Primary patient-derived lung adenocarcinoma cell culture challenges the association of cancer stem cells with epithelial-to-mesenchymal transition

The cancer stem cell (CSC) and epithelial-to-mesenchymal transition (EMT) models have been closely associated and used to describe both the formation of metastasis and therapy resistance. We established a primary lung cell culture from a patient in a clinically rare and unique situation of primary resistant disease. This culture consisted of two biologically profoundly distinct adenocarcinoma cell subpopulations, which differed phenotypically and genotypically. One subpopulation initiated and sustained in spheroid cell culture (LT22s) whereas the other subpopulation was only capable of growth and proliferation under adherent conditions (LT22a). In contrast to our expectations, LT22s were strongly associated with the epithelial phenotype, and expressed additionally CSC markers ALDH1 and CD133, whereas the LT22a was characterized as mesenchymal with lack of CSC markers. The LT22s cells also demonstrated an invasive behavior and mimicked gland formation. Finally, LT22s were more resistant to Cisplatin than LT22a cells. We demonstrate a primary lung adenocarcinoma cell culture derived from a patient with resistant disease, with epithelial aggressive subpopulation of cells associated with stem cell features and therapy resistance. Our findings challenge the current model associating CSC and disease resistance mainly to mesenchymal cells and may have important clinical implications.


Tumor cell isolation in detail
Tumor tissues were cut into small (3-5 mm) pieces and collected in gentleMACS Ctubes (Miltenyi Biotec) containing PBS and Collagenase B 2 mg/mL (Roche). C-tubes were then connected to the gentleMACS dissociator and tumor dissociation was run with the program for human tumor number 1. Minced tumors were incubated in the Ctubes using a MACSmix Tube Rotator (Milteny Biotec) under continuous rotation for one hour at 37°C. Following incubation, a second dissociation step was performed on the gentleMACS using the human tumor program number 2. The resulting single cell suspension was then filtered through a 70 µm cell strainer and centrifuged at 300g for 5 min. Red blood cells were lysed using ammonium chloride solution (Stemcell Technologies) and cells were then transferred to ultra-low attachment flasks (Corning, Acton, MA, USA) with CSC media.

Immunohistochemistry (IHC)
After deparaffinization and rehydration, sections were pretreated with low pH retrieval solution (Dako, Vienna, Austria) for 10 min in the microwave. For the immunostaining the Ultravision LP Detection System based on horse radish peroxidase (HRP) Polymer and 3,3'-Diaminobenzidine (DAB) Plus Chromogen (Thermo Scientific, Waltham, MA USA) was used according to manufacturer's protocol. Briefly, after blocking with ultra V block for 5 min, sections were incubated with the antibody of interest. The signal was enhanced with the incubation of a primary antibody enhancer. Sections were first incubated with HRP polymer and then with DAB plus solution. The sections were counterstained with an attenuated HE staining and mounted with Aquatex (Merck, Darmstadt, Germany).

Quantitative real time polymerase chain reaction (qRT-PCR)
mRNA was isolated from 5*10 5 -1*10 6 cells with the RNeasy Mini Kit (Qiagen, Hiden, Germany) according to the manufacturer's protocol. The isolated RNA was eluted in 30 µL RNase-free water. Reverse transcription of 1 µg of RNA into cDNA was done with the QuantiTect reverse transcription kit (Qiagen) according to the manufacturer's instructions. mRNA expression was investigated by qRT-PCR with SYBR Green assay (Roche, Vienna, Austria) on a Light Cycler 480 (Roche) using 96 well plates.
Reactions were performed in a total volume of 20 µL containing 1x Mastermix SYBR green I (Roche), 25 µM of each primer and 20 ng cDNA. A PCR program was run for 45 cycles starting with the denaturation step at 95°C for 10 sec, then 60°C for 20 sec, and an elongation step at 72°C for 15 sec. For quality control, it ended with a melting step of the product. A non-amplification control and a non-target control were included in each plate as technical controls. qRT-PCR reactions were performed in duplicates and cycle threshold values were averaged. Calculation of expression values was done using the qBase plus software (Biogazelle, Gent, Belgium) 1 .
Glycerinaldehyd-3-phosphat-Dehydrogenase (GAPDH) and TATA-Box Binding Protein (TBP) were determined as appropriate reference genes using the geNorm module in qBase plus and were used to normalize gene expression levels. The gene expression levels in spheres were compared to expression in adherent cells. Primers are summarized in the Supplementary Table S3.

Copy number profiling in detail
Depending on the DNA concentrations, 50-100 ng of DNA from sorted cell fractions

Authentication of cell populations
In order to verify the same origin of different cell populations, passages and tumor samples, STR profiling and analysis of mitochondrial DNA (mtDNA) was performed.
Mitochondrial genomic sequences were extracted from whole genome sequencing data obtain from the copy number profiling assay and haplotypes were compared for each sample. For STR analysis 0.7 ng of extracted DNA were amplified with the PowerPlex 16HS System (Promega, Mannheim, Germany) according to manufacturer´s instruction on a thermocycler MyCycler (Biorad, Vienna, Austria). In 6 this analysis 16 STR loci can be evaluated such as Penta E, D18S51, D21S11,   TH01, D3S1358, FGA, TPOX, D8S1179, vWA, Amelogenin, Penta D, CSF1PO, D16S539, D7S820, D13S317 and D5S818. The amplified fragments were detected with a capillary electrophoresis on the 3730 Genetic Analyzer (Applied Biosystem, Vienna, Austria).