Experimental methods summary (3): animal models
1. Tumor cell proliferation
2. Tumor cell metastasis
2.1. In vitro invasion models
2.2. In vivo metastasis models
3. Tumor cell drug resistance
3.1. Establishing a resistant cell line
3.2. Establishing a resistant nude-mouse xenograft model
The examples below address proliferation, metastasis and drug resistance.
Cell preparation: Expand cells infected with GeneA knockdown lentivirus to the required number. Groups: blank control, negative control and experimental group.
Use male BALB/c nude mice, six weeks old, 10 per group, and acclimate for one week before tumor-cell injection. Digest and centrifuge XXX cells, prepare a single-cell suspension, count and suspend an appropriate number in PBS, then inoculate subcutaneously in the flank. Inoculate 2×106 cells in 100 μL per mouse. Measure tumor volume every five days. After 30 days, administer pentobarbital sodium intraperitoneally at 80 mg/kg; photograph anesthetized mice laterally against a blue background with the inoculation site uppermost. Euthanize by cervical dislocation, remove and weigh the tumor, and photograph it against a blue background. Divide it: fix one part in 4% paraformaldehyde for pathology and store the other at −80°C.
Tumor metastasis models include in vitro invasion models, used to study invasion of adjacent tissues, and in vivo metastasis models, used to study formation of distant lesions.
Example: establishment of an invasive glioma animal model.
Method: Use immunodeficient BALB/c nude mice, 10 per group, and stereotactically inoculate isolated, characterized GFP-transfected glioma stem cells intracranially. After anesthesia and a midline scalp incision, remove periosteum and drill at the coordinates stated in the source: 0.5 cm posterior to the coronal suture and 2.5 cm right of the sagittal suture. Slowly inject 2 μL containing 1×104 cells per mouse into the striatum at 2.5–3 mm depth. At selected time points, euthanize some animals for fluorescence and pathological comparison, and assess in vivo growth and stem-cell markers. The source coordinates are reproduced as written and require verification before use.
Example: a breast-cancer xenograft model established by subcutaneous inoculation in the scapular region to examine effects of Zhuanggu Zhentong capsules on integrin αvβ3 (ITG-αvβ3) and bone sialoprotein (BSP), markers associated with bone metastasis.
Method: Digest logarithmically growing breast cancer cells MDA-MB-231 with 0.25% trypsin, collect a single-cell suspension, count with a hemocytometer and adjust to 2×107 cells/mL. Inoculate 0.2 mL per nude mouse subcutaneously above the right forelimb scapula. A soft raised area appears, largely resolves after two days, and a firm rice-sized nodule is palpable after 10 days. Assess the effect of Zhuanggu Zhentong capsules on IGT-αvβ3 and BSP expression in xenograft tissue. The source uses both ITG and IGT spellings; verify the intended identifier.
Example: orthotopic transplantation to establish a nude-mouse metastasis model of human clear-cell renal carcinoma and observe tumor pathology.
Method: Inject logarithmically growing 786-0 cells (human clear-cell renal carcinoma) subcutaneously into nude mice. At approximately 1.5 cm tumor diameter, remove and passage between mice by the same method (two mice per passage). After 3–4 passages, cut the xenograft into 1 mm3 pieces and implant under the renal capsule to establish a surgical orthotopic implantation (SOI) metastasis model. When mice are moribund, euthanize by cervical dislocation, culture lung metastases and passage with trypsin 4–5 times to obtain a relatively pure population named 786-0-LM1. In parallel, inject 786-0 cell suspension under the renal capsule to establish an orthotopic cell-injection model.
This example induces resistance using paclitaxel in human esophageal squamous carcinoma EC109 cells, combining intermittent high-dose exposure with increasing time. Expose logarithmically growing EC109 cells for two hours to 0.625 μg/mL paclitaxel (stated as 12.5 times its IC50 for EC109). Discard the drug-containing medium, wash three times with PBS at 4°C, treat with 0.25% trypsin and centrifuge at 800 r/min for 4 min. Resuspend and reseed. After 24 hours, remove dying cells, expand surviving cells and, once 70–80% confluent and growing well, passage three times. Repeat twice for three total drug exposures of EC109 to obtain EC109/Taxol. Use MTT to compare sensitivity of parental and resistant cells to cytotoxic compounds.
Collect parental EC109 and resistant EC109/Taxol cells under sterile conditions, dilute to 5×107 cells/mL in sterile saline, and inoculate EC109 and EC109/Taxol separately subcutaneously in the right axilla of nude mice at 1×107 cells per mouse. When tumors reach 1 cm diameter, record time to tumor formation and the tumor formation rate for both cell types.
Lifeint provides EV research and application services from sample preparation, isolation and characterization to omics, functional studies, modification and scale-up. It jointly established a biomedical technology centre with Xiamen University School of Pharmaceutical Sciences, built process-development laboratories to GMP standards, and contributed to an EV expert consensus. Its service records cover more than 300 plant species.
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