B16 xenograft model (subcutaneous and metastatic)
Metastatic melanoma is the deadliest form of skin cancer. The five-year survival rate for patients with advanced stage disease is 18 percent, per the American Academy of Dermatology (AAD). Preclinical xenograft animal models can provide insight into in vivo treatment responses in humans as well as help finding new therapeutics for metastatic melanoma. The B16 cell line was isolated from the skin cells of a mouse (Mus musculus) with melanoma. B16 is an excellent host for skin cancer studies as well as for experimental cellular and molecular biology. Targeting the Src/STAT3 pathway is viewed as a novel approach for melanoma management. A 2014 study by Fu et al. published in Experimental Dermatology investigated the involvement of STAT3 signaling in the anti-melanoma action of atractylenolide II (AT-II), using the B16 xenograft model. According to the article, AT-II significantly inhibits tumor growth in the B16 xenograft mouse model and blocks the activation/phosphorylation of STAT3 and Src in the xenografts. An Annals of Oncology article by Sanmamed et al. (2016) reviewed melanoma models, including the B16 model, that are appropriate for studying immunotherapy and checkpoint blockers in murine xenografts. They highlighted the importance of replicating the human tumor microenvironment, especially in regards to a functioning human immune system. Similarly, Kuzu et al. (2015) published a Cancer Growth Metastasis study reviewing patient-derived tumor xenografts (PDTXs), genetically engineered mouse models (GEMMs), and syngeneic models in melanoma research. They reviewed B16 cells in context of syngeneic models; B16 is commonly used because they form tumors after chemical induction and have an array of subclones varying in invasion, proliferation and metastasis. Lastly, Yoshiura et al. (2009) used the B16 model to study the anticancer effects of vaccination with Tpit/E cells, whole endothelial cells that are predicted to function as type of immunotherapy. Results demonstrated that vaccination of B16 xenografts with Tpit/E cells resulted in inhibition of subcutaneous tumor growth as well as appearance of lung metastases. The B16 cell line (mouse melanoma) is used to create the CDX (Cell Line Derived Xenograft) B16 xenograft mouse model. The B16 melanoma model is widely used to screen anti-proliferation therapeutics (e.g. simvastin, paclitaxel).
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Basic study design
- Cells used for injection are maintained at exponential phase growth prior to inoculation.
- Upon start of the study, the B16 cells are collected by trypsinization and the cell count viability is determined using a trypan blue exclusion assay (required: 98% cell viability). The cell suspension is adjusted to the required density for injection.
- All mice (severe combined immunodeficient SCID mice, 8-10 w.o.) receive a single subcutaneous injection in the hind leg. Each 100 µL injection contains one million cells of the B16 Matrigel cell suspension.
- Injection sites are palpated three times a week until it is determined tumors are established. Tumors are measured with digital calipers until they reach an ideal average size of 50-150 mm3.
- Animal randomization into the treatment cohorts and administration of the compound of interest is performed following the treatment schedule.
- Daily tumor measurements and mouse weights (3 times weekly) are recorded.
- Animals are euthanized when tumor size reaches the predetermined size limit or 2,000 cu millimeters.
- Necropsy is performed as defined for termination of experiment.
- All tumors are excised, weighed and then documented via digital imaging.
- Standard gross necropsies are performed and tissues collected for downstream analysis.
- Tumors and tissues are stabilized in RNAlater, snap frozen in LN2, prepared for histology or nucleic acid isolated for expression analysis.
- Animals were housed in a pathogen-free animal facility in accordance with the Guide for Care and Use of Laboratory Animals and the regulations of the Institutional Animal Care and Use Committee (IACUC).
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 utilizing a luciferase expressing cell line to monitor orthotopic tumor growth is exhibited below. The same ideology of tumor observation 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 fluorescence was analyzed 10 minutes after intraperitoneal luciferin injection.
View full details of the case study by clicking here.
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.
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 B16 xenograft model:
- B16 Tumor Growth Delay (TGD; latency)
- B16 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)
- B16 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