concentration of 20% and added to the cells. After a 1 h incubation at 37°C, the supernatant containing non-adherent cells was collected and temporarily maintained at 37°C, while the adherent cells were detached using trypsin-EDTA. The cell suspension and supernatant were mixed in the tubes and subsequently centrifuged at 300 × g for 5 min. The pellet was then resuspended in either PBS or serum-free DMEM. Cell viability was subsequently assessed using the trypan blue exclusion method. Equal volumes of cell suspension and 0.4% trypan blue solution were mixed and the mixture was loaded onto a hemocytometer. Viable (unstained) and non-viable (blue-stained) cells were counted under a light microscope and the cell death rate (as a percentage) was calculated using the following formula: Cell death rate (%)=number of blue-stained cells/total number of cells ×100. At least five independent wells were analyzed per treatment group.

All quantitative data are presented as mean ± standard deviation (SD). For in vivo experiments, n=5 mice per group; for in vitro assays, at least n=4 independent replicates per group unless otherwise indicated. Two-tailed, unpaired Student's t-tests were used for single pairwise comparisons and one-way analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) test was used for ≥3-group comparisons. Exact P-values are reported on the graphs and/or in the figure legends; where thresholds are shown, significance is denoted as *P<0.05, **P<0.01 and *P<0.001; ‘ns’ indicates P≥0.05. Error bars on all graphs represent SD and all ‘±’ values denote SD. P<0.05 was considered to indicate a statistically significant difference.

The procedure for generating and purifying the murine anti-gicerin antibody is summarized in Fig. 1A. BALB/c mice were immunized with recombinant gicerin protein, followed by IgG purification from serum. The IgG fraction was subsequently affinity-purified using a Sepharose column conjugated with gicerin protein. SDS-PAGE analysis revealed a single band at ~150 kDa in the final purified fraction, indicating that a highly pure IgG preparation was obtained. Furthermore, ELISA demonstrated that the purified antibody exhibited strong binding activity to gicerin protein, with an ELISA titer of 12,800 when compared to pre-immune IgG.

The neutralizing activity of anti-gicerin IgG was evaluated using a cell aggregation assay. When anti-gicerin IgG was added to the suspension culture of gicerin-expressing L929 cells, a marked inhibition of cell-cell aggregation was observed (Fig. 1Ba and Bb). Dose-response curves were generated by plotting the aggregation index against the logarithmic antibody concentrations (Fig. 1Bc). Nonlinear regression analysis determined that the half-maximal inhibitory concentration (IC₅₀) of anti-gicerin IgG was 49.5 ng/ml, indicating its potent neutralizing effect on cell adhesion.

Immunofluorescence analysis revealed the presence of distinct membrane-associated signals in NUGC-4 cells, confirming robust gicerin expression (Fig. 2A). As a limitation of this study, it was unable to perform co-localization staining with another inner membrane protein. This was due to the poor adherence of NUGC-4 cells, which made them susceptible to detachment during the multiple steps required for double immunofluorescence staining, such as repeated washing, antibody incubation and prolonged handling. Western blot analysis demonstrated that gicerin was expressed in the membrane fractions of B16 and HUVEC cells, both of which are known to endogenously express gicerin, similarly to the recombinant protein. In addition, gicerin expression was also detected in NUGC-4 cells (Fig. 2B). Notably, doublet bands were observed at ~120 kDa, indicating that NUGC-4 cells express both the S- and the L-isoforms of gicerin (19,20).

In the subcutaneous tumor model, tumor masses derived from cells treated with anti-gicerin antibody exhibited markedly suppressed tumor growth compared with the control group (P <0.01; Fig. 3A). Immunofluorescence analysis further revealed strong gicerin expression on the membranes of tumor cells and in the endothelial cells of newly formed blood vessels within tumors located in the cervicodorsal region of the implanted mice (Fig. 3B). Moreover, histopathological analysis revealed that tumors in the control group exhibited extensive invasion into the dermis, muscle and adipose tissue. In addition, epithelial-like arrangements of tumor cells were observed in the control group, thereby revealing histological features consistent with poorly differentiated adenocarcinoma. By contrast, tumors in the anti-gicerin antibody-treated group were more circumscribed and exhibited markedly reduced local invasiveness. Furthermore, frequent and prominent focal necrotic lesions were observed in tumors from animals administered the anti-gicerin antibody. Within these tumors, cellular degeneration, structural disintegration, pyknosis and karyorrhexis were also frequently noted (Fig. 3C).

In the tail-vein injection model, mice that received pre-immune IgG-treated cells were found to develop multiple tumor nodules in the lung parenchyma, often accompanied by hemorrhagic changes (Fig. 4). By contrast, mice injected with anti-gicerin antibody-treated cells exhibited markedly fewer metastatic lesions, indicating substantial inhibition of hematogenous spread.

To evaluate the role of gicerin in mediating cell adhesion, culture dishes were coated with recombinant gicerin protein and seeded with NUGC-4 cells. A substantial number of cells adhered to the gicerin-coated surface under control conditions. However, in the presence of anti-gicerin antibody, cell adhesion to the gicerin-coated areas was markedly inhibited, as evidenced by a significant reduction in the number of adherent cells (Fig. 5A). These findings demonstrated that the anti-gicerin antibody effectively neutralizes the homophilic binding activity of gicerin, thereby suppressing gicerin-mediated adhesion of NUGC-4 cells.

When HUVECs were used as a feeder layer and NUGC-4 cells were seeded, a substantial number of NUGC-4 cells adhered to the surface of HUVECs. Immunofluorescence staining subsequently revealed that gicerin was positively expressed in the monolayer of HUVEC cells, as well as in the NUGC-4 cells adhering to them (Fig. 5B). In this experiment, two-color staining was attempted to visualize HUVECs, which served as the feeder layer and the NUGC-4 cells adhered to them. However, successful fluorescence-based visualization could not be achieved. The reason for this was that the adhesion of NUGC-4 cells to HUVECs was weak and during the double-staining procedure, where gicerin on HUVECs was labeled red with rhodamine and gicerin on NUGC-4 cells was labeled green using a FITC-conjugated antibody, the NUGC-4 cells detached. Therefore, it was not possible to perform dual staining with different dyes. In this condition, NUGC-4 cells adhered to the surface of HUVECs with a small, rounded morphology. Based on this morphology and the observed fluorescence, both NUGC-4 cells and HUVECs were confirmed to express gicerin, as evidenced by the green fluorescence emitted by both cell types. Notably, the fluorescence intensity was stronger in NUGC-4 cells. These findings clearly demonstrate gicerin positivity in both cell types through their morphological and fluorescence characteristics.

The addition of an anti-gicerin antibody markedly decreased the number of NUGC-4 cells that adhered to HUVECs compared with the addition of pre-immune IgG, demonstrating that the anti-gicerin antibody could inhibit the adhesion of NUGC-4 cells to vascular endothelial cells (Fig. 5C).

To examine the involvement of gicerin in the migratory potential of NUGC-4 cells, an in vitro scratch assay were performed. Notably, NUGC-4 cells treated with pre-immune IgG exhibited markedly greater migration into the scratched area compared with those treated with anti-gicerin IgG (Fig. 5D). By day 4, the scratched area was completely closed in the pre-immune IgG group, whereas cell migration was markedly delayed in the group treated with anti-gicerin antibody. These findings suggested that gicerin expression facilitates the migration of NUGC-4 cells.

When pre-immune IgG was used, the addition of fresh serum did not induce cell death. By contrast, significant levels of cell death were observed when fresh murine serum was added to NUGC-4 cells preincubated with anti-gicerin IgG; however, no cell death occurred when heat-inactivated serum was used (Fig. 6A). Taken together, these results suggested that complement activity in the serum, engaged by the anti-gicerin IgG bound to NUGC-4 cells, is responsible for inducing cell death (Fig. 6B). The doubling time of NUGC-4 cells was 23.4 h. When pre-immune IgG was added, the doubling time was 24.7 h and when anti-gicerin IgG was added, it was 22.5 h (Fig. 6C). These findings suggest that the addition of anti-gicerin IgG does not affect the proliferation of gastric cancer cells: no significant difference between pre-immune IgG and anti-gicerin IgG groups; (P<0.05).