Intr oduction Resu lts , ZhiYuan (William) Lin , Taunia Closson , Olga

ELK3-51
B1G5
RV202
24E10
C11
RCK105
B56
D1H2
EF5
BrdU
Vimentin
E-cadherin
Pan-keratin
CK7
Ki67
Histone H3
Ir191, Ir193
Yb176
Er168
Dy164
Dy162
Gd158
Gd156
Sm154
Eu151
Metal
201192B
3176016A
3168007B
3164020A
3162027A
3158021A
3156023A
Cat. No.
Nuclear
Nuclear
Nuclear
Cytoplasmic and
membranous
Cytoplasmic and
membranous
Cytoplasmic and
membranous
Cytoplasmic and
membranous
Nuclear
Cytoplasmic and nuclear
Staining
Diagnosis
Ductal ADC, PD
Ductal ADC, WD
Mucinous cyst ADC & Inv. ADSQ
Ductal ADC, MD
Stagea
T3N1b
T3N0
T3N1b
T4N0
K-ras mutationb
K12, CGT
K12, GTT
Wild type
K12, GAT
Toll-free: +1 866 359 4354 • fluidigm.com
South San Francisco, CA 94080 USA
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Fluidigm Corporation
Abbreviations: OCIP, Ontario Cancer Institute Pancreas; ADC, adenocarcinoma; ADSQ,
adenosquamous carcinoma; WD, well-differentiated; MD, moderately differentiated; PD,
poorly differentiated.
aTNM classification of tumors of the exocrine pancreas: T3, tumor extends directly into any
of the following: duodenum, bile duct, peripancreatic tissues; T4, tumor extends directly
into any of the following: stomach, spleen, colon, adjacent large vessels; N0, no regional
lymph node metastasis; N1b, metastasis in multiple regional lymph nodes.
bMutation occurs in "codon 12, base transition."
OCIP No.
23
28
34
51
Table 2. Primary pancreatic cancer specimens [4]
Intercalator
AB Clone
Antibodies/
Reagent
Table 1. Analytes and targets
Mass cytometry [1] uniquely enables high-dimensional,
single-cell proteomic analysis for systems-level discovery and
comprehensive functional profiling applications. At the core of
the technology are an inductively-coupled plasma ion source
and a fast elemental analyzer designed for metal-conjugated
affinity reagents [2].
Imaging mass cytometry (IMC) is projected to bring
quantitative measurement into histo-cytometry while offering
analysis of more than 50 biomarkers and resolution down to 1
µm. The methodology uses tissue sections (FFPE or frozen)
stained with a cocktail of antigen-specific antibodies
conjugated to different metal isotopes. The immunostained
and dried samples are inserted into an ablation chamber
where a small-spot-size pulsed laser scans the tissue. Using
mass cytometry, isotopes associated with each spot are
detected and indexed against the source location, yielding an
intensity map of the target proteins throughout the tissue or
the region of interest. The new platform instrument will
provide researchers with the ability to quantitatively
interrogate tissue sections with a high degree of multiplicity
while
retaining
spatial
information
on
cellular
microenvironment [3].
We have reported that hypoxia is a major adverse prognostic
factor in pancreatic cancer [4], and the results show striking
association between hypoxia and aggressive growth and
metastasis
formation.
To
better
understand
the
heterogeneous distribution of hypoxia in pancreatic cancer
tissue and its consequences, we applied this advanced
technology to human orthotopic primary pancreatic cancer
xenograft tissues to understand the complexity of pancreatic
cancer biology, specifically hypoxic tumor microenvironment.
IMC enables the study of single-cell heterogeneity, signaling
pathways and function in complex pancreatic cancer patient
tissue samples.
Introduction
e-
NO2
N R
O2
.BIND TO
MACROMOLECULES
ANTIBODY DETECTION
O2
Vimentin
E-cadherin
Vimentin
E‐cadherin
•
•
•
•
BrdU/Ki67/Histone H3
IMC
IMC
IMC
EF5/E‐cadherin/Histone H3
IMC
EF5/Ki‐67/Histone H3
IMC
Pan‐keratin/E‐cadherin/HistoneH3
IMC
EF5/Pan‐keratin/Histone H3
EF5/BrdU/Histone H3
IMC
Pan‐keratin/E‐cadherin/Histone H3
IMC
BrdU/Ki67/Histone H3
IMC
IMC results for EF5, Ki67, pankeratin, E-cadherin and histone
H3 of OCIP51 liver metastasis.
Image size: 800x600 µm.
Immunofluorescence images
(left) and IMC results (right) for
EF5 and BrdU of OCIP51 liver
metastasis. Sections for IF and
IMC are cut from the same
block, but not sequential.
Spontaneous formation of liver
metastases (arrows) from
OCIP51, grown at the orthotopic
site (left) [4]. Hematoxylin and
eosin (H&E) staining of OCIP51
liver metastasis (right).
Figure 10. IMC results for EF5, BrdU, Ki67, pan-keratin, E-cadherin
and histone H3 of OCIP51 primary tumor. Image size: 800x1,000 µm.
EF5/Pan‐keratin/Histone H3
IMC
EF5/BrdU/Histone H3
IMC
EF5/E‐cadherin/HistoneH3
EF5/Ki‐67/Histone H3
EF5‐Cy5/BrdU‐AF488/DAPI
100 µm
IF
H&E stain
®
Figure 8. IMC results for EF5, BrdU,
Ki67, E-cadherin, pan-keratin and
histone H3 of the mucinous
adenocarcinoma OCIP34.
Image size: 800x400 µm.
Figure 9. OCIP51 liver metastasis.
EF5/E‐cadherin/Histone H3 Pan‐keratin/E‐cadherin/Histone H3
EF5/BrdU/Histone H3
We thank all members of the Proteomics R&D team and the Reagent
Development team at Fluidigm for their contributions to this study.
Hypoxia is identified using EF5 in human orthotopic pancreatic cancer
xenografts by IMC. Obtained results are comparable to IF hypoxia
detection.
The cells within the hypoxic regions showed epithelial features similar to
the nonhypoxic regions, including maintenance of apical/basal polarity,
membrane localization of E-cadherin. Positive staining for vimentin in the
hypoxic region indicates epithelial-mesenchymal transition.
A striking loss of BrdU incorporation in the EF5-positive regions indicates
that hypoxia causes cell-cycle arrest in these models.
Expand antibody panel to include signaling pathway biomarkers.
Conclusion and future work
Figure 7. IMC results for pan-keratin, CK7,
EF5, E-cadherin and vimentin of OCIP28.
Image size: 800x200 µm.
E‐cadherin/Vimentin/HistoneH3
EF5/E‐cadherin/Histone H3
EF5/Pan‐keratin/Histone H3
Pan‐keratin/CK7/Histone H3
Figure 6. IMC results for pan-keratin, CK7 and
E-cadherin of OCIP28. Image size: 400x100 µm.
E‐cadherin/Pan‐keratin/Histone H3
Pan‐keratin/CK7/Histone H3
Figure 5. IMC results for pan-keratin, CK7, EF5 and
E-cadherin of OCIP23. Image size: 800x200 µm.
Pan‐keratin/E‐cadherin/Histone H3
CK7/E‐cadherin/Histone H3
E‐cadherin/Vimentin/Histone H3
Pan‐keratin/CK7/Histone H3
Figure 4. IMC results for pan-keratin, CK7, EF5 and
E-cadherin of OCIP23. Image size: 400x100 µm.
E‐cadherin/Pan‐keratin/Histone H3
EF5/Pan‐keratin/Histone H3
EF5/E‐cadherin/Histone H3
1. Bandura, D.R., Baranov, V.I., Ornatsky, O.I. et al. “Mass cytometry:
technique for real time single cell multitarget immunoassay based on
inductively coupled plasma time-of-flight mass spectrometry.” Anal.
Chem. 81 (2009): 6813–22.
2. Ornatsky, O., Bandura, D.R., Baranov, V.I. et al. “Highly multiparametric
analysis by mass cytometry.” J. Immunol. Methods 361 (2010): 1–20.
3. Giesen, C., Wang, H.A. et al. “Highly multiplexed imaging of tumor
tissues with subcellular resolution by mass cytometry.” Supplementary
figures. Nat. Methods 11 (2014): 417–22.
4. Chang, Q., Jurisica, I., Do, T., Hedley, D.W. “Hypoxia predicts aggressive
growth and spontaneous metastasis formation from orthotopically grown
primary xenografts of human pancreatic cancer.” Cancer Res. 71 (2011):
3110–20.
References
Figure 3. IMC results for EF5, E-cadherin, vimentin and histone H3 of the
mucinous adenocarcinoma OCIP34. Image size: 800x200 µm.
Histone H3
E‐cadherin
Histone H3
EF5
Figure 2. IMC results for EF5, E-cadherin, vimentin and histone H3 of the
mucinous adenocarcinoma OCIP34. Image size: 400x100 µm.
Histone H3
E-cadherin
Histone H3
EF5
Figure 1. Hypoxia detection using nitroimidazole probes.
EF5, pimonidazole
R=CH2CONHCF2CF3
N
REACTIVE
PRODUCTS
TISSUE FIXATION OF EF5 UNDER HYPOXIA
Results
Pan‐keratin/CK7/Histone H3
Qing Chang1, Zhi Yuan (William) Lin1, Taunia Closson1, Olga Ornatsky1, Sasha Loboda1, Vladimir Baranov1, David Hedley2
1Fluidigm Canada Inc., 2Ontario Cancer Institute/Princess Margaret Hospital, Canada
Assessment of tumor hypoxia and its consequences in human orthotopic primary pancreatic cancer xenografts
using imaging mass cytometry