Cell lines and co-cultures
The human mammary carcinoma cell line MCF-7 and the human lung fibroblast cell line MRC-5 were obtained from the ATCC (Rockville, MD, USA). Collagen type I stock solution (10 mg/ml, rat tail) were purchased from BD Biosciences (Bedford, MA, USA); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma (St. Louis, MO, USA); Cell culture media consisted of Dulbecco’s Modified Eagles Essential Medium (DMEM, Invitrogen, Carlsbad, CA, USA) to which 10 % fetal bovine serum (FBS, Sigma, St. Louis, MO, USA) and 1 % pen/strep (P/S, Sigma, St. Louis, MO, USA); Dimethylsulfoxide (DMSO) were purchased from ATCC (Rockville, MD, USA); 48 and 96-well cell culture clusters and other plastic disposables were purchased from Corning Inc. (Corning, NY, USA). Transforming growth factor beta 1 (TGF-β1) (Sigma, St. Louis, MO, USA) was added to some cultures to transform a fraction of the MRC-5 fibroblasts into cancer-associated fibroblasts (CAFs).
Type I collagen was selected as the 3-D scaffold for cell culture because it resembles the extracellular matrix milieu of invasive breast carcinoma better than other polymers. Collagen solutions were diluted from 10 mg/ml and neutralized with DMEM medium and 1 N sodium hydroxide to 2.0 mg/ml. Cells were introduced to a particular sample at this time, before matrix polymerization. 600 μl volumes of collagen solution were dispensed to a 48-well plate and incubated at 37 °C as the collagen polymerized over 30 min. After polymerization, 300 μl of complete DMEM was applied to the top of the gelled samples. With or without cells, the total fluid volume added to each sample was 600 μl so that matrix stiffness was not influenced by medium volume.
Co-cultures were constructed by mixing 60,000 MCF-7 epithelial cells and 200,000 MRC-5 fibroblast cells (1:3 ratio) into 600 μl of collagen matrix to mimic conditions of residual disease in the post-surgical human breast. Many researchers have used the 3/1 ratio breast cancer cell lines (i.e. MCF-7) to fibroblast (i.e. MRC-5) to examine breast cancer. However, we wanted to examine the post-surgical environment, thus looking at the influence of a microenivironment in a post-surgical region [17–20]. Values were chosen after evaluating the sustainability and proliferation responses of cell samples. At higher initial cell densities, MCF-7 cells proliferated continuously and overtook samples. At lower initial cell densities, MCF-7 cells had difficulty sustaining growth. The initial density of MRC-5 cells was optimized in our previous work for the sample volume. The same initial cell densities were seeded in samples where each cell type was cultured independently. Cultures were maintained for 5 days and the medium was changed every 2–3 days.
Sample irradiation
Samples were irradiated by placing them in a 6 MV photon field generated by a linear accelerator (Varian Medical System, Palo Alto, CA, USA) as described previously [4]. Briefly, a uniform dose was delivered with the gantry set to 0° and a 100 cm source-to-axis distance. An 8 × 8 cm2 field size was selected to ensure cell-culture samples were uniformly irradiated. Dosimetric-quality solid water was placed above and below the well plates to provide proper build-up dose. All samples were taken from the incubator and placed in a thermally isolating container for transportation to and from the clinic. Samples were removed from the container, exposed to ionizing radiation (IR) at room temperature in a single dose, and returned to the incubator within 30 min. The standard clinical dose fraction for breast treatment of 180 cGy was delivered to samples at a rate of 400 cGy/min. Other samples were exposed at the same dose rate but at a reduced fraction size of 90 cGy or an accelerated fraction size of 360 cGy. All sample irradiations were delivered in 5 fractions, once each 24 h over 5 consecutive days, so the total dose was 450, 900, or 1800 cGy. Dose delivery verification was conducted following clinical standards.
MTT assays
In order to approximate the metabolic activity of irradiated cells, the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used. This sensitive, quantitative and reliable assay measures the conversion of MTT substrate produced in cells into a formazan salt by cellular dehydrogenase [21]. MTT assesses cellular metabolism based on the ability of the mitochondrial succinate-tetrazolium reductase system to convert the MTT into formazan. Viable cells reduce the water-soluble yellow-colored MTT to a water-insoluble purple colored formazan product in proportion to their metabolic rate. The amount of colored formazan product formed, as determined spectrophotometrically after dissolving the formazan crystals in DMSO, is proportional to the net metabolic activity of cells with functioning mitochondria in the sample. The MTT assay is a common tool used in radiobiological studies to assess metabolic activity [17]. We applied it to in-vitro cell cultures for the assessment of cellular metabolism, survival and proliferation as described below.
We were unable to reliably use MTT assays in 3-D cultures, so we performed 2-D culture studies to measure metabolic activity and 3-D cultures to measure cell proliferation. Then we compared them to establish a calibration in which either measurement described cell proliferation.
Cells harvested from culture flasks by trypsinization were resuspended in new phenol free media, plated at different cell densities in 96-well culture plates, and incubated at 37 °C in 5 % CO2. The cell medium was replaced every 2–3 days. The cell monolayer is treated for 4 h with MTT dissolved in PBS (concentration 5 mg/ml) at 37 °C and 5 % CO2. After incubation, the MTT solution is removed and 100 μl DMSO is added to each well of the 96-well plate to dissolve the formazan crystals. Plates were shaken for 10 min to ensure adequate solubilization. Assessment of metabolic activity was recorded as relative colorimetric changes measured at 570 nm. Samples were read in an Emax reader (Molecular Devices, Wokingham, UK) with Softmax PRO version 4.3 software. Control-sample wells for absorbance readings contained no cells or medium but MTT solution was added as per experimental wells on the plate. They were removed after incubation and DMSO was added. All experiments were performed at least 3 times and the results were averaged.
Immunofluorescence staining (IF)
MRC-5 and MCF-7 cells were mixed in type I collagen at a final collagen concentration of 2 mg/ml to form 3-D co-cultures. Samples were maintained in DMEM containing 10 % FBS and 1 % P/S at 37 °C in a humidified atmosphere containing 5 % CO2. To quantify the cell growth in collagen gel following mechanical testing, each sample was washed with Phosphate-Buffered Saline (PBS, Lonza, Wackersville, MD, USA) before being fixed with 4 % paraformaldehyde at 4 °C overnight. Samples were then washed and permeabilized in 0.02 % Triton X-100 in PBS for 15 mins followed by 5 % non-fat milk blocking for 2 h. Gels were then incubated with primary monoclonal α-SMA antibody, consisting of Bovine Serum Albumin (BSA, Sigma), PBS and Tween 20 (T, Sigma), (1:100 in 1 % BSA/PBS/T) overnight at 4 °C and secondary antibody (1:200 in 1 % BSA/PBS/T) at room temperature for 2 h. Cell nuclei were stained with TO-PRO 3 (1:1000 in PBS) for 20 mins. Sections were mounted between two glass coverslips with anti-photobleaching reagent.
Confocal microscopy
Immunofluorescence-stained 3-D cultures were examined with a Leica SP2 laser scanning confocal microscope (Leica, Heidelberg, Germany) with Hg lamp, helium/neon laser, and the associated software (Leica Confocal Software Version 2.00). A 488 nm excitation wavelength beam was applied for Fluorescein isothiocyanate (FITC) mapping, and a 633 nm excitation wavelength beam was used to find cell nuclei via TO-PRO 3 mapping. For each sample, a 20X objective captured a series of images along the z-coordinate (sample depth) at 3–5 μm depth increments. We used a confocal microscopy system (Microradiance; Bio-Rad, Philadelphia, PA, USA) consisting of a 25-Mw argon ion laser emitting at 488 and 514 nm and a 1-Mw green helium–neon laser emitting at 546 nm attached to a BX-50 microscope (Olympus Imaging America, Inc., Center Valley, PA, USA).
Automated area quantification for cell counting
MCF-7 cells were found to aggregate in clusters as cells proliferated in the 3-D collagen matrix. Their cellular morphology was obviously different from the MRC-5 cells, and so the two were easily distinguished. MCF-7 cells present relatively rounded and less spread morphologies while the MRC-5 cells appeared as long-tailed bodies expressing alpha-smooth muscle actin (α-SMA) with distinct nuclei, as shown in Fig. 7. These imaging techniques were used to quantify proliferation rates for each cell type.
The numbers and areas of cells occupied by each type were counted using automated segmentation software developed for the project was applied to digital microscope images (Qayyum and Kwak). To differentiate MRC-5 and MCF-7 cells in co-culture samples, both TO-PRO3 red and FITC green fluorescent (GF) images were recorded and merged. In the MCF-7 cell growth assay, a nucleus size exclusion strategy was applied during the cell counting procedure. MRC-5 cells displayed a more elongated nucleus than MCF-7 cells. We collected the circular regions and then analyzed the MCF-7 cell number and area. We calibrated the automated image counting method with a hand counting method to validate accuracy. Coupling information from IF image analysis with MTT assays, we estimated viable cell numbers during the five-day experiment to estimate cell proliferation rates.
Mechanical testing
The protocol for mechanical testing is detailed in our previous work [4]. Briefly, samples were tested using a TA.XT Plus Texture Analyzer System with a 1 kg load cell (Texture Technologies Corp., Scarsdale, NY, USA) and a compressive indentation procedure within 1 h following delivery of IR. The fluid medium was removed from the top of the sample before testing with a 5-mm-diameter spherical indenter. The indenter was pressed into the sample surface under quasi-static conditions (indenter velocity = 10−1 mm/min) and to a depth of 2.5 mm. Force (F) and displacement (d) were recorded by the instrument to estimate sample stiffness. Force-displacement plots show how force sensed by the indenter varied with indenter depth during loading and unloading phases. Stiffness k is defined as the slope F(d)/d near d = 0.
Statistical analysis
Analysis of variance (ANOVA) was performed using R software to determine the significance of differences at α = 0.05 level of significance. Observed differences among mean sample groups were evaluated with post-hoc analysis, including Tukey HSD. This method was similar to earlier publication [4].