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Frame et al. Investigating models for prostate cancer research

collected every 6 min). Segmentation and tracking of cytometry also shows the separation of the two
every cell was carried out, during which each cell track populations [Figure 5B]. After selection, QPI was
is assigned an identification number [Figure 4B]. The carried out [Figure 5C] and an analysis using the CAT
movement of every cell was tracked and measured over was completed. A QPI signature was established for
time, and tracks were observed as 2D [Figure 4C] and each cell type [Figure 5D and E], indicating that CB
3D representations [Figure 4D]. The representations cells had a larger mass and size [Figure 5D and E]
demonstrate that the automated tracking procedure than the TA cells. The TA cells had a higher value
used by the Cell Analysis Toolbox (CAT) software is relating to cell sphericity compared to the CB cells
capable of following individual cells. For example [Figure 5F] Significantly, once these parameters were
with BPH-1 cells a doublet of cells circled round and established, a heterogeneous (unselected) culture of
round each other, which is observed as a spiral over primary prostate epithelial cells was analyzed. The
time [Figure 4E]. Morphological measurements (e.g. area measurement from the ptychographic signatures
area, thickness, volume, radius, sphericity) and kinetic of each cell type was applied to the images of the
measurements (speed, displacement, meandering mixed culture and the software was able to identify TA
index) can be extracted from CAT. Of note, cells in the and CB cells within the culture [Figure 5G].
primary cultures are significantly larger on average
than all the cell lines [Figure 4F]. In addition, most of DISCUSSION
the cell lines were much less motile than the primary
culture apart from P4E6, which is the closest cell These studies highlight that the use of a single cell line
line to a primary culture [Supplementary Video 1]. is insufficient to make a conclusion about efficacy and
Even though the primary cells are significantly more mechanism of action of a treatment. In addition, using a
motile, which can be measured as mean speed of a panel of cell lines may also not be a great improvement
track [Figure 4G], they actually show slower growth because results from experiments in cell lines have
and proliferation than the cell lines. Indeed, there is a been seen here and in other studies to be quite different
range of growth and proliferation rates in all cell lines from primary cells [26-28] . Cells in primary cultures have
measured. The growth and proliferation rate is one compensatory signaling pathways that have been lost
example of a unique QPI measurement that takes into in cell lines, and so an inhibitor that works well in cell
account the whole population rather than individual lines may be less effective or ineffective in primary
cells and in this case is represented as total dry mass cultures . This is one explanation for such high
[28]
over time [Figure 4H]. attrition rate in the drug pipeline; weak, incomplete,
[17]
unrepresentative or inappropriate models. Also, it
Ptychographic label-free imaging can distinguish has previously been shown that the DNA methylation
between cell populations in heterogeneous primary profile is quite different in cell lines compared to
epithelial cell cultures. primary cells and indeed between different primary cell
subpopulations [29,30] , thus impacting how cells respond
Although ptychographic QPI can measure detailed to various treatments. Indeed, epigenetic changes
morphological and kinetic measurements to distinguish can also be induced in response to treatments such
between different cell populations, the power of as radiation, which relates to radioresistance and
the technique is to harness these individual cell radiosensitization . The use of primary cells from
[31]
measurements to take into account cell heterogeneity. both normal and cancerous patient tissue as part of
We sought to determine whether QPI can distinguish the drug pipeline may be at least part of the solution.
between cell populations within a primary prostate Indeed, use of this model, in vitro primary cell culture,
epithelial cell culture. We already know that within was critical in the development of an oncolytic
these cultures, which have a predominantly basal adenovirus for prostate cancer [32-34] , which is currently
epithelial cell phenotype, there are three subtypes; in clinical trials.
rare stem-like cells - CD133 /α β integrin , TA cells -
hi
+
2 1
CD133 /α β integrin and CB cells - α β integrin . First, Results presented here and previous studies
-
lo
hi
2 1
2 1
we enriched for TA and CB cells using rapid collagen looking at cancer stem-like cells suggest that a
[15]
adherence to select the TA cells (which also contains combination treatment of Vorinostat and radiation
the rare stem cell population). Immunofluorescence may be more effective in treating prostate cancer
staining highlights the high expression of α β integrin than radiation alone. Since Vorinostat is already
2 1
in TA cells and the low expression in CB cells clinically-approved , a move to clinical trials for
[35]
[Figure 5A]. Staining of the whole population shows the combination treatment could be swift. However,
a mixture of cells with different fluorescent intensities. before this could happen, a prognostic indicator and/
Staining the cells with CD49b and analyzing by flow or a measurement output, other than overall survival,
Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ December 6, 2017 309