Longitudinal bioluminescence imaging to monitor mammary tumor growth and treatment response using the chick chorioallantoic membrane model

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Cell lines and cell culture

MDA-MB-231 triple-negative breast cancer cells (ATCC HTB-26) and estrogen-positive MCF-7 breast cancer cells (ATCC HTB-22) were obtained from ATCC and cultured in DMEM with 10% fetal bovine serum (FBS; Gibco Life Technologies, Grand Island, NY, USA). MCF-10A (CRL-10317), was obtained from ATCC and cultured in DMEM with horse serum (5%), EGF (20 ng/mL), insulin (10 µg /ml), hydrocortisone (0.5 mg/ml) and cholera toxin (100 ng/ml). These cells were cultured at 37°C in a humidified environment and were supplemented with 5% CO2. For all experiments, cells were between 70 and 80% confluent at the start of the experiment and cell viability, as assessed by trypan blue, was greater than 90%. All cell lines were bioengineered to express firefly luciferase using lentivirus transduction. Neomycin-resistant bioluminescent plasmid expressing firefly luciferase was purchased from Addgene (plasmid #21471)14. For in-house viral particle formation, HEK293 T cells were transfected with turbofectin, plasmid, gag and env genes. After 24 and 48 h of incubation, the medium rich in viral particles was collected, centrifuged and stored at -80°C. On the day of infection, the cells were treated with the viral particles and polybrene (Sigma- Aldrich TR-1003-G). Luciferase-positive cells were selected using neomycin, G418 (700 µg/mL). Confirmation of luciferase expression was determined by bioluminescence imaging using an IVIS Lumina (Caliper Lifesciences, Waltham, MA).

Formation of 3D tumor spheres

The tumor spheres were prepared two days before the xenograft on the chicken embryo CAM. The number of cells per sphere was optimized for each cell line based on BLI tumor growth measured over a 9-day period (Supplementary Fig. 1A–H). 2.5×105 cells per sphere for MDA-MB-231 and 5 × 10 cells4 cells per sphere for MCF7 were used for all experiments. The cells were resuspended in 50 μL of Matrigel® and seeded in the form of droplets on a 10 cm culture plate. The cell/matrigel spheres were incubated for half an hour at 37°C to solidify, then incubated for 2 days under standard cell culture conditions.

Ex ovo chicken chorioallantoic membrane test

Fertilized eggs were obtained from the Poultry Research Center (AB, Canada) and incubated at 37°C with rotation for three days. On the third day of embryonic development (EDD), the eggs were cracked as previously describedten. Tumor xenografts were performed on new EDD. The xenograft was performed using spheres derived from mammary cell lines. Before planting, the spheres were washed with PBS (once) and the baseline BLI was measured. For BLI measurements, ~20 µL of luciferin (15 mg/mL) was added topically to spheroids, incubated for 5 min, and imaged on an IVIS Lumina (Caliper Lifesciences, Waltham, MA) using the setting of BLI optical imaging for 10 s. The sphere was placed on a marked area of ​​the CAM. Tumor growth was monitored on days 2, 4, 7, and 9 after transplantation using IVIS imaging. At the endpoint, day 9 after engraftment, tumors were resected, fixed, and processed for immunohistochemistry.

Screening for anti-angiogenic drugs

Axitinib was purchased from SelleckChem (Houston, TX). Axitinib was diluted in DMSO and stored at -20°C at a stock concentration of 10 mM. Chicken embryo tumor sphere xenografts were topically treated with 30 μL of axitinib (10 μM) or control (DMSO) on days 4 and 7 post-transplantation. Tumor growth was monitored using IVIS imaging and at the endpoint, day 9 post-transplant, tumors were resected, fixed and processed for immunohistochemistry. Similarly, Bevacizumab was also purchased from SelleckChem (Houston, TX). Chicken embryo spheres were treated with 10.5 µL bevacizumab (7.5 mg/kg) or control on days 4 and 7 after engraftment and tumor growth was monitored by IVIS.

Immunohistochemistry

Hematoxylin and eosin (H&E) staining was performed for histological analysis of tumor tissues. Tumors grown on the chick CAM were removed, fixed in 4% PFA, stored in ethanol, and then embedded in paraffin. Embedded tumors were sectioned at 5 μm, baked overnight at 37°C, followed by deparaffinization with xylene and rehydration using an ethanol gradient (100%, 95%, 70%, 50%) followed by 1X phosphate buffered saline (PBS) wash. For H&E staining, sections were then stained with the Hematoxylin and Eosin kit according to the manufacturer’s instructions (Vector Labs, Cat No H-3502). For Ki67 immunostaining, tissue sections were deparaffinized and dehydrated as mentioned above. Then, heat-induced antigen retrieval was performed at 95°C for 40 min in 10 mmol/L citrate buffer (pH 6). After heating, slides were cooled to room temperature and washed in 1X PBS. Sections were permeabilized in 0.1% Triton X-100 in PBS, then blocked for 20 min in 10% goat serum in PBS for 20 min. (Vector Labs, Cat No. S-1000). Slides were washed with PBS and incubated with polyclonal rabbit antibody Ki67 1:1000 (Abcam, Cat No. ab15580) for 1 h at room temperature. Excess antibodies were removed by washing three times with 1XPBS. Slides were incubated for 1 h at room temperature with biotinylated goat anti-rabbit IgG (1:200, Vector Labs), washed with PBS, then horseradish peroxidase (HRP) streptavidin for 30 min at room temperature and stained with DAB (3–3′-diaminobenzidine) substrate (Vector Labs, SK-4100). Slides were counterstained with Harris’s hematoxylin, dehydrated, and mounted with Vecta mounting medium (Vector Labs).

Data analysis

Data was collected, processed in Excel, and analyzed using GraphPad Prism 9.1.2. Data passing the Kolmogorov–Smirnov normality test were analyzed by Student’s two-tailed unpaired t-test or Tukey’s one-way ANOVA multiple comparison test. If normality failed, data were analyzed using the Mann-Whitney nonparametric test or the Kruskal-Wallis ANOVA test. Data were normalized to the BLI reading of each individual tumor at D0. Angiogenesis analysis was performed using ImageJ® software. The raw image was imported into ImageJ and converted to an 8-bit image. The ellipse drawing feature was used to position an ellipse over the entire tumor area. The ellipse was then subtracted using the “clear” function to remove the tumor sphere. The “Threshold” image was set to default (black and white) and adjusted. An ellipse area of ​​x=200 and y=200 pixels surrounding the subtracted tumor was selected and pixels outside the 200 × 200 region were removed using the “clear” function. Pixels in the 200 × 200 region represented tumor penetrating vasculature. The “particle analysis” function was performed on this area so that the vessels surrounding the tumor were quantified (total pixels). All data presented are from an average of at least three independent experiments, unless otherwise stated. The statistical tests used are noted in the figure legend. Error bars represent the standard error of the mean (SEM). The statistical significance of the different groups was determined by a p-value less than 0.05 and scored accordingly. Graphs were prepared in GraphPad Prism and figures were prepared in Adobe Illustrator CC 2022.

Ethics approval

All experimental protocols performed in this study were in accordance with the institutional protocol and study approval was obtained by the UBC Institutional Biosafety Review Board (BIO#B17-0151). The study was conducted in accordance with ARRIVE guidelines.

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