Book of abstract 2008

Quantification of viability in organotypic spheroids of human malignant
glioma for drug testing
Philip C. De Witt Hamer and Cornelis J. F. Van Noorden
Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
The prognosis for patients with glioblastoma multiforme (GBM) remains poor, despite
surgical resection, radiotherapy and temozolomide chemotherapy. To screen for novel
therapeutics established cell lines or primary cell cultures are customarily used. However,
the correlation between in vitro and in vivo responses is poor. Organotypic spheroids
are grown from primary explants from surgical specimens and provide a more complex
biological system than monolayer cell cultures, that maintains cell-cell interactions,
extracellular matrix and cellular heterogeneity. We found that the cancer genomic
profiles of 5 original GBMs and their OSs compared better than that of their primary cell
culture using whole genome array comparative genetic hybridization, thus proving that
the OS model is genetically more representative for GBM1. Accurate and reproducible
quantification of therapeutic responses in the OS model has been lacking. For this
purpose, lactate dehydrogenase (LDH) activity was demonstrated in cryostat sections of
spheroids2. Calibrated digital image acquisition of the stained cryostat sections enabled
demarcation of LDH-active and LDH-inactive tissue areas by thresholding at specific
absorbance values. The viability index (VI) was calculated as ratio of LDH-active areas
and total spheroid tissue areas. Duplicate staining and processing on the same tissue
showed good correlation and therefore reproducibility. As a positive control to reduce
viability, sodium azide incubation of spheroids induced reduction in VI to almost zero.
However, standard therapy such as temozolomide and other positive controls such as
cyanide did not show any effect on viability indicating either lack of diffusion and/or
multidrug resistance of the cancer cells. We also determined sample size requirements for
a valid screening strategy: how many spheroids per experimental group and how many
sections per spheroid are required to detect one-third reduction in the VI after treatment?
Because of the large biological variation of the VI (20%), at least 12 spheroids per group
and 1 section per spheroid are required3. We conclude that quantification of viability in
cryostat sections of OSs from GBM can be performed reliably and reproducibly with this
approach. Furthermore, OSs represent GBM genetics better than primary cell cultures.
Unfortunately, the large number of spheroids prevents high-throughput screening and
the lack of response to treatment can either be an artefact of culture conditions or a
phenomenon of glioma cells.
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References:
1. De Witt Hamer PC, Van Tilborg AAG, Eijk PP, Sminia P, Troost D, Van Noorden CJF, IJlstra B and
Leenstra S (2007) The genomic profile of human malignant glioma is altered early in primary cell culture
and preserved in spheroids. Oncogene, publication ahead of print. doi: 10.1038/sj.onc.1210850.
2. De Witt Hamer PC, Jonker A, Leenstra S, Ruijter JM, and Van Noorden CJF (2005) Quantification of
viability in organotypic multicellular spheroids of human malignant glioma using lactate dehydrogenase
activity: a rapid and reliable automated assay. J. Histochem. Cytochem. 53:23-34.
3. De Witt Hamer PC, Leenstra S, Van Noorden CJF and Zwinderman AH (2008) Cancer spheroids for
screening of experimental therapies: how many spheroids and sections are required? J. Microsc. in press.
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