Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoid generation from mouse mammary tumors captures the genetic heterogeneity of clinically relevant copy number alterations

“…Mice were sacrificed according to IUCAC guidelines with CO2 asphyxiation and secondary cervical dislocation. Tumors were dissected and organoids generated following the protocol described previously [11]. For Figure 2a, tumors were dissected into quadrants, and samples taken as described in schema.…”

“…Single cells were strained and harvested after Tryple™ Express (Gibco™; cat: 12605036) digestion and visual verification of single cell dissociation. Organoid invasion assay was performed as described previously [11]. Collagenase was used to digest 3D collagen I gels to isolate invasive organoids.…”

“…Organoids were then randomly split into a control group which were grown in suspension and an experimental group grown in 3D culture. Growing organoids in 3D culture allows ex vivo assessment of their invasive potential through collective migration into collagen [11][12][13]. In brief, the organoids were either kept in media suspension or embedded in 3D collagen I gels and assessed for invasion (Fig.…”

“…We reasoned if FGFR1 copy number is amplified in metastatic lesions, it could potentially be amplified starting at the earliest stages of metastasis, invasion out of the primary tumor. To test whether FGFR1 copy number amplification is associated with invasion, we generated mammary tumor organoids derived from MMTV-PyMT mice using differential centrifugation [11][12][13]. Organoids were then randomly split into a control group which were grown in suspension and an experimental group grown in 3D culture.…”

“…Notably, in all but PAK1, tissue copy number varied significantly from pooled organoid samples (Mann-Whitney, p<0.05). Pooled organoid samples include cancerous mammary epithelium and exclude any stroma, muscle, or other cells that may be included in tissue samples [11, 13]. Thus, copy number reads from pooled organoid samples provide a more accurate representation of cancer epithelial cells within a tumor instead of a mixture of epithelial, stromal and immune cells.…”