RKO Xenograft Model

RKO xenograft model

Colorectal cancer is the second primary cause of cancer-related deaths in men, accounting for nearly 50,000 fatalities annually in the United States, as per the American Cancer Society (ACS).  Xenograft models aid in understanding the cellular and molecular mechanisms, contributing to the development of novel anti-cancer drugs. The RKO cell line was isolated form adhesive epithelial cells of a male patient with colorectal carcinoma. RKO is a poorly differentiated colon carcinoma cell line established by M.Brattain. The RKO cell line contains wild-type p53 but lacks h-TRβ. The RKO cell line is tumorigenic in nude mice and expresses urokinase receptor (u-PAR). According to a 2015 study in World Journal of Gastroenterology (He et al.), RKO cell line is utilized as the model for studying NOB1 gene because of its relatively short doubling time as well as an established genetic profile. Results demonstrated that NOB1 silencing via small RNA interference (lenti-virus mediated) inhibits colorectal cancer tumor growth by altering pathways including BAX and WNT, which affect apoptosis, cell proliferation and angiogenesis. A 2011 study by Yang et al. (Cancer Research) used the RKO model to examine the antitumor activity of Vemurafenib, a BRAF inhibitor, in colorectal cancer models with BRAF mutations. BRAF is part of the RAS-RAF pathway which is involved in differentiation, survival and cell proliferation. RKO was chosen for its de novo vemurafenib resistance. Data demonstrated that combination of vemurafenib with cetuximab and/or irinotecan, capecitabin and/or bevacizumab, or erlotib resulted in increased survival and antitumor activity. Lastly, Dang et al. published a study (Cancer Research, 2006) using RKO cells and xenograft model to examine the role of HIF-1α in nonhypoxia-mediated tumor cell proliferation. Results demonstrated that HIF-1α does promote in vitro and in vivo nonhypoxia-mediated proliferation but only contributes to hypoxic survival in vitro. Data also showed that VEGF disruption resulted in growth delay and expansion of hypoxic compartments while HIF-1α did not similarly affect the expanded compartments. The RKO cell line is used to create the CDX (Cell Line Derived Xenograft) RKO xenograft mouse model. The RKO xenograft model enables the in vivo efficacy of immunotherapies (e.g. pembrolizumab, ipilimumab) and other targeted therapies (e.g. NOB1 inhibition).

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Basic study design

  1. Each athymic BALB/c (nu/nu) mouse (10 weeks old) receives a single s.c. injection into the hind leg.  The cell inoculation contains 1 x 106 cells (in 100 µL injection volume) of the Matrigel+RKO cell suspension.
  2. The mice are observed until tumors are established.  Tumors are expected to reach 50-150 mmbefore dosing begins.  Animals are randomized into treatment groups.
  3. Tumors (daily) and body weights (tri-weekly) are recorded.
  4. The study ends as tumors reach maximum tumor size limits (2,000 cu. millimeters).  Tumors are resected from the animal and weighed.  A standard necropsy is performed; tissues are collected following the customer’s request.
  5. Tumors and/or tissues are frozen in niquid nitrogen, prepared for histology in 10% NBF or stabilized in RNAlater reagent.
  6. Animals are housed in a pathogen-free facility following the Guide for Care and Use of Laboratory Animals, along with regulations of the Institutional Animal Care and Use Committee (IACUC).

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RKO 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 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 RKO xenograft model:

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