70 m cell strainer

PBS-perfused lungs were inflated with 2 ml of digestion cocktail containing 50 U/ml dispase (Corning), 250 U/ml collagenase type I (Worthington), 5 U/ml elastase (Worthington), and 60 U/ml DNAse I (Roche). The trachea was clipped distally and lungs were dissected in a petri dish on ice to remove extrapulmonary airways (trachea and main bronchi). Lung lobes were placed in a C tube (Miltenyi) containing 3 ml of digestion cocktail, and the m_lung_01 program was run on gentleMACS (Miltenyi). C tubes were placed in a rotating incubation oven at 37 °C for 30 min. The m_lung_02 program was run again, and the tubes were placed on ice for the next steps. The lung cells were passed through a 70 µm cell strainer (Corning) and centrifuged at 400 g for 5 min. The pellet was resuspended in a red blood lysis buffer solution (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA), incubated for 2 min on ice, and washed with EasySep buffer (STEMCELL Technologies) at 400 g for 5 min. To isolate mesenchymal cells (Fig. 2, Supplementary Fig. 2), a milder digestion cocktail was used containing only 375 U/ml collagenase and 60 U/ml DNAse I. In all experiments, lung single-cell suspensions from several mice were pooled before fluorescence-activated cell sorting was performed.

Before extraction, lungs were perfused with 30 ml normal saline through right ventricle of the heart under 2% isoflurane inhalation anesthesia. Lung tissues were harvested from mice and filled with Ca⁺⁺ and Mg⁺⁺ free Hank’s balanced salt solution (HBSS) (Corning; Corning, NY).
They were sliced into small pieces by sterile surgical blade and were digested in HBSS supplemented with 100 U/ml of collagenase I (Worthington Biochemical, Lakewood, NJ) for 30 min at 37 °C with periodic shaking. The resultant fragments were meshed by a 70 µm cell strainer (Corning) and a 10 ml syringe plunger and washed with HBSS. The remaining red blood cells were lysed with lysing buffer (BD Biosciences, San Jose, CA). The numbers of isolated lung cells were counted using a microscope (Eclipse TS100; Nikon, Tokyo, Japan).

BM cells were collected from WT mice (control), young non-tumor-bearing SKO and DKO-Osx1-Cre-tdTomato mice at different time points (SKO yBM, DKO yBM respectively), as well as at the time of tumor detection (SKO BMT, DKO BMT respectively). Mice were sacrificed using CO₂, femurs and tibiae from both legs were cleaned from connective tissue, and the BM was flushed out using a syringe with 27 Gauge needle. Red blood cells were lysed using a hypotonic treatment (water dilution). The cells were then centrifuged at 2000 rpm for 10 min, filtered using 70 µM cell strainer (Corning, Massachusetts, USA) and plated at an appropriate density after one passage (p1) [31 ].

h-pES10 cells were washed once with phosphate buffered saline (PBS), then trypsinized using TrypLE Select. Cells were centrifuged and resuspended in fresh human ESC medium containing 10 µg/mL Hoechst 33342 (Sigma Aldrich, St. Lois, MO, USA) and incubated at 37 °C for 30 min. Cells were then centrifuged and resuspended in PBS with 10% KSR and 10 µM ROCKi, filtered through a 70 µm cell strainer (Corning, Glendale, AZ, USA), and sorted by DNA content in BD FACS Aria III (BD Biosciences) using a 405 nm laser. Sorted cells were plated in 6-well plates with fresh growth medium containing 10 µM ROCKi.

Single-cell suspensions from frozen CRC lesions were prepared by mechanical dissociation and enzymatic digestion. These lesions were kept at -80°C in an ultra-low temperature freezer at the Biological Resource Center, Taizhou Hospital of Zhejiang Province (National Human Genetic Resources Platform of China).
After taking them out from the freezer, the lesions were balanced at room temperature for 5 ~ 10 min, washed three times with HBSS buffer (Thermo Fisher Scientific, Grand Island, NY, USA), sectioned into small pieces (5 ~ 6 mm) and washed with HBSS buffer again. After dissociation, small lesion pieces were digested in HBSS buffer containing 2% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), collagenase type IV (1 mg/ml, Thermo Fisher Scientific, Grand Island, NY, USA), and hyaluronidase (10 ng/mL, Merck, St. Louis, MO, USA) at 37°C for 4 h (28 (link)). After digestion, the cells were filtered with a 70 µm cell strainer (Corning, Durham, NC, USA) to obtain single cell suspension that was used for flow cytometry analysis.