Page 30 - Read Online

Page 10 of 15 Zhou et al. J Cancer Metastasis Treat 2018;4:41 I http://dx.doi.org/10.20517/2394-4722.2018.16

As in U251 and U251-NS cultures, monosomy-7 cell in U251-EFEMP1 (withdrawal of Dox) is a result of
Chr7-MS following proliferation of 2-Chr7 (2n). Withdrawal of Dox-induced EFEMP1 from U251-EFEMP1
(+Dox) mainly caused a 3-fold decrease of monosomy-7 cell percentage from 9% to 3%, suggesting a pro-
CIN effect of Dox or its induced ectopic EFEMP1. The latter has shown an InCIN effect in TMC and STIC
populations as described above. FISH analyses showed that the percentage of monosomy-7 in single-cell
lines of U251-EFEMP1 (withdrawal of Dox) without or with Dox-treatments were increased by four- and
two-fold, respectively, compared with their parental line [Figure 4B]. Hence in 2-Chr7 (2n) cells of low
tumorigenicity and low CIN rate, EFEMP1 has also played the role of InCIN.

DISCUSSION
TH is a hallmark of the most malignant glioma, glioblastoma multiforme (GBM) where “M” stands for
“multiforme” based on the degree of tumor cell diversity assessed solely with histopathology, both between
different tumors and, within the overall cell population of any given individual tumor. If they do not
succumb to their original tumor, most patients with GBM go on to experience tumor recurrence, despite
surgical resection, post-operative radiation and chemotherapy. There is no histological or cytogenetic
difference between primary and recurrent GBM (regardless of multiplicity of treatments and recurrences).
Most GBMs (about 80%) show loss of chromosome 10 (monosomy 10) , with activation of PI3K-mediated
[38]
growth signaling as a result of loss of tumor suppressor PTEN leading to aggressive growth . The other most
[39]
commonly seen numerical chromosome aberration in GBM is gain of Chr7 (trisomy/polysomy 7) . Chr7
[40]
copy number variation, including monosomy 7, occurs in both high- and low-grade gliomas, and appears
to be associated with invasive and proliferative cell phenotypes [40-44] . Through FISH analysis of individual
cells within glioma tissue and CQ-PCR analysis of whole tissue, we showed increased Chr7-defined cell
diversity in comparison to non-tumoral tissues of brain, and the positive relation of this diversity to the
malignant nature and behavior of these tumors. The grade-dependent increase of trisomy-7 cells may
have functional implications, e.g., a high proliferative phenotype, as also suggested by other studies .
[40]
Comparing grade II and III gliomas with oligodendroglia components, the observation of high percentage
of cells with 5 copies Ch7 and low percentage of cells with 1 copy of Chr7 in AO could be functionally
significant with increase of malignant phenotype due to increase of CIN rate, which requires further study
with larger sample sizes of AO.

From analyzing the distributions of Chr7-defined subpopulations in GBM-derived cell line U251 and its clonal
subculture line U251-NS under both in vitro and in vivo conditions, overall our findings support the idea that MS
rate increased by the dominating tumor cell subpopulation in U251 [Figure 2] and U251-NS [Figure 3A] in response
to decrease of two and three dimensional cell densities, respectively, and in U251-EFEMP1 (+Dox) in forming
soft agar colonies [Figure 4B]. Our conclusion of increasing MS rate is not from the direct measurement. The
increase of MS rate of the dominating TMC subpopulation in U251 was concluded based on a serial reduction
of its percentage along with increase of the minor STIC subpopulation [Figure 2A] and decrease of cell viability
[Figure 2B] due to decrease of cell plating density. Given the same culture conditions that were unfavorable
to monosomy-7, less supportive to STIC, and favorable to TMC, results from this experiment undermines the
impact from cell plating density on each subpopulation’s proliferation and/or death rate which may affect the
state of TH. In contrast, it highlights the immediate impact from the dramatic decrease of local extracellular
factors. In U251-NS, where STIC was the key cells, similar results was observed suggesting increase of MS rate due to
decrease of cell density [Figure 3A]. Base on mouse survival that is negatively related to the speed of tumor growth,
monosomy-7 cells remain slow-growing under in vivo conditions. The increase of monosomy-7 percentage in
i.c. xenograft of U251-NS compared to that of in vitro culture suggests less apoptotic rate of monosomy-7 cells
in conditions of in vivo vs. in vitro. The significant increase of monosomy-7 cell portion in xenografts from
decrease of inoculum size could be mainly caused by an increase of MS rate of STIC in responding to dramatic
decrease in concentration of local extracellular factors playing roles as InCIN, including EFEMP1.