GoKawiilClinical cohorts
All patients in the M0/M1 case–control cohort were diagnosed with colorectal adenocarcinoma at the Institute of Pathology, Faculty of Medicine, LMU Munich, and were subsequently identified through a systematic database search in collaboration with the Munich Cancer Registry. Patients in the M1 group were histologically or radiologically diagnosed with local recurrence or distant metastasis to any site within a 5-year observation period after resection of a colorectal adenocarcinoma. To build the M0 group, a patient who did not progress within the same time frame was pair-matched to each M1 patient according to sex, age, disease stage, tumour grade and primary tumour site. Available patient and tumour characteristics, as well as survival data, were collected. Patients with secondary malignancies were excluded. The availability of sufficient analysable tumour tissue limited patient numbers to 82 per group. Clinicopathological details are listed in Supplementary Table 1. For the UICC stage II cohort, anonymized CRC specimens from patients who underwent surgical resection at LMU Munich between 1994 and 2017 were obtained from the archives of the Institute of Pathology LMU. Follow-up data were recorded prospectively by the Munich Cancer Registry. Specimens were anonymized. Clinicopathological details are listed in Supplementary Table 2. The studies were approved by the Institutional Ethics Committee of the Medical Faculty of the LMU (approval no. 18-105-UE) and carried out according to the Declaration of Helsinki. For both cohorts, TROP2 expression was detected on 4-µm-thick formalin-fixed paraffin-embedded sections using anti-TROP2 antibody (Abcam; ab227691; clone SP295) at a 1:100 dilution on a Ventana BenchMark ULTRA autostainer instrument. Normal human tonsil epithelia were included as positive controls in each staining run.
PDOs and tissues were established from tumours or normal mucosa obtained at the University Hospital Heidelberg (ethical approval nos. S-136/2021 and S-871/2021), in accordance with ethical guidelines and regulations. All patients provided written informed consent. Clinical features and genetic driver mutations (identified by whole-exome sequencing) are provided in Supplementary Table 4.
Kaplan–Meier plots of disease-free survival and relapse-free survival hazard ratios in 1,336 patients with CRC from 16 publicly available datasets were generated using the Kaplan–Meier Plotter tool (https://kmplot.com/analysis/). The automatic best cut-off was used, incorporating data from a previous study53. Results shown in Extended Data Fig. 1h,i were generated using SubtypeExploreR (https://subtypeexplorer.qub.ac.uk/).
Animal experiments
All mouse experiments were approved by the local authorities of Regierungspraesidium Karlsruhe under animal protocols G-148-20, G-27-22, G-159-22 and G-164-22. Tumour growth was monitored regularly, and mice were euthanized when tumours reached the maximum permitted tumour size or when humane end point criteria were met. These limits were defined in accordance with the approved animal protocols and were not exceeded in any of the experiments. The mice were housed at the DKFZ animal facilities in accordance with the local and latest standards with a 12 h dark and light cycle, a constant temperature (20–24 °C) and humidity (45–65%). They were provided with a rodent-specific diet and water ad libitum.
Tacstd2 CreERT2 transgene generation
The TAG stop codon of the Tacstd2 gene was replaced with a P2A-CreERT2-FRT-NeoR-FRT cassette. This CreER knock-in allele was generated by homologous recombination of a bacterial artificial chromosome clone in embryonic stem cells. Embryonic stem cells were selected with neomycin for 12 days. Insertion of the donor sequence was verified by PCR with the KAPA Long Range PCR Kit (Roche; KK3502) according to the manufacturer’s guidelines. The PCR primers are detailed in Supplementary Table 5. The positive embryonic stem clone was injected into C57BL/6N blastocysts, followed by embryo transfer into RjOrl:SWISS recipient female mice. The neomycin resistance cassette was removed by crossing the founder mouse with the FLPe mouse strain.
Virus production
Lenti-X 293T cells (Takara Bio; 632180) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) GlutaMAX (Thermo Fisher Scientific; 31966021) with 10% fetal bovine serum(Thermo Fisher Scientific; A5256701) and 1% penicillin–streptomycin (Sigma; P4458-100ML) up to a confluency of 70%. The Lenti-X cells were transfected using polyethyleneimine (PolyScience; 23966-1) with plasmid of interest (Supplementary Table 7), pMD2.G and psPAX2 plasmids in a molar ratio of 1:1:1. Supernatant was changed 12–24 h after transfection. After centrifugation, the supernatant was removed, the lentiviral pellets were resuspended in ice-cold phosphate buffered saline (PBS) and aliquots were stored at −80 °C.
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