snail cooperates with krasg12d to increase stem cell ...€¦ · 2014-06-18 · blue (tb) stains...

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Snail Cooperates with KrasG12D in vivo to Increase Stem Cell Factor and Enhance Mast Cell

Infiltration

Lawrence M. Knab1,2, Kazumi Ebine1,4, Christina R. Chow1,3, Sania S. Raza1,3, Vaibhav Sahai1,

Akash P. Patel1, Krishan Kumar1,4, David J. Bentrem2,3,4, Paul J. Grippo2,3, Hidayatullah G.

Munshi1,3,4,*

1Division of Hematology/Oncology, Department of Medicine, and 2Division of Surgical Oncology,

Department of Surgery, Feinberg School of Medicine, Northwestern University; 3Robert H. Lurie

Comprehensive Cancer Center of Northwestern University, Chicago, IL 60611. 4Jesse Brown

VA Medical Center, Chicago, IL 60612

Running Title: Snail promotes pancreatic inflammation

Key Words: Snail, inflammation, pancreatic cancer, mast cells, stem cell factor

Conflict of Interests: The authors have no conflict of interests.

*To whom correspondence should be addressed:

Hidayatullah G. Munshi, MD

Northwestern University Feinberg School of Medicine

Lurie Building, Room 3-117

303 E Superior Street

Chicago Illinois 60611

p (312) 503-2301

f (312) 503-0386

[email protected]

on February 17, 2021. © 2014 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 18, 2014; DOI: 10.1158/1541-7786.MCR-14-0111

http://mcr.aacrjournals.org/

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ABSTRACT

Pancreatic ductal adenocarcinoma (PDAC) is associated with a pronounced fibro-inflammatory

stromal reaction that contributes to tumor progression. A critical step in invasion and metastasis

is the epithelial to mesenchymal transition (EMT), which can be regulated by the Snail family of

transcription factors. Overexpression of Snail (Snai1) and mutant KrasG12D in the pancreas of

transgenic mice, using an elastase (EL) promoter, resulted in fibrosis. To identify how Snail

modulates inflammation in the pancreas, we examined the effect of expressing Snail in EL-

KrasG12D mice (KrasG12D/Snail) on mast cell infiltration, which has been linked to PDAC

progression. Using this animal model system, it was demonstrated that there are increased

numbers of mast cells in the pancreas of KrasG12D/Snail mice compared to control KrasG12D

mice. In addition, it was revealed that human primary PDAC tumors with increased Snail

expression are associated with increased mast cell infiltration and that Snail expression in these

clinical specimens positively correlated with the expression of stem cell factor (SCF/KITLG), a

cytokine known to regulate mast cell migration. Concomitantly, SCF levels are increased in the

KrasG12D/Snail mice compared to control mice. Moreover, overexpression of Snail in PDAC cells

increased SCF levels and that the media conditioned by Snail-expressing PDAC cells promoted

mast cell migration. Finally, inhibition of SCF using a neutralizing antibody significantly

attenuated Snail-induced migration of mast cells.

Implications: Together, these results elucidate how the EMT regulator Snail contributes to

inflammation associated with PDAC tumors.

INTRODUCTION




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Pancreatic cancer, the fourth leading cause of cancer-related death in the US (1, 2), is

associated with a pronounced stromal reaction composed of collagen-rich extracellular matrix,

pancreatic stellate cells, and inflammatory cells (3-5). It has also been shown that there are

dynamic changes in the number and function of these inflammatory cells during pancreatic

cancer progression (6, 7). We previously published that mast cells, which have been extensively

studied in allergic reaction and autoimmunity (8), are increased in human PDAC tumors and

contribute to pancreatic cancer progression (9). A recent report showed that mast cells

modulate proliferation and activation of pancreatic stellate cells (10), which are key mediators of

pancreatic fibrosis in vivo (11, 12). Significantly, chronic pancreatitis, which is associated with

ongoing inflammation and fibrosis (13), is a risk factor for pancreatic cancer in humans and

facilitates pancreatic cancer development in transgenic mouse models (14, 15).

Epithelial-mesenchymal transition (EMT) has been identified as a key step in tumor

invasion and metastasis, enabling cells to disrupt normal tissue architecture and invade

surrounding structures (16, 17). One of the key regulators of EMT is the transcription factor

Snail (Snai1), which is associated with higher-grade PDAC tumors and is upregulated in >75%

of human PDAC tumors (18, 19). Significantly, Snail also promotes fibrosis in vivo (20, 21).

Transgenic overexpression of Snail in the kidney was sufficient to induce fibrosis in mice (20),

while ablation of Snail in the liver attenuated chemical-induced fibrosis (21). We also recently

showed that overexpression of Snail with mutant KrasG12D (KrasG12D/Snail) in mouse pancreas

promoted fibrosis (22). Importantly, Snail can also modulate inflammatory signaling in vivo

through upregulation of chemokines and cytokines (21, 23, 24). Snail overexpression in

keratinocytes increased cytokines and chemokines in vitro (23), while Snail overexpression in

epidermal keratinocytes in a transgenic mouse model promoted cutaneous inflammation that

was associated with increased cytokine production (24).

In this report, we examined the effect of Snail expression in epithelial cells on mast cell

infiltration in vivo and in vitro. We show that there are increased numbers of mast cells in the




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pancreas of KrasG12D/Snail mice compared to control mice (KrasG12D). We also show that human

pancreatic tumors with increased Snail expression are associated with increased mast cell

infiltration. To further understand how Snail increases mast cell infiltration, we examined in

human PDAC tumor samples the association between Snail levels and cytokines previously

shown to regulate mast cell migration. We show that Snail expression in human PDAC tumors

positively correlates with expression of stem cell factor (SCF). Additionally, we show that SCF

levels are increased in the KrasG12D/Snail mice compared to KrasG12D mice. Moreover, we

demonstrate that in vitro overexpressing Snail in PDAC cells increases SCF levels and that the

media conditioned by Snail-expressing PDAC cells increases mast cell migration. Significantly,

inhibiting SCF using neutralizing antibody blocks Snail-induced migration of mast cells. Overall,

these results increase our understanding of how the EMT regulator Snail contributes to

pancreatic cancer progression through inflammatory cell infiltration.

MATERIALS AND METHODS

Antibodies/Reagents - Reagents for trichrome, chloroacetate esterase (CAE) and toluidine

blue (TB) stains were purchased from Sigma. The TβRI inhibitor SB431542 was obtained from

Calbiochem. FOXP3 (FJK-16a) antibody was from eBioscience, Snail (C15D3) and Slug

(C19G7) antibodies were purchased from Cell Signaling, F4/80 (123105) antibody was from

BioLegend, Gr-1 (Ly6G/Ly6C clone RB6-8C5) and CD45 (30-F11) antibodies were purchased

from BD Bioscience, and CD3 (ab 5690) antibody was purchased from Abcam. Tryptase (sc-

33676) and �-tubulin (sc-8035) antibodies were from Santa Cruz Biotechnology. Secondary

antibodies were purchased from Sigma. Function blocking antibody against SCF (AF-255-NA)

and isotype-matched IgG antibody were purchased from R&D Systems. The SCF quantikine

ELISA kit (DCK00) was obtained from R&D Systems.




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Transgenic Mice - All animal work was conducted in compliance with the Northwestern

University IACUC guidelines. TRE-Snail transgenic mice were created by the Transgenic Core

Facility at Northwestern University and have been described previously (22). The TRE-Snail

mice were crossed with EL-tTA mice, kindly provided by Dr. Eric Sandgren (25), to generate EL-

tTA/TRE-Snail (Snail) bigenic mice. In EL-tTa mice, the transactivator tTa is expressed

downstream of elastase (EL) promoter, thus enabling targeting of Snail to pancreatic acinar and

centroacinar cells (25, 26). The bigenic mice were further crossed with EL-KrasG12D mice, which

express constitutively active mutant Kras in the pancreatic acinar cells (25-27), to generate

KrasG12D/Snail trigenic mice (22). Notably, these mice at the point of histological examination at

3-4 months of age do not develop overt pancreatic neoplasms but instead demonstrate

increased acinar-ductal metaplasia, proliferation and increased fibrosis (22).

Immunohistochemistry - Pancreatic tissue specimens from KrasG12D/Snail and control

KrasG12D were stained for leukocytes using CD45 antibody, T-cells using CD3 antibody, T-

regulatory cells using FOXP3 antibody, and macrophages using F4/80 antibody. The tissue

sections were also stained with anti-Gr-1 antibody, which identifies both granulocytes and

myeloid derived suppressor cells (28). Mast cells were identified with CAE, TB or tryptase stains

(8-10). Antigen retrieval was performed as previously described (22, 27, 29). Photographs for

quantitative comparison were taken using a Carl Zeiss Axioskop 40 microscope and camera

(22, 27).

Human PDAC tissue analysis - Pancreatic tissue was obtained from patients with pancreatic

adenocarcinoma on an IRB-approved protocol and de-identified. The specimens were stained

for mast cells using CAE and tryptase staining. In addition, RNA was isolated from human

PDAC tumors previously stored in RNAlater and analyzed for Snail, tryptase and cytokines by

quantitative RT-PCR (30-32).




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Cell culture - AsPC1 and CD18/HPAF-II cells were obtained from American Type Culture

Collection (Manassas, VA), while human mast cell line HMC-1 has been described previously

(33, 34). AsPC1 cells were last authenticated by STR profiling at the Johns Hopkins Genetic

Resources Core Facility in 2010, while CD18 cells were authenticated by STR profiling in 2013.

AsPC1 and CD18 cells expressing Snail or Slug were generated as detailed previously (31, 35).

AsPC1-vector, AsPC1-Snail, AsPC1-Slug, CD18-vector, and CD18-Snail cells have not been

previously authenticated.

Conditioned Media - Cells expressing control vector or Snail were allowed to condition the

media for 72 hours to generate Vector- and Snail-conditioned media (VCM and SnCM,

respectively).

Immunoblotting - Immunoblotting for Snail, Slug and α-tubulin was done as previously

described (27, 31).

Quantitative Real Time-PCR analysis - Reverse transcription of mRNA to cDNA was

performed using Taqman Reverse Transcription reagents from Applied Biosystems.

Quantitative gene expression was performed with gene specific Taqman probes, TaqMan

Universal PCR Master Mix and the 7500 Fast Real-time PCR System from Applied Biosystems.

The data were then quantified with the comparative CT method for relative gene expression (29).

Cytokine protein expression - SCF in the media conditioned by control cells or cells

expressing Snail or Slug was measured using the Quantikine ELISA kit purchased from R&D

systems.

Mast cell migration assay - HMC-1 mast cells (2 x 105) were added to the upper chamber of

an 8 μm uncoated Boyden chamber with media conditioned by control cells or Snail-expressing

cells in the lower chamber. The HMC-1 cells were allowed to migrate over 18 hours into the




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lower chamber, collected and counted (10). Neutralizing antibody or isotype-matched control

antibody was added to the lower chamber. All migration assays were performed in triplicate and

repeated a minimum of three times.

Statistical Analysis - In vivo and in vitro results were compared using t-test analysis. Error bars

represent standard error of the mean. All analyses were performed on GraphPad Prism 5 for

Mac OS X.

RESULTS

Snail expression in EL-KrasG12D mice increases inflammation and promotes mast cell

infiltration. Recently, we showed that over-expression of Snail and mutant Kras (KrasG12D) in

mouse pancreas using an elastase (EL) promoter enhanced collagen-rich stromal reaction

without causing overt pancreatic cancer [(22) and Fig. 1A]. Since inflammation plays a

significant role in PDAC development and progression (26, 36), we evaluated the effect of Snail

on inflammatory cell infiltration in our EL-KrasG12D mouse model by staining for leukocytes using

CD45 antibody. As shown in Fig. 1B, there was a significant increase in leukocyte infiltration,

which were primarily located in the pancreatic stroma, in the KrasG12D/Snail mice compared to

the KrasG12D mice. Previously, we published that increased infiltration of mast cells in human

PDAC tumors was associated with worse prognosis (9). Since increased Snail expression in

human PDAC tumors is also associated with more advanced disease (18, 19), we examined the

effect of Snail expression in the EL-KrasG12D mice on mast cell infiltration using chloroacetate

esterase (CAE) and toluidine blue (TB) stains to identify mast cells. As shown in Fig. 1C, there

were increased numbers of CAE(+) cells in pancreas from KrasG12D/Snail mice compared to

KrasG12D mice, indicating that Snail promotes infiltration of mast cells in the EL-KrasG12D mice.

Consistent with the CAE stain, TB staining also showed that there were increased numbers of




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mast cells in the KrasG12D/Snail mice compared to the KrasG12D (Fig. 1D). Similar to the

leukocytes, the mast cells were also primarily located in the pancreatic stroma. We also

examined the extent to which Snail affected mast cell infiltration following induction of

pancreatitis using cerulein treatment. As shown in Supplementary Fig. S1, there was a trend

towards increased infiltration of mast cells in KrasG12D/Snail mice compared to KrasG12D mice

following induction of pancreatitis.

Effect of Snail expression in EL-KrasG12D mice on infiltration of CD3(+) cells, FOXP3(+)

cells, Gr-1(+) cells and F4/80(+) cells. In an effort to further characterize the inflammatory cell

infiltration into the pancreatic microenvironment, we evaluated the effect of Snail expression in

the EL-KrasG12D mice on CD3(+) cells to identify T-cells; FOXP3 (+) cells to identify T-regulatory

cells; Gr-1(+) cells to identify granulocytes and also myeloid-derived suppressor cells; and

F4/80(+) cells to identify macrophages. There was no significant difference in the overall

number of T-cells (Fig. 2A) or in the number of T-regulatory cells (Fig. 2B) in pancreas from

KrasG12D/Snail mice compared to KrasG12D mice. However, there was a statistically significant

increase in the number of Gr-1(+) cells and macrophages in the pancreas of KrasG12D/Snail mice

compared to KrasG12D mice (Figs. 2C,D).

Snail expression in human PDAC tumors correlates with mast cell activation marker

tryptase and with the cytokine stem cell factor. To understand how Snail expression

increases mast cell infiltration in the EL-KrasG12D mice, we examined the effect of Snail

expression on cytokine expression using human PDAC tissue samples. Initially, we examined

the relationship between Snail expression in human PDAC tumors and tryptase, which is

primarily expressed in mast cells (37). We show that both CAE and tryptase stain for mast cells

in human PDAC tumors (Fig. 3A). Because of lack of a commercial antibody to detect

endogenous Snail protein, we examined the relationship between Snail at the mRNA level and

mast cells using tryptase mRNA as a surrogate for mast cells (37). As shown in Fig. 3B, tumor




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samples with increased Snail mRNA demonstrate increased tryptase mRNA levels. We next

examined the relationship between Snail in PDAC tumor samples and cytokines known to be

involved in mast cell migration (8). Specifically, we examined the relationship between Snail

mRNA expression and expression of stem cell factor (SCF), granulocyte macrophage-colony

stimulating factor (GM-CSF), interleukin-8 (IL-8) and chemokine (C-C motif) ligand 5 (CCL5)

mRNA levels. There was very little GM-CSF mRNA expression in our human tumor samples

(data not shown). Although there was no correlation between expression of Snail mRNA and

CCL5 mRNA or between expression of Snail mRNA and IL-8 mRNA levels, there was a

significant correlation between Snail mRNA and SCF mRNA expression in human PDAC

samples (Fig. 3C).

Snail expression in EL-KrasG12D mice increases SCF expression. Since Snail mRNA levels

in human PDAC tumors correlates with SCF mRNA levels, we examined the effect of Snail

expression in EL-KrasG12D mice on SCF levels. As shown in Fig. 4A, there was a significant

increase in SCF mRNA expression in the KrasG12D/Snail mice compared to KrasG12D mice. We

also examined the effect of Snail expression in the EL-KrasG12D mice on CCL5 and CXCL1, the

mouse homologue for IL-8. Consistent with our human PDAC tumor samples, there was not a

significant increase in CCL5 (Fig. 4B) or CXCL1 (Fig. 4C) mRNA expression in the pancreas of

KrasG12D/Snail mice compared to KrasG12D mice.

Snail expression in human PDAC cells in vitro increases SCF production and mast cell

migration. We next examined whether Snail expression in vitro also results in increased SCF

levels. Snail expression in the PDAC cell lines AsPC1 (Fig. 5) and CD18 (Supplementary Fig.

S2) significantly increased SCF mRNA levels. Moreover, the media conditioned by Snail-

expressing PDAC cells (SnCM) demonstrated increased levels of SCF protein (Fig. 5B and

Supplemental Fig. S2B). Since we have previously reported that Snail expression in AsPC1

cells increases TGF-β signaling (22), we examined the effect of blocking TGF-β signaling on




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Snail-induced SCF expression. As shown in Fig. 5C, treatment with the TGF-β type I receptor

inhibitor SB431542 significantly attenuated Snail-induced SCF mRNA levels.

We next examined the effect of Snail expression in PDAC cells on migration of the

human mast cell line HMC-1 cells using a transwell migration assay (9, 10) and media

conditioned by PDAC-V cells (VCM) and PDAC-Sn cells (SnCM). Relative to migration towards

media conditioned by PDAC-V cells, there was increased migration of mast cells towards media

conditioned by Snail-expressing PDAC cells (Fig. 5D and Supplementary Fig. S2C). Finally, we

examined the effect of blocking SCF on the ability of HMC-1 mast cells to migrate towards

media conditioned by control cells or Snail-expressing cells. As shown in Fig. 5E and

Supplementary Fig. 2SD, treatment with the function-blocking anti-SCF antibody decreased

migration of HMC-1 cells towards media conditioned by Snail-expressing PDAC cells without

affecting migration of HMC-1 cells towards media conditioned by control PDAC cells. Overall,

these results demonstrate that Snail increases expression of SCF to regulate mast cell

migration in vitro and suggest that the increased infiltration of mast cells in Snail-expressing

lesions is mediated by SCF.

DISCUSSION

There is significant interplay between EMT and inflammation in regulating cancer

progression. It was previously shown that inflammatory signaling could induce EMT. For

example, Snail activity is increased via stabilization at the protein level in response to TNF-�-

driven NF-�B signaling (38). Conversely, Snail can also modulate inflammatory signaling in vivo

through upregulation of chemokines and cytokines (21, 23, 24). Significantly, Snail-related

protein Slug (Snai2) can also modulate inflammatory signaling in vivo through upregulation of

chemokines and cytokines (23, 39). Snail and Slug overexpression in keratinocytes increases

production of cytokines IL-6, IL-8 and the chemokine CXCL1 (23). Snail overexpression in




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epidermal keratinocytes in a transgenic mouse model promotes cutaneous inflammation that is

associated with increased IL-6 production (24). Snail ablation in turn attenuates the

inflammatory response in a chemical-induced liver fibrosis model (21), while Slug ablation

attenuates cutaneous inflammatory response following ultraviolet radiation (39). In this report,

we show that Snail expression in the mouse pancreas also enhances inflammation. Snail

expression in mouse pancreas increases infiltration of mast cells, and Snail expression in

pancreatic cancer cells induces expression of the cytokine SCF to promote mast cell migration.

Additionally, we show that Snail expression in human tumors correlates with the mast cell

marker tryptase and SCF, suggesting that Snail may also promote infiltration of mast cells in

human PDAC tumors by increasing SCF expression.

Inflammation plays a critical role in pancreatic cancer development and progression.

Acute pancreatitis can accelerate the progression of precursor pancreatic intraepithelial

neoplastic lesions to PDAC in mutant K-ras-driven mouse models of pancreatic cancer (40, 41).

Importantly, although expression of embryonic mutant K-ras is sufficient for tumor initiation in

various mouse models of pancreatic cancer (42, 43), expression of mutant K-ras in adult mouse

pancreas does not result in any obvious phenotypic changes (15). However, induction of chronic

pancreatitis promotes PDAC development in adult mice expressing mutant K-ras (14, 15). We

have recently published that Snail expression in EL-KrasG12D mice resulted in more advanced

acinar-ductal metaplasia that was associated with increased proliferation and fibrosis (22). It is

possible that some of the phenotypic changes that we recently reported in the KrasG12D/Snail

mice may have been due to increased infiltration of inflammatory cells. Significantly, it is

increasingly recognized that mast cells contribute to pancreatic cancer progression. Increased

numbers of mast cells in human PDAC tumors are associated with poor prognosis (9, 10).

Moreover, there is significant cross-talk between cancer cells and mast cells resulting in

pancreatic cancer progression. Pancreatic cancer cells in vitro can increase migration and




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activation of mast cells that can in turn increase proliferation of pancreatic cancer cells (9).

Recently, it was also shown that blocking mast cell migration and function in vivo reduces PDAC

growth and increases survival of mice bearing PDAC tumors (10).

We also show that Snail-induced migration of mast cells was mediated by SCF. We

have also found that Slug can induce SCF expression in PDAC cells (Supplementary Fig. S3).

Although we are still in the process of determining the mechanism by which Snail and Slug

increase SCF, our preliminary studies indicate that Snail increases SCF through TGF-β

signaling. Significantly, we have recently shown that TGF-β2 expression is induced by Snail in

PDAC cells and that increased TGF-β signaling in KrasG12D/Snail is associated with increased

fibrosis and stellate cell activation (22). Notably, mast cells also promote proliferation and

activation of stellate cells (10), which are key regulators of fibrosis in vivo (11, 12). The

increased activation of stellate cells was shown to be due to increased TGF-β2 production (10).

Thus, it is possible that the increased number of mast cells in our Snail-expressing mice may

also have contributed to the stellate cell activation and fibrosis that we have previously reported

to be present in the KrasG12D/Snail mice (22).

Overall, we demonstrate that Snail in the presence of the oncogenic KrasG12D induces

inflammation that is associated with increased mast cell infiltration. Moreover, we show that

Snail expression in human PDAC tumors is also associated with evidence of increased mast

cell infiltration. Mechanistically, Snail increases SCF production to promote mast cell migration.

These results increase our understanding of the interplay between the EMT regulator Snail and

inflammation in pancreatic cancer progression.




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GRANT SUPPORT

This research was supported by grant R01CA126888 (H.G.M.) from the NCI, a Merit award

I01BX001363 (H.G.M.) from the Department of Veterans Affairs, and the Rosenberg Family

Fund (H.G.M.). This research was also supported by the training grants T32CA070085 (C.R.C)

and T32CA079447 (S.S.R.) from the NCI.

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FIGURE LEGENDS

Figure 1: Snail expression in EL-KrasG12D mice increases inflammation and promotes

mast cell infiltration. The TRE-Snail mice were crossed with EL-tTA mice to generate Snail

(EL-tTA+/TRE-Snail+) mice. The Snail mice were crossed with EL-KrasG12D mice to generate

mice expressing both Snail and KrasG12D in the pancreas (EL-Kras+/EL-tTA+/TRE-Snail+ =

KrasG12D/Snail) or littermate control mice that expressed only KrasG12D (EL-Kras+/EL-tTA+/TRE-

Snail- or EL-Kras+/EL-tTA-/TRE-Snail- = KrasG12D). A. Pancreas from KrasG12D and

KrasG12D/Snail mice were analyzed for Snail expression by immunofluorescence using DAPI to

counterstain nuclei. Representative pancreatic sections from 3-month old KrasG12D and

KrasG12D/Snail mice were analyzed for fibrosis using trichrome staining (blue=fibrosis). B.

Pancreatic tissue samples from 3-month old KrasG12D and KrasG12D/Snail mice were collected

and stained for leukocytes using CD45 antibody, and the number of leukocytes averaged over 8

high-power fields were counted. C,D. Pancreatic tissue samples from 3-month old KrasG12D and

KrasG12D/Snail mice were collected and stained for mast cells using chloracetate esterase (CAE;

C) and toluidine blue (TB; D) and the total number of mast cells per section was counted. The p-

values were calculated using unpaired t-test. Scale bars, 50 μm.

Figure 2: Effect of Snail expression in EL-KrasG12D mice on infiltration of CD3(+) cells,

FOXP3(+) cells, Gr-1(+) cells and F4/80(+) cells. A-D. Pancreatic tissue samples from 3-

month old KrasG12D and KrasG12D/Snail mice were collected and stained for T-cells using CD3

antibody and the number of T-cells averaged over 8 high-powered fields were counted (A). T-

regulatory cells were stained using FOXP3 antibody, and the total number of T-regulatory cells

per section was counted (B). The sections were stained with Gr-1 antibody to identify

granulocytes/myeloid derived suppressor cells and the total number of Gr-1(+) cells per section




17

was counted (C). Macrophages were stained with F4/80 antibody, and the total number of

macrophages per section was counted (D). The p-values were calculated using unpaired t-test.

Scale bars, 50 μm.

Figure 3: Snail expression in human PDAC tumors correlates with mast cell activation

marker tryptase and with the cytokine stem cell factor. De-identified human PDAC samples

were procured on an IRB-approved protocol. A. Formalin-fixed paraffin-embedded tumors were

stained with CAE and immunostained with anti-tryptase antibody to identify mast cells. Scale

bars, 50 μm. B,C. The mRNA samples from human PDAC tumors were processed for Snail,

tryptase, chemokine (C-C motif) ligand 5 (CCL5), interleukin 8 (IL-8) and stem cell factor (SCF)

mRNA by qRT-PCR and normalized using GAPDH mRNA levels and the relationship between

Snail and tryptase, CCL5, IL-8 or SCF was analyzed using Pearson correlation coefficient.

Figure 4: Snail expression in EL-KrasG12D mice increases SCF expression. A-C. The mRNA

samples from pancreatic tissue of 3-month old KrasG12D and KrasG12D/Snail mice were

processed for stem cell factor (SCF; A), chemokine (C-C motif) ligand 5 (CCL5; B), and the

mouse functional homologue to IL-8 chemokine (C-X-C motif) ligand 1 (CXCL1; C) mRNA by

qRT-PCR and normalized using GAPDH mRNA levels. The p-values were calculated using

unpaired t-test.

Figure 5: Snail expression in PDAC cells increases SCF production and mast cell

migration. A. Western blot of AsPC1 pancreatic cancer cells expressing control vector (V) or

Snail (Sn). B. The mRNA samples from AsPC1-V and AsPC1-Sn cells were analyzed for stem




18

cell factor (SCF) and GAPDH using real-time PCR, and the mRNA levels normalized to the

levels present in vector-expressing control cells (*** p<0.001). AsPC1-VCM and AsPC1-SnCM

were generated as detailed in Materials and Methods. SCF protein levels in the conditioned

media were determined using the human Quantikine ELISA kit DCK00 purchased from R&D

Systems (**, p<0.01). C. AsPC1-V and AsPC1-Sn cells were treated with either DMSO or TGF-

β type I receptor inhibitor SB431542 for 72 hours, and the effect on stem cell factor (SCF)

mRNA level was determined by qRT-PCR and normalized using GAPDH mRNA levels. The

mRNA levels were normalized to the levels present in DMSO-treated AsPC1-V cells (*, p<0.05).

The p-values were calculated using paired t-test. E. HMC-1 mast cells (2 x 105) were added to

the upper chamber of an 8 μm uncoated Boyden chamber with either AsPC1-VCM or AsPC1-

SnCM supplemented with 1% serum in the lower chamber. HMC-1 cells were allowed to migrate

into the lower chamber, collected and counted. The p-values were calculated using paired t-test.

E. HMC-1 mast cells (2 x 105) were added to the upper chamber of an 8 μm uncoated Boyden

chamber with either AsPC1-VCM or AsPC1-SnCM in the lower chamber. A neutralizing antibody

against SCF (AF-255-NA) or an isotype-matched IgG antibody was added to the lower

chamber. HMC-1 cells were allowed to migrate over 18 hours into the lower chamber, collected

and counted.*, p <0.05. The p-values were calculated using paired t-test.




Published OnlineFirst June 18, 2014.Mol Cancer Res Lawrence M. Knab, Kazumi Ebine, Christina R. Chow, et al. Factor and Enhance Mast Cell Infiltration

in vivo to Increase Stem CellG12DSnail Cooperates with Kras

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