H6621 cell culture medium, supplements kit and gelatin-based coating solution were purchased from Cell Biologics (Chicago, IL, USA). DMEM, sodium pyruvate solution, BSA, fatty acid-free BSA, poly-l-lysine, puromycin, HEPES solution and JumpStart Taq DNA polymerase were purchased from Sigma-Aldrich (St.Louis, MO, USA). RPMI 1640 medium (without l-glutamine) was from Lonza (Basel, Switzerland). FBS, trypsin-EDTA solution, l-glutamine, penicillin/streptomycin (P/S), OptiMEM, and F-12 (Ham's nutrient medium) GIBCO™, RevertAid reverse transcriptase, RiboLock RNase inhibitor, random hexamer primers, dNTPs, GeneRuler 100-bp Plus DNA Ladder, HEPES, the bicinchoninic acid protein assay reagent kit Pierce™ and Alexa Fluor™ Red Fluorescent Control siRNA were from Thermo Fisher Scientific (Waltham, MA, USA). Sphingosine 1-phosphate (S1P) was from Biomol (Plymouth, PA). Primary antibodies against S1P1, S1P3, TG, NIS, TPO, and Hsc70 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). HRP-conjugated goat anti-rabbit IgG and the Aurum total RNA isolation kit were from Bio-Rad (Hercules, CA, USA). Primary antibodies against VEGFR2, HIF-1α, p21waf1/cip1, p27kip1, cdk6, MMP2, ERK1/2, pERK1/2, E-cad, N-Cad, TSHR, and HRP-conjugated anti-rat and anti-mouse IgG were from Cell Signaling Technology (Denver, MA, USA). Primary antibody against STIM1, ORAI1, and calpain activity and MTT proliferation assay kits were purchased from Abcam (Cambridge, MA). Cell culture plastic ware and human collagen type IV were from Becton Dickinson (Franklin Lakes, NJ, USA), and Transwell inserts for migration assays were from Corning, Inc. (Corning, NY). All the chemicals and reagents used were of molecular biology and reagent grades. Fura-2 AM was from Molecular Probes (Eugene, OR). Thapsigargin was from Alexis Corporation (San Diego, CA). KAPA Probe Fast Master Mix was from Kapa Biosystems (Boston, MA), and the Universal Probe Library probes were from Roche (Basel Switzerland).
Cell Culture - Human primary thyroid epithelial cells were purchased from Cell Biologics (Chicago, IL, USA). The cells were cultured in complete human epithelial cell medium with supplements kit (H6621, cell biologics, Chicago, IL, USA). The cells were plated on gelatin-coated plates as per manufacturer’s instructions. The human ML-1 follicular thyroid cancer cells were cultured in DMEM with 10% FBS, 1% penicillin/streptomycin (P/S), and 1% L-glutamine. FTC-133 thyroid follicular cancer cells were purchased from Banca Biologica e Cell Factory (Genova, Italy). The cells were cultured in DMEM and F-12 (Ham’s) medium (1:1) with 10% FBS and 1% L-glutamine and 1% P/S. The C643 anaplastic thyroid cancer cells were provided by Dr Nils-Erik Heldin (Karolinska Institute, Stockholm, Sweden) and cultured in DMEM with 10% FBS, 1% L-glutamine, and 1% penicillin/streptomycin. The anaplastic thyroid cancer THJ-16T cells were acquired from Dr John Copland (Mayo Clinic, FL, USA). The cells were cultured in RPMI 1640 with 10% FBS, 1% penicillin/streptomycin, 1 mM sodium pyruvate, and 25.03 mM HEPES. The cell cultures were maintained in a water saturated atmosphere supplemented by 5% CO2 and 95% air at 37 C° in the incubators.
Generation of stable cell lines - Cells were grown on 12-well plates. The transduction was performed according to the manufacturer’s instructions using non-targeting shRNA lentivirus particles, and STIM1- or ORAI1-targeting lentiviral particles (Sigma, St. Louis, MO, USA). The sequences are provided in Supplementary Table.1. After 48 hours, the medium was changed to the medium containing 0.5 µg/ml Puromycin. The cells were cultured with puromycin containing medium hereafter. The knock-down of STIM1 and ORAI1 was measured on mRNA level by quantitative-PCR and on protein level by western blotting, respectively.
Transient transfections − 4 million cells were pelleted and re-suspended in 400 µl OptiMEM together with 20 µg of the control siRNA, siSTIM1, pYFP, pSTIM1 or pORAI1 plasmids. The cells were electroporated at 975 µF and 240 V and were grown in respective media for 48 hours before the start of the experiments.
Qualitative end-point PCR and quantitative realtime PCR - RNA was extracted with Aurum™ Total RNA Mini Kit (Bio-Rad; CA, USA) or by TRI reagent (Sigma Aldrich; St. Louis, MO, USA) according to the manufacturer’s instructions. RNA integrity was checked by gel electrophoresis and RNA concentration and purity was determined with Nanodrop 2000 (Thermo Fisher Scientific; Waltham, MA) and NanoVue Plus (Healthcare Bio-Sciences AB; Uppsala, SE). cDNA samples were prepared with RevertAid reverse transcriptase and SuperScript IV ® Reverse Transcriptase (Thermo Fisher Scientific, MA, USA) from equal amounts of RNA. Reaction mixtures lacking either reverse transcriptase or RNA were used as negative controls. For primers information see supplementary Table. 1. Quantitative real-time PCR assays were designed with the Universal Probe Library (UPL) Assay Design Center (www.rocheappliedscience.com). PBGD was used as a reference gene. RT-PCR was performed with KAPA Probe Fast Master Mix and the StepOnePlus Real-Time PCR System (Applied Biosystems™, Thermo fisher scientific, MA, USA) using the relative standard curve method.
Western Blotting - The whole cell lysates and for the Western blotting were performed as described elsewhere [16]. In some experiments, equal amounts of proteins were loaded on 10% Fast Cast Stain-free sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. Blotting was performed using PVDF membranes on BioRad Transblot system. After blocking, the blots were probed overnight with primary antibodies. Proteins were detected with enhanced chemiluminescence (ECL; Thermo Scientific, 242 Waltham, MA). Densitometric analysis was performed using the ImageJ program for image analysis (National Institutes of Health, Bethesda, MD), and the results were corrected for protein loading by normalization with Hsc70 expression.
Measurements of [Ca 2+ ] i in Single Cells - Cells were processed and analyzed as described elsewhere [17]. A HAMAMATSU digital camera C10600 ORCA-R2 with controller (Photonics K.K.) was used to capture fluorescence images at 1–3 s to avoid bleaching. The images were acquired and processed using the Axon Imaging Workbench 6 software (INDEC BioSystems, Santa Clara, CA). The intracellular free calcium concentrations were calculated as ratio F340/380.
Invasion Assays - Invasion assays were performed as described elsewhere [18]. Cells were serum starved by growing in 0.2% FAF-BSA-containing serum-free medium (SFM) overnight before the start of the invasion assays. In these experiments, the cells were stimulated with S1P and allowed to migrate towards 10% lipid-stripped FBS (LS-FBS) for 16h. In other experiments, cells were allowed to invade towards 10% FBS-containing culture medium for 16h. The non-migrated cells were removed with a cotton swab. The migrated cells were fixed in 2% paraformaldehyde for 10 min and then stained with 0.1% crystal violet in 20% methanol for 5 min. The membranes were washed with PBS and water and allowed to dry overnight. The cells were counted at 40X magnification in a straight line bisecting the membrane.
Zymography - ML-1 MOCK and STIM1-KD cells were grown on 35-mm plates up to 80% confluency. The medium was changed to SFM medium for overnight. Next day, the medium was changed to fresh SFM medium (1 ml on each plate) and the cells were stimulated with or without S1P (100 nM) for 6 h. After 6 h, the media were collected. Equal volumes of medium were mixed with loading buffer (0.1 m Tris-phosphate buffer, pH 6.8, containing 20% glycerol, 6% SDS, and 0.04% bromphenol blue). The samples were electrophoresed with the 10% SDS gels containing gelatin (2.65 mg/ml). The gels were incubated in Zymo buffer (50 mm Tris-HCl containing 2.5% Tween 80 and 0.02 NaN3, pH 7.5) for 30 min. The gels were then incubated in Zymo buffer containing 1 µm ZnCl2 and 5 mm CaCl2 for 30 min. For gelatinolytic activity gels were incubated at 37°C for overnight in buffer containing 50 mm Tris-HCl, 5 mm CaCl2, 1 µm ZnCl2, and 0.02% NaN3 (pH 7.5). The gelatin degradation was visualized under UV light, and after that, the gels were stained with Coomassie Blue R250 for 1–2 h. The gelatinolytic activity was visualized as clear bands against blue background on stained gels. The clear bands after destaining for 30 min, as shown in (Figure. 5E) were quantified by the program ImageJ. The data were normalized with the respective total protein concentrations of the respective cells in the culture plates.
Calpain activity assays − 2 million cells were grown on 100-mm plates. The cells were serum-starved overnight and stimulated with or without S1P (100 nM) for 6 h. Next, the cells were detached and washed three times with PBS. Thereafter, calpain activity assays were performed according to the manufacturer’s instructions (Abcam, Cambridge, MA). The samples were analyzed at an excitation of 400 nm and emission at 505 nm using a fluorescence analyzer. The results were normalized with the respective total protein concentrations of each culture plate and presented as % calpain activity.
Proliferation Assays - The [3H] thymidine incorporation method was used to study the proliferation of the ML-1 MOCK, STIM1-KD and ORAI1-KD cells. 50,000 cells were seeded on 35-mm plates and allowed to grow for 24 h. Four hours prior to the end of each experiment, 0.4 µCi/ml [3H]thymidine was added. The cells were washed three times with PBS, incubated for 10 min with 5% trichloric acetic acid, and then incubated for 10 min with 0.1 m NaOH. The samples were transferred into scintillation tubes and high sample load scintillation cocktail Optiphase Hisafe 3 was added. The radioactivity was measured using a Wallac 1414 liquid scintillation counter.
MTT proliferation assay method 10,000 ML-1 MOCK and STIM1-KD cells were grown on 96 well plate for overnight and treated with indicated concentration of respective drugs for 24 h. Thereafter, the assays were performed according to the manufacturer’s instructions. The absorbances were measured at 590 nm using a spectrophotometer.
FACS Analysis − 0.5 million ML-1 Mock, STIM1-KD and ORAI1-KD cells were grown overnight on 35-mm plates. The cells were detached with EDTA-trypsin solution and were centrifuged. The cell pellets were suspended in 500 µl of propidium iodide solution (0.05 mg/ml propidium iodide, 3.8 µM sodium citrate, 0.1% Triton X-100 in PBS) and incubated for 15 min at room temperature. The samples were then processed by flow cytometry using FACSCalibur and CellQuest Pro software (BD Biosciences, San Jose, CA). ModFit LT 4.1 software was used to calculate the percentage of cells in each phase of cell cycle.
Iodine uptake Assays - The uptake of iodide in ML-1 MOCK and STIM1-KD cells was determined by a nonradioactive, fast and highly reproducible spectrophotometric method based on the catalytic effect of iodide on reduction of yellow cerium (IV) to colorless cerium (III) in the presence of arsenious acid (Sandell-kolthoff reaction). The assay was performed exactly as described elsewhere [19]. In some experiments, the cells were pre-incubated with 1 mU/ml for overnight before the start of the assay.
Synthesis and modification of MPDA - The mesoporous polydopamine (MPDA) particles were prepared by a templated synthesis method as described elsewhere [20]. The surface of the obtained MPDA nanoparticles were modified by N,N-dimethylethylenediamine (DMEA) presenting a tertiary amine group for facilitating the siRNA loading [21]. 1 mg of MPDA nanoparticles were suspended in 2 ml of Tris buffer (pH.8.5). After the introduction of DMEA (25.6 mg), the mixture was stirred for 1 hour at room temperature. The DMEA modified MPDA nanoparticles were isolated from the suspension by centrifugation.
siRNA loading of DMEA modified MPDA nanoparticles − 0.1 mg siRNA STIM1, non-target control siRNA or control siRNA-Alexa Fluro 555 were mixed with 1mg DMEA modified MPDA in 1 ml of 2-(N-morpholino) ethanesulfonic acid (MES) buffer (final concentration 10 mM, pH 5.0) through continuous vibration mixing for 40 min. The siRNA loaded DMEA modified MPDA nanoparticles were separated by centrifugation at 11 000 rpm for 15 min and stored at -20⁰C. 50,000 ML-1 cells were grown on polylysine coated glass bottom 35 mm plate for overnight. Next day, 5 µM control siRNA loaded or siRNA STIM1 loaded nanoparticles were added to respective culture plates. The cells were grown for 96 h to 120 h. The cells were washed, and the coverslips were mounted with DAPI containing mounting media ProLong™ Gold Antifade mountant with DAPI. The laser scanning confocal microscopy was performed using a Leica TCS SP8 X microscope (Leica Microsystems GmbH, Wetzlar, Germany) with 63x oil immersion magnification and a HC PL APO CS2 objective.
Zebrafish embryo xenograft - The procedure to carry-out xenotransplantation in zebrafish embryos was modified from as described elsewhere [22, 23]. Adult zebrafish (Danio rerio, casper strain (roy-/-; mitfa-/-) [24] were maintained according to standard procedures [25]. The embryos were collected after natural spawning in breeding tanks. Experimentation with zebrafish was performed under license ESAVI/9339/04.10.07/2016. The zebrafish embryos were cultured in E3-medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) supplemented with 0.2 mM phenylthiourea (PTU, Sigma-Aldrich) at 33°C. Two days post-fertilization, the embryos were anesthesized with MS-222 (200 mg/l, Sigma-Aldrich) and mounted into low-melting point agarose for tumor transplantation.
Prior to transplantation, ML-1 cells were labelled with 5µM CellTracker Green CMFDA (Thermofisher Scientific), trypsinized and washed with PBS. Approximately 5–10 nl of ML-1 cell suspension was microinjected into yolk or pericardial cavity of the embryo using CellTramVario (Eppendorf), Injectman Ni2 (Eppendorf) micromanipulator and self-made borosilicate glass needles pulled from glass capillaries (TW100-4, World Precision Instruments Ltd., Sarasota, FL) using micropipette puller (PB-7, Narishige, Tokyo, Japan). After injections, the agarose gel was broken and embryos were released, washed with E3 and cultured in E3-PTU at 33°C. On 1 day post-injection (1 dpi), the successfully xenografed healthy embryos were selected to the experiment and placed into 12-well or 96-well plates (1 embryo per well). The embryos were anesthesized and imaged at 1 dpi and 4 dpi using Zeiss AxioZoom fluorescence stereomicroscope of Nikon Eclipse widefield microscope.
The size of the tumor was determined by manually measuring the fluorescent tumor area using FIJI software (ImageJ version 1.49 k) [26]. Samples having significant malformations, died embryos and images where embryo was not laterally oriented or out-of-focus were excluded from the analysis. Samples were not blinded.
STIM1 staining of patient samples - The use of tissue samples and related data was approved by Auria Biobank (decision AB19-2988) and the Hospital District of Southwest Finland and Turku University Hospital (decision T12/011/20). The samples were from both female and male patients (9 anaplastic thyroid carcinoma, 29 follicular thyroid carcinoma, and 138 papillary thyroid carcinoma). The age distribution of the patients was 18–88 years of age. Thyroid tissue was prepared according to standard histology practice, using buffered formalin (pH 7.0) for fixation and embedding tissue into paraffin blocks. Tissue microarrays (TMAs) were prepared from tumour area of each patient. As controls, normal thyroid tissue samples (control) were obtained from 104 patients. Tissue sections were cut and stained with anti-STIM1 antibody (1/200; Abcam 57834) using Labvision Autostainer (Thermo-Fisher Scientific, Fremont, CA, USA) and detected with an Orion 2 step detection system using goat anti mouse/rabbit HRP + WellMed T100 HRP. Final images were acquired at 20X magnification using the case viewer software (HISTECH Ltd, Hungary).
High throughput data mining and Gene expression analyses - The clinical and transcriptomic data of the Thyroid Carcinoma (THCA) cohort were downloaded from the GDC database (https://portal.gdc.cancer.gov/projects/TCGA-THCA). We included 502 thyroid tumors and 58 adjacent normal samples for transcriptome analysis. Among those tumor samples, 353 were papillary thyroid tumors while 106 were follicular thyroid tumors. To assess gene expression levels in normal thyroid samples, we retrieved gene expression data (as raw count values) of 563 thyroid samples from the Genotype-Tissue Expression (GTEx) (https://gtexportal.org/home) for comparative analysis of gene expression. Only genes that express in at least 50% of the samples were assessed. DESeq2 was used for differential expression analysis between thyroid cancer groups and their matched para-cancerous samples [27]. A Benjamini-Hochberg adjusted p-value of < 0.05 was considered as statistical significance. The expression profiles of genes of interest (STIM1 and ORAI1) were also extracted from both retrieved datasets after performing variance stabilizing (VST) transformation for comparative analyses between cancerous tissues, cancer adjacent normal tissues, and controls.
Statistical analysis - The results are presented as means ± the standard error of the mean from at least three independent measurements. Student's t test was used when two means were compared. One-way analysis of variance and Bonferroni's post hoc test were applied when three or more means were compared. Kruskal-Wallis test, Kolmogorov-Smirnov test and Fisher exact test were used to analyze non-normally distributed data. GraphPad Prism 8 program (GraphPad Software Inc., San Diego, CA) was used for the statistical analyses. p values < 0.05 were considered statistically significant.