Patient material
Tissue Microarray (TMA) The TMA contained 846 tumor biopsies from 412 patients retrieved from patients who underwent surgery for SI-NET at Sahlgrenska University Hospital in the years 1986 to 2013.
Details of the construction of the tissue microarray have previously been described [
20]. The diagnosis of all tumors was revalidated by staining all tumors for hematoxylin and eosin, synaptophysin and chromogranin A, and reviewed by board certified pathologist (O.N.).
SI-NET biobank Fresh-frozen tissue biopsies were collected at the time of surgery from the Endocrine unit of the Surgery department at Sahlgrenska University Hospital from 1986 to 2019. Resected tissue from SI-NET patients was evaluated in the operating theatre and material was collected from primary tumors, lymph node and liver metastases. The extent of tissue sampled depended on tumor spread and size of resected tumors. Consistently, the largest intestinal tumor and the most adjacent lymph-node metastasis were sampled if present. Starting in 2010, measurements in mm of all 3 dimensions were obtained and documented in a local register coupled to the biobank. From 2010 to the present, tissue has been sampled by a group of three lab technicians in a standardized manner. Tumor volume (mm^3) based on measurements was estimated as = ((width (mm) * height (mm) * depth (mm)) / 2.
Cell lines and patient-derived tumor cells The GOT1 cell line was established from a liver metastasis of a midgut neuroendocrine tumor [
21] and was cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS), 5 µg/mL insulin and 5 µg/mL transferrin. The P-STS cell line was a gift from Professor R Pfragner. It was established from the primary tumor, described as a grade 3 NET located in the terminal ileum [
22], and was cultured in M199: Ham’s F12 (1:1) supplemented with 10% FBS. The cell lines were regularly tested for
Mycoplasma species (Eurofins GATC Biotech GmbH) The identity of the cell lines was validated by STR analysis [
23]. MCF10A was purchased from ATCC (CRL-10,317) and was cultured as previously described in Debnath et al. [
24]. Patient-derived tumor cells were established from SI-NET samples stored in the local biobank and prepared as previously described [
25]. All patient-derived tumor cells were cultured RPMI1640 supplemented with 4% FBS, 5 µg/mL insulin and 5 µg/mL transferrin. All culture media contained 100 IU/mL penicillin and 100 µg/mL streptomycin.
Drugs and experimental methods
Drugs and reagents Nonanoic acid, 1-nonanol and decanoic acid were purchased from Sigma-Aldrich and dissolved in DMSO. Octreotide Hospira, 100 mg/ml injection solution was purchased from Pfizer.
Immunohistochemistry (IHC) and Scoring of TMA IHC was performed on cell lines GOT1, P-STS and MCF10A, patient tumor tissue and TMAs. Paraffin embedded cell blocks from all cell lines were created using a Cellient automated cell block system (Hologic). Paraffin embedded tissue blocks from those patient tumors used for primary cell cultures were obtained from Sahlgrenska University Hospital. These blocks were prepared for routine clinical histopathology. TMAs were generated as described above. Sections (3–4 μm) from paraffin blocks were placed on glass slides and treated in Dako PT-Link using EnVision FLEX Target Retrieval Solution (high pH). The following primary antibodies were used: anti-chromogranin A (PHE5, Millipore and ab68271, Abcam), anti-synaptophysin (Sy38, Dako), anti- OR51E1 (LS-A1854, LSBio) and anti-OMP (B-6, Santa Cruz). IHC staining was performed in a Dako Autostainer Link using EnVision FLEX according to the manufacturer’s instructions (DakoCytomation). EnVision FLEX+ (LINKER) rabbit or mouse was used for all staining’s except anti-chromogranin A (PHE5) Positive and negative controls were included in each run. Relative expression of OR51E1 and OMP on tumors present on the TMA was evaluated by assigning a score of 0–3 to the relative staining intensity, with 0 being negative staining and 3 the highest staining present on the TMA. This evaluation was performed by two blinded independent observers with an initial 87.6% agreement in assessment of OR51E1 score and 90.3% agreements in assessment of OMP score. Samples with differing assessments were reviewed again by both observers and a final score was agreed upon. Only overall staining intensity was assessed and subcellular staining was not assessed.
Metabolic activity The cells were seeded onto black non-optical 96-well cell culture plates (Nunc, Thermo Fisher Scientific) and 24 h was allowed for cell attachment before start of the experiments. The cells were treated with a range of 0-3000 µM nonanoic acid or 1-nonanol. To measure the metabolic activity after 3 days treatment the assay plates were incubated with AlamarBlue (Thermo Fisher Scientific) for 6 h at 37 °C, and then analyzed by a fluorescence plate reader (Wallac 1420, PerkinElmer; ex. 560 nm and em. 640 nm). All experiments were performed in triplicate with 3 technical replicates.
Proliferation GOT1, P-STS and MCF10A cells were treated with nonanoic acid, 1-nonanol or DMSO vehicle for at least two doubling times. Cells cultivated without drug were included as a control. For GOT1 this represents three weeks, for P-STS 10 days and for MCF10A 5 days. CyQUANT® Cell Proliferation Assay Kit (Invitrogen) was used according to manufacturer’s instructions to quantify DNA content reflecting cell amount. All experiments were performed in triplicate with 3 technical replicates.
Serotonin quantification GOT1 cells were cultured with medium containing nonanoic acid (300 or 750 µM) or medium with vehicle (DMSO). After 24 h exposure the medium was collected and serotonin was quantified using ELISA according to the manufacturer’s instructions (ab133053, Abcam). Medium with vehicle was included as a control. Experiments were performed three times with 3 replicates.
Cell cycle analysis For analyses of cell cycle s-phases, GOT1 cells were cultured in the presence of 300 µM nonanoic acid or medium with vehicle (0.1% DMSO) for 3 weeks. One million cells per ml were lysed and stained for 30 min at 37 °C in modified Vindelöv’s solution (20 mM Tris, 100 mM NaCl, 1 µg/mL 7-AAD, 20 µg/mL RNase, and 0.1% NP40 adjusted to pH 8.0) followed by the analysis of DNA content using the FL3 channel with a BD Accuri C6 flow cytometer. Experiments were performed three times with 3 replicates.
Western blot analysis Western blot was performed on GOT1 cells cultured with 300 µM nonanoic acid for 1, 2 or 3 weeks. Whole-cell lysates were prepared by adding ice-cold RIPA lysis buffer (Thermo fischer) and Protease Inhibitor Cocktail Set III (cat. no.: 539,134; EMD Biosciences, La Jolla, CA, USA). A total of 20 µg cell lysate was run on 10% or 4–12% NuPAGE Bis–Tris polyacrylamide gels (Invitrogen) and transferred to PVDF membranes (Invitrogen). The membranes were probed using the antibody anti-synaptophysin (DAK-SYNAP, Dako). Membranes were stripped with ReBlot Plus Strong Antibody Stripping Solution (Millipore, Temecula, CA, USA) and re-probed with antibody against β-actin (cat. no. MAB 8226; Abcam) to estimate the amount of protein transferred. Blotted proteins were visualized using HRP-conjugated secondary antibodies and chemiluminescence detection (Super Signal West Dura Extended Duration Substrate; Thermo, Rockford, IL, USA). The chemiluminescence signals were detected with an image reader (LAS 4000; Fujifilm, Tokyo, Japan) and quantified using MultiGauge version 3.1 software (Fujifilm). 5 replicates for 3 experiments were included. Relative arbitrary densitometric units (ADU) was normalized according to loading control (β-actin) and calculated as = (synaptofysin ADU)/ (β-actin ADU). Relative change in synapthofysin expression was calculated as = (nonanoic acid treatment normalized ADU)/ (mean DMSO control normalized ADU). Controls are presented as = (individual normalized control ADU / mean normalized control ADU).
RNA sequencing and RT-PCR GOT1 cells were cultured for three days with 300 µM nonanoic acid or medium with vehicle (0.1% DMSO).Cells were harvested and RNA extracted using RNeasy mini kit (Qiagen). RNA sequencing was performed at the bioinformatics core facility, Sahlgrenska academy, University of Gothenburg. Alignment, identification and quantification of transcripts enabling pairwise comparisons were performed using the Partek flow software. RNA from three replicate experiments of GOT1 cells exposed to 300nM Nonanoic acid or vehicle (DMSO) for three days were reverse-transcribed using Thermoscientific RevertAid RT kit (Thermofisher). PCR reactions in triplicate were set up using predesigned KiCqStart SYBR® Green primers for the genes ACTB, GAPDH, MDK, PCSK1, SCL18A1, SCL18A2, TPH1, VGF (Sigma-Aldrich) and SYBR™ Green PCR Master Mix (Applied Biosystems). The PCR reactions were subjected to PCR cycling conditions recommended by Applied Biosystems in a 7500 Fast Real-Time PCR System (Applied Biosystems). Expression values were calculated as mean values relative to ACTB and GAPDH (2–(Ct (target gene)) – (Ct (ACTB+GAPDH/2))). Statistical significance between experimental conditions was calculated using paired Student’s t-test (GraphPad Prism 8) and a p value of < 0.05 was considered as significant.