Estrogen pellet

All mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanyang Technological University (NTU). In the experimental lung metastasis model, a total of 5 × 10⁵ MCF7-C3 or 231-C3 cells were injected into the tail vein of 6-8-week-old female BALB/c nude mice (BioLasco Company, Taiwan). After the mice were sacrificed at the designed times, various tissues were examined for micrometastases using an MVX10 Fluorescence MacroZoom System (Olympus, Japan) equipped with FRET filters (Ex = 436 ± 10 nm; diachronic mirror = 455 nm; Em₁ = 480 ± 20 nm and Em₂ = 535 ± 15 nm).
Orthotopic xenograft models were generated by injecting of 1 × 10⁶ MCF7 or MDA-MB-231 cells in 100 μL of Matrigel (BD Biosciences, USA) into the mammary fat pad of nude mice. For mice injected with MCF7 cells, one estrogen pellet (1.7 mg per pellet, Innovative Research of America, USA) was subcutaneously implanted into each mouse 4 days prior to cell injection. Metastatic 231-C3 series cell lines were isolated from micrometastases or macrometastases on lung by dissection under imaging system followed by collagenase IV digestion (Sigma) (Figure 7A).

One week before injection of tumor cells, mice were ovarectomized and implanted with an estrogen pellet (0.36 mg/pellet, 60‐day release, Innovative Research of America, Sarasota, FL, USA) using a precision trochar, as per the manufacturer’s instructions. Tumor cells were injected in the fourth and/or ninth mammary fat pad of female 8–20 weeks old NSG mice. For MDA‐MB‐231 tumor development, 5 × 10⁵ MDA‐MB‐231 cells in sterile PBS were injected. For MDA‐MB‐231 and T47D mixed tumors, a total of 1 × 10⁶ cells was injected in a ratio of 10:1 Cre⁺:reporter⁺ cells in sterile PBS with 50% growth factor‐reduced Matrigel (BD Biosciences, Franklin Lakes, NJ, USA).

Animal experiments were approved by the UTSW Institutional Animal Care and Use Committee (IACUC protocol 2018–102359). An estrogen pellet (0.25 mg/pellet, 21-day release; Innovative Research of America) was implanted s.c. in the mouse dorsum one day before tumor inoculation. One million MCF-7_RBKO cells were mixed in PBS:matrigel (1:1) and then injected s.c. into 6-week-old female nude mice. For PDXs, ER+/RB1-deleted PDX fragments were implanted s.c. into 6-week-old female NOD scid gamma (NSG) mice. Once tumors reached ≈200 mm³, mice were randomized to receive treatment with vehicle (5% DMSO, 40% PEG-300 and 5% Tween-80 in sterilized water), fulvestrant (5 mg/mouse/week, s.c.), pemrametostat (200 mg/kg/day, via orogastric gavage). or combination both drugs. Tumor size was serially measured with calipers and calculated every three days with the formula: volume = width² × length/2. At the end of the treatment, tumors were harvested and then snap frozen in liquid N₂ or fixed in 10% neutral-buffered formalin.

All mouse studies were conducted using animal protocols approved by the Institutional Animal Care and Use Committee of the National Taiwan University Medical College in accordance with institutional guidelines. An estrogen pellet (0.72 mg 90-day release, Innovative Research of America, Sarasota, Florida) was implanted on 5-week-old female BALB/cAnN.Cg-Foxn1nu/CrlNarl nude mice (National Laboratory Animal Center, Taipei, Taiwan) 2 days before 2 × 10⁶ MCF-7 cell inoculation. When the average tumor size reached 500 mm³, the mice were randomized according to their tumor size into six treatment groups: phosphate-buffered saline (PBS; control), tamoxifen (50 mg/kg), alpelisib (35 mg/kg), buparlisib (25 mg/kg), alpelisib (35 mg/kg) + tamoxifen (50 mg/kg), and buparlisib (25 mg/kg) + tamoxifen (50 mg/kg). These treatments were administered by gavage feeding once daily for 6 days per week. The tumor size was measured weekly using a caliper and calculated as 0.5 × longest length × (width)². The mice were sacrificed after 7 weeks of treatment, and the tumors were harvested for immunohistochemical (IHC) staining. IHC images were acquired using a Leica DMR microscope (Heidelberg, Germany) at our institution.

Three days prior to tumor cell implantation, an estrogen pellet (0.72mg/90 day release, Innovative Research of America, Sarasota, FL) was placed subcutaneously in the back of the neck of female Nu/Nu mice. Mice were injected subcutaneously in the lower dorsal region on both sides with 1x10⁶ MCF-7-HER2 cells suspended in 50 μL of Matrigel (Corning Inc., Tewksbury, MA) and monitored for recovery following the procedure. Two tumors grew on each mouse and a total of seven animals were used in the study. Once tumors reached a pinch-measurement length of 1 cm, mice were euthanized and tumor and muscle samples were resected and cut into 4–7 mm thick slices in preparation for staining. As has been reported in prior studies, only the cut surface of tumor slices were used for imaging.