Dmi8 system

The transfected cells were fixed in 4% paraformaldehyde solution on the 12-well-plates, stained with 0.05 μg/mL Nile red (Sigma, USA) for 10 min, washed with phosphate-buffered saline twice, and then stained with DAPI (Beyotime, Shanghai, China). Images of the cells were acquired with a Leica DMi8 system.

The organoid culture was first approved by the Institutional Review Board of Nanjing Drum Tower Hospital. The organoid culture method was described previously.^[28] Briefly, ≈1 cm³ GC tissues from three different GC patients were minced, washed with 1× chelating buffer (5.6 × 10⁻³m Na2HPO4, 8.0 × 10⁻³m KH2PO4, 96.2 × 10⁻³m NaCl, 1.6 × 10⁻³m KCl, 43.4 × 10⁻³m sucrose, 54.9 × 10⁻³m d‐sorbitol, and 0.5 × 10⁻³m d,l‐dithiothreitol (pH = 7)), and cut into 20–50 small pieces. The glands were pressed and then centrifuged for 5 min at 200× g and 4 °C. ≈100 glands per 50 µL of basement matrix were seeded in one well of a 24‐well plate warmed to 37 °C. 500 µL of medium containing growth factors (50 ng mL⁻¹ EGF, 100 ng mL⁻¹ noggin, 1 µg mL⁻¹ R‐spondin1, 50% Wnt‐conditioned medium, 200 ng mL⁻¹ FGF10, 1 × 10⁻⁹m gastrin, 2 × 10⁻⁶m TGF‐beta inhibitor, and 10 × 10⁻⁶m RHOKi) was carefully added to each well. After organoids formed, they were transfected with ABL overexpression or corresponding control lentiviral vectors for 8 h. Then, the organoids were seeded in 24‐well plates, cultured for one week, and treated with or without DDP (1 µg mL⁻¹) for 24 h, and organoid images were acquired with a LEICA DMi8 system. Then, the organoids were fixed, and the expression of ABL, cleaved caspase 3, and Ki67 was detected using the methods described below.

To explore the presence of efferocytosis mediated by macrophages in PDAC. According to the manufacturer's instructions, Multicolor IHC staining was performed using a Multiple Immunofluorescence Kit (AiFang biological, AFIHC035). Briefly, the paraffin‐embedded tissues were dewaxed, and then the antigen retrieval was performed by sodium citrate at 98°C for 20 min. After infiltrating in 3% H₂O₂ for 15 min, the tissues were blocked by 5% BSA. Then, the tissues were incubated by primary antibody at 4°C overnight. Then, the tissues were incubated by secondary antibody for 30 min and stained by TSA for 5 min. For each target, the steps of antigen retrieval, blocking, incubation of primary antibody, incubation of secondary antibody, and TSA staining are required. Finally, the nuclei were stained with DAPI. PBS was used to wash tissues between each step. These images were obtained by the LEICA DMi8 system and analysed by the ImageJ software.

For CCK8 assay, after the cells were transfected for 48 h, the cells were plated at a density of 2,000 cells per well in 96-well-plates. After 72 h, cell viability was determined using CCK-8 assay according to the manufacturer's instructions (APExBIO, Houston, TX, USA).
For clonogenic assay, after the cells were transfected for 48 h, the cells were seeded at a density of 500 cells per well in 12-well-plates and incubated at 37°C for 10–14 days. Then, cells were fixed with methanol for 30 min and stained with crystal violet (Beyotime, Shanghai, China) for 30 min.
For EdU assay, the cells were plated at a density of 10,000 cells per well in 96-well-plates, and the cells were transfected for 48 h. The cell immunofluorescence was determined by the EdU kit according to the manufacturer's instructions (RiboBio, Guangzhou, China). Images of the cells were acquired with a Leica DMi8 system.

Apoptosis was assessed using the TUNEL Apoptosis Detection Kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. Cell nuclei were stained with DAPI. Confocal images of cells were sequentially acquired with a LEICA DMi8 system. Cells undergoing apoptosis in tumor tissue slides were also detected as described above.

To detect cells with nuclease-induced mutations and test the functionality of the gRNAs, a surrogate reporter system (Figure 3B) was adopted.³⁸ (link) Briefly, double-stranded, linear oligonucleotides representing E1A target regions, each carrying clusters of 2–3 individual target sites in close proximity to each other, were inserted into the EcoR1 and BamHI sites of vector RVO1 (PNA Bio, Newbury Park, CA, USA), giving rise to vectors RV01-1, -3, and -4 (carrying the target sites for gRNAs 1, 3, and 4, respectively); RV01-2 and -9 (carrying the target sites for gRNAs 2 and 9, respectively); RV01-5, -6, and -8 (carrying the target sites for gRNAs 5, 6, and 8, respectively); and RV01-7 and -10 (carrying the target sites for gRNAs 7 and 10, respectively). The final RV01-based vectors were sequenced using primers RV01 sequencing FW and RV (Table S1). The inserted target sequences are listed in Table S2. 2e+04 to 5e+04 HeLa cells were seeded into the wells of a 96-well plate and transfected with 100 ng of reporter vectors using Lipofectamine 2000 (Thermo Fisher Scientific), followed by transduction with the recombinant Cas9/gRNA-expressing vectors. 48 h post transduction, pictures were acquired with a Leica DMi8 system and analyzed with the microscope software platform LAS X Life Science (Leica, Wetzlar, Germany).

For wound-healing assay, the transfected cells were grown to confluence in a 12-well-plate. Next, the cells were cultured in RPMI-1640 with 1% FBS for 12 h. The confluent monolayer was then disrupted with a cell scraper and filmed at the indicated hours via Leica DMi8 system. The rate of wound closure was calculated as the ratio of the average distance between the two wound edges and the total cell duration of migration.
For Transwell assay, the transfected cells in 100 μL of serum-free medium were seeded in the upper chambers coated with or without 50 μL of Matrigel (BD Biosciences), and 600 μL of culture medium containing 10% FBS was placed in the lower chambers. After 12 h of incubation at 37°C, cells that migrated to the bottom of the membrane were fixed with 4% paraformaldehyde for 30 min, stained with crystal violet (Beyotime, Shanghai, China) for 30 min, and imaged.