Introduction and expression of PIK3CAE545K in a papillary thyroid cancer BRAFV600E cell line leads to a dedifferentiated aggressive phenotype

Cell lines and culture conditions

The LAM1 cell line, a gift from Dr. John Copland (Mayo Clinic, Jacksonville, Florida) which is a confirmed BRAFV600E PTC cell line, was used for this study [11]. The LAM1 cell line was cultured in DMEM/F12 media supplemented with 10% FBS (GIBCO), penicillin (100 µg/mL) and streptomycin (100 µg/mL) (Invitrogen). Short tandem repeat (STR) profiling was carried out at The Centre for Advanced Genomics as described previously [12]. PCI6A and JHU029 are both head and neck squamous cell cancer cell lines and both carry canonical hot spot mutations in PIK3CA. PCI6A carries an E545K mutation and JHU029 has a H1047L. For the purpose of this study, PCI6A was used as a positive control for the expression of the E545K mutation and JHU029 was used to confirm the specificity of the mutant primer qPCR.

Transfection of a PIK3CAE545K expression vector into the LAM1 cell line

The most frequent PIK3CA variant occurring in ATC is the E545K variant [9], providing the rationale for our study. PIK3CA exon9 E545K was a gift from Bert Vogelstein (Addgene plasmid #16,642). This expression vector [13] was used to transfect LAM1 parental cells (PTC BRAFV600E) to generate a stable PIK3CAE545K mutant for use in over-expression studies. The mutant cell line was denoted as LAM1:PIK3CAE545K. A parallel transfection was performed using an empty vector control plasmid (denoted as LAM1EV). The parental cell line was transfected using Lipofectamine 3000 Reagent (Cat no. L3000001, Thermo Fisher Scientific) according to the standard protocol using 5 µg of plasmid DNA. Cells were incubated overnight, and transfection media was replaced the following day with normal cell line media. The following day, geneticin (G418) was added for selection at a concentration of 300 µg/mL to select against untransfected cells. The concentration of G418 used was predetermined for the LAM1 cell line using a standard kill curve.

Immunoblotting

Immunoblotting was completed as described previously with 20 µg protein loaded per well (Pinto et al. 2018). PI3 kinase (p110α) (Cat no. 4249) and α-tubulin (Cat no. 2125) antibodies were obtained from Cell Signaling Technology. Membranes were incubated with peroxidase-conjugated anti-rabbit secondary antibodies diluted 1:5000 for 1 h at room temperature. Detection of target proteins was performed using Luminata Forte Western HRP substrate (EMD Millipore). α-tubulin was used as a loading control.

Growth curves

Cells were seeded at a density of 2400 cells per well into 96-well plates. Plates were then read using PrestoBlue (Thermo Fisher Scientific) at 0, 24, 48 and 72 h. Fluorescence readings were completed using a BioTek Synergy Microplate Reader with 560 nm excitation and 590 nm emission wavelengths. An unpaired Student’s, two-tail t-test was used for statistical analysis, where comparisons were made between LAM1EV and LAM1:PIK3CAE545K cell lines for each time point. P values < 0.05 were considered to represent statistically significant differences. Statistical analysis is displayed for the 72-h time point when compared to the control at the same time point.

Dose–response curves (Etoposide)

Etoposide is a chemotherapy used clinically to treat ATC, and functions by inhibiting the enzyme topoisomerase II. Cell lines were seeded into 96-well plates at a density of 2400 cells per well and incubated overnight at 37 °C and 5% CO2. After 24 h, cells were treated with Etoposide (Cat no E1383; Sigma Aldrich) using a concentration range of 0.125 to 32 µM. After 72 h, plates were read by incubating cells with PrestoBlue for 1 h at 37 °C. After incubation with PrestoBlue, fluorescence readings were completed using a BioTek Synergy Microplate Reader with 560 nm excitation and 590 nm emission wavelengths. To generate dose–response curves, raw fluorescence data was loaded into Prism® 8 GraphPad Software, where the values were then normalized to the untreated control samples and the average viability for each concentration tested was calculated. In order to determine the half-maximal inhibitory concentration (IC50), the normalized relative fluorescence units (RFU) of the Etoposide-treated samples were calculated as a percentage of the mean RFU of the untreated samples. Drug concentrations were then transformed to a logarithmic scale (Log10) and IC50 values were calculated using non-linear regression (curve fit) (Prism8).

Clonogenic assay

LAM1EV and LAM1:PIK3CAE545K cells were seeded into six-well tissue culture plates at a density of 1,000 cells per well. Cells incubated at 37 °C for 10 days based on colony formation of the parental cells with cell media changed every three days. Cells were washed with PBS and fixed with 100% chilled methanol for 15 min. Following fixation, cells were stained with 0.5% crystal violet in 10% methanol/1 × PBS for a total of 10 min. Brightfield microscopy was used to quantify colony formation, with a colony defined as being ≥ 50 cells. For quantification, three representative fields per well were counted for the number of colonies and this was completed for both the parental and mutant cell lines. LAM1:PIK3CAE545K cells were compared to the parental control line and p values < 0.05 were considered to represent statistically significant differences in colony formation (Student’s t-test).

Clonogenic assay: radiation response

LAMEV and LAM1:PIK3CAE545K cells were seeded at a density of 250 (0 Gy), 500 (1 Gy), 1000 (2 Gy) and 2000 (4 Gy) cells per well in six-well plates, with respective doses of radiation applied after 24 h (with three biological replicates). Cells were incubated at 37 °C for a one-week period with cell media changed every three days. Cells were washed with PBS and fixed with 100% chilled methanol for 15 min. Following fixation, cells were stained with 0.5% crystal violet in 10% methanol/1 × PBS for 10 min. Stained colonies were quantified and counted using brightfield microscopy with a colony defined as being ≥ 50 cells. The plating efficiency (PE) of cells that were untreated was calculated. The survival fraction was calculated and defined as the number of colonies that arose following radiation treatment, and expressed in terms of PE [14].

Cell migration assay

Cell migration for LAM1EV and LAM1:PIK3CAE545K cell lines were quantified using a 24-well cell migration plate and fluorometric analysis (Cat no. CBA-100; Cell Biolabs Inc). The initial cell suspension was placed into the upper chamber containing serum free media at a density of 5 × 104 cells per chamber, with three biological replicates. Cells were incubated at 37 °C to allow migratory cells to pass through the polycarbonate membrane and cling to the bottom of the chamber. Cells that stayed in the upper chamber were considered to be non-migratory. Migratory cells were then dissociated from the membrane of the chamber using the Cell Detachment Buffer provided in the cell migration plate kit. Cells which migrated through were lysed and quantified using CyQuant GR Fluorescent Dye provided in the same kit. Fluorescence readings were completed using a BioTek Synergy Microplate Reader and representative images were collected using brightfield microscopy.

Hind flank model to study thyroid cancer disease progression

Mice were maintained and handled in accordance with the Western University AUP 2015–045 protocol. We used the cell lines LAM1EV and LAM1:PIK3CAE545K for the generation of cell line xenograft hind flank models of disease (n = 5 per group). Cell lines were treated with plasmocin for two weeks prior to use in mouse models and screened for mycoplasma before use. Both cell lines were cultured in DMEM/F12 media supplemented with 10% heat inactivated FBS without antibiotics for two weeks before initiation of experimentation. A total of 1 × 106 cells were injected subcutaneously into the hind flank of each athymic nude mouse. Mice were weighed twice weekly and tumour dimensions (length and width) were measured once weekly when tumours were palpable using digital calipers. Individual tumour volumes were calculated using the formula: [length × (width)2] × 0.52 [15]. Mice were sacrificed at 14 weeks post-injection. Tumours were dissected, weights were recorded in milligrams (mg) (Additional file 2: Table S2) and stored in formalin for FFPE block preparation and IHC studies.

Immunohistochemistry

Immunohistochemistry (IHC) staining was carried out in the Molecular Pathology Core Facility (Western University) where 4-µM sections were cut and tested for expression of thyroid-related markers. Primary antibodies were used according to the manufacturer’s instructions: TTF-1 (Abcam; ab76013), PAX8 (Abcam; ab76013), Beta Catenin (Cell Signalling; 8480S), Ki67 (Cell Signalling; 9027S) and Thyroglobulin (Agilent; IR50961-2). Slides were scanned using an Aperio ScanScope slide scanner.

Statistical analysis

A Student’s unpaired, two-tail t-test was performed using Prism® 8 Graphpad Software Macintosh Version (by Software MacKiev © 1994–2014 GraphPad Software, Inc). P values < 0.05 were considered statistically significant. P values are defined as not significant p > 0.05, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001 and **** represents p < 0.0001.

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