| In the monolayer assay the anti-proliferative and cytotoxic activity of test compounds is determined in permanent tumor cell lines. It is mainly used as a rapid initial screen to select the most promising compounds based on potency and selectivity. | This includes drug combination studies with Combination Index (CI) determination according to Chou-Talalay. ONCOTEST has established more than 40 unique cell lines directly from patient-derived tumor xenografts. |
Propidium Iodide (PI) Assay:
Between 4,000 and 20,000 exponentially growing cells are seeded per well in 96-well microtiter plates. 24 hours later, test compounds are added by a liquid handling robotic system and treatment is continued for 4 days. After cell permeabilization, PI is used to quantify viable cells. Compounds can be tested in different concentrations and replicates according to customer needs. Growth inhibition/cytotoxicity is expressed by individual and mean IC50 and IC70values, calculated either by two-point curve fit or non-linear regression analysis based on the concentration-effect curve. Data summaries are presented by an IC50 and IC70 mean graph (similar to the NCI analysis), or by a respective heat map. Alternative cytotoxicity assays like the XTT or Alamar Blue assay are available for individual studies upon request.
Compare Analysis:
Profiling novel compounds in ONCOTEST's 42 cell line panel as described below allows subsequent Compare Analysis. The test compounds unique IC50/70 activity profile is compared with activity profiles of more than 100 anti-cancer agents with known mechanisms-of-action (MoA) including many approved cancer drugs. Similar activity profiles are indicative of a similar MoA. Reference drugs used for Compare Analysis include alkylating agents, anti-metabolites, broadspectrum and target- specific protein kinase inhibitors (e.g. HER family, ARK-A/B, PLKs, PKCs, VEGFR, PDGFR, CDKs), inhibitors of DNA, RNA or protein synthesis including mTOR inhibitors, inhibitors of class 1 and 2 histone deacetylases (HDAC), inhibitors of heat-shock protein 90 (HSP90), mitotic kinesins and tubulin interacting drugs, proteasome as well as topoisomerase I/II inhibitors.
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We offer testing in a large collection of individual cell lines and defined cancer cell line panels. Currently 5 different panels are available, representing all major tumor histologies including niche tumors or patients of different ethiology. All routinely used cell lines are qualified as authentic/unique by genomic fingerprinting (STR) analysis.
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Based on a low passage number and thus avoiding the selection of undifferentiated subclones, xenografts re-established from the cell line resemble the original patient-derived xenograft in histology and chemosensitivity. Most ONCOTEST cell lines show relatively slow in vitro growth with doubling times between 30 and 48 hours. As a consequence, compound treatment of up to 4 days is most suitable.
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| Immunohistological characterization of cytokeratin profiles (table on the left): Comparison of matched pairs of tumor xenografts reestablished from the respective cell lines and the corresponding patient-derived xenografts using tissue microarrays (TMAs) demonstrated nearly 100% identity for expression of selected cytokeratins (ck)5, 7, 13, hepatocyte-specific antigen (HSA), desmin, ttf 1, CEA and CD10.
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Chemosensitivity of Original vs. Cell Line-Derived Xenograft: Comparison of matched pairs of tumor xenografts reestablished from the respective cell lines and the corresponding patient-derived xenografts with regard to the anti tumor activity of 5 standard-of-care compounds.
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