Download pilot project 1

Secretome Analyses from Staged Pancreatic Cancer Cell Lines
Rowena S. Chu, Steven L. Miller, David L. Wells, Christopher S. Sakoda, Lianji Jin, PhD and Jason A. Bush, PhD
Department of Biology, California State University, Fresno
Figure 1. (Upper) Pancreatic duct
cells are nourished by secretions
from surrounding acinar cells.
A)
(Lower)
Known gene mutations
correlated with specific PanIN
pre-malignant stages.
Abstract
Pancreatic ductal adenocarcinoma is one of the most lethal cancers; its aggressive nature and late onset of physical
symptoms lead to severe prognoses. Recognition of disease biomarkers is imperative and has great potential for early detection of the
disease. Pancreatic ductal adenocarcinoma has the lowest 5-year survival of any cancer; its aggressive nature and late onset of physical
symptoms lead to poor prognoses. Pancreatic cancer primarily occurs in the exocrine portion of the organ, with fewer occurrences in the
endocrine section. Approximately 95% of cancerous exocrine tumors (carcinomas) are derived from ductal cells, while a smaller fraction of
tumors are from acinar cells. To tackle this problem, we are attempting to evaluate the secreted protein profile of pancreatic cancer cell
lines derived from different stages. Critical to the success of this workflow is the derivation of cells that are sustainable without overt
morphological changes in peptide- and protein-free media conditions or dramatic necrosis over a 72 hr profile. We have successfully
adapted ten human pancreatic cancer cell lines into serum free environments and pursue collection of conditioned media by a
combination of ultracentrifugation, molecular weight cutoff, protein precipitation, gel-based separation, and tryptic digestion followed by
MALDI-ToF-MS protein identification. Preliminary data suggested significant cellular autolysis that liberated cytosolic proteins such as betaactin. To minimize cytosolic contamination, cell lines were subsequently cultured from low-serum to Matrigel™ in a serum-free media to
phenocopy the ductal environment typical for these cells. Consistent with the model that advanced stage cancer has increased secretory
function, earlier stage pancreatic cancer cell lines showed generally lower protein secretion while later stage cell lines showed generally
increased secretion. A catalog of proteins is being compiled in ProteinScape (Bruker Daltonics) and validated biochemically including
positive identifications for Glutathione S-Transferase pi (GSTP1) and Galectin-3 (GAL3)—two proteins that have been correlated with
tumor secretions from prostate and breast cancer, respectively. Taken together, our reproducible workflow demonstrates the utility of
assessing the secretome fraction from cultured cancer cells.
Background
 Pancreatic cancer (PC) is the 5th leading cause of cancer death in the U.S.; it is difficult to
detect, resistant to treatment, and usually diagnosed after metastasis
 In 2008, the ACS estimated that 37,680 people in the U.S. alone was diagnosed with
pancreatic cancer, and of that population, only 9% was predicted to survive [1]
 Pancreatic cancer research is funded at shockingly low levels; it receives 10% of the funding
of breast cancer research but causes 78% of the mortality [2]
 Early detection is essential for successful treatment of PC; studies have shown that the cancer
can be inherited as part of a familial cancer syndrome (Palladin) [3]
 A current area of interest for early PC detection is in identifying biomarkers in the cancer cell
secretome [4]. Discovery of up- or down-regulated proteins in cancer can serve as diagnostic
biomarkers and provide clues to cancer progression [5]
Figure 2. Depiction of the cancer
cell secretome through a crosssection of the pancreatic duct.
 Every cell leaves a record of each physiological state, typically through waste emitted into the
blood or through signals to other cells [6]. Significant research has shown specific markers
expressed by the tumor types formed in PC, yet very few markers have been identified [7]
SECRETOME
Intens. [a.u.]
Capan-1
Results
Table 1. In vitro pancreatic cancer cell lines adapted to protein
and peptide free media (Cellgro Complete, Mediatech). Cells
are magnified up to 400X.
SFM
Figure 3. Methodology
Flowchart
PC Cell Lines
Adapt to
SFM
Early
MATRIGEL
BxPc-3
Mid
Collect Media
24, 48, & 72 hrs.
Panc 3.27
Panc 4.03
Panc 10.05
Mid
TCA/Sarcoysl
Protein ppt.
Secretome
Protein
Pellets
Mid
Mid
1D Gels
PL45
2D Gels
Mid
In-Gel Tryptic Digests
Capan-1
Mid-Late
MALDI-TOF ANALYSIS
Capan-2
HPAF-II
Late
Late
Tissue
Culture
&
Adaptation to Matrigel
& SFM
 Cells are grown to
70-75% confluency
 Secretome media is
collected from 24-72
hours
TCA/Sarcoysl Protein
Precipitation
 Protein
is
concentrated through
Centricon®
devices
(Millipore, Billerica, MA)
and precipitated using
100% TCA and 100mM
N-lauryl
sarcosine
sodium. Pellets are
washed with THF and
solubilized
with
a
standard
extraction
buffer
1D
&
2D-Gel
Electrophoresis
 Samples
are
separated on 10% TrisGlycine/4-12% Bis-Tris
gels
 SyproRuby® stained
overnight
In-gel Trypsin Digests
 Band/spots digested
for 18 hours and
spotted
onto
the
AnchorChip MTP plate
(Bruker
Daltonics,
Fremont, CA)
 Peptide samples read
at RP mode and
analyzed with MASCOT
1581.865
3
2
HPAF-II
1491.810
BRCA2
K-ras
1
842.509
DPC4
Her-2/neu
p16
p53
BRCA2
kRAS
GAPDH*
Cell Line
AsPc-1 BxPc-3 P10.05 P3.27 P4.03 PL45 Cap-1 Cap-2 HPAF
+
+++
++
++
+
++
+
+
+
++
+
N/A
+++
++
+
+
+++
+++
++
N/A
+
+++
++
++
++
+++
+++
++
N/A
+++
+
++
+
++
++
++
+++
N/A
++
++
+++
+++
+++
+++
+++
+++
+++
+++
+++
1.
2.
3.
4.
5.
6.
7.
American Cancer Society. Cancer Facts and Figures, 2007. Atlanta, GA.
Lomberk, G. Patient Advocacy. Pancreatology 2008;8(4-5):420-421
Pogue-Geile, K.L. et al. Pallindin Mutation Causes Familial Pancreatic Cancer and Suggests a New Cancer Mechanism. PLoS Medicine
2006;3(12):1-13
Hanas, Jay S. et al. Biomarker Identification in Human Pancreatic Cancer Sera. Pancreas 2008;(36)1:61-69.
Bhattacharyya, S. et al. Diagnosis of Pancreatic Cancer Using Serum Proteomic Profiling. Neoplasia 2004;6(5):674-686
Mark Aspinall-O'Dea, Eithne Costello, MD. The pancreatic cancer proteome - recent advances and future promise. Proteomics 2007;
1(9):1066-1079
Kim, Y.W. et al. Characterization of Clones of a Human Pancreatic Adenocarcinoma Cell Line Representing Different Stages of
Differentiation. Pancreas 1989;(4):353-362
1839.028
1716.864
1275.725 1441.695
1971.122
2647.152
3047.598
3489.454
0
500
1000
1500
2000
2500
3000
3500
m /z
P4.03
x104
568.156
2.5
BxPc-3
B.
2.0
Figure 4. (Upper) RT-PCR of genetic markers from pancreatic cancer cell lines. (Lower)
A high level of expression is indicated by multiple plus signs and darker green shades,
and no expression with a minus sign. GAPDH was used as a control.
1.5
1230.784
1.0
Table 2. Positive protein identifications from in-gel tryptic digests of 1D and 2D gels using MALDI-TOF
(Autoflex II ToF/ToF) and Mascot analysis (searching NCBInr db).
0.5
1676.918
PROTEIN
ACCESSION
NUMBER
%
COVERAGE
PEPTIDE
MATCHES
CELL
LINE
SIZE
(DA)
1548.809
1966.010
1386.878
842.509
GEL
0.0
Actin, beta [ACTB]
14250401
58%
21
Panc 3.27
40978
1D
Actin, gamma [ACTG1]
809561
26%
11
AsPc-1
40992
1D
Alpha 2,3-Sialyltransferase III [ST3GAL3]
27448437
16%
2
BxPc-3
20874
2D
Cytidine deaminase [CDA]
115392103
43%
5
AsPc-1
23910
1D
500
x104
1000
1500
2000
2500
3000
3500
m /z
615.412
4
1741.956
3
Ferritin heavy polypeptide [FTH1]
114621872
30%
5
HPAF-II
28405
2D
Galectin-3 binding protein isoform 3 [GAL3]
114670746
19%
11
Panc 3.27
64671
1D
Glutathione S-transferase pi [GSTP1]
197097848
39%
6
Panc 3.27
23375
1D
Heterochromatin protein 1 [HP1]
168984313
28%
4
HPAF-II
5048
2D
Inositol monophosphatase [IMPA1]
194214900
24%
5
Panc 4.03
46803
2D
Mindbomb homolog 2 [MIB2]
122890283
22%
10
HPAF-II
99434
2D
Palladin [PALLD]
194224103
22%
2
HPAF-II
19731
2D
Serine kinase [STK24]
Literature Cited
Controls
HEK NBE NLF
++
+++ ++
N/A N/A
++
+
++
N/A N/A N/A
N/A N/A N/A
N/A N/A N/A
+++ N/A N/A
Intens. [a.u.]
FBS
4
Intens. [a.u.]
AsPc-1
Stage
A.
Figure 6. MS spectra of Nidogen-1
(ECM
component),
MIB2
(Overexpression
in
certain
cancers), and Palladin (Pancreatic
cancer susceptibility).
615.354
Gene
Cell Line
x104
109086857
10%
5
Panc 3.27
43469
1D
2
Figure 5. Analysis of secreted proteins
from pancreatic cancer cell lines.
Protein-free medium conditioned for
24-72 hrs was concentrated by a
TCA/sarcoysl precipitation method.
Protein bands/spots from 1D (A) and
2D (B) gels were excised, destained,
and identified by MALDI-MS (Table 1).
Conclusions & Future Directions
 Pancreatic cancer cells show minimal perturbations when adapted to growth in serum-free medium and Matrigel substrate.
 We have developed a workflow that can enrich for secretomes and have identified secreted proteins associated with pancreatic cancer.
Future work includes:
 Validation of positive identifications via immunoblotting
 Perform LC-MS/MS analyses on secretome
1
2760.269
1372.787
3087.478
842.509
1229.883
2039.990
2943.464
0
500
1000
1500
2000
2500
3000
3500
m /z
Acknowledgements
This research was funded in part by the College of Science & Math at CSU
Fresno, a CSUPERB Faculty Seed grant and the Robert & Norma Craig
Foundation. Competitive travel awards were generously provided by
CSUPERB and USHUPO.