Cell lines and reagents

KKU-M213 and KKU-M156 human iCCA cell lines, purchased from the Japanese Collection of Research Bioresources (JCRB) or the American Type Culture Collection (ATCC), were used for the experiments. Cell Lines Service (Eppelheim, Germany) performed cell line authentication. Both cell lines were grown in Dulbecco’s modified Eagle medium (DMEM), (Gibco, Grand Island, NY, USA) supplemented with 10% Fetal Bovine Serum (FBS), Antibiotic-Antimycotic, Sodium Pyruvate and Hepes, (Gibco, Grand Island, NY, USA). Cells were cultured at 37 °C in a 5% CO2 humidified atmosphere. Cell lines, before use, were tested mycoplasma-free using the MycoFluor™ Mycoplasma detection Kit (ThermoFisher Scientific, Waltham, MA, USA). Crenigacestat (LY3039478, Selleckchem Chemicals, Houston, TX, USA) was used to treat iCCA cell lines in vitro and in animal models in vivo. Stock solutions were prepared in Dimethylsulfoxide (DMSO) (ThermoFisher Scientific, Waltham, MA, USA) and aliquots were stored at -80 °C.

Establishment of iCCA hCAF culture from fresh human iCCA tissues

Approval of the study was granted by the local Ethics Committee, Istituto Tumori “Giovanni Paolo II” (Bari, Italy) (protocol number: 145; date of release: March 2022); in compliance with the Helsinki Declaration. Informed consent was obtained from all individuals. After surgical resection, iCCA tissue specimens were immediately stored in MACS tissue storage solution (Miltenyi Biotec, Bergisch Gladbach, Germany) and processed for hCAFs isolation as previously reported [23]. iCCA hCAFs were isolated from three patients. We performed enzymatic and mechanical digestion of iCCA tissue fragments in HBSS solution with 50–200 U/mL collagenase Type IV (Thermo Fisher Scientific, Waltham, MA, USA), 3 mM CaCl2, and Antibiotic–Antimycotic (Thermo Fisher Scientific, Milan, Italy) at 37 °C by rotation for 2 h or more as needed. The resulting cells were harvested by recovering the volume of digestion and washed with PBS. The cell population harvested was cultured in complete IMDM (Iscove’s Modified Dulbecco’s Medium) with 20% FBS and Antibiotic–Antimycotic at 37 °C in a 5% CO2 humidified atmosphere to obtain hCAF cultures.

Viability and proliferation assay on iCCA cell in co-culture with hCAFs

For the co-culture, 15 × 103 iCCA cells were seeded onto 24-well plates with complete DMEM medium. Additionally, 20 × 103 hCAFs were seeded onto transwell inserts with 0.4 μm pore size (Corning, Bedford, MA, USA) in complete IMDM medium. After 24 h, the transwell inserts with seeded hCAFs were transferred to 24-well plates for co-culturing with iCCA cells. As control, iCCA cells without hCAFs in transwell inserts were prepared. iCCA cell cultures and hCAFs/iCCA co-cultures prepared in this way were treated with vehicle and Crenigacestat (5 µM) in IMDM + 1% FBS for 72 h. At the end of treatment, iCCA cell viability in the presence or absence of hCAFs was determined with the CyQUANT™ XTT Cell Viability kit (ThermoFisher Scientific, Waltham, MA, USA) and for proliferation by Trypan Blue cell counting. Data are expressed as the mean ± SD of three independent experiments with three hCAFs.

Transwell migration assay

KKU-M213 or KKU-M156 cell migration was induced for 18 h, after 72 h of treatment. In detail, we developed three different co-culture approaches: pre-treating for 72 h 15 × 103 iCCA cells onto transwell inserts with 0.4 μm pore size or 40 × 103 hCAFs onto 24-well plates separately, or both cell types simultaneously in co-culture with Crenigacestat (5 µM) or vehicle. In each case, after the treatment, we evaluated the iCCA cell migration in co-culture with 40 × 103 hCAFs. As described for the vitality assay, also for the study of this cell functional aspect, we used a control condition, namely the migration of iCCA cells without hCAFs seeded in the bottom of the wells. Cell migration was allowed by using transwell inserts suitable for 24-well plates, with 6.5 mm internal diameter, and 8 μm pore size (Corning, NY, USA). The membrane of these transwell inserts had been coated with rat tail collagen I (final concentration 10 µg/mL) on the lower surface for 2 h at room temperature. 20 × 103 iCCA cells were suspended in 200 µL of serum-free IMDM medium and loaded onto the top chamber of transwell inserts. Cells were allowed to migrate for 18 h in IMDM medium + 10% FBS at 37 °C and 5% CO2. After incubation, the migrated cells were fixed in 4% PFA (pH 7.2 in PBS) and stained with crystal violet for 10 min. Five fields per membrane were captured in bright field at 10× magnification. The analysis was performed on the mean number of migrated cells/field. Data are expressed as the mean ± SD of three independent experiments with three hCAFs.

3D homo-spheroid and hetero-spheroid cultures

Three-dimensional (3D) cell culture models were set up using the hanging drop method as previously described [24]. Briefly, cells were suspended, at a concentration of 1 × 105 cells/ml, in medium with 0.24% methylcellulose (Sigma, St. Louis, MO, USA). Forty drops of 25 µl each were pipetted onto the lid of 100 mm dishes. In this way each drop and thus each spheroid consisted of 2.5 × 103 cells. Homo-spheroids were developed starting from a single cell type, KKU-M213 or KKU-M156 cells. Instead, hetero-spheroids were developed by respecting the 1:3 rate for KKU-M213 or KKU-M156 cells: hCAFs. After 3 days of incubation at 37 °C and 5% CO2, spheroids were transferred into different culture supports depending on the cell function assay. Each experimental condition was performed three times and hetero-spheroids were replicated with three hCAFs. Values are presented as mean ± SD.

Flow Cytometric analysis of cell cycle

For cell cycle analysis, the recovered spheroids were cultured on an ultralow-attachment culture plate (Corning, Bedford, MA, USA) and treated with vehicle or Crenigacestat (5µM) twice for 5 days. The cell cycle was induced on cell suspensions obtained after disruption of the homo- and hetero-spheroids. Briefly, floating spheroids were disrupted using Tryple™ Select (Gibco, life Technologies corporation, NY, USA) plus 1mM EDTA pH 8.0 (Invitrogen by ThermoFisher Scientific, USA) and pipetting every 10 min. 1 × 106 cells were washed once in cold PBS and fixed with cold 70% ethanol at 4 °C overnight. The fixed cells were then washed twice in PBS, the supernatant was discarded, and the cells were treated with of RNase A (Sigma, United States; 100 µg/mL) for 15 min at 37 °C. Propidium iodide; Sigma, United States; 200 µL of 50 µg/mL stock) was then added to the cells and incubated for 30 min at 4 °C in the dark. The DNA content of the cells was determined by flow cytometry.

Spheroid viability assay

For the viability assay, the spheroids were embedded in engineered hydrogel networks made of bovine skin hydrolyzed collagen and Corning® Matrigel® Matrix; the pH was neutralized with 0.5 M acetic acid. To prevent the spheroids from settling and sticking to the bottom of the well, a drop of matrix was placed in the well and allowed to dry for 20 min before embedding the samples into the matrix. In addition, spheroids were treated with vehicle and Crenigacestat (5µM). After 5 days of treatment, cell viability was assayed using the CellTiter-Blue™ Viability Assay (Promega, Tokyo, Japan).

Spheroid invasion assay

The spheroids were embedded in an engineered hydrogel network. The bovine collagen type I solution and Corning® Matrigel® were mixed and neutralized with 0.5 M acetic acid to obtain a matrix suitable for the invasion assay. Fresh medium was added to the final gel solution, to simulate cell embedding. Spheroids were treated with Crenigacestat (5µM) or vehicle for 72 h. Images were captured in bright field at 10× magnification. The analysis was performed on the area of invasion ratio of treated spheroids/untreated spheroids.

Spheroid immunofluorescence

For immunofluorescence staining, the spheroids were fixed in a 4% PFA solution at 4 °C for 30 min, and then washed twice in 0.1% Triton X-100 in TBS. After washing, samples were permeabilized with 0.5% Triton X-100 in TBS at room temperature. Then, the spheroids were incubated with anti-FAP (1:150, Abcam, Cambridge, UK), anti-EPCAM antibodies (1:800, Cell Signaling Technologies, MA, USA) in an antibody dilution buffer (2% bovine serum albumin/0,1% Triton X-100 in TBS solution) and anti-Ki67 (1:250, Abcam, Cambridge, UK), overnight at 4 °C in rotation. The following day, after washing, samples were incubated with secondary goat anti-rabbit immunoglobulin G H&L (1:500 Alexa Fluor 594, Thermo Fisher Scientific, Waltham, MA, USA) for FAP, with secondary goat anti-mouse immunoglobulin G H&L (1:50 Alexa Fluor 488, Thermo Fisher Scientific, Waltham, MA, USA) for EPCAM and with secondary goat anti-rabbit immunoglobulin G H&L (1:50 Alexa Fluor 488, Thermo Fisher Scientific, Waltham, MA, USA) for Ki67. The incubation with secondary antibodies lasted 3 h at room temperature in the dark. After further washing, nuclei staining was performed with 0.5 ng/ml of PureBlu DAPI Nuclear Staining Dye (Bio-Rad Laboratories, USA) in 0.1% Triton X-100 in TBS, incubating for 1 h at room temperature in the dark. Spheroids were put on the slides and covered with ProLong™ Diamond antifade mounting medium (Invitrogen by ThermoFisher Scientific, USA). Confocal images and movies of spheroids were acquired using the Nikon Ti2-E Inverted Research Microscope equipped for confocal imaging in conjunction with a Nikon A1rSi Laser Point Scanning Confocal System, Plan Fluor Ph 20X objective and NIS-Elements “AR” 5.0 software and improved by deconvolution method based on Richardson-Lucy Algorithm.

In vivo study

Housing and all procedures involving the mice were performed according to the protocol approved by the Ethics Committee (Protocol number 257/2023-PR, date of release 29/03/2023) at Biogem Animal House in Ariano Irpino (Avellino, Italy) following the National Academy of Sciences Guidelines. Two million KKU-M213 cell lines or KKU-M213:hCAFs, respecting the 1:1 ratio, were subcutaneously injected into the flanks of 4–5-week-old female CD1 nude mice. Drinking water was supplied ad libitum. Each mouse was offered a complete daily pellet diet (GLP 4RF21, Mucedola) throughout the study. The analytical certificates of animal food and water were retained at Biogem premises. Each mouse was monitored daily for clinical signs and mortality, and body weight was recorded twice a week. The tumor volume was monitored weekly by a caliper and evaluated with the formula (mm3) = [length (mm) × width (mm)2]/2, where width and length are the shortest and longest diameters. When the tumor masses volume reached approximately 70–100 mm3, the mice were randomly subdivided into 2 experimental groups of six animals and administered Crenigacestat (8 mg/kg) or vehicle by oral gavage every 2 days for 20 days. At the end of the study, mice were sacrificed by cervical dislocation, and tumor samples were collected and sectioned for immunohistochemical, hematoxylin-eosin and trichrome staining or for RNA extraction.

Histological staining method

Tumor specimens were fixed in 4% paraformaldehyde and embedded in paraffin using standard procedures. To analyze the grade of tissue fibrosis Masson’s trichrome staining with the Mallory trichrome acc. McFarlane kit (DIAPATH) was performed, following the manufacturer’s instructions. The degree of fibrosis was classified according to the adapted METAVIR score as previously reported [19]. The images were acquired with the Eclipse Ti2 microscope (Nikon Inc., Melville, NY, USA).

RNA extraction

Total RNA from explanted tumor masses was isolated with the miRNeasy mini kit (Qiagen, Hilden, Germany) in combination with the TissueLyser homogenizer (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The RNA concentration was determined with the Qubit™˝ฏ RNA HS Assay kit (Thermo Fisher Scientific, Waltham, MA, USA) on a Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA quality was evaluated using the High Sensitivity RNA ScreenTape (Agilent Technologies, Palo Alto, CA, USA) on an Agilent 4200 TapeStation system (Agilent Technologies).

Whole transcriptome profiling

Total RNA samples were reverse transcribed using the Ion Torrent™ NGS Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Target region amplification was performed using the Ion AmpliSeq Transcriptome Human Gene Expression core panel (Thermo Fisher Scientific, Waltham, MA, USA) on the Ion Chef System. The barcoded libraries were quantified by qPCR with the Ion Library TaqMan Quantitation kit (Thermo Fisher Scientific, Waltham, MA, USA). Finally, libraries were templated onto the Ion Chef and sequenced using a 540 chip on the Ion GeneStudio S5 Prime system (Thermo Fisher Scientific, Waltham, MA, USA).

Sequencing data are available under accession number GSE273905 at the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?&acc=GSE273905).

Quantitative real-time PCR

cDNA was reverse transcribed using the iScript Reverse Transcription Supermix (Bio-Rad Laboratories) according to the manufacturer’s instructions. Quantitative PCR reactions were performed using SsoAdvanced SYBR green (Bio-Rad Laboratories) and the primers sequences for CCNE2 forward, 5-TCAAGACGAAGTAGCCGTTTAC-3′; reverse, 5-TGACATCCTGGGTAGTTTTCCTC-3′, CCND1 forward, 5-GCTGCGAAGTGGAAACCATC-3′; reverse, 5-CCTCCTTCTGCACACATTTGAA-3′, and Hs_GAPDH_1_SG QuantiTect Primer Assay ID: QT00079247 (Qiagen). The CFX96 System (Biorad, Hercules, CA, USA) was used for Real-Time PCR. Comparative real-time PCR was performed in triplicate, including no-template controls. Relative expression was calculated using the 2−ΔΔCt method.

Bioinformatics and statistical analyses

Ion Torrent Suite Server v5.16.1 (Thermo Fisher Scientific, Waltham, MA, USA) software was used to generate the transcription data as raw read counts using the ampliSeqRNA plugin with default settings. Downstream analyses were performed with Transcriptome Analysis Console 4.0 software (Thermo Fisher Scientific, Waltham, MA, USA). DEGs were identified with the Limma eBayes method using a 1.5 threshold of fold-change and p-value ≤ 0.05. Hierarchical clustering was generated with Alt Analyze 2.1.3 software [25]. Canonical pathways, biological processes and molecular networks associated with DEGs were analyzed with Ingenuity Pathway Analysis (IPA) software (Qiagen, USA). Biological and technical replicates were analyzed with the most appropriate statistical tests (i.e. t test or ANOVA). For in vivo studies, Mann-Whitney U Test was performed. A p-value ≤ 0.05 was considered statistically significant. Statistical analysis and graphs were generated using GraphPad Prism 5.0 software (La Jolla, CA, USA).