Mice were housed under specific pathogen–free conditions, and all experimental procedures were approved by the Vanderbilt University Institutional Animal Care and Use Committee and were conducted in an Association for Assessment and Accreditation of Laboratory Animal Care–accredited facility.

4T1 tumor establishment. Female BALB/cJ mice (8 to 12 weeks old) weighing between 18 and 20 g were obtained from the Jackson Laboratory. Animals were housed at 22 ± 5°C in a 12-hour light/dark cycle and fed rodent chow and water freely. Orthotopic mammary fat pad implantation was performed as follows: 30,000 4T1-luc or 4T1-mCherry cells were injected (50-μl cell suspension in PBS) in the right fourth mammary gland with a 30-gauge needle (BD Biosciences, San Jose, CA, USA), under anesthesia by continuous inhalation of 2% isoflurane gas for 5 to 10 min. Injections were performed under sterile conditions. Bioluminescence images of primary tumor and subsequent metastasis were captured using an IVIS Lumina III imaging system (PerkinElmer Inc.) 5 min after intraperitoneal injection of d-luciferin (150 mg/kg; 100 μl). Mice were euthanized at humane end points.

Tumor resection. Nineteen days after injection of 4T1 cells, primary tumors were resected. Hairs from the resection site were removed 24 hours before surgery using Nair hair removal cream. Mice were kept under anesthesia during surgery. Mice received subcutaneous injections of ketoprofen (2 mg/kg) immediately before surgery and 24 hours after surgery. The surgical site was cleaned with iodine and ethanol. An incision adjacent to the tumor was made with surgical sharp-blunt–type scissors, and the tumor was gently teased away from the skin with needle-nose forceps and removed. After removal of the tumor, the incision was sutured with 4-0 silk nonabsorbable surgical sutures. Upon completion of the surgery, mice were moved to the recovery area under slightly warm conditions (under a heat lamp). Mice were brought back to the mouse facility after they had resumed consciousness and were monitored every alternate day for labored breathing, metastatic nodules, and primary tumor recurrence.

Treatment. To test the efficacy of ETL on the progression of metastases, mice were injected with 100 μl of ST, NL, or ETL (50 μg of TRAIL per injection) through the tail vein. Mice were divided into three groups based on intensity of BLI or tumor size on day 16. Mice were given three doses: 48 hours before the tumor is resected (day 19), immediately after the surgery, and 72 hours after the tumor resection. The entire in vivo schematics is shown in Fig. 3A, where the days marked in red indicate the three injection time points.

Hematological and histological evaluation of mice. Tail bleeding was performed at different time points for quantitation of mCherry-positive disseminated 4T1 cells in peripheral blood. Red blood cell (RBC) lysis was done using RBC lysis buffer for 20 min, and flow cytometry was performed to monitor mCherry-positive cells. Mice were euthanized by CO2 asphyxiation followed by terminal blood collection via cardiac puncture. Body and organ weights were recorded; then, tissues were fixed in 10% neutral buffered formalin immediately following euthanasia of the mice. Complete blood counts were performed on the Forcyte Hematology Analyzer (Oxford Science), and serum chemistry was performed on the ACE Alera (Alfa Wassermann) clinical chemistry analyzer. Spleens were evaluated histologically on the basis of the organ weight data.

Primary tumors were evenly bisected perpendicular to the skin surface, unbiased sectioning of the liver was performed, lungs were embedded whole on the thoracic pluck, and one tibia and one femur were evaluated. Formalin-fixed tissues were routinely processed using a standard 8-hour processing cycle of graded alcohols, xylenes, and paraffin wax, embedded and sectioned at 4 to 5 μm, floated on a water bath, and mounted on hydrophilic glass slides. Bone decalcification was performed using Immunocal (StatLab, McKinney, TX). H&E staining was performed on the Gemini autostainer (Thermo Fisher Scientific, Waltham, MA). All histopathologic interpretation was conducted by a board-certified veterinary pathologist under masked conditions. Photomicrographs were captured on an Olympus BX43 microscope (Olympus Corporation, Tokyo, Japan) equipped with a SPOT Flex camera and software setup (Diagnostic Instruments Inc., Sterling Heights, MI). Quantification of intratumoral necrotic regions was conducted using QuPath, an open-source digital pathology platform.

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