2.9. In Vivo Cell Derived Xenograft Tumour Model

Female BALB/c nude mice (5 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). MDA-MB-231 cells (1 × 10⁷) in 100 μL of PBS were injected into the back fat pad of 12 BALB/c nude mice. The tumour size was measured with callipers. The tumour volume was calculated as 0.5 × (longest measurement) × (shortest measurement)². When the tumour diameter reached 2–3 cm, the tumours were harvested and placed in a sterile environment. The tumours were then cut into 1.5 mm × 1.5 mm uniform tumour blocks and inoculated separately into the backs of another 50 mice. After 1–2 weeks of inoculation, when the tumour volume reached 100–200 mm³, the tumour-bearing nude mice were randomly divided into groups according to the tumour volume. The experimental animals were divided into five groups with 10 mice in each group: glucose plus sorbitol aqueous solution as the blank control group; paclitaxel plus gemcitabine as an active control [48]; and gefitinib and irinotecan alone and combined as the experimental groups. The grouping and drug administration information are shown in Table 2. The doses of paclitaxel and gemcitabine were converted with reference to the actual usage in ClinicalTrials.gov (ClinicalTrials.gov Identifier: {"type":"clinical-trial","attrs":{"text":"NCT01287624","term_id":"NCT01287624"}}NCT01287624). The doses of gefitinib and irinotecan were based on the results of a preliminary dose tolerance experiment involving the administration of each drug alone. Tumour size and body weight were measured every other day for 14 days from the start of the drug treatment cycle. On day 14, the tumour size and mouse body weight were measured. Subsequently, the mice were sacrificed, and the subcutaneous tumours were harvested. The tumours were weighed and photographed. Three specimens were randomly selected from each group for haematoxylin-eosin (H&E, Procell, Wuhan, Hubei, China) staining. The Servicebio Institutional Animal Use Committee (IACUC) approved all of the experimental protocols (animal ethics permit NO. 2018006, approval date: 13 September 2018).

Grouping of xenotransplantation experiments and drug treatment information.

^a intraperitoneal injection; ^b gavage.

For histological analyses, tumour tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin sections were stained with H&E for pathological analysis. Three tumour samples were taken from each group for staining.