Accutase (cat. no. A6964), heparin (cat. no. H3149), gelatin (cat. no. G2500), laminin (cat. no. L2020), and extracellular matrix (ECM) gel (cat. no. E1270) were sourced from Sigma-Aldrich. Basic fibroblast growth factor (bFGF, cat. no. CYT-218) and epidermal growth factor (EGF, cat. no. CYT-217) were acquired from Prospecbio. Trypsin (cat. no. SH30042.01), RPMI-1640 (cat. no. SH30027.01), DME/F-12 (cat. no. SH30023.01), and antibiotics (cat. no. SV30079.01) were obtained from HyClone. B-27 supplement (cat. no. 17504-044) and fetal bovine serum (FBS, cat. no. A56708-01) were sourced from Gibco. Antibodies for vimentin (cat. no. A11952), E-cadherin (cat. no. A11492), and N-cadherin (cat. no. A0433) were acquired from ABclonal. Antibodies for β-actin (cat. no. 4967), CD44 (cat. no. 37259), CD133 (cat. no. 64326), ALDH1A1 (cat. no. 12035), SOX2 (cat. no. 3579), NANOG (cat. no. 3580), OCT4 (cat. no. 2750), CypA (cat. no. 2175), CD147 (cat. no. 13287), STAT3 (cat. no. 9139), phospho-STAT3 (cat. no. 9145), AKT (cat. no. 9272), phospho-AKT (cat. no. 4060), ERK1/2 (cat. no. 9102), and phospho-ERK1/2 (cat. no. 9101) were all sourced from Cell Signaling Technology.

The AGS human gastric cancer cell line (KCLB no. 21739), sourced from the Korean Cell Line Bank (Seoul, South Korea), was maintained under specific conditions as follows: For adherent cell growth, RPMI-1640 medium containing 10% FBS and 1% antibiotics was utilized. Subculturing of adherent cells was performed using trypsin. For tumorsphere cell propagation, DME/F-12 medium containing 20 ng/ml bFGF, 20 ng/ml EGF, B-27 supplement, 5 µg/ml heparin, and 1% antibiotics was employed. Tumorsphere cells were subcultured by dissociating them with Accutase (13,15). The cells were incubated at a constant temperature of 37°C in a humidified environment with 5% CO₂.

CypA-specific siRNA (siCypA) was synthesized by Bioneer (Daejeon, South Korea). The sequences for siCypA were designed as follows: the sense sequence was 5′-GCUCGCAGUAUCCUAGAAU-3′ and the antisense sequence was 5′-AUUCUAGGAUACUGCGAGC-3′ (16). Non-targeting siRNA control was procured from Santa Cruz Biotechnology. To introduce the siRNAs into AGS GCSCs, dissociated AGS tumorsphere cells were seeded in culture plates using serum-free medium and transfected with siRNAs (100 nM) using Lipofectamine^™ 2000 (Invitrogen). Confirmation of CypA knockdown was carried out through Western blotting.

AGS GCSCs, either non-silenced or CypA-silenced, were plated at a quantity of 3×10³ cells per well in 96-well plates, using serum-free medium supplemented with bFGF and EGF, and cultured for 72 h. Tumorspheres with a diameter greater than 60 µm were detected and enumerated using an optical microscope. To assess cell proliferation, the CellTiter-Glo^® 2.0 Cell Viability Assay kit (Promega) was utilized. Luminescence signals were measured with a microplate reader (13).

AGS GCSCs, either non-silenced or CypA-silenced, were seeded at varying densities ranging from 5 to 200 cells per well in 96-well plates with serum-free medium containing bFGF and EGF. Following a 7-day incubation period, the presence and quantity of tumorspheres exceeding 60 µm in diameter in each well were examined using light microscopy (Olympus). To determine the rate of tumorsphere formation, data analysis was conducted using Extreme Limiting Dilution Analysis (ELDA) tool, accessible at http://bioinf.wehi.edu.au/software/elda/. This analysis was performed on November 16, 2023 (14).

AGS GCSCs, with or without CypA silencing, were cultured in 60-mm culture dishes at a quantity of 2×10⁵ cells per well in serum-free medium containing bFGF and EGF for 72 h. Following incubation, cells were collected and stained in accordance with the manufacturer's instructions, using either Muse^® Cell Cycle (cat. no. MCH100106) or Annexin V & Dead Cell reagent (cat. no. MCH100105) (Luminex). Subsequently, cell cycle distribution and the proportion of apoptotic cells were assessed using the Muse^® Cell Analyzer, operated with MuseSoft_V1.8.0.3 software (13).

Wounds were created using the ibidi culture insert system (ibidi GmbH). After placing a culture insert into each well of a laminin-coated 24-well plate, non-silenced or CypA-silenced AGS GCSCs (15×10⁴ cells/70 µl) were inoculated into each insert using serum-free medium containing bFGF and EGF. After a 24-h incubation for cell attachment, the culture inserts were removed. Afterwards, cell migration was monitored by light microscopy for 0, 2, 4, and 6 h, and the gap area was measured (16).

Cell invasion was evaluated utilizing a Transwell system equipped with polycarbonate membrane inserts with an 8.0 µm pore size (SPL Life Sciences, Pocheon, South Korea). Gelatin (1 mg/ml) and ECM gel (3 mg/ml) coatings were applied to the bottom and upper surfaces of the filter, respectively. AGS GCSCs, either non-silenced or CypA-silenced, were seeded at a quantity of 5×10⁴ cells per well in the filter chamber using serum-free culture medium containing bFGF and EGF and allowed to culture for 24 h. Following staining with hematoxylin and eosin, cells that had invaded were observed and quantified using an optical microscope (16).

Equal quantities of cell extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transfer to polyvinylidene difluoride membranes. The membranes were next incubated with 5% skim milk for blocking before being immunolabeled with primary antibodies, with dilutions ranging from 1:2,000 to 1:10,000. Detection of the immunolabeling was carried out using an enhanced chemiluminescence kit (DoGenBio, Seoul, South Korea), following incubation with secondary antibodies conjugated to horseradish peroxidase at a dilution of 1:3,000. Band intensities were quantified utilizing ImageJ 1.5 tool. Levels of expression were measured by comparing the ratio of each target protein relative to β-actin (13).

The CAM assay serves as a widely adopted in vivo model, valued for its speed, simplicity, and inherent immunodeficiency, particularly in assessing angiogenesis, tumorigenesis, toxicology, and drug delivery (17,18). In this study, non-silenced or CypA-silenced AGS GCSCs were utilized, with a total of 2×10⁶ cells per egg, mixed with ECM gel at a ratio of 40 µl per egg, and then transplanted into the CAM of fertilized eggs. Following transplantation, the eggs underwent incubation in a humidified environment for 7 days. At the end of this incubation period, the tumors that developed in the CAM were collected, and their weight and diameter were recorded (13).

The data are presented as the average ± standard deviation derived from a minimum of three separate experiments. Statistical evaluation was conducted using one-way ANOVA followed by Tukey's post-hoc test, employing SPSS version 9.0 software. A P-value below 0.05 denotes statistical relevance.

We have previously demonstrated that GCSCs can be selectively enriched from GC cell lines as tumorspheres under serum-free medium supplemented with bFGF and EGF (13,15). The stem-like characteristics of GC cells are accompanied by upregulation of stemness-related factors. AGS tumorsphere cells grown in these cancer stem cell culture conditions significantly overexpressed key GCSC markers, such as CD44, CD133, ALDH1A1, NANOG, SOX2, and OCT4, compared to AGS adherent cells cultured in 10% serum-supplemented conditions (Fig. 1A). These data indicate that AGS tumorsphere cells have stem-like properties, and the serum-free spheroid culture can enhance the expansion of the AGS-derived GCSC population. Furthermore, in AGS tumorsphere cells, the levels of CypA and CD147 expression were higher than in AGS adherent cells, indicating that the CypA/CD147 pathway might be crucial for the maintenance of GCSCs (Fig. 1A). Therefore, all experiments in this study using AGS GCSCs were performed under serum-free tumorsphere culture conditions selective for cancer stem cells.

To investigate the role of CypA in GCSCs, we performed genetic knockdown of CypA (gene name: peptidylprolyl isomerase A, PPIA) using RNA interference and then analyzed phenotypic changes. AGS-derived GCSCs were transfected with either siRNA targeting CypA (siCypA) or a non-targeting siRNA control. As depicted in Fig. 1B, the significant reduction of CypA expression by siCypA was confirmed by Western blotting.

Initially, we evaluated the influence of CypA knockdown on the proliferation of GCSCs derived from AGS cells through an ATP-monitoring luminescence assay. CypA knockdown demonstrated a notable inhibitory effect on AGS GCSC proliferation, as illustrated in Fig. 2A. Tumorsphere formation capability stands as a distinctive trait of cancer stem cells (5,6). Furthermore, silencing CypA resulted in a decrease in both the quantity and size of tumorspheres generated by AGS GCSCs, as depicted in Fig. 2B. The impact of CypA knockdown on the tumorsphere-forming ability of AGS GCSCs was further evaluated by limiting dilution assay (LDA). CypA-silenced AGS GCSCs exhibited a 3-fold lower frequency of tumorsphere formation than non-silenced control cells (Fig. 2C).

Next, we investigated whether inhibition of AGS GCSC proliferation by CypA knockdown was associated with regulation of cell cycle and apoptosis using flow cytometry. CypA knockdown increased G0/G1 and S phase cell populations and decreased G2/M phase cell populations in comparison to control cells (Fig. 2D). Additionally, reducing CypA levels led to an elevated percentage of apoptotic cells relative to the control group, as indicated in Fig. 2E. These findings underscore that silencing CypA not only restrains the proliferation and tumorsphere-forming capacity of GCSCs derived from AGS cells but also arrests the cell cycle at G0/G1 and S phases, along with inducing apoptosis. Consequently, these results suggest a favorable role for CypA in the proliferation and sustenance of GCSCs.

We further assessed whether CypA knockdown affects key metastatic functions such as migration and invasion of AGS-derived GCSCs. As shown in the results from the wound closure assay, CypA-silenced AGS GCSCs showed reduced migratory capacity compared to non-silenced control cells (Fig. 3A). In addition, Transwell invasion assay revealed that CypA knockdown markedly inhibited the invasiveness of AGS GCSCs (Fig. 3B).

The high metastatic capacity of GCSCs is closely associated with the promotion of epithelial-mesenchymal transition (EMT) (5,19–21). We investigated whether CypA silencing affects the expression of key EMT-related proteins in AGS GCSCs. Western blotting results showed that CypA knockdown increased the levels of the epithelial cell marker E-cadherin, while decreasing the levels of mesenchymal cell markers, including vimentin and N-cadherin (Fig. 3C). This indicates that CypA silencing downregulates EMT. Therefore, these results suggest that CypA may upregulate the metastatic ability of GCSCs by promoting EMT.

Accumulating evidence has shown that CypA/CD147 interaction promotes cancer cell proliferation, metastasis, stemness, and resistance to therapies via the activation of key downstream oncogenic signaling pathways (10,22–24). Thus, we examined whether CypA silencing affects the expression levels of CD147 and downstream signaling effectors mediated by the CypA/CD147 axis, including ERK1/2, AKT, and signal transducer and activator of transcription 3 (STAT3), in AGS-derived GCSCs. As a result, CD147 expression was significantly suppressed by CypA knockdown (Fig. 4A). Moreover, CypA silencing markedly inhibited the levels of phosphorylated STAT3, AKT, and ERK1/2 relative to their total protein levels (Fig. 4A). These results indicate that CypA knockdown downregulates CypA/CD147-mediated downstream signaling pathways by reducing CD147 expression in AGS GCSCs.

Next, we evaluated the impact of CypA knockdown on the expression of major stem cell regulators. As shown in Fig. 4B, CypA knockdown effectively suppressed the expression of key stem cell surface markers such as CD133 and CD44, the specific cytoplasmic stem cell marker ALDH1A1, and master transcriptional regulators of stem cells such as OCT4, SOX2, and NANOG in AGS GCSCs. Taken together, these data imply that CypA may enhance the stem-like features of GC by upregulating stemness-related signaling and regulators through interaction with CD147.

To analyze the impact of CypA knockdown on the in vivo tumor-forming potential of AGS-derived GCSCs, we performed a chick embryo chorioallantoic membrane (CAM) assay. Non-silenced control cells or CypA-silenced cells were mixed with ECM gel, implanted on the CAM surface, and cultured for 7 days. The weight and size of the developed tumor were then calculated. In the control group, tumors averaged 6.6 mg in weight and 3.2 mm in diameter, whereas tumors in the CypA knockdown group averaged 2.4 mg in weight and 2.0 mm in diameter (Fig. 5). These findings indicate that CypA silencing significantly suppressed in vivo tumor growth derived by AGS GCSCs. Therefore, CypA may play a crucial role in sustaining the tumorigenic ability of GCSCs.