wu hypoxia

Hypoxia and Circulating Tumor Cells in

Pancreatic CancerDouglas Wu

Lambowitz lab

Pancreatic cancer

http://i.usatoday.net/yourlife/_photos/2011/10/05/Jobs-battle-with-pancreatic-cancer-RRESHAC-x-large.jpg

- Low survival rate - Highly metastatic

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coimplantationwithmyoepithelial cells reversed the invasivenessof breast carcinoma xenografts (Hu et al., 2008). Similarly, inovarian carcinomas, the mesothelial cell layer that lines perito-neal and pleural organs serves as an obstacle to further dissem-ination that can be overcomeby carcinoma cell-exerted,myosin-dependent traction forces that physically displace mesothelialcells (Iwanicki et al., 2011). Moreover, modulation of ECM stiff-ness, achieved by altering collagen crosslinking, affects breastcarcinoma progression via altered integrin signaling (Leventalet al., 2009).

At a cell-biological level, most types of carcinomas can invadeas cohesivemulticellular units through a process termed ‘‘collec-

Figure 1. The Invasion-Metastasis CascadeClinically detectable metastases represent theend products of a complex series of cell-biologicalevents, which are collectively termed the invasion-metastasis cascade. During metastatic progres-sion, tumor cells exit their primary sites of growth(local invasion, intravasation), translocate sys-temically (survival in the circulation, arrest at adistant organ site, extravasation), and adapt tosurvive and thrive in the foreign microenviron-ments of distant tissues (micrometastasis forma-tion, metastatic colonization). Carcinoma cells aredepicted in red.

Figure 2. Stromal Cells Play Vital Rolesduring the Invasion-Metastasis CascadeMetastatic progression is not an exclusively cell-autonomous process. Indeed, carcinoma cellsenlist nonneoplastic stromal cells to aid in eachstep of the invasion-metastasis cascade. Exam-ples of the roles of stromal cells during metastasisare illustrated. Carcinoma cells are depicted inred. Angptl4, angiopoietin-like 4; CSF-1, colony-stimulating factor 1; EGF, epidermal growthfactor; IL-4, interleukin 4; MMP-9, matrix metal-loproteinase 9; OPN, osteopontin; SDF-1, stromalcell-derived factor 1.

tive invasion.’’ Alternatively, individualtumor cells may invade via two distinctprograms: the protease-, stress-fiber-,and integrin-dependent ‘‘mesenchymalinvasion’’ program or the protease-,stress-fiber-, and integrin-independent,Rho/ROCK-dependent ‘‘amoeboid inva-sion’’ program (Friedl and Wolf, 2003).

Indeed, differential expression of molecules that enable eithermesenchymal or amoeboid invasion can be observed in signa-tures of local invasiveness derived from mammary carcinomamodels (Wang et al., 2004).Tumor cells can apparently interconvert between these

various invasion strategies in response to changing microenvi-ronmental conditions. This has caused some to propose thatrobust suppression of single-cell invasion requires concomitantinhibition of the mesenchymal and amoeboid invasion programs(Friedl and Wolf, 2003). Indeed, certain regulators of invasionfunction as pleiotropically acting factors that simultaneouslymodulate components of both pathways. For example, the

276 Cell 147, October 14, 2011 ª2011 Elsevier Inc.

Metastasis

Valastyan and Weignburg, Cell. 2013

Circulating tumor cells

http://www.verastem.com/research/ http://www.eurostemcell.org/de/node/16637

Cancer stem cells

http://www.eurostemcell.org/de/node/16637

Epithelial-mesenchymal transition promotes invasion

Peinado, et al. Nat Rev. 2007 http://blogs.scientificamerican.com/guest-blog/2013/10/30/the-hallmarks-of-cancer-6-tissue-invasion-and-metastasis/

http://blogs.scientificamerican.com/guest-blog/2013/10/30/the-hallmarks-of-cancer-6-tissue-invasion-and-metastasis/

Hypoxic environment promotes EMT

Peinado & Cano. Nature Cell Biology. 2008http://cascadeprodrug.com/main/img_1271875515_14862_1283460123_mod_618_316.jpg

http://cascadeprodrug.com/main/img_1271875515_14862_1283460123_mod_618_316.jpg

Peinado & Cano. Nature Cell Biology. 2008

Hypoxic environment promotes EMT

Normoxia

HIF1-a

Hydroxyprolination

Ubiquitination by VHL

Proteasome

Hypoxia

HIF1-a

Hydroxyprolination inhibited

No ubiquitination by VHL

Transcription

Hypoxic environment promotes CTC production, intravasation and tumorigenicity

Hypothesis

Hypothesis: Hypoxic environment promotes CTC production, intravasation and tumorigenicity

Challenge: No clear definition of CTCs

Article

Single-Cell RNA Sequencing Identifies ExtracellularMatrix Gene Expression by Pancreatic Circulating TumorCells

Graphical Abstract

HighlightsPancreatic 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-

atic tumor metastasis

AuthorsDavid T. Ting, Ben S. Wittner, ..., Shya-

mala Maheswaran, Daniel A. Haber

[email protected](S.M.),[email protected] (D.A.H.)

In BriefCirculating 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 NumbersGSE51372

GSE60407

GSE51827

Ting et al., 2014, Cell Reports 8, 1905–1918September 25, 2014 ª2014 The Authorshttp://dx.doi.org/10.1016/j.celrep.2014.08.029

Ting et al.. Cell Reports. 2014

To achieve deep RNA-sequencing profiles of CTCs at thesingle-cell level, we applied an inertial focusing-enhancedmicro-fluidic device, the CTC-iChip, which allows high-efficiency nega-tive depletion of normal blood cells, leaving CTCs in solutionwhere they can be individually selected and analyzed as singlecells (Ozkumur et al., 2013). This antigen-agnostic isolation ofCTCs enables the characterization of CTCs with both epithelialand mesenchymal characteristics. Further, the high quality ofRNA purified from viable, untagged CTCs is particularly wellsuited for detailed transcriptome analysis. We applied theCTC-iChip to the pancreatic cancer mouse model that allowsfor simultaneous analysis of primary tumor and CTCs, with theshared driver mutations across different animals facilitating theidentification of CTC-specific heterogeneity. Here, we presenta comprehensive transcriptome analysis of CTCs at the single-cell level, pointing to distinct cell subsets within CTC popula-tions. Notably, we have identified the unexpected abundantexpression of extracellular matrix (ECM) genes in mouse pancre-atic CTCs and across human CTCs of pancreatic, breast, andprostate origin. Consistent with the importance of tumorstroma-derived ECM signaling in targeting cancer cell metas-tasis (Zhang et al., 2013), the cell-autonomous expression of

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 tothe dissemination of cancer to distalorgans.

RESULTS

Isolation of Mouse Pancreatic CTCsThe CTC-iChip combines initial hydrody-namic size-based separation of all nucle-ated cells (leukocytes [WBCs] and CTCs)away from red blood cells, platelets,and plasma, with subsequent inertialfocusing of the nucleated cells into a sin-gle streamline to achieve high-efficiencyin-line magnetic sorting. While tumorepitopes are highly variable, WBC cell-surface markers are well established;applying magnetic-conjugated anti-WBCto this very high-throughput microfluidiccell-separation device can thus excludethe vast majority of WBCs to reveal a

small number of untagged CTCs (Figure 1A). Whole-blood label-ing using 100 anti-CD45 beads per WBC achieved >103 deple-tion in normal mice, mice bearing orthotopic tumors, and theKPC mice (Figure 1B).We first tested the efficacy of the CTC-iChip using a GFP-

tagged mouse PDAC cell line (NB508). CTC recovery throughthe CTC-iChip was measured to be 95% (mean ± 3% SD), us-ing GFP-tagged NB508 cells spiked into whole mouse blood.Applying the CTC-iChip to orthotopic tumors derived frompancreatic inoculation of GFP-tagged NB508 cells generated>1,000 CTCs/ml in all three mice tested (Figure 1C). Finally,CTC analysis of blood specimens from KPC mice bearingendogenous tumors, using dual immunofluorescent stainingof cells with the epithelial marker pan-cytokeratin (CK) andthe leukocyte marker CD45, revealed a median 118 CTCs/ml(mean 429 CTCs/ml; range, 0–1,694) (Figures 1C and 1D). NoCK-positive cells were detected in seven healthy controlmice. The majority of CD45-positive cells that remained inthe product after blood processing through the microfluidic de-vice retained immunomagnetic beads on their surface. Thus,the untagged cells constituting CTCs were readily distin-guished from WBCs in the final CTC-iChip product (Figure 1D),

1906 Cell Reports 8, 1905–1918, September 25, 2014 ª2014 The Authors

CTC-iChip

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SubtypeCTC EMT+CTC plateletCTC epithelial


CTC heterogeneity


Article

Single-Cell RNA Sequencing Identifies ExtracellularMatrix Gene Expression by Pancreatic Circulating TumorCells

Graphical Abstract

HighlightsPancreatic 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-

atic tumor metastasis

AuthorsDavid T. Ting, Ben S. Wittner, ..., Shya-

mala Maheswaran, Daniel A. Haber

[email protected](S.M.),[email protected] (D.A.H.)

In BriefCirculating 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 NumbersGSE51372

GSE60407

GSE51827

Ting et al., 2014, Cell Reports 8, 1905–1918September 25, 2014 ª2014 The Authorshttp://dx.doi.org/10.1016/j.celrep.2014.08.029

p−value = 0.000382

Hif1a

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60

90

norm

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d m

ean

coun

t

annotationcirculating tumor cells (n=109)primary tumor (TuGMP3) (n=38)

HIF1-a mRNA is more abundant in CTCs

Yasuda et al. BBRC. 2014

• HIF1-a can be post-transcriptionally regulated through its 3’ UTR

Thanks to Pubmed’s GEO Datasets!

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


Hypoxia signaling is up-regulated in CTCs

Hypothesis

Aim 1: Investigate up-regulation of molecules in the hypoxia signaling pathway.


• KPC mice A R T I C L E

Figure 1. Targeting endogenous KrasG12D and Trp53R172H expression to the mouse pancreas

A: Endogenous alleles of KrasG12D and Trp53R172H are conditionally activated in the pancreata of LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (triple mutant)mice. Specific PCR analysis of genomic DNA from the pancreata, but not the tails, of triple mutant mice reveals the expected “1LoxP” recombinationproduct for each targeted allele.B: Survival of LSL-KrasG12D/+;LSL- Trp53R172H/+;Pdx-1-Cre mice is significantly decreased. Kaplan-Meier curves reveal a median survival in triple mutant miceof approximately 5 months, significantly less than wild-type (WT), LSL-Trp53R172H/+;Pdx-1-Cre, and LSL-KrasG12D/+;Pdx-1-Cre mice (p < 0.001, log-rank test, foreach pairwise combination).

lesions in these young animals, with demonstrated rearrange- and frequently contained mucin, as demonstrated by Alcianblue staining (Figure 2N). Metastases to the liver (Figures 2Iment of the mutant alleles, were observed infrequently (data

not shown). A similar stage and degree of disease burden oc- and 2J) and lungs (Figures 2K and 2L) were morphologicallysimilar to the pancreatic primaries.curs in animals expressing only endogenous KrasG12D (Hingor-

ani et al., 2003). Thus, invasive tumors did not appear to de- Some tumors also contained minor, poorly differentiated, orundifferentiated components with anaplastic (n = 7/25; Supple-velop in utero or even in the immediate postnatal period, a

latency that suggests the requirement for additional genetic mental Figure S1A) or sarcomatoid features (n = 2/25; Supple-mental Figures S1B and S1C). Noninvasive cystic papillaryevents for disease progression.

A significant disease burden did become apparent in animals neoplasms (n = 3/25; Supplemental Figure S1D) were occa-sionally noted, as were areas of focal adenosquamous mor-by 10 weeks of age at the earliest. Importantly, the full spec-

trum of preinvasive lesions was apparent in these mice (Figure phology (n = 3/25, Supplemental Figures S1E and S1F), an un-common form of human pancreatic ductal cancer comprising2G and data not shown). Histologic analyses of primary pan-

creatic carcinomas from triple mutant animals revealed a pre- 3%–4% of reported cases (Kardon et al., 2001). Finally, severalanimals developed esophageal papillomas (Supplemental Fig-dominant moderately well-differentiated to well-differentiated

morphology organized in a glandular architecture (Figure 2H) in ure S1G) and hyperplasias, or papillomatosis, of the biliary tree(Supplemental Figures S1H and S1I), likely reflecting expres-the majority of mice (n = 22/25; Table 1), as is observed in the

human disease. The carcinomas expressed CK-19 (Figure 2O) sion of Pdx-1 in the developing foregut endoderm.

CANCER CELL : MAY 2005 471

Hingorani , et al. CANCER CELL. 2005

Tumor model


http://www.cancerresearchuk.org/prod_consump/groups/cr_common/@cah/@gen/documents/image/crukmig_1000img-12231.jpg






is shown in log10.








(white circle).


RESULTS





1. Immunofluorescent staining

2. Hypoxia microarray

Aim 1: Approach

http://www.cancerresearchuk.org/prod_consump/groups/cr_common/@cah/@gen/documents/image/crukmig_1000img-12231.jpg

treated (48 h) or normoxia-treated control cells were seeded at aconcentration of 105 cells per cm2 of 24-well plates. Afterwards thecells were exposed to hypoxia (Migration measured underHypoxia) or normoxia (Migration measured under Hypoxia) foradditional 48 h and the number of transmigrated cells wascounted. The percentage of transmigrated cells was normalized tothe percentage of cell vitality evaluated by an MTT assay at theendpoint of the experiment.

Statistical analysisQuantitative data are presented as the mean 6 SD. Data were

analyzed using the Student’s t test for statistical significance.P,0.05 was considered statistically significant.

Results

Expression of hypoxia-, EMT- and CSC-markers inpancreatic cancer tissue

To study co-expression of hypoxia- and EMT-markers weperformed double immunofluorescence staining of patient-derivedfrozen tissue samples of pancreatic ductal adenocarcinoma. InHIF-1a positive regions E-cadherin was down-regulated and Slugup-regulated revealing tumor-hypoxia-induced EMT (Fig. 1A). Insome tumor areas, cells with either Vimentin or E-cadherinexpression were observed in close proximity (Fig. 1B). Thisindicates that surrounding stromal cells are positive for Vimentinand negative for E-cadherin. Most interestingly, hypoxic regionspositive for the hypoxia marker HIF-2a showed co-staining withCD133, suggesting that tumor hypoxia is associated withexpression of CSC markers (Fig. 1C). These data confirm thathypoxia-driven EMT occurs in tumor tissue of patients withpancreatic cancer and CSC-positive tumor cells are present inhypoxic tumor microenvironments.

Hypoxia induces HIF-1a signaling and morphologicalchanges in vitro

For more detailed evaluation of hypoxia-induced EMT and theinfluence to differentiated and CSC-like pancreatic cancer cells weused five established human cell lines of PDA. According to thedegree of differentiation of the primary tumor, mutations in K-rasor p53, colony- and spheroid-forming capacity, ALDH activity,tumorigenicity in mice and expression of E-cadherin andVimentin we classified these cell lines as CSChigh or CSClow

(Table 1). Hypoxia was induced by incubation of cells in a gasmixture of 1% O2, 5% CO2 and 94% N2. This resulted in fast up-regulation of HIF-

1a and its target gene VEGF within 2 hours in both, CSClow

and CSChigh cells as examined by Western blot analysis. Incontrast, cells cultured under normoxic conditions did not up-regulate these proteins (Fig. 2A, Data not shown). CSClow cells hadan expression peak between 6 and 12 hours, which graduallydeclined over time but was still visible at 72 h. In contrast, CSChigh

cells had an earlier peak between 4 and 6 h, which was diminishedto very low levels already at 24 hours and was undetectable at72 h in both cell lines. In contrast, VEGF expression was steadilyenhanced during a period of 72 h. In line with the observedhypoxia-related signaling the number of cells with a fibroblastoid-like phenotype increased within 72 h from 20 to 28% in CSClow

cells and from 28 to 33% in CSChigh cells (Fig. 2C). Induction ofthe percentage of spindle-shaped cells was even more pronouncedand higher in CSChigh cells upon TGF-b, a well known-inducer ofEMT, which was used as positive control. Similar morphologicalchanges upon exposure to hypoxia were observed in Capan-1,Capan-2 and AsPC-1 cancer cells (Figure S1). These data suggest

that in vitro induction of hypoxia induces EMT in both cellpopulations - more differentiated and CSC-like pancreatic cancercells, but the effect is faster and stronger in CSChigh cells.

Hypoxia up-regulates EMT-related protein expressionFor further evaluation of cell-type specific effects of hypoxia-

induced EMT we examined expression of proteins involved inEMT in the two CSClow cell lines BxPc-3 and Capan-2 and in thethree CSChigh cell lines MIA-PaCa2, AsPC-1 and Capan-1. Afterexposure to normoxia or hypoxia for 48 h we labeled the cells withspecific antibodies and analyzed fluorescence by double immuno-fluorescence microscopy. While both CSClow cell lines had strongbasal expression of E-cadherin but low expression of Vimentin, asexpected, hypoxia induces downregulation of E-cadherin and

Figure 1. Co-expression of hypoxia, EMT and CSC markers inpancreatic cancer tissues. (A) Double immunofluorescence stainingsof tumor samples from patients with pancreatic cancer. Nuclearexpression of HIF-1a (green, arrow) and membrane expression of E-cadherin (red, arrow) or Slug (red, arrow) is shown. Yellow color onmerged images indicates co-expression of E-cadherin or Slug in HIF-1a-positive cells. (B) Membrane expression of E-cadherin (red), cytoplasmicexpression of Vimentin (green) along with Dapi-staining of nuclei (blue).White squares indicate Vimentin-positive cells within the tumor massthat are negative for E-cadherin or vice versa. Bar: 100 mm. Twofoldmagnifications of the areas surrounded by white squares are shown onthe left. (C) Nuclear expression of HIF-2a and membrane expression ofCD133 is shown as single staining and as merged staining in which theyellow color indicates double-positivity.doi:10.1371/journal.pone.0046391.g001

Hypoxia-Induced EMT in Pancreatic Cancer

PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e46391

Aim 1: Immunofluorescence stain

Salnikov, et al. PLoS One. 2013

treated (48 h) or normoxia-treated control cells were seeded at aconcentration of 105 cells per cm2 of 24-well plates. Afterwards thecells were exposed to hypoxia (Migration measured underHypoxia) or normoxia (Migration measured under Hypoxia) foradditional 48 h and the number of transmigrated cells wascounted. The percentage of transmigrated cells was normalized tothe percentage of cell vitality evaluated by an MTT assay at theendpoint of the experiment.

Statistical analysisQuantitative data are presented as the mean 6 SD. Data were

analyzed using the Student’s t test for statistical significance.P,0.05 was considered statistically significant.

Results

Expression of hypoxia-, EMT- and CSC-markers inpancreatic cancer tissue

To study co-expression of hypoxia- and EMT-markers weperformed double immunofluorescence staining of patient-derivedfrozen tissue samples of pancreatic ductal adenocarcinoma. InHIF-1a positive regions E-cadherin was down-regulated and Slugup-regulated revealing tumor-hypoxia-induced EMT (Fig. 1A). Insome tumor areas, cells with either Vimentin or E-cadherinexpression were observed in close proximity (Fig. 1B). Thisindicates that surrounding stromal cells are positive for Vimentinand negative for E-cadherin. Most interestingly, hypoxic regionspositive for the hypoxia marker HIF-2a showed co-staining withCD133, suggesting that tumor hypoxia is associated withexpression of CSC markers (Fig. 1C). These data confirm thathypoxia-driven EMT occurs in tumor tissue of patients withpancreatic cancer and CSC-positive tumor cells are present inhypoxic tumor microenvironments.

Hypoxia induces HIF-1a signaling and morphologicalchanges in vitro

For more detailed evaluation of hypoxia-induced EMT and theinfluence to differentiated and CSC-like pancreatic cancer cells weused five established human cell lines of PDA. According to thedegree of differentiation of the primary tumor, mutations in K-rasor p53, colony- and spheroid-forming capacity, ALDH activity,tumorigenicity in mice and expression of E-cadherin andVimentin we classified these cell lines as CSChigh or CSClow

(Table 1). Hypoxia was induced by incubation of cells in a gasmixture of 1% O2, 5% CO2 and 94% N2. This resulted in fast up-regulation of HIF-

1a and its target gene VEGF within 2 hours in both, CSClow

and CSChigh cells as examined by Western blot analysis. Incontrast, cells cultured under normoxic conditions did not up-regulate these proteins (Fig. 2A, Data not shown). CSClow cells hadan expression peak between 6 and 12 hours, which graduallydeclined over time but was still visible at 72 h. In contrast, CSChigh

cells had an earlier peak between 4 and 6 h, which was diminishedto very low levels already at 24 hours and was undetectable at72 h in both cell lines. In contrast, VEGF expression was steadilyenhanced during a period of 72 h. In line with the observedhypoxia-related signaling the number of cells with a fibroblastoid-like phenotype increased within 72 h from 20 to 28% in CSClow

cells and from 28 to 33% in CSChigh cells (Fig. 2C). Induction ofthe percentage of spindle-shaped cells was even more pronouncedand higher in CSChigh cells upon TGF-b, a well known-inducer ofEMT, which was used as positive control. Similar morphologicalchanges upon exposure to hypoxia were observed in Capan-1,Capan-2 and AsPC-1 cancer cells (Figure S1). These data suggest

that in vitro induction of hypoxia induces EMT in both cellpopulations - more differentiated and CSC-like pancreatic cancercells, but the effect is faster and stronger in CSChigh cells.

Hypoxia up-regulates EMT-related protein expressionFor further evaluation of cell-type specific effects of hypoxia-

induced EMT we examined expression of proteins involved inEMT in the two CSClow cell lines BxPc-3 and Capan-2 and in thethree CSChigh cell lines MIA-PaCa2, AsPC-1 and Capan-1. Afterexposure to normoxia or hypoxia for 48 h we labeled the cells withspecific antibodies and analyzed fluorescence by double immuno-fluorescence microscopy. While both CSClow cell lines had strongbasal expression of E-cadherin but low expression of Vimentin, asexpected, hypoxia induces downregulation of E-cadherin and

Figure 1. Co-expression of hypoxia, EMT and CSC markers inpancreatic cancer tissues. (A) Double immunofluorescence stainingsof tumor samples from patients with pancreatic cancer. Nuclearexpression of HIF-1a (green, arrow) and membrane expression of E-cadherin (red, arrow) or Slug (red, arrow) is shown. Yellow color onmerged images indicates co-expression of E-cadherin or Slug in HIF-1a-positive cells. (B) Membrane expression of E-cadherin (red), cytoplasmicexpression of Vimentin (green) along with Dapi-staining of nuclei (blue).White squares indicate Vimentin-positive cells within the tumor massthat are negative for E-cadherin or vice versa. Bar: 100 mm. Twofoldmagnifications of the areas surrounded by white squares are shown onthe left. (C) Nuclear expression of HIF-2a and membrane expression ofCD133 is shown as single staining and as merged staining in which theyellow color indicates double-positivity.doi:10.1371/journal.pone.0046391.g001

Hypoxia-Induced EMT in Pancreatic Cancer

PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e46391

Salnikov, et al. PLoS One. 2013


Expected results in CTCs

VimentinHIF1-aDAPI


1. Immunofluorescence staining shows HIF1-a in CTCs

2. Downstream targets also up-regulated


Predictions

HIF1-a is a master regulator in hypoxia signaling

HIF1-aSurvival - bcl2 - Vegfa

Proliferation - Igf2 Immmunosuppress

- Mif - Il10

Metabolism - Ldha - Pdk1

Motility - Met - Mmp

Angiogenesis - Vegfa

EMT - Snai2 - Twist1

ECM modulation - Mmp

Single cell RNA-seq of CTCs had active hypoxia signaling

Downstream targets of HIF1-a are also up-regulated

p−value = 0.0436 p−value = 0.00225 p−value = 0.256 p−value = 0.000142 p−value = 0.00254 p−value = 0.00145

p−value = 1.72e−05 p−value = 0.0195 p−value = 0.0315 p−value = 0.192 p−value = 0.00205 p−value = 1.03e−08

Bcl2 Igf2 Il10 Ldha Met Mif

Mmp14 Pdk1 Snai2 Twist1 Vegfa Vim0

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annotationcirculating tumor cells (n=109)primary tumor (TuGMP3) (n=38)

MP2.1MP2.11MP2.17

MP2.18MP2.2

MP2.20

MP2.21MP2.24MP2.26MP2.30

MP2.32

MP2.36

MP2.4MP3.15MP3.17

MP3.2

MP3.21MP3.3MP3.5MP3.8MP3.9MP4.1MP4.13MP4.14MP4.17MP4.20MP4.22MP4.24MP4.28MP4.29MP4.3MP4.31MP4.32MP4.4MP4.6MP4.7MP4.8MP6.10

MP6.11

MP6.15MP6.16MP6.17

MP6.18

MP6.19MP6.2MP6.20MP6.21MP6.3MP6.4MP6.5

MP6.6

MP6.7MP6.9

MP7.1

MP7.12

MP7.13

MP7.16

MP7.18

MP7.20MP7.21

MP7.25

MP7.29

MP7.3

MP7.30MP7.31

MP7.33MP7.34MP7.37MP7.4MP7.40

MP7.41MP7.42MP7.7

MP7.8MP7.9

TuGMP3.1.051413TuGMP3.14.051413TuGMP3.15.051413TuGMP3.16.051413TuGMP3.17.051413TuGMP3.19.051413TuGMP3.2.051413TuGMP3.20.051413TuGMP3.22.051413TuGMP3.24.051413TuGMP3.25.051413TuGMP3.26.051413TuGMP3.31.051413TuGMP3.34.051413TuGMP3.35.051413TuGMP3.4.051413TuGMP3.5.051413TuGMP3.6.051413TuGMP3.7.051413TuGMP3.8.051413

Bcl2

Igf2

Il10

Ldha

Met

MifMmp14

Pdk1

Snai2

Twist1Vegfa

Vim

−4

0

4

0.0 2.5 5.0 7.5 10.0PC1

PC2

Cell type

aa

circulating tumor cellsprimary tumor (TuGMP3)

Single cell RNA-seq of CTCs had active hypoxia signaling

Biplot: HIF1-a downstream targets introduce variations between CTCs and primary tumor cells

Summary• CTCs show EMT markers with

hypoxia phenotype • Downstream targets of HIF1-a up-

regulated in CTCs


1. Investigate up-regulation of molecules in the hypoxia signaling pathway in CTC.



Specific aims


Loss of HIF1-a reduces CTCs production

Hypothesis

Aim 2: Identify the effects of hypoxia on CTC biogenesis

HIF1a knockdown MIA PaCa-2 cells

!Xenograft






is shown in log10.








(white circle).


RESULTS





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

Schwab et al. BCR. 2012

• Tumor size• correlate to previous study

Days post injection

Percenttumors<500

mm3 log-rank p<0.0001

medianWT, 64 daysKO, 127 days

WTKO

Days after cell transplant

Tumorvolume,mm3

KOWT

ATumorvolume,mm3

Tumorweight,g

p=0.0091

*

Tumorburden,%

BW

p=0.0343

*

WT WT WTKO KO KO

p=0.0068

21 31 2 3

WTTumors

KOTumors

HIF-1α110

76

160

CRM

MTEC,0.5% O2WT KO

D E

Vegf Pgk1 Glut1

MeanfolddecreaseinKO

FKi67

p=0.03 * *p=0.002

caspase-3

%ofpositivecells

B C

Figure 3 Deletion of Hif1a decreases primary tumor growth. (A) Wild-type (WT) or knockout (KO) cells (n = 50,000) were transplanted intoFVB/Nj mice. All tumors were harvested at day 56 to evaluate tumor weight, volume and burden (percentage tumor weight/total body weight,BW) (n = 10 recipients/genotype, P < 0.05, unpaired Student’s t-test). (B) The growth rate of WT and KO tumors following transplant of 50,000cells. Best-fit curves were established based on a polynomial fit algorithm using GraphPad Prism 4.0 software. Data in (A) and (B) arerepresentative of seven independent experiments (> 60 recipients/genotype). (C) When 500 WT and KO cells were implanted into the mammaryfat pad, the median time until 50% of recipients developed tumors > 500 mm3 was 64 days for WT mice and 127 days for KO mice (n = 14recipients/genotype, P < 0.001, logrank test). (D) Western blot for HIF-1a in three independent tumors (500 to 750 mm3) per genotype. WT andKO cells that were cultured for 6 hours under hypoxic conditions served as positive and negative controls, respectively. CRM, cross-reactivematerial. MTEC, mammary tumor epithelial cell. (E) Mean fold change ± SEM in expression of HIF-1 targets in KO tumors as determined by qRT-PCR (n = 5 tumors/genotype). Decreased gene expression in KO tumors is presented as a negative fold-change relative to WT tumors. (F) Anincrease in Ki67+ cells in KO tumors is balanced by an increase in caspase 3-positive cells (n = 5 tumors/genotype, *P < 0.05, Student’s t-test).Representative immunostaining images are shown in Additional file 2 Figure S4.

Schwab et al. Breast Cancer Research 2012, 14:R6http://breast-cancer-research.com/content/14/1/R6

Page 10 of 25

Aim 2: PredictionsHIF1-a knockdown cells tumor xenograft shows

slower growth rate

Published Expected

0

25

50

75

100

Cou

nt

SubtypeCTC EMT+CTC plateletCTC epithelial

• CTC count• Decrease in HIF1-a knockout

• Change in CTC subtypes • Decrease in CTC EMT+ subtype

Aim 2: PredictionsMice implanted with HIF1-a knockdown cells

have fewer CTCs

• 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

1. Investigate up-regulation of molecules in the hypoxia signaling pathway in CTC.



Specific aims


Aim 3: Investigate hypoxia-mediated enhanced tumorigenicity of CTCs.

Loss of HIF1-a reduces CTC tumorigenicity

Hypothesis

!Xenograft






is shown in log10.








(white circle).


RESULTS





!Xenograft

Serial dilution

Aim 3: Approcah


MIA PaCa-2 cells

!Xenograft






is shown in log10.








(white circle).


RESULTS





Serial dilution

Aim 3: Approcah


MIA PaCa-2 cells

3D soft fibrin matrix

assay / invasive assay

Tumour-initiating cells (TICs) are a self-renewing, highlytumorigenic subpopulation of cancer cells. They play acritical role in the initiation and progression of cancer1.

These tumorigenic cells exhibit high chemo-resistance toconventional chemotherapeutic drug treatment and thereforeare speculated to be key players in cancer relapses afterchemotherapy2. However, the concept of TICs has beencontroversial. Past reports show that a high percentage (425%)of human melanoma cells can generate a tumour in a NOD-SCIDinterleukin-2 receptor-g chain null (IL2rg! /! ) mouse3,4,suggesting that there is no hierarchy of clonal repopulation inmelanoma. We recently developed a mechanical method ofselecting TICs from cancer cell lines and primary cancer cells byculturing single cancer cells in soft fibrin gels5. Remarkably, inaddition to being able to generate local primary tumours in wild-type syngeneic mice, when injected in tail veins, as few as ten ofsuch cells can generate distant metastatic colonization and growtumours in the lungs of wild-type non-syngeneic mice. Therefore,we functionally define these soft-fibrin-gel-selected melanomacells as tumour-repopulating cells (TRCs) based on their highefficiency in repopulating tumours in wild-type syngeneic andnon-syngeneic mice. Soon after our report, three other groupsindependently provide strong experimental evidence in mice thatTRCs do exist in brain6, skin7 and intestinal8 tumours. In vivoimaging of unperturbed tumours further confirmed the existenceof TRCs9,10. However, the underlying mechanisms of howTRCs maintain their self-renewing capability remain elusive.In the current study, we demonstrate that melanoma TRCsexhibit plasticity in mechanical stiffening, histone 3 lysineresidue 9 (H3K9) methylation, Sox2 expression and self-renewal. Three-dimensional (3D) soft fibrin matrices promote

H3K9 demethylation and increase Sox2 expression and self-renewal, whereas stiff ones exert opposite effects.

ResultsSelf-renewal plasticity of TRCs. It is known that soft substratescan sustain self-renewal of mouse embryonic stem cells11 andsubstrate rigidity can regulate the fate of mesenchymal stemcells12, indicating that rigidity of extracellular matrix plays animportant role in the maintenance and regulation of stem cellproperties. As TRCs are selected from a population of melanomacells that are usually cultured on rigid plastic, we asked whatwould happen if we plated these TRCs back to rigid substrates. Todetermine the effect of substrate rigidity on the gene expression,we cultured TRCs on rigid plastic for 1, 3, 5 and 7 days, andquantified their Sox2 expression. TRCs gradually lost Sox2expression in both mRNA and protein levels along with theculture time on plastic (Fig. 1a,b and Supplementary Fig. 18c,d).Sox2 expression of TRCs dramatically decreased after 1 day andwas as low as that of control cells after 3 days on plastic. Otherstem cell genes Bmi-1, C-kit, Nestin and Tert, which areupregulated in TRCs5, were also downregulated after culture onplastic (Supplementary Fig. 1).

To examine the functional consequences of the loss of Sox2 andother stem cell genes, we re-plated those TRCs back into 90-Pasoft fibrin gels after culture on rigid substrates for 1, 3, 5 and 7days, respectively. The growth rate of spheroids in fibrin matricessuccessively decreased with the culture time of TRCs on plastic(Fig. 1c), which is not a result of the increased apoptosis rate(Supplementary Fig. 2). Moreover, colony number was alsodecreased (Supplementary Fig. 3). After 7-day culture on plastic,

Sox2Con

trol

TRC1 d

ay3 d

ays

5 day

s

7 day

s

Contro

lTRC

1 day

3 day

s

5 day

s

7 day

s

GAPDH

GAPDH

TRC1 day3 days5 days7 days

*

** **

*

** *

**

*

**

*Control

800700600500

Col

ony

size

(×1

03 µ

m3 )

400300200100

01 2Culture time on plastic (day) Culture time of TRC on plastic (day)

Control753100

0.05Cel

l stif

fnes

s (k

Pa)

0.1

0.15

0.2

0.250.15 kPa0.6 kPa8 kPaGlass

3 4 5

Sox2

200180 * * Sox2

*

160140

Rel

ativ

e ex

pres

sion

141210

86420

Figure 1 | Inhibition of Sox2 expression and self-renewal of TRCs on 2D rigid substrates. (a) Sox2 expression at both mRNA level (top panel) andprotein level (bottom panel). Control: B16-F1 cells cultured on plastic. TRC: Control B16-F1 cells were cultured in 90-Pa fibrin gels for 5 days. 1 day, 3 days,5 days or 7 days: TRCs were seeded on 2D rigid dishes for 1, 3, 5 or 7 days. Images are representatives of three independent RT–PCRs and two independentwestern blotting experiments. (b) Quantification of Sox2 expression by real time RT–PCR. Mean±s.e.m.; n¼ 3 independent experiments. *Po0.05.(c) 2D rigid substrates inhibit TRC growth. Significant differences between Control and TRC, or 1 or 3 days from Day 1 to 5 in the 90-Pa fibrin gels(all Po0.05, except TRC at day 2, where P¼0.068, 1 day at day 2, where P¼0.52, and 3 days at Day 2 and Day 3, where P40.15). No differencesbetween Control and 5 or 7 days from Day 1–5 (all P40.06, except 5 days at Day 1 and Day 3, where Po0.04). Each data point was averaged fromat least 30 colonies. *Po0.05 between TRC and all other groups. (d) 2D rigid substrates promote stiffening of TRCs with substrate rigidity. B16-F1 cellscultured on plastic were used as a control. All were compared with ‘0.15 kPa’ in each group. Mean±s.e.m.; *Po0.01. Each data point was averaged from4150 cells from two independent experiments. All the statistics were conducted using Student’s t-test throughout the manuscript unless otherwisespecified.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5619

2 NATURE COMMUNICATIONS | 5:4619 | DOI: 10.1038/ncomms5619 | www.nature.com/naturecommunications

& 2014 Macmillan Publishers Limited. All rights reserved.

Aim 3: Investigate hypoxia-mediated enhanced extravasation of CTCs.

Alternative: 3D soft fibrin matrix assay

Tan, et al. Nature Comm. 2014

• Decrease in tumorigenicity from CTCs collected from mice implanted with HIF1-a knockdown MIA PaCa-2 cells

Aim 3: Expected result

Hypoxia

HIF1-a

EMT

Macroscopic Metastasis

Summary

Modified Peinado & Cano. Nature Cell Biology. 2008

wu hypoxia

Science