3. Figure 1
Clinically
end prod
events, w
metastas
sion, tum
(local inv
temically
distant o
survive a
ments of
tion, met
depicted
tive inv
tumor c
program
and int
invasion
stress-fi
Rho/RO
sion’’ p
Metastasis
Valastyan and Weignburg, Cell. 2013
10. Hypothesis: Hypoxic environment promotes CTC
production, intravasation and tumorigenicity
Challenge: No clear definition of CTCs
Single-Cell RNA Sequencing Identifie
Matrix Gene Expression by Pancreat
Cells
Graphical Abstract
Highlights
Pancreatic CTCs can be enriched with antigen-agnostic micro-
fluidic technology
Single-cell RNA sequencing of pancreatic CTCs reveals three
distinct CTC populations
Extracellular matrix genes are highly expressed in mouse and
human CTCs
The extracellular matrix protein SPARC contributes to pancre-
Au
Da
ma
Co
ma
(S.
ha
In
Cir
ric
bu
de
flu
se
fyi
ge
ca
ex
ric
to
Ac
GS
GS
GS
Ting et al.. Cell Reports. 2014
Figure 1. CTC Single-Cell Isolation
(A) Schematic of the CTC-iChip-negative inertial
focusing device system.
(B) Mouse WBC depletion consistency between
normal and cancer mouse models. WBC depletion
is shown in log10.
(C) CTC enumeration by immunofluorescent
staining (CK+/CD45-/DAPI+) from normal and
cancer mice. Bar represents mean.
(D) Representative image of CK-positive CTCs.
DAPI (blue), CK (red), and CD45 (green). Scale bar,
20 mm. Bright-field image highlighting lack of im-
munomagnetic anti-CD45 beads on CK+ CTCs
(white circle).
ECM genes by CTCs may contribute to
the dissemination of cancer to distal
organs.
RESULTS
CTC-iChip
11. 0
25
50
75
100
Count
Subtype
CTC EMT+
CTC platelet
CTC epithelial
Ting et al.. Cell Reports. 2014
CTC heterogeneity
Hypothesis: Hypoxic environment promotes CTC
production, intravasation and tumorigenicity
Article
Single-Cell RNA Sequencing Identifies Extracellular
Matrix Gene Expression by Pancreatic Circulating Tumor
Cells
Graphical Abstract
Highlights
Pancreatic CTCs can be enriched with antigen-agnostic micro-
Authors
David T. Ting, Ben S. Wittner, ..., Shya-
mala Maheswaran, Daniel A. Haber
Correspondence
maheswaran@helix.mgh.harvard.edu
(S.M.),
haber@helix.mgh.harvard.edu (D.A.H.)
In Brief
Circulating tumor cells (CTCs) are en-
riched for the precursors of metastasis,
but their composition has not been fully
defined. Ting et al. have utilized a micro-
fluidic device to perform single-cell RNA
sequencing of pancreatic CTCs, identi-
fying three distinct populations that sug-
gest multiple paths in the metastatic
cascade. Extracellular matrix gene
expression in particular was highly en-
riched in CTCs, pointing to a contribution
to distal spread of cancer.
Accession Numbers
GSE51372
13. 1. Investigate up-regulation of molecules in the hypoxia
signaling pathway in CTCs.
2. Identify the effects of hypoxia on CTC biogenesis.
3. Investigate hypoxia-mediated enhanced tumorigenicity
of CTCs.
Specific aims
Hypothesis: Hypoxic environment promotes CTC
production, intravasation and tumorigenicity
14. Hypoxia signaling is up-regulated in
CTCs
Hypothesis
Aim 1: Investigate up-regulation of
molecules in the hypoxia signaling pathway.
15. Aim 1: Investigate up-regulation of
molecules in the hypoxia signaling pathway.
• KPC mice
A R T I C
Hingorani , et al. CANCER CELL. 2005
Tumor model
16. Ting et al.. Cell Reports. 2014
http://www.cancerresearchuk.org/prod_consump/groups/
cr_common/@cah/@gen/documents/image/
To achieve deep RNA-sequencing profiles of CTCs at the
single-cell level, we applied an inertial focusing-enhanced micro-
fluidic device, the CTC-iChip, which allows high-efficiency nega-
tive depletion of normal blood cells, leaving CTCs in solution
where they can be individually selected and analyzed as single
cells (Ozkumur et al., 2013). This antigen-agnostic isolation of
CTCs enables the characterization of CTCs with both epithelial
and mesenchymal characteristics. Further, the high quality of
Figure 1. CTC Single-C
(A) Schematic of the CTC
focusing device system.
(B) Mouse WBC depletion
normal and cancer mouse
is shown in log10.
(C) CTC enumeration b
staining (CK+/CD45-/DAP
cancer mice. Bar represen
(D) Representative image
DAPI (blue), CK (red), and C
20 mm. Bright-field image
munomagnetic anti-CD45
(white circle).
ECM genes by CTCs
the dissemination of
organs.
RESULTS
Isolation of Mouse P
The CTC-iChip combi
namic size-based sep
ated cells (leukocytes
away from red bloo
and plasma, with s
focusing of the nuclea
gle streamline to achi
in-line magnetic sor
epitopes are highly v
surface markers are
applying magnetic-co
to this very high-throu
cell-separation device
the vast majority of
small number of untagged CTCs (Figure 1A).
ing using 100 anti-CD45 beads per WBC ac
tion in normal mice, mice bearing orthotop
KPC mice (Figure 1B).
We first tested the efficacy of the CTC-i
tagged mouse PDAC cell line (NB508). CTC
the CTC-iChip was measured to be 95% (m
ing GFP-tagged NB508 cells spiked into w
1. Immunofluorescent
staining
2. Hypoxia microarray
Aim 1: Approach
20. 1. Immunofluorescence staining shows HIF1-a in CTCs
2. Downstream targets also up-regulated
Aim 1: Investigate up-regulation of
molecules in the hypoxia signaling pathway.
Predictions
24. Summary
• CTCs show EMT markers with
hypoxia phenotype
• Downstream targets of HIF1-a up-
regulated in CTCs
Aim 1: Investigate up-regulation of
molecules in the hypoxia signaling pathway.
25. 1. Investigate up-regulation of molecules in the hypoxia
signaling pathway in CTC.
2. Identify the effects of hypoxia on CTC biogenesis.
3. Investigate hypoxia-mediated enhanced tumorigenicity
of CTCs.
Specific aims
Hypothesis: Hypoxic environment promotes CTC
production, intravasation and tumorigenicity
26. Loss of HIF1-a reduces CTCs production
Hypothesis
Aim 2: Identify the effects of hypoxia on
CTC biogenesis
27. HIF1a knockdown
MIA PaCa-2 cells
!
Xenograft
Figure 1. CTC Single-Cell Isolation
(A) Schematic of the CTC-iChip-negative inertial
focusing device system.
(B) Mouse WBC depletion consistency between
normal and cancer mouse models. WBC depletion
is shown in log10.
(C) CTC enumeration by immunofluorescent
staining (CK+/CD45-/DAPI+) from normal and
cancer mice. Bar represents mean.
(D) Representative image of CK-positive CTCs.
DAPI (blue), CK (red), and CD45 (green). Scale bar,
20 mm. Bright-field image highlighting lack of im-
munomagnetic anti-CD45 beads on CK+ CTCs
(white circle).
1. Single cell RNA-seq
2. CTC count
3. Tumor Size
Schwab et al. Breast Cancer Research 2012
Yang and Kang. Yonsei Med J. 2008
MIA PaCa-2 cells
Aim 2: Approach
28. Schwab et al. BCR. 2012
• Tumor size
• correlate to previous study
Percenttumors<500
mm3
log-rank p<0.0001
median
WT, 64 days
KO, 127 days
WT
KO
Tumorvolume,mm3
KO
WT
A
Tumorvolume,mm3
Tumorweight,g
p=0.0091
*
Tumorburden,%BW
p=0.0343
*
WT WT WTKO KO KO
p=0.0068
B C
ab et al. Breast Cancer Research 2012, 14:R6
/breast-cancer-research.com/content/14/1/R6
Page 10 of
Aim 2: Predictions
HIF1-a knockdown cells tumor xenograft shows
slower growth rate
29. Published Expected
0
25
50
75
100
Count
Subtype
CTC EMT+
CTC platelet
CTC epithelial
• CTC count
• Decrease in HIF1-a knockout
• Change in CTC subtypes
• Decrease in CTC EMT+ subtype
Aim 2: Predictions
Mice implanted with HIF1-a knockdown cells
have fewer CTCs
30. • Tumor size decreases
• Fewer CTCs
• Change in CTC count is due to loss of
EMT+ subtype
Summary
Aim 2: Identify the effects of hypoxia
on CTC biogenesis
31. 1. Investigate up-regulation of molecules in the hypoxia
signaling pathway in CTC.
2. Identify the effects of hypoxia on CTC biogenesis.
3. Investigate hypoxia-mediated enhanced tumorigenicity
of CTCs.
Specific aims
Hypothesis: Hypoxic environment promotes CTC
production, intravasation and tumorigenicity
32. Aim 3: Investigate hypoxia-mediated
enhanced tumorigenicity of CTCs.
Loss of HIF1-a reduces CTC tumorigenicity
Hypothesis
33. !
Xenograft
Figure 1. CTC Single-Cell Isolation
(A) Schematic of the CTC-iChip-negative inertial
focusing device system.
(B) Mouse WBC depletion consistency between
normal and cancer mouse models. WBC depletion
is shown in log10.
(C) CTC enumeration by immunofluorescent
staining (CK+/CD45-/DAPI+) from normal and
cancer mice. Bar represents mean.
(D) Representative image of CK-positive CTCs.
DAPI (blue), CK (red), and CD45 (green). Scale bar,
20 mm. Bright-field image highlighting lack of im-
munomagnetic anti-CD45 beads on CK+ CTCs
(white circle).
!
Xenograft
Serial
dilution
Aim 3: Approcah
HIF1a knockdown
MIA PaCa-2 cells
MIA PaCa-2 cells
34. !
Xenograft
Figure 1. CTC Single-Cell Isolation
(A) Schematic of the CTC-iChip-negative inertial
focusing device system.
(B) Mouse WBC depletion consistency between
normal and cancer mouse models. WBC depletion
is shown in log10.
(C) CTC enumeration by immunofluorescent
staining (CK+/CD45-/DAPI+) from normal and
cancer mice. Bar represents mean.
(D) Representative image of CK-positive CTCs.
DAPI (blue), CK (red), and CD45 (green). Scale bar,
20 mm. Bright-field image highlighting lack of im-
munomagnetic anti-CD45 beads on CK+ CTCs
(white circle).
Serial
dilution
Aim 3: Approcah
HIF1a knockdown
MIA PaCa-2 cells
MIA PaCa-2 cells
3D soft
fibrin matrix
assay /
invasive
assay
35. C
GAPDH
TRC
1 day
3 days
5 days
7 days
*
*
Control
800
700
600
500
Colonysize(×103µm3)
400
300
200
100
0
1 2
Culture time on plastic (day)
0
0
0.05
Cellstiffness(kPa)
0.1
0.15
0.2
0.25
3 4 5
Rel
6
4
2
0
ition of Sox2 expression and self-renewal of TRCs on 2D rigid subs
Aim 3: Investigate hypoxia-mediated
enhanced extravasation of CTCs.
Alternative: 3D soft fibrin matrix assay
Tan, et al. Nature Comm. 2014
36. • Decrease in tumorigenicity from CTCs
collected from mice implanted with HIF1-
a knockdown MIA PaCa-2 cells
Aim 3: Expected result
