RAGE signaling inhibition prevents the malignant phenotypes of pancreatic ductal adenocarcinoma cells

Abstract

Receptor for advanced glycation end-products (RAGE) is implicated in immune and inflammatory diseases as well as the malignant phenotypes of cancer. Blocking RAGE signaling is thought to reduce its impact on disease states. In this study, we investigated the effect of RAGE signaling inhibitors (inhRAGE) on the malignant phenotypes of pancreatic ductal adenocarcinoma (PDAC). Four PDAC cell lines were used. quantitative PCR (qPCR) was performed to evaluate RAGE expression. Assays for cell proliferation, migration, and invasion were performed with or without inhRAGE and the addition of several ligands. The effects of inhRAGE were examined by creating ectopic and orthotopic xenograft tumor models, and the resected specimens were subjected to immunohistochemical analysis. RAGE expression was confirmed in all the cell lines. inhRAGE significantly reduced the migration and invasion abilities of the cells exposed to high-mobility group B1 (HMGB1) or S100B, which are tumor- and inflammation-associated RAGE ligands, without inhibiting cellular proliferation. In both the ectopic and orthotopic models, inhRAGE treatment significantly inhibited tumor growth. Immunostaining confirmed the suppression of angiogenesis and inflammation. inhRAGE inhibits the malignant phenotypes of PDAC, suggesting that inhRAGE may be a novel therapeutic agent for the condition.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-48913-5.

Introduction

Globally, pancreatic ductal adenocarcinoma (PDAC) ranks sixth in terms of cancer-related mortality and is characterized by a poor prognosis and high mortality rate despite recent improvements in therapy regimens and surgical methods¹. Currently, surgical resection combined with preoperative or postoperative chemotherapy is considered the best treatment for long-term survival². However, many cases are difficult to cure because of local or distant metastasis at the time of diagnosis, and even in cases in which radical resection is performed, early recurrence is often observed. Therefore, a multidisciplinary approach is essential for the treatment of PDAC. The tumor microenvironment, which is composed of a dense and vast fibrous stroma coupled with a highly heterogeneous population of cancer-associated fibroblasts, endothelial cells, and immune cells, plays a crucial role in PDAC³.

Receptor for advanced glycation end-products (RAGE) was originally discovered as a receptor for advanced glycation end-products (AGEs) produced by non-enzymatic glycation reactions between proteins and reducing sugars⁴. RAGE is a pattern recognition and intracellular signal-transducing receptor that recognizes AGEs and various ligands, including HMGB1, S100 proteins, and lipopolysaccharides. It is involved in cell proliferation and metastasis and is known to be expressed not only in PDAC cells but also in various cancer cells, such as colorectal and prostate cancer^5–7. In RAGE signaling, the cytoplasmic tail of RAGE (ctRAGE) binds to Diaphanous-1 (Diaph1). To comprehensively capture the breadth of RAGE signaling, small-molecule antagonists of ctRAGE-Diaph1 interactions have recently been developed, including inhRAGE (Compound 11). However, the role of inhRAGE in the suppression of cancer development remains unclear.

Therefore, in this study, we examined the effectiveness of inhRAGE in the malignant phenotypes of PDAC using both in vitro and in vivo approaches.

Materials and methods

Cell cultures

Four human pancreatic cancer cell lines, AsPC-1, BxPC-3, MIAPaCa-2, and PANC-1 were obtained from the American Type Culture Collection (Rockville, Maryland, USA). AsPC-1 cells were maintained in the Roswell Park Memorial Institute (RPMI) −1640 medium with 1 mM sodium pyruvate. BxPC-3 cells were maintained in the same medium, whereas PANC-1 and MIAPaCa-2 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM). Both of these media were supplemented with 100 µg/ml streptomycin, 20 U/ml penicillin, and 10% fetal bovine serum. Each cell line was maintained at 37 ˚C and 5% CO₂.

Reagents

The ctRAGE/Diaph1 inhibitor (compound 11, C19H16N2O3; inhRAGE) was chemically synthesized by Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Recombinant human HMGB1 and S100B were purchased from Abcam (Cambridge, UK) and Calbiochem (San Diego, CA, USA), respectively.

Real-time reverse transcription PCR

Total RNA was extracted from each cell line using the TRIzol reagent (Thermo Fisher Scientific, Tokyo, Japan). cDNA was synthesized using an AffinityScript QPCR cDNA Synthesis Kit (Agilent Technologies, Tex, USA). Using the TaqMan technique (50 °C for 2 min, 95 °C for 10 min, and 40 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s) and the SYBR Green PCR Kit (QuantiTect, QIAGEN, Germany), real-time quantitative PCR was carried out. Using the ΔΔCt method, the relative abundance of transcripts was normalized based on the expression of β-actin.

Cell proliferation assay

Cell proliferation assay was performed using the CellTiter 96^® Non-Radioactive Cell Proliferation Assay (Promega, Wisconsin, USA, catalog number: G4000). Briefly, 96-well plates (Corning, New York, USA) were used. PANC-1 and MIAPaCa-2 were seeded at a density of 1.2 × 10⁴ cells per well in 100 µL of DMEM with or without 0.1–10 µM of inhRAGE. AsPC-1 and BxPC-3 were seeded at a density of 1.2 × 10⁴ cells per well in 100 µL of RPMI-1640 with or without 0.1–10 µM of inhRAGE. Each plate was incubated for 0–96 h at 37 ˚C and 5% CO₂, and thereafter 15 µL of dye solution was added to each well. Following this, each plate was incubated for 4 h. After incubation, 100 µL of the solubilization solution/stop mix was applied to each well. The plates were once again incubated overnight in a sealed container at room temperature under a humidified atmosphere. The absorbance was recorded at a wavelength of 490 nm in each well using an automated 96-well plate reader.

Cell migration/invasion assay

The inhibitory potential of inhRAGE on the migrative/invasive capacity of the human pancreatic cancer cell lines was assessed using a transwell migration/invasion assay, as described previously with some modifications^8,9. For this purpose, 24-well Boyden chambers (8 μm pore size) (Corning, NY, USA) were used. PANC-1 and MIAPaCa-2 were seeded at a density of 2 × 10⁵ cells in the upper chamber in 500 µL of DMEM with or without 0.1–10 µM of inhRAGE, and the lower chambers received 750 µL of DMEM with 10% Fetal Bovine Serum (FBS). Experiments were performed on the AsPC-1 and BxPC-3 cells using the same volume of RPMI-1640 as the medium. Each cell line was incubated at 37 ˚C and 5% CO₂ for 16 h, and the cells were fixed with 3.7% formaldehyde, followed by methanol for 20 min. After two washes with PBS, the cells were stained with 1% crystal violet for 15 min. Noninvasive cells that were still present on the membrane surface were scraped away using a cotton swab. Using an x20 magnification optical microscope (EVOS, AMG, AMEX-1200, USA), the migrating cells at the bottom membrane were counted in each of the six different fields of the membrane.

Subsequently, ligands were added to the assay. BxPC-3 were seeded at a density of 2 × 10⁵ cells in the upper chamber in 500 µL of RPMI-1640 with 10 µM of inhRAGE, and 750 µL of RPMI-1640 with 0.1% FBS and 0–100 ng/ml of HMGB1 or 0–10 µg/ml of S100B as a ligand was added to the lower chambers. Each cell line was incubated at 37 ˚C and 5% CO₂ for 16 h, treated in the same manner as above, and the migrated cells at the bottom membrane were counted.

For the cell invasion assay, 24-well Boyden chambers (8 μm pore size) (Greiner Bio-One International Gmbh, Kremsmünster, Austria) were used. Before seeding each cell into the top chamber, 100 µL of 100 µM Matrigel (Corning Inc, NY, USA) in DMEM or RPMI-1640 was overlaid on the top of the membranes. Using the same method as for the cell migration assay, each cell line was seeded at a density of 2 × 10⁵ cells in the upper chamber in 500 µL of the corresponding medium with or without 0.1–10 µM of inhRAGE, and 750 µL of the corresponding medium containing 10% FBS was added to the lower chambers. After 24 h of incubation, the invasive cells were stained and counted using the same method as for the migration assay.

All experiments were performed in triplicate.

Rac1 pull-down assay

Rac1 activity was evaluated using a Rac1/Cdc42 Activation Assay Kit (non-radioactive) (Merck Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. Briefly, cells were lysed on ice in Mg²⁺ lysis/wash buffer (Merck Millipore, Burlington, MA, USA) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (Thermo Scientific, Waltham, MA, USA). Total protein concentration was measured using a BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA).

The pull-down products and an aliquot of total lysate (20 µg) were mixed with Laemmli reducing sample buffer, heated at 95 °C for 5 min, and separated by 12.5% SDS-PAGE (ATTO Corp., Tokyo, Japan). Proteins were then transferred onto PVDF membranes (Merck Millipore, Burlington, MA, USA). After blocking with 3% skim milk in PBS for 30 min, the membranes were incubated overnight at 4 °C with an anti-Rac1 antibody (mouse monoclonal IgG, clone 23A8, 1:1,000; Merck Millipore, Burlington, MA, USA). The membranes were subsequently incubated with goat anti-mouse IgG IRDye 680RD secondary antibody (1:10,000; LI-COR Biosciences, Lincoln, NE, USA), and protein bands were visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). An aliquot of the total lysate was analyzed by western blotting as an input control.

Ectopic and orthotopic xenograft mouse models

For the experiments, 4-week-old female BALB/c-nu/nu mice (Charles River Laboratories Japan, Inc., Kanagawa, Japan) were used. All the mice were treated and housed in accordance with the Fundamental Guidelines for the Proper Conduct of Animal Experiments and Related Activities at Academic Research Institutions, administered by Japan’s Ministry of Education, Culture, Sports, Science, and Technology. All animal experiments were conducted in compliance with the ARRIVE guidelines, including study design, sample size determination, randomization, animal procedures, and statistical analyses, and were approved by the Animal Experiment Committee of Kanazawa University (AP-204153).

First, 3.0 × 10⁶ cells of BxPC-3 were injected subcutaneously into the back of each mouse. Three days after the injection, the mice were randomly divided into two groups: inhRAGE (n = 7) and control (n = 7). Mice in the control group received only 100 µL of PBS daily, whereas mice in the inhRAGE group received 100 µg/body of inhRAGE intraperitoneally (i.p.) daily. No predefined exclusion criteria were established. However, one mouse in the control group was excluded from the analysis due to failure of tumor establishment. The final number of mice analyzed was n = 7 in the inhRAGE group and n = 6 in the control group. The body weight and tumor volume of each mouse were measured twice a week. The tumor volume was calculated as previously reported, using V = length × width²/2¹⁰. The animals were sacrificed by cervical dislocation 9 weeks after the start of each administration.

To create an orthotopic xenograft mouse model, the mice were anesthetized by intraperitoneal administration of medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg). A 1 cm transverse incision was made on the left flank of each mouse, the pancreas was exposed, and 3.0 × 10⁶ BxPC-3 cells were injected into the tail of the pancreas using a 29G needle. The incision was closed using a 6 − 0 polypropylene suture (PROLENE; Ethicon, Inc., NJ, USA). Three days after the injection, the mice were randomly assigned to two groups: control (n = 13) and inhRAGE (n = 13). Mice in the control group received only 100 µL of PBS daily, whereas mice in the inhRAGE group received 100 µg/body of inhRAGE i.p. daily. No exclusion criteria were applied, and the data from all the mice were included in the analysis. No predefined exclusion criteria were established. However, one mouse in the control group died immediately following cell inoculation and was therefore excluded from the analysis. The final number of mice analyzed was n = 13 in the inhRAGE group and n = 12 in the control group. The body weights of all the mice were measured twice per week. The mice were sacrificed by cervical dislocation 6 weeks after the start of each administration, and the tumor volume and weight were measured.

Immunohistochemistry

Tumors resected from the mice were immunostained using the method described in a previous study¹¹. Resected tumor specimens were preserved in 10% neutral-buffered formalin before being embedded in paraffin. Each section was deparaffinized and pretreated by autoclaving in a 10% citrate buffer for 15 min at 120 °C. Following treatment with a protein-blocking serum (Dako, Kyoto, Japan) for 10 min, the sections were incubated for 30 min with 2% skim milk to inhibit any non-specific reactions. Primary antibodies were then applied to the sections and incubated overnight at 4 °C; primary antibodies against CD68 (Abcam, ab125212, UK), CD31 (Abcam, ab28364, UK) and S100B (Abcam, ab218515, UK) were used. After washing the sections with PBS, immunoreactivity was detected using the EnVision reagent (Dako). Each slide was developed with diaminobenzidine, followed by contrast staining with hematoxylin. Each section was examined with an Olympus DP70-SET microscope digital camera, UPLFLN 100X objective lens, and the ‘Cell Scans Standard’ software.

Statistics

Statistical analysis was performed using Prism for macOS (version 10.11) and Student’s t-tests (two-tailed), Kruskal-Wallis tests, and two-way analysis of variance. All the data are presented as means ± standard error of the mean (SEM). Statistical significance was set at p < 0.05.

Results

RAGE gene expression

To assess RAGE expression, quantitative PCR (qPCR) was performed on four PDAC cell lines with differing degrees of differentiation. All four cell lines (AsPC-1, BxPC-3, PANC-1, and MIAPaCa-2) showed RAGE gene expression according to the qPCR analysis, and a trend showing a correlation between the expression level and the type of tumor differentiation was observed. BxPC-3 (moderately differentiated) and AsPC-1 (highly differentiated) cells showed high RAGE expression. In contrast, PANC-1 (poorly differentiated) and MIAPaCa-2 (undifferentiated) cells showed lower RAGE expression, particularly PANC-1, which was significantly less (Fig. 1).

Fig. 1.

In each cell line, RAGE gene expression was observed. Four cell lines with different degrees of differentiation were used. RAGE expression was verified in each cell line. The highly differentiated cell lines (AsPC-1 and BxPC-3) showed higher RAGE gene expression than the less-differentiated cell lines (PANC-1 and MIAPaCa-2). All experiments were conducted in triplicates. Data are presented as the mean ± SEM. ∗p < 0.05; ∗∗p < 0.01. RAGE: receptor for advanced glycation end-products.

Inhibitory effect of inhRAGE on the proliferation, migration, and invasion of pancreatic cancer cell lines in vitro

Next, we examined whether inhRAGE inhibits the growth, migration, and invasion of pancreatic cancer cell lines. In the cellular proliferation assays, cells were seeded at a density of 1.2 × 10⁴ cells per well, and experiments were performed using three plates per condition and repeated twice independently. Under these conditions, the addition of inhRAGE did not affect the proliferation of any of the cell lines (Fig. 2). Conversely, all the cell lines exhibited a concentration-dependent inhibitory effect of inhRAGE on cell migration (Fig. 3). Furthermore, in the matrigel cell invasion assay, treatment with inhRAGE significantly inhibited the invasion of all the cell lines in a concentration-dependent manner (Fig. 4).

Fig. 2.

inhRAGE was not involved in cellular proliferation. For this experiment, 1.2 × 10⁴ cells of each cell line with or without 0.1–10 µM of inhRAGE were incubated for 0–96 h and absorbance was measured at a wavelength of 490 nm. All experiments were conducted in triplicates and repeated twice independently. Data are presented as the mean ± SEM.

Fig. 3.

inhRAGE inhibited cellular migration. (A) Using 1% crystal violet staining, light microscopy observations were made of each cell line’s migration following a 16-hour incubation period. Six fields were randomly assigned images, and the number of migrated cells in each field was counted. (B) The migrated cell count for each field is displayed using bar graphs. The experiment was conducted in triplicate. Data are presented as the mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Fig. 4.

inhRAGE inhibited cellular invasion. (A) Using 1% crystal violet staining, light microscopy observations were made for the invasion of each cell line after a 24-hour incubation period. Six fields were randomly assigned images, and the number of invading cells in each field was counted. (B) The invading cell count for each field is displayed using bar graphs. All experiments were conducted in triplicates. Data are presented as the mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

To further validate whether the inhibitory effects of inhRAGE on cell migration are mediated through RAGE-dependent signaling, additional experiments were performed. Knockdown of AGER using siRNA significantly reduced AGER mRNA expression, whereas treatment with inhRAGE did not alter AGER expression (Supplementary Figure S1). Both AGER knockdown and inhRAGE treatment suppressed Rac1 activation, a downstream signaling molecule of RAGE (Fig. 5A). Furthermore, HMGB1-induced cell migration was significantly attenuated by AGER knockdown in migration assays (Fig. 5B, C). Similar results were obtained in MIAPaCa-2 cells, confirming that suppression of PDAC cell migration is mediated through RAGE-dependent signaling (Supplementary Figure S2).

Fig. 5.

Genetic and pharmacological inhibition of RAGE suppresses Rac1 activation and ligand-induced PDAC cell migration. (A) Rac1 activation was examined using a pull-down assay. Active Rac1 (Rac1-GTP) was isolated from cell lysates using PAK-PBD agarose beads and detected by western blotting with an anti-Rac1 antibody. Cells were treated with HMGB1 (100 ng/mL) in the presence or absence of the RAGE inhibitor (inhRAGE, 10 µM) or AGER siRNA. Total Rac1 levels in whole-cell lysates were analyzed as input controls. (B) Transwell migration assay demonstrating that HMGB1 stimulation enhances migration of BxPC-3 cells, whereas AGER knockdown significantly suppresses HMGB1-induced cell migration. In these experiments, the DMSO control exhibited the same behavior as the CTRL siRNA (not shown). (C) Quantification of migrated cells shown in (B). (D, F) Using 1% crystal violet dye, light microscopy observations were made of each cell line’s migration following an 18-hour incubation period. Six fields were randomly assigned images, and the number of migrated cells in each field was counted. (E, G) Bar graphs are used to indicate the number of migrated cells per field. HMGB1 (0–100 ng/ml) or S100B (0–10 µg/ml) were added to the lower chambers of the Boyden chambers (8 µM pore size) with or without inhRAGE (10 µM) treatment. The experiment was conducted in triplicate. Data are presented as the mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001. AGER, advanced glycation end-product receptor; RAGE, receptor for advanced glycation end-products; HMGB1, high mobility group box 1.

Next, we investigated whether stimulation of RAGE by tumor- and inflammation-associated ligands enhances cell migration using BxPC-3 cells. BxPC-3 cells were selected for this experiment because they exhibited the highest RAGE mRNA expression among the four PDAC cell lines examined by qRT-PCR and showed the greatest migratory activity in the cell migration assay. BxPC-3 cell migration was improved by adding HMGB1 or S100B to the lower chamber of the transwell plate in a concentration-dependent manner. Furthermore, cell migration to the upper chamber was suppressed by inhRAGE (Fig. 5D-G).

Inhibitory effect of inhRAGE on the growth of pancreatic cancer cell lines in ectopic and orthotopic xenografted models

We examined the inhibitory effects of inhRAGE on the growth of pancreatic cancer cell lines in ectopic and orthotopic xenograft mouse models. There were no adverse effects of inhRAGE and no mice died as a result of inhRAGE administration. Following the transplantation of BxPC-3 cells, the tumor volume at nine weeks was considerably lower in the inhRAGE group (429.1 ± 113.4 mm³) than in the control group (1553.3 ± 286.5 mm³) (p < 0.01) using in vivo ectopic xenograft models. However, there was no substantial difference in the body weight between the inhRAGE group (22.67 ± 0.62 g) and the control group (21.72 ± 1.19 g) (p = 0.78) (Fig. 6A, B). Similarly, in the orthotopic xenograft model, the tumor volume was significantly smaller in the inhRAGE group (234.3 ± 45.3 mm³) than in the control group (768.1 ± 199.4 mm³) (p < 0.01), although there was no difference in body weight (inhRAGE group: 19.91 ± 0.84 g, control group: 21.45 ± 0.93 g, p = 0.17) at 6 weeks following transplantation (Fig. 6C).

Fig. 6.

inhRAGE had an inhibitory effect on tumor growth and angiogenesis in ectopic and orthotopic xenograft mouse models. (A) In this experiment, 3.0 × 10⁶ of BxPC-3 cells were transplanted subcutaneously into the back of mice, who were then intraperitoneally injected with inhRAGE (inhRAGE group; 100 µg/body) or PBS (control group). Photographs of the tumors excised from the ectopic xenografted mice sacrificed at 9 weeks are shown. (B) Body weights and tumor volumes were measured twice a week in the inhRAGE and control groups. (C) Body weight trends and tumor volumes at the point of sacrificial death in the inhRAGE and control groups of orthotopic xenografted mice are shown. Following this, 3.0 × 10⁶ of BxPC-3 was injected into the pancreatic tail under general anesthesia and inhRAGE (inhRAGE group; 100 µg/body) or PBS (control group) was then administered intraperitoneally. Body weight was measured twice a week. After 6 weeks, the mice were sacrificed and the tumor volumes were measured. (D) Immunohistochemical staining for CD68, a macrophage marker, in tumor tissues and quantitative analysis of CD68-positive cells. (E, F) Immunohistochemical staining for CD31, the endothelial marker, and S100B, the inflammatory marker. Data are presented as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01. RAGE, receptor for advanced glycation end-products; inhRAGE, inhibitor of RAGE signaling.

Additionally, inhRAGE treatment significantly reduced the number of CD68-positive macrophages in the tumor microenvironment. We also observed a decrease in CD31-positive endothelial cells and inflammatory S100B expression in tumor tissues resected from the ectopic xenograft mouse models (Fig. 6D-F).

Discussion

This is the first study to investigate the effects of RAGE signaling pathway inhibitors on the malignant phenotypes of cancers. In this study, four human PDAC cell lines representing distinct degrees of differentiation were utilized to examine whether differentiation status affects RAGE expression and its contribution to malignant potential. In vitro experiments revealed that inhRAGE significantly suppressed cell migration and invasion without affecting proliferation in all RAGE-expressing cell lines.

RAGE signaling has been implicated in tumor progression in various malignancies, particularly through its interaction with high mobility group box 1 (HMGB1). Takeuchi et al. reported that HMGB1 stimulation activates downstream RAGE-dependent pathways, including NF-κB, Cdc42, and Rac1, thereby enhancing proliferation, migration, and invasion in human fibrosarcoma HT1080 cells¹². In addition to its direct effects on tumor cells, extracellular HMGB1 can bind to RAGE expressed on inflammatory and endothelial cells, activating MAPK/NF-κB signaling and inducing pro-inflammatory mediators such as TNF-α and interleukins, which further promote tumor progression and metastasis¹³. Consistent with these findings, HMGB1–RAGE signaling has been shown to enhance tumor growth and invasiveness in glioma, melanoma, and glioblastoma models^14–16. Collectively, these observations highlight the broader oncogenic role of RAGE signaling and support its biological relevance in PDAC.

In the present study, additional genetic validation experiments further supported the involvement of RAGE signaling in PDAC cell migration. Knockdown of AGER suppressed Rac1 activation and attenuated HMGB1-induced cell migration, indicating that the pro-migratory effects of HMGB1 are mediated, at least in part, through RAGE-dependent Rac1 signaling pathways. In addition, Rac1 is known to interact with multiple signaling pathways involved in cytoskeletal remodeling, cell motility, and tumor invasion, suggesting that inhibition of the RAGE–Diaph1–Rac1 axis may influence broader signaling networks that contribute to PDAC progression. These findings provide mechanistic support for the role of RAGE signaling in promoting malignant phenotypes of PDAC cells and support the specificity of pharmacological RAGE inhibition.

Collectively, these results provide genetic and signaling evidence supporting target engagement of RAGE inhibition in PDAC cells. While EMT, stemness, or metabolic reprogramming were not examined in the present study, these processes have been reported to be influenced by RAGE signaling in other malignancies and warrant further investigation in PDAC.

Moreover, treatment with inhRAGE suppressed PDAC tumor growth in both ectopic and orthotopic xenograft models. Systemic treatment with inhRAGE has also been found to inhibit angiogenesis and inflammatory reactions inside implanted tumor tissues, which comprise a critical tumor microenvironment surrounding PDAC cells. Previous studies have found a positive correlation between RAGE expression and the metastatic properties of pancreatic cancer cells¹⁷. However, no studies have examined the use of RAGE inhibitors, including inhibitors of RAGE signaling pathways.

In other strategies targeting RAGE, antagonists, such as azeliragon (in phase 3 clinical trials), FPS-ZM1, low-molecular-weight heparin, and papaverine, have been reported to block RAGE ligand binding to its extracellular V-type domain^18–21. They can also inhibit the intracellular signal transduction of RAGE. In addition, soluble RAGE-type molecules acting as decoy-type receptors against RAGE may be beneficial in preventing RAGE ligands from interacting with cell-surface RAGE. These approaches for blocking RAGE signaling pathways or combination therapy with RAGE signaling inhibitors such as inhRAGE are expected to inhibit the malignant phenotypes of PDAC in the future.

It has previously been reported that patients with severe postoperative surgical-site infectious complications after surgery for PDAC, such as pancreatic fistula, have a significantly worse prognosis²². Additionally, this study showed that tumor- and inflammation-related proteins (HMGB1) and inflammatory mediators (S100B) are involved in the activation of cell migration and invasion via RAGE in PDAC cells. Treatment with inhRAGE also improved the intra- and extra-tumoral microenvironments of PDAC. In particular, immunohistochemical analysis of tumor tissues from the xenograft models demonstrated a significant decrease in CD68-positive macrophage infiltration following inhRAGE treatment. We also observed a reduction in CD31-positive endothelial cells and decreased expression of the inflammatory mediator S100B. These findings suggest that the antitumor effect of inhRAGE in vivo may be mediated, at least in part, through modulation of the tumor microenvironment, including suppression of inflammatory cell infiltration and tumor-associated angiogenesis, rather than through a direct inhibitory effect on cancer cell proliferation. These results support the theory that inflammation associated with severe complications after surgery worsens the prognosis of PDAC.

In this study, not only ectopic but also orthotopic xenografted models were used. In patient-derived tumor transplantation models, orthotopic xenograft models have been created for various carcinomas, such as breast, colon, stomach, and pancreatic cancer^23–26. Orthotopic xenograft models have the advantage that the hemodynamic behavior of the tumor can be observed in a manner that is specific to each organ, metastases can be assessed, and orthotopic transplantation of tumor tissue can cause metastasis similar to that observed in patients. This model better reproduces the behavior of the tumor in the patient, including metastasis, than the subcutaneous transplantation model and is closer to the clinical condition. In this study, an orthotopic xenograft model was used to evaluate the results in a more clinically relevant manner.

The current study has several limitations, as described below. This study used cell lines rather than patient-derived cancer cells. The effect of inhRAGE may be limited by varying degrees of RAGE expression in each cell line. However, the adverse effects of inhRAGE in humans remain unknown. This was also a small-scale animal study.

In conclusion, RAGE expression was detected in PDAC cell lines. The ligands HMGB1 and S100B promoted migration and invasion, whereas inhRAGE treatment inhibited them. In the ectopic/orthotopic xenograft model, inhRAGE inhibited tumor growth. These results suggest that treatment with inhRAGE may be a novel therapy for the malignant phenotypes of PDAC.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

H.M., S.M., Y.Y. and S.Y. designed and conceived this study. H.M., S.M. and T.A. collected data. T.T., R.T., S.T., M.O. S.N. and I.M. were involved in planning and supervised the work. H.M, S.M., Y.Y. and S.Y analyzed and interpreted the results. H.M. wrote the main manuscript text. H.M. and S.M. prepared all figures. All authors discussed the results and commented on the manuscript.

Funding

This study was supported by KAKENHI 23K15491.

Data availability

The data that support the findings of this study are available from the corresponding author, upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Disclosure

I certify that all my affiliations or financial involvement with any organization or financial conflict with the subject matter discussed in the manuscript are completely disclosed in the acknowledgement section of the manuscript.