A375 Xenograft Model

A375 Xenograft Altogen Labs

A375 xenograft model (subcutaneous and metastatic)

The A375 cell line (human melanoma) is used to create the CDX (Cell Line Derived Xenograft) A375 xenograft mouse model.

Basic study design

  1. A375 cells are maintained in the exponential growth phase prior to injection.
  2. A375 cells are then prepared for injection by trypsinization.  The percentage of viable cells are determined using the trypan blue exclusion protocol (98% cell viability required). The appropriate density of the cell suspension is adjusted.
  3. Each mouse (NOD/SCID, 10-12 weeks old) receive subcutaneous injections in the hind leg flank .  Each injection contains one million cells of the matrigel A375 cell suspension in a volume of 100 microliters.
  4. Three time weekly, the injection sites are palpated until tumors are established. Digital calipers are used to measure tumor growth until an average size of 50-150 mm3 is reached.
  5. Animals are then randomized into the necessary number of treatment cohorts.  Administration of the test compound is performed according to the treatment schedule.
  6. Tumors are measured per specified days and mouse weights are recorded 3 times weekly.
  7. When tumor size reaches the pre-determined size limit or 2,000 cu millimeters, the animals are euthanized.
  8. Tumors are excised from the animal, weighed and then documented by digital imaging.
  9. Gross necropsies are performed and specified tissues are collected for downstream analysis.
  10. The collected tumors and tissues can stabilized in RNAlater, snap frozen in LN2, prepared for histology or RNA isolated for gene expression analysis.

Metastatic Model

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.

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A375 Xenograft Model

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 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 the A375 xenograft model:

  • A375 Tumor Growth Delay (TGD; latency)
  • A375 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)
  • A375 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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A375 Xenograft Model