Primary cell culture of canine mammary gland tumors
Canine mammary gland tumors were obtained from the College of Veterinary Medicine at Chonnam National University and local hospitals for primary cell culture. Tumor fragments ~ (1 cm × 1 cm) were dissociated and washed with D-PBS (LB001-02, Welgene, Korea) supplemented with 1% penicillin and streptomycin (P/S, LS202-02, Welgene, South Korea). Gentle MACS Dissociator (130-093-235, Miltenyi Biotec, Germany), enzymatic solution mixed with tissues, was used to dissociate them, and the cells were then rotated for 10 min in CO2 incubator at 37 °C, at 5%, utilizing MACs mix Tube Rotation. Then, Cells were separated using a 70 μm mesh filter and then centrifuged at 400 x g for 10 min. The supernatant was removed, and the cell pellet was resuspended and maintained in DMEM/F-12 (Welgene, Seoul, South Korea) or F media (Puricellmania, Seoul, South Korea) supplemented with 10% FBS (Hyclone, UT, USA) and 1% P/S solution. To eliminate fibroblasts, the differential trypsinization method was described in a previous study [3]. HeLa and MDCK cells were purchased from ATCC, and TP53-knockout fibroblasts were kindly provided by Professor H.G. Kim [30]. All cells were incubated at 37 °C with 5% CO2. All cells used in the experiment have passage numbers ranging from 10 to 30.
Cell growth assay
MGT cell lines were seeded in triplicate in a 6-well plate with 2 × 104 cells/well for cell growth assay. Cell counts were performed on days 1, 3, and 5 following the manufacturer’s instructions using an ADAM-MC cell counter (NanoEntek, Seoul, South Korea).
Soft agar colony formation assay
The bottom layer of a 6-well plate was made up of a 0.6% agar solution in DMEM/F-12 containing 20% FBS, and the top layer was seeded with a single-cell suspension of 5 × 104 cells using a 0.3% agar solution in DMEM/F-12. 2 weeks later, the formation of spheres was observed using CELENA® S digital microscopy (Logos Biosystems, Anyang, Korea). Quantification of colonies involved randomly selecting three distinct areas for monitoring in each well, and individual colonies larger than 20 μm were counted.
RNA sequencing analysis
RNA was extracted using the RiboEx reagent (GeneAll, Seoul, South Korea). RNA was prepared for the mRNA sequencing library using the MGIEasy RNA Directional Library Prep Kit (MGI) according to the manufacturer’s instructions. The products are then purified and enhanced by PCR to form the final cDNA library. The double-stranded library is measured using the QauntiFluor ONE dsDNA System (Promega). The library is followed by the cleanup of circularization products. The QauntiFluor ssDNA System (Promega) was used to quantify the library. The constructed library was then sequenced on the MGIseq system (MGI). All datasets created and analyzed in this study are deposited in the NCBI’s Gene Expression Omnibus (GSE230271).
Bioinformatics analysis
Genes with P < 0.05 were selected for cluster analysis, complete linkage clustering was carried out using the Cluster 3.0 program, and Java Tree View was used for visualization. Using the GraphPad Prism 8 program (GraphPad Software Inc.), a volcano plot was created to display the differentially expressed genes in tumor samples. GSEA (v4.2.3, available at http://www.broad.mit.edu/gsea/) was used to analyze significant biological pathways.
H&E staining and histological analysis
Canine MGT tissues were preserved in 10% neutral formalin for 3–4 days at 20 °C. Following sample washing, cutting, and dehydration, an automatic embedding machine was used to perform paraffin embedding. Tissues were sectioned using a microtome with 3 μm thickness and were mounted on slides and then stained with hematoxylin and eosin (H&E). The slides were examined using standard light microscopy. Nuclear and cellular pleomorphism, mitotic index, the presence of a necrosis region, and lymph node metastasis were graded based on H&E staining for histological classification and grade evaluation of tissues [13].
Cell viability assay
Cells were inoculated with 1 × 103 cells per well in triplicate in 96-well plates and cultured for 24 h until attachment. The cells were then treated with various concentrations of BYL719 (PI3K inhibitor, S2814, Selleckchem, USA). After 72 h, cell viability was measured using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan), following the manufacturer’s instructions. At 450 nm, the fluorescence value was measured using a Synergy HTX multi-mode reader (Biotek, USA). The IC50 values were calculated by GraphPad Prism 8 software (GraphPad Software Inc.).
Immunofluorescence (IF)
An immunofluorescence assay was performed to detect the expression of the α-SMA (1:200, mouse anti-alpha-smooth muscle actin, M0851, Dako, USA), Vimentin (1:200, mouse anti-vimentin, M0725, Dako, USA), Cytokeratin (1:200,mouse anti-cytokeratin, M3515, Dako, USA) in the MGT cell lines. MGT cell lines were seeded in 24-well plates with placed 12 mm coverslips (SPL, Korea). Cells were fixed in 4% paraformaldehyde solution for 15 min at room temperature, permeabilized in 0.2% Triton X-100 (9036-19-5, Sigma-Aldrich) for 10 min, and blocked with 5% bovine serum albumin (BSA) (9048-46-8, Sigma-Aldrich) in D-PBS for 1 h. The cells were incubated with primary antibodies diluted in D-PBS (1:200) with 5% BSA overnight at 4 °C, then incubated with the secondary antibody (1:400) for 2 h at 37℃. Each stage was washed three times for 5 min with D-PBS. All coverslips were mounted with a mounting solution with DAPI (101098-044, Vector laboratories, CA, USA). Slides were imaged with confocal microscopy (Carl Zeiss, Germany).
Western blot analysis
MGT cell lines were lysed in RIPA buffer (89,900, Thermo Fisher Scientific) supplemented with phosphatase inhibitor cocktail 2 (ApexBio, USA). A bicinchoninic acid (BCA) Protein Assay Kit (23,227, Thermo Fisher Scientific) was used to determine protein content. Proteins were separated on 10% sodium dodecyl sulfate–polyacrylamide gel and then transferred to polyvinylidene difluoride membranes. These membranes were blocked with 5% skim milk in phosphate-buffered saline with Tween®20 (9005-64-5, Sigma) (PBS-T) at room temperature for 1 h and then incubated with corresponding primary antibodies (1:1,000) at 4 °C overnight with moderate shaking. After washing with PBST, membranes were incubated with an appropriate secondary antibody (1:1,000) at room temperature for 1 h. Membranes were then visualized using chemiluminescence (iBright™ 1500, Invitrogen, Thermo Scientific, USA) following the manufacturer’s protocol. Primary antibodies included: Rabbit anti-Vimentin (1:1,000, #5741, Cell signaling, USA), Mouse anti-E-cadherin (1:1,000, #14,472, Cell signaling, USA), Mouse anti-N-cadherin (1:1,000, sc-59,987, Santa Cruz Biotechnology, USA), Mouse anti-Cytokeratin 5 (1:1,000, sc-32,921, Santa Cruz Biotechnology, USA), mouse anti-α-SMA (1:1,000, M0851, Dako, USA), rabbit anti-Akt (1:1,000, #9272, Cell signaling, USA), rabbit anti-p-Akt (1:1,000, #9271, Cell signaling, USA), and mouse anti-GAPDH (1:10,000, #2118, Cell signaling, USA).
Quantitative reverse transcription-polymerase chain reaction
Total RNA was extracted with RiboEx reagent (GeneAll, South Korea) and purified with a Hybrid-R kit (GeneAll, South Korea) as directed by the manufacturer. A RevertAid First-Strand cDNA Synthesis kit mRNA (Thermo Fisher Scientific, USA) was used to synthesize cDNA from 500 ng of mRNA. SYBR Premix Ex Taq I™ (Takara Bio, Japan) was used for PCR on a CFX96 real-time PCR Detection System (Bio-Rad, South Korea). Results of quantitative reverse transcription-PCR (qRT-PCR) were evaluated as Ct values and quantified using the 2−ΔΔCt method. GAPDH was used as a reference gene to analyze the canine’s gene quantitatively. Primer sequences used for qRT-PCR amplification were as follows: ACTA2 (a-SMA) F: 5’-CATCACCAACTGGGACGACA-3’, R: 5’-GTACATGGCTGGGACGTTGA-3’; VIM (Vimentin) F: 5’-GCGGGAGAAGATGTTGACAATG-3’, R: 5’-CGCAGCCACGCTTTCATATT-3’; GAPDH F: 5’-ATGGTGAAGGTCGGAGTCAA-3’, R: 5’-ATCACCCCATTTGATGTTGG-3’; CDH1 (E-cadherin1) F: 5’-AAATCACATCCTACACCGCC-3’, R: 5’-ATTAACCTCCAGCCAACCG-3’.
DNA extraction and sequence analysis
Genomic DNA was isolated from cultured cell lines using the G-DEX™ IIc Genomic DNA Extraction kit (iNtRON Biotechnology Inc., 17231, Seoul, Korea), following the manufacturer’s prescribed protocols. To detect the c.3140A > G (H1047R) missense mutation in the coding sequence of canine PIK3CA, the following sequencing primer sets, originated from a previous study [12], were used: F: 5’- CTG GAA TGC CAG AAC TAC AAT C -3′; R: 5’- CTG TTC ATG GAT TGT GCA ATT CC -3′. PCR products were electrophoresed, and specific bands were excised from the agarose gel. Amplified PCR products were extracted from the gel using an EZ-Pure™ Gel Extraction Kit. Ver. 2 (Enzynomics, Daejeon, Korea) DNA samples were submitted to Solgent Inc. (Daejeon, Korea) for sequencing using an ABI PRISM 3730XL Analyzer.
Mycoplasma detection
Mycoplasma contamination in cell lines was assessed using a LookOut® Mycoplasma PCR Detection Kit (MP0035, Sigma-Aldrich, USA) following the manufacturer’s instructions. Briefly, the supernatant from cultured cells was collected, and a PCR reaction was performed after mixing it with PCR premix and primer mix. PCR products were analyzed by electrophoresis on a 1.2% agarose gel, and the presence of mycoplasma DNA band was visualized under UV light using the Invitrogen™ iBright™ CL750 Imaging System (Thermo Scientific, USA).
Tumorigenicity assays, tumor growth in immunodeficient mice
For tumorigenicity assays, three 4-week-old female BALB/c nude mice were utilized. Each mouse was inoculated subcutaneously in the left inguinal mammary fat pad with a cell suspension consisting of 3 × 106 MGT cells in 100 µL D-PBS mixed with 100 µL Matrigel. Tumor growth was monitored and measured weekly. After 9 weeks, euthanasia was performed using 1% isoflurane to ensure the mice were unconscious and free of pain prior to sacrifice. The tumor masses were collected and fixed in 4% paraformaldehyde for histological analysis. All procedures involving animals were approved by the Chonnam National University’s Animal Care Committee and complied with the guidelines and regulations set by the Republic of Korea government (CNU IACUC-YB-2023-105).
Statistical analysis
Microsoft Excel 2013 (Microsoft Inc., Redmond, WA, USA), SPSS 20 (SPSS Inc., Chicago, IL, USA), and GraphPad Prism 8 software (GraphPad Software Inc., La Jolla, CA, USA) were used for statistical analyses. P-value < 0.05 indicated statistically significant. The heatmaps were made using Java TreeView (version 1.1.6r4). Significant differences between and among groups were determined by a two-tailed t-test and one‐way ANOVA, followed by Tukey’s multiple comparison test.
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