DNA alterations in Cd133+ and Cd133- tumour cells enriched from intra-operative human colon tumour biopsies

Twenty-seven patients diagnosed with colon cancer selected for curative surgery and who did not receive any chemotherapy treatment prior to biopsy collection, according to institutional guidelines at the time, were included in this study (2013–2014). Written, informed consent was obtained from all patients. The study protocol was approved by the Board of Ethics at the University of Gothenburg (permit number: 365–05). Patient characteristics, after exclusion of one patient whose tumour was later confirmed to be an adenoma, are shown in Table 1. Two tumour biopsies were collected from each tumour; one was immediately frozen in liquid nitrogen and the other placed in tissue storage solution for cell separation.

The biopsy designated for cell separation was kept in pre-chilled MACS Tissue Storage Solution and used within 24 h. The samples were dissociated into single cell suspensions using Tumour Dissociation Kit (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) by mechanical dissociation and enzymatic degradation of extracellular matrix. The single cells were incubated with Dead Cell Removal Microbeads for 15 min at room temperature and separated with MACS LS Column; the labelled cells were collected as the effluent fraction. Cells were washed with 9 ml stock solution (1.25 ml 20X Binding Buffer Stock Miltenyi Biotec diluted up to 25 ml with ddH₂O) and centrifuged at 300G for 10 min and labelled with CD133 antibodies conjugated to ferromagnetic beads (CD133 Microbeads, 130–050-801, MACS Miltenyi Biotec) for 30 min at 4 °C. After incubation, cells were washed with 9 ml MACS Buffer, centrifuged at 300G for 10 min, separated using MACS LS column and kept on ice until further analyses (Fig. 1). CD133 negative cells were used in a second step for separation labelled with EpCAM antibodies (CD326, 130–061-101, MACS Miltenyi Biotec) for 30 min at 4 °C and washed with MACS Buffer before separation. Cells were passed through MACS LS column. The negative fraction was collected as the effluent (CD133−/EpCAM-) and positive cells were flushed out of the column and collected as EpCAM positive fraction (CD133−/EpCAM+). Cells were used immediately for downstream analysis after separation of CD133+, CD133−/EpCAM+ and CD133−/EpCAM- fractions.

There are some technical issues to consider with studies of genomic alterations in small amount of cells as performed in the present study. One such matter is that CD133+ cells are few within tumours. Therefore, whole genome amplification (WGA) was necessary before microarray analyses, which may affect amplification and interpretations of microarray results [7]. We chose to use a Multiple Displacement Amplification (MDA) technique that was recommended for CGH microarrays (Agilent) in detection of amplifications and deletions of DNA. Also, the EpCAM+/Cd133- cell fraction was collected by additional round of enrichment with column elusion, which may reduce recovery. However, unselective difference in recovery of cell fractions should not impact seriously on the possibility to perform qualitative comparisons. The DNA quality was checked along preparations on all samples.

Biopsies from four random patients were separated with CD133 antibody as described above (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) and the two fractions obtained, CD133+ and CD133-, were used for immunocytochemistry. The cells were attached to a glass slide using cytospin centrifugation at 1000 rpm for 4 min. The slides were placed in methanol at −20 °C for 20 min for fixation. After this step, the MACH2 staining protocol was followed (Histolab, Gothenburg, Sweden). Briefly, peroxide was blocked with peroxidised 1 reagent (Histolab, Gothenburg, Sweden) for 5 min. After washing with TBS wash buffer (Histolab, Gothenburg, Sweden), cells were covered with Background Sniper (Histolab, Gothenburg, Sweden) for 10 min to avoid non-specific binding. TBS wash buffer was used in all washing steps. Cells were incubated with CD133 primary antibody (AC133, 130–090-422, Miltenyi Biotec, 15 μg/ml) overnight at 4 °C, washed, and incubated with MACH2 30 min for secondary ALP detection. After washing, cells were covered with Warp red chromogen (prepared according to data sheet) for 7 min and counter stained with hematoxylin for 2 min. The slides were washed with tap water and mounted using aqueous mounting medium. Mouse IgG1 (Dako 0931, 15 μg/ml) was used as negative control.

DNA and RNA were isolated from CD133+ and CD133−/EpCAM+ cells according to Allprep DNA/RNA micro Kit (Qiagen, Hilden, Germany) and from tumour tissue with Allprep DNA/RNA mini kit (Qiagen, Hilden, Germany). Briefly, cells were lysed and homogenized with RLT Buffer and transferred to an Allprep DNA spin column for binding of genomic DNA. The flow through was mixed with 70% ethanol and transferred to an RNeasyMinElute spin column for total RNA binding. To elute the RNA, 12 μl of RNase-free water were used and DNA was eluted with 50 μl of Buffer EB pre-heated at 70 °C. RNA quality and quantity were measured using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Only RNA with RIN value ≥6.5 was used in further analyses. RNA samples were stored at −80 °C and DNA at −20 °C.

Gene expression levels of genes related with stemness were measured by Q-PCR in the CD133+ and CD133−/EpCAM+ cell fractions from randomly selected patients. Genes tested were BMP7 (CD133+ n = 16, CD133−/EpCAM+ n = 17), PROM1 (gene for CD133; CD133+ n = 10, CD133−/EpCAM+ n = 12), and POU5F1/Oct4 (CD133+ n = 12, CD133−/EpCAM + =13). All three genes were not tested in all samples due to small sample size, poor RNA quality and/or low amount of RNA. GAPDH was used as a housekeeping gene, confirmed separately [8], and run for all samples. cDNA synthesis was performed using QuantiTect Reverse Transcription kit (Qiagen, Hilden, Germany). Q-PCRs were run in LightCycler 1.5 using LightCycler FastStart DNA Master plus SYBR Green I kit (Roche Diagnostics, Basel, Switzerland) with final primer concentration 0.5 mM for each gene. Primer information is described by Lönnroth et al. [9]. For each Q-PCR, 2 μl cDNA were used with the following PCR conditions: Activation for 10 min at 95 °C and denaturation for 10 s at 95 °C, 20 °C/s were the same for all reactions. Annealing: 7 s 58 °C (PROM1); 4 s 64 °C (BMP7, GAPDH); 5 s 66 °C (OCT4B1). Extension and cycle numbers: 22 s 72 °C, 40 cycles (PROM1); 5 s 72 °C, 45 cycles (BMP7); 20 s 72 °C, 40 cycles (OCT4B1); 5 s 72 °C, 40 cycles (GAPDH). PCR efficiency and slope of standard curve for GAPDH was 88.97% and −3.62, BMP7 83.44% and −3.79, PROM1 76.42% and −3.77, and OCT4B1 91.01% and −3.60. Q-PCR results were calculated according to the relative standard curve method and all samples were in the range of the standard curve. Negative controls were negative. Results were analysed with ANOVA followed by Fisher PLSD and are presented as mean units/units of GAPDH ± SEM. P ≤ 0.05 was considered statistically significant in two-tailed tests.

DNA from CD133+ and CD133−/EpCAM+ populations as well as reference DNA (Agilent Euro male, #5190–3796) for CGH array analysis were amplified using the REPLI-g Single Cell Kit (Qiagen, Hilden, Germany) according to manufacturer’s protocol. Briefly, 2.5 μl of template DNA was incubated with 2.5 μl buffer D1 for 3 min. Neutralization buffer, N1, was added and REPLI-g master mix added after neutralization. The mixture was incubated for 2 h at 30 °C. DNA polymerase was inactivated at 65 °C for 3 min. Reference DNA was amplified in 8 aliquots that were pooled and used as reference DNA in the array CGH. DNA was stored at −20 °C.

DNA was purified using GFX PCR DNA and Gel Band Purification kit (Illustra, GE Healthcare, Little Chalfont, UK) according to manufacturer’s instructions. DNA samples were mixed with 500 μl of Capture Buffer type 3, loaded onto a GFX MicroSpin column and centrifuged at 16000G for 1 min. The column was washed with 500 μl Wash buffer type 1 and centrifuged (16000G, 1 min); this washing step was repeated to achieve high purity. DNA was eluted with 30 μl Elution buffer type 6 after 1 min incubation. DNA quality was checked on a 2% agarose gel and additional tests were run using 2200 Tape station (Agilent Technologies, Santa Clara, USA). DNA quantity was measured using Qubit® assay (Thermo Fischer Scientific, Waltham, MA, USA). Two patients were excluded from further analysis due to poor DNA quality. DNA was stored at −20 °C.

Q-PCR assays were performed at 4 regions of chromosome 19p to study if the deletions at chromosome 19p detected in cell fractions also were detected in corresponding tumour tissue. Well-known colon cancer alterations were also evaluated with Q-PCR; 1 region at chromosome 13 and 1 region at chromosome 20 as well as 1 region at chromosome 10 that is known to be unaffected in colorectal cancer (control) in DNA from tumour tissue biopsies. Primers were designed by TATAA Biocenter AB (Gothenburg, Sweden) with PrimerBlast (http://​www.​ncbi.​nlm.​nih.​gov/​tools/​primer-blast/​), primer sequences for chromosome 10, 13 and 20 are described elsewhere [10] and for chromosome 19 in Additional file 1: Table S1. The Q-PCR assays were validated on gBlocks to estimate the PCR efficiency of the assay. A seven point standard curve was generated with four replicates in each point and run in ten-fold dilution steps. The dilution series covered a template concentration between 2 × 10⁷ and 20 copies/reactions. LPA carriers were added to the dilution series to avoid unspecific interactions of target. Q-PCR analysis was performed with TATAA SYBR® GrandMaster® mix (TATAA Biocenter AB, #TA01) in 10 μl reactions on CFX384 instrument (Bio-Rad, Hercules, CA, USA). Two replicates of the DNA control (Agilent Euro male, #5190–3796) with concentration 20 ng/reaction were measured together with the standards. Raw Q-PCR data was analysed with GenEx software (version 6, MultiD Analyses AB), standard curve and limit of quantification were generated. Specificity control (amplicon size) for PCR products from designed assays was performed on capillary electrophoresis instrument (Fragment Analyser, Advanced Analytical Technologies, Ankeny, IA, USA) according to manufacturer’s instructions. Results were analysed according to comparative Cq method with the region at chromosome 10 and reference DNA (Agilent Euro male, #5190–3796) as standards. Results between 0.90–1.10 were regarded as not altered.

CD133 protein expression was evaluated in 4 samples by immunocytochemistry to demonstrate that CD133+ and CD133- cell fractions were separated into two cell populations with different expression of CD133. The results display CD133 protein expression in 63% (range 51–74%) of enriched cells in the CD133+ fraction and <2% (range 0–3%) of CD133- cells (Fig. 2a).

Transcript levels of known cancer stem cell genes indicated that the CD133+ cell population expressed higher levels of such genes. PROM1 (PROM1 = CD133 gene) displayed increased expression compared to cells in the CD133−/EpCAM+ population (p = 0.018) (Fig. 2b). CD133+ cell samples also showed higher expression of another cancer stem cell gene, BMP7, compared to CD133−/EpCAM+ cell samples (p = 0.0081). OCT4 gene expression was below detection limit in all cell samples, except 5 samples (2 CD133+, 3 CD133−/EpCAM+; all from different patient tumour biopsies).

The number of DNA alterations in the two cell fractions, CD133+ and CD133−/EpCAM+, displayed great heterogeneity; in the CD133+ cell population DNA alterations in the 20 patients ranged from 6 to 230 per patient (amplifications 3–18, deletions 3–212), while a range of 4–278 DNA alterations per patient (amplifications 2–17, deletions 2–261) were seen in the CD133−/EpCAM+ cell population.

Overall array CGH results indicated that deletions corresponded to 87% of DNA alterations in all samples; thus more common than amplifications. The total number of significant alterations (2285) in all samples was equally distributed between the two cell populations; 51% was from CD133+ population and 49% from the CD133−/EpCAM+ population (Table 2). Deletions detected in both CD133+ and CD133−/EpCAM+ [(shared deletions) and found in more than 50% (10 patients) of evaluated patients], were located on chromosome 1, 2, 7, 8, 10, 12, 14, 15, 16, 18, and 19. Amplifications detected in both CD133+ and CD133−/EpCAM+ cells [(shared amplifications) and found in more than 10 patients] were located on chromosome 3 and 14 (both related to deletions in the Agilent Euro male reference DNA) (Table 3). A list of shared deletions is supplied as Additional file 2: Table S2. Deletion of chromosome 19p occurred in 27 samples (10 CD133+ and 17 CD133−/EpCAM+) representing 18 patients (Fig. 3). A complete gene list of chromosome 19p deletion is added as Additional file 3: Table S3.

Amplifications were more common in CD133+ cells (83%) than in CD133−/EpCAM+ cells (17%); while deletions were more equally distributed between the cell populations, CD133+ 42% and CD133−/EpCAM+ 58% (Table 2). No significant differences in number (Fig. 4) or size (bp) of alterations between CD133+ and CD133−/EpCAM+ were detected (Table 2), except for chromosome 1, 7 and 19, which showed significantly larger alterations in CD133−/EpCAM+ population (Table 4 and Fig. 3). There were 8 alterations detected specific for CD133−/EpCAM+ cell population located at 8q24.3, 11q12.2-q13.3, 11q12.3, 11q13.4, 11q23.3, 16q13, 17p13.2 and 17q21.2. For the CD133+ cell population, 3 deletions were detected only located on 1q42.12, 8p11.22 and 12q13.13-q14.1 (Table 5).

DNA alterations in tumour tissue were detected with Q-PCR of selected by known genomic regions and on chromosome 19 in CRC. Amplifications were detected in chromosome 13 and 20 in 55% (chr13 n = 11, chr20 n = 11) of the samples (n = 20). Two deletions were detected in chromosome 20 and five deletions in chromosome 13. In chromosome 19p, a deletion in the specific region was detected in 6 of 20 patients (total 11 deletions in 80 reactions; 4 Q-PCR assays × 20 samples). Several tumour samples displayed amplification in tumour tissue at chromosome 19p (50%, n = 40 of 80 reactions, representing 10 of 20 patients) (Fig. 2).