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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 5 of 15

based on the percentages of four Chr7-defined cells shown in Figure 1A, to compare the degree of tumor
subpopulation diversity between different grades of gliomas. As shown in Figure 1B, gliomas of all grades
presented significantly higher value of H-index compared with non-tumoral brain tissues from patients
with epilepsy. Furthermore, grade III AO and IV GBM both showed significantly higher values of H-index
compared with grade II OG, due to significant increase of cells carrying 5 and 3 copies Chr7, respectively.

To determine if the observed increase in cells with 3 copies of Chr7 are trisomy-7 or triploid cells, we
performed CQ-PCR for CNV of EGFR on 7p in reference to three single-copy genes (ERC2, SPAG16 and
SPOCK1) on three different chromosomes (3p, 2q and 5q, respectively) in a larger set of human glioma
samples, including OG and GBM tissues used in FISH analyses. As shown in Figure 1C, most of GBMs (78%)
showed significantly (on the average of 1.3-fold) higher copies of EGFR compared with that of OGs. About
22% of GBM showed very high copies of EGFR, with 7%, on the average 9-fold higher and 15%, on the average
26-fold higher, from focal EGFR amplification. As reported previously, this is due to extrachromosomal
oncogene amplification or double minute chromosome (DM) . In contrast to overall increase of EGFR
[34]
CNV, CQ-PCR showed overall decrease of PTEN gene copy, with an average of less than 1 ratio of PTEN to
one of the three reference genes. Taking together, data from FISH and CQ-PCR are consistent with increase
of trisomy-7 population in GBM as compared with OG.

Low cell-plating density caused increase of CIN rate
We have previously presented two GBM heterogeneity models where variations in Chr7 or DM status
characterized tumor subpopulations functionally defined as TMC and STIC, which were enriched by certain
in vitro culture conditions, known as serum-adherent (SA) and neurosphere (NS) conditions, respectively [13,14] .
The dynamic state of tumor sub population diversity was stabilized with one dominant subpopulation over
long-term passages at high cell-plating densities without changing culture conditions. However, under the
same culture conditions, single-cell cultures, derived from single cell or soft agar colony, presented not only
diverse cell populations, but also higher degrees of heterogeneity compared with their parental cultures.
Examples are Chr7-defined subpopulations in single-cell SA and NS lines of four established GBM cell lines
(U251, A172, LN229, and T98G) , as well as DM-defined subpopulations in single-cell NS line of a GBM-
[13]
derived primary culture 51A . The explanation of this phenomenon would be an increase of CIN rate,
[14]
shown by increase of MS rate of the subpopulation-defining chromosome or DM, due to loss, and dramatic
weakening, of inhibitors of CIN (InCIN) in initial and subsequent cell divisions of single-cell lines.

To test the hypothesis that regulation of CIN rate is paracrine-mediated, Chr7-FISH was carried out in U251
derived from serial decrease of cell-plating density from that normally used in cell passages (~10,000 cells/cm ).
2
U251 cells at above 90% and about 40% confluence from plating with 10,000 and 1000 cells in a 24-well plate were
fixed 2 days later for FISH analysis. Cells from three selected wells, each containing 16 colonies one week after
seeding 50 cells per well in a 24-well plate, were detached by trypsin-EDTA and passed into a 35-mm dish and
cultured for two days prior to FISH analysis. The percentages of cells with Chr7 copy of 1, 2 & 4, and 3 & 6 of U251
derived from various cell-plating density were plotted in the left panel of Figure 2A, and the H-index calculated
based on the percentages of these populations was shown in the right panel of Figure 2A.

We have shown previously that TMC in U251 carrying 3 copies of Chr7, 2 normal, 1 with q-arm deletion,
denoted as 3-Chr7 (2n, 1d), and STIC carrying 2 copies of Chr7, 1 normal, 1 with q-arm deletion, denoted
as 2-Chr7 (1n, 1d). Counting of whole chromosome number (WCN) for 145 metaphase nuclei of U251 and
U251-NS showed that the majority (87%) have aneuploid karyotypes with a modal chromosome number of
50. Hence both cells with 2 or 3-copies of Chr7, which were differentially enriched in U251-NS and U251,
respectively, had near diploid karyotypes. The small portion (4%-5%) of cells with 4 and 6-copies of Ch7 were
therefore considered as transient tetraploid stages of STIC and TMC, respectively, as shown in Figure 2A.
Monosomy-7 cells with 1 copy of Chr7 in U251 were also near diploid.