Inhibition of adipose tissue-derived fatty acid binding protein suppresses pancreatic cancer progression and metastasis

Inhibition of adipose tissue-derived fatty acid binding protein suppresses pancreatic cancer progression and metastasis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Inhibition of adipose tissue-derived fatty acid binding protein suppresses pancreatic cancer progression and metastasis Shuhei Shinoda, Naohiko Nakamura, Kazuho Inoko, Mizuho Sato-Dahlman, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5404541/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, and obesity is a known risk factor for PDAC. Fatty acid binding protein 4 (FABP4) is noted to be higher in obese patients, and linked to the progression of obesity-related cancers. This study aimed to elucidate the role of FABP4 and the anticancer effect of FABP4 inhibition in PDAC using preclinical mouse models. Methods In mouse PDAC cells derived from genetic pancreatic cancer model with KRASG12D and p53 mutation, and human PDAC cell lines, we assessed cell viability, cellular proliferation, apoptosis, and invasion capability after FABP4 and/or FABP4 inhibitor (HTS01037) treatment. The antitumor effect of FABP4 inhibition was evaluated with syngeneic PDAC tumor in FABP4 null (AKO) mice as well as syngeneic and xenogeneic subcutaneous tumor models in mice treated with HTS01037. HTS01037 treatment was also tested in orthotopic as well as liver metastasis models. We analyzed epithelial-mesenchymal transition (EMT) and cancer stemness makers in vitro and vivo samples. In addition, efficacy of combination therapy of gemcitabine (GEM) plus HTS01037 was assessed in the syngeneic model. Results In vitro , HTS010137 suppressed FABP4-induced cell viability in human and murine PDAC cells. FABP4 increased cellular proliferation, and HTS01037 reversed the changes and increased apoptosis. FABP4 promoted migration and invasive potency, and increased EMT and stemness markers that were associated with up-regulation of EMT activating transcription factor ZEB1. Both FABP4 knockout and inhibition with HTS01037 suppressed the syngeneic subcutaneous tumor growth with reduction of EMT and stemness. Similar to the syngeneic tumors, the xenogeneic tumor growth was inhibited by HTS01037 treatment. HTS01037 showed significant anticancer and antimetastatic effect which improved the survivals in the orthotopic model. HTS01037 also attenuated development and growth of liver metastases in the liver metastasis model. Moreover, HTS01037 enhanced the efficacy of GEM to PDAC in vitro and in vivo . Conclusion FABP4 promoted the PDAC progression and FABP4 inhibition showed significant anticancer effect by suppressing cellular proliferation, EMT, and cancer stemness. FABP4 inhibitor has a promising translational value for PDAC treatment and can be a critical therapeutic option in PDAC patients. Pancreatic ductal adenocarcinoma fatty acid binding protein 4 metastasis obesity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive cancer with a dismal 5-year survival rate of < 10% and is estimated to become the 2nd leading cause of cancer related mortality by 2030 in the United States 1 2 3 4 . While primary tumor invasion and distant metastasis are representing features and the leading causes of mortality among PDAC patients 5 , the exact reasons that lead to the local aggressive advancement and distant metastasis have not been fully understood. Many PDAC risk factors (e.g. age, sex, gene mutations, smoking, chronic pancreatitis, diabetes) have been reported, and clinical data indicated that obesity and accumulation of adipose tissue (AT) were correlated to PDAC incidence 3 6 7 . Considering with the increased prevalence of obesity worldwide, there is an urgent need to identify therapeutic targets for PDAC related to obesity. Recent studies suggest that multiple factors including inflammation, adipokines, and lipid metabolism that are deeply associated with AT could affect the incidence and progression of PDAC 8 9 10 11 12 . However, the key mechanisms of how the AT changes affect PDAC progression have not been determined, and it therefore is still unclear if inhibition of the AT-derived factors have a potential clinical value for the treatment of PDAC patients. Fatty acid-binding proteins (FABPs), a family of intracellular lipid chaperones, are small water-soluble proteins that can affect lipid fluxes, signaling and metabolism 13 14 . Among 12 FABP family members, FABP 4 (alternatively called adipocyte protein 2, aP2) is mainly released from adipocytes and macrophages that consist of AT, and plays an important role in the development of insulin resistance and metabolically driven chronic inflammation 13 14 . Higher levels of circulating FABP4 have been reported in obese subjects 15 , and recent studies indicated that FABP4 is linked to the development, invasion, and metastasis of multiple kinds of cancers known as an obesity-related malignancy 16 17 18 19 . Most importantly, higher FABP4 expression in the tumors by immunohistochemistry was correlated with poor prognosis in PDAC patients 20 , suggesting that FABP4 can be involved in PDAC progression. However, the mechanism of FABP4-induced PDAC progression and the in vivo anticancer effect of FABP4 inhibition in PDAC has not been comprehensively elucidated. Recently, we reported that FABP4 increased cell proliferation and downregulation of reactive oxygen species (ROS) activity in PDAC cells 21 . The cascades of cancer progression consist of multi-factorial events associated with cell proliferation, apoptosis, and cell cycle 22 . Furthermore, epithelial–mesenchymal transition (EMT), especially the intermediate states between epithelial and fully mesenchymal condition referred to as partial or hybrid EMT 23 , as well as cancer stemness contribute to cancer invasion and early-stage dissemination as well as therapeutic resistance of PDAC 24 25 . In this context, we hypothesized that exogenous FABP4 can trigger the protumor-progression pathways in PDAC and FABP4 inhibition may suppress local tumor growth and distant metastasis via multiple mechanisms, such as cell proliferation, EMT, and stemness. We have already identified and characterized HTS01037 (HTS), an inhibitor of fatty acid binding and a competitive antagonist of protein-protein interactions mediated by FABP4 26 . The aim of present study was to elucidate the impact of exogenous FABP4 on the PDAC progression and identify the anticancer mechanisms of FABP4 inhibition in preclinical mouse models to determine the potential of FABP4 inhibitor for PDAC treatment in clinical setting. Methods Cell Lines KPC cells, murine pancreatic cancer cell line with KRASG12D and p53 mutation isolated from genetic pancreatic cancer model with KRASG12D and p53 mutation (KPC mice) 27 with C57BL/6J background, was used in all experiments. KPC cell is kindly provided by Dr. Ashok Saluja (Sylvester Comprehensive Cancer Center, University of Miami, Miami, Florida, U.S). We also used human pancreatic cancer cell lines; PANC-1 (CRL-1469), CAPAN-2 (HTB-80), CFPAC-1 (CRL-1918), and MIA PaCa-2 (CRL-1420, ATCC, Manassas, Virginia), and human gastrointestinal cancer cell lines; HT29 (HTB-38, ATCC) and GIST-T1 (provided by Dr. Yujiro Hayashi, Ph.D. Mayo Clinic, Rochester, MN). KPC, PANC-1, CFPAC-1, MIA PaCa-2, and GIST-T1 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and a 1% penicillin-streptomycin mixture (100 IU/mL and 100 µg/mL, respectively). CAPAN-2 and HT29 cells maintained in McCOY’S 5A Medium and OE19 cells in RPMI1640 Medium with 10% FBS and a 1% penicillin-streptomycin mixture. All cells were cultured at 37°C in a humidified incubator with 5% CO 2 . Reagents Recombinant FABP4 protein was purchased from Abcam (ab133145; Cambridge, MA, USA), and HTS01037 (HTS) was purchased from Cayman chemical (Cayman-10699; Ann Arbor, Michigan, USA). Cell viability and proliferation Cellular proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Cell suspensions (3000 cells/100 µl) were added to each well in a 96-multiwell culture plate and incubated at 37°C for 24 hours. HTS (0.03, 0.06, 0.12, 0.24, 0.49, 0.98, 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250 µM), FABP4 (100 ng/ml) and FABP4 + HTS (30 µM) were subsequently added to each well, and the cells were incubated for a further 48 hours. At the end of the culture period, 10 µl of CellTiter-96®Aqueous One-Solution Cell Proliferation Assay MTS reagent (Promega, Madison, WI, USA) was added to each 100 µl of culture media, which were incubated for 2 hours. Absorbance at 490 nm was measured with a multimode reader and the results were expressed as the percentage viable with respect to the untreated control. Cell cycle/ Apoptosis/ Reactive Oxygen Species (ROS) analysis KPC cells were seeded into 100 mm dishes and cultured with phosphate-buffered saline (PBS) as a control or FABP4 (100 ng/ml) or FABP4 + HTS (30 µM) for 48 hours. The assays were performed as previously described 21 . Scratch Assay KPC Cells were seeded into 6-well plate for a confluence at around 90% with DMEM containing 10% FBS. Wound was scratched using 200 µL sterile tips and the cells were cocultured with 0.1% FBS DMEM containing recombinant FABP4 (0, 50, 100, and 200 ng/ml). At the same time, 30 µM of HTS was added into the medium. The area of wound was observed under the microscope every 24 hours continuously and the area dimension at the same place in the wound was calculated by Image J software. The motility of cells was measured by the formula percentage of wound area that was calculated by the ratio of the wound area at 24 hours or 48 hours to the area at 0 hours in each group. Invasion Assay Invasion assay was performed using the CytoSelect 24-well Cell Invasion Assay kit (Cell Biolabs, Inc.) according to the manufacturer’s protocol. 5 x 10 5 cells (KPC or PANC-1 cells) were seeded in the cell culture upper insert after the rehydration of the basal membrane in FBS-free DMEM media. The lower well of the invasion plate was filled with DMEM media supplemented with 5% FBS. 200 ng/ml FABP4 was added to the both upper inserts and lower wells. HTS (30 µM) was added the upper chamber only. The setup was incubated for 24 hours in a standard tissue culture incubator. After the extraction and staining of the invaded cells from the basal membrane, the lysates from the extracted cells were analyzed at OD 560 using FLUOstar Omega microplate reader (BMG LABTECH). Gemcitabine (GEM) and HTS combination therapy in vitro Cell viability was evaluated by MTS assay as same protocol of cellular proliferation assay. KPC or PANC-1 cells suspensions (5000 cells/100 µl) were added to each well in a 96-multiwell culture plate and incubated at 37°C for 24 hours. GEM (0, 20, 50, 100, 200 µM) and HTS (30 µM) were subsequently added to each well, and the cells were incubated for a further 48 hours. The results were expressed as the percentage viable with respect to the untreated control. Immunoblotting Protein was extracted with Lysis buffer and 40 µg protein of each sample was applied per lane of a 4 to 20% gradient gel (Bio-Rad). Electrophoresis was performed at 0.02 A at room temperature with SDS loading buffer. The gels were transferred onto polyvinylidenedifluoride membranes in transfer buffer using Trans-Blot Turbo (Bio-Rad). After blocking with Intercept Blocking Buffer (LI-COR) for 1 hour at room temperature, the membranes were probed with a primary antibody overnight at 4°C. Antibodies against the following proteins were used: polyclonal rabbit anti-vimentin (Proteintech, 10366-1-AP, 1:1000); monoclonal mouse anti-CDH1 (Abcam, ab231303, 1:1000); monoclonal rabbit β-actin (Cell Signaling, 13E5, 1:1000). Membranes were washed and incubated with secondary antibody conjugated to Li-Cor IRDye for 1 hour and visualized using Odyssey infrared imaging (Li-Cor Biosciences). RNA Isolation and Quantitative Reverse-Transcription Polymerase Chain Reaction (RT-PCR) Total RNA was isolated using the RNeasy Mini Kit (Qiagen, 74106) from samples and reverse-transcribed with PrimeScript RT Master Mix (Takara, RR036A) using the manufacturers’ protocols. Quantitative RT-PCR was performed using a Roche LightCycler 480 II and PowerUp SYBR Green Master Mix (Thermo Fisher, A25742). Gene expression was calculated by LightCycler Software (Roche). Primer sequences are described in Supplementary Table 1. Immunofluorescence Tumors were fixed with formalin for hematoxylin eosin stain (HE) and immunostaining. In immunohistochemical analysis, after deparaffinization and rehydration, antigen retrieval was applied by steamer for 30 minutes with citrate buffer (Sigma-Aldrich, C9999) and the endogenous peroxidase was destroyed by 3% hydrogen peroxide. Blocking was carried out for 30 minutes at room temperature using the blocking reagent from Immunoperoxidase Secondary Detection System (Millipore, DAB500). Primary antibodies were reacted at 4°C overnight and then the secondary antibody with fluorescence was incubated for 1 hour at room temperature. The following primary antibodies were used: polyclonal rabbit anti-vimentin (Proteintech, 10366-1-AP, 1:200); monoclonal mouse anti-E-cadherin (Abcam, ab231303, 1:200); monoclonal rabbit anti-CD133 (Abcam, ab271092, 1:200). Slides were stained with 4′,6-diamidino-2-phenylindole (DAPI). All images were acquired on a Leica LAS X microscope. Animal Studies All experimental procedures using animals were approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC, #1911-37568A). Whole body FABP4 null (AKO) mice, FABP4 +/- heterozygous (WT), and C57BL/6J mice were bred and maintained in the animal facility of the University of Minnesota with authorized protocol from the IACUC. FABP4 null mice with subcutaneous syngeneic tumor : After feeding a high-saturated-fat diet (F3282; BioServe, Flemington, NJ) for 12 weeks after weaning, mice were injected with KPC cells (2 × 10 6 cells) into the flank of the AKO mice (n = 6) and WT mice (n = 9). Tumor volume was measured three times weekly. The tumor volume was calculated using the following formula: estimated tumor volume = (shortest diameter 2 × longest diameter)/2. At the end of experiment, mice were euthanized in accordance with the guidelines of the IACUC. Syngeneic tumor mouse model treated by HTS : KPC cells (2 × 10 6 cells) were injected subcutaneously into the backs of the C57BL/6J mice. When the average estimated tumor volume reached to 140 mm³ (14 days after KPC cells inoculation), the mice were divided into three groups: Control (n = 7), HTS 1.5mg/kg (n = 7), and HTS 5mg/kg (n = 7) groups. HTS groups of mice received HTS by intraperitoneal (i.p.) administration, and control group of mice received PBS alone at day 0. The growth of subcutaneous tumors in each group was observed until day 11. In experiments for assessment of EMT and stemness markers, we also collected the tumors after 2 days of PBS or HTS 5 mg/kg treatment. A protocol of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, serum biochemistry, and serum HTS concentration analysis is provided in Supplementary Materials. To assess antitumor effect of HTS in AKO mice, treatment of PBS (n = 3) or HTS 5 mg/kg i.p. (n = 4) were performed in AKO mice with KPC cell subcutaneous tumors that were established by same condition as above. The tumor volume in each group was observed until day 13 after the treatment. Measurement for FABP4 level The subcutaneous tumors from AKO and WT mice were excised at day 28 and processed for intratumoral FABP4 analysis. Blood samples were collected simultaneously during subcutaneous tumor removal. Tumor and serum FABP4 were assessed with mouse FABP4 ELISA (ab277426, Abcam) according to the manufacturer’s instructions. TUNEL assay Tumors from syngeneic mice model injected with vehicle alone or HTS 5 mg/kg were excised and processed at day 2 and apoptotic tumor cells were detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using the ApopTag Fluorescein In Situ Apoptosis Detection Kit (Sigma Aldrich) according to the manufacturer’s protocol. All slides were scanned using a Leica DM5500 B (Leica microsystems, Buffalo grove, IL, USA). After choosing the random 3 area, the stain-positive area was measured using ImageJ software (National Institute of Health, Maryland, USA). In experiments for assessment of EMT and stemness markers, we also collected the tumors after 2 days of vehicle alone or HTS 5mg/kg treatment. At the end of experiment, mice were euthanized in accordance with the guidelines of the IACUC. Serum biochemistry Blood samples were collected at day 11 from syngeneic tumor mouse model treated by HTS. Serum biochemistries were analyzed by IDEXX BioAnalytics (Columbia, MO, USA). Serum HTS concentration analysis To analyze the serum HTS concentration in syngeneic tumor mouse model, blood samples were collected 20 min after receiving vehicle control and HTS 5mg/kg intraperitoneal administration. Serum HTS levels were measured by using high performance liquid chromatography (HPLC) system. Dr. Steven Carmella and Dr. Stephen Hecht developed a reasonable HPLC system and assay method for HTS. Reverse phase HPLC system with UV detection at 331 nm was used for the HTS. We used S-Phenylmercapturic acid (SPMA) as the internal standard. Orthotopic tumor mouse model Orthotopic tumors were generated by injecting 1 × 10 6 KPC cells in 75% Matrigel (Corning Life Sciences, Corning, NY) with PBS directly into the pancreas tail of C57BL/6J mice. The next day of inoculation, the orthotopic mice received i.p. injection of HTS 5mg/kg (n = 5) or PBS (n = 5), and euthanized 8 days after KPC orthotopically inoculation. The volume of orthotopic tumor was calculated using the formula mentioned before. For the survival analysis using the orthotopic model, we orthotopically injected 1 × 10 5 KPC-Luc/RFP cells and performed the treatment of HTS 5 mg/kg (n = 6) or PBS (n = 6) after 14 days of the cell inoculation. The orthotopic tumor growth was monitored by bioluminescence imaging with in vivo imaging system (IVIS) spectrum (Supplementary Materials), and the survival rate and incidence of distant metastases were assessed in each group. Liver metastasis mouse model The liver metastasis model was established by direct injection of 1 × 10 6 KPC cells into spleen of C57BL/6J mice. HTS 5 mg/kg (n = 6) or PBS (n = 6) were injected 30 minutes before the KPC injection into the spleen. The mice were euthanized 14 days after the KPC inoculation, and presence of liver tumors were analyzed. In another experiment using the liver metastasis model, we started the treatment of HTS 5 mg/kg (n = 5) or PBS (n = 5) after seven days of intrasplenic injection of KPC-Luc/RFP cells (1 × 10⁶) and continued the treatment every five days. Liver metastases in the HTS and control groups were observed by IVIS at day 0 and 14, and all the mice were euthanized at day 21 after the treatment. IVIS spectrum Bioluminescent imaging was performed using a highly sensitive, IVIS Spectrum in vivo imaging system (PerkinElmer). For all in vivo imaging, D-luciferin solution was intraperitoneally injected (75 mg/kg) in all mice for 5 minutes. Mice were then anesthetized with 2% isoflurane prior and during imaging with IVIS spectrum. Bioluminescence of D-luciferin carried KPC-Luc/RFP tumor cells was quantified using Living Image. GEM and HTS combination therapy in syngeneic tumor mouse model : KPC cells (1 × 10⁶ cells) were injected subcutaneously into the backs of the C57BL/6J mice. After 7 days of the inoculation, the mice were divided into three groups: Control (PBS i.p. n = 6), GEM (50 mg/kg i.p. n = 6), and GEM + HTS (50 mg/kg and 5 mg/kg i.p. n = 6) groups. The tumor volume was measured every three days using the formula mentioned before. Xenograft mouse models CAPAN-2, CFPAC-1, MIA PaCa-2, or PANC-1 cells (3 × 10⁶ cells) were injected subcutaneously into the backs of nude mice (male. 6–8 weeks of age). When the average estimated subcutaneous tumor volume reached to around 80–100 mm³, the mice received PBP (n = 3–4) or HTS 5mg/kg (n = 3–4) i.p. treatment (Day 0). The growth of subcutaneous tumors in each group was observed until day 14 and the tumor volume was measured using the formula mentioned before. Statistical Analysis Data are shown as mean ± standard deviation (SD). Statistical analysis was performed using student’s t-tests or a non-parametric Wilcoxon test. For all tests, differences with P < 0.05 were considered significant. All statistical analyses were performed using JMP 9.0 software (SAS Institute, Cary, NC, USA). Results FABP4 inhibitor attenuated FABP4-induced cell growth in human pancreatic cancer cells To evaluate the effect of extracellularly added FABP4 and FABP4 inhibitor (HTS) in human PDAC cells, we assessed the cell viability after the treatment of FABP4 or FABP4 + HTS by the MTS assay (Fig. 1 A-D). In CAPAN-2, CFPAC-1, and MIA PaCa-2 cells, FABP4 significantly induced the increased cell viability. HTS treatment (30 µM) reverted the FABP4-induced cell viability in CAPAN-2 and CFPAC-1, and significantly suppressed PANC-1 cell viability. While significant suppression for cell viability was not observed in MIA PaCa-2 with 30 µM HTS, the high dose of HTS (60 µM) significantly inhibited the cell viability (Supplementary Fig. 1A). In other gastrointestinal cancers, such as gastrointestinal stromal tumor (GIST-T1) and colorectal cancer (HT29), FABP4 did not increased the cell viability and HTS showed no significant effect (Fig. 1 E and F), suggesting HTS had a specific suppressive effect for FABP4-induced cell growth in human PDAC. FABP4 inhibitor suppressed cell proliferation and induced apoptosis of the mouse pancreatic cancer cells Next, mouse pancreatic cancer cells derived from genetic pancreatic cancer model with KRASG12D and p53 mutation (KPC cells) 27 were used to evaluate the anticancer mechanisms of FABP4 inhibition in vitro and in vivo. We assessed whether FABP4 and its inhibitor can affect the KPC cell viability, proliferation and cell cycle. In the MTS assay, the viability of KPC cells was decreased in a dose-dependent manner when treated with HTS (Fig. 2 A. The IC50 value of HTS was 25.6 µM). While FABP4 treatment significantly increased the KPC cell viability, HTS treatment (30 µM) significantly suppressed the cell viability (Fig. 2 B). In the cell cycle analysis (Fig. 2 C), FABP4 treatment decreased the percentage of G1 phase cells and this change was totally reverted by addition of HTS. These results suggested that FABP4 significantly facilitated the G1 phase cells to entry into the S and G2 phases (release from G1 arrest), and HTS inhibited the activation of cell cycle induced by FABP4. To further characterize the mechanisms of HTS, apoptotic cell death was assessed by flow cytometry using propidium iodide (PI) and Annexin V staining (Fig. 2 D). The percentages of late-stage apoptotic cells (= PI positive cells) were increased by HTS, compared to the FABP4 treatment (Fig. 2 E), indicating that HTS reduced the cell viability of pancreatic cancer cells by not only suppressed proliferation but also increased apoptosis. FABP4 promoted migration and invasion, and induced partial EMT and stemness in vitro To determine whether FABP4 regulates migration and invasion capability in pancreatic cancer cells, we performed scratch assay and invasion assay. In the scratch assay, exogenous FABP4 promoted the migration of KPC cells in dose dependent manner (Fig. 3 A). FABP4 also facilitated the cell migration in time dependent manner that was cancelled by the treatment of HTS (Fig. 3 B-D. Representative images are shown in Supplementary Fig. 1B). In the invasion assay using KPC and PANC-1 (human pancreatic cancer) cell lines, exogenous FABP4 significantly enhanced the invasive capability and the FABP4-induced invasion was completely eliminated by HTS (Fig. 3 E and F). Next, we assessed the effect of FABP4 for induction of EMT phenotype in KPC cells. In Western blotting, the expression of vimentin was gradually increased after the FABP4 exposure (Fig. 3 G). In contrast, FABP4 tended to induce decreased expression of CDH1 (Fig. 3 H). The mRNA expression of vimentin was also increased and CDH1 was decreased by FABP4 exposure in KPC cells (Supplementary Fig. 1C and D). These results mean that FABP4 induces partial EMT in PDAC cells. Regarding with EMT activating transcription factors, the mRNA levels of TWIST1, SNAIL1, and ZEB1 in KPC cells tended to increase after FABP4 exposure (Fig. 3 I-K). Especially, the ZEB1 level was significantly up-regulated by FABP4 exposure. Therefore, we focused on ZEB1 activity as a promising transcription factor that could associate with FABP4-induced EMT. In the KPC and PANC-1 cells, the ZEB1 level was significantly increased by FABP4 exposure and the ZEB1 up-regulation was inhibited by HTS (Fig. 3 L and M). Because ZEB1 activity was also reported to be involved in cancer stemness of pancreatic cancer 28 , we assessed stemness marker in KPC cells by RT-PCR. FABP4 exposure induced significant up-regulation of the stemness markers CD133 and CD44 that were completely attenuated by HTS (Fig. 3 N and O). Aldehyde dehydrogenase 1 (ALDH1) was also increased after FABP4 exposure and SOX2 tended to be suppressed by HTS treatment (Supplementary Fig. 1E and F). These results suggested that HTS suppressed PDAC invasion as well as partial EMT and stemness phenotype associated with activation of ZEB1. FABP4 inhibition reduced tumor growth in mouse syngeneic subcutaneous PDAC tumor Syngeneic subcutaneous KPC cell tumor growth was evaluated in whole body FABP4 null (AKO) and FABP4 +/- heterozygous (WT) mice 29 (Fig. 4 A). The FABP4 knockout significantly inhibited the tumor growth at day 28 after the KPC cells inoculation (Fig. 4 B). Both of the serum and intratumor FABP4 levels in the AKO mice were barely detectable and significantly lower than that in the WT mice (Fig. 4 C and D), indicating that host derived exogenous FABP4 contributed the PDAC tumor growth. Next, antitumor effect and appropriate dose of HTS were assessed in C57BL/6J mice with subcutaneous tumor of KPC cells (Fig. 4 E). The mice received HTS at two different doses (1.5mgkg and 5mg/kg) and control group of mice received vehicle alone at day 0. HTS 5mg/kg significantly suppressed the tumor growth at day 11 compared to control. (Fig. 4 F). Neither of the HTS-treated groups showed any serious side effects, such as weight loss (Fig. 4 G) or altered serum biochemistries (Table 1 ). The serum HTS concentration 20 min after i.p, administration was sufficient to inhibit the FABP4 activity (Table 1 ). In the TUNEL assay using the tumors at day 2 after the treatment (Fig. 4 H and I), tumors treated with HTS 5mg/kg significantly showed higher number of apoptotic cells compared to control (Fig. 4 J). In addition, we assessed the antitumor effect of HTS in AKO mice in order to rule out the possibility that HTS inhibited tumor progression by inhibiting other FABPs (Fig. 4 K). In AKO mice, HTS 5mg/kg did not suppress syngeneic tumor growth significantly, compared to vehicle treatment (Fig. 4 L), indicating the antitumor effect of HTS was mainly caused by inhibition of exogenous FABP4. Body weight change was not observed in the both groups at the end of experiment (Fig. 4 M) Table 1 Serum biochemistry after FABP4 inhibitor administration Control HTS 5mg/kg Total protein 5.3 ± 0.5 4.8 ± 0.2 Albumin (g/dL) 2.5 ± 0.5 2.6 ± 0.1 Total Bilirubin (mg/dL) 0.2 ± 0.1 0.2 ± 0.0 AST (U/L) 188 ± 53 180 ± 24 ALT (U/L) 29 ± 10 35 ± 15 BUN (mg/dL) 19.3 ± 4.5 20.7 ± 3.1 Creatinine (mg/dL) 0.17 ± 0.06 0.10 ± 0.00 HTS (µM) 7.9 ± 2.8 HTS: HTS01037, AST: Aspartate aminotransferase, ALT: Alanine transaminase, BUN: blood urea nitrogen. EMT and stemness markers in subcutaneous tumors were decreased by FABP4 inhibition To analyze the intratumor expression of EMT and stemness markers at earlier time point after treatment, subcutaneous tumors were harvested at day 2 after i.p. injection of HTS or vehicle, and mRNA levels were measured (Fig. 5 A). The intratumor mRNA level of vimentin in the HTS group was significantly lower than that in the control group (Fig. 5 B), while CDH1 level didn’t show significant change in this time point between the groups (Fig. 5 C). The FABP4 inhibitor group showed significantly lower ZEB1 level compared to the control group. However, there was no significant difference in the levels of TWIST1 and SNAIL1 between the groups (Fig. 5 D-F). These results suggested that the suppression of EMT associated with ZEB1 activity could be involved in the antitumor mechanism of HTS. As for stemness markers, the intratumor mRNA level of ALDH1 in the HTS group was significantly lower than that in the control group (Fig. 5 G). The SOX2 level also tended to be suppressed by HTS treatment (Supplementary Fig. 1G). Furthermore, we assessed IL22 expression in the subcutaneous tumors in both the HTS treated and AKO mice because IL22 promotes EMT and stem cell features of PDAC, and is deeply associated with PDAC progression 30 . The tumors treated with HTS (day 11) showed significantly suppressed mRNA level of IL22 (Fig. 5 H). In the AKO mice (Fig. 5 I), the intratumor mRNA and protein level of IL22 were significantly suppressed compared to those in WT mice (Fig. 5 J and K). FABP4 inhibitor suppressed orthotopic tumor growth and metastasis by reducing EMT and stemness We evaluated whether HTS treatment could suppress the progression of orthotopic tumors (Fig. 6 A). The HTS treatment next day of KPC cell inoculation significantly inhibited orthotopic tumor growth (Fig. 6 B and C). In the hematoxylin eosin stain (HE) staining, orthotopic tumors in the HTS group showed less tumor invasion into the normal pancreatic parenchyma, compared to the tumors in the control (Fig. 6 D). In western blotting, the intratumor CDH1 expression in the HTS group was significantly higher than that in the control group (Fig. 6 E and F). Immunofluorescence of orthotopic tumors also evaluated decreased expression of vimentin (Fig. 6 G) and increased expression of CDH1 (Fig. 6 H) in the HTS group. In the staining of stemness markers, CD133 expression was observed in the marginal area of tumor in the control group. In contrast, the expression of CD133 seemed to be suppressed in the HTS group (Fig. 6 I). These results indicated that the FABP4 inhibitor prevent partial EMT and stemness in the orthotopic tumors. Next, we performed HTS treatment after orthotopic KPC-Luc/RFP tumor establishment and assess the survival rate (Fig. 6 J). The chronological imaging of vivo imaging system (IVIS) showed that all the mice in the control exhibited tumor progression from day 0 to day 7 after the treatment, whereas the three mice in the HTS group showed tumor regression and the two mice kept stable disease (Fig. 6 K). In addition, the HTS group showed significantly higher survival rate after the treatment compared to the control group (Fig. 6 L). Most importantly, the two and one control mice developed microscopic liver and lung metastases, respectively (Fig. 6 M), but the HTS group didn’t develop any distant metastases when the mice were euthanized at day 25 after the treatment. We also analyzed the mRNA levels of luciferase in liver tissues of the orthotopic model to assess the existence of micro-liver metastasis of KPC-Luc/RFP cells. The luciferase levels of the liver in the HTS group were significantly lower than that in the control group (Supplementary Fig. 1H). HTS successfully inhibited the cancer dissemination from the primary orthotopic tumors and improved the prognosis of mice. FABP4 inhibitor showed antimetastatic effect in liver metastatic mouse model We determined the inhibitory effect of HTS for metastasis development using a liver metastasis model. The HTS treatment was performed 30 minutes before the intrasplenic inoculation of KPC cells, and the incidence of liver metastasis was measured at day 14 after the treatment (Fig. 7 A). The representative images of macroscopic liver and HE staining in each group are shown in Fig. 7 B. Although most of mice exhibited macroscopic liver metastases in the control group, only 8.3% of mice in the HTS group showed macroscopic liver tumors (Fig. 7 C). The liver weight in the control group was significantly increased due to metastasis formation, compared to those in the HTS group (Fig. 7 D). We choose randomly one slice of the liver specimen from the HE staining slide and microscopically measured the number and area ratio of liver tumors in the whole slice. In the HTS group, the microscopic number and the area ratio of metastasis lesion were significantly decreased, compared to the control group (Fig. 7 E and F). Furthermore, we used another treatment schedule for the liver metastasis mouse model, where we started HTS treatment after establishment of liver metastasis. After seven days of the intrasplenic inoculation of KPC-Luc/RFP cells, mice underwent HTS treatment every five days during 2 weeks, and metastasis progression was assessed by IVIS (Fig. 7 G). Before starting the treatment, we have histologically confirmed the establishment of liver metastases at day 0 (Fig. 7 H). IVIS images revealed that all the mice in the control group showed liver metastasis progression at day 14. In contrast, the HTS group tended to show less progression of liver metastases, although a few mice showed increased area of luciferase expression in the liver at day 14 (Fig. 7 I). The macroscopic liver metastasis formation at day 21 was relatively reduced in the HTS group compared to the control (Fig. 7 J). These suggested that FABP4 inhibitor could have inhibitory effect for metastasis development and growth in the liver. FABP4 inhibitor enhanced gemcitabine chemosensitivity in PDAC To evaluate the translational value of HTS, we assessed whether HTS can affect chemosensitivity to gemcitabine (GEM) in PDAC. In MTS assay, viability of KPC cells was decreased by GEM treatment in dose dependent manner, and the decreased cell viability was increased by FABP4 exposure (Supplementary Fig. 1I). When HTS was combined with GEM treatment, HTS significantly enhanced the killing effect of GEM in both KPC and PANC-1 cell lines (Fig. 8 A and B). Because ROS activity is known to be associated with chemosensitivity for GEM, ROS activity in KPC cells was examined. While the cells exposed by FABP4 showed significantly lower ROS activity than cells without treatment, HTS treatment significantly increased ROS activity (Fig. 8 C). In vivo, we used the mouse model with syngeneic subcutaneous tumor of KPC cells to assess additional effect of HTS to GEM chemotherapy (Fig. 8 D). The individual changes of tumor volume after the treatment in the control, GEM, and GEM plus HTS groups are shown in Fig. 8 E-G. In the GEM only group, the tumor growth was suppressed but some mice showed tumor regrowth from day 3 to day 6. The GEM plus HTS combination therapy showed more enhanced antitumor effect than GEM alone and significantly suppressed tumor growth compared to the control group (Fig. 8 H). Thus, HTS could enhance the GEM chemosensitivity by suppressing EMT and stemness as well as increasing ROS activity in the tumors. FABP4 inhibitor suppressed human PDAC xenograft tumor growth in mice We evaluated antitumor effect of HTS in human PDAC xenograft subcutaneous tumors of nude mice. After the tumor establishment using human pancreatic cancer cells, HTS treatment (5mg/kg) was performed at day 0 and the tumor growth was observed (Fig. 9 A). Individual tumor growth and relative tumor volume in control and HTS groups were shown in Fig. 9 B-D (CAPAN-2), E-G (CFPAC-1), H-J (MIA PaCa-2), and K-M (PANC-1), respectively. In the xenograft models, HTS treatment significantly suppressed CAPAN-2 and CFPAC-1 tumor growth. While the antitumor effect of HTS in MIA PaCa-2 tumors was slightly weaker than that in other tumors, significant difference between the groups was observed at day 19 after the treatment. In contrast, PANC-1 tumors were significantly suppressed by HTS at day 6 and some tumors in the HTS group were shrank down at day 10 (macroscopic findings of tumors at day 10 were shown in Supplementary Fig. 1J). HTS demonstrated the significant anticancer effect in syngeneic as well as xenograft PDAC mouse models, suggesting FABP4 inhibitor has the translational value to human study. Discussion Obesity is one of key factors to stimulate PDAC progression and is associated with a higher risk of PDAC incidence 31 32 . To reduce the relative risk of PDAC, bariatric surgery is considered to be one of the most effective ways for obese patients 33 . We have reported that FABP4 level in obese patients was reduced by bariatric surgery but not intensive lifestyle modification and medical management in patients with type 2 diabetes mellitus 34 . Interestingly, the reduction of FABP4 levels did not show significant correlation with the percentage of body weight loss after the interventions 34 . These suggest that FABP4 might be an independent risk factor for PDAC and FABP4 inhibition can directly impact on PDAC progression beyond improvement of overweight. In this study, exogenous FABP4 induced not only tumor cell growth but also local invasion and metastasis driven by induction of EMT and cancer stem cell phenotype in human and mouse PDAC cells. In vivo, FABP4 inhibition significantly suppressed the progression and distant metastasis, and furthermore enhanced the chemosensitivity of PDAC. FABP4 inhibitor exhibited significant anticancer effect in non-obese preclinical mouse models, suggesting FABP4 inhibitor has promising therapeutic effect in obese as well as non-obese PDAC patients. Biologically, FABP4 is an intracellular and extracellular lipid protein that bind a variety of hydrophobic ligands including fatty acids 14 35 . FABP4 is involved in glucose and lipid metabolism, cell proliferation, and apoptotic process in some cancers 36 37 38 . However, the role of FABP4 in PDAC progression has not completely elucidated. The data in this study showed that exogenous FABP4 increased the PDAC cell proliferation, facilitated the entry of G1 phase cells into the S/G2 cells, while HTS reverted the effect of FABP4 on cell cycle and induced the apoptosis as well. Exogenous FABP4 also increased invasion and migration capability directly in PDAC cells. While it has been known that FABP4 plays important roles in the storage, transportation, and solubilization of fatty acid, and promotes cells proliferation/growth, and invasion 16 39 , our results indicated that FABP4 seems to behave as an adipokine-like stimulant that can directly promote cancer progression. In vivo experiments revealed that FABP4 inhibition suppressed the local tumor progression as well as distant metastases. Interestingly, FABP4 was barely detectable in the serum and subcutaneous tumors in AKO mice. This indicates that release of FABP4 from PDAC cells was minimal in vivo and the main source of FABP4 impacting the tumor growth is supposed to be exogenous FABP4 in the circulation produced by ATs adjacent to tumor. On the other hand, the subcutaneous tumor growth was strongly suppressed by HTS 5mg/kg than FABP4 knockout. Initially, we suspected the enhanced antitumor effect of HTS might be caused by inhibiting other FABPs including FABP5 40 . However, HTS did not show significant antitumor effect in AKO mice. This result means that inhibition of exogenous FABP4 is the only significant mechanism for the antitumor function of HTS. The FABP4 knockout inhibited the primary tumor growth, and the FABP4 inhibitor furthermore suppressed the local invasion and distant metastasis by inhibiting EMT and stemness pathway. The EMT occurs in a gradual manner characterized by several cellular states expressing different levels of epithelial and mesenchymal markers, which called as partial EMT 23 . We revealed retention of CDH1 expression and reduction of vimentin expression in the tumors at several time point after the HTS treatment, indicating that HTS inhibited the process of partial EMT. Because the partial activation of EMT is a critical process for the initiation of the metastatic cascade 41 42 , the FABP4 inhibitor markedly prevented the metastatic cascade by inhibiting the partial EMT, and improved the survival in the mouse models. In addition, FABP4 induced an EMT-activator ZEB1 up-regulation and HTS attenuated the ZEB1 up-regulation in vitro and vivo. Our data suggest that the ZEB1 up-regulation by FABP4 was deeply associated with the induction of partial EMT as well as cancer stemness. The ZEB1 activation, but not other EMT-related transcription factors such as SNAIL1 and TWIST1, was reported to be essential for tumor initiation, invasion, metastasis, and stemness properties in PDAC 28 43 . These evidences supported our findings that ZEB1 is a key transcription factor that can induce partial EMT and cancer stemness in the FABP4-related PDAC progression. In addition to EMT, cancer stemness are associated with progression and metastasis of PDAC 23 44 . Importantly, the partial EMT phenotype is involved in increased cancer stemness that is associated with chemosensitivity 23 45 . We observed the direct suppression of partial EMT and cancer stemness by HTS. Previous reports showed that ZEB1 depletion significantly reduced stemness in the PDAC mouse model 28 , and chemo-resistant PANC-1 cells with EMT characteristics were resensitized to chemotherapy by ZEB1 knockdown 44 . These results indicated that FABP4-induced EMT and stemness associated with ZEB1 activation could affect chemosensitivity in PDAC. Our data revealed that HTS enhanced the cytocidal effect of GEM in PDAC cells, and GEM plus HTS combination therapy showed significantly enhanced antitumor effect in subcutaneous tumors, suggesting that HTS could lead the GEM sensitivity through inhibition of partial EMT and stemness by ZEB1 down-regulation. Interestingly, FABP4 also reduced ROS activity in mouse as well as human PDAC cells 21 . Because the accumulation of intracellular ROS induces chemosensitivity and cell death 46 , up-regulation of ROS activity by HTS might partially contribute the GEM sensitivity and enhanced the anticancer effect. Considering that HTS treatment suppressed tumor growth without serious side effect in not only syngeneic mouse PDAC model but also human PDAC xenograft model in mice, HTS could be a possible candidate for combination therapy with conventional anticancer agents for PDAC patients. In summary, FABP4 plays an important role in tumor growth and progression associated with cell proliferation, EMT, and stemness in PDAC. FABP4 inhibitor showed significant anticancer effect and enhanced chemosensitivity to GEM. The present study suggested that FABP4 is a very promising therapeutic target and FABP4 inhibitor can have the translational value in the clinical setting for the better management of PDAC patients. Abbreviations AKO, whole body FABP4 null; ALDH1, aldehyde dehydrogenase 1; EMT, epithelial–mesenchymal transition; FABPs, fatty acid-binding proteins; GEM, gemcitabine; HE, hematoxylin and eosin; HTS, HTS01037; IC50, half maximal inhibitory concentration; IVIS, in vivo imaging system; Luc, luciferase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; NRF2, nuclear factor E2-related factor 2; PBS, phosphate-buffered saline; PDAC, pancreatic ductal adenocarcinoma; PI, propidium iodide; ROS, reactive oxygen species; RT-PCR, reverse-transcription polymerase chain reaction; SD, standard deviation; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling, WT, FABP4 +/- heterozygous Declarations Acknowledgments Shuhei Shinoda and Naohiko Nakamura contributed equally to this work. We thank Dr. Ashok Saluja for providing KPC cells and giving advices for the experiments with this cell line. Authorship contributions SS and NN contributed equally to this work. MY, SI, DB, SS, and NN contributed conception and design of the study. SS and NN performed experiments and acquired data. SS, NN, KI, and MSD were involved in the development of the methodology. SC and SH performed analysis and interpretation of data. MY, SI, and DB provided supervision. SS and NN wrote draft of the manuscript. MY, SI, and DB revised the manuscript critically for important intellectual content. All authors commented on and approved the final version of the manuscript submitted for publication. Funding This work was supported by grants from UMN Department of Surgery Research Support (to MY) and Eugene C. and Gail V. Sit Chair in Pancreatic and Gastrointestinal Cancer Research Endowment Fund. Ethics approval and consent to participate All experimental procedures using animals were approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC, #1911-37568A). Consent for publication Not applicable. Availability of data and materials All data, analytic methods, and study materials are available to other researchers. Competing interests The authors have declared that no conflict of interest exists. References Hidalgo M, Cascinu S, Kleeff J, et al. Addressing the challenges of pancreatic cancer: future directions for improving outcomes. Pancreatology. 2015;15:8–18. Rahib L, Smith BD, Aizenberg R, et al. Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States. Cancer Res. 2014;74:2913–21. Park W, Chawla A, O'Reilly EM. Pancreat Cancer: Rev JAMA. 2021;326:851–62. Khalaf N, El-Serag HB, Abrams HR, et al. Burden of Pancreatic Cancer: From Epidemiology to Practice. Clin Gastroenterol Hepatol. 2021;19:876–84. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–74. Wood LD, Canto MI, Jaffee EM, et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5404541","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":375824849,"identity":"40d2a60a-132c-4c7b-bcbc-5b7550b7b6b9","order_by":0,"name":"Shuhei Shinoda","email":"","orcid":"","institution":"Yamaguchi University Graduate school of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shuhei","middleName":"","lastName":"Shinoda","suffix":""},{"id":375824850,"identity":"c0a5be40-4ea9-4565-9862-6fd54d4bab0d","order_by":1,"name":"Naohiko Nakamura","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Naohiko","middleName":"","lastName":"Nakamura","suffix":""},{"id":375824851,"identity":"49b7f718-40af-4eea-a255-1fb03d3f76f7","order_by":2,"name":"Kazuho Inoko","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Kazuho","middleName":"","lastName":"Inoko","suffix":""},{"id":375824852,"identity":"da507875-e2b2-492d-95ef-75dc2327f3fd","order_by":3,"name":"Mizuho Sato-Dahlman","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Mizuho","middleName":"","lastName":"Sato-Dahlman","suffix":""},{"id":375824853,"identity":"bddbe27e-e0d3-4859-8bde-c0453c9cd22a","order_by":4,"name":"Steven Carmella","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Steven","middleName":"","lastName":"Carmella","suffix":""},{"id":375824854,"identity":"10520026-30d3-4469-b7a5-6b5fec1d8b01","order_by":5,"name":"Stephen Hecht","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Stephen","middleName":"","lastName":"Hecht","suffix":""},{"id":375824855,"identity":"0255ea76-dc2f-4e63-9c60-6cd618e48e57","order_by":6,"name":"David A Bernlohr","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"A","lastName":"Bernlohr","suffix":""},{"id":375824856,"identity":"ff249ffb-5975-4113-87aa-0a200e9774bc","order_by":7,"name":"Sayeed Ikramuddin","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Sayeed","middleName":"","lastName":"Ikramuddin","suffix":""},{"id":375824857,"identity":"a9cdf387-0c09-48d1-b035-c65a65f9cb24","order_by":8,"name":"Masato Yamamoto","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYNACAwk5fgiLmVgtFRbGkg2kaTlTkWhwgFgtBsdPJ3662SaRYHwjO+0BQ4V1YgNBLWdyN0vntknkmd3I3W7AcCadCC0HcjeAtBQDtWyTYGw7TISW8283/wZqSdw8A6TlHzFagIZL55yRSNwgAdLSQIQWyRtvt1nnVEgYS5x5u00i4Vi6MUEtfOdzN9/OMaiT428H2vKhxlqWoBaFA8i8BELKQUCeoKGjYBSMglEwCgBrjUM7i+HLXQAAAABJRU5ErkJggg==","orcid":"","institution":"University of Minnesota","correspondingAuthor":true,"prefix":"","firstName":"Masato","middleName":"","lastName":"Yamamoto","suffix":""}],"badges":[],"createdAt":"2024-11-06 16:53:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5404541/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5404541/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71056485,"identity":"4d9d2a45-e643-4132-8570-3896289326b5","added_by":"auto","created_at":"2024-12-10 16:17:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":148464,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFABP4 increases cell viability and FABP4 inhibitor cancels FABP4-induced cell viability in human pancreatic cancer cells \u003c/strong\u003e(A-D) Cell viability (n = 3) after FABP4 (100 ng/ml) or FABP4 + FABP4 inhibitor HTS01037 (HTS; 30 μM) treatment for 48 hours was analyzed by MTS assay in human pancreatic cancer cells; CAPAN-2, CFPAC-1, PANC-1, and MIA PaCa-2. (E, F) The viability of other gastrointestinal cancer cells (GIST-T1 and HT29) was also assessed after the treatment. Results are presented as mean ± SD. (* P \u0026lt; .05)\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5404541/v1/0efe32473e5e7bc670f0d70a.png"},{"id":71055561,"identity":"9d56e4b4-ea5e-43d5-a483-983490461f1c","added_by":"auto","created_at":"2024-12-10 16:09:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":174293,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFABP4 inhibitor suppresses proliferation and induces apoptosis of mouse pancreatic cancer cells. \u003c/strong\u003e(A) Cell viability was examined for KPC cells (mouse pancreatic cancer) treated with HTS01037 (HTS) for 48 hours (n = 3) by MTS assay. Based on four-parameter logistic model, half maximal inhibitory concentration (IC50) of HTS was calculated as 25.6 μM. (B) KPC cells were incubated with FABP4 (100 ng/ml), HTS (30 μM), or FABP4 + HTS for 48 hours (n = 3), and cell viability was assessed by MTS assay. (C) Cell cycle was examined for KPC cells treated with vehicle control (PBS), FABP4 (100 ng/ml), or FABP4 + HTS (30 μM) for 48 hours by flow cytometry (n = 3). FABP4 decreased the percentage of G1 phase cells and increased the percentage of G2/M phase cells. These changes were totally reverted by addition of HTS (FABP4 + HTS). (D) Effect of HTS on apoptosis was examined in KPC cells. Cells were incubated with the FABP4 (100 ng/ml) or FABP4 + HTS (30 μM) for 48 hours. Cells were stained with Annexin V and propidium iodide (PI), and examined percentage of PI positive cells by flow cytometry (n = 3). (E) HTS increased percentage of PI positive cells that means late-stage apoptotic cells. Results are presented as mean ± SD. (* P \u0026lt; .05)\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5404541/v1/dbbfe365e431a91b36f12d32.png"},{"id":71055566,"identity":"4173566f-eec2-4a5c-921d-ae051ee8734c","added_by":"auto","created_at":"2024-12-10 16:09:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":273842,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFABP4 increases migration and invasion capability, and changes expression of epithelial–mesenchymal transition (EMT) and stemness markers in pancreatic cancer cells.\u003c/strong\u003e (A-D) The cell motility of KPC cell was measured by scratch assay. The formula percentage of wound area was calculated by the ratio of the wound area to the area before treatment in each group. The wound area ratio after different concentration of FABP4 for 24 hours (A) and chronological change of wound area after FABP4 with or without HTS01037 (HTS; 30 μM) (B-D) were evaluated (n = 3). (E, F) Invasive capability in KPC and PANC-1 cells after FABP4 (200 ng/ml) or FABP4 + HTS (30 μM) treatment for 24 hours was quantified by Cell Invasion Assay kit (n = 4). (G, H) EMT markers in KPC cells were analyzed by immunoblotting. FABP4 (100 ng/ml) exposure induced elevated vimentin expression in time dependent manner and tended to decrease expression of CDH1. (I-K) mRNA levels of EMT-related transcription factors were examined after FABP4 (200 ng/ml, 3 hours) exposure in KPC cells (n = 4) by RT-PCR. (L, M) mRNA level of ZEB1 was increased after FABP4 (200 ng/ml, 3 hours) exposure, and the FABP4-madiated ZEB1 up-regulation was inhibited by HTS (30 μM) in KPC and PANC-1 cells (n = 5). (N, O) mRNA levels of stemness markers (CD133 and CD44) after FABP4 (200 ng/ml) or FABP4 + HTS (30 μM) treatment for 24 hours were evaluated in KPC cells (n = 5). Results are presented as mean ± SD. (* P \u0026lt; .05)\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5404541/v1/96c43cc9461f4625ef68870e.png"},{"id":71055559,"identity":"6424ea71-d03e-44d2-995b-cd3affbad4fa","added_by":"auto","created_at":"2024-12-10 16:09:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":375461,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFABP4 inhibition suppresses syngeneic subcutaneous PDAC tumor growth.\u003c/strong\u003e (A) Syngeneic subcutaneous tumors were established by injecting KPC cells in whole body FABP4 null (AKO, n = 6) and FABP4 +/- heterozygous (WT, n = 9) mice, and the tumor growth was observed until day 28 after the inoculation of KPC cells. Before inoculating KPC cells, the AKO and WT mice fed a high-saturated-fat diet for 12 weeks after weaning. (B) The average tumor volumes at day 28 of the AKO mice and WT mice were 744.0 ± 273.2 and 1186.7 ± 328.1 mm³, respectively. (C) Individual tumor volume in the AKO and WT groups. (D, E) Serum and intratumoral FABP4 levels at day 28 were measured in the AKO and WT mice by ELISA. (F) Syngeneic subcutaneous tumors were established by injecting KPC cells in C57BL/6J mice. After the tumor establishment,HTS01037 (HTS; 1.5 or 5 mg/kg) or PBS were injected intraperitoneally (i.p.) \u0026nbsp;at day 0 and the tumor growth was observed until day 11 (n = 7 mice per cohort). (G) KPC tumor growth was significantly suppressed by HTS 5 mg /kg, compared to control. The average tumor volumes at day 11 of the control, HTS 1.5mgkg and 5mg/kg were 479.4 ±391.4, 408.1 ±423.1, and 200.4 ±117.5 mm³, respectively (H) Individual tumor volume in the control, HTS 1.5mgkg and 5mg/kg groups. (I) There was no difference in body weight at day 11 after the treatment in each group (n = 4). (J, K) Subcutaneous tumor samples were collected after 2 days of PBS or HTS 5 mg/kg treatment to assess the apoptotic cells by TUNEL assay. (L) After choosing the random 3 area of TUNAL assay in control and HTS 5mg/kg groups, the positive area was scored (n = 4 mice per cohort, ×20 magnifications). (M) AKO mice with syngeneic subcutaneous KPC tumors were treated with PBS (n = 3) or HTS 5mg/kg i.p. (n = 4). The tumor growth was observed until day 13 after the treatment. (N) Relative tumor volume in the AKO and AKO + HTS groups. There was no significant difference in tumor growth between the control and HTS groups. (O) Individual tumor volume in in the AKO and AKO + HTS groups. (P) The Body weight in the both groups did not change at the end of experiment (day 13).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5404541/v1/fce5b63f2576d74dbc65a15d.png"},{"id":71056483,"identity":"9b7e92bf-08c3-438b-bf73-08e7080800a9","added_by":"auto","created_at":"2024-12-10 16:17:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":177131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFABP4 inhibitor decreases expression of epithelial–mesenchymal transition (EMT) and stemness markers in subcutaneous tumors \u003c/strong\u003e(A) KPC subcutaneous tumors in C57BL/6J mice were harvested at day 2 (n = 4 mice per cohort) or day 11 (n = 5 mice per cohort) after intraperitoneal (i.p.) treatment of HTS01037 (HTS; 5 mg/kg) or PBS to assess expression of EMT and stemness markers. (B, C) Intratumoral mRNA levels of EMT markers were compared between control and HTS groups (day 2) by RT-PCR. (D-F) Intratumoral mRNA levels of EMT-related transcription factors such as TWIST1, SNAIL1 and ZWB1 were assessed (day 2). (G) Intratumoral mRNA levels of stemness maker, ALDH1, were compared between control and HTS groups (day 2). (H) mRNA levels of IL22, that promotes EMT and stem cell features, were assessed in subcutaneous tumors (day 11) of control and HTS groups. (I) Subcutaneous tumors of whole body FABP4 null mice (AKO, n = 9) and FABP4 +/- heterozygous (WT, n = 6) were harvested at day 28. (J, K) mRNA and protein levels of IL22 were measured by RT-PCR and ELISA, respectively. Results are presented as mean ± SD. (* P \u0026lt; .05)\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5404541/v1/9e300f33061ee388f85c476b.png"},{"id":71056482,"identity":"1b586418-c040-4bfa-b88d-2424e813e763","added_by":"auto","created_at":"2024-12-10 16:17:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1015921,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFABP4 inhibitor shows anticancer effect with inhibition of epithelial–mesenchymal transition (EMT) and stemness in orthotopic tumor mouse model.\u003c/strong\u003e (A) Orthotopic tumors were generated by injecting KPC cells in 75% Matrigel directly into the pancreas tail of C57BL/6J mice. One day after orthotopically inoculation of KPC cells, the mice received HTS01037 (HTS 5 mg/kg, n = 5) or PBS (n = 5) intraperitoneally (i.p.), and euthanized at day 8. (B, C) Macroscopic finding and mean volume of tumors at day 8 were compared between control and HTS groups. The mean volume at day 8 in the control and HTS groups was 605.7 ±115.3 and 130 ±58.5 mm³, respectively. (D) Hematoxylin and eosin (HE) staining revealed orthotopic tumors of HTS group had less tumor invasion into the normal pancreatic parenchyma. (E, F) CDH1 expression of orthotopic tumor was analyzed by immunoblot and the expression in control and HTS groups was quantified. (G-I) EMT (vimentin and CDH1) and stemness (CD133) markers of orthotopic tumor samples at day 8 were assessed by immunohistochemical analysis. Immunofluorescence (green) showed decreased intratumoral expression of vimentin and CD133, and increased expression of CDH1 in HTS group. (J) In the survival analysis using the orthotopic mouse model, we orthotopically injected KPC-Luc/RFP cells and performed i.p. treatment of HTS 5 mg/kg (n = 6) or PBS (n = 6) after 14 days of the cell inoculation. The orthotopic tumor growth was monitored by in vivo imaging system (IVIS) at day 0, 4, and 7 after the treatment. (K) Orthotopic tumor progression at day 7 was suppressed in HTS group compared to control in IVIS. (L) Survival rates showed significant difference between control and HTS groups (p = .03; Log-rank test). (M) Some control mice developed microscopic liver and lung metastases at day 25 after orthotopic injection of KPC-Luc/RFP cells. No distant metastasis was detected in HTS group. Results are presented as mean ± SD. (* P \u0026lt; .05)\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5404541/v1/f9268d1cd0ebb5ebc0911c32.png"},{"id":71056484,"identity":"1f0c79d1-9040-47e9-92a6-ac338813fc98","added_by":"auto","created_at":"2024-12-10 16:17:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":852689,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFABP4 inhibitor shows antimetastatic effect in liver metastasis mouse model.\u003c/strong\u003e (A) To establish liver metastasis, KPC cells were injected into the spleen of C57BL/6J mice. HTS01037 (HTS 5 mg/kg, n = 6) or PBS (n = 6) were intraperitoneally (i.p.) injected 30 minutes before the KPC inoculation and mice euthanized at day 14. (B) HTS treatment decreased macroscopic liver metastasis formation at day 14 (arrow). Hematoxylin and eosin (HE) staining of the liver specimens showed decreased aria of liver metastases in HTS group. (C-F) Incidence of liver metastasis, liver weight, and microscopic number and area ratio of liver metastasis at day 14 were evaluated in control and HTS groups. One slice of the liver specimen was randomly chosen from the HE staining slide and microscopical number and area ratio of liver tumors were calculated in the whole slice. (G) In another experiment using the liver metastasis mouse model, HTS 5 mg/kg (n = 5) or PBS (n = 5) i.p. treatment started seven days after intrasplenic injection of KPC-Luc/RFP cells and progression of liver metastases were observed by in vivo imaging system (IVIS). The treatment was continued every five days until the mice were euthanized at day 21. (H) Before starting the treatment, establishment of micro-liver metastases was histologically confirmed at day 0. (I) Progression of liver metastasis in control and HTS group was assessed by IVIS at day 0 and 14 after the treatment. (J) Macroscopical findings of liver at day 21 showed less progression of liver metastases in HTS group. Results are presented as mean ± SD. (* P \u0026lt; .05)\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5404541/v1/141f33f9bc1c06acd4ba9186.png"},{"id":71055563,"identity":"08ef8dec-797b-495a-b7f0-eddff4d12b14","added_by":"auto","created_at":"2024-12-10 16:09:53","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":852689,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFABP4 inhibitor enhances anticancer effect of gemcitabine (GEM) in vitro and vivo.\u003c/strong\u003e (A, B) Cell viability after GEM monotherapy and HTS01037 (HTS) + GEM combination therapy was assessed in mouse (KPC) and human pancreatic cancer (PANC-1) cells by MTS assay (n = 5). (C) KPC cells were incubated with FABP4 (100ng/ml) or FABP4 + HTS (30 μM) for 48 hours, and ROS activity was quantified by hydrogen peroxide/peroxidase assay (n = 3). (D) Syngeneic tumors were established by injecting KPC cells subcutaneously into C57BL/6J mice. After 7 days of inoculation of KPC cells, mice received treatment with PBS (n = 6), GEM (50 mg/kg, n = 6), or GEM + HTS (50 mg/kg and 5 mg/kg, n = 6) intraperitoneally (i.p.). (E-G) Individual tumor volume ratio in control, GEM, and GEM + HTS groups were measured every three days after the treatment. (H) GEM + HTS combination treatment significantly suppressed tumor growth compared to control. Results are presented as mean ± SD. (* P \u0026lt; .05)\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-5404541/v1/a43beffe0d1cc58701fc7774.png"},{"id":71055567,"identity":"804303f0-08fb-431e-8a57-7bfea0cda0ab","added_by":"auto","created_at":"2024-12-10 16:09:53","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":314732,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFABP4 inhibitor suppresses xenograft PDAC tumor growth.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) For xenograft tumors, human pancreatic cancer cells (CAPAN-2. CFPA-1, MIA PaCa-2, and PANC-1 (3 × 10⁶ cells)) were injected subcutaneously into the backs of nude mice. After the tumor establishment, the mice received PBP or HTS01037 (HTS 5mg/kg) intraperitoneal (i.p.) treatment at day 0. (B, C) Individual CAPAN-2 tumor volume in control and HTS groups were observed until day 13 after the treatment. (D) Relative volume of CAPAN-2 tumor was compared between the groups. (E, F) Individual CFPAC-1 tumor volume in control and HTS groups were observed until day 13 after the treatment. (G) Relative volume of CFPAC-1 tumor was compared between the groups. \u0026nbsp;(H, I) Individual MIA PaCa-2 tumor volume in control and HTS groups were observed until day 19 after the treatment. (J) Relative volume of MIA PaCa-2 tumor was compared between the groups. (K, L) Individual PANC-1 tumor volume in control and HTS groups were observed until day 8 after the treatment. (M) Relative volume of PANC-1 tumor was compared between the groups. Results are presented as mean ± SD. (* P \u0026lt; .05)\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-5404541/v1/6424c69a6bede1c3db37497a.png"},{"id":78098630,"identity":"c9b835bb-223b-4693-8e74-b8082f7869be","added_by":"auto","created_at":"2025-03-10 00:46:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6110109,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5404541/v1/289cb5f7-a74e-4785-8a6d-516960b0af25.pdf"},{"id":71055565,"identity":"80f9ec2c-1763-44e3-9896-d3abbde152bd","added_by":"auto","created_at":"2024-12-10 16:09:53","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":568837,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialsandMethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-5404541/v1/b956a44d3dc51ac484576aed.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Inhibition of adipose tissue-derived fatty acid binding protein suppresses pancreatic cancer progression and metastasis","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePancreatic ductal adenocarcinoma (PDAC) is the most aggressive cancer with a dismal 5-year survival rate of \u0026lt;\u0026thinsp;10% and is estimated to become the 2nd leading cause of cancer related mortality by 2030 in the United States \u003csup\u003e1 2 3 4\u003c/sup\u003e. While primary tumor invasion and distant metastasis are representing features and the leading causes of mortality among PDAC patients \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, the exact reasons that lead to the local aggressive advancement and distant metastasis have not been fully understood. Many PDAC risk factors (e.g. age, sex, gene mutations, smoking, chronic pancreatitis, diabetes) have been reported, and clinical data indicated that obesity and accumulation of adipose tissue (AT) were correlated to PDAC incidence\u003csup\u003e3 6 7\u003c/sup\u003e. Considering with the increased prevalence of obesity worldwide, there is an urgent need to identify therapeutic targets for PDAC related to obesity. Recent studies suggest that multiple factors including inflammation, adipokines, and lipid metabolism that are deeply associated with AT could affect the incidence and progression of PDAC \u003csup\u003e8 9 10 11 12\u003c/sup\u003e. However, the key mechanisms of how the AT changes affect PDAC progression have not been determined, and it therefore is still unclear if inhibition of the AT-derived factors have a potential clinical value for the treatment of PDAC patients.\u003c/p\u003e \u003cp\u003eFatty acid-binding proteins (FABPs), a family of intracellular lipid chaperones, are small water-soluble proteins that can affect lipid fluxes, signaling and metabolism\u003csup\u003e13 14\u003c/sup\u003e. Among 12 FABP family members, FABP 4 (alternatively called adipocyte protein 2, aP2) is mainly released from adipocytes and macrophages that consist of AT, and plays an important role in the development of insulin resistance and metabolically driven chronic inflammation\u003csup\u003e13 14\u003c/sup\u003e. Higher levels of circulating FABP4 have been reported in obese subjects\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, and recent studies indicated that FABP4 is linked to the development, invasion, and metastasis of multiple kinds of cancers known as an obesity-related malignancy \u003csup\u003e16 17 18 19\u003c/sup\u003e. Most importantly, higher FABP4 expression in the tumors by immunohistochemistry was correlated with poor prognosis in PDAC patients \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, suggesting that FABP4 can be involved in PDAC progression. However, the mechanism of FABP4-induced PDAC progression and the in vivo anticancer effect of FABP4 inhibition in PDAC has not been comprehensively elucidated. Recently, we reported that FABP4 increased cell proliferation and downregulation of reactive oxygen species (ROS) activity in PDAC cells \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The cascades of cancer progression consist of multi-factorial events associated with cell proliferation, apoptosis, and cell cycle \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Furthermore, epithelial\u0026ndash;mesenchymal transition (EMT), especially the intermediate states between epithelial and fully mesenchymal condition referred to as partial or hybrid EMT \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, as well as cancer stemness contribute to cancer invasion and early-stage dissemination as well as therapeutic resistance of PDAC \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In this context, we hypothesized that exogenous FABP4 can trigger the protumor-progression pathways in PDAC and FABP4 inhibition may suppress local tumor growth and distant metastasis via multiple mechanisms, such as cell proliferation, EMT, and stemness. We have already identified and characterized HTS01037 (HTS), an inhibitor of fatty acid binding and a competitive antagonist of protein-protein interactions mediated by FABP4 \u003csup\u003e26\u003c/sup\u003e. The aim of present study was to elucidate the impact of exogenous FABP4 on the PDAC progression and identify the anticancer mechanisms of FABP4 inhibition in preclinical mouse models to determine the potential of FABP4 inhibitor for PDAC treatment in clinical setting.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Lines\u003c/h2\u003e \u003cp\u003eKPC cells, murine pancreatic cancer cell line with KRASG12D and p53 mutation isolated from genetic pancreatic cancer model with KRASG12D and p53 mutation (KPC mice) \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e with C57BL/6J background, was used in all experiments. KPC cell is kindly provided by Dr. Ashok Saluja (Sylvester Comprehensive Cancer Center, University of Miami, Miami, Florida, U.S). We also used human pancreatic cancer cell lines; PANC-1 (CRL-1469), CAPAN-2 (HTB-80), CFPAC-1 (CRL-1918), and MIA PaCa-2 (CRL-1420, ATCC, Manassas, Virginia), and human gastrointestinal cancer cell lines; HT29 (HTB-38, ATCC) and GIST-T1 (provided by Dr. Yujiro Hayashi, Ph.D. Mayo Clinic, Rochester, MN). KPC, PANC-1, CFPAC-1, MIA PaCa-2, and GIST-T1 cells were maintained in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and a 1% penicillin-streptomycin mixture (100 IU/mL and 100 \u0026micro;g/mL, respectively). CAPAN-2 and HT29 cells maintained in McCOY\u0026rsquo;S 5A Medium and OE19 cells in RPMI1640 Medium with 10% FBS and a 1% penicillin-streptomycin mixture. All cells were cultured at 37\u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReagents\u003c/h3\u003e\n\u003cp\u003eRecombinant FABP4 protein was purchased from Abcam (ab133145; Cambridge, MA, USA), and HTS01037 (HTS) was purchased from Cayman chemical (Cayman-10699; Ann Arbor, Michigan, USA).\u003c/p\u003e\n\u003ch3\u003eCell viability and proliferation\u003c/h3\u003e\n\u003cp\u003eCellular proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Cell suspensions (3000 cells/100 \u0026micro;l) were added to each well in a 96-multiwell culture plate and incubated at 37\u0026deg;C for 24 hours. HTS (0.03, 0.06, 0.12, 0.24, 0.49, 0.98, 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250 \u0026micro;M), FABP4 (100 ng/ml) and FABP4\u0026thinsp;+\u0026thinsp;HTS (30 \u0026micro;M) were subsequently added to each well, and the cells were incubated for a further 48 hours. At the end of the culture period, 10 \u0026micro;l of CellTiter-96\u0026reg;Aqueous One-Solution Cell Proliferation Assay MTS reagent (Promega, Madison, WI, USA) was added to each 100 \u0026micro;l of culture media, which were incubated for 2 hours. Absorbance at 490 nm was measured with a multimode reader and the results were expressed as the percentage viable with respect to the untreated control.\u003c/p\u003e\n\u003ch3\u003e\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cb\u003eCell cycle/ Apoptosis/ Reactive Oxygen Species (ROS) analysis\u003c/b\u003e\u003c/div\u003e \u003cp\u003eKPC cells were seeded into 100 mm dishes and cultured with phosphate-buffered saline (PBS) as a control or FABP4 (100 ng/ml) or FABP4\u0026thinsp;+\u0026thinsp;HTS (30 \u0026micro;M) for 48 hours. The assays were performed as previously described\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eScratch Assay\u003c/h3\u003e\n\u003cp\u003eKPC Cells were seeded into 6-well plate for a confluence at around 90% with DMEM containing 10% FBS. Wound was scratched using 200 \u0026micro;L sterile tips and the cells were cocultured with 0.1% FBS DMEM containing recombinant FABP4 (0, 50, 100, and 200 ng/ml). At the same time, 30 \u0026micro;M of HTS was added into the medium. The area of wound was observed under the microscope every 24 hours continuously and the area dimension at the same place in the wound was calculated by Image J software. The motility of cells was measured by the formula percentage of wound area that was calculated by the ratio of the wound area at 24 hours or 48 hours to the area at 0 hours in each group.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eInvasion Assay\u003c/h2\u003e \u003cp\u003eInvasion assay was performed using the CytoSelect 24-well Cell Invasion Assay kit (Cell Biolabs, Inc.) according to the manufacturer\u0026rsquo;s protocol. 5 x 10\u003csup\u003e5\u003c/sup\u003e cells (KPC or PANC-1 cells) were seeded in the cell culture upper insert after the rehydration of the basal membrane in FBS-free DMEM media. The lower well of the invasion plate was filled with DMEM media supplemented with 5% FBS. 200 ng/ml FABP4 was added to the both upper inserts and lower wells. HTS (30 \u0026micro;M) was added the upper chamber only. The setup was incubated for 24 hours in a standard tissue culture incubator. After the extraction and staining of the invaded cells from the basal membrane, the lysates from the extracted cells were analyzed at OD 560 using FLUOstar Omega microplate reader (BMG LABTECH).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGemcitabine (GEM) and HTS combination therapy in vitro\u003c/h3\u003e\n\u003cp\u003eCell viability was evaluated by MTS assay as same protocol of cellular proliferation assay. KPC or PANC-1 cells suspensions (5000 cells/100 \u0026micro;l) were added to each well in a 96-multiwell culture plate and incubated at 37\u0026deg;C for 24 hours. GEM (0, 20, 50, 100, 200 \u0026micro;M) and HTS (30 \u0026micro;M) were subsequently added to each well, and the cells were incubated for a further 48 hours. The results were expressed as the percentage viable with respect to the untreated control.\u003c/p\u003e\n\u003ch3\u003eImmunoblotting\u003c/h3\u003e\n\u003cp\u003eProtein was extracted with Lysis buffer and 40 \u0026micro;g protein of each sample was applied per lane of a 4 to 20% gradient gel (Bio-Rad). Electrophoresis was performed at 0.02 A at room temperature with SDS loading buffer. The gels were transferred onto polyvinylidenedifluoride membranes in transfer buffer using Trans-Blot Turbo (Bio-Rad). After blocking with Intercept Blocking Buffer (LI-COR) for 1 hour at room temperature, the membranes were probed with a primary antibody overnight at 4\u0026deg;C. Antibodies against the following proteins were used: polyclonal rabbit anti-vimentin (Proteintech, 10366-1-AP, 1:1000); monoclonal mouse anti-CDH1 (Abcam, ab231303, 1:1000); monoclonal rabbit β-actin (Cell Signaling, 13E5, 1:1000). Membranes were washed and incubated with secondary antibody conjugated to Li-Cor IRDye for 1 hour and visualized using Odyssey infrared imaging (Li-Cor Biosciences).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA Isolation and Quantitative Reverse-Transcription Polymerase Chain Reaction (RT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated using the RNeasy Mini Kit (Qiagen, 74106) from samples and reverse-transcribed with PrimeScript RT Master Mix (Takara, RR036A) using the manufacturers\u0026rsquo; protocols. Quantitative RT-PCR was performed using a Roche LightCycler 480 II and PowerUp SYBR Green Master Mix (Thermo Fisher, A25742). Gene expression was calculated by LightCycler Software (Roche). Primer sequences are described in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eTumors were fixed with formalin for hematoxylin eosin stain (HE) and immunostaining. In immunohistochemical analysis, after deparaffinization and rehydration, antigen retrieval was applied by steamer for 30 minutes with citrate buffer (Sigma-Aldrich, C9999) and the endogenous peroxidase was destroyed by 3% hydrogen peroxide. Blocking was carried out for 30 minutes at room temperature using the blocking reagent from Immunoperoxidase Secondary Detection System (Millipore, DAB500). Primary antibodies were reacted at 4\u0026deg;C overnight and then the secondary antibody with fluorescence was incubated for 1 hour at room temperature. The following primary antibodies were used: polyclonal rabbit anti-vimentin (Proteintech, 10366-1-AP, 1:200); monoclonal mouse anti-E-cadherin (Abcam, ab231303, 1:200); monoclonal rabbit anti-CD133 (Abcam, ab271092, 1:200). Slides were stained with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI). All images were acquired on a Leica LAS X microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnimal Studies\u003c/h2\u003e \u003cp\u003e All experimental procedures using animals were approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC, #1911-37568A). Whole body FABP4 null (AKO) mice, FABP4 +/- heterozygous (WT), and C57BL/6J mice were bred and maintained in the animal facility of the University of Minnesota with authorized protocol from the IACUC.\u003c/p\u003e \u003cp\u003e\u003cb\u003eFABP4 null mice with subcutaneous syngeneic tumor\u003c/b\u003e: After feeding a high-saturated-fat diet (F3282; BioServe, Flemington, NJ) for 12 weeks after weaning, mice were injected with KPC cells (2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells) into the flank of the AKO mice (n\u0026thinsp;=\u0026thinsp;6) and WT mice (n\u0026thinsp;=\u0026thinsp;9). Tumor volume was measured three times weekly. The tumor volume was calculated using the following formula: estimated tumor volume = (shortest diameter\u003csup\u003e2\u003c/sup\u003e \u0026times; longest diameter)/2. At the end of experiment, mice were euthanized in accordance with the guidelines of the IACUC.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSyngeneic tumor mouse model treated by HTS\u003c/b\u003e: KPC cells (2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells) were injected subcutaneously into the backs of the C57BL/6J mice. When the average estimated tumor volume reached to 140 mm\u0026sup3; (14 days after KPC cells inoculation), the mice were divided into three groups: Control (n\u0026thinsp;=\u0026thinsp;7), HTS 1.5mg/kg (n\u0026thinsp;=\u0026thinsp;7), and HTS 5mg/kg (n\u0026thinsp;=\u0026thinsp;7) groups. HTS groups of mice received HTS by intraperitoneal (i.p.) administration, and control group of mice received PBS alone at day 0. The growth of subcutaneous tumors in each group was observed until day 11. In experiments for assessment of EMT and stemness markers, we also collected the tumors after 2 days of PBS or HTS 5 mg/kg treatment. A protocol of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, serum biochemistry, and serum HTS concentration analysis is provided in Supplementary Materials. To assess antitumor effect of HTS in AKO mice, treatment of PBS (n\u0026thinsp;=\u0026thinsp;3) or HTS 5 mg/kg i.p. (n\u0026thinsp;=\u0026thinsp;4) were performed in AKO mice with KPC cell subcutaneous tumors that were established by same condition as above. The tumor volume in each group was observed until day 13 after the treatment.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMeasurement for FABP4 level\u003c/strong\u003e \u003cp\u003eThe subcutaneous tumors from AKO and WT mice were excised at day 28 and processed for intratumoral FABP4 analysis. Blood samples were collected simultaneously during subcutaneous tumor removal. Tumor and serum FABP4 were assessed with mouse FABP4 ELISA (ab277426, Abcam) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTUNEL assay\u003c/strong\u003e \u003cp\u003eTumors from syngeneic mice model injected with vehicle alone or HTS 5 mg/kg were excised and processed at day 2 and apoptotic tumor cells were detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using the ApopTag Fluorescein In Situ Apoptosis Detection Kit (Sigma Aldrich) according to the manufacturer\u0026rsquo;s protocol. All slides were scanned using a Leica DM5500 B (Leica microsystems, Buffalo grove, IL, USA). After choosing the random 3 area, the stain-positive area was measured using ImageJ software (National Institute of Health, Maryland, USA). In experiments for assessment of EMT and stemness markers, we also collected the tumors after 2 days of vehicle alone or HTS 5mg/kg treatment. At the end of experiment, mice were euthanized in accordance with the guidelines of the IACUC.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSerum biochemistry\u003c/strong\u003e \u003cp\u003eBlood samples were collected at day 11 from syngeneic tumor mouse model treated by HTS. Serum biochemistries were analyzed by IDEXX BioAnalytics (Columbia, MO, USA).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSerum HTS concentration analysis\u003c/strong\u003e \u003cp\u003eTo analyze the serum HTS concentration in syngeneic tumor mouse model, blood samples were collected 20 min after receiving vehicle control and HTS 5mg/kg intraperitoneal administration. Serum HTS levels were measured by using high performance liquid chromatography (HPLC) system. Dr. Steven Carmella and Dr. Stephen Hecht developed a reasonable HPLC system and assay method for HTS. Reverse phase HPLC system with UV detection at 331 nm was used for the HTS. We used S-Phenylmercapturic acid (SPMA) as the internal standard.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eOrthotopic tumor mouse model\u003c/strong\u003e \u003cp\u003eOrthotopic tumors were generated by injecting 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e KPC cells in 75% Matrigel (Corning Life Sciences, Corning, NY) with PBS directly into the pancreas tail of C57BL/6J mice. The next day of inoculation, the orthotopic mice received i.p. injection of HTS 5mg/kg (n\u0026thinsp;=\u0026thinsp;5) or PBS (n\u0026thinsp;=\u0026thinsp;5), and euthanized 8 days after KPC orthotopically inoculation. The volume of orthotopic tumor was calculated using the formula mentioned before. For the survival analysis using the orthotopic model, we orthotopically injected 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e KPC-Luc/RFP cells and performed the treatment of HTS 5 mg/kg (n\u0026thinsp;=\u0026thinsp;6) or PBS (n\u0026thinsp;=\u0026thinsp;6) after 14 days of the cell inoculation. The orthotopic tumor growth was monitored by bioluminescence imaging with in vivo imaging system (IVIS) spectrum (Supplementary Materials), and the survival rate and incidence of distant metastases were assessed in each group.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eLiver metastasis mouse model\u003c/strong\u003e \u003cp\u003eThe liver metastasis model was established by direct injection of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e KPC cells into spleen of C57BL/6J mice. HTS 5 mg/kg (n\u0026thinsp;=\u0026thinsp;6) or PBS (n\u0026thinsp;=\u0026thinsp;6) were injected 30 minutes before the KPC injection into the spleen. The mice were euthanized 14 days after the KPC inoculation, and presence of liver tumors were analyzed. In another experiment using the liver metastasis model, we started the treatment of HTS 5 mg/kg (n\u0026thinsp;=\u0026thinsp;5) or PBS (n\u0026thinsp;=\u0026thinsp;5) after seven days of intrasplenic injection of KPC-Luc/RFP cells (1 \u0026times; 10⁶) and continued the treatment every five days. Liver metastases in the HTS and control groups were observed by IVIS at day 0 and 14, and all the mice were euthanized at day 21 after the treatment.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eIVIS spectrum\u003c/strong\u003e \u003cp\u003eBioluminescent imaging was performed using a highly sensitive, IVIS Spectrum in vivo imaging system (PerkinElmer). For all in vivo imaging, D-luciferin solution was intraperitoneally injected (75 mg/kg) in all mice for 5 minutes. Mice were then anesthetized with 2% isoflurane prior and during imaging with IVIS spectrum. Bioluminescence of D-luciferin carried KPC-Luc/RFP tumor cells was quantified using Living Image.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGEM and HTS combination therapy in syngeneic tumor mouse model\u003c/b\u003e: KPC cells (1 \u0026times; 10⁶ cells) were injected subcutaneously into the backs of the C57BL/6J mice. After 7 days of the inoculation, the mice were divided into three groups: Control (PBS i.p. n\u0026thinsp;=\u0026thinsp;6), GEM (50 mg/kg i.p. n\u0026thinsp;=\u0026thinsp;6), and GEM\u0026thinsp;+\u0026thinsp;HTS (50 mg/kg and 5 mg/kg i.p. n\u0026thinsp;=\u0026thinsp;6) groups. The tumor volume was measured every three days using the formula mentioned before.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eXenograft mouse models\u003c/strong\u003e \u003cp\u003eCAPAN-2, CFPAC-1, MIA PaCa-2, or PANC-1 cells (3 \u0026times; 10⁶ cells) were injected subcutaneously into the backs of nude mice (male. 6\u0026ndash;8 weeks of age). When the average estimated subcutaneous tumor volume reached to around 80\u0026ndash;100 mm\u0026sup3;, the mice received PBP (n\u0026thinsp;=\u0026thinsp;3\u0026ndash;4) or HTS 5mg/kg (n\u0026thinsp;=\u0026thinsp;3\u0026ndash;4) i.p. treatment (Day 0). The growth of subcutaneous tumors in each group was observed until day 14 and the tumor volume was measured using the formula mentioned before.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analysis was performed using student\u0026rsquo;s t-tests or a non-parametric Wilcoxon test. For all tests, differences with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant. All statistical analyses were performed using JMP 9.0 software (SAS Institute, Cary, NC, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFABP4 inhibitor attenuated FABP4-induced cell growth in human pancreatic cancer cells\u003c/h2\u003e \u003cp\u003eTo evaluate the effect of extracellularly added FABP4 and FABP4 inhibitor (HTS) in human PDAC cells, we assessed the cell viability after the treatment of FABP4 or FABP4\u0026thinsp;+\u0026thinsp;HTS by the MTS assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D). In CAPAN-2, CFPAC-1, and MIA PaCa-2 cells, FABP4 significantly induced the increased cell viability. HTS treatment (30 \u0026micro;M) reverted the FABP4-induced cell viability in CAPAN-2 and CFPAC-1, and significantly suppressed PANC-1 cell viability. While significant suppression for cell viability was not observed in MIA PaCa-2 with 30 \u0026micro;M HTS, the high dose of HTS (60 \u0026micro;M) significantly inhibited the cell viability (Supplementary Fig.\u0026nbsp;1A). In other gastrointestinal cancers, such as gastrointestinal stromal tumor (GIST-T1) and colorectal cancer (HT29), FABP4 did not increased the cell viability and HTS showed no significant effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and F), suggesting HTS had a specific suppressive effect for FABP4-induced cell growth in human PDAC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFABP4 inhibitor suppressed cell proliferation and induced apoptosis of the mouse pancreatic cancer cells\u003c/h2\u003e \u003cp\u003eNext, mouse pancreatic cancer cells derived from genetic pancreatic cancer model with KRASG12D and p53 mutation (KPC cells) \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e were used to evaluate the anticancer mechanisms of FABP4 inhibition in vitro and in vivo. We assessed whether FABP4 and its inhibitor can affect the KPC cell viability, proliferation and cell cycle. In the MTS assay, the viability of KPC cells was decreased in a dose-dependent manner when treated with HTS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. The IC50 value of HTS was 25.6 \u0026micro;M). While FABP4 treatment significantly increased the KPC cell viability, HTS treatment (30 \u0026micro;M) significantly suppressed the cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In the cell cycle analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), FABP4 treatment decreased the percentage of G1 phase cells and this change was totally reverted by addition of HTS. These results suggested that FABP4 significantly facilitated the G1 phase cells to entry into the S and G2 phases (release from G1 arrest), and HTS inhibited the activation of cell cycle induced by FABP4. To further characterize the mechanisms of HTS, apoptotic cell death was assessed by flow cytometry using propidium iodide (PI) and Annexin V staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The percentages of late-stage apoptotic cells (=\u0026thinsp;PI positive cells) were increased by HTS, compared to the FABP4 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), indicating that HTS reduced the cell viability of pancreatic cancer cells by not only suppressed proliferation but also increased apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFABP4 promoted migration and invasion, and induced partial EMT and stemness in vitro\u003c/h2\u003e \u003cp\u003eTo determine whether FABP4 regulates migration and invasion capability in pancreatic cancer cells, we performed scratch assay and invasion assay. In the scratch assay, exogenous FABP4 promoted the migration of KPC cells in dose dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). FABP4 also facilitated the cell migration in time dependent manner that was cancelled by the treatment of HTS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D. Representative images are shown in Supplementary Fig.\u0026nbsp;1B). In the invasion assay using KPC and PANC-1 (human pancreatic cancer) cell lines, exogenous FABP4 significantly enhanced the invasive capability and the FABP4-induced invasion was completely eliminated by HTS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F). Next, we assessed the effect of FABP4 for induction of EMT phenotype in KPC cells. In Western blotting, the expression of vimentin was gradually increased after the FABP4 exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). In contrast, FABP4 tended to induce decreased expression of CDH1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). The mRNA expression of vimentin was also increased and CDH1 was decreased by FABP4 exposure in KPC cells (Supplementary Fig.\u0026nbsp;1C and D). These results mean that FABP4 induces partial EMT in PDAC cells. Regarding with EMT activating transcription factors, the mRNA levels of TWIST1, SNAIL1, and ZEB1 in KPC cells tended to increase after FABP4 exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-K). Especially, the ZEB1 level was significantly up-regulated by FABP4 exposure. Therefore, we focused on ZEB1 activity as a promising transcription factor that could associate with FABP4-induced EMT. In the KPC and PANC-1 cells, the ZEB1 level was significantly increased by FABP4 exposure and the ZEB1 up-regulation was inhibited by HTS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL and M). Because ZEB1 activity was also reported to be involved in cancer stemness of pancreatic cancer \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, we assessed stemness marker in KPC cells by RT-PCR. FABP4 exposure induced significant up-regulation of the stemness markers CD133 and CD44 that were completely attenuated by HTS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN and O). Aldehyde dehydrogenase 1 (ALDH1) was also increased after FABP4 exposure and SOX2 tended to be suppressed by HTS treatment (Supplementary Fig.\u0026nbsp;1E and F). These results suggested that HTS suppressed PDAC invasion as well as partial EMT and stemness phenotype associated with activation of ZEB1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eFABP4 inhibition reduced tumor growth in mouse syngeneic subcutaneous PDAC tumor\u003c/h2\u003e \u003cp\u003eSyngeneic subcutaneous KPC cell tumor growth was evaluated in whole body FABP4 null (AKO) and FABP4 +/- heterozygous (WT) mice \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The FABP4 knockout significantly inhibited the tumor growth at day 28 after the KPC cells inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Both of the serum and intratumor FABP4 levels in the AKO mice were barely detectable and significantly lower than that in the WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D), indicating that host derived exogenous FABP4 contributed the PDAC tumor growth. Next, antitumor effect and appropriate dose of HTS were assessed in C57BL/6J mice with subcutaneous tumor of KPC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). The mice received HTS at two different doses (1.5mgkg and 5mg/kg) and control group of mice received vehicle alone at day 0. HTS 5mg/kg significantly suppressed the tumor growth at day 11 compared to control. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Neither of the HTS-treated groups showed any serious side effects, such as weight loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG) or altered serum biochemistries (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The serum HTS concentration 20 min after i.p, administration was sufficient to inhibit the FABP4 activity (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the TUNEL assay using the tumors at day 2 after the treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH and I), tumors treated with HTS 5mg/kg significantly showed higher number of apoptotic cells compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). In addition, we assessed the antitumor effect of HTS in AKO mice in order to rule out the possibility that HTS inhibited tumor progression by inhibiting other FABPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK). In AKO mice, HTS 5mg/kg did not suppress syngeneic tumor growth significantly, compared to vehicle treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL), indicating the antitumor effect of HTS was mainly caused by inhibition of exogenous FABP4. Body weight change was not observed in the both groups at the end of experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSerum biochemistry after FABP4 inhibitor administration\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHTS 5mg/kg\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlbumin (g/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Bilirubin (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAST (U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e188\u0026thinsp;\u0026plusmn;\u0026thinsp;53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e180\u0026thinsp;\u0026plusmn;\u0026thinsp;24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALT (U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e29\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e35\u0026thinsp;\u0026plusmn;\u0026thinsp;15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBUN (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e19.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e20.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCreatinine (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHTS (\u0026micro;M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eHTS: HTS01037, AST: Aspartate aminotransferase, ALT: Alanine transaminase, BUN: blood urea nitrogen.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEMT and stemness markers in subcutaneous tumors were decreased by FABP4 inhibition\u003c/h2\u003e \u003cp\u003eTo analyze the intratumor expression of EMT and stemness markers at earlier time point after treatment, subcutaneous tumors were harvested at day 2 after i.p. injection of HTS or vehicle, and mRNA levels were measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The intratumor mRNA level of vimentin in the HTS group was significantly lower than that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), while CDH1 level didn\u0026rsquo;t show significant change in this time point between the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The FABP4 inhibitor group showed significantly lower ZEB1 level compared to the control group. However, there was no significant difference in the levels of TWIST1 and SNAIL1 between the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F). These results suggested that the suppression of EMT associated with ZEB1 activity could be involved in the antitumor mechanism of HTS. As for stemness markers, the intratumor mRNA level of ALDH1 in the HTS group was significantly lower than that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). The SOX2 level also tended to be suppressed by HTS treatment (Supplementary Fig.\u0026nbsp;1G). Furthermore, we assessed IL22 expression in the subcutaneous tumors in both the HTS treated and AKO mice because IL22 promotes EMT and stem cell features of PDAC, and is deeply associated with PDAC progression \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The tumors treated with HTS (day 11) showed significantly suppressed mRNA level of IL22 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). In the AKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI), the intratumor mRNA and protein level of IL22 were significantly suppressed compared to those in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ and K).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eFABP4 inhibitor suppressed orthotopic tumor growth and metastasis by reducing EMT and stemness\u003c/h2\u003e \u003cp\u003eWe evaluated whether HTS treatment could suppress the progression of orthotopic tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The HTS treatment next day of KPC cell inoculation significantly inhibited orthotopic tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and C). In the hematoxylin eosin stain (HE) staining, orthotopic tumors in the HTS group showed less tumor invasion into the normal pancreatic parenchyma, compared to the tumors in the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). In western blotting, the intratumor CDH1 expression in the HTS group was significantly higher than that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and F). Immunofluorescence of orthotopic tumors also evaluated decreased expression of vimentin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG) and increased expression of CDH1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH) in the HTS group. In the staining of stemness markers, CD133 expression was observed in the marginal area of tumor in the control group. In contrast, the expression of CD133 seemed to be suppressed in the HTS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). These results indicated that the FABP4 inhibitor prevent partial EMT and stemness in the orthotopic tumors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we performed HTS treatment after orthotopic KPC-Luc/RFP tumor establishment and assess the survival rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). The chronological imaging of vivo imaging system (IVIS) showed that all the mice in the control exhibited tumor progression from day 0 to day 7 after the treatment, whereas the three mice in the HTS group showed tumor regression and the two mice kept stable disease (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). In addition, the HTS group showed significantly higher survival rate after the treatment compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). Most importantly, the two and one control mice developed microscopic liver and lung metastases, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM), but the HTS group didn\u0026rsquo;t develop any distant metastases when the mice were euthanized at day 25 after the treatment. We also analyzed the mRNA levels of luciferase in liver tissues of the orthotopic model to assess the existence of micro-liver metastasis of KPC-Luc/RFP cells. The luciferase levels of the liver in the HTS group were significantly lower than that in the control group (Supplementary Fig.\u0026nbsp;1H). HTS successfully inhibited the cancer dissemination from the primary orthotopic tumors and improved the prognosis of mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eFABP4 inhibitor showed antimetastatic effect in liver metastatic mouse model\u003c/h2\u003e \u003cp\u003eWe determined the inhibitory effect of HTS for metastasis development using a liver metastasis model. The HTS treatment was performed 30 minutes before the intrasplenic inoculation of KPC cells, and the incidence of liver metastasis was measured at day 14 after the treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The representative images of macroscopic liver and HE staining in each group are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB. Although most of mice exhibited macroscopic liver metastases in the control group, only 8.3% of mice in the HTS group showed macroscopic liver tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). The liver weight in the control group was significantly increased due to metastasis formation, compared to those in the HTS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). We choose randomly one slice of the liver specimen from the HE staining slide and microscopically measured the number and area ratio of liver tumors in the whole slice. In the HTS group, the microscopic number and the area ratio of metastasis lesion were significantly decreased, compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE and F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, we used another treatment schedule for the liver metastasis mouse model, where we started HTS treatment after establishment of liver metastasis. After seven days of the intrasplenic inoculation of KPC-Luc/RFP cells, mice underwent HTS treatment every five days during 2 weeks, and metastasis progression was assessed by IVIS (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). Before starting the treatment, we have histologically confirmed the establishment of liver metastases at day 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). IVIS images revealed that all the mice in the control group showed liver metastasis progression at day 14. In contrast, the HTS group tended to show less progression of liver metastases, although a few mice showed increased area of luciferase expression in the liver at day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). The macroscopic liver metastasis formation at day 21 was relatively reduced in the HTS group compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ). These suggested that FABP4 inhibitor could have inhibitory effect for metastasis development and growth in the liver.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eFABP4 inhibitor enhanced gemcitabine chemosensitivity in PDAC\u003c/h2\u003e \u003cp\u003eTo evaluate the translational value of HTS, we assessed whether HTS can affect chemosensitivity to gemcitabine (GEM) in PDAC. In MTS assay, viability of KPC cells was decreased by GEM treatment in dose dependent manner, and the decreased cell viability was increased by FABP4 exposure (Supplementary Fig.\u0026nbsp;1I). When HTS was combined with GEM treatment, HTS significantly enhanced the killing effect of GEM in both KPC and PANC-1 cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and B). Because ROS activity is known to be associated with chemosensitivity for GEM, ROS activity in KPC cells was examined. While the cells exposed by FABP4 showed significantly lower ROS activity than cells without treatment, HTS treatment significantly increased ROS activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). In vivo, we used the mouse model with syngeneic subcutaneous tumor of KPC cells to assess additional effect of HTS to GEM chemotherapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). The individual changes of tumor volume after the treatment in the control, GEM, and GEM plus HTS groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE-G. In the GEM only group, the tumor growth was suppressed but some mice showed tumor regrowth from day 3 to day 6. The GEM plus HTS combination therapy showed more enhanced antitumor effect than GEM alone and significantly suppressed tumor growth compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH). Thus, HTS could enhance the GEM chemosensitivity by suppressing EMT and stemness as well as increasing ROS activity in the tumors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eFABP4 inhibitor suppressed human PDAC xenograft tumor growth in mice\u003c/h2\u003e \u003cp\u003eWe evaluated antitumor effect of HTS in human PDAC xenograft subcutaneous tumors of nude mice. After the tumor establishment using human pancreatic cancer cells, HTS treatment (5mg/kg) was performed at day 0 and the tumor growth was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). Individual tumor growth and relative tumor volume in control and HTS groups were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB-D (CAPAN-2), E-G (CFPAC-1), H-J (MIA PaCa-2), and K-M (PANC-1), respectively. In the xenograft models, HTS treatment significantly suppressed CAPAN-2 and CFPAC-1 tumor growth. While the antitumor effect of HTS in MIA PaCa-2 tumors was slightly weaker than that in other tumors, significant difference between the groups was observed at day 19 after the treatment. In contrast, PANC-1 tumors were significantly suppressed by HTS at day 6 and some tumors in the HTS group were shrank down at day 10 (macroscopic findings of tumors at day 10 were shown in Supplementary Fig.\u0026nbsp;1J). HTS demonstrated the significant anticancer effect in syngeneic as well as xenograft PDAC mouse models, suggesting FABP4 inhibitor has the translational value to human study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eObesity is one of key factors to stimulate PDAC progression and is associated with a higher risk of PDAC incidence \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. To reduce the relative risk of PDAC, bariatric surgery is considered to be one of the most effective ways for obese patients \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. We have reported that FABP4 level in obese patients was reduced by bariatric surgery but not intensive lifestyle modification and medical management in patients with type 2 diabetes mellitus \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Interestingly, the reduction of FABP4 levels did not show significant correlation with the percentage of body weight loss after the interventions \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. These suggest that FABP4 might be an independent risk factor for PDAC and FABP4 inhibition can directly impact on PDAC progression beyond improvement of overweight. In this study, exogenous FABP4 induced not only tumor cell growth but also local invasion and metastasis driven by induction of EMT and cancer stem cell phenotype in human and mouse PDAC cells. In vivo, FABP4 inhibition significantly suppressed the progression and distant metastasis, and furthermore enhanced the chemosensitivity of PDAC. FABP4 inhibitor exhibited significant anticancer effect in non-obese preclinical mouse models, suggesting FABP4 inhibitor has promising therapeutic effect in obese as well as non-obese PDAC patients.\u003c/p\u003e \u003cp\u003eBiologically, FABP4 is an intracellular and extracellular lipid protein that bind a variety of hydrophobic ligands including fatty acids \u003csup\u003e14 35\u003c/sup\u003e. FABP4 is involved in glucose and lipid metabolism, cell proliferation, and apoptotic process in some cancers \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. However, the role of FABP4 in PDAC progression has not completely elucidated. The data in this study showed that exogenous FABP4 increased the PDAC cell proliferation, facilitated the entry of G1 phase cells into the S/G2 cells, while HTS reverted the effect of FABP4 on cell cycle and induced the apoptosis as well. Exogenous FABP4 also increased invasion and migration capability directly in PDAC cells. While it has been known that FABP4 plays important roles in the storage, transportation, and solubilization of fatty acid, and promotes cells proliferation/growth, and invasion \u003csup\u003e16 39\u003c/sup\u003e, our results indicated that FABP4 seems to behave as an adipokine-like stimulant that can directly promote cancer progression. In vivo experiments revealed that FABP4 inhibition suppressed the local tumor progression as well as distant metastases. Interestingly, FABP4 was barely detectable in the serum and subcutaneous tumors in AKO mice. This indicates that release of FABP4 from PDAC cells was minimal in vivo and the main source of FABP4 impacting the tumor growth is supposed to be exogenous FABP4 in the circulation produced by ATs adjacent to tumor. On the other hand, the subcutaneous tumor growth was strongly suppressed by HTS 5mg/kg than FABP4 knockout. Initially, we suspected the enhanced antitumor effect of HTS might be caused by inhibiting other FABPs including FABP5 \u003csup\u003e40\u003c/sup\u003e. However, HTS did not show significant antitumor effect in AKO mice. This result means that inhibition of exogenous FABP4 is the only significant mechanism for the antitumor function of HTS.\u003c/p\u003e \u003cp\u003eThe FABP4 knockout inhibited the primary tumor growth, and the FABP4 inhibitor furthermore suppressed the local invasion and distant metastasis by inhibiting EMT and stemness pathway. The EMT occurs in a gradual manner characterized by several cellular states expressing different levels of epithelial and mesenchymal markers, which called as partial EMT \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. We revealed retention of CDH1 expression and reduction of vimentin expression in the tumors at several time point after the HTS treatment, indicating that HTS inhibited the process of partial EMT. Because the partial activation of EMT is a critical process for the initiation of the metastatic cascade \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, the FABP4 inhibitor markedly prevented the metastatic cascade by inhibiting the partial EMT, and improved the survival in the mouse models. In addition, FABP4 induced an EMT-activator ZEB1 up-regulation and HTS attenuated the ZEB1 up-regulation in vitro and vivo. Our data suggest that the ZEB1 up-regulation by FABP4 was deeply associated with the induction of partial EMT as well as cancer stemness. The ZEB1 activation, but not other EMT-related transcription factors such as SNAIL1 and TWIST1, was reported to be essential for tumor initiation, invasion, metastasis, and stemness properties in PDAC \u003csup\u003e28 43\u003c/sup\u003e. These evidences supported our findings that ZEB1 is a key transcription factor that can induce partial EMT and cancer stemness in the FABP4-related PDAC progression.\u003c/p\u003e \u003cp\u003eIn addition to EMT, cancer stemness are associated with progression and metastasis of PDAC \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Importantly, the partial EMT phenotype is involved in increased cancer stemness that is associated with chemosensitivity \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. We observed the direct suppression of partial EMT and cancer stemness by HTS. Previous reports showed that ZEB1 depletion significantly reduced stemness in the PDAC mouse model \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, and chemo-resistant PANC-1 cells with EMT characteristics were resensitized to chemotherapy by ZEB1 knockdown \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. These results indicated that FABP4-induced EMT and stemness associated with ZEB1 activation could affect chemosensitivity in PDAC. Our data revealed that HTS enhanced the cytocidal effect of GEM in PDAC cells, and GEM plus HTS combination therapy showed significantly enhanced antitumor effect in subcutaneous tumors, suggesting that HTS could lead the GEM sensitivity through inhibition of partial EMT and stemness by ZEB1 down-regulation. Interestingly, FABP4 also reduced ROS activity in mouse as well as human PDAC cells \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Because the accumulation of intracellular ROS induces chemosensitivity and cell death \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, up-regulation of ROS activity by HTS might partially contribute the GEM sensitivity and enhanced the anticancer effect. Considering that HTS treatment suppressed tumor growth without serious side effect in not only syngeneic mouse PDAC model but also human PDAC xenograft model in mice, HTS could be a possible candidate for combination therapy with conventional anticancer agents for PDAC patients.\u003c/p\u003e \u003cp\u003eIn summary, FABP4 plays an important role in tumor growth and progression associated with cell proliferation, EMT, and stemness in PDAC. FABP4 inhibitor showed significant anticancer effect and enhanced chemosensitivity to GEM. The present study suggested that FABP4 is a very promising therapeutic target and FABP4 inhibitor can have the translational value in the clinical setting for the better management of PDAC patients.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAKO, whole body FABP4 null; ALDH1, aldehyde dehydrogenase 1; EMT, epithelial\u0026ndash;mesenchymal transition; FABPs, fatty acid-binding proteins; GEM, gemcitabine; HE, hematoxylin and eosin; HTS, HTS01037; IC50, half maximal inhibitory concentration; IVIS, in vivo imaging system; Luc, luciferase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; NRF2, nuclear factor E2-related factor 2; PBS, phosphate-buffered saline; PDAC, pancreatic ductal adenocarcinoma; PI, propidium iodide; ROS, reactive oxygen species; RT-PCR, reverse-transcription polymerase chain reaction; SD, standard deviation; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling, \u0026nbsp;WT, FABP4 +/- heterozygous\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShuhei Shinoda and Naohiko Nakamura contributed equally to this work.\u0026nbsp;We thank Dr. Ashok Saluja for providing KPC cells and giving advices for the experiments with this cell line.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSS and NN contributed equally to this work. MY, SI, DB, SS, and NN contributed conception and design of the study. SS and NN performed experiments and acquired data. SS, NN, KI, and MSD\u0026nbsp;were involved in the development of the methodology. SC and SH performed analysis and interpretation of data. MY, SI, and DB provided supervision. SS and NN wrote draft of the manuscript.\u0026nbsp;MY, SI, and DB revised the manuscript critically for important intellectual content. All authors commented on and approved the final version of the manuscript submitted for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from UMN Department of Surgery Research Support (to MY) and Eugene C. and Gail V. Sit Chair in Pancreatic and Gastrointestinal Cancer Research Endowment Fund.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures using animals were approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC, #1911-37568A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data, analytic methods, and study materials are available to other researchers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no conflict of interest exists.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHidalgo M, Cascinu S, Kleeff J, et al. 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PLoS ONE. 2011;6:e16186.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXue D, Zhou X, Qiu J. Emerging role of NRF2 in ROS-mediated tumor chemoresistance. Biomed Pharmacother. 2020;131:110676.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pancreatic ductal adenocarcinoma, fatty acid binding protein 4, metastasis, obesity","lastPublishedDoi":"10.21203/rs.3.rs-5404541/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5404541/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, and obesity is a known risk factor for PDAC. Fatty acid binding protein 4 (FABP4) is noted to be higher in obese patients, and linked to the progression of obesity-related cancers. This study aimed to elucidate the role of FABP4 and the anticancer effect of FABP4 inhibition in PDAC using preclinical mouse models.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn mouse PDAC cells derived from genetic pancreatic cancer model with KRASG12D and p53 mutation, and human PDAC cell lines, we assessed cell viability, cellular proliferation, apoptosis, and invasion capability after FABP4 and/or FABP4 inhibitor (HTS01037) treatment. The antitumor effect of FABP4 inhibition was evaluated with syngeneic PDAC tumor in FABP4 null (AKO) mice as well as syngeneic and xenogeneic subcutaneous tumor models in mice treated with HTS01037. HTS01037 treatment was also tested in orthotopic as well as liver metastasis models. We analyzed epithelial-mesenchymal transition (EMT) and cancer stemness makers \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003evivo\u003c/em\u003e samples. In addition, efficacy of combination therapy of gemcitabine (GEM) plus HTS01037 was assessed in the syngeneic model.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e, HTS010137 suppressed FABP4-induced cell viability in human and murine PDAC cells. FABP4 increased cellular proliferation, and HTS01037 reversed the changes and increased apoptosis. FABP4 promoted migration and invasive potency, and increased EMT and stemness markers that were associated with up-regulation of EMT activating transcription factor ZEB1. Both FABP4 knockout and inhibition with HTS01037 suppressed the syngeneic subcutaneous tumor growth with reduction of EMT and stemness. Similar to the syngeneic tumors, the xenogeneic tumor growth was inhibited by HTS01037 treatment. HTS01037 showed significant anticancer and antimetastatic effect which improved the survivals in the orthotopic model. HTS01037 also attenuated development and growth of liver metastases in the liver metastasis model. Moreover, HTS01037 enhanced the efficacy of GEM to PDAC \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eFABP4 promoted the PDAC progression and FABP4 inhibition showed significant anticancer effect by suppressing cellular proliferation, EMT, and cancer stemness. FABP4 inhibitor has a promising translational value for PDAC treatment and can be a critical therapeutic option in PDAC patients.\u003c/p\u003e","manuscriptTitle":"Inhibition of adipose tissue-derived fatty acid binding protein suppresses pancreatic cancer progression and metastasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-10 16:09:48","doi":"10.21203/rs.3.rs-5404541/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aea647b5-78cd-4897-baf5-c1d407ea990a","owner":[],"postedDate":"December 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-15T14:08:28+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-10 16:09:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5404541","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5404541","identity":"rs-5404541","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}