Fig. 3.

Suspension culture supplemented with basement membrane-rich extract facilitates stable long-term culture and efficient lentiviral transduction for in vivo metastasis studies. a Using the 2% Matrigel suspension culture method, a starting input of 1 million tumor organoid cells (n = 3 mice, each line is a biological replicate, each dot is a passage) were expanded and passaged for 2 weeks. Right, image of >300 million MMTV-PyMT cells expanded from 1 million initial cells after 2 weeks. b Percent viability and mean fold growth by viable cell number of MMTV-PyMT cells at passage number 2, 5, or 7 in 2% Matrigel suspension culture. n = 4 mice. No significant differences were detected between passages for growth or viability (unpaired t-tests, p > 0.05). c Immunofluorescence for basal cell marker Keratin-14 in MMTV-PyMT organoids freshly isolated from primary tumors (passage 0) or after 4 passages in 2% Matrigel suspension culture. Representative images from n = 2 mice. d Log₂ tumor/normal copy number variations from low coverage whole genome sequencing (plotted as 100 kilobase bins) of MMTV-PyMT organoids from the same mouse shortly after isolation (passage 0) or after 10 passages in 2% Matrigel suspension culture. e Heatmap of passaged log₂ copy number ratios (tumor/normal) minus passage 0 log₂ copy number ratios (mean per chromosome). f Passaged log₂ copy number ratios (tumor/normal) minus passage 0 log₂ copy number ratios (mean per chromosome). Each dot is a chromosome, n = 4 mice, n = 6 passages. Red line = median. g Left, schematic of lentiviral transduction of organoids in suspension. MMTV-PyMT tumor organoids were lentivirally transduced with PGK-EGFP at MOI ~10 in suspension. Then tumor organoids were lentivirally transduced with non-targeting (sgCTRL) or EGFP targeting (sgEGFP) lentiCRISPRv2 constructs. Right, DIC and GFP images after 10 days of puromycin selection showing effective EGFP knockout. h MMTV-PyMT organoids were cultured in suspension +2% Matrigel and transduced with a GFP encoding lentivirus as in G. After puromycin selection, PyMT-GFP clusters were orthotopically transplanted into the mammary fat pads of NSG mice. Tumors were collected 6 weeks later. n = 10 mice, n = 20 tumors. i MMTV-PyMT organoids were cultured in suspension +2% Matrigel and transduced with GFP encoding lentivirus as in G. After puromycin selection, PyMT-GFP clusters were injected by tail vein into NSG mice. 3 weeks later lungs were harvested to assess metastatic outgrowth via stereomicroscopy. n = 8 mice