60 day release 17β estradiol pellets

The animal study was reviewed and approved by the Ethics Committee of Shandong University Cheeloo College of Medicine, the approval number is 21002. Female BALB/c nude mice (4-6 weeks old; NBRI of Nanjing University, Nanjing, China) were implanted subcutaneously with 0.36 mg of 60-day release 17β-estradiol pellets (Innovative Research, TX). Caski cells transfected with shNC, shER-α36, NC-LV and NC-ER-α36 were collected, 5 × 10⁶ cells were subcutaneously injected into the axilla of each mouse. The tumor size was measured by Vernier calliper once every 2 days, and tumor volumes were calculated using the equation: length × width² × 0.5. Three weeks post-injection, these mice were sacrificed and tumors were removed, photographed and weighed.

All animal studies were approved by the Institutional Animal Care and Use Committee of Dongguk University (No: IACUC-2017-010-1). In total, 1 × 10⁷ cells (MCF-7/tamR/shNC and MCF-7/tamR/shMMP1) in a 100-μL suspension [1:1 mix of PBS and Matrigel (BD Biosciences, San Jose, CA, USA)] were implanted subcutaneously into 6-week-old female BALB/c nude mice (Orient Bio, Sungnam, Korea), followed by implantation of 60-day release 17β-estradiol pellets (0.72 mg/pellet total dose; Innovative Research of America, Sonnasota, FL, USA). After 4 weeks, the test group was intraperitoneally injected with 100 μL (1 mg/kg) of tam in corn oil (Sigma-Aldrich, St. Louis, MO, USA) five times per week for 4 weeks, whereas the control group was treated with only corn oil. Tumor volume (length × width² × 0.5) was measured each week. After eight weeks of cell injection, xenografted tumors were collected. The tissues were fixed in 4% paraformaldehyde and embedded in paraffin blocks for histological analysis.

Female BALB/c nude mice were implanted with 0.72 mg 60-day release 17β-estradiol pellets (Innovative Research of America, Sarasota, FL, USA). After 6 days, 1 × 10⁷ BT-474 cells were injected into the mammary fat pad in a 1:1 PBS:matrigel suspension (BD matrigel; BD Biosciences, San Jose, CA, USA). When tumor volumes reached an average of about 100 mm³, the mice were randomly divided into groups of 10 mice each. Treatments consisted of twice weekly intravenous injection of different anti-ErbB2 mAbs for four consecutive weeks. Control mice were given vehicle (IgG) alone. Tumors were measured with digital calipers, and tumor volumes were calculated by the formula: volume=length × (width)²/2. Animal procedures were performed under an approved protocol.

Six‐week‐old NOD/SCID mice were injected under their fat pads with MCF‐7 cells (1 × 10⁶) or MCF‐7 cells (1 × 10⁶) mixed with CAFs (3 × 10⁶) as previously described.^[25, ^70] To support the estrogen‐dependent MCF‐7 tumor growth, 17β‐estradiol 60‐day release pellets (Innovative Research of America) (1.7 mg per mouse) were subcutaneously implanted into mice 3 days prior to tumor injection. Mice were treated with docetaxel at a dose of 10 mg kg⁻¹ by intraperitoneal injection weekly when the tumor reached a diameter of 3 mm.^[25] Tumor size was measured every 4 days with calipers, and the volume was calculated as 0.5 × length × width². After 5 weeks of docetaxel treatment, positron emission tomography/computed tomography (PET/CT) scanning was performed. Next, the xenografts were harvested and subjected to further analysis.
To qualify ALDH1⁺ tumor cells in xenografts, the xenografts were dissociated by collagenase type I (1.5 mg mL⁻¹) and collagenase type III (1.5 mg mL⁻¹) at 37 °C with agitation for 30 min in DMEM containing 10% FBS. Single cell suspensions were obtained by filtration through a 40 µm filter. Tumor cells were isolated from xenografts using the CD326 (EpCAM) Tumor Cell Enrichment and Detection Kit (Cat# 130‐090‐500, Miltenyi Biotec) before ALDH1 staining.
All mice were kept in biosafety level laboratories at the Animal Experiment Center of Sun Yat‐Sen University.

All animal studies were approved by the Korea University Animal Care and Use Committee (# KOREA-2019-0129). First, 17-β-estradiol 60-day release pellets (Innovative Research of America) were transplanted into the left flank of athymic nude Foxn1nu female mice (Envigo, MA, USA) 1 day before tumor inoculation. After that, 5 × 10⁶ of BT474 cells and 5 × 10⁶ of LR-BT474 cells mixed with 50% matrigel (Corning cat. 356231, New York, NY, USA) were subcutaneously implanted into the right flank of athymic nude mice. When tumors reached approximately 150 mm³, animals were randomized to treatment groups: vehicle, 17-DMAG, lapatinib, and combination of 17-DMAG and lapatinib (n = 4, group). 17-DMAG was administered by intraperitoneal injection three times per week for 5 weeks at 5 mg/kg. Lapatinib was administered for 5 weeks at 75 mg/kg by oral gavage daily during the study period. Using a digital caliper, the same investigator serially measured vertical tumor size twice a week. Tumor size was calculated according to the following formula: TV (mm 3) = (length [mm] × (width [mm] 2)/2. Animal weights were also measured twice a week.