Establishment and characterization of a new human gallbladder cancer cell line, OCUG-2

Abstract

BACKGROUND

Gallbladder cancer (GBC) is a highly aggressive malignant tumor originating from the biliary tract. As one of the most common malignancies in the biliary system, GBC is particularly challenging due to its tendency to remain asymptomatic, which often results in delayed diagnoses even at advanced stages. Combined with its invasive potential and poor response to conventional therapies, GBC has a high mortality rate, highlighting the critical need for innovative therapeutic approaches. Identifying molecular biomarkers for early detection and discovering novel therapeutic targets might be essential to improving outcomes of patients with GBC.

AIM

To establish a novel GBC cell line to investigate the molecular mechanisms underlying GBC progression and evaluate potential therapeutic targets.

METHODS

We developed a unique GBC cell line, named OCUG-2, derived from a metastatic peritoneal implant, and verified its authenticity using short tandem repeat (STR) profiling. RT-PCR and RNA sequencing (RNA-seq) were performed to assess gene expression profiles, with functional enrichment analyzed through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The MTT cell proliferation assay and an invasion assay were performed to evaluate response to nine inhibitors. Immunohistochemistry (IHC) was conducted on 34 GBC samples to analyze insulin-like growth factor 1 receptor (IGF1R) expression.

RESULTS

OCUG-2 cells displayed adhesive growth with dendritic morphology and a 30-hour doubling time. Subcutaneous inoculation of OCUG-2 cells into mice confirmed their tumorigenic potential. STR analysis authenticated the cell line, and there was high mRNA and protein expression of IGF1R in OCUG-2 cells. The IGF1R inhibitor picropodophyllotoxin significantly inhibited OCUG-2 cell proliferation, yielding an IC₅₀ of 0.49 μM. RNA-seq analysis identified gene fusions, and GO/KEGG functional enrichment analyses revealed pathways implicated in cancer progression. IHC analysis showed IGF1R positivity in 18 of 34 GBC cases, with significant association between IGF1R expression and poor prognosis. In invasion assays, an IGF1R inhibitor effectively reduced OCUG-2 cell invasiveness.

CONCLUSION

IGF1R might be a promising target for GBC. The newly established OCUG-2 cell line serves as a valuable model for investigating the molecular mechanisms of GBC and evaluating therapeutic strategies.

Key Words: Gallbladder cancer; Cell line; Insulin-like growth factor 1 receptor; Molecular target; Mouse model

Core Tip: We established a new gallbladder cancer (GBC) cell line derived from a metastatic peritoneal implant of GBC. These cells, named OCUG-2, showed adhesive growth with a dendritic morphology. Doubling time of OCUG-2 cells was 30 hours. OCUG-2 cells were positive for insulin-like growth factor 1 receptor (IGF1R), and an IGF1R inhibitor reduced the proliferation of OCUG-2 cells. Immunohistochemistry found IGF1R positivity in 18 of 34 cases of GBC, and IGF1R expression was associated with poor prognosis. These findings suggest that IGF1R may be a promising target for GBC and OCUG-2 might be useful for analysis of GBC.

INTRODUCTION

Gallbladder cancer (GBC) accounts for approximately two-thirds of all biliary tract cancers[1]. Most early-stage GBC cases are identified incidentally by cholecystectomy performed for gallbladder stones, and they have relatively good prognosis. By contrast, patients with GBC diagnosed at a late stage show poor survival[1-3], possibly because these tumors present limited sensitivity to chemotherapy. The development of a novel therapy based on the biological behaviors of GBC is urgently being sought to improve the prognosis of late-stage GBC; however, the mechanisms responsible for the characteristic progression of GBC have not been clearly understood. While GBC cell lines would be useful in studying the biological behavior of GBC[4,5], the number of GBC cell lines remains small. To the best of our knowledge, only nine GBC cell lines, have been developed to date, namely OCUG-1, NOZ, TYGBK-1, TYGBK-8, G-415, EH-GB2, ZJU-0430, and TJGBC2[6-9].

Insulin-like growth factor 1 receptor (IGF1R), a tyrosine kinase receptor, is crucial for various biological functions such as cell proliferation, differentiation, and metabolism. Dysregulated expression of molecules in the IGF1R pathways has been detected across a spectrum of cancers, including brain tumors, head and neck cancer, breast cancer, and gastrointestinal cancer[10]. Despite promising preclinical evidence supporting the potential of IGF1R-targeted therapies as anticancer treatments, clinical trials have yet to yield definitive results[11]. Research on the expression of IGF1R in GBC is scarce, and the role of IGF1R-targeted therapies in GBC remains uncertain.

In this study, we established a new GBC cell line, OCUG-2, and introduce further evidence suggesting that IGF1R-targeted therapies hold promise for patients with GBC.

MATERIALS AND METHODS

Patient and cell culture

The cell line was isolated from metastatic peritoneal implants of GBC in a 78-year-old male. This tumor was cultured in Dulbecco's Modified Eagle Medium (DMEM; Fujifilm Wako Pure Chemical Co., Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Nichirei Biosciences, Inc., Tokyo, Japan), 0.5 mmol/L sodium pyruvate (Sigma-Aldrich, St. Louis, MO, United States), and a penicillin-streptomycin solution (Fujifilm Wako Pure Chemical Corporation). Adherent cell lines, identified within the tumor tissue culture, were termed OCUG-2. OCUG-2 cells were harvested using trypsin (Fujifilm Wako Pure Chemical Co.). The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂. The OCUG-2 cells had been maintained for over 24 months and had undergone more than 100 passages. All experiments utilized cells confirmed to be free from mycoplasma contamination. This study was approved by the Osaka Metropolitan University Ethics Committee (Reference No. 0924, 2022-110). Informed consent was obtained from the patient. This study was conducted according to the principles of the Declaration of Helsinki.

Tumorigenicity

A suspension of approximately 1 × 10⁷ OCUG-2 cells was subcutaneously inoculated into BALB/c nude mice (provided by Oriental Kobo, Osaka, Japan). After 6 weeks, tumor incidence was assessed. The subcutaneous tumors were fixed in 10% formalin for paraffin embedding and sectioning. All animal experiments were conducted in accordance with the guidelines of the Ethics Committee.

Growth kinetics

Fifty thousand OCUG-2 cells were seeded in 24-well plates with 1 mL DMEM containing 10% FBS, and cell numbers were counted every 24 hours for 11 days with the Coulter Counter Z2 (Beckman Coulter, Inc., Sykesville, MD, United States). The doubling time was determined from the growth curve.

Short tandem repeat analysis

To authenticate the cell line, short tandem repeat (STR) profiling was conducted by the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan).

RT-PCR

Total cellular RNA was extracted from OCUM-2 cells using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, United States). Subsequently, 0.4 μg RNA was reverse transcribed into cDNA using the ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). Then, the synthesized cDNA was amplified by PCR in 40 cycles, each cycle consisting of an annealing step at 60 °C for 30 seconds, an extension step at 72 °C for 60 seconds, and a final extension step at 72 °C for 7 minutes. PCR amplification was performed in a thermal cycler using AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific).

Growth inhibition assay

Twelve molecular target drugs were employed: Picropodophyllotoxin (IGF1R inhibitor; Sigma), CP-673451 (PDGFR inhibitor; MedChemExpress, Monmouth Junction, NJ, United States), afatinib (EGFR inhibitor; MedChemExpress), SU11274 (c-Met inhibitor; Selleckchem, Houston, TX, United States), infigratinib (FGFR1/2/3 inhibitor; MedChemExpress), FGF401 (FGFR4 inhibitor; Cayman Chemical Co., Ann Arbor, MI, United States), sapitinib (ERBB1/2/3 inhibitor; MedChemExpress), trastuzumab (HER2 inhibitor; Nippon Kayaku, Tokyo, Japan), ramucirumab (VEGFR2 inhibitor; Eli Lilly, Indianapolis, IN, United States), panitumumab (EGFR inhibitor; Takeda Pharmaceuticals, Tokyo, Japan), cetuximab (EGFR inhibitor; Merck, Rahway, NJ, United States), and bevacizumab (VEGF inhibitor; Pfizer Inc., New York, NY, United States). The growth effects of these drugs on OCUG-2 cells were assessed using the MTT cell proliferation assay (Dojindo, Kumamoto, Japan) after a 72-hour incubation in 96-well plates.

Invasion assay

Prior to initiating the Transwell assay, the membrane was pre-coated with Matrigel to simulate the extracellular matrix. Cells were subsequently suspended in serum-free DMEM and seeded into the upper compartment, which contained a membrane with 12-μm pores (Corning Inc., Corning, NY, United States). The lower compartment contained DMEM supplemented with 10% FBS, serving as a chemoattractant, along with nine molecular target drugs: Panitumumab, cetuximab, bevacizumab, ramucirumab, SU11274, infigratinib, FGF401, sapitinib, and trastuzumab. After incubation for 72 hours, cells that migrated to the lower compartment were stained with Diff-Quick (Sysmex Co., Kobe, Japan) and counted.

Immunohistochemistry

A total of 34 GBC tissue samples were obtained from Osaka Metropolitan University Hospital (Osaka, Japan), with all patients providing written informed consent. The tissue slides were subjected to heating for deparaffinization. After blocking the endogenous peroxidase activity, samples underwent overnight incubation at 4 °C with anti-IGF1R antibody (1:200; Santa Cruz Biotechnology, Dallas, TX, United States). This step was followed by incubation with a biotinylated secondary antibody and application of streptavidin-peroxidase complex. Counterstaining was performed using Mayer's hematoxylin. Immunohistochemistry (IHC) staining results were evaluated based on both the intensity and area of positive staining, with two independent pathologists conducting reviews independently. Positive cell percentages were scored as: < 5%, 0; 5%-25%, 1; 26%-50%, 2; 51%-75%, 3; and 76%-100%, 4. Staining intensity was graded as follows: 0 for no visible staining, 1 for pale yellow, 2 for tan, and 3 for brown. The final IHC score for each sample was calculated by multiplying the scores of positive cell percentage and staining intensity. A score of ≥ 6 was deemed indicative of positive expression.

RNA sequencing

Total RNA was extracted from OCUG-2 cells. Then, cDNA libraries were generated and sequenced on the Illumina NovaSeq 6000 platform (Illumina, Inc., San Diego, CA, United States), producing 150 bp paired-end reads. Clean RNA sequencing (RNA-seq) reads were mapped to the human reference genome (GRCh38) using the STAR aligner (version 2.7.10b), incorporating gene annotation data from the National Center for Biotechnology Information (NCBI, Bethesda, MD, United States) RefSeq database obtained from NCBI. Gene fusions were detected using STAR-Fusion (version 1.10.0) and Arriba (version 2.4.0). The results were obtained by taking the intersection of the findings from both tools.

Functional enrichment analysis

Biological functional enrichment was analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with the R package "ClusterProfiler", utilizing the “org.Hs.eg.db” package for human gene annotation. The cutoff criterion for significance was defined as P < 0.05.

Statistical analyses

Each experiment was carried out independently and in triplicate, except when noted otherwise. Data are presented as the mean ± SD. The graphs and statistical analyses were conducted using RStudio version 2023.06.0+421 and Python 2.7.3. Standard two-tailed unpaired student’s t-tests or Fisher's exact test were employed for statistical evaluations. P < 0.05 was considered statistically significant. The significance levels are denoted as follows: Not significant (NS; P > 0.05, ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001). The statistical methods used in this study were reviewed by Tomoya Sano, a biomedical statistician from Osaka Metropolitan University Graduate School of Medicine Department of Gastroenterological Surgery, and by Qiang Wang from Osaka Metropolitan University Graduate School of Medicine.

RESULTS

Primary gallbladder tumor and establishment of a new GBC cell line, OCUG-2

A computed tomography scan delineated the site of the primary gall bladder tumor (Figure 1A) from the patient whose metastatic peritoneal implants we harvested to establish the novel GBC cell line, OCUG-2. Histopathological analysis of the peritoneal tumor revealed it to be poorly differentiated (Figure 1B). The OCUG-2 cells did not require immortalization procedures. STR analysis demonstrated that approximately 70% of the loci matched those in STR profile databases, identifying OCUG-2 as a unique cell line originating from GBC (Supplemental Figure 1; Table 1). Under microscopic examination, OCUG-2 cells displayed a dendritic morphology (Figure 2A) and were found to proliferate with a doubling time of 30 hours (Figure 2B). Histological examination of the xenografted tumors following the subcutaneous injection of 1.0 × 10⁷ OCUG-2 cells into mice revealed a poorly differentiated tumor with moderate stromal cells (Figure 2C).

Figure 1 Computed tomography scan and histopathological findings of a patient with gall bladder cancer. A: Computed tomography (CT) findings of the primary tumor. CT scan image of the transverse and coronal planes display gallbladder carcinoma (arrows); B: Hematoxylin and eosin staining of the metastatic peritoneal tumor of patients with gallbladder cancer. The tumor showed poorly differentiated growth with moderate stromal cells.

Figure 2 Morphologic findings of OCUG-2 cells. A: Photomicrography of living OCUG-2 cells; B: Growth curve of OCUG-2 cells; C: Hematoxylin and eosin (H&E)-stained section of subcutaneous tumors induced by OCUG-2 cell injection in nude mice. Tumor histology was consistent with that of the original primary tumor, as shown by H&E staining. Scale bar, 100 μm.

Table 1 Short tandem repeat analysis.

Microsatellite (chromosome number)	OCUG-2 cells
D3S1358 (3)	16
TH01 (11)	7, 9
D21S11 (21)	30
D18S51 (18)	13
Penta E (15)	12
D5S818 (5)	12
D13S317 (13)	12
D7S820 (7)	9, 12
D16S539 (16)	9, 10
CSF1PO (5)	12
Penta D (21)	11
Amelogenin (X Y)	X
vWA (12)	16
D8S1179 (8)	13, 15
TPOX (2)	8, 12
FGA (4)	20, 21

Expression of growth factor receptors in OCUG-2 cells

In OCUG-2 cells, the mRNA expression of FGFR1, FGFR3, FGFR4, VEGFR2, PDGF-Rβ, EGFR, ERBB2, ERBB3, IGF1R, FMS-like tyrosine kinase 3, and c-Met was detected, whereas the expression of FGFR2, VEGFR1, VEGFR3, PDGFRα, ERBB4, c-Kit, and EGFR was not observed (Figure 3A).

Figure 3 Expression of growth factor receptors and growth kinetics. A: mRNA expression of growth factor receptors EGFR, HER2, ERBB3, ERBB4, PDGFR-α, PDGFR-β, insulin-like growth factor 1 receptor, FMS-like tyrosine kinase, FGFR1, FGFR2, FGFR3, FGFR4, VEGFR1, VEGFR2, VEGFR3, c-Kit, and c-Met; B: Growth curves of OCUG-2 cells treated with panitumumab, cetuximab, bevacizumab, ramucirumab, CP673451, picropodophyllin, SU11274, infigratinib, FGF401, afatinib, sapitinib, and trastuzumab. Growth of OCUG-2 cells was significantly decreased by picropodophyllotoxin.

Effects of molecular target drugs on the proliferation of OCUG-2 cells

Among the tested compounds, only the IGF1R inhibitor picropodophyllotoxin exhibited potent inhibitory effects on tumor growth, with an IC₅₀ of 0.49 μM in OCUG-2 cells. CP673451 and afatinib demonstrated moderate inhibition of OCUG-2 cell proliferation, with IC₅₀ values of 2.6 μM and 3.1 μM, respectively. The inhibitory effects of SU11274 (IC₅₀ = 10.5 μM), infigratinib (IC₅₀ = 10.6 μM), FGF401 (IC₅₀ = 32.7 μM), and sapitinib (IC₅₀ = 21.1 μM) on OCUG-2 cell growth were notably weak, while the remaining drugs exhibited no significant inhibitory effect at all (Figure 3B).

Effects of molecular target drugs on the invasion of OCUG-2 cells

In the invasion assays, nine molecularly targeted drugs were evaluated following their performance in growth inhibition assessments: Panitumumab, cetuximab, bevacizumab, ramucirumab, SU11274, infigratinib, FGF401, sapitinib, and trastuzumab. Of these, SU11274 and FGF401 demonstrated notable inhibitory effects on tumor invasion (Figure 4A). The inhibitory effects of infigratinib and sapitinib were comparably modest, while the other compounds appeared to exert no significant inhibitory effect on the invasion capabilities of OCUG-2 cells.

Figure 4 Invasion ability of OCUG-2 cells and insulin-like growth factor 1 receptor staining of gall bladder tumors. A: Effects of various targeted drugs on the invasion ability of OCUG-2 cells. Invasion ratio of OCUG-2 cells post-treatment compared to controls. Photograph (200 ×) of OCUG-2 invasion cells treated with control or SU11274; B: Immunohistochemical staining of insulin-like growth factor 1 receptor (IGF1R). IGF1R expression was found on cancer cell membrane. Scale bar, 100 μm. Kaplan-Meier survival analysis comparing overall survival and disease-free survival in IGF1R-positive and IGF1R-negative gallbladder cancer patients. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001.

Expression of IGF1R in 34 GBC tissues

Analysis of GBC tissues from the 34 patients revealed that 17 cases had positive IGF1R expression, while the other 17 were negative for IGF1R expression. Results of IHC facilitated the division of these patients into two distinct groups. Analysis of clinical data revealed no significant distinction between the groups with IGF1R-positive and IGF1R-negative expression (Table 2); however, Kaplan-Meier survival analysis demonstrated a significant difference (P = 0.038) in overall survival between patients with positive and negative IGF1R expression (Figure 4B). Univariate regression analysis for overall survival indicated that factors such as chemotherapy status and tumor-node-metastasis staging, together with IGF1R expression, were associated with the overall survival of patients with GBC, while multivariate regression analysis identified IGF1R expression as the sole factor significantly associated with overall survival (Table 3).

Table 2 Correlation between clinicopathologic features and insulin-like growth factor 1 receptor expression in gallbladder cancer.

Feature	IGF1R-positive (n = 17)	IGF1R-negative (n = 17)	P value
Sex
Male	6	10
Female	11	7	0.17
Chemotherapy
Yes	11	10
No	6	7	0.72
T stage
T1/T2	8	10
T3/T4	9	7	0.49
N stage
Negative	8	12
Positive	9	5	0.16
M stage
Negative	14	16
Positive	3	1	0.28
Clinical stage
I/II	6	10
III/IV	11	7	0.16

IGF1R: Insulin-like growth factor 1 receptor.

Table 3 Univariate and multivariate regression analyses of overall survival.

Feature	Univariate analysis		Multivariate analysis
	Hazard ratio (95%CI)	P value	Hazard ratio (95%CI)	P value
Sex	0.658 (0.259-1.672)	NS
Chemotherapy	3.039 (1.077-8.578)	0.03	1.969 (0.594-6.517)	NS
T	1.771 (1.165-2.692)	0.005
N	2.833 (1.2-6.687)	0.01
M	4.905 (1.516-15.88)	0.003
Stage	1.767 (1.146-2.723)	0.006	1.595 (0.950-2.679)	0.077
IGF1R expression	2.654 (1.102-6.913)	0.04	3.302 (1.208-9.023)	0.02

CI: Confidence interval; IGF1R: Insulin-like growth factor 1 receptor; NS: Not significant.

Transcriptomic profiling characteristics of OCUG-2 cells

RNA-seq of OCUG-2 cells yielded 133346877 paired sequences. SnpEff initially identified 509702 variants, and after stringent filtering to remove instances where the reference matched the alternative, indicating non-variants, a total of 507657 variants were retained for in-depth analysis. Among these, 311622 variants, accounting for 61.4%, were classified as known variants. The analysis further revealed 5186 multi-allelic Variant Call Format entries, underscoring a variant rate of one variant every 6083 bases and emphasizing the rich genomic variation within the OCUG-2 cells. The variant breakdown included 377093 single nucleotide polymorphisms, 69993 insertions, and 60571 deletions (Figure 5A). Among the characterized variant effects, 221030 (2.2%) were identified as having high impact, while 208325 (2.0%) exhibited low impact. Most of the effects, 95%, were categorized as modifiers, with a smaller fraction, 0.48%, being moderate. Missense mutations accounted for 47.1% of these effects. Additionally, nonsense mutations made up 1.1%, and silent mutations represented 51.9 of the total variant effects. Notably, the most prevalent nucleotide substitutions in OCUG-2 cells were A↔G and C↔T transitions (Figure 5B).

Figure 5 RNA-sequencing characterization of OCUG-2 cells. A: Circular graph depicting single nucleotide polymorphisms and indel variations in the OCUG-2 genome; B: Mutation spectrum of single nucleotide variants in the OCUG-2 genome; C: Gene Ontology analysis and the significantly enriched Kyoto Encyclopedia of Genes and Genomes pathways of the top 20 mutation genes selected based on q-values. GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Fourteen potential fusions were detected in OCUG-2 cells. Notably, protocadherin alpha 3 (PCDHA3)-PCDHAC2, ERC1-phosphodiesterase 3A, and PCDHGB2-PCDHGA8 fusions were identified as in-frame (Table 4). Specifically, the tetraspanin 14-AL392185.1 fusion spanned two chromosomes (chromosome 10 and chromosome 9) but was characterized as out-of-frame. The analyses of functional biological processes and KEGG pathway enrichments for mutated genes in OCUG-2 cells are presented in Figure 5C.

Table 4 Candidate gene fusions detected in RNA sequencing data of OCUG-2 cells.

Gene fusion	Breakpoint1	Breakpoint2	Confidence	Reading frame
LEPROT: LEPR	chr1:65425378	chr1:65565546	High	Out-of-frame
LEPROT: LEPR	chr1:65430048	chr1:65565546	Medium	Out-of-frame
TSPAN14: AL392185.1	chr10:80504778	chr9:95602159	High	Out-of-frame
TSPAN14: AL392185.1	chr10:80489314	chr9:95602159	High	Out-of-frame
TSPAN14: AL392185.1	chr10:80506862	chr9:95602874	Medium
TSPAN14: AL392185.1	chr10:80504778	chr9:95600165	Medium	Out-of-frame
RNF115: ITGA10	chr1:145823772	chr1:145907465	High	Out-of-frame
PCDHA3: PCDHAC2	chr5:140803591	chr5:140978949	High	In-frame
AC092807.3: DDAH1	chr1:85496166	chr1:85358847	High
AC092807.3: DDAH1	chr1:85494729	chr1:85358847	Medium
AC092807.3: DDAH1	chr1:85577984	chr1:85358847	Medium
ERC1: PDE3A	chr12:1141787	chr12:20556660	High	In-frame
TVP23C: CDRT4	chr17:15540433	chr17:15440285	High	Out-of-frame
PRR16: AC114284.1	chr5:120464645	chr5:120789954	High	Out-of-frame
PCDHGB2: PCDHGA8	chr5:141362556	chr5:141494807	Medium	In-frame
MYO9A: PKM	chr15:72117680	chr15:72219110	Medium
RASSF8: SSPN	chr12:26055446	chr12:26224293	Medium	Out-of-frame
RASSF8: SSPN	chr12:26055446	chr12:26179996	Medium	Out-of-frame
AC104123.1: CAST	chr5:96379652	chr5:96675539	Medium
MGST1: AC007529.2	chr12:16383604	chr12:16661766	Medium
LDLRAD3: PRR5L	chr11:36098461	chr11:36296290	Medium	Out-of-frame

DISCUSSION

We previously reported the establishment a GBC cell line named OCUG-1. In this study, we successfully established a novel GBC cell line named OCUG-2, derived from the metastatic peritoneal implants of a patient with GBC. OCUG-2 cells exhibit an adhesive growth pattern with a dendritic morphology. Subcutaneous tumors in mice inoculated with OCUG-2 cells displayed poor differentiation, which resembled the histopathological features of the original primary GBC, suggesting the validity of the OCUG-2 cell line as a representation of primary gallbladder tumors. STR analysis demonstrated that OCUG-2 did not match any STR profile databases of reported cell lines, indicating that it is an original cell line. These findings might suggest that OCUG-2 is a valuable and unique cell line for the characterization of GBC.

OCUG-2 cells showed high mRNA expression of IGF1R and positive expression of IGF1R. The IGF1R inhibitor picropodophyllotoxin significantly reduced the proliferation of OCUG-2 cells, with an IC₅₀ of 0.49 μM, suggesting that IGF1R is a key oncogene in OCUG-2 cells. IHC found IGF1R positivity in 18 of 34 GBC cases, and IGF1R expression was significantly associated with poor prognosis of patients with GBC. Multivariate regression analysis identified IGF1R expression as an independent prognostic factor. Kornprat et al[12] previously reported that IGF1R expression in 57 cases of GBC was correlated with lymph node metastases and hepatic metastases. These findings suggest that IGF1R signaling may be a promising target for GBC.

The amelogenin gene, located in the Xp22.1-Xp22.3 region of the X chromosome and the Yp11.2 region of the Y chromosome, is utilized in sex-typing tests. Observations across global populations have revealed variable rates of amelogenin on the Y chromosome (AMELY) dropout: 8.3% in Sri Lankans[13], 3.6% in Indian-Malays, 0.88% in Malays[14], and 0.02% in Australians[15]. Although the exact frequency of AMELY deletion in the Japanese population remains undetermined, occurrences have been documented[16]. Despite the common observation of Y nullisomy in various human malignancies[17], RNA-seq analysis in this study detected the expression of genes located on the Y chromosome in OCUG-2 cells, confirming the chromosome's presence. These findings suggest that the AMELY dropout observed in this study may be inherent to the patient from whom the OCUG-2 cell line was derived.

KEGG pathway analysis of mutated genes in OCUG-2 cells indicated human papillomavirus (HPV), Shigellosis, and Salmonella infections as three of the top four enriched terms. While HPV infection is reportedly linked with anogenital and oropharyngeal cancer[18], the association between HPV infection and GBC has not been documented. Kirkegård et al[19] conducted a cohort study involving 83008 women who underwent cervical conization, which served as an indicator of chronic HPV infection. The authors observed increased risks of certain types of cancer, including those of the gallbladder and biliary tract, with standardized incidence ratios of 1.3. Unlike the case with esophageal tumors[20,21], however, direct evidence connecting the presence of HPV DNA to GBC is still lacking. By contrast, an association of Salmonella infection with GBC has been confirmed[22,23]. Pathways such as MAPK/extracellular signal-regulated kinase, PI3K/Akt/mTOR, CREB/SP-1, and BSG (CD147) have been proposed to mediate Salmonella-induced gallbladder carcinogenesis[24]. In our study, the detection of protein serine/threonine kinase activity from GO analysis indicated that the PI3K/Akt, Ras/MAPK/mTOR, and protein kinase C signaling pathways may play pivotal roles in OCUG-2 cells.

CONCLUSION

In summary, we successfully established OCUG-2, a novel cell line derived from the peritoneal metastases of a patient with GBC. IGF1R signaling is presumed to be one of promising targets for OCUG-2 cells. Thus, OCUG-2 cells might be useful for the analysis of characteristic features of GBC.

ACKNOWLEDGEMENTS

We would like to acknowledge the Research Support Platform at Osaka Metropolitan University Graduate School of Medicine for providing the necessary infrastructure for our study.