Cancer Model and Animal ProtocolsEngineering of a Bioluminescent Mammary Gland Tumor Cell Line

The medullary breast adenocarcinoma cell line originally isolated as a spontaneous tumor from C57BL/6 mouse was obtained from CH3 BioSystems LLC (Amherst, NY, USA). It was modified to stably express the Luciferase reporter gene. In brief, the coding sequence of the luciferase reporter gene luc2 (Photinus pyralis), which has been codon optimized for mammalian expression, was amplified by PCR from the pGL4.50[luc2/CMV/Hygro] plasmid template (Cat.# E1310, Promega, Madison, WA). Flanking XhoI restriction sites were added for the subcloning into the SalI site of the pLENTI PGK DEST Vector (Plasmid 9065, Addgene Cambridge, MA). Lentiviral particles were obtained in the HEK293T cell line and used to infect E0771 cells. Blasticidin (5 µg/ml) resistant cells were amplified and tested for luciferase expression and bioluminescence in vitro. Then cells were maintained as described in [10]. To control for luciferase expression, cells grown at 70–80% confluency were washed twice with PBS1X and scraped in 300 µl of lysis buffer (Tris HCl 50 mM, pH 7.4, with 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 1X Complete protease inhibitor (Roche)). For Western blot analysis, nearly equal amounts of proteins (20 µg) were separated on 8–14% SDS–PAGE and transferred onto nitrocellulose membrane. Membrane was blocked with milk 3% in 1× PBS, Tween-20 0.1%, and incubated overnight at 4 °C with anti-Luciferase (sc-57,604; 1/1000, Santa Cruz Biotechnology) and (reprobed) with anti-actin, (A-1978; 1/1000, Sigma). Secondary horseradish peroxidase (HRP), conjugated anti-Mouse and anti-Rabbit antibodies were from Jackson ImmunoResearch. Signals were acquired using the (Amersham Imager 600).

In Vivo Studies

Mice breeding and maintenance were done in the accredited IGBMC/ICS animal house (C67-2018-37), in compliance with French and EU regulations on the use of laboratory animals for research. Animal experiments were approved by the ethical committee Com’Eth (Comité d’Ethique pour l’Expérimentation Animale, Strasbourg, France) and the French ministry of Higher Education and Research (#9177-2017030811336376v4). This project included the longitudinal evaluation of orthotopic tumor grafts in immunocompetent mice. In total we used 34 C57BL/6 mice aged from 18 to 24 weeks-old and evaluated tumor growth using 3 different modalities.

Mice were injected with 200,000 luciferase-expressing E0771 cells in the 4th left mammary gland at Day 0. The mice were then followed-up at Day 4, 7, 11, 14, 18 and 21. Depilation was done before injection and before each imaging session.

Mice were anesthetized using isoflurane. Induction was achieved with an isoflurane concentration of 3%, followed by maintenance at 1% in oxygen at a flow rate of 0.5-1 l/min.

All animals were euthanized at day 21 or when tumor longest diameter, assessed by caliper, was higher than 6 mm.

Caliper Measurement

For each tumor, the longest diameter tumor L and its perpendicular diameter W were measured using a digital caliper. The tumor volume was estimated with the following formula:

$$Vo} = \frac \times L \times W \times \left( \right)/2$$

High-frequency Ultrasound Imaging Protocol

After caliper measurement, mice were scanned with hand-held high-frequency ultrasound probe (Vevo 3100, Fujifilm, VisualSonics). No anesthesia was required at that point.

Animal handling adhered to the National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs) guidelines to ensure welfare-centric practices. Initially, the mouse was gently secured by holding the base of the tail with the thumb and forefinger, allowing it to stabilize itself using its forelimbs on a non-slip surface. Subsequently, a scruff restraint was employed to carefully expose the animal’s ventral side. While skin shaving was not required for ultrasound, it was needed for the BLI procedure. A layer of ultrasound gel, pre-warmed to 37 °C, was applied to the skin over the fourth left mammary gland to facilitate optimal acoustic contact. The ultrasound probe was then delicately positioned on the gland. All handling and ultrasonic scanning procedures could be performed by a single operator. The duration of each examination was calculated on images metadata, to assess efficiency of the procedure.

Tumor echogenicity was recorded (hyperechoic/isoechoic to the surrounding gland, slightly hypoechoic or strongly hypoechoic). Tumor heterogeneity was defined as areas of abnormal signal in the tumor, assessed semi-quantitatively as absent, minimal (< 10% of the tumor volume), significative (10–50%) or extensive (> 50%).

For tumor measurements, one image was taken in the plane of the longest diameter and another in the perpendicular plane. On each image, two perpendicular measurements were done, resulting in 4 measurements: L and W for tumor length and width, H1 and H2 for the measurements of tumor height in each of the two perpendicular images. Maximal subcutaneous tissue thickness between probe and tumor was also measured on the available images. To measure inter-reader variability, images from ten distinct individuals at different timepoints were independently evaluated by two readers of different level of experience. Finally, the tumor volume was estimated with the following formula (ellipsoid volume formula):

$$Vo} = \frac \times L \times W \times \left( \right)/2$$

Interval growth rate was defined as the change of volume between two consecutive timepoints normalized by the tumor volume at the first timepoint. For categorization purpose, stability was considered to be a change of less than 5%.

Growth pattern described the evolution of tumor volume over the entire follow-up period. The growth patterns were defined a posteriori, based on follow-up data clustering, as fast-growing, slow-growing, delayed-fast-growing and regressing tumors.

Bioluminescence Imaging

Immediately after HFUS assessment, mice were injected intraperitoneally with 250 µL of D-Luciferin (15 mg/ml, XenoLight, Perkin Elmer). BLI scans were taken with a IVIS spectrum In Vivo Imaging System (PerkinElmer). For BLI acquisition, mice were anesthetized using 1.5–3% isoflurane.

With animals in dorsal decubitus, BLI images were sequentially acquired during 20 min, with an exposure time of 1 to 4 s, a field of view of 12.5 cm.

Analyses were conducted by capturing regions of interest (ROIs) from each image, from different timepoints after injection, quantifying the total flux and radiance, which represents the amount of light emitted per unit area and solid angle, as well as the variability of the signal and determination of the peak signal.

Tumor Tissue Processing

After animal sacrifice, tumors were excised, their long axis measured and their weight was determined using a calibrated analytical balance, and reference tumor volume (VolREF) was extrapolated from tissue mass assuming a density of 1 mg/mm3. Tumors were then fixed in formaldehyde and embedded in paraffin and cut.

Hematoxylin and Eosin Staining and Tumor Cellularity Evaluation

Sections underwent hematoxylin and eosin (H&E) staining for assessment of general tumor structure, margins, necrosis and evaluation of tumor cellularity.

Evaluation of tumor cellularity has been described previously [11]. In our study, it involved the selection of a representative axial slice, tumor contouring and semi-automatic tumor cell counting using QuPath [12] algorithm. Tumor cells detection was based on the optical density using hematoxylin staining, with optimization of the sigma function and the minimum nuclear area, to reduce nuclear fragmentation and exclude small immune cells. The absolute number of tumor cells then normalized by the surface area of the contoured tumor to generate a tumor density. The tumor density was then multiplied by the surface area of the total tumor using the greatest linear dimension as the diameter.

These analyses were conducted on all tumors, except on fragmented, highly heterogeneous or irregular-shaped tumors, tumors with improper staining or other artifacts preventing a correct evaluation of tumor cellularity.

Immunofluorescence Labeling

Sections also underwent immunofluorescence labeling targeting pancytokeratin, CD4, CD8 and CD34 for evaluation of the epithelial, immune and vascular components, respectively. Spatial distribution of markers and the number of positively stained cells per unit area were assessed.

Statistical Analysis

Correlation analysis between imaging modalities, caliper and histological assessment, were conducted using Pearson test. For correlation between imaging (HFUS-derived tumor volume and BLI tumor brightness) and histology, imaging assessment was done the same day of mouse sacrifice. To assess inter-reader variability, two metrics were used: root mean square deviation (RMSD), that quantify the average magnitude of the difference between the measurements, and intraclass correlation coefficient (ICC) to assess the degree of agreement between readers.

Student test was used to compare quantitative values, such as the density of positively stained cells for immunofluorescence. A 2-tailed p-value of < 0.05 was considered statistically significant. Linear correlation was calculated with Pearson’s method (ρ).

The analyses utilized the following Python libraries: pandas (v.1.5.1), statsmodels (v.0.13.2) and matplotlib (v.3.5.1).