BxPC3 xenograft model (subcutaneous and metastatic)
Pancreatic ductal adenocarcinoma remains an incurable disease with a poor prognosis and life expectancy. Malignant pancreatic cancer is a significant cause of cancer-related mortality. The BxPC-3 tumorigenic epithelial cell line was initially derived from a 61-year-old female patient with pancreatic adenocarcinoma. Proliferator-activated receptor-γ (PPAR-γ) is a nuclear receptor that inhibits various cancer cell processes, including pancreatic cancer cell proliferation. A 2014 study by Ninomiya et al. published in Oncology Letters investigated the inhibitory effect of pioglitazone on the proliferation of the BxPC-3 pancreatic cancer cell line in vitro and in vivo. The article reported that pioglitazone induces CEA mRNA expression, suppresses IL 8 and COX 2 mRNA expression in vitro, and inhibits BxPC-3 xenograft growth. These findings indicate that pioglitazone treatment blocks the proliferation and metastasis of pancreatic cancer cells and could be useful for the patients in preventing pancreatic cancer metastases. A 2007 Molecular Cancer Therapeutics study by Yip-Schneider et al. used the BxPC3 model to investigate the antitumor effects of combination therapy with sulindac, a COX inhibitor, and LC-1, an analogue of the NF-κB inhibition-associated compound parthenolide. Results indicated the treatment had evidence for preclinical efficacy and that antitumor effects are mediated through cyclin D1 levels. A 2010 Molecular Cancer Therapy article (Yu et al.) used the BxPC-3 xenograft model to study the therapeutic efficacy of GLV-1h68, a replication-competent vaccinia virus. Results demonstrated that the virus was able to successfully and selectively infect tumor cells, replicate, and lyse the tumor cells. Combination therapy with cisplatin or gemcitabine also enhanced and accelerated antitumor effects; the mechanism is suggested to be related to a proinflammatory immune response. Finally, Lu et al. (2008) used the BxPC-3 cell model to characterize the mechanism of erlotinib, a small molecule EGFR tyrosine kinase inhibitor. Findings demonstrated that both in vitro and in vivo erlotinib treatment inhibited growth with increased apoptosis and decreased angiogenesis. The BxPC-3 cell line (human pancreas) is used to create the CDX (Cell Line Derived Xenograft) BxPC-3 xenograft mouse model. The BxPC-3 pancreatic tumor model is a well-established model to investigate gemcitabine or small molecule tyrosine kinase inhibitors (TKIs), such as erlotinib.
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Basic study design
- BxPC-3 cells used for injection are maintained at exponential growth up to injection.
- The BxPC-3 cells are trypsinized and then cell count and viability is determined using a trypan blue exclusion assay (98% viability required). Cell suspension concentration is adjusted to the appropriate density.
- Each mouse (NCr-nu/nu mice, 10 weeks old) receives a subcutaneous injection into the flank of one hind leg containing 1 million cells, in a volume of 100 uL of the matrigel & BxPC3 cell suspension.
- Injection sites are palpated multiple times weekly until tumor establishment. Tumors are measured with digital calipers to establish an average size of 50-150 mm3.
- Animals are then randomized into predetermined treatment cohorts the compound of interest is administered according to the treatment schedule.
- Tumors are measured daily and recorded, and mouse weights are recorded 3 times weekly.
- Animals are euthanized as tumor size reaches near 2,000 cu millimeters or the predetermined study size limit.
- Necropsy is performed as defined in the termination of experiment.
- Tumors excision, weight and an image is documented.
- Tissues are collected via standard necropsies for downstream analysis.
- All tumors and tissues can be frozen in LN2, prepared for histology or stabilized in RNAlater for gene expression analysis.
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).
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 BxPC-3 xenograft model:
- BxPC-3 Tumor Growth Delay (TGD; latency)
- BxPC-3 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)
- BxPC-3 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