Modelling reoxygenation effects in non-small cell lung cancer cell lines and showing epithelial-mesenchymal transition

Cell lines and culture conditions

We have used the following NSCLC cell lines, NCI-H460, HCC827, NCI-H1299, NCI-H1703 and SK-MES-1, which were gifted by Professor Derek Richard (QUT, Brisbane). All of the NSCLC cell lines were STR profiled and authenticated. This gave us the confidence that our cell lines are correctly identified, and not cross contaminated with other cells. Cells were cultured under standard conditions in humidified incubators at 37 °C, 20% O2, 5% CO2 (normoxia) or in a hypoxic chamber at 1–2% O2, 5% CO2, (94% N2). To avoid the influence of culture media, all culture media conditions were standardised to RPMI-1640-Glutamax (Life Technologies, Inc) supplemented with 10% foetal bovine serum (FBS) (Life Technologies, Inc) and 1% Penicillin–Streptomycin (Life Technologies, Inc).

Optimisation of culture conditions to investigate hypoxia and reoxygenation effects

To determine the effects of oxygenation on NSCLC cell lines, cells were incubated at different oxygen concentrations: normoxia (20% oxygen) or hypoxia (1% O2) for 14 days to capture in-vivo conditions of CTCs as they enter circulation. In addition, cells maintained in hypoxia were further exposed to a pulse of reoxygenation (normoxia) for 4 h prior to collection. This is to model exposure of CTCs to relatively high O2 levels while in circulation (Bartkowiak et al. 2020) (Fig. 1). NSCLC cell lines were cultured in replicates of three in T75 flasks (Greiner CELLSTAR™ TC), and cells were collected at full density for downstream experiments.

Fig. 1figure 1

Illustration of the cell culture regimen to study the effects of different oxygen conditions (hypoxia/reoxygenation) in five NSCLC cell lines, NCI-H460, HCC827, NCI-H1299, NCI-H1703 and SK-MES-1

STR profiling and mycoplasma testing

To detect cross-contamination in NSCLC cell lines, we used a short tandem repeat (STR) profiling technique on the GenePrint® 10 System (Promega, USA) according to the manufacturer’s instructions at the Genomics Research Centre (GRC; Brisbane, Australia). The NSCLC cell lines were processed using the Applied Biosystems® 3500 Genetic Analyzer. Data were analysed using GeneMapper® v5.0 software (Applied Biosystems). Appropriate positive and negative controls were run and confirmed for each sample. Furthermore, mycoplasma testing was also performed in all NSCLC cell lines using the MycoAlert Mycoplasma detection system (Lonza, Switzerland) at the Genomics Research Centre (GRC; Brisbane, Australia). All cell lines were negative for mycoplasma.

MTT assay for cell proliferation

To determine cell proliferation, NSCLC cell lines were seeded in 24 replicates at 2500cells/well in 96-well flat-bottomed plates (Grenier Bio-One CELLSTAR®). Cells were left to settle overnight in a standard incubator at 37 °C, 20% O2, 5% CO2 for normoxic conditions and 1–2% O2, 5% CO2, (94% N2) for hypoxic conditions. At 24, 48 and 72 h time points, cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/ml; Promega, Shanghai, China) at a final concentration of 0.5 mg/ml for 4 h as per manufacturer’s instructions. Absorbance was measured using a CLARIOstar Plus microplate reader (BMG Labtech, Germany) at 490 nm absorbance used as the reference wavelength. The assay was performed in three biological replicates.

Colony-forming assay

The soft agar colony formation assay was performed according to the previously described protocol (Borowicz et al. 2014). Colonies were stained with crystal violet solution and imaged with a Nikon Eclipse TS2 microscope. Colonies were defined as groups of more than 20 cells when counted manually.

Sphere formation assay

Post trypsinisation, cells were spun down at 1000g for 5 min to obtain a pellet. The cell pellet was resuspended in warmed 1 × concentration of Happy Cell® ASM (Vale Life Sciences) diluted with RPMI-1640-Glutamax supplemented with FBS, and Pen Strep described above. Seeding density was optimised to 0.5 × 106/well for each cell line and plated in 96 well Ultra-Low Attachment plates (Corning). Cells were incubated for 72 h in normoxia and hypoxia (1% O2) and visualised with a Nikon Eclipse TS2 microscope.

RNA extraction and cDNA synthesis

For quantitative mRNA analysis, total RNA (106 cells) was extracted using the RNeasy Mini Kit following the manufacturer’s protocol. RNA extraction procedure comprised of an on-column DNase treatment using RNase-free DNase (Qiagen, Germany), as described by the manufacturer. Total RNA was eluted in 25 μL nuclease-free water. The RNA concentration and quality were assessed using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, USA). Purity of samples was estimated by measuring A260/280 and A260/230 ratios of spectrophotometric absorbance to check for copurified contaminants during RNA isolation. cDNA synthesis and genomic DNA elimination were conducted using the RT2 First Strand Kit (Qiagen, Germany) according to the manufacturer’s instructions, using 400 ng total RNA for each sample. Negative reverse transcription (without the reverse transcriptase enzyme) was conducted to prove that the samples are devoid of genomic DNA.

EMT array

Human Epithelial to Mesenchymal Transition (EMT) RT2 Profiler PCR Array (Qiagen, Germany) was used to investigate the expression of multiple EMT-related genes in NSCLC cell lines under normoxia and hypoxic conditions. The epithelial to mesenchymal transition RT2 Profiler PCR Array includes cell surface receptor, extracellular matrix and cytoskeletal genes mediating cell adhesion, migration, motility, and morphogenesis. The array also includes genes controlling cell differentiation, development, growth, proliferation and signal transduction and transcription factor associated with the process of EMT. The expression of 84 genes was quantified by qPCR in cells grown in normoxia and hypoxia for 14 days and compared to the same cell types subjected to a pulse of reoxygenation for 4 h. All analyses were performed using the web-based PCR Array Data Analysis Software tool provided on the Gene Globe Data Portal (GeneGlobe 2020). Arithmetic means of the housekeeping genes B2M and RPLP0 were used for normalisation, and fold change was used to analyse differences in gene expression.

Quantitative real-time PCR (qPCR)

HIF-1α expression levels were tested using primers (fw_GCACAGGCCACATTCACG, rev_TGAAGATTCAACCGGTTTAAGGA) obtained from the literature (Wei et al. 2013). Human β-actin (Forward: CAC CAT TGG CAA TGA GCG GTT C; Reverse: AGG TCT TTG CGG ATG TCC ACG T) was used for normalisation of total RNA input per qPCR reaction. Total RNA (20 ng) was used with PowerUp™ SYBR® Green Master Mix (Applied Biosystems, Thermo Fisher Scientific, USA). qPCR conditions were as follows: 50 °C for 2 min; 95 °C for 10 min; 40 cycles at 95 °C for 15 s and 60 °C for 1 min; and a final melting curve analysis with the following conditions: 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. qPCR reaction per NSCLC cell line under a condition was run in duplicate using the QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems™).

Western blot analysis

Total protein concentration from cell lysates was quantified using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of proteins (25 µg) were loaded into 10% SDS-PAGE gels and run at 100 V for 90 min. Proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane at 100 V for 90 min at 4 °C. The membrane was blocked for 1 h at room temperature (RT) using 5% bovine serum albumin (BSA). Membranes were incubated overnight at 4 °C with the following primary antibodies: EGFR (Cell signaling—#4267), HIF-1α (Sigma-Aldrich- # HPA000907), AKT (Cell signaling—#4691P), p-AKT (Cell signaling—#4060), Vimentin—(Santa Cruz—-#sc32322), Pan-Cytokeratin (Thermo Fisher—#ma513203), 4EBP1—(Cell signaling—#9644), p4EBP1—(Cell signaling #2855). Following primary antibody incubation, membranes were incubated with anti-rabbit IgG-HRP secondary antibody (Cell signaling—#7074) or anti-mouse IgG-HRP secondary antibody (Cell signaling—#7076) for 1 h at RT. The membranes were incubated with Pierce ECL Western Blotting substrate (Thermo Fisher Scientific) and imaged using ChemiDoc XRS + System (Bio-Rad Laboratories).

Statistical analysis

All statistical analyses were performed using GraphPad Prism 8 software version 8.43 (GraphPad Software Inc., La Jolla, CA, USA). For cell proliferation assay and colony-forming assay, an unpaired two-tailed Student’s t test was performed. For analysing the EMT array and qPCR, a One-Way Analysis of Variance (ANOVA) with Multiple Comparisons was performed. Data are shown as mean ± standard deviation unless otherwise specified.

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