GoKawiilSelection and prioritization of extracellular tumour-derived genes
Candidate extracellular tumour-derived genes were identified using a stepwise filtering and prioritization strategy integrating single-cell transcriptomic analyses, protein subcellular localization annotations, functional dependency data, domain-based functional annotation and literature-based curation (Extended Data Fig. 1a).
Publicly available scRNA-seq datasets from human14 and mouse15 PDAC were independently analysed. Within each dataset, malignant or premalignant epithelial populations were defined. Differential gene expression analysis was performed comparing malignant or premalignant cells to non-malignant epithelial cells. Genes were retained if they met stringent significance and effect size thresholds (adjusted P value ≤ 0.05, log 2 fold change > 4 for mouse datasets and adjusted P value ≤ 0.05, log 2 fold change > 3 for human) and showed predominant expression within malignant or premalignant epithelial compartments (Extended Data Fig. 1a). Gene lists derived from human and mouse datasets were subsequently merged to generate an initial cross-species candidate pool.
To enrich for factors with extracellular activity, protein subcellular compartment annotations were obtained from the Compartments database53. Enrichment analysis was performed, and genes annotated to the enriched term ‘extracellular region part’ were retained. These genes were intersected with the differentially expressed gene sets to further refine the candidate list.
To exclude genes whose perturbation would be expected to strongly impair tumour cell viability under standard in vitro conditions, candidates were evaluated using CRISPR–Cas9 dependency scores from the DepMap database across 46 human pancreatic cancer cell lines. Mean CRISPR dependency scores were calculated for each gene, and genes with average scores between −0.15 and 0.15 were retained, indicating minimal effects on cell-intrinsic fitness (Extended Data Fig. 1b).
Pathway- and domain-level analyses were performed to characterize the biological programmes represented within the candidate pool (Extended Data Fig. 1c,d). These enrichment analyses were used to provide biological context and to guide a literature-informed selection of the final gene set (Extended Data Fig. 1e).
The selection rationale for each gene is summarized in Supplementary Table 1.
Lentiviral vector construction and production
A comprehensive protocol for generating PC/CRISPR lentiviral vectors and library pools is available on the Addgene website, as part of the Pro-Code vector kit (Addgene 1000000197) and has been described previously12,54. Below, we provide a brief overview of the procedure. For selecting sgRNA sequences targeting each gene, we used the Brie CRISPR library for mouse genes55. A complete list of the oligonucleotides used for the experiments in this paper is provided in Supplementary Table 3. Library cloning was performed using nuclear PC/CRISPR lentiviral vectors (Addgene 1000000197), while validations utilized lentiCRISPR v2 (Addgene 5296156). Library cloning was conducted in a 96-well plate format, where constructs were annealed, ligated, and transformed individually but processed in parallel, as outlined in the Pro-Code vector kit protocol. Oligonucleotides were prepared by resuspending them to 100 µM in water. For annealing, forward and reverse oligos were mixed to a final concentration of 2 µM, combined with 10× NEBuffer 2.1 and water. Next, they were heated to 95 °C for 5 min, followed by gradual cooling to room temperature. The PC/CRISPR lentiviral vector was digested with BbsI-HF (NEB), per the manufacturer’s instructions, and purified using Qiagen PCR purification columns. Annealed oligos were ligated into the digested vector backbone by combining 50 ng of digested plasmid with 6–8 ng of annealed oligos, incubating at room temperature for 10 min with Quick Ligase (NEB). Next, 5 µl of the ligation reaction was transformed into 50 µl of TOP10 chemically competent bacteria. After incubating on ice for 30 min, the bacteria were heat-shocked at 42 °C for 30 s, cooled on ice for 2 min, and plated on LB ampicillin agar plates overnight at 37 °C. Colonies were picked, cultured, and plasmid DNA was extracted using the Zymo ZR Plasmid MiniPrep Classic Kit. The sgRNA sequence was confirmed via Sanger sequencing. For cloning into the lentiCRISPR v2 backbone, a similar process was followed. However, sgRNAs were inserted into the BsmBI site.
Lentiviral vector production was performed as previously described57 and detailed in the Pro-Code_Kit_Methodology.pdf on the Addgene website. In brief, HEK293T cells were seeded at 500,000 cells per well in 6-well plates and incubated at 37 °C with 5% CO 2 . After 24 h, cells were transfected using calcium phosphate with third-generation lentiviral packaging plasmids and the transfer plasmid (pVSV (1 µg), pMDLg/pRRE (2 µg), pRSV-REV (1 µg), and PC/CRISPR vector (6 µg)). The plasmids were first mixed with 2.5 M CaCl 2 , vortexed, and incubated for 10 min. Then, 2× HBS solution (281 mM NaCl, 100 mM HEPES, 1.5 mM Na 2 HPO 4 , pH 7.05) was added dropwise with gentle vortexing. The transfection mixture was applied to cells, and the medium was replaced after 14 h. Supernatants were collected 30 h after medium replacement, filtered through a 0.22 µm PVDF disc filter, and stored at −80 °C.
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