Cytokeratin 18

NOD scid gamma (NSG) mice were purchased from Jackson Laboratory. Eight week old female mice were injected unilaterally with 2.5×10⁶ cells in 200 µL of 50∶50 Matrigel/Collagen I into the fourth abdominal fat pad by subcutaneous injection at the base of the nipple. Tumor growth was monitored externally using vernier calipers for up to 30 weeks and animals sacrificed when tumors reached 10% of body weight. Necropsies were performed to identify macro-metastases. Primary tumors and organs were harvested, paraffin embedded, sectioned and stained with hematoxylin and eosin or antibodies against cytokeratin 18 (CK18), epidermal growth factor receptor (EGFR), and Her2. Pathology processing and staining of harvested mouse tissues was performed at the Lombardi Comprehensive Cancer via Science Exchange, Inc. Slides were analyzed by a pathologist to confirm the presence of metastases. RNA was isolated from three independent metastases and primary xenograft tumors for analysis. Note: No estrogen pellets were required for growth of estrogen receptor alpha positive cell lines, MCF7 and BT474.

Blood samples were obtained at 08:00 AM following overnight fasting of 12 h and ensuring a minimum of 12 h since the last use of antihypertensive drugs. The included variables were fasting plasma glucose (FPG), uric acid, creatinine, total proteins, total cholesterol (TC), and triglycerides (TG). All concentrations were expressed in mg/dL except for total protein levels, which were given in g/dL.
We evaluated the serum protein profile using capillary electrophoresis with a Capillarys 2 analyzer (Sebia, Lisses, France). The concentrations of alpha-, beta-, and gamma-globulins were expressed in g/dL. The alpha band includes some acute phase proteins, such as CRP. We measured wide-range CRP (wrCRP) concentrations by immunoturbidimetry in an Advia 2400 Clinical Chemistry System (Siemens, Munich, Germany) and the levels were expressed in mg/dL. The beta band includes inflammation-linked proteins such as those related to iron (Fe) homeostasis, coagulation, lipid metabolism, beta-2-microglobulin, and complement compounds. We evaluated fibrinogen levels by the Clauss method using the coagulation analyzer ACL TOP® 350 (Werfen Company, Bedford, MA, USA) and concentrations were expressed in mg/dL. Beta-2-microglobulin levels were measured by nephelometry employing a Dimension Vista® system (Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA) and concentrations were expressed in mg/dL. The gamma band mainly includes the immunoglobulins whose quantitative and qualitative evaluation allows the characterization of the immuno-inflammatory response [21 ,22 (link),23 (link),24 (link)].
Because of its relevance in inflammatory states, we included IL-6 as a pro-inflammatory cytokine. IL-6 levels were measured by a chemiluminescent immunoassay (Immulite-2000 System, Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA) and concentrations were expressed as pg/mL. Tissue polypeptide-specific (TPS) antigen levels were measured by a chemiluminescent immunoassay (Immulite-TPS; Siemens Medical Solutions Diagnostics, Gwynedd, UK) that detects cytokeratin-18 fragments, generated by cytolysis or epithelial apoptosis and concentrations were expressed in U/L. ESR is an acute phase reactant that depends mostly on the concentration of circulating acute-phase proteins, specifically fibrinogen. We measured the ESR employing an automated TEST-1 device (Alifax, Padua, Italy) that was validated using the reference Westergren method. ESR was expressed in mm/h. [25 (link),26 (link),27 (link)].

The 293T cells were cultivated in the medium composed of Dulbecco’s modified Eagle medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 2% penicillin/streptomycin (Gibco), maintained at 37 °C in 5% CO₂. The process for lentivirus generation of shRNA was performed as previously reported [22 (link)]. Briefly, when the confluence of cultured 293T cells reached 80%, pLKO.1-shSOCS1, psPAX2 and pMD2.G were co-transfected into 293T cells through TransIntro™ EL Transfection Reagent (TransGen Biotech, Beijing, China) for generating lentiviral particles (shSOCS1) in accordance with the manufacturer’s instructions. The 293T cells were co-transfected with pLKO.1-TRC, psPAX2 and pMD2G as the non-interfered control group (shNC). They were all measured by the serial dilution method for the infection titer of concentrated lentivirus particles.
Buffalo mammary epithelial cells (BuMECs) were isolated from the mammary gland tissue of lactating Binglangjiang buffalo (60 d postpartum) and purified based on the differential sensitivity of the cells to trypsin digestion as previously described by our group [22 (link),23 (link)]. The BuMECs purified and identified by cytokeratin 18 (Sigma, Louis, MO, USA) were cultivated and expanded to passage five in a basal medium composed of DMEM (Gibco), 10% fetal bovine serum (Gibco), 100 μg/mL penicillin/streptomycin (Gibco) and various cytokines, including 5 μg/mL insulin (Sigma), 2 μg/mL hydrocortisone (Sigma) and 100 ng/mL epidermal growth factor (Sigma), and maintained at 37 °C under 5% CO₂. When the cell confluence reached 80% in a 6-well cell culture plate, the cells were cultured for 24 h in a basal medium supplemented with 2 μg/mL prolactin (Sigma). Subsequently, these cells were transfected with EGFP-SOCS1 (3 μg), EGFP-CEBPA (3 μg), siCEBPA (60 nM) and the corresponding negative controls (EGFP and siNC) according to the manufacturer’s protocol of TransIntro^TM EL transfection reagent (TransGen Biotech, Beijing, China). At the same time, they were transduced into shSOCS1 and shNC to knock down SOCS1. Through pre-experiments, it was determined that the best results were obtained when the cells were collected 48 h after treatment. Therefore, the cells of each treatment group were harvested 48 h later for gene and protein expression analysis.