Balb c nu nu female mice

All the animal experiments were approved by the Institutional Animal Care and Use Committee of Okayama University and conducted in compliance with a protocol reviewed by the committee. BALB/c nu/nu female mice were purchased from CLEA Japan Inc. (Tokyo, Japan) at 4 weeks of age. Twelve mice, four mice per group, were inoculated subcutaneously with NMFH-1 cells at a concentration of 5 × 10⁶ cells in 100 μl of RPMI on the right buttock under general anesthesia performed with 2% isoflurane. Four control mice received 100 μl RPMI by the same route. Tumor sizes were measured after one week of injection and then measured once a week. Blood samples were obtained by cardiac puncture at three, five, and seven weeks after injection under general anesthesia. The animals were sacrificed after blood sampling. The blood samples were stored at 4 °C in CAPIJECT micro collection tubes (TERUMO, Tokyo, Japan), and blood processing was done within 2 h of sampling as previously described for patient serum samples.

BALB/c-nu/nu female mice (aged 5–6 weeks; CLEA Japan, Tokyo, Japan) were maintained under pathogen-free conditions. For the cell line tumor model, mice were inoculated subcutaneously with HER3/RH7777 (1 × 10⁶) and RH7777 (1 × 10⁶) cells in the left and right thighs, respectively, under isoflurane anesthesia. The mice were used for biodistribution and PET study at about 2 weeks after inoculation, when the tumors were 5–10 mm in diameter. For CTOS tumor model, approximately 1000 CTOS C45s of diameter 40–100 μm were suspended in 50 μL of Matrigel Matrix GFR (BD Biosciences) and transplanted subcutaneously into the flanks of NOD/SCID mice (NOD.CB17-Prkdc SCID /J, Charles River Japan, Yokohama, Japan). The CTOS tumor-bearing mice were used for further experiments once the tumor diameter reached 10–15 mm, which occurred 3–6 weeks after transplantation. General conditions of the mice were monitored at least twice a week.

Organoid-derived tumor tissues induced by DMBA treatment after subcutaneous injection into BALB/c-nu/nu female mice (5 weeks of age; CLEA Japan, Inc., Tokyo, Japan) were previously harvested and cryopreserved (Supplementary Figure S1A) (Naruse et al., 2020 (link)). In the present study, the tumor tissue samples were used for whole genome sequencing (WES).