1) An immunofluorescence protocol was optimized to differentiate pancreatic circulating tumor cells (CTCs) from leukocytes in blood using cell line models. 2) Parameters such as fixation buffer, permeabilization time and concentration, antibody incubation times and concentrations, and washing steps were altered. 3) The optimized protocol improved consistency of staining for cytokeratin, a CTC marker, and CD45, a leukocyte marker, allowing differentiation of cell types captured using a microfluidic device from patient blood.

1. Immunofluorescent Differentiation of Pancreatic Cancer Cells and Leukocytes:
A Cell Line Model for Identifying Circulating Tumor Cell Populations in Patient Blood
Capturing and Identifying Circulating
Tumor Cells is Key to Understanding Cancer
Progression
Metastasis is responsible for
most deaths due to cancer
• Metastasis is the process by which cells
from primary tumors enter into the
bloodstream and form new tumors at
distant sites (Fig. 1)
• The mechanism of metastasis is poorly
understood
Basic IF protocol
Fixation: “freezes” cells in their current state so they stop growing, but retain structure
Permeabilization: allows antibodies to enter into cells to bind to target proteins
Blocking: reduces non-specific binding of antibodies
Primary antibody: binds to target protein; may or may not be attached to a fluorophore
Secondary antibody: (if primary antibody is not fluorophore-conjugated) binds to
primary antibody; is attached to a fluorophore
Washing: removes buffers between steps and reduces background fluorescence signal
Microscopy was Used to Visualize Cells to Assess Immunofluorescence
Staining
Jaeda E. J. Patton,1 Marie E. Godla,2 Conor N. Gruber,1 Fredrik I. Thege2 & Brian J. Kirby3, 4
Biology [1]; Biomedical Engineering [2]; Sibley School of Mechanical and Aerospace Engineering [3]; Department of Medicine, Division of Hematology and Medical Oncology [4]
Final Optimized Protocol
Optimization of the immunofluorescence protocol focused
on addressing three main challenges:
Protocol Optimization Was Achieved
Primarily by Altering Fixation and
Permeabilization Techniques
My thanks to Marie Godla, Fredrik Thege, and Brian Kirby for all of their
invaluable help and mentorship on this project. Thanks also to Ian Cardle,
Conor Gruber, and all other members of the Kirby group.
Funding was provided by the National Cancer Institute through the Cornell
Center for Microenvironment and Metastasis.
Figure 1. Model of metastasis. Image
source: National Cancer Institute
1. National Cancer Institute. (April 2013). SEER Stat Fact Sheet: Pancreas
Cancer. Retrieved from http://seer.cancer.gov/statfacts/html/pancreas.html
2. Maheswaran, S. & Haber, D. A. (2010). Circulating Tumor Cells: a window
into cancer biology and metastasis. Current Opinion in Genetics and
Development, 20(1), 96–99.
3. Thege, F. I., Lannin, T. B., Santana, S. M., Saha, T. N., Rhim, A. D., & Kirby,
B. J. (2012). Mutliplexed microfluidic immunocapture of circulating pancreas
cells for the early detection of pancreatic carcinogenesis [PowerPoint slides].
Retrieved from
http://www.kirbylab.com/wiki/bin/view/Main/ThegeBMESTalk2013
4. Kirby, B. J., Jodari, M., Loftus, M. S., Gakhar, G., Pratt, E. D., Chanel-Vos,
C., … Giannakakou, P. (2012). Functional Characterization of Circulating
Tumor Cells with a Prostate-Cancer-Specific Microfluidic Device. PLoS ONE,
7(4), e35976. doi:10.1371/journal.pone.0035976
A
B
C
B
C
Figure 3. CK-CF543 (red) and DAPI (blue) staining. CK-CF543 signal was
inconsistent in PANC1s, making it difficult to distinguish between U937s and
PANC1s (A). CK-CF543 signal was more consistent in BxPC3s than in PANC1s
(B). CK-CF543 signal was stronger with 2% PFA in PHEM fixation than with
2% PFA in PBS (C). Image C captured by C. N. Gruber.
Figure 4. CD45-AF488 (green) and DAPI (blue) staining. With permeabilization
with Triton X, virtually no CD45-AF488 signal was seen (A). This was true
even with antibody incubation time increased from 1 hour to overnight (B).
Without permeabilization, CD45-AF488 signal increased dramatically (C).
A PANC1 U937 PANC1 + U937
PANC1 BxPC3 U937
BxPC3
PFA in PBS
BxPC3
PFA in PHEM
U937
PFA in PHEM
BxPC3 U937 BxPC3 + U937
BxPC3 U937 BxPC3 + U937
BxPC3
w/ Triton X
BxPC3
w/o Triton X
A B
Figure 5. CK-CF543 (red), CD45-AF488 (green), and DAPI (blue) staining. When Triton X (permeabilizaton
buffer) was left in during CK-CF543 incubation, CD45-AF488 signal was inconsistent in U937s (A).
Removal of Triton X during CK-CF543 incubation along with decreased Triton X concentration and fewer
washing steps increased consistency of CD45-AF488 signal (B). Images captured by F. I. Thege.
U937BxPC3 BxPC3 + U937
Longer Triton X
BxPC3 + U937
Shorter Triton X
An Immunofluorescence Protocol was
Optimized for Identifying CTCs in Blood
References
Acknowledgements
CTCs have great potential for
pancreatic cancer treatment
• Pancreatic cancer is difficult to detect
early-stage
• 5-year survivorship is only 6.0%
• CTCs could aid in determining best
treatment options for patients
The geometrically enhanced differential immunocapture (GEDI)
device captures CTCs
• Blood is flowed through a geometrically optimized microarray of antibody-coated
posts (Fig. 2)
• CTCs bump into and stick to posts
• The device also captures leukocytes, which must be differentiated from CTCs
Immunofluorescence (IF) is a technique for differentiating
between CTCs and leukocytes
• IF uses fluorophore-conjugated antibodies to stain for specific proteins in cells (Fig. 2)
• IF staining for the epithelial and leukocytic proteins allows us to differentiate between
GEDI device-captured CTCs and leukocytes
Cell lines were used to model pancreatic CTCs and leukocytes
• PANC1 and BxPC3 for pancreatic CTCs
• U937 for leukocytes
Cytokeratin (CK) antibody was used as a CTC marker
CD45 antibody was used as a leukocytic marker
Several parameters were optimized
• Model CTC cell line
• Incubation time and concentration for fixation, permeabilization, and blocking
buffers, and antibody solutions
• Fixation buffer solvent
• Number/intensity of washing steps
• Order of protocol steps
Fixation
2% PFA in PHEM
15 min.
Blocking
10% NGS in PBS
30 min.
CD45
4 μg/ml in 10% NGS
1 hr.
Permeabilization
0.15% Triton X in 10% NGS
15 min.
CK
5 μg/ml in 10% NGS
1 hr.
DAPI
1 μg/ml in DI H2O
15 min.
Wash 3x
Wash 2x
Wash 1x
Wash 2x
Wash 2x
Challenge Solution
Cells, especially leukocytes, were
being washed away during staining
(data not shown)
 Decreased number and
intensity of washing steps
CK fluorescence signal was
low/inconsistent (Fig. 3A, B)
 Switched from PANC1 to
BxPC3 cell line to model
CTCs (Fig. 3B)
 Switched from fixing with
4% PFA in PBS to 2% PFA
in PHEM (Fig. 3C)
CD45 fluorescence signal was
low/inconsistent (Fig. 4A, B; 5A)
 Did not permeabilize before
CD45 staining (Fig. 4C)
 Decreased Triton X
concentration during
permeabilization (Fig. 5B)
 Removed Triton X before
CK staining (Fig. 5B)
Limitations of the Protocol
Cell lines are not perfect models of CTCs and leukocytes found in
patient blood
• Cell lines have different mutations and morphology than patient-
derived cells, and therefore might stain differently
• The staining protocol will be tested in the future on cells
captured from patient blood
Stress induced by the staining protocol could limit the kinds of
analyses that are able to be performed on GEDI device-captured
CTCs
Circulating tumor cells (CTCs)
are critical to metastasis
• CTCs are cells from primary tumors that
have entered the bloodstream, but have
not yet formed metastatic tumors (Fig.
1)
• CTCs provides a non-invasive means for
early cancer detection and development
of personalized treatment
• CTCs can also improve our
understanding of metastasis
Figure 2. Analysis of CTCs using the GEDI device.