Page 26 - Read Online

Page 6 of 15 Zhou et al. J Cancer Metastasis Treat 2018;4:41 I http://dx.doi.org/10.20517/2394-4722.2018.16

A B

Figure 2. Increase in population diversity and decrease of cell viability both correlate with decrease in cell plating density. (A) Chr7-FISH
was carried out in U251 cultures plated at various cell densities and in SA1, a clonal line of U251 established in serum-containing medium.
Based on near diploid karyotypes of cells in U251, cells carrying 1, 2 & 4, and 3 & 6 copies of Chr7 were denoted as monosomy-7 cells,
[13]
STIC and TMC, respectively, as described previously . Shannon diversity index was calculated based on the percentages of these three
cell subpopulations, as described previously [22] ; (B) colony formation rate from one week culture of 50 cells of U251 in 35, 60, and 100 mm
dishes in 3, 4, and 10 mL of medium, respectively, in 4-6 replicates

As shown in Figure 2A, decreasing cell plating density of U251 cultures in the same surface area and volume
of culture medium caused a gradual decrease in percentage of TMC (67%, 55%, 45%, and 40%) along with
a gradual increase in percentage of STIC (31%, 40%, 48%, 53%), leading to a gradual increase in population
diversity, as shown by increase of H-index value. SA1 is a single-cell line of U251 formed and expanded in
SA conditions. Its CGH profile confirmed its origin from a TMC in U251 . As shown in Figure 2A left
[13]
panel, the percentage of STIC (53%) in SA1 was slightly higher than that of TMC (40%). While in its parental
culture, TMC was the dominating population (average 77%), based on analyses of four different passages.
Because there was no change in culture conditions from SA to NS, which is favorable or against the growth
of STIC or TMC, respectively, the observed increase in percentages of STIC and corresponding decrease of
TMC would mostly due to increase of Chr7-MS rate by TMC in responding to decrease of paracrine effect
of InCIN from decrease of cell plating density.

We then analyzed colony formation rate of U251 in SA conditions, by plating 50 cells of U251 in 35, 60 and
100 mm dishes with nearly 3-fold serial increase of surface areas from 10 to 28, and to 79 cm . As shown in
2
Figure 2B, there was a near 3-fold of serial decrease of colony formation rates, which is not related to changes
in volume of culture medium (from 3 to 4, and to 10 mL), but to cell plating density. Clearly, it is more to the
change of cell density that changed cell viability in colony formation assay. According to notion that most
aneuploid cells from chromosomal MS are nonviable, decrease of cell survival would be consistent with
increase of MS rate, in response to decrease of cell-plating density in above described colony formation assay.

Taken together, results of FISH analysis showed increase in population diversity, and colony formation assay
showed decrease in colony formation rate due to increase of two dimensional cell density of U251. Overall
our data are consistent with paracrine control of cancer cell CIN rate, by local concentration of extracellular
factors secreted by its self as well as its neighboring cancer cells.

Extracellular control of CIN in maintenance of TH
The above described increase of CIN rate in establishment of single-cell lines and a negative association
between population diversity and cell-plating density suggest paracrine control of cancer cell CIN rate,
with InCIN acting in the extracellular compartment. This conclusion was supported by FISH analyses of
intracranial (i.c.) xenografts derived from U251-NS with different inoculum sizes. U251-NS is a single-cell
line of U251 with 90% STIC under NS-conditions which did not support the growth of TMC . The small
[35]
(1%-2%) portion of TMC in U251-NS is likely from Chr7-MS of STIC as demonstrated in mathematical