Calu-6 xenograft model
Lung cancer is the leading cause of cancer-related death worldwide among both women and men. It affects smokers 15 to 30 times more often than people who do not smoke and is the most preventable cancer, per the American Cancer Society. Xenograft animal models are useful for cancer research, aiding scientists in finding novel therapies for patients with lung malignancies. The Calu-6 epithelial cell line is isolated from lung tissue of a 61-year-old Caucasian female with anaplastic carcinoma and is a suitable host for lung cancer research. Also, Calu-6 cells are instrumental in the development of safer and more efficient inhaled therapeutics. A 2016 study published in Oncotarget, demonstrates that an increase in the levels of G protein-coupled receptor (GPR171) is vital for lung cancer tumor progression both in vitro and in vivo. The study indicates that anti-GPR171 antibody treatment inhibits proliferation of lung carcinoma cells in the Calu-6 xenograft model. Therefore, GPR171 is a promising target for the development of antineoplastic drugs. The Calu-6 cell line (human lung) is used to create the CDX (Cell Line Derived Xenograft) Calu-6 xenograft mouse model. The Calu-6 tumor model is an established xenograft model used to study the anti-tumor activity of therapeutics, such as bevacizumab, docetaxel, cisplatin and motesanib.
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Basic study design
1. Calu-6 cells used for injection are maintained under conditions of exponential growth prior to injection
2. Calu-6 cells are prepared for injection using trypsinization and counting viable cells using a trypan blue exclusion test (98% cell viability required). Cell suspension is adjusted to appropriate density.
3. Each mouse (athymic BALB/C (nu/nu), 12 w.o.) receives a single subcutaneous injection in a single hind leg. Each injection contains one million cells in a volume of 100 uL of the matrigel/Calu-6 cell suspension.
4. Injection sites are palpated three times weekly until tumors are established. Tumors are measured via digital calipers until they reach an average size of 50-150 mm3.
5. Animals are randomized into predetermined treatment cohorts and the administration of the compound of interest is performed according to the treatment schedule.
6. Tumors are then measured daily and the mouse weights are recorded up to 3 times a week.
7. Animals are then euthanized when tumor size reaches the predetermined size limit (or approximately 2,000 cu millimeters).
8. A necropsy is performed as defined in the agreed upon termination of experiment.
9. Tumors are excised from the mice, weighed and then documented by digital imaging.
10. Tissues are collected by a standard gross necropsy for downstream analysis.
11. Tumors/tissues can be immersed in RNAlater for stabilization, snap frozen in LN2 or prepared for histology.
Xenograft animal models are used to assess the effectiveness of experimental test compounds against specific types of cancer. New medicines are tested on staged tumor growths that have been engrafted via subcutaneous or orthotopic inoculation in an immunocompromised mouse or rat model. All clinically approved anti-cancer agents have been evaluated with conventional preclinical in vivo models. Xenograft studies can be highly complex, starting with the selection of the appropriate animal model, choice of tumorigenic cell line, administration method, dosing, analysis of tumor growth rates and tumor analysis (histology, mRNA and protein expression levels).
Altogen Labs provides an array of laboratory services using over 30 standard Cell Line Derived Xenograft (CDX) models and over 20 PDX models. Researchers investigating the role of specific proteins or gene products in regulating tumor growth can benefit from development of protein overexpression (genetically engineered to ectopically express proteins, tumor suppressors, or oncogenes) and RNAi cell lines with long term gene silencing. Altogen Labs provides quantitative gene expression analysis of mRNA expression (RT-PCR) and protein expression analysis using the WES system (ProteinSimple).
The dosing of the experimental compound of interest is initiated, for a staged study, when the mean tumor size reaches a specified volume (typically 50-100 mm3). In an unstaged study, the dosing of the compound of interest is initiated immediately after xenografting. Mice are dosed once or twice a day for 28 days (or other desired study duration) via the chosen route of administration. Tumor volume (mm3) is calculated via the “(W x W x L) / 2” formula, where W is tumor width and L is tumor length.
Animal handling and maintenance at the Altogen Labs facility is IACUC-regulated and GLP-compliant. Following acclimatization to the vivarium environment, mice are sorted according to body mass. The animals are examined daily for tumor appearance and clinical signs. We provide detailed experimental procedures, health reports and data (all-inclusive report is provided to the client that includes methods, results, discussion and raw data along with statistical analysis). Additional services available include collection of tissue, histology, isolation of total protein or RNA and analysis of gene expression. Our animal facilities have the flexibility to use specialized food or water systems for inducible gene expression systems.
Following options are available for the Calu-6 xenograft model:
- Calu-6 Tumor Growth Delay (TGD; latency)
- Calu-6 Tumor Growth Inhibition (TGI)
- Dosing frequency and duration of dose administration
- Dosing route (intravenous, intratracheal, continuous infusion, intraperitoneal, intratumoral, oral gavage, topical, intramuscular, subcutaneous, intranasal, using cutting-edge micro-injection techniques and pump-controlled IV injection)
- Calu-6 tumor immunohistochemistry
- Alternative cell engraftment sites (orthotopic transplantation, tail vein injection and left ventricular injection for metastasis studies, injection into the mammary fat pad, intraperitoneal injection)
- Blood chemistry analysis
- Toxicity and survival (optional: performing a broad health observation program)
- Gross necropsies and histopathology
- Positive control group employing cyclophosphamide, at a dosage of 50 mg/kg administered by intramuscular injection to the control group daily for the study duration
- Lipid distribution and metabolic assays
- Imaging studies: Fluorescence-based whole body imaging, MRI