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The P8119 cell line represents a mesenchymal tumor cell derived from a mammary adenocarcinoma that spontaneously arose in a MMTV-PyMY (mouse mammary tumor virus promoter driven Polyoma middle T-antigen) transgenic C57BL/6 female mouse. This cell line carries the Polyoma virus middle T oncogene, however its expression is down-regulated. (L Ellies, personal communication)

Py8119 cells are, to a degree, spindle-shaped and do not form discrete colonies in culture. (PubMed: 24368187)

This cell line is very robust and forms aggressive mesenchymal tumors in vivo. The tumors are not metastasized when injected orthotopically, however tail vein injection results in tumors in multiple sites including lung, liver and bone. (L Ellies, personal communication)

Py8119 express mesenchymal markers N-cadherin, vimentin, Slug (SNAI2) and cytokeratin 14 and is negative for expression of estrogen receptor, progesterone receptor and HER2 (human epidermal growth factor receptor 2).           (PubMed: 24368187)

The Py8119 cell line exhibits a more undifferentiated phenotype and grows more aggressively in cell culture in comparison to the more differentiated and epithelial-like CRL-3279, Py230 cells, which were also derived from a MMTV-PyMY transgenic C57BL/6 female mouse            (PubMed: 22531600)

• Fetal bovine serum (FBS; ATCC 30-2020) for a final concentration of 5%

Volumes used in this protocol are for 75 cm² flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

1. Remove and discard culture medium. Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) (ATCC 30-2200) or 0.25% (w/v) Trypsin - 0.53 mM EDTA (ATCC 30-2101) solution to remove all traces of serum which contains trypsin inhibitor.
2. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).

   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.

1. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
2. Transfer cell suspension to a centrifuge tube and spin at approximately 125 xg for 5 to 10 minutes.
3. Discard supernatant. Resuspend the cell pellet in fresh growth medium.
4. Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 1 X 10⁴ to 5 X 10⁴ viable cells/cm2 is recommended.
5. Incubate cultures at 37°C. Subculture when cell density reaches between 2 X 10⁵ and 3 X 10⁵ cells/cm2.

Subcultivation Ratio: 1:5 to 1:10 is recommended.

Medium Renewal: 2 to 3 times a week

Guy CT, et al. Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Mol. Cell Biol. 12: 954-961, 1992. PubMed: 1312220

Maglione JE, et al. Transgenic Polyoma middle-T mice model premalignant mammary disease. Cancer Res. 61: 8298-8305, 2001. PubMed: 11719463

Gibby K, et al. Early vascular deficits are correlated with delayed mammary tumorigenesis in the MMTV-PyMT transgenic mouse following genetic ablation of the NG2 proteoglycan. Breast Cancer Res. 14: R67, 2012. PubMed: 22531600

Biswas T, et al. Attenuation of TGF-β signaling supports tumor progression of a mesenchymal-like mammary tumor cell line in a syngeneic murine model. Cancer Lett 346: 129-138, 2014. PubMed: 24368187