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Zhou et al. J Cancer Metastasis Treat 2018;4:41 I http://dx.doi.org/10.20517/2394-4722.2018.16 Page 3 of 15

Understanding such “Yin” and “Yang” reciprocal aspects of CIN could facilitate development of therapeutic
strategies, which could potentially prevent cancer recurrence.

This study attempts to explore this possibility, by studying a cell line model of GBM in which two tumor
subpopulations have been functionally characterized as stem-like tumor initiating cell (STIC) and tumor
mass-forming cell (TMC), defined by different copies of chromosome 7 (Chr7), and their inter-conversions
via MS of Chr7 . We further studied our prior finding of changes in the steady state of Chr7-defined
[13]
subpopulations in response to microenvironmental cues and an extracellular protein named fibulin-3, or
EGF containing fibulin-like extracellular matrix protein 1 (EFEMP1) .
[32]

METHODS
Ethics statement for human tissues
Tumors from Tissue Bank of UC Irvine and University of Arkansas for Medical Science were included in
this study, with Institutional Review Board approval, as reported previously .
[33]
Cell cultures
The human glioma cell line U251 (also known as U251HF) was obtained from M.D. Anderson Cancer
Center, University of Texas. U251-NS is a single-cell line of neural sphere culture of U251 established at
UC Irvine Brain Tumor Research Laboratory. Characterization of U251 with phenotypes defined as tumor
mass-forming cells (TMC) and U251-NS as stem-like tumor initiating cells (STIC) and their Short Tandem
Repeat (STR) profiles were reported previously . EFEMP1 and Empty/pTRIPZ lentiviral vectors and their
[13]
transduced glioma cells (U251 and U251-NS) were described by Hu et al. .
[32]
U251 (including those infected by lentiviral vectors) was grown in monolayer cultures in DMEM/F12
supplemented with 5% bovine serum, respectively, while U251-NS (including those infected by lentiviral
vectors) were grown in 1% agar-coated plates in DMEM/F12 supplemented with epidermal growth factor
(EGF, 20 ng/mL), basic fibroblast growth factor (FGF, 10 ng/mL), and 1% B27 (Invitrogen, Carlsbad, CA).
U251-NS was attached in fibronectin (10 mg/mL)-coated plates prior to FISH analysis.

Fluorescence in situ hybridization
The methods for fluorescence in situ hybridization analyses on glioma specimens, glioma xenografts from
[32]
intracranial models of mice, and cell cultures were reported previously . Briefly, metaphase-spread slides
were obtained by exposing exponentially growing cells to nacadozole solution (100 µg/mL final, Sigma) for 1 h.
Then the cells were trypsinized (0.25% trypsin/EDTA, Invitrogen) to collect cell pellets, which were treated
with a hypotonic solution (phosphate buffer) for 5 min at 37 ˚C. The cell pellets were fixed (methanol:glacial
acetic acid = 3:1) for at least 30 min. Finally, the cell suspensions were dropped onto slides to get metaphase
chromosome spreads. Cryosections (7 mm) of human glioma and epilepsy brain tissue frozen specimens,
and mice brain with i.c. xenografts of glioma cells were fixed with 100% methanol for 5 min. The slides were
further treated with 0.3% sodium citrate solution for 10 min in a pressure cooker, and rinsed with water
briefly. FISH analyses on glioma cells and tissues were performed using Vysis LSI EGFR SpectrumOrange/
CEP 7 SpectrumGreen Probes (Abbott Molecular Inc) following the manufacturer's instructions. Cells were
counted on slides using a Nikon Eclipse TS100/TS100F fluorescent microscope with a 100× lens.

The numbers of Chr7 centromeres per nucleus, detected by the FISH CEP7 probe, were counted and the
percentages of cells with different copies of Chr7 were determined based on counting of more than 250 nuclei
per sample of tumors or cell cultures. These data were used to establish the level of tumor heterogeneity with
regard to Chr7-defined cell subpopulations. The Shannon diversity index (H) was calculated to show the
degree of diversity with regard to Chr7-tumor cell subpopulations as described previously .
[22]