Hs578T xenograft model
Breast cancer remains the second primary cause of cancer deaths in females as well as the most commonly diagnosed malignancy in women. Nearly 252,000 women annually in the United States are diagnosed with breast cancer with greater than 40,000 of the cases turning fatal, as per the National Breast Cancer Foundation. Well-characterized breast cancer cell lines have proven to be essential tools for investigating the biological characteristics of carcinomas. Preclinical studies of the Hs578T mouse xenograft model are invaluable in examining cellular response to therapeutic drugs and vital for patients with advanced stage disease. The Hs578T triple-negative breast cancer (TNBC) epithelial cell line is established from a carcinoma of the human breast of a 74-year-old Caucasian female patient. TNBC comprises nearly 10–15 percent of all breast malignancies and has a poor outcome compared to the other types of breast cancer, as per a 2010 article published in Breast Disease. Studies that use the Hs578T xenograft model include the 2016 Nature study by Kim et al. which identified an oncogenic signaling axis involving TrkC, which through downstream regulators promotes primary tumor growth, tumor self-renewal, metastasis, epithelial to mesenchymal transition (EMT) and autocrine-mediated maintenance of the mesenchymal state. Zhang et al. (2014) used the Hs578T mouse xenograft as a TNBC model to examine efficacy and resistance for inhibition of mTOR, which is commonly activated in TNBC patients. They tested two mTOR inhibitors, sirolimus (rapamycin) and temsirolimus (CCI-779), and concluded the need for combination therapy as the inhibitors were successful in limiting growth but not eradicating tumors. Finally, Chen et al. (2018) used the Hs548T xenograft model to verify the role of Contactin 1 (CNTN1) in breast cancer, as it had been implicated in invasion and metastasis in other cancer types. They concluded that CNTN1 promotes proliferation, cell cycle progression, colony formation, migration and invasion in vitro and enhances tumor growth in vivo, making CNTN1 a target for further mechanistic and therapeutic studies. The Hs578T cell line is used to create the CDX (Cell Line Derived Xenograft) Hs578T xenograft mouse model. The Hs578T xenograft model enables studies that focus on therapeutics targeting expression mechanisms that control aneuploidy (reduction of Mps1 gene) or degradation of the basement membrane and extracellular matrix that allow higher invasion (MMP-1 and MMP-3 genes).
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Basic study design
1. Prior to collection, all cells are maintained at exponential growth levels.
2. The cells are collected by trypsinizing in the flasks. Cell count is then determined and viability established by trypan blue exclusion (98% min viability). Suspensions are adjusted to density required for injection.
3. One million cells (Matrigel + Hs578T suspension), in a volume of 100 µL is injected s.c. into rear flank of each mouse (NOD/SCID or athymic BALB/C, 10 to 12 weeks).
4. Injection sites observed until tumors are established. Then, calipers are used to determine tumor average sizes (50-150 mm3).
5. Post sorting (randomization) into treatment groups the compounds (test articles) are administered according to the agreed upon treatment table.
6. Daily tumor measurements will be logged and mouse weights documented (up to 3 times a week).
7. As the study’s tumor size upper limit is reached (or 2,000 cubic millimeters), the animals are euthanized following established practices.
8. End of study necropsies are performed. Tumors are removed from the study animals and weights recorded. The tumor is then documented using digital imaging technology.
9. A list of predetermined tissues are collected by standard gross necropsy and either 1) snap frozen, 2) submersed in RNAlater, 3) nucleic acids isolated, or 4) prepared for histological analysis.
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.
Xenograft animal models are used to assess the effectiveness of drugs 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.
Following options are available for the Hs578T xenograft model:
- Hs578T Tumor Growth Delay (TGD; latency)
- Hs578T 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)
- Hs578T 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