Autotaxin inhibitor IOA-289 reduces gastrointestinal cancer progression in preclinical models

Human colorectal adenocarcinoma HT-29 and Caco-2 cells, hepatocellular carcinoma HLE cells, cholangiocarcinoma RBE and KKU-M213 cells and pancreatic carcinoma PANC-1 and MIA PaCa-2 cells were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA, USA). The hepatocellular carcinoma cell line HLF was purchased from JCRB Cell Bank (Japan). All cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 1 mM pyruvate, 25 mM HEPES, 100 U/mL penicillin–streptomycin (all from Thermo Fisher Scientific, Waltham, MA, USA), and maintained in a humidified atmosphere at 37 °C containing 5% CO₂. The medium was changed every three days. ATX inhibitor IOA-289 was a kind gift from iOnctura.

Cells were seeded in 96-well plates at a density of 2000 cells/well in 10% FBS supplemented medium. After 24 h, medium was removed, the cells were washed with PBS and cultured in the presence or absence of FBS and of different concentrations of IOA-289. After the time set for the experiment (24, 48 and 72 h), the cells were fixed with a 4% PFA solution and stained with 0.02% crystal violet for 10 min. Subsequently the plates were washed with water to remove excess dye. The cell-bound dye was redissolved in 1% SDS and the optical density was measured at λ = 570 with a iMark™ Microplate Absorbance Reader (Bio-rad).

Spheroids were created using the hanging drop method. For all the experiments, cells were suspended, at a concentration of 50,000 cells/ml, in medium with 0,24% of methylcellulose (Sigma) and supplemented with 2% FBS, in the absence or presence of increasing concentration of IOA-289. Twenty-five microliter drops were pipetted onto the lid of 100 mm dishes, that were inverted over dishes containing 5 ml of cell culture medium to avoid drying. After 3 days of incubation at 37 °C and 10% CO₂, spheroids were transferred by pipetting onto a low-attachment 6-well culture plate (Corning). Images of 10–20 spheroids per experiment were acquired with a NIKON Eclipse Ti2 microscope and spheroids size was measured with ImageJ software (National Institute of Health, USA).

The culture surface of a 96-well plate was coated with a thin layer of Matrigel (Corning) (30 µl/well) and incubated for 20 min at 37 °C to allow it to gel. Cells were trypsinized and filtered through a 40 µm mesh to make a single cells suspension. Two microliters of medium containing 2000 cells were mixed with 28 µl of matrigel and layered on top of the previously coated wells. After gelification for 20 min at 37 °C, 90 µl of 10% FBS complete medium were added to the wells. After 24 h, the 10% FBS medium was removed and replaced by 2% FBS medium with 3,9 and 12 µM IOA-289. Medium was changed every 2–3 days. Colonies had been generated after 10–14 days of culture and cell clusters (with at least 50 cells) were counted microscopically without staining using a NIKON Eclipse Ti2 microscope.

Cell cycle analysis was performed using a Cell-Clock Assay Kit (Biocolor Life Science Assays, County Antrim, UK.) on cell lines treated with 3,9 and 12 µM IOA-289 for 24 h. Briefly, 2.0 × 10⁵ cells/well were seeded on 24-well plates and incubated at 370C, 5% C02, to > 80% confluence. The cells were then treated with fresh medium containing IOA-289. After 24 h, the cell cycle phases were visualized using the kit according to the manufacturer’s instructions. Images were analysed by ImageJ software to determine the ratio of cells in each cell cycle phase.

Cells were seeded in a 12-well plate in 10% FBS medium and grown to reach a 80–90% confluence. Then, a scratch was made on the cell monolayer using a p200 pipette tip. The plate was washed with sterile PBS to remove the debris and markings were created on the outer bottom of the plate with a tip marker, to be used as reference points. Each cell line was incubated with medium without FBS, containing increasing concentrations of IOA-289 (1–3-9–12 µM). Images were acquired at 0, 24 and 48 h using a confocal microscope Nikon Eclipse Ti2 in bright field microscopy and Image-J software was used to measure the scratched area. Cell migratory ability for wound-healing was assessed by applying the following formula: [(wound area at 0 h) – (wound area at indicated time)] / (wound area at 0 h).

Cell culture inserts with 8 µM pores (Corning, inc.) were coated with 50 µl of a 10 mg/ml Collagen type 1 solution (Gibco, USA). 1.5 × 10⁴ cells were plated in the upper chamber in serum-free medium, in the absence or presence of 12 µM IOA-289, while the bottom wells were filled with complete medium. The cells were allowed to migrate across the membrane for at least 16 h. After incubation, the cells were fixed with PFA 4% for 10’ and stained with 0.02% crystal violet for 10’. Excess dye was washed off with tap water and the non-migrated cells were scraped off from the upper surface of the membrane with a cotton swab. Cell migratory ability was assessed by counting the average number of cells in at least 5 fields at 10X magnification.

To detect early apoptosis by immunofluorescence, the Annexin V-FITC kit (Miltenyi Biotech, Germany) was used. Cells were seeded and grown on Nunc Lab-Tek II Chamber Slide. After washing with PBS, cells were incubated with IOA-289 3, 9 and 12 µM in serum-free medium. After 2 h cells were washed with Binding Buffer (BB) and stained with a solution of Annexin V-FITC antibody in BB for 15’. After two washes with BB cells were fixed with a 4% paraformaldehyde solution and a coverslip was mounted onto the slide using a DAPI Ready Made Solution with Antifade (Merck, US). Images were acquired using the Nikon Eclipse Ti2 confocal microscope and analyzed with ImageJ software.

Flow cytometry analysis of cell apoptosis following treatment with 3, 9 and 12 µM IOA-289 for 24 h was done using the Annexin V-FITC kit, according to the manufacturer’s instructions. Briefly, adherent cells were detached using StemPro Accutase (Thermo Fisher Scientific, Waltham, MA, USA) and mixed with any floating cells; 10⁶ cells were washed with 1 ml of Binding Buffer (BB). The pellet was resuspended with 100 µl of BB, 10 µl of Annexin V-FITC were added and incubated for 15 min in the dark. After another wash with BB, the cell pellet was resuspended with 500 μL of BB, and 5 μL of propidium iodide were added prior to analysis with the Navios flow cytometer, using Kaluza software for quantification and graph plotting (Beckman Coulter).

The Human Apoptosis Antibody Array Kit (ab134001, Abcam) was used to detect the apoptosis pathway. Briefly, cells were lysed and each membrane was incubated at 4° overnight with 500 µg of total extracted protein. Membranes were then washed and incubated overnight at 4° with Biotin-conjugated Anti-Cytokines. After a incubation of the membranes with HRP-streptavidin for 2 h at room temperature, chemiluminescence was detected using ChemiDoc XRS + (Bio-Rad, USA). The images were analyzed with Image Lab 5.2.1.

ENPP2 mRNA expression was evaluated in a dataset of CRC, CCA, HCC and PC patients from TGCA using the GEPIA tool (http://​gepia2.​cancer-pku.​cn/​) (REF: Tang, Z. et al. (2019) GEPIA2: an enhanced web server for large-scale expression profiling and interactive analysis. Nucleic Acids Res, https://​doi.​org/​10.​1093/​nar/​gkz430.).

In order to evaluate ENPP2 gene expression levels in patients with cholangiocarcinoma (CCA), hepatocellular carcinoma (HCC), colorectal cancer (CRC), and pancreatic cancer (PC), we used data from TCGA database comparing them with peritumoral counterpart and GTEx database. We found that ENPP2 expression was upregulated in CCA (p > 0.05), HCC (p < 0.01) and PC (p < 0.01). Instead, in CRC, ENPP2 gene expression resulted downregulated in comparison with normal tissue (p < 0.01) (Fig. 1).

To evaluate the anti-proliferative/cytotoxic effects of IOA-289, we treated different cell lines developed from cholangiocarcinoma (CCA), hepatocellular carcinoma (HCC), colorectal cancer (CRC), and pancreatic cancer (PC) with increasing concentration of IOA-289. After the drug treatment, attached cells were fixed and stained with crystal violet dye. This screening method is generally used to evaluate the effect of chemotherapeutics or other anti-cancer drugs on cell survival and proliferation. In our first experiments, IOA-289 was administered to cells for 72 h at concentrations ranging from 10 µM up to 50 µM, in culture medium supplemented with 10% FBS. IOA-289 showed a significant (p < 0.05) cytotoxic effect only at the highest concentrations (30 to 50 µM, Fig. 2) in all the cell lines used: KKU-M213,HLE, HT-29, and PANC-1 cells. To test the hypothesis that the presence of serum, and consequently endogenous LPA, could interfere with the IOA-289 activity, we treated eight cancer cell lines with IOA-289 at concentration ranging from 1 µM up to 12 µM, in the presence or absence of 10% FBS. As reported in Fig. 3, IOA-289 displayed a significant (p < 0.05) cytotoxic effect in a dose-dependent manner even at lower concentrations (3 to 12 µM). This effect, already evident after 48 h, was strengthened after 72 h of incubation. Thus, IOA-289 showed cytotoxic effect in cells cultured in the absence of FBS but not in those with 10% FBS present in the medium; these effects were consistent for all eight cancer models, and were observed at each repetition (at least three independent experimental repetitions). These results suggest that IOA-289 exerted a strong anti-proliferative effects in all the models and also that FBS interferes with its activity. To further expand our preliminary observations, we challenged HLE, KKU-M213, HT-29 and Panc-1 cells with 9 µM of IOA-289 in the absence or presence of increasing concentrations of FBS. In all the cell models, the presence of FBS significantly (p < 0.05) reduced the activity of IOA-289, starting at an FBS concentration of 2%, while the IOA-289 effect was completely abolished at 5% FBS. In Panc-1 cells, FBS blocked drug effectiveness even at the lowest concentration (Fig. 4). In conclusion, IOA-289 selectively inhibits LPA, leading to an anti-proliferative effect, but in presence of FBS the drug effectiveness is blocked, likely due to supraphysiological levels of endogenous LPA. Further confirmation of these results was obtained with tests of spheroid formation and clonogenicity. As can be seen in Fig. 5, IOA-289 proved very effective in inhibiting the formation and growth of spheroids, in particular in the HLE and KKU-M213 cell lines, starting at the low concentration of 3 uM. In PANC-1 cells an inhibitory effect is, instead, observable only at a concentration of 12 uM. Furthermore, in all cell lines analysed, IOA-289 proved equally effective in inhibiting, in a dose-dependent manner, the ability of cells to form colonies starting from single cells (Fig. 6). In particular, in KKU-M213, HLE and HT-29 cells the formation of colonies with at least 50 cells was completely inhibited at the 12 uM concentration of IOA-289. Determination of the number of viable cells by colorimetry confirmed the inhibitory effect of IOA-289 on proliferation. Analysis of the cell cycle in all the cell lines examined showed that the effect of IOA-289 treatment on cell growth was to cause an arrest in S phase, particularly evident in KKU-M213, HLE and Panc-1 cells (Fig. 7).

Tumor progression is characterized by an enhanced cell motility and invasiveness. To expand our knowledge of the effects of IOA-289 on HLE, KKU-m213, HT-29 and Panc-1 cells, we assessed their migratory activity when treated with IOA-289, by both a wound healing and a transwell migration assay. Cells were seeded in a 12-well plate and, when a confluent monolayer was reached, an artificial gap was generated between the cells using a pipette tip. Scratch closure by migrating cells was observed after 48 h and 72 h. As reported in Fig. 8A, IOA-289 significantly (p < 0.05) reduced cell migration and wound closure of KKUM-213 cells treated with 9 \(\mathrm{\mu M}\) IOA-289 (achieving an approximately 50% reduction in wound closure), compared to control, already after 24 h and 48 h of treatment. In HLE cells a concentration of 3 µM IOA-289 is already sufficient to cause a 40% inhibition of cell migration, whereas in PANC-1 cells a substantial reduction (60%) in migration was observed with the highest concentration of inhibitor, at 12 µ. In HT-29 cells 9 and 12 µM of IOA-289 significantly (p < 0.05) inhibited wound closure. Similarly, the transwell assay showed that IOA-289 treatment significantly inhibited the migratory capacity of all the tested cancer cell lines (Fig. 8B). In conclusion, IOA-289 can inhibit cancer cell migration.

To further explore the activity of IOA-289 on gastrointestinal cancer cells, we investigated its effectiveness on inducing cell apoptosis. Following incubation with IOA-289, cells underwent morphological changes, featuring cell shrinkage and cytoplasmic vacuolization (Fig. 9A). An early event in apoptosis is the loss of plasma membrane asymmetry, resulting in the translocation of phosphatidylserine (PS) from the inner to the outer plasma membrane leaflet. Exposed PS on the cell surface can be used to measure apoptosis based on the binding with Annexin V. KKU-M213, HLE, HT-29, and PANC-1 cells were incubated with increasing concentrations of IOA-289 (3, 9 and 12 µM). After 4 h, cells were stained with a FITC-Annexin V antibody and fixed. In KKU-m213, HT-29 and Panc-1, IOA-289 significantly (p < 0.05) induced cell apoptosis at 3, 9 and 12 µM in a dose dependent manner, whereas in HLE cells the same effect was seen at 9 and 12 µM (Fig. 9B). To corroborate these results the same cancer cell lines were treated with the same concentrations of IOA-289 for 24 h and apoptosis/necrosis was analyzed by flow cytometry. A general increase of apoptosis and necrosis of KKU-M213, PANC-1, HLE, and HT-29 cells was observed, in a dose-dependent manner, consistently with the previously observed decrease of cell proliferation (Fig. 10). In addition, a human apoptosis antibody array was used to identify the differential expression of 43 apoptosis-related proteins in cells treated with 12 µM IOA-289 for 24 h as compared to untreated cells. As shown in Fig. 11, IOA-289 may promote cell apoptosis through the upregulation of pro-apoptotic proteins such as Bim, Caspase-3 and -8,, DR6, Fas, Fas-L, p53,SMAC and others.