J Clin Neurol.  2015 Apr;11(2):172-177. 10.3988/jcn.2015.11.2.172.

Preclinical Assessment of the Anticancer Drug Response of Plexiform Neurofibroma Tissue Using Primary Cultures

• Jiang W
• Mautner VF
• Friedrich
• Kluwe L

• KMID: 2242662
• DOI: http://doi.org/10.3988/jcn.2015.11.2.172

Abstract

BACKGROUND AND PURPOSE
Individualized drug testing for tumors using a strategy analogous to antibiotic tests for infectious diseases would be highly desirable for personalized and individualized cancer care.
METHODS
Primary cultures containing tumor and nontumor stromal cells were utilized in a novel strategy to test drug responses with respect to both efficacy and specificity. The strategy tested in this pilot study was implemented using four primary cultures derived from plexiform neurofibromas. Responses to two cytotoxic drugs (nilotinib and imatinib) were measured by following dose-dependent changes in the proportions of tumor and nontumor cells, determined by staining them with cell-type-specific antibodies. The viability of the cultured cells and the cytotoxic effect of the drugs were also measured using proliferation and cytotoxicity assays.
RESULTS
The total number of cells decreased after the drug treatment, in accordance with the observed reduction in proliferation and increased cytotoxic effect upon incubation with the two anticancer drugs. The proportions of Schwann cells and fibroblasts changed dose-dependently, although the patterns of change varied between the tumor samples (from different sources) and between the two drugs. The highly variable in vitro drug responses probably reflect the large variations in the responses of tumors to therapies between individual patients in vivo.
CONCLUSIONS
These preliminary results suggest that the concept of assessing in vitro drug responses using primary cultures is feasible, but demands the extensive further development of an application for preclinical drug selection and drug discovery.

Keyword

personalized medicine; drug selection; preclinical test; specificity; primary culture; in vitro testing

MeSH Terms

Antibodies
Cells, Cultured
Communicable Diseases
Drug Discovery
Fibroblasts
Humans
Precision Medicine
Neurofibroma, Plexiform*
Pilot Projects
Schwann Cells
Sensitivity and Specificity
Stromal Cells
Antibodies

Figure

• 

  Fig. 1 Immunostaining with antibodies raised against S100 (green in A, C, and G) for Schwann cells and CD90 (red in B, D, and G) for fibroblasts. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; blue). A and B: Plexiform neurofibroma (PNF)-derived Schwann cells and fibroblasts without drug treatment stained with S100 and CD90, respectively. C and D: PNF-derived Schwann cells and fibroblasts treated with 20 µM nilotinib for 5 days and stained with S100 and CD90, respectively. E and F: Immunofluorescence pictures before and after calculation of cell numbers using ImageJ software. G: Superimposed S100, CD90, and DAPI staining, revealing cells that are immunonegative for both S100 and CD90.

• 

  Fig. 2 Proportions of tumor cells (solid line) and nontumor cells (broken line) in cultures derived from four unrelated plexiform neurofibroma tumors after treatment with two anticancer drugs (nilotinib: A, C, E, and G; and imatinib: B, D, F, and H) at various concentrations. IC50 (concentration at 50% of maximum viability) and CC50 (concentration at 50% maximum cytotoxicity) were determined for all cells in separate cultures derived from the corresponding tumors.

• 

  Fig. 3 Illustration of the concept of a genetic preclinical drug test. A resected tumor will be subjected to cell culture and genetic screening for alterations. The cultured cells will be treated with various drugs, and the number of living tumor cells will then be quantified by measuring tumor-specific genetic alterations. The total number of cells can be quantified by measuring a universal DNA sequence, from which the amount of nontumor cells can be derived. dPCR: digital PCR, FISH: fluorescence in situ hybridization.

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