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CTC ISOLATION AND ESTABLISHMENT OF THE CTC-MCC-41 CELL LINE
We previously described the generation of the colon CTC line named CTC-MCC-41 (6) that
has been now growing in culture for three years with a constant doubling time of 20 hrs in
Medium 2 (RPMI1640, Growth factors: EGF and FGF-2, Insulin-Transferrin-Selenium
supplement, L-Glutamine). This CTC line was established from CTCs isolated from a patient
with colon cancer and a CTC count higher than 300 using the CellSearch system for CTC
detection (6) (supplementary Figure S1). Briefly, 10 mL of blood was collected in an EDTA
tube and was immediately used for CTC isolation by depletion of hematopoietic CD45positive cells using the RosetteSep Human Circulating Epithelial Tumor Cell Enrichment
cocktail (StemCell Technology, Vancouver, Canada) (7). Isolated CTCs were then plated in
24-well non-adherent plates and cultured in hypoxic conditions (2% O2) at 37°C in
Dulbecco’s modified Eagle’s medium/F12 with 20 g/mL insulin, 1% N2 complement, 20
ng/mL epithelial growth factor, 2mM L-Glutamine, 10 ng/mL fibroblast growth factor-2
(FGF2) and 2% fetal calf serum (FCS) (Medium 1). After a few weeks, CTCs were switched
to Medium 2 to improve CTC growth in normoxic conditions (5% CO2). Single colonies from
the CD45-negative population developed during the first weeks of cell culture and were
transferred in new 24-well plates and then in T25 flasks for culture expansion. In these
conditions, CTCs could be quickly expanded and after a few months we obtained billions of
tumor cells.
CLINICOPATHOLOGIC CHARACTERISTICS OF THE PATIENT AND TUMOR GIVING
RISE TO CTC-MCC-41
Standard histopathologic analysis of diagnostic biopsies performed on the primary tumor and
one lymph node revealed a poorly differentiated adenocarcinoma with a lot of isolated single
cells that lost their adhesive properties. The primary tumor was also characterized as KRAS
wild-type (codons 13 and 12) and BRAF mutated (V600E mutation) according to the
standard-of-care analyses (6). In addition, the MSI status of the tumor was determined as
stable using multiplex PCR approach.
Clinically, the patient had a widespread disease at diagnosis with numerous abdominal and
mediastinal metastatic lymph nodes as well as metastatic lesions in the liver. The primary
tumor was located in the transverse colon. No bone metastasis was detected during the cancer
follow-up (6).
HT-29 CELL LINE
For our experiments, HT-29 cells (ATCC® HTB-38™; a human colorectal adenocarcinoma
cell line that was established in 1964 by J. Fogh from the primary tumor of a 44-year-old
Caucasian female) were maintained in Dulbecco's modified Eagle's medium (# 31966-021,
Gibco, ThermoFisher) supplemented with 10% FCS and penicillin/gentamycin and cultured at
37°C and 5% CO2 in a humidified incubator.
RNA ISOLATION
The RNeasy Mini Kit (ref.74106; Qiagen) was used to extract total RNA from each cell
sample, according to the manufacturers’ recommended protocol. Total RNA quantity and
purity were determined by using a NanoDrop® ND-1000 spectrophotometer (NanoDrop NDThermo Fisher Scientific, Wilmington, DE, USA). RNA integrity was assessed by using the
Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, http://www.agilent.com).
CTC-MCC-41 cells were prepared at passages P6, P8 and P10. We indeed decided to take
early passages of this CTC line to keep the characteristics of the CTCs as they were in the
colon cancer patient. In parallel, HT-29 cells from the ATCC (purchased at passage P128)
were prepared at passages P130, P132 and P134. All RNA samples were stored at -80°C until
use.
COMPLEMENTARY RNA PREPARATION AND MICROARRAY HYBRIDATION
Total RNA (200 ng) was used to prepare cRNA using the Affymetrix 3’ IVT express protocol
(ref.901229). An oligo-dT primer with a T7 promoter sequence was used to synthesize firststrand cDNA. After second strand synthesis, the complete cDNA was amplified by in vitro
transcription (linear amplification) with a T7 RNA polymerase. A biotinylated nucleotide
analog was incorporated during the in vitro transcription step. Amplified RNA (aRNA) was
quantified with a NanoDrop ND-1000 spectrophotometer (NanoDrop ND-Thermo Fisher
Scientific, Wilmington, DE, USA). RNAs from Bacillus subtilis genes (lys, phe, thr, and dap)
included in the GeneChip Eukaryotic Poly-A RNA Control Kit (Affymetrix, Santa Clara, CA)
were amplified and labeled in the same conditions and used as positive controls. After
fragmentation, 12 µg of labeled anti-sense aRNA was hybridized to HG-U133 plus 2.0
GeneChip arrays (AffymetrixTM). In total, six chips (three chips for CTC-MCC-41 cell
samples and three chips for HT-29 cell samples) were used for the microarray hybridization
experiments.
BIOINFORMATIC ANALYSIS
Known genes and their corresponding expression values were uploaded in the software and
each gene symbol was mapped to its corresponding gene object in the Ingenuity Pathways
Knowledge Base. Gene networks were algorithmically generated based on their connectivity
and scored. This score is a numerical value used to rank networks according to how relevant
they are to the genes in the input dataset; however, it may not be an indication of the network
quality or significance. The score takes into account (i) the number of focus genes in the
network and (ii) the size of the network to estimate the network relevance to the original list
of focus genes. To identify molecular pathways, data were arranged by relation type using
Pathway Studio 9.0 (Ariadne Genomics; Rockville, MD, USA). This program integrates
relevant information on the imported genes, thus allowing the identification of biological
pathways, gene regulation networks and protein interaction maps.
REVERSE TRANSCRIPTION-QUANTITATIVE PCR ANALYSIS
Reverse transcription (RT) was performed as recommended by the manufacturer (Invitrogen)
in a 20 μl reaction volume that included 2 μg of RNA, Superscript III (ref. 18080, Invitrogen),
oligo-dT primer, dNTP mixture, MgCl2 and RNase inhibitor. Quantitative PCR (qPCR) was
then performed using the Brilliant III Ultra-Fast SYBR® Green Master Mix, (600882, Agilent
technologies) with 1 μl of 1/100 dilution of the RT reaction product and 6µM of each primer
(SIGMA Genosys) in a total volume of 10 μl. PCR amplification was carried out using a
LightCycler 480 apparatus with the following program: 1 cycle of 95°C for 3min; 40 cycles
of 95°C for 5s, 60°C for 10s; 1 cycle 95°C for 1min, 55°C for 5s, 95°C for 30s and 37°C for
30s. Gene expression levels were normalized to the expression of the housekeeping gene 2globulin, using the following formula: 100/2ΔΔCt, where ΔΔCt = ΔCt unknown - ΔCt
positive control. Each sample was analyzed in triplicate and multiple water blanks were
included. The primer sequences are shown in (Supplementary data, Table S1).