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. 2011 Feb;56(2):239-49.
doi: 10.1002/pbc.22801. Epub 2010 Oct 4.

National Cancer Institute Pediatric Preclinical Testing Program: Model Description for in Vitro Cytotoxicity Testing

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Free PMC article

National Cancer Institute Pediatric Preclinical Testing Program: Model Description for in Vitro Cytotoxicity Testing

Min H Kang et al. Pediatr Blood Cancer. .
Free PMC article

Abstract

Background: The National Cancer Institute (NCI) has established the Pediatric Preclinical Testing Program (PPTP) for testing drugs against in vitro and in vivo childhood cancer models to aid in the prioritization of drugs considered for early phase pediatric clinical trials.

Procedures: In vitro cytotoxicity testing employs a semi-automated fluorescence-based digital imaging cytotoxicity assay (DIMSCAN) that has a 4-log dynamic range of detection. Curve fitting of the fractional survival data of the cell lines in response to various concentrations of the agents was used to calculate relative IC(50) , absolute IC(50) , and Y(min) values. The panel of 23 pediatric cancer cell lines included leukemia (n = 6), lymphoma (n = 2), rhabdomyosarcoma (n = 4), brain tumors (n = 3), Ewing family of tumors (EFT, n = 4), and neuroblastoma (n = 4). The doubling times obtained using DIMSCAN were incorporated into data analyses to estimate the relationship between input cell numbers and final cell number.

Results: We report in vitro activity data for three drugs (vincristine, melphalan, and etoposide) that are commonly used for pediatric cancer and for the mTOR inhibitor rapamycin, an agent that is currently under preclinical investigation for cancer. To date, the PPTP has completed in vitro testing of 39 investigational and approved agents for single drug activity and two investigational agents in combination with various "standard" chemotherapy drugs.

Conclusions: This robust in vitro cytotoxicity testing system for pediatric cancers will enable comparisons to response data for novel agents obtained from xenograft studies and from clinical trials.

Conflict of interest statement

Conflict of Interest Statement

C Patrick Reynolds, MD PhD is a co-inventor on patents related to the DIMSCAN system that have been licensed by Children’s Hospital Los Angeles to BioImaging, Solutions Inc.

Figures

Fig. 1
Fig. 1
Representative graphs from Kaleidagraph software for a) rapamycin dose-response in Rh-41 showing the differences of absolute IC50 vs relative IC50, and b) melphalan dose-response in TC-71. Relative IC50 is determined at the concentration required to induce 50% effect between the baseline and maximum (black solid line and arrow). Absolute IC50 is the concentration that is inhibitory for 50% of cells (red dotted line and arrow). Dose (in a log scale) is presented on the x axis. Instead of zero for a control concentration, we used 0.001 nM to enable graphing the zero dose values on this scale. The figures illustrate the typical plateau effect of an anti-mitotic agent (a), for which the absolute IC50 and relative IC50 values are different, and a cytotoxic agent (b), for which the absolute IC50 and relative IC50 values are similar. In vitro cytotoxicity terms are in accordance with NIH Chemical Genomics Center terminology (http://www.ncgc.nih.gov/guidance/section11.html).
Fig. 2
Fig. 2
Dose-response curves to vincristine (0.003 – 10 nM, a), melphalan (0.01 – 100 μM, b), etoposide (0.01 – 100 μM, c), and rapamycin (0.01 – 100 nM, d) obtained by DIMSCAN analysis of the PPTP in vitro panel. The two plots for CCRF-CEM represent the independent testing of this cell line by two different technicians (● technician #1, ○ technician #2) that are used for quality control of the data.
Fig. 3
Fig. 3
Relative sensitivity to vincristine, melphalan, etoposide, and rapamycin is represented as a ratio of the median relative IC50 of the panel to the individual relative IC50 for each line, with <1.00 (left) denoting resistance and >1.00 (right) denoting sensitivity relative to the rest of the panel. This method of defining sensitivity is similar to the “mean graph” used by the NCI in other studies [46], and was employed here as part of our effort to determine the best method of evaluating and calibrating the PPTP in vitro models.
Fig. 4
Fig. 4
Relative I/O of each cell line in response to vincristine, melphalan, etoposide, and rapamycin. Values close to 0 are consistent with cytostasis, values close to 1 indicate no treatment effect, and values close to -1 indicate primarily cytotoxicity. In using results obtained for the Relative I/O metric, we categorize results, for example, a value of < −0.8 would indicate a strong cytotoxic effect, while a value of > 0.8 would indicate no treatment effect. Values between −0.2 and +0.2 likely indicate a primarily cytostatic effect. Other values could be assigned to intermediate categories (for example, modest cytostasis for 0.2 to 0.8 or moderate cytotoxicity for −0.2 to −0.8).

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