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British Journal of Cancer logoLink to British Journal of Cancer
. 2024 Sep 20;131(9):1555–1566. doi: 10.1038/s41416-024-02859-5

Dual-modified antisense oligonucleotides targeting oncogenic protocadherin to treat gastric cancer

Mitsuro Kanda 1,✉, Yuuya Kasahara 2, Dai Shimizu 1, Takahiro Shinozuka 1, Masahiro Sasahara 1, Shunsuke Nakamura 1, Yohei Iguchi 3, Masahisa Katsuno 3,4, Yasuhiro Kodera 1, Satoshi Obika 5
PMCID: PMC11519331  PMID: 39304746

Abstract

Background

The objective of this study was to develop an innovative treatment strategy utilizing antisense oligonucleotides (ASOs) that target the gene encoding protocadherin alpha 11 (PCDHA11) and to elucidate the role of PCDHA11 in gastric cancer cells.

Methods

We designed and screened 54 amido-bridged nucleic acid (AmNA)-modified ASOs, selecting them based on PCDHA11-knockdown efficacy, in vitro and in vivo activity, and off-target effects. We assessed the impact of AmNA-modified anti-PCDHA11 ASOs on cellular functions and signaling pathways, and investigated the effects of Pcdha11 deficiency in mice.

Results

AmNA-modified anti-PCDHA11 ASOs significantly reduced the proliferation of gastric cancer cells and other solid tumors, whereas overexpression of PCDHA11 enhanced cell proliferation. The selected ASOs inhibited cellular functions related to the metastatic potential of gastric cancer cells, including migration, invasiveness, spheroid formation, and cancer stemness. Our findings revealed that AmNA-modified anti-PCDHA11 ASOs disrupted the AKT/mTOR, Wnt/β-catenin, and JAK/STAT signaling pathways. In mouse models of peritoneal metastasis (gastric and pancreatic cancer), systemic metastasis, and established subcutaneous tumors, administration of AmNA-modified anti-PCDHA11 ASOs inhibited tumor growth. ASO treatment induced reversible, dose- and sequence-dependent liver damage. Pcdha11-deficient mice demonstrated normal reproductive, organ, and motor functions.

Conclusions

AmNA-modified anti-PCDHA11 ASOs offer a promising therapeutic strategy for the treatment of gastric cancer and other solid malignancies.

Subject terms: Molecular medicine, Gastric cancer, Targeted therapies

Introduction

Gastric cancer is a significant global health concern, ranking as the fifth most prevalent cancer worldwide and the fourth leading cause of cancer-related mortality [1]. Despite aggressive surgical interventions, over 50% of patients undergoing curative resection experience disease recurrence in the form of metastatic disease [2]. Although systemic chemotherapy options for metastatic or recurrent gastric cancer have gradually expanded, primarily through the use of molecular targeted agents, therapeutic outcomes are limited by refractoriness and resistance [2–4]. Therefore, the development of novel therapeutic approaches is highly desirable.

Intercellular adhesion is crucial for maintaining epithelial tissue integrity and homeostasis [5]. During the initiation and progression of epithelial tumors, the deregulation of adhesion molecule expression, such as E-cadherin, disrupts epithelial structures and is associated with the epithelial-to-mesenchymal transition [6, 7]. Ongoing research into cadherin signaling in malignant tumors strongly suggests that molecular targeting represents a viable strategy for developing more effective therapies [8, 9]. Consequently, we conducted transcriptome and bioinformatics analyses of cell surface antigens and discovered that the gene encoding protocadherin alpha 11 (PCDHA11), a member of the cadherin superfamily of cell adhesion regulators [10], is specifically overexpressed in gastric cancer tissues with metastatic potential. While PCDHA11 physiologically functions to maintain neuronal connections in the brain, its role in malignant tumors remains unknown [11].

The exponential advancement of novel molecular targeting cancer therapies utilizing antisense oligonucleotides (ASOs) selectively targets RNA sequences. Anticipating the clinical application of ASOs, we developed a next-generation ASO modified by incorporating amido-bridged nucleic acids (AmNAs) with phosphorothioate-linked structures that bind to mRNAs with higher affinities [12]. AmNA is an artificial nucleic acid featuring an amide cross-linked sugar moiety [12]. It exhibits high double-strand formation ability, high nuclease resistance, and reduced hepatotoxicity [13]. The candidate modalities for molecular targeted cancer therapy include small molecule compounds, mid-molecular nucleic acid drugs, and high-molecular antibodies. Small molecule compounds often exhibit low target specificity and are prone to adverse events. Although antibody drugs possess high target specificity and can inhibit protein-protein interactions, there is no available evidence showing that the functional domain of PCDHA11 is stably presented outside the plasma membrane, posing a challenge for an antibody-based approach. The most fundamental goal of inhibiting protein function for disease treatment is to prevent the intracellular production of proteins, and nucleic acid drugs can be logically designed to match the target sequence. Therefore, we reasoned that ASOs are the ideal modality for efficiently inhibiting PCDHA11. In this study, we provide two lines of evidence elucidating the function of PCDHA11 in gastric cancer cells and demonstrate that AmNA-modified PCDHA11-specific ASOs show promise for treating gastric cancer and other solid malignancies.

Materials and methods

Transcriptome analysis

To investigate candidate molecules with crucial functions linked to metastatic potential, we performed global expression profiling on surgically resected primary gastric cancer tissues, adjacent noncancerous gastric mucosa, and hepatic metastatic tissues from four patients with gastric cancer and synchronous hepatic metastasis. This analysis was conducted using the HiSeq platform (Illumina, San Diego, CA, USA) [14].

PCDHA11 expression in cell lines and clinical samples

Quantitative reverse-transcription PCR (qRT-PCR) was conducted to quantify PCDHA11 mRNA expression using specific primers (Supplementary Table 1). Immunohistochemical (IHC) analysis was performed with a rabbit polyclonal PCDHA11 antibody (orb445197; biorbyt, Cambridge, UK) to examine 100 sections from patients with pStage II or III gastric cancer (approval # 2014-0043), as previously described [15]. The efficacy of PCDHA11 knockdown was assessed via western blotting, utilizing a rabbit anti-PCDHA11 polyclonal antibody (1:500 dilution, STJ193665, St John’s Laboratory, London, UK). External validation cohorts were generated using global cohort data from The Cancer Genome Atlas (TCGA), accessed through the open-source c-BioPortal (https://www.cbioportal.org/), and the Kaplan-Meier Plotter (http://kmplot.com/analysis/) [16].

Design and synthesis of AmNA-modified anti-PCDHA11 ASOs

The loop structure of PCDHA11 mRNA, to which ASOs bind with high affinity, was predicted using RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) and mfold (http://unafold.rna.albany.edu/?q=mfold) [17]. We designed 54 sequences of varying lengths, considering sequence identities with Mus musculus PCDHA11 mRNA and avoiding sequences associated with hepatotoxicity. AmNA-modified ASOs were synthesized and purified by Gene Design, Inc. (Osaka, Japan).

In vitro transfection using ASOs

Transfection of gastric cancer cell lines with ASOs was performed using the Ca2+ enrichment medium method [18]. Cells were cultured in a 24-well plate (5000 cells per well) and transiently transfected the next day with AmNA-modified ASOs (25–400 nmol/L) in 9 mM CaCl2.

Functional analyses

We used a Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) for measuring cell proliferation. We employed a wound-healing assay to measure cell migration and BioCoat Matrigel invasion chambers (BD Biosciences, Bedford, MA, USA) to assess cell invasiveness as previously described. Caspase activity was assessed using a Caspase-Glo 3/7 Assay System (Promega, Madison, WI, USA) according to the manufacture’s protocol. Apoptotic cells were visualized using an annexin V-Alexa Fluor 568 conjugate (A13202, Thermo Fisher Scientific, Waltham, MA, USA). Spheroid cultures were analyzed using PrimeSurface96U multiwell plates (Sumitomo Bakelite, Tokyo, Japan). Aldehyde dehydrogenase (ALDH) levels, a surrogate marker of stem/progenitor cells, were estimated using the ALDEFLOUR fluorescent reagent system (Stem Cell Technologies, Vancouver, BC, Canada). ALDH-positive cells were detected using a FACS Calibur system (BD Biosciences, Franklin Lakes, NJ, USA) [19]. We evaluated the adhesion of cancer cells to human primary mesothelial cells using a Vybrant Cell Adhesion Assay Kit (Thermo Fisher Scientific).

Forced expression and stable knockout of PCDHA11

To analyze the effects of PCDHA11 overexpression, KATO-III cells (1 × 105) were transfected with a PCDHA11 expression vector (RC215523; OriGene Technologies, Rockville, MD, USA) or a control empty vector (PS100001; OriGene Technologies) using a Neon electroporation system (Thermo Fisher Scientific). Genome editing was used to generate stable PCDHA11-KO HGC-27 cell lines, and edited sequences were confirmed using Sanger sequencing. The cell morphology of PCDHA11-stable knockout cells was compared to that of the parental HGC-27 cells utilizing the BZ-X810 imaging system (Keyence, Osaka, Japan).

Intracellular signaling analysis

To determine candidate signaling molecules, phosphorylation of 1006 unique sites among 409 proteins in cells was quantified using the PTMScan Direct Multi-Pathway Enrichment Kit (Cell Signaling Technology, Danvers, MA, USA). Protein expression and phosphorylation were assessed using a capillary electrophoresis method (Simple Western; ProteinSimple, San Jose, CA, USA) [17]. Antibodies used for this purpose are listed in Supplementary Table 2.

Mouse models

The Animal Research Committee of Nagoya University approved experiments using animals, performed according to ARRIVE guidelines (approval number M230102-002). First, we evaluated the effects of intraperitoneal administration of AmNA-modified anti-PCDHA11 ASOs in mouse models of peritoneal metastasis. Cells lines developed from gastric cancer (MKN1) or pancreatic cancer (AsPC-1) were implanted into the abdominal cavities of Nod-SCID mice (males, 6-weeks old). Mice (n = 4, each condition) were intraperitoneally injected once after implantation each week for 4 weeks with 500 μL of glucose, 0.6 mg (approximately 30 mg/kg) of hPCDHA11-3716 in the presence of 15 mM CaCl2. Second, parental HGC-27 (gastric cancer cell line) or stable PCDHA11-KO cells were implanted into the abdominal cavities of Nod-SCID mice (males, 6-weeks old), and their macroscopic appearance were observed 4 weeks after implantation. Third, the effects of 3 additional AmNA-modified anti-PCDHA11 ASOs (hPCDHA11-2255, -3319, and -3969; doses, 0.6 and 2.0 mg) were evaluated. Third, systemic mouse models of metastasis were established to evaluate the effects of ASOs on controlling hematogenous metastasis. The tail veins of Nod-SCID mice (males, 6-weeks old; n = 5, each condition) were injected with 1 × 106 of MKN45 cells (untransfected control, transfected ASO-NEG, or transfected hPCDHA11-3969, 400 nM). Mice were killed 6 weeks after implantation.

Last, we used mouse models of subcutaneous tumors to evaluate the effects of ASOs in mice harboring established tumors. HGC-27 cells (5 × 106 per injection) were subcutaneously injected into the both flanks of male nude mice (BULB/cSlc-nu/nu), 6-weeks old. When tumor volumes reached an average volume = 50 mm3, mice were randomized into groups (n = 3, 6 nodules each). Glucose (control); ASO-NEG; 0.6 mg of hPCDHA11-2255, -3319, or -3969 were intraperitoneally injected once weekly for 4 weeks in 15 mM CaCl2.

Toxicity of AmNA-modified anti-PCDHA11 ASOs

To assess the toxicity of ASOs, BALBcnu/nu mice (males, 6-weeks old, n = 4, each condition) underwent blood tests once each week for 2 weeks after intraperitoneal administration of 500 μL glucose; 0.2 mg NEG-ASO; and 0.2 mg, 0.6 mg or 2.0 mg of AmNA-modified anti-PCDHA11 ASOs (hPCDHA11-2255, -3319, or -3969) in 15 mM CaCl2. Blood tests were performed 3 weeks after treatment ceased. The appearance of the skin, food consumption, and body weights of each group were monitored. The mice were sacrificed 4 weeks after treatment initiation, and macroscopic and pathological characteristics of the liver, kidney, and brain were obtained.

In silico and microarray analysis of off-target effects of AmNA-modified anti-PCDHA11 ASOs

Sequence searches allowing for mismatches, insertions, or deletions were performed using GGGenome (https://GGGenome.dbcls.jp/) [20, 21]. Total RNAs extracted from HGC-27 cells transfected with mock, ASO-NEG, or AmNA-modified anti-PCDHA11 ASOs (hPCDHA11-2255, -3319 and -3969) were subjected to microarray analysis using a Whole Human Genome DNA microarray (Agilent, Santa Clara, CA, USA). The results of microarray and in silico analyses were combined to identify candidate genes potentially subject to off-target effects of AmNA-modified anti-PCDHA11 ASOs.

Generation of Pcdha11-deficient mice

The CRISPR/Cas9 system was used to generate Pcdha11−/− mice maintained under specific pathogen-free conditions at the Laboratory Animal Science of Nagoya University Graduate School of Medicine. Appearance, body weight, development of major organs, and blood tests were subsequently evaluated. The rotarod test (Economex Rotarod; Columbus Instruments, Columbus, OH, USA) was employed to assess motor ability and general motor coordination [22].

Statistical analysis

JMP 16 software (SAS Institute Inc., Cary, NC) was used for statistical analyses, and P < 0.05 indicates a significant difference between datasets.

Results

Transcriptome analysis identifies PCDHA11 as a candidate for molecular targeting

Transcriptome analysis was performed as the first step to list the candidate genes regulating the characteristics of refractory cancers that may serve as potential new therapeutic targets. Among 57,749 gene transcripts, global expression analysis identified 21 candidate genes exhibiting stepwise upregulation in primary gastric cancer tumors and their metastatic progeny compared with normal gastric tissues. One such gene encodes a transmembrane or extracellular matrix protein that may be involved in intercellular adhesion (Supplementary Table 3).

A literature review of gene functions and preliminary experiments directed our focus toward PCDHA11 for several reasons. Expression analysis using our institutional cohort of 230 patients, conducted to verify the reproducibility of transcriptome data from a small sample size (n = 4 for each group), revealed that elevated PCDHA11 expression in gastric cancer tissues correlated with poor prognosis. These findings were corroborated by external validation databases, including The Cancer Genome Atlas (TCGA) and Kaplan-Meier Plotter [16]. The oligonucleotide sequence of PCDHA11 is accessible at http://www.ncbi.nlm.nih.gov/. Notably, there have been no previous reports on the oncological roles of PCDHA11, rendering it a compelling target for drug discovery research. Our preliminary data indicated that increased PCDHA11 expression was observed in several solid tumors, and siRNA-mediated PCDHA11 knockdown inhibited the growth of gastric cancer cells.

PCDHA11 expression as a potential biomarker

The mean level of PCDHA11 mRNA was significantly higher in 300 gastric cancer tissues compared with those of the corresponding adjacent normal tissues (Fig. 1a). Patients were classified into high or low PCDHA11 expression groups according to the median value of PCDHA11 mRNA levels in primary gastric cancer tissues. High PCDHA11 expression significantly associated with pathological T4, undifferentiated tumor type, pathological invasive growth, presence of peritoneal metastasis, and advanced disease stage (Supplementary Table S4). The high-PCDHA11 group experienced shorter overall and disease-free survival times than the low-PCDHA11 group (Fig. 1b and Supplementary Fig. 1A). Similar results were obtained using two external validation datasets (Fig. 1b). Multivariable analysis identified high PCDHA11 expression as an independent prognostic factor for overall survival (Supplementary Table 5). The high- PCDHA11 group suffered a greater prevalence of overall and peritoneal recurrences compared with the low- PCDHA11 group (Supplementary Fig. 1B).

Fig. 1. Clinical relevance of PCDHA11 expression.

Fig. 1

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a Gastric cancer tissues expressed higher mean levels of PCDHA11 mRNA than corresponding normal adjacent tissues. *P < 0.05 between the two groups. b Prognosis and PCDHA11 expression in the institutional, TCGA, and Kaplan-Meier-plotter cohorts. c Immunohistochemical analysis of PCDHA11 expression in stage II or III gastric cancer. PCDHA11 expression at the primary cancer component was associated with greater frequency of disease recurrence. *P < 0.05 between the two groups. d qRT-PCR analysis of PCDHA11 mRNA in the indicated human gastric cancer cell lines and representative results of PCR array showing positive correlations between PCDHA11 and IL1RN, SNAI1, TGFB1, and ZEB1 mRNA.

Figure 1c shows representative sections with positive or undetectable PCDHA11 expression. Among 100 patients with stage II or III gastric cancer, 52 expressed PCDHA11 at the primary cancer component. The incidence of postoperative recurrences was significantly higher in the PCDHA11-positive group compared with that of the PCDHA11-negative group (14.6% and 55.8%, respectively) (Fig. 1c).

PCDHA11 expression and pcr analysis of gastric cancer cell lines

qRT-PCR analysis of 14 human gastric cancer cell lines revealed that PCDHA11 mRNA was differentially expressed compared with the control normal epithelial cell line FHs74 (Fig. 1d). Levels of snail family transcriptional repressor 1 (Snai1), transforming growth factor beta 1 (Tgfb1), and zinc finger E-box binding homeobox 1 (Zeb1) mRNAs correlated positively with those of PCDHA11 mRNA, whereas there was an inverse correlation between the levels of interleukin 1 receptor antagonist (Il1rbn) and PCDHA11 mRNAs (Fig. 1d).

Design and screening of AmNA-modified anti-PCDHA11 ASOs

Our anti-PCDHA11 ASOs included flanking regions of AmNA-modified nucleotides, and all phosphate groups were phosphorothioated (Fig. 2a). We designed 54 sequences (Supplementary Table 6). To screen for optimal ASOs, we compared their abilities to inhibit PCDHA11 mRNA expression in gastric cancer cell lines. Thus, we treated MKN1 cells (expressed high levels of PCDHA11 mRNA) with 400 nM each of candidate ASOs (Supplementary Fig. 2A). First, hPCDHA11-2382 and -3716 were selected for further testing. Concentration-dependent knockdown efficacies of those two ASOs were achieved in MKN1 cells (Fig. 2b).

Fig. 2. Effects of candidate ASOs, hPCDHA11-2382 and -3716, on knockdown of PCDHA11 mRNA expression, cellular functions of cancer cells and in vivo tumor growth.

Fig. 2

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a Structures of AmNA-modified anti-PCDHA11ASOs. b Concentration-dependent knockdown efficacy of PCDHA11 mRNA expression by hPCDHA11-2382 and -3716. c The proliferation rate of gastric cancer cells was attenuated by hPCDHA11-2382 and -3716. *P < 0.05 between ASO-NEG and AmNA-modified anti-PCDHA11 ASOs at Day 6. Invasiveness (d) and migration (e) of gastric cancer cell lines were attenuated when transfected with hPCDHA11-2382 and 3716. *P < 0.05 between ASO-NEG and AmNA-modified anti-PCDHA11 ASOs. f Intraperitoneal administration of hPCDHA11-3716 in mouse models of peritoneal metastasis of gastric and pancreatic cancers. In both models, 4 weeks administration of hPCDHA11-3716 inhibited the growth of peritoneal nodules. *P < 0.05 between ASO-NEG and hPCDHA11-3716. Error bars indicate the standard deviation.

In vitro and in vivo inhibitory effects of hPCDHA11-2382 and -3716 on the aggressiveness of cancer cells

hPCDHA11-2382 and hPCDHA11-3716 inhibited the proliferative potential of MKN1 (differentiated gastric cancer, Fig. 2c) and SW1116 (colon cancer, Supplementary Fig. 2B) cells. Alterations of functions associated with the metastatic potential of gastric cancer cells such as migration and invasiveness were determined using the candidate ASO-transfectants. The invasiveness and migration of HGC-27 cells transfected with hPCDHA11-2382 or hPCDHA11-3716 were significantly decreased compared with that of the untransfected control and NEG-ASO cells (Fig. 2d, e).

We used MKN1 (gastric cancer) and AsPC-1 (pancreatic cancer) cell lines to compare the effects of intraperitoneal administration of hPCDHA11-3716 (0.6 mg) compared with that of mock-transfected cells and cells transfected with ASO-NEG. After 4-weeks treatment, macroscopic observations revealed few peritoneal nodules in the hPCDHA11-3716 groups, whereas the numbers and sizes of tumor nodules on the omentum and mesenteric tissues increased in mice treated with vehicle and ASO-NEG both in MKN1 and AsPC-1 cells (Fig. 2f). Quantitative analysis revealed inhibition of tumor growth in mice treated with hPCDHA11-3716 compared with those treated with vehicle and ASO-NEG both in MKN1 and AsPC-1 cells (Fig. 2f).

Evaluation of in vitro and in vivo inhibitory effects of hPCDHA11-2255, hPCDHA11-3319, and hPCDHA11-3969

In search of ASOs with higher activity, we decided to test another three candidates, hPCDHA11-2255, 3319, and -3969. Target sites for hPCDHA11-3969 was identified according to the predicted loop structure of PCDHA11 mRNA (Fig. 3a). Concentration-dependent knockdown efficacies of those three ASOs were confirmed in HGC-27 cells (Fig. 3b). Inhibition of proliferation was observed in poorly differentiated gastric cancer (HGC-27), pancreatic cancer (AsPC-1), esophageal squamous cell carcinoma (TT), breast cancer (MCF7 and SKBR3), and lung adenocarcinoma (H1975) cells transfected with hPCDHA11-2255, -3319, and -3969 (Fig. 3c and Supplementary Fig. 3A). Furthermore, the levels of PCDHA11 protein expression in cells treated with hPCDHA11-2255, 3319, and -3969 were significantly reduced (Fig. 3d).

Fig. 3. Design and influence of hPCDHA11-2255, -3319 and -3969 on PCDHA11 mRNA and protein expression, in vitro and in vivo cancer cell growth.

Fig. 3

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a The predicted loop structure of hPCDHA11-3969. b Concentration-dependent knockdown efficacy of PCDHA11 mRNA expression by hPCDHA11-2255, -3319 and -3969. c The proliferation rate of gastric cancer cells was attenuated by hPCDHA11-2255, -3319 and -3969. *P < 0.05 between ASO-NEG and AmNA-modified anti-PCDHA11 ASOs at Day 6. d Confirmation of knockdown of PCDHA11 protein expression by hPCDHA11-2255, -3319 and -3969. e Intraperitoneal administration of hPCDHA11-3319 and -3969 attenuated the growth of peritoneal metastasis of gastric cancer cells. *P < 0.05 between ASO-NEG and AmNA-modified anti-PCDHA11 ASOs. f Knockdown of PCDHA11 expression decreased the formation of systemic metastasis of gastric cancer cells injected into the tail vein. Microscopic findings are shown (400× magnification). g Treatment effects of AmNA-modified anti-PCDHA11 ASOs in mouse subcutaneous xenograft models of gastric cancer. *P < 0.05 between ASO-NEG and AmNA-modified anti-PCDHA11 ASOs at day 28. Error bars indicate the standard deviation.

The in vivo antitumor effects of hPCDHA11-2255, -3319, and -3969 were evaluated using mouse models of intraperitoneal metastasis. Only sparse peritoneal nodules were observed in engrafted mice treated with 0.2 mg of hPCDHA11-3319 and -3969 (Fig. 3e). Upon increasing the ASO dose to 0.6 mg, hPCDHA11-2255, -3319, and -3969 demonstrated a significant inhibitory effect on the formation of peritoneal nodules (Supplementary Fig. 3B). Subsequently, we investigated the impact of PCDHA11 knockdown on the establishment of systemic metastasis. Based on prior reports and our preliminary experiments, we utilized MKN45 cells, from which mouse models of systemic metastasis can be consistently established via intravenous tail injection of cancer cells. One mouse in the ASO-NEG group succumbed during the study, and six weeks after cancer cell implantation, the PCDHA11 knockdown group exhibited fewer tumors metastasized to the lungs and skin compared to untransfected controls and the ASO-NEG-transfected group (Fig. 3f). We further explored whether ASO administration exerted a therapeutic effect when initiated after tumor formation. HGC-27 cells were subcutaneously implanted in mice, and those with a nodule of ≥50 mm³ were randomly assigned to treatment groups. The growth rates of subcutaneous tumors were attenuated by weekly administration of hPCDHA11-3319 and -3969 (Fig. 3g).

Target specificity of hPCDHA11-2255, -3319, and -3969

We overexpressed PCDHA11 in KATO-III cells, which expressed low levels of endogenous PCDHA11 mRNA. Compared with the control cells transfected with the empty vector, forced expression of PCDHA11 increased the proliferation of KATO-III cells (Fig. 4a). Next, HGC-27 cells were subjected to knockout experiments, because they expressed the highest relative levels of PCDHA11 mRNA. A stable PCDHA11-knockout HGC-27 cell was isolated, and direct sequence analysis verified genome editing (Supplementary Fig. 4A). No reference sequence was identified through sequence analysis. Given that all alleles exhibited mutations, including insertions from the knockout vector, genome editing events were deemed to be homozygous. No alterations in cell morphology were observed in the stable PCDHA11-knockout HGC-27 cells (Supplementary Fig. 4B). A significant inhibitory effect of ASOs was observed in the parental cells, while little inhibition of proliferation was induced by ASOs in the HGC-27/PCDHA11-knockout cells (Fig. 4b). ASO-mediated knockdown of PCDHA11, particularly by hPCDHA11-3969, increased caspase activities compared with untransfected HGC-27 cells. In contrast, this reaction was only modest in the HGC-27/PCDHA11-knockout cells (Fig. 4c). Formation of the peritoneal nodules was decreased in mice implanted HGC-27/PCDHA11-knockout cells compared to those implanted parental HGC-27 cells (Fig. 4d).

Fig. 4. Specificity and influence of AmNA-modified anti-PCDHA11 ASOs on caspase activity, apoptosis, cancer stemness, and functions associated with metastatic potential.

Fig. 4

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a Proliferation assay of KATO-III cell transfected with empty and PCDHA11-overexpressing vectors. *P < 0.05 between the empty vector and overexpression vector at day 5. b Little inhibitory effects of AmNA-modified anti-PCDHA11 ASOs on proliferation of Pcdha1-knockout cells. c In HGC-27 parental cells, hPCDHA11-2255 and -3969 increased caspase activities compared to controls, whereas little changes in caspase activities by AmNA-modified anti-PCDHA11 ASOs were observed in Pcdha1-knockout cells. d Growth of parental and PCDHA11-knockout HGC-27 xenografts in mouse peritoneal metastasis models. *P < 0.05. e Annexin V staining revealed that the proportion of apoptotic cells detected was significantly increased by AmNA-modified anti-PCDHA11 ASOs. Effects of UV irradiation served as a positive control. *P < 0.05. f Comparison of spheroid formation by gastric cancer cells. g Proportions of ALDH-positive living gastric cancer cells (right upper panels) were decreased by hPCDHA11-3969. h Proportion of cancer cells adhering to human primary mesothelial cells was decreased by hPCDHA11-2255, -3319 and -3969. *P < 0.05 between ASO-NEG and AmNA-modified anti-PCDHA11 ASOs. Error bars indicate the standard deviation.

Influence of AmNA-modified Anti-PCDHA11 ASOs on cellular functions including apoptosis, cancer stemness and adhesion to the mesothelial cells

We next evaluated the effects of AmNA-modified anti-PCDHA11 ASOs on apoptosis and cancer stem cell properties to unravel the mechanism by which the ASOs inhibited cancer cell proliferation. Annexin V assay data revealed that HGC-27 cells transfected with hPCDHA11-3969 harbored more annexin V-positive cells compared with untransfected or ASO-NEG cells, indicating that cells transfected hPCDHA11-3969 were more susceptible to apoptosis (Fig. 4e).

When we employed a spheroid cell culture assay to assess cancer cell stemness, we found that hPCDHA11-3969 significantly inhibited spheroid formation, indicating the stemness phenotype (Fig. 4f). The ALDH assay was used to detect the presence of subpopulations exhibiting cancer stem cell-like properties vs control cells. The percentage of HGC-27 cells expressing the stemness marker ALDH was decreased by hPCDHA11-3969 compared with that of the untransfected control and NEG-ASO cells (Fig. 4g). Adhesion to mesothelial cells is an important function required for cancer cells that mediates peritoneal metastasis formation. The ratio of cells adhering to human mesothelial cells was significantly suppressed by AmNA-modified anti-PCDHA11 ASOs (Fig. 4h).

Safety profile of AmNA-modified anti-PCDHA11 ASOs

The toxicities of weekly administration of hPCDHA11-2255, -3319, and -3969 were evaluated. Intraperitoneal injection of 0.2 mg, 0.6 mg or 2.0 mg of ASOs was performed once weekly for two weeks, and blood tests were performed three times (Fig. 5a). Significant skin signs around injection sites were not observed. Mice administered hPCDHA11-ASOs (all doses) exhibited no significant loss of body weight (Fig. 5b), reduced activity, or impaired oral intake. Mice administered hPCDHA11-ASOs produced elevated levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) in a dose-dependent manner (Fig. 5c, Supplementary Fig. 5a, b). The remarkable elevation of AST and ALT levels were observed in mice administered hPCDHA11-3969. For all sequences and concentrations, AST and ALT values, which were elevated after administration, decreased 3 weeks after terminating their administration. Particularly, AST and ALT values recovered close to baseline in mice treated with 0.2 mg of hPCDHA11-2255 and -3319 (Fig. 5c). These findings indicate that hepatotoxicity induced by AmNA-modified anti-PCDHA11 ASOs was reversible and sequence dependent. The elevation of total bilirubin levels was not observed at any dose of ASOs. Administration of hPCDHA11-ASOs (all doses) did not induce detectable renal dysfunction and elevation of amylase levels (Fig. 5d, Supplementary Figs. 5A, B); and the livers, kidneys, and brains did not exhibit significant changes in appearance, volume, and hematoxylin and eosin staining (Fig. 5e).

Fig. 5. Toxicities of hPCDHA11-2255, -3319 and -3969.

Fig. 5

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a Toxicity test treatment schedule. b Changes in body weight after administration of 0.6 mg ASOs. c Liver function after administration of 0.6 mg ASOs. d Serum amylase levels and renal function after administration of 0.6 mg ASOs. e Appearance; histological findings; and volumes of the liver, kidney, and brain after administration of 0.6 mg ASOs. Error bars indicate the standard deviation.

Potential off-target effects of AmNA-modified anti-PCDHA11 ASOs

Results of in silico analysis using GGGenome for mouse pre-spliced RNA indicated that 0 and 29 identical sequences with one or two mismatches were targeted by hPCDHA11-2255, 0 and 41 identical sequences with one or two mismatches were targeted by hPCDHA11-3319, and 2 and 98 identical sequences with one or two mismatches were targeted by hPCDHA11-3969 (Supplementary Table 7). Next, we performed microarray analysis using human gastric cancer cell (HGC-27) to detect the effects of the ASOs on candidate off-target genes in human, which were defined by the relative expression levels as follows: (1) ≥ 2-fold increase or ≤0.5-fold decrease compared with mock (untreated) cells, (2) no significant change (<0.5-fold or <2-fold) in ASO-NEG gastric cancer cells compared with the mock transfectants. Combining microarray data and bioinformatics data from in silico analysis for human pre-spliced RNA revealed that hPCDHA11-2255 had two identical matched, 2 single mismatches and 6 sequences with 2 mismatches. Furthermore, hPCDHA11-3319 had two identical matched, 2 single mismatches and 12 sequences with 2 mismatches. hPCDHA11-3969 lacked identical and single mismatches but had 47 sequences with 2 mismatches (Supplementary Table 8). Information on genes at potential risk of off-target effects of hPCDHA11-3969 were listed in Supplementary Table 9.

Effects of AmNA-modified anti-PCDHA11 ASOs on signal transduction pathways

The numerous cadherin superfamily members exhibit diverse activities in cancer cells. Using the transcriptome analysis data, we reviewed the results regarding cadherin-related molecules. Among them (n = 93), the mRNA levels of 10 were significantly upregulated in gastric cancer tissues with distant metastases. PCDHA11 was the most highly expressed, showing unique behavior among protocadherins (Fig. 6a).

Fig. 6. Effect of AmNA-modified anti-PCDHA11 ASOs on signal transduction pathways.

Fig. 6

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a Volcano plot of expression levels of cadherin superfamily members in gastric cancer tissues with distant metastasis and those of candidate ligands. b Results of the western blot digital imaging analysis. c Hypothetical working model of AmNA-modified anti-PCDHA11 ASOs.

Understanding the mechanism of action of AmNA-modified anti-PCDHA11 ASOs requires investigating ASO-induced changes in intracellular signaling. Therefore, we evaluated the expression and phosphorylation status of cancer-related signaling pathways. We used hPCDHA11-2255 and hPCDHA11-3319, which were found to be sequences with a low off-target risk. AmNA-modified anti-PCDHA11 ASOs suppressed the phosphorylation of FAK and PKCα as well as a subsequent decrease of the phosphorylation of AKT and mTOR. β-catenin signaling was also inactivated by knockdown of PCDHA11 via decreased phosphorylation of its upstream, the Axin1-TEAD1 pathway. Moreover, AmNA-modified anti-PCDHA11 ASOs inhibited the phosphorylation of the cell-cycle and DNA synthesis regulators CDC43 and CDC73, and western blots analysis detected inhibition of the phosphorylation of c-Raf-MEK1/2 signal as well as that of JAK1-STAT3 in cells transfected with ASOs (Fig. 6b). Our proposed working model of the mechanism of AmNA-modified anti-PCDHA11 ASOs action in cancer cells is shown in Fig. 6c.

Generation and characterization of Pcdha11−/− mice

To identify the pathophysiological functions of PCDHA11, we generated Pcdha11+/+, Pcdha11+/−, and Pcdha11−/− mice. Their genomes were electrophoretically fractionated (Fig. 7a). Knockout of 1 or both Pcdha11 alleles (Pcdha11+/− and Pcdha11−/− mice) were not embryonic lethal and did not effect appearance (Fig. 7b) or body weight (Supplementary Fig. 6a) and did not cause developmental abnormalities of the liver, lung, or brain (Fig. 7b). Moreover, neither the Pcdha11+/− or Pcdha11−/− mice exhibited dysfunctional motor coordination or motor learning, as measured by the rotarod test (Fig. 7c). Both genotypes were not associated with detectable abnormalities in the blood counts (Fig. 7d), liver function (Fig. 7e), renal function (Fig. 7f), and metabolic parameters (Supplementary Fig. 6B).

Fig. 7. Characterization of Pcdha11-deficient mice.

Fig. 7

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a Genotyping of Pcdha11+/+, Pcdha11+/−, and Pcdha11−/− mice. b Gross appearance and major organs. Microscopic findings (×400 magnification). c Rotarod test of motor coordination and learning administered to Pcdha11+/+, Pcdha11+/−, and Pcdha11−/− mice. Blood counts (d), liver functions (e), and renal functions (f) of Pcdha11+/+, Pcdha11+/−, and Pcdha11−/− mice. Error bars indicate the standard deviation.

Discussion

Here we designed and synthesized ASOs targeting the mRNA encoding the transmembrane protein PCDHA11 that mediates intercellular adhesion, which is implicated in intercellular adhesion and identified as an overexpressed gene in gastric cancer tissues with distant metastases. Our primary objective was to identify ASOs with potent anti-cancer activity; therefore, we initially assessed the efficacy of candidate ASOs through knockdown efficiency, in vitro inhibition of cell proliferation, and in vivo effectiveness. The preliminary in vivo dosage was determined based on a prior study of AmNA-modified ASOs [17]. Subsequently, we performed toxicity assessments on the selected ASOs to address potential issues for future clinical applications. Our findings suggest that targeting PCDHA11 with ASOs offers a promising therapeutic strategy for gastric cancer and potentially other malignancies.

This study elucidates that PCDHA11 represents a compelling new target for solid tumors, including gastric cancer, and that AmNA-modified anti-PCDHA11 offers a promising therapeutic modality. Particularly, the encouraging outcomes observed in peritoneal metastasis models provide optimism for advancing the treatment of gastric cancer. Peritoneal metastasis is a particularly grievous manifestation of gastric cancer progression, notwithstanding concerted efforts to enhance systemic chemotherapy efficacy [23, 24]. A significant limitation of systemic chemotherapy is the inadequate delivery of therapeutic agents to peritoneal tumors. The elevated molecular weight of AmNA-modified anti-PCDHA11 ASO, approximately seven times greater than that of paclitaxel, supports its potential for intraperitoneal administration. This high molecular weight facilitates prolonged retention within the abdominal cavity, enabling direct interaction with cancer cells while reducing systemic exposure that could lead to toxicity [25].

Given that our transcriptome analysis was preliminary, based on only four samples per patient group, we subsequently conducted a PCDHA11 expression analysis on an expanded cohort. We found a significant increase in PCDHA11 mRNA expression levels in 300 primary gastric cancer tissues compared with noncancerous gastric tissues. Furthermore, tissue levels of PCDHA11 mRNA associated with adverse prognosis were reproduced by our analysis of two independent external validation datasets, and statistical analyses identified high PCDHA11 mRNA expression as an independent prognostic factor for survival of patients with gastric cancer. Moreover, IHC analysis showed that PCDHA11 levels were significantly associated with postoperative disease recurrence. This finding suggests that tissue expression of PCDHA11 may serve as a valuable biomarker for risk stratification regarding recurrence and could significantly enhance the management of gastric cancer patients by guiding the selection of adjuvant therapies post-surgery. Notably, in patients with metastatic gastric cancer, our results underscore the potential of PCDHA11 as a therapeutic target and highlight the utility of its expression as a companion diagnostic tool to identify individuals who might be particularly responsive to treatment with anti-PCDHA11 ASOs.

Similar to classical cadherins (e.g. E-cadherin), PCDHA11, a single-pass transmembrane glycoprotein, may function as a receptor and a ligand, because it homophilically interacts with itself and with high affinity at intercellular connections [10, 26–28]. In normal human cells, αE-catenin, β-catenin, and p120-catenin bind the intracellular domain of PCDHA11 and are linked to the cytoskeleton [11]. In normal human tissues, PCDHA11 levels are relatively abundant in cranial nervous system tissues, endocrine tissues (such as the parathyroid and pituitary) and pancreatic tissues, while it is rarely expressed in heart, kidney, lung, digestive tract, liver, and mammary gland [27]. Moreover, in normal cells, protocadherins interfere with intracellular β-catenin and GSK3β/mTOR signaling [29, 30].

Numerous studies report aberrant expression of cadherin superfamily members in malignant tumors [31]. The most well-recognized knowledge is that reduced expression of E-cadherin contributes to the epithelial-mesenchymal transition of cancer cells and promotes cancer metastasis [32]. However, not all members of the cadherin superfamily exhibit the same tumor-suppressive functions as E-cadherins. In fact, elevated expression of N-cadherin has been linked to cancer progression [9, 31]. Despite their structural and physiological similarities [9, 33], procadherins exhibit divergent roles in cancer. Specifically, some procadherins function as tumor suppressors, while others promote tumorigenesis. For instance, protocadherins PCDH8 and PCDH10 are known to act as tumor suppressors, whereas PCDH7 and PCDHA11 function as oncogenic promoters [31]. Interestingly, our transcriptome data show diverse expression patterns of protocadherins, among which PCDHA11 was the most highly up-regulated in cancer tissues. Although transcriptomic analysis using PCDHA11-KO cells would be beneficial for a more comprehensive understanding, our data suggest that PCDHA11 functions as a cancer-promoting factor, displaying distinct properties among protocadherins.

Our ASO-knockdown experiments demonstrate that AmNA-modified anti-PCDHA11 antisense oligonucleotides (ASOs) modulate cellular functions at various stages of metastatic progression, including invasion, migration, and adhesion. Furthermore, these ASOs inhibit spheroid formation and stemness, which are pivotal in facilitating cancer progression by enabling tumor cells to persist in hypoxic conditions and acquire resistance to anticancer agents [34]. ASOs targeting PCDHA11 disrupt intracellular signaling pathways distinct from those affected by receptor tyrosine kinases, such as HER2 and VEGFR2, and operate through mechanisms different from those of immune checkpoint inhibitors. Due to these divergent mechanisms, PCDHA11-targeted ASOs may offer therapeutic benefits for patients who are resistant to existing molecularly targeted therapies for gastric cancer, potentially providing an additive effect when used in combination with other treatments.

To assess the efficacy of ASOs in metastatic cancers, a systemic mouse model of metastasis was established via tail vein injection of cancer cells. In the PCDHA11-knockdown cohort, tumor formation in the skin and lungs was notably suppressed due to reduced hematogenous metastasis. The subsequent phase of this study will involve evaluating the therapeutic efficacy of intravenously administered ASOs. Additionally, we examined whether ASOs could exert therapeutic effects in mice with substantial cancer burdens. Administration of ASOs led to a deceleration in the growth rates of established subcutaneous tumors; however, tumor regression was not observed. This finding indicates the necessity of enhancing therapeutic efficacy through combination therapy with cytotoxic antitumor agents or immunotherapy. Importantly, ASOs demonstrated in vivo activity without the need for transfection-enhancing reagents, achieved by adjusting the CaCl2 concentration. Consequently, a Ca2+-enriched medium was developed to amplify oligonucleotide activity, independent of their net charge and structural modifications, thereby improving in vivo silencing efficacy [18]. This approach offers a significant advantage for clinical applications, as conventional in vitro transfection reagents are not universally applicable.

The most common and severe adverse effect of ASOs is hepatotoxicity [35, 36]. Our study demonstrated that liver damage was induced by AmNA-modified anti-PCDHA11 ASOs in a dose- and sequence-dependent manner. Notably, the hepatotoxicity caused by these ASOs, even when pronounced, did not compromise the overall health of the mice. Furthermore, this liver damage was reversible, with the mice fully recovering within three weeks. Although mice are generally more susceptible to ASO-induced hepatotoxicity compared to humans, the hepatotoxicity observed with hPCDHA11-3969 was alarmingly high. This was particularly concerning given that hPCDHA11-3969 also exhibited potent inhibition of cell proliferation. It was validated using the in vitro Caspase-Glo 3/7 Assay System [37], recognized as a surrogate for in vivo tissue cytotoxicity, which also yielded elevated values for hPCDHA11-3969. However, further investigations are warranted. Off-target risk assessment indicated that hPCDHA11-3969 maintained specificity up to a single mismatch but suppressed the expression of 47 genes with two mismatches. This off-target activity is likely the primary contributor to the severe hepatotoxicity observed, necessitating a cautious interpretation of hPCDHA11-3969’s activity data, especially considering the suppression of eight spliced RNAs and 50 pre-spliced RNAs due to two mismatches. Conversely, in vivo experiments identified hPCDHA11-2255 as one of the less active sequences among the candidates. Thus, hPCDHA11-2255, in its current form, is not suitable for future clinical applications. It will require chemical modification, activity enhancement through delivery mechanisms, or an alternative sequence to improve its efficacy. Given the current treatment efficacy, off-target profile, and toxicity data, hPCDHA11-3319 emerges as the most promising ASO among the 54 sequences initially screened. To advance toward clinical application, optimization through nucleotide adjustment, refinement of surrounding sequences, and chemical modifications to mitigate hepatotoxicity should be pursued.

To elucidate the mechanism of action of AmNA-modified anti-PCDHA11 ASOs, we conducted a comprehensive analysis of intracellular signaling pathways. Our investigation revealed critical downstream signals mediated by PCDHA11, uncovering potentially intricate interactions between PCDHA11 and various signaling pathways. These interactions likely contribute to the enhancement of stemness, metastasis, and survival in gastric cancer cells. Protocadherin proteins, functioning as cell adhesion molecules at the intersection of signaling pathways, are essential for the regulation of cellular differentiation, tissue regeneration, and maintenance. Notably, members of the protocadherin family exhibit diverse roles in different cancer types. For instance, PCDH20 acts as a tumor suppressor by antagonizing the Wnt/β-catenin pathway, whereas the loss of PCDH17 may activate the EGFR/MEK/ERK pathway, thereby accelerating the progression of liver cancer [29, 38]. Our findings suggest that the unique role of PCDHA11 in cancer cells is supported by complex interactions with the AKT/mTOR, RAS/MEK, Wnt/β-catenin, and JAK/STAT pathways. These insights provide a deeper understanding of the molecular underpinnings of PCDHA11’s function in cancer, which may inform the development of targeted therapeutic strategies. A transcriptome analysis utilizing PCDHA11-KO cells is warranted for a more comprehensive understanding.

The embryos of N-cadherin homodeficient mice die because of abnormal heart development. In contrast, Pcdha11−/− knockout mice were infertile. The potential safety of targeting PCDHA11 is indicated by our findings that such mice did not exhibit abnormal reproduction, development, metabolism, or motor functions. These observations enhance our understanding of the physiological roles of PCDHA11 and the potential effects of PCDHA11-targeted therapy. Crossing these knockout mice into a transgenic cancer model would be highly valuable to assess whether the genetic loss of PCDHA11 also results in reduced tumor growth and metastasis. Prior to conducting preclinical toxicity tests in rats and monkeys, it is imperative to optimize delivery tools, administration routes, intervals, and dosages.

Oligonucleotide-based therapeutics are of intermediate size with much improved selectivity towards the target and induce fewer off-target effects than small molecules [35, 39]. The first transcript-targeted therapies authorized for use in clinical practice are focused against genetic diseases [40]. In contrast, the use of ASOs remains an area of active investigation for cancer treatment, mainly due to insufficient delivery, low transfection efficacy, and abnormally high levels of RNase H expressed by cancer cells [41, 42]. The strengths of our AmNA-ASOs are as follows: (1) oligonucleotide sequences with enhanced target specificity, lack of significant sequence similarity with the mouse counterpart of PCDHA11, (2) chemical modifications extend the half-life of therapeutic ASOs to prolong exposure of cancer cells, (3) higher binding affinity to target molecules that enhance drug efficacy, (4) higher nuclease resistance attributed to the phosphorothioate-linked structure of AmNA, and (5) a simple transfection employing calcium-enrichment of culture media [43, 44]. Despite these efforts, significant challenges remain in applying ASOs to cancer therapy. These include maintaining their activity in blood and ascitic fluid, ensuring penetration into solid tumor tissues, and facilitating uptake into cancer cells. Ongoing endeavors to enhance ASO activity and delivery are essential. Additionally, considering combination therapies with other anticancer drugs and delivery tools that can mitigate these limitations will be crucial for advancing ASO-based cancer treatments.

We acknowledge several limitations in our study. Upstream factors that stimulate PCDHA11 activity were not identified. The frequency, administration route, optimal dose, and optimal duration of treatment, as well as the survival benefits of AmNA-modified anti-PCDHA11 ASOs, were not determined. The small sample sizes of mice (n = 4 or n = 5) and the exclusive use of male mice limit the significance of our findings. Additionally, tumor samples from mice post-ASO administration were not preserved, and IHC data for tumor nodules are unavailable. The use of patient-derived xenograft models, which was not incorporated in this study, may provide further insights into the oncological involvement of PCDHA11. Lastly, the reduction of hepatotoxicity and enhancement of treatment efficacy remain crucial challenges for the clinical application of these ASOs.

In conclusion, AmNA-modified anti-PCDHA11ASOs represent a promising strategy for treating gastric cancer and other solid tumors by modulating cellular functions associated with cancer metastasis.

Supplementary information

Supplemental Materials (1.3MB, pdf)

Acknowledgements

We thank Edanz Group (www.edanzediting.com/ac) and Springer Nature Author Services for editing a draft of this paper.

Author contributions

Study concept and design: M.K. (Mitsuro Kanda), Y.K. (Yuuya Kasahara) and S.O. Acquisition of the data: Y.K. (Yuuya Kasahara), S.N., T.S., M.S., Y.I. and M.K. (Masahisa Katsuno). Statistical analysis: D.S. Management of data acquisition: M.K. (Mitsuro Kanda) and Y.K. (Yuuya Kasahara). Analysis of the present data: S.N., T.S., M.S. D.S. and Y.I. Critical interpretation of the present data: M.K. (Mitsuro Kanda) and Y.K. (Yuuya Kasahara). Drafting of the paper: M.K. (Mitsuro Kanda). Critical revision of the paper for important intellectual content: Y.K. (Yuuya Kasahara), S.N., T.S., M.S., D.S., Y.I. and M.K. (Masahisa Katsuno), S.O. and Y.K. (Yasuhiro Kodera). Obtained funding: M.K. (Mitsuro Kanda), Y.K. (Yuuya Kasahara) and S.O. Technical or material support: Y.K. (Yuuya Kasahara) and S.O. Study supervision: Y.K. (Yasuhiro Kodera).

Funding

This work was supported by the Japan Agency for Medical Research and Development (AMED; JP20lm02030005, JP21ak0101154, JP22ak0101154 and JP23ak0101154) and a Grant-in-Aid for Scientific Research (20K21629).

Data availability

Data sources and handling of the publicly available datasets used in this study are described in the Materials and Methods. The other data generated in this study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

The Institutional Review Board of Nagoya University, Japan, approved this study, and written informed consent was obtained from all patients (approval number 2014-0043). The Animal Research Committee of Nagoya University approved experiments using animals, performed according to ARRIVE guidelines (approval number M230102-002).

Footnotes

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

Supplementary information

The online version contains supplementary material available at 10.1038/s41416-024-02859-5.

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Associated Data

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

Supplementary Materials

Supplemental Materials (1.3MB, pdf)

Data Availability Statement

Data sources and handling of the publicly available datasets used in this study are described in the Materials and Methods. The other data generated in this study are available from the corresponding author upon reasonable request.

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