T-47D Xenograft Model













T-47D xenograft model

Breast cancer is responsible for over 40,000 deaths annually in the United States, as per the American Cancer Society (ACS). Xenograft studies are based on direct implantation of tumor cell lines into various organs of immunodeficient mice. Well-characterized cancer cell lines are valuable preclinical models for breast cancer research as well as for the evaluation of the efficacy of a potential drug candidate. Cancer cell lines serve as potent tools for life sciences, aiding in drug development. The T-47D hypotriploid epithelial cell line is derived from ductal carcinoma of the breast of a 54-year-old female patient and is a suitable transfection host. The T-47D cell line has been routinely utilized in breast cancer research. Examples include the 2017 Nature study by Riggio et al. that examined the mechanisms behind AKT1 and AKT2 isoforms in promoting breast cancer progression; results showed in vitro AKT1 promoted proliferation and growth and inhibited cell migration while AKT2 promoted cell migration and invasion. This study highlighted the importance of differentiating between isoforms and concluded that AKT2 upregulation would make a reasonable target due to its link to metastasis which in turn contributes to poor prognosis. A 2016 study by Puchalapalli et al. used the T47D model for its estrogen receptor positive (ER+) phenotype in order to determine the optimal mouse model for studying spontaneous metastasis and concluded that NOD scid gamma (NSG) mice are a better model compared to nude mice as NSG mice demonstrate more severe tumor growth, metastases, and metastatic burden. Finally, a 2006 Cancer Research article (Hartman et al.) used the T47D xenograft model to test the proliferative effects of estrogen receptor β (ER-β), which has reported antiproliferative effects as opposed to ER-α which has estrogen-mediated proliferative effects on normal and tumor breast cells. Results demonstrated that the introduction of ER-β into the xenograft model reduced tumor growth, intramural blood vessels. VEGF expression, and platelet-derived growth factor β (PDGFβ), suggesting that the mechanism of action of growth inhibition is via angiogenesis inhibition.

The T-47D cell line is used to create the CDX (Cell Line Derived Xenograft) T-47D xenograft mouse model. The T-47D xenograft model is estrogen receptor positive and progesterone receptor positive and lends itself to anti-tumor studies taking into consideration receptor status (e.g. quinoline) and angiogenesis (e.g. ERβ replacement therapy).

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Basic study design

  1. T-47D cells are specifically maintained under exponential growth for in vivo studies.  The cells are collected via trypsinization and viability and cell counts are determined.  Concentration of the cell suspension is diluted to the working injection volume.
  2. Mice (NOD/SCID, 10-12 weeks) receive a s.c. injection in the hind leg that contains one million cells (Matrigel plus T-47D cells; vol = 100 µ).
  3. Injections are monitored until tumor establishment.  Digital calipers are used to measure tumors; the in-life part of the study begins as tumors reach an average size of 50-150 mm3, at which point the animals are randomized to form study groups.
  4. The dosing schedule is followed for administration of the compound of interest
  5. Tumor measurements occur daily, along with body weights being recorded 3 times a week.  At the end of study, necropsies are performed and samples are collected according to the tissue collection sheet.  All tumors are resected from the mice and weighed.  Tumor and tissue samples are frozen in liquid N2, prepared in 10% NBF for histology or isolated for genetic analysis.

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T-47D Xenograft Model

Animal handling and maintenance at the Altogen Labs facility is IACUC-regulated and GLP-compliant. Following acclimation 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.

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 T-47D xenograft model:

  • T-47D Tumor Growth Delay (TGD; latency)
  • T-47D 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)
  • T-47D 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

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T-47D Xenograft Model