Nod cb17 prkdcscid j mice

Immunodeficient NOD.CB17-Prkdc scid/J mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). The colony was housed at the animal housing and experimentation service of the University of Cantabria, under aseptic conditions and veterinary control. The animal experimental protocol was approved by the Research Ethics Committee of the University and the Health Council of Cantabria, as established by current regulations. Surgery was performed on 8-week-old mice, weighing 24–48 g, after intraperitoneal anesthesia and antibiotic. Through a lateral approach to the femur, a complete open osteotomy was performed at the level of the middle third of the right femur by cutting with a number 15 scalpel. The fracture was stabilized by retrograde nailing from the knee (intercondylar region) with a 27 G needle. Subsequently, the periosteum was cauterized at 800 °C with a portable bipolar electrocautery to slow down the bone healing process. Sedation was reversed and postoperative analgesia was administered. The animals were sacrificed 4 weeks after surgery to extract the femurs under study (Figure 1).

8-week-old female NOD.CB17-Prkdc^scid/J mice (Jackson Laboratories) were used for orthotopic injections of modified 4T1 tumor cells. We had 10 mice per group for the p120-catenin WT and S/T6A injected groups and 4 mice per group in 4T1 parental and shp120 injected controls. Following anesthesia, a small incision was made to expose mammary gland number 4, and 1×10⁴ 4T1 cells per mouse were injected into the mammary fat pad. Weekly caliper measurements were used to calculate tumor volume for primary tumor growth rates. Tumor volume was estimated using Volume = (Length x Width²)/ 2, a common calculation to approximate tumor volume of mammary fat pad tumors, an approximation for the volume of a sphere (4/3 x π x r³). Mice were sacrificed 28 days post injection as this timepoint was shown to have established metastases based on previous experiments. At the time of sacrifice, the primary mammary tumors were formalin-fixed, paraffin-embedded, and sectioned for immunohistochemical analysis. Lungs were collected and filled with Bouin’s solution to facilitate surface tumor detection. Lung tissues were then formalin-fixed, paraffin-embedded, and sectioned for immunohistochemical analysis.

All animal experiments were performed according to a protocol approved by the committee for control and supervision of experiments on animals (CPCSEA) (Reg. No: 326/CPCSEA) and the Institutional Animal Ethics Committee (IAEC) of RGCB (Reg. No. IAEC/618). An approximate 1 × 10⁵ volume of sorted cells of SP1, SP2, MP1, and, MP2 fractions collected in culture medium was resuspended in 100 μL of DPBS mixed with 100 μL of phenol-free, growth-factor-reduced Matrigel (BD Biosciences; 356231). Each fraction was subcutaneously injected in one of the 4th or 9th abdominal mammary glands of 6–7-week-old female NOD.CB17-Prkdcscid/J mice (The Jackson Laboratory, ME, USA) in groups of 4 animals per fraction. As a control for the enrichment of tumor-initiating cells (T-ICs) in sorted fractions, a Hoechst-dye-treated and unsorted whole-cell population was injected at dilutions of 1 × 10⁵ into the NOD/SCID mice. The tumor growth was followed up weekly by palpation and monitored until a size range of 1.0–1.5 cm was reached or the health of the mouse rapidly deteriorated. Upon termination of the experiments, the animals were killed as per CPCSEA guidelines, checked for tumors or metastases (white spots on organs), and tumors were immediately excised and processed for immunohistochemistry.

NOD.CB17-Prkdc^scid/J mice (Jackson Laboratory, Bar Harbor, Maine) were housed in pathogen-free conditions and used to test the tumorigenicity of BL cell lines. Immunodeficient mice were injected subcutaneously in one flank with 1×10⁷ EBV-negative Akata cells, and in the other flank with Kem I cells (n = 12) or Wp-R BL cells Oku (n = 16) or Sal (n = 14) and monitored until tumors reached 2 cm, at which time mice were sacrificed. The data were analyzed using student's T test as well as by Mann-Whitney U test, both of which yielded equivalent results, determining that the differences in time to tumor formation between Kem I and Oku or Sal were highly significant (p<0.001).