II.1. CELL CULTURE
MCF7 (ATCC® HTB-22™), MDA-MB-231 (ATCC® HTB-26™) and 293 HEK-293 (ATCC® CRL-1573™) cell lines were acquire from ATCC company (Manassas, Virginia, United States).
MCF7 are epithelial cells isolated from the breast tissue of a 69-year-old, white, female patient with metastatic adenocarcinoma with Pleural effusion. The MCF7 line retains several characteristics of differentiated mammary epithelium including the ability to process estradiol via cytoplasmic estrogen receptors and the capability of forming domes. The cells express the WNT7B oncogene[50].
The MDA-MB-231 cell line was isolated at M D Anderson from a pleural effusion of a patient with invasive ductal carcinoma and is commonly used to model late-stage breast cancer[73]. This cell line is ER, PR, and E-cadherin negative and expresses mutated p53. In microarray profiling, the MDA-MB-231 cell genome clusters with the basal subtype of breast cancer. Since the cells also lack the growth factor receptor HER2, they represent a good model of triple-negative breast cancer. MDA-MB-231 cells are invasive in vitro and when implanted orthotopically produce xenografts that spontaneously metastasize to lymph nodes[73].
The Pa00 cell line was derived in the laboratory from the sample of a patient with metastatic breast cancer with ascitic effusion [47], in which we found a small population of cells that express stem cell cancer markers, as can be seen in the results of the present article.
Pa00, MCF7 and MDA-MB-231 cells were maintained in DMEM (Lonza. Basel, Switzerland) 10% FBS (Lonza), 1% Glutamine (Lonza) (0,2M) and 1% Penicillin/streptomycin (Lonza) (100 units+100 ug/10ul) in a 5% CO2 humidified incubator at 37ºC.
II.2. STAIN WITH DDAO AND SORTING
Cells for cell cycle dynamic assays were plated (100 000 cells). Next, these cells were washed with PBS, disaggregated and then incubated at 37ºC with Cell Trace ® Far-Red-DDAO-SE (DDAO-SE, Molecular probe ref C34564. Eugene, Oregon, United States), at the concentration recommended in the product data sheet. After 10 min, an aliquot of freshly labeled cells (approx. 200 000 cells) was fixed with 0,5% of paraformaldehyde to use as a positive control for the experiment. Then, we centrifuged the cells at 180g for five minutes and we suspended it in the culture medium to grow under standard conditions. Eight days in vitro later, CM-spf cells were disaggregated and sorted by In-fluxTM (Becton Dickinson. Franklin Lakes, New Jersey, United States) sorting equipment depending on their fluorescent retaining labelling level. Positive cells were considered slowly dividing cells, and thus potential tumour initiating cells.
II.3. CYTOMETRY ASSAY
Cytometry assays were performed with a MACSQuant Analyzer 10 cytometer (Miltenyi Biotec ref 130-096-343. Bergisch Gladbach, Germany). Samples were first washed in PBS and incubated with Miltenyi Biotec FcR blocking Reagent (human), in 100 microliters of sample. The immunostaining was performed per the standard protocol recommended by the commercial houses of the different antibodies. The antibodies and the working dilutions used were as follows: AntiBCRP-FITC 5D3 Chemicon (Temecula, California, United States) and AntiBCRP-PE 5D3 Chemicon incubated for 20 minutes at a 1:10 dilution; while AntiEpCAM- PE Clon HEA-125, AntiAC133-PE Clon AC133, AntiCD133-PE 293C3, Anti CD44-FITC and CD24-PE, all from Miltenyi Biotec, were incubated 10 minutes at a 1:10 dilution. Sorting experiments were performed with a BD Influx™ cell sorter. FITC or PE are used as abbreviation of Fluorescein isothiocyanate and Phycoerythrin respectively.
II.4. GENE EXPRESSION
For gene expression assays, total RNA from cultured cells was extracted using the RNeasy Mini kit (Qiagen, Hilden, Germany) and used immediately for reverse transcriptase reactions or stored at 80ºC until use. RNA concentration was measured in a Nanodrop spectrophotometer (Thermo Fisher,Waltham, MA, USA). cDNA synthesis was performed using the RevertAid First Strand cDNA Synthesis kit (Fermentas, Waltham, MA, USA). The reaction was prepared according to the manufacturer’s instructions. Quantitative PCR reaction was performed for CDKN1A (also known as p21 inhibitor) using the KiCqStart SYBR Green kit (Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. KiCqStart SYBR Green predesigned primers (Sigma, St. Louis, MO, USA) were employed (H_CDKN1A_1: Forward primer 5' TGTAAAACGACGGCCAGT and Reverse primer 5' CAGGAAACAGCTATGACC). Primers were used in a final concentration of 300 nM, and 10 ng of cDNA were used per well, for a total volume of 10 µl. All cDNA samples were measured in triplicate in a 96-well plate covered with adhesive seals in the thermocycler Roche LightCycler 480 (Roche, Basel, Switzerland). Reactions started with 10 min at 95ºC, followed by 45 cycles of 15 s at 95ºC, 1 min at 60ºC and 10 s at 72ºC. The 2-DCT method was used for calculating the normalized mRNA expression. Fold increase relation over untreated controls has been used to graphicaly represent the decrease of gene expression. GADPH was used as housekeeping gene in quantitative PCR assay.
II.5. XENOGRAFTS
FOXn1nu females, at 1 month of age were obtained from Charles River International Laboratories (Wilmington, Massachusetts, United States) to use in these experiments. Animals were housed and bred under 20-25 ºC, 50-60% humidity and a 12 hour light-dark cycle. All experiments were performed in accordance with relevant guidelines and regulations and the animals were treated in accordance with the approval of the local ethics committee (University of Castilla-La Mancha PI081746). The experiments were performed as previously described[30]. In short, untreated cells (control) and treated cells (8nM PEDF or CTE-PEDF) suspended in PBS were injected subcutaneously into both flanks of immunocompromised mice. A total of 1200, 5000 or 3*106 cells (depending of the experiment) were injected in a final volume of 200 uL of a 1:1 dilution with MatrigelTM Basement Membrane Matrix (Becton Dickinson) with a 25 gauge-needle. The experimental unit is a single animal and a total of 28 animals were used. The mice were randomly assigned to the control and treatment groups via tail numbering that was also used as the order for successive xenograft injections. In the first experiment with animals (Figure 1) a total of 11 animals were used; with four animals in the control group and seven animals in the treatment group. For the second animal experiment (Figure 5) a total of nine animals were used, with three animals in each group (control, PEDF or CTE-PEDF treated injected cells). For the last animal experiment (Figure 6) a total of eight animals were used, with two injections each (one per flank, treated cells in the right flank and untreated cells in the left flank). There is an extensive track record in the published literature of using athymic nude mice and mouse xenograft models with human tumour cell lines to increase understanding of the factors affecting tumour growth[87]. The number of mice in each experiment was chosen based on this well-detailed experience, including experience in PDX (patient derived xenograft) models[88]. Possible confounding factors between groups were minimized by placing the mice from each group in different boxes (3-4 animals per cage, corresponding to animals of the same experimental group: control or treatment). All investigators responsible for the animal experiments in the team were aware of the group assignments at the different phases of the experiment (during assignment, conduct of the experiment, evaluation of results and data analysis).
Tumour growth was monitored weekly with a caliper. The final tumour volume was calculated as V= 2 x L1 x L2 x π/6. Tumours were mechanically and chemically dissociated with collagenase and trypsin at 37ºC. They were then washed with PBS and seeded in DMEM medium, 10% FBS, 1% glutamine (200 mM), 1% Penicillin/streptomycin, 0.5% EGF and 0.04% FGF in a 5% CO2 humidified incubator at 37ºC 12h before the start of other experiments. The outcome measures assessed in the different experiments were: tumour size over time, markers expressed by tumour cells, and rates of resistance in dose-response assays in cell culture of these xenograft cells.
The criteria used to include and exclude both animals (during the experiment) and data points (during the analysis) were established a priori. Specifically, those mice that suffered harassment or mistreatment by their peers in the cage for reasons beyond the control of the caretakers and showed significant deterioration or premature deaths not directly linked to the experiment were to be excluded from the study. Those animals would be mentioned although not considered for cellular and molecular analyses. However, no such circumstance was observed throughout the experiments and therefore no mice were excluded in these experiments.
II.6. PEDF and Cter-PEDF, CTE-PEDF PRODUCTION
PEDF and the carboxy-terminal part of PEDF protein (Cter-PEDF) were cloned into pcDNA™3.1/myc-His A, B, & C Mammalian Expression Vectors (InvitrogenTM. Carlsbad, California, United States) following the protocol previously described by Sánchez-Sánchez and coworkers[89]. After cloning, vectors were checked by sequencing (3130 Applied Biosystems. Foster City, California, United States). Serine 227 was mutated into a glutamate (E) residue using ser227glu-dw (5’-CCA AGT AGA AAT CCT CGA GCT CAG TCT TTC TGG AGT-3’) and ser227glu-up (5-GTT TGA CTC CAG AAA GAC TGA GCT CGA GGA TTT CTA-3) primers. After mutagenesis, the Cter-PEDF peptide with this glutamic change was named CTE-PEDF. Proteins were produced by HEK-293-T cells after transfecting with phosphate calcium. Conditioned mediums were collected after 3 DIV, quantified by western-blot using anti-c-myc mouse monoclonal IgG1 (Santa Cruz Biotechnology. Dallas, Texas, United States), and anti-phosphoserine clone 4A4, (Millipore. Burlington, Massachusetts, United States) and stored at -20ºC or purified using GE Healthcare Life Sciences™ HisTrap™ FF Crude columns (Thermo Fisher Scientific. Waltham, Massachusetts, United States).
II.7. CHRONIC TREATMENT WITH PEDF AND CTE-PEDF
Cells were treated with PEDF or CTE-PEDF at a final concentration of 8 nM in the medium. The acute treatment consists of a single treatment: two hours of peptide exposure before chemotherapy treatment. Chronic treatment consists of six peptide treatments week (culture medium with CTE once per week), totalling six weeks of peptide exposure before chemotherapy treatment.
II.8. ACUTE TREATMENT WITH PEDF AND CTE-PEDF
Cells were plated in a 24-well plate at a final concentration of 15 000 cells/well in a total volume of 200 microliters. Half of the plate was plated with CTE-PEDF medium. The next day, radiotherapy to 2 Gy, 4 Gy and 6 Gy was applied by a Siemens PRIMUS X6MV LINAC. Field size 10 × 10 cm2, depth 10 cm, SSD = 100 cm, Siemens PRIMUS X6MV.
II.9. DOSE-RESPONSE CURVE
Cells were plated in a 24-well plate at a final concentration of 15 000 cells/well in a total volume of 200 microliters or 200 cells/well in a 96-well plate for sorted cells. Half of the plate was plated with CTE-PEDF medium. The next day, the drug was added in decreasing concentrations 4 nM, 2 nM, 1 nM, 0.5 nM, 0.25 nM. These were cultured for 3 days under drug exposure in in vitro conditions, and then developed with an MTT assay or methyl purple assay.
II.10. MTT ASSAY
The cell culture media was removed, after which 100 microliters/well of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added. After 30 minutes of incubation, the supernatant was removed, and the precipitated crystals were dissolved in 100 microliters of DMSO. The plate was read in a spectrophotometer at 540 nm.
II.11. METHYL PURPLE ASSAY
This method[33] is used to quantify surviving cells. 5 000 cells/well were seeded in 24-well plates, in a final volume of 250 µL. The next day, cells were treated with increasing doses of chemotherapeutic agents and stored in a humidified incubator at 37ºC, 5% CO2 for 4 days. Then, cells were fixed with 0.5% glutaraldehyde (Sigma. St. Louis, Missouri, United States) for 10 minutes. Next, cells were stained with 0.1% crystal violet for 20 minutes. After several washes with PBS, 10% acetic acid was used to solubilize the sample. Finally, a spectrophotometric reading was performed at a wavelength of 590 nm. The IC50 value was determined as the dose necessary to eliminate 50% of the cell population, obtained through logarithmic regressions made with the DE.0 plus v 1.0 program.
II.12. HISTOLOGY
Cryostat sectioning slides were performed using a cryostat (Microm HM 550, Thermo Scientific). Tumour samples were fixed with formaldehyde at 4%, placed in sucrose 30% overnight, included into Tissue-Tek® OCT™ (Sakura ® Finetek USA. Torrance, California, United States) and then frozen with liquid nitrogen. Slides (12µm) were stained with hematoxylin and mounted with Dako Ultramount Aqueous Permanent Mounting Medium (Agilent Technologies. Santa Clara, California, United States) and analyzed with a Leica-DMRXA-photomicroscope (Leica. Wetzlar, Germany).