4T1 xenograft model (subcutaneous and metastatic)
Breast cancer remains the second leading cause of cancer deaths in females worldwide. Although necessary improvements in breast cancer diagnosis and management have been made, early detection and anti-metastatic treatment of breast cancer remain a crucial challenge. Preclinical studies of the 4T1 metastatic mouse xenograft model can help overcome these limitations, which is critical for patients with advanced stage disease. The 4T1 tumorigenic epithelial cell line is derived from the mammary gland tumor of a mouse and is resistant to 6-thioguanine without mutagen treatment. According to a 1992 article in Cancer Research, when injected into BALB/c mice, the 4T1 cell line can spontaneously metastasize to both the lung and liver and form visible nodules in these organs. The 4T1 cell line is particularly useful for stage IV human breast cancer research since tumor from this cell line mimics human breast cancer and readily metastasizes through mice, making a suitable breast cancer model. Liu et al. (2014) used an orthotopic 4TI xenograft model to demonstrate inhibition of the Fas signaling cascade suppresses tumor metastasis and growth and reduces Fas-initiated inflammation in breast cancer, thereby establishing Fas signaling as a potential therapeutic target. A Cell study (Aceto et al., 2014) used a 4T1 xenograft model to define that circulating tumor cell clusters (CTC clusters) present in cancer patients’ blood originate from primary tumors and greatly contribute to metastatic spread of the cancer. Gao et al. (2011) used a murine 4T1 model to test breast cancer formation and metastasis after treatment with doxorubicin (DOX) delivered via pH-sensitive micelles. They found that a polymeric micelle formed with both poly(L-lactide) (PLLA) (Mn 3000)-b-poly(ethylene glycol) (PEG) (Mn 2000)-folate and poly(L-histidine) (PHis) (Mn 4700)-b-PEG (Mn 2000) loaded with DOX prevented metastasis and prevented tumor growth, thereby reporting a novel method for the safer delivery of a known chemotherapy agent. The 4T1 cell line is routinely used to create the CDX (Cell Line Derived Xenograft) 4T1 xenograft mouse model. Upon implantation, the production of colony stimulation factors, cytokines, chemokines and angiogenesis factors allows the 4T1 tumor cells to metastasize efficiently. The 4T1 metastatic model enables the study of late-stage breast cancer therapeutic agents.
Download Altogen Labs 4T1 Xenograft Model PowerPoint Presentation:
Basic study design
- Flasks of 4T1 cells are maintained under conditions of exponential growth prior to injection.
- 4T1 cells are prepared for injection by trypsinization and viable cell counts are determined using trypan blue exclusion (98% cell viability required). Cell suspension adjusted to appropriate density.
- Each mouse (NOD/SCID, 10-12 weeks old) receive a subcutaneous injection of one million cells in the flank of the hind leg in a volume of 100 microliters of the Matrigel 4T1 cell suspension.
- The injection sites are palpated three times weekly until tumors are established. Tumors are then measured using digital calipers until they reach an average size of 50-150 mm3.
- Animals are randomized into treatment cohorts and administration of the compound of interest is performed according to the treatment schedule.
- Tumors are measured daily and mouse weights are recorded 2-3 times weekly.
- Animals are euthanized when tumor size reaches 2,000 mm3 or the predetermined size limit per approved IACUC protocol.
- Necropsy and tissue collection are performed as defined for termination of experiment.
- Tumors are excised, weighed and documented by digital imaging.
- Standard gross necropsies are performed and tissues are collected for downstream analysis.
- Tumors and tissues can be snap frozen in LN2, stabilized in RNAlater reagent or prepared for histology.
CDX models are mouse xenografts used in pre-clinical therapeutic studies. However, as primary tumors proliferate they invade surrounding tissue, become circulatory, survive in circulation, implant in foreign parenchyma and proliferate in the distant tissue. This result leads to an extremely high percentage of death in cancer patients due to metastasis. Metastatic tumor mouse models are utilized to develop novel therapeutic agents that target metastasis (anti-metastatic therapeutics).
To create a metastatic model, the cell line of interest is transfected with vectors containing green fluorescent protein (GFP) or luciferase. Maintained under antibiotic selection, only cells containing the integrated vector will survive. The new cell line clones are capable of stably expressing the gene of interest and are used in metastatic mouse model studies. Although each new cell line clone may contain its own inherent difficulties, the new cell line contains the ability to track internal tumor progression via bioluminescence (luciferase fluorescence after injecting luciferin) or fluorescence (GFP). Internal orthotopic and metastatic tumor growth (not palpable) can now be measured throughout the study, enabling a researcher to gain more insight and additional data in contrast to relying on end of study tumor weight measurements.
Case Study: U87-luc Xenograft Model
An example of Altogen Labs contract research study using a luciferase expressing U87-luc cell line to monitor in vivo tumor growth. The same concept of tumor observation using in vivo imaging is incorporated in metastatic tumor models.
Luciferase expressing U87-luc cells were implanted and tumors allowed to grow. Tumor growth was monitored in a Night Owl (Berthold Technologies) imaging system 10 minutes after an intraperitoneal (IP) injection of the luciferin substrate. As seen in the example below, luciferase expression (measured as photons emitted) in the U87-luc model grants the researcher a visual image and quantifiable metric for orthotopic or metastatic tumor progression.
Figure 1. Luciferase expression in U87-luc orthotopic model. Control and implanted glioma mouse model luminescence was analyzed 10 minutes after intraperitoneal luciferin injection.
View full details of the case study by clicking here.
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).
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).
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 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. Our animal facilities have the flexibility to use specialized food or water systems for inducible gene expression systems.
Following options are available for 4T1 xenograft model:
- 4T1 Tumor Growth Delay (TGD; latency)
- 4T1 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)
- 4T1 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