HL-60 xenograft model
Leukemia is cancer of the blood-forming tissues that affects the blood and bone marrow. Acute myeloid leukemia (AML) comprises nearly 90 percent of all adult acute leukemias. Despite significant improvements in leukemia management and diagnosis, new treatment options are required. The tumorigenic HL-60 cell line was isolated from the peripheral blood tissue of a 36-year-old female patient with acute promyelocytic leukemia (APL) in 1977. The HL-60 cell line has proven to be a potent model for studies of human myeloid cell differentiation and differentiation in general. A 2015 article published in Environmental Toxicology (Chen et al.) investigates the cytotoxic effects of irinotecan HCl (CPT-11) in HL-60 cells in vitro and tumor development in the HL-60 leukemia xenograft model in vivo. The article demonstrates that CPT-11 inhibits tumor growth in both in vivo and in vitro leukemia models and could be a promising agent for the treatment of APL. A 1997 Clinical Cancer Research article by Perentesis et al. used the HL-60 model to show that the genetically engineered recombinant diphtheria toxin human granulocyte macrophage colony-stimulating factor chimeric fusion protein (DTetGMCSF) showed anti-leukemia activity with limited toxicity; today GM-CSF is known as Leukine™ (sargramostim) and is used to complement leukemia therapy. Lin et al. (2015) released a study outlining how rutin, a citrus derived glycoside, inhibits tumor growth in a HL-60 xenograft model which provides grounds for its further study. The HL-60 cell line (human leukemia) is used to create the CDX (Cell Line Derived Xenograft) HL-60 xenograft mouse model. The HL-60 xenograft model is a relevant preclinical model enabling screening studies of acute myeloid leukemia (AML) chemotherapeutics and anticancer prodrugs such as irinotecan.
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Basic study design
1. Cell growth is maintained in exponential phase before collection.
2. HL-60 cells are collected for inoculation, cell count is determined and viability is checked with a trypan blue test (min 98% viability). Cell suspensions are adjusted to the required density for animal inoculation.
3. One million cells is inoculated subcutaneously (s.c) into the hind leg of each mouse (NOD/SCID or athymic BALB/C, 10-12 weeks). The volume of the injection is 100 µL (Matrigel+HL-60).
4. The injected cells are observed until palpated tumors are observed. Calipers (digital) are used for tumor measurements; average sizes of 50-150 mm3.
5. The test compounds are administered according to an agreed upon treatment schedule after randomization of the animals into appropriate groups.
6. Daily measurements (tumor) are recorded and mouse weights documented at least 3 times weekly.
7. As the end tumor size limit is reached (or 2,000 mm3), animals are euthanized.
8. A necropsy is performed as requested for the end of the study.
9. Tumors are removed from the mice, weights are recorded, and tumors are documented (digital imaging).
10. Tissues from a predetermined list are collected (snap frozen or submersed in RNAlater, isolation of nucleic acids is performed or the tissue is prepared for histological analysis) by performing a standard gross necropsy.
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 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 theHL-60 xenograft model:
- HL-60 Tumor Growth Delay (TGD; latency)
- HL-60 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)
- HL-60 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