Bio-Computational Study of a Prostate Cancer Cell Line Using
Morphometric Analysis of the Nucleus and Nucleolus
The morphology of nuclear and cellular structures reflects tissue phenotype during normal development and in the disease states. The present gold standard of diagnosis of prostate cancer is characterization of 4um tissue biopsy sections using the
standard staining techniques for prostate clinical histology and pathology, hematoxylin/eosin and Feulgen. The purpose of this experiment is to use nuclear and nuceloli area, volume, and position information to provide insight on how the morphology
of nuclear and cellular structures reflect the cellular phenotypes of a prostate cancer cell line, PC3, when in the epithelial (EPI) and induced epithelial-to-mesenchymal (EMT) states. This may further assist in the early detection and differential
diagnosis of prostate cancer, especially in the early metastic phase. We will explore the extension of research on semi- automated machine classification of prostate cancer states. This will include 3 dimensional morphometric characteristics of whole
nuclei, nucleoli position information as well as nuclei/nuceloli relationships via simple fluorescent stains for DNA, RNA and nuclear envelope antibody staining. The images collected from these stains are then converted and analyzed by software
programs including Farsight, custom ImageJ plugins and the Statistics Online Computational Resource (SOCR). This analysis will lead to algorithms for the morphometric analysis of the collected imagery. 3D confocal microscopy will be compared
to standard fluorescent wide-field microscopy using segmentation and image analysis of characteristics such as size, number of nucleoli and distance to the perimeter of the nuclear envelope. Currently, imagery and statistics for 3D nuclear volume,
curvature, and fractal dimension have been collected for matched EPI and EMT cell cultures, with additional data on nucleoli expected. Preliminary analysis has shown differences between the two conditions, and more data will be collected to discern
the additional morphometric characteristics of the nuclei and nucleoli to increase the statistical power of the patterns shown to date.
Abstract
Brian Athey, Ivo Dinov, Ken Pienta, Walter Meixner, Alex Ade, Alex Kalinin, Ari Allyn-Feuer, Gordon Fon,
and John Mathew
Department of Computational Medicine and Bioinformatics, University of Michigan Medical School
Objectives
Conclusions So Far
Results in Progress
Methods
The next major goal in the experiment will be to develop
fluorescent probes to correlate gene expression levels and gene
locations in nuclear heterochromatin or euchromatin
compartments to further characterize expression differences
between the two cell lines.
Example of FISH Probe Design
Section of Intron 4 (Provided by Galaxy and UCSC Genome
Browser)
Probes by Bio-Search Technologies Stellaris:
actaattgttggtgctatctag
tttgaccaggctatttaaactt
gagtgtcagcatgttaaacatt
gctcactgcagccttgaactcctagactcaagccatcttcccacccagtagggctacggatgtacactaccatgc
ccagctgatttttttttaatttttgttttaattttttgtagagacaaaggggtcttgctatgttcccaggctggtgtctaac
tcctggccttaagtgatcctcccaacgtggcctcccaaagtgctggtattacaggtgtgagccactgcaactgac
ctatgtggttcttttgataggagagactaattgttggtgctatctagcacacactgtgtgtagacatcttgttaaat
agaaaatagatttatgggtatgactatgaagagtctaattccccaaaccacacacacaactctatctacgtttgac
caggctatttaaacttaactgcagagtgtcagcatgttaaacattgatttacataaaatgatagctgcccacttt
cttgtaaatgttataaaaactgtagagattaactaaaaaatgcacacagaagtttgctttcagttccacaagggtag
tttatttttgttataaaaacagtattccccactttcttagataccagatctctgcccagattttacccagtttcatcttgct
gctctctaatctcctatgtatgtaatatactttgaccatttaaatatgtattaagaca
Long Term GoalIntroduction
The objective of this experiment is to show that the
morphology of nuclear and cellular structures can be used to
make predictions of the type of prostate cancer, specifically
the differences in Epthelial PC3 cells and PC3 having
undergone the epithelial-to-mesenchymal transition. This
will expand on the work already done (1) but will consist of
using 3D imagery which may provide better results. The
hypothesis for this experiment is that there would be a
difference in morphology of the nucleus and nucleolus.
These differences could be used as another bio-marker in
combination with the Gleason scoring system, the present
standard diagnostic test in prostate biopsies.
• Use of simple fluorescent stains for DNA, RNA, and
Nuclear envelope antibody staining
• Analyzing of acquired images using Farsight, custom,
ImageJ plugins and the Statistics Online
Computational Resource (SOCR)
• Use of segmentation and image analysis of nuclear
characteristics to compare 3D confocal and two-
photon microscopy to standard fluorescent wide-field
microscopy.
From left to right: DAPI prolong Gold, Anti-
Fibrillarin, Ethidium Bromide
From left to right: DAPI, Nuclear Mask from
DAPI Channel, Image J Plugin to segment
connected and isolated nuclear masks
Results from plugin Segmentation
DAPI and FibrillarinNUP-98 and Fibrillarin
PC3 EMT from bottom left to top right:
Ethidium, DAPI, Fibrillarin
PC3 EMI from bottom left to top right: Ethidium,
DAPI, Fibrillarin
In the early 1970’s Dr. Donald F. Gleason and his lab used
stained sections of carcinoma cells to create a histologic
pattern arrangement of these cells, this became the basis of
the Gleason grading system. The score from the Gleason
gradient can give information on the tumor size and its
pathologic stage. This can be used to help to make a
prediction tumor aggressiveness and assist in prognosis
and treatment options.of the pathological stage. The search
for additional biomarkers to detect earlier changes in
tissue, and particularly early cellular changes, has
continued. One avenue which has shown promise has been
detecting morphometric changes in nuclear structure from
the Epi to EMT state of PC3 cells using 2-dimesional
microscopy (1). The morphometric measurements from
these images could be used to provide information to
doctors about the possibility of future metastatic disease.
(1) Verdone, J. E., Parsana, P., Veltri, R. W. and Pienta, K. J. (2015), “Epithelial–
mesenchymal transition in prostate cancer is associated with quantifiable changes in
nuclear structure.” Prostate, 75: 218–224. doi: 10.1002/pros.22908
Humphrey, Peter A. “Gleason Grading and prognostic factors in carcinoma of the
prostate.” Modern Pathology 17 (2004): 292-306. Web. 17 April 2015.
References
Currently the analysis of the PC3 Epi vs. MSC
nuclear/nucleolus images are not yet completed.
The initial results support the hypothesis: that
there is a statistical difference in the nucleolus
and nucleus.
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