Myeloid-Derived Suppressor Cells in the Development of ...€¦ · the development of lung cancer using two experimental models: a lung cancer model in CC10 transgenic mice (CC10Tg)

Research Article

Myeloid-Derived Suppressor Cells in the Developmentof Lung Cancer

Myrna L. Ortiz1, Lily Lu1, Indu Ramachandran1,2, and Dmitry I. Gabrilovich1,2

AbstractMyeloid-derived suppressor cells (MDSC) are widely implicated in immune suppression associated with tumor

progression and chronic inflammation. However, very little is known about their possible role in tumordevelopment. Here, we evaluated the role of MDSC in two experimental models of lung cancer: inflamma-tion-associated lung cancer caused by chemical carcinogen urethane in combination with exposure to cigarettesmoke; and a transgenic CC10Tg model not associated with inflammation. Exposure of mice to cigarette smokealone resulted in significant accumulation in various organs of cells with typical MDSC phenotype(Gr-1þCD11bþ). However, these cells lacked immunosuppressive activity and could not be defined as MDSC.When cigarette smoke was combined with a single dose of urethane, it led to the development of tumor lesions inlungs within 4 months. By that time, Gr-1þCD11bþ cells accumulated in the spleen and lung and had potentimmunosuppressive activity, and thus could be defined as MDSC. In the CC10Tg model, accumulation ofimmunosuppressive MDSC was observed only at 4 months of age, after the appearance of tumor lesions in thelungs. Accumulation of MDSC in both models was abrogated in S100A9 knockout mice. This resulted in adramatic improvement in survival of mice in both models. Thus, cigarette smoke results in the expansion ofimmature myeloid cells lacking suppressive activity. Accumulation of bona fide MDSC in both models wasobserved only after the development of tumor lesions. However, MDSC played a major role in tumor progressionand survival, which suggests that their targetingmay provide clinical benefits in lung cancer.Cancer Immunol Res;2(1); 50–58. �2013 AACR.

IntroductionMyeloid-derived suppressor cells (MDSC) are one of the

major factors that negatively regulate immune responses incancer (1, 2). In mice, these cells are defined as Gr-1þCD11bþ

cells comprising pathologically activated CD11bþLy6CloLy6Gþ

immature granulocytes, CD11bþLy6ChiLy6G�monocytes, anda small proportion of myeloid precursors. The main charac-teristic of these cells is their potent ability to suppress T-cellfunction. In addition, MDSC promote tumor progressionthrough a number of different other mechanisms (3). The roleof MDSC in tumor progression is well established. In contrast,the contribution of these cells to tumor development is notclear. Some cells with a typical MDSC phenotype are present inhealthy individuals and control mice. However, these cells lackimmunosuppressive activity and largely represent immaturemyeloid cells (IMC). Therefore, the term IMC was coined todistinguish these cells fromMDSC. In tumor-bearing mice, the

accumulation of MDSC is usually observed after the tumor hasbecome palpable (4). In patients with cancer, a strong asso-ciation between stage of cancer and presence of MDSC hasbeen established in various types of cancer (5–9). Proinflam-matory factors were always considered a critical element ofMDSC accumulation in cancer (10). In recent years, amplereports have described the appearance of cells with an MDSCphenotype and function in mice and human patients withvarious chronic infections and inflammation (11). These obser-vations raised the possibility that MDSC can contribute direct-ly to the development of tumors, as it is known that inflam-mation plays an important role in cancer development (12).

In this study, we tested possible contributions of MDSC tothe development of lung cancer using two experimentalmodels: a lung cancer model in CC10 transgenic mice(CC10Tg) and a lung cancer model caused by the injectionof carcinogen urethane in combination with cigarettesmoke. The mCC10TAg transgene was created by fusing thecoding sequences of the SV40 TAg gene with the mCC10promoter (13). This promoter targets transgene expressionspecifically to the proximal pulmonary lung epithelial Claracells. The Clara cells are the nonciliated secretory cells of thepulmonary epithelium characterized by a large apical domeshape and abundant endoplasmic reticulum. At 2 months ofage, CC10Tg mice display areas of hyperplasia of the bron-chiolar epithelium. At 3 months of age, a number of tumorfoci are observed, and after 4 months, the majority of thelung is composed of transformed cells (13).

Authors' Affiliations: 1H. Lee Moffitt Cancer Center, Tampa, Florida; and2The Wistar Institute, Philadelphia, Pennsylvania

Current address for L. Lu: National Cancer Institute, NIH, Bethesda,Maryland.

Corresponding Author: Dmitry I. Gabrilovich, The Wistar Institute, 3600Spruce Street, Philadelphia, PA 19104. Phone: 215-495-6955; Fax: 215-573-2456; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-13-0129

�2013 American Association for Cancer Research.

CancerImmunology

Research

Cancer Immunol Res; 2(1) January 201450

on October 23, 2020. © 2014 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Published OnlineFirst October 18, 2013; DOI: 10.1158/2326-6066.CIR-13-0129

http://cancerimmunolres.aacrjournals.org/

Smoking is the leading cause of lung cancer. Each cigarettecontains a complex mixture of polycyclic aromatic hydrocar-bons along with other lung carcinogens, tumor promoters, andcocarcinogens (14). Tobacco-associated carcinogens by them-selves may not recapitulate the exact nature of tobacco's effecton tumor development. In addition to carcinogens, smokingcauses chronic inflammation. The local lung inflammationinitiated by cigarette smoke persists after patients havestopped smoking, generating smoking-independent oxidantstress, thus explaining the persistence and progression of thedisease after smoking has been discontinued (15). The mostappropriate model to recapitulate the complexity of the effectof cigarette smoke on mice is a smoking chamber in whichmice are exposed to tobacco smoke for prolonged periods oftime. This approach, however, requires a very long exposure ofmice to smoke and results in the development of lung canceronly in a proportion of mice (16). Therefore, cigarette smoke iscombined with the use of chemical carcinogens. In this study,we used urethane, a tobacco-related carcinogen known toinduce lung cancer (17, 18).To clarify the role of MDSC in the development of lung

cancer, we used S100A9 knockoutmice. S100A9was implicatedin the abnormal differentiation of myeloid cells in cancer andthe accumulation of MDSC (5, 19–22). The expansion of MDSCwas significantly reduced in S100A9-deficient tumor-bearingmice and mice treated with complete Freund's adjuvant (CFA;refs. 21, 22). Consistent with these findings, dendritic cellsderived fromS100A9-deficientmice induced stronger responseof allogeneic T cells (23).Using these experimental models, we tested the hypoth-

esis that MDSC are directly involved in the development ofexperimental lung cancer. Our results demonstrated thatalthough immunosuppressive bona fide MDSC are criticallyimportant for tumor progression, and their presence nega-tively affects the survival of mice, the initial phase of tumordevelopment is not associated with immunosuppressiveMDSC. In our smoking model, cigarette smoke causedstrong accumulation of IMC lacking immunosuppressiveactivity. MDSC accumulated in these mice only after tumorlesions became detectable. In the transgenic model of lungcancer, MDSC accumulation was also only evident aftertumor development.

Materials and MethodsMiceAll animal experiments were approved by the University of

South Florida (Tampa, FL) Institutional Animal Care and UseCommittee (IACUC). Mice were housed in pathogen-free facil-ities. FVB/N and C57BL/6 mice were obtained from theNational Cancer Institute (Frederick, MD). S100A9KO miceon C57BL/6 background were described previously (24). Thesemice were backcrossed to FVB/N background for 10 genera-tions. CC10Tg mice were obtained from Dr. F. DeMayo (BaylorCollege of Medicine, Houston, TX) and described previously(13). These mice were on C57BL/6 background. CC10Tg werecrossed with C57BL/6 S100A9KO mice for six generationswith subsequent backcross to S100A9KO mice. CC10Tgþ/�

S100A9�/� mice were used in the experiments. The control

group comprised CC10Tgþ/�S100A9þ/þ mice from the samegeneration of backcross.

Lung carcinogenesis modelExposure of mice to cigarette smoke was performed using a

whole-body smoke inhalation system (Teague TE-10C; TeagueEnterprises). Mice were exposed to an average of 250 mg/m3

total suspended particulate (TSP) of cigarette smoke for 2hours/day, 5 days/week during 2 to 4 months using thestandard Federal Trade Commission (FTC) method: a 2-sec-ond 35 cm3 puff once per minute for a total of nine puffs. TSPconcentration was characterized daily by collecting particleson 25-mm Pallflex membrane filters. Standard reference cigar-ettes 3R4F were purchased from the Tobacco ResearchInstitute (University of Kentucky, Lexington, KY) and used inthis study. At the end of week 3 of smoke exposure, somemice received a single intraperitoneal injection of urethane (1.5g/kg). Cigarette smoke continued for 3 to 4moremonths. After3 or 4months of exposure to smoke, mice were euthanized andthe presence ofmyeloid cells and the size and number of tumorlesions were evaluated.

Preparation of single-cell suspensions and antibodystaining

Lungs were thoroughly minced and digested in RPMI-1640medium containing collagenase XI (2 mg/mL; Sigma-Aldrich)at 37�C for 15 minutes. Samples were resuspended in calcium-and magnesium-free PBS-EDTA at room temperature. Redblood cells were lysed with ammonium–chloride–potassiumbuffer, washed in PBS-EDTA, and passed through a 50-mm cellstrainer. After single-cell preparation, cells were counted andstained with the following antibodies: Gr1-APC, CD11b-PEy7,F480-PE, CD11c-FITC, Ly6C-FITC, and Ly6C-PE. All antibodieswere from BD Biosciences. To obtain single-cell suspensionfrom lymph nodes and spleens, tissues were smashed througha 70-mm strainer without the use of collagenase digestion.Peripheral blood was collected from the submandibular vein.

T-cell–suppressive activity of MDSCIn experiments with CC10Tg mice on C57BL/6 background,

the effect of MDSC on antigen nonspecific (CD3/CD28 anti-bodies inducible) and antigen-specific (OVA-derived peptide-specific OT-1 CD8þ T cells) proliferation were measured.Briefly, T cells obtained either from wild-type (WT) C57BL/6mice or from C57BL/6 OT-1 mice were labeled with carboxy-fluorescein diacetate succinimidyl ester (CFSE) and mixedwith irradiated (20 Gy) syngeneic splenocytes at a 1:4 ratio.MDSC were added at different ratios and cells were stimulatedwith either 1 mg/mLCD3 and 0.5 mg/mLCD28 antibodies or 0.5mg/mLOT-1–specific peptide SIINFEKL. Proliferation of T cellswas assessed 72 hours later by flow cytometry. Experimentswere performed in triplicate.

In experiments with urethane/cigarette smoke models onFVB/N background, antigen-specific T-cell proliferation wasassessed using allogeneicmixed leukocyte reaction. Briefly, 105

splenocytes from BALB/c mice (responders) were mixed with105 T cell–depleted and irradiated (20 Gy) splenocytes fromFVB/N mice (stimulators) in U-bottom 96-well plates.

MDSC and the Development of Lung Cancer

www.aacrjournals.org Cancer Immunol Res; 2(1) January 2014 51




Fluorescence-activated cell sorting (FACS)–purified Gr-1þ

cells were added into wells at different ratios. On day 4, cellswere pulsed with 3H-thymidine (1 mCi/well; GE Healthcare) for18 hrs. 3H-thymidine uptake was counted using a liquid scin-tillation counter and expressed as counts per minute (cpm).

Evaluation of lung tumorsWhole lungs were flushed with PBS and fixed in 4% para-

formaldehyde for at least 24 hours and paraffin embedded.Paraffin-embedded lungs were serially sectioned transversallyat 4 mm and histologically examined with hematoxylin andeosin (H&E) stain.

Evaluation of myeloid cells by flow cytometrySeveral populations of myeloid cells were evaluated in this

study: (i) IMC/MDSC defined as Gr-1þCD11bþ cells, includingsubset of CD11bþLy6CloLy6Gþ granulocytic and CD11bþ

Ly6ChiLy6G� monocytic cells; (ii) macrophages (M�), whichwere defined as Gr-1�CD11bþF4/80þ cells; and (iii) dendriticcells, defined as Gr-1�F4/80�CD11cþ cells.

Statistical analysisStatistical analysis was performed using unpaired two-tailed

Student t test with significance determined at P < 0.05. For theanalysis of survival, log-rank Mantel–Cox analysis was used.

ResultsMyeloid cells in spontaneous model of lung cancer

To assess the potential role of MDSC/IMC in the develop-ment of lung cancer in oncogene-driven transgenic model, weused CC10Tg C57BL/6 mice. Consistent with a previous report(13), these mice develop multiple tumor lesions in the lungsby 4 months of age (Fig. 1A). CC10Tg mice had a significantlyhigher proportion of Gr-1þCD11bþ cells in the peripheralblood at 2.5 to 3 months of age (Fig. 1B), and higher presenceof these cells in the spleens and lymphnodes at 3 to 4months ofage (Fig. 1C and D). No differences in the proportion of Gr-1þCD11bþ cells among CD45þ hematopoietic cells in the lungwere found at that time between WT and CC10Tg mice (Fig.1E). In contrast, the proportion of M� in the lungs increasedsignificantly in CC10Tg mice at 3 months of age (Fig. 1F).

1 2 2.5 30

5

10

15

20

Months

% o

f cells

WTCC10

Gr-1+CD11b+ cells in blood

**

1 2 3 40

2

4

6

Gr-1+CD11b+ cells in spleen

Months

% o

f ce

lls

WTCC10

*

1 2 3 40.0

0.2

0.4

0.6

Gr-1 +CD11b+ cells in LN

Months

% o

f cells

WT

CC10*

*

1 2 3 40

5

10

15

20

Gr-1+CD11b+ cells in lung

Months

% o

f C

D45

+cells WT

CC10

1 2 3 40

10

20

30

Gr-1–F4/80+CD11b+ cells in lung

Months

% o

f C

D4

5+

ce

lls WTCC10

*

*

A B

C D

F

WT CC10 WT CC10 E

Figure 1. The accumulation of myeloid cells in a spontaneous lung cancer model. A, lung tissues from CC10Tg mice at 4 months of age were harvested andstained with H&E. Images (magnification, �20) are representative of lung tissues from 5 mice. B–D, percentages of Gr-1þCD11bþ cells in peripheralblood (B), spleen (C), and lymph nodes (LN; D) from CC10Tg mice and WT mice at indicated ages. Representative FACS data are shown in top. E and F,the proportions of MDSC (E) and macrophages (F) among CD45þ hematopoietic cells isolated from lungs. In all panels mean and SEM from 3 to 6 mice pergroup are shown. �, P < 0.05 between CC10Tg and WT mice.

Ortiz et al.

Cancer Immunol Res; 2(1) January 2014 Cancer Immunology Research52




Thus, Gr-1þCD11bþ cells increased in peripheral blood,spleens, and lymph nodes of CC10Tg mice around the timeof the appearances of the first tumor lesions (3 months) andexpanded further by 4 months. We previously found that lungGr-1þCD11bþ cells from4-month-oldCC10Tgmice hadpotentimmunosuppressive activity (25).We askedwhether the spleenGr-1þCD11bþ cells from 3-month-old mice had immunosup-pressive activity. The cells were tested on their ability to inhibitCD3/CD28–inducible T-cell proliferation (Fig. 2A) and the

proliferation of OT-1 T cells in response to the specificOVA-derived peptide—SIINFEKL (Fig. 2B). In both cases, noinhibition of T-cell proliferation was observed (Fig. 2A and B).

To establish a possible role of MDSC in the natural pro-gression of lung tumors in CC10Tg mice, we crossed CC10Tgmice with S100A9KO mice. In contrast with CC10Tg mice,double-transgenic S100A9KO � CC10Tg mice had no upregu-lation of Gr-1þCD11bþ cells in the peripheral blood (Fig. 3A).Survival of S100A9KO � CC10Tg mice was significantly

Figure 2. Evaluation of myeloidcell-suppressive activity. Gr-1þCD11bþ cells were isolated fromthe spleens of CC10Tg mice at 3months of age and cocultured withCFSE-labeled splenocytes fromWT CD45.1þ congenic C57BL/6mice stimulated with anti-CD3/CD28 antibody (A) or with CFSE-labeled CD45.2þ OT-I T cellsstimulated with the specificpeptide SIINFEKL (B). IrradiatedCD45.1þ splenocytes were usedas antigen-presenting cells. After 3days of cultures, T-cell proliferationwas analyzed by CFSE dilution.Typical examples of threeperformed experiments are shown.

1 2 2.5 30

5

10

15

20

Months

% o

f cells

CC10/S100A9KO

S100A9KO

120 140 160 180 2000

20

40

60

80

100CC10

CC10/S100A9

Days

Perc

enta

ge s

urv

ival

P = 0.0001

A B Figure 3. Effect of S100A9 deletionon the survival of CC10Tg mice.A, the percentages of Gr-1þCD11bþ cells in the blood fromCC10Tg mice (WT) and CC10Tg �S100A9KO. Analyses wereperformed at the indicated timepoints. Each group included 3mice. B, survival of CC10Tg (n¼ 8)and CC10Tg � S100A9 (n ¼ 9)mice.






(P ¼ 0.0001) higher than that of the single-transgenic CC10Tgmice (Fig. 3B). These results indicate thatMDSC accumulationin the transgenic model of lung cancer did not precede theformation of lung tumors, but was rather the result of tumordevelopment. However, MDSC significantly affect tumor pro-gression, which wasmanifested in better survival of S100A9KOmice.

Exposure of mice to cigarette smoke induces expansionof myeloid cells in mice

To better clarify the role of MDSC in lung cancer directlyassociated with inflammation, we used the cigarette smokemodel in FVB/Nmice. First, we asked whether cigarette smokealone affected the presence of different populations of myeloidcells. Cigarette smoke did not change the total number of

Gr1+CD11b+ cells

% o

f ce

lls%

of

ce

lls

% o

f ce

lls

% o

f ce

lls%

of

ce

lls

% o

f ce

lls%

of

ce

lls

Control Smoking

0

5

10

15

20P = 0.01

Gr1+CD11b+ cells

Control Smoking0

5

10

15

20P = 0.003

F4/80+ cells

Control Smoking

0

2

4

6

8

ns

F4/80+ cells

Control Smoking0

2

4

6

8

10

ns

CD11c+ cells

Control Smoking

0.0

0.2

0.4

0.6

0.8

P = 0.03

1 month

3 months

CD11c+ cells

Control Smoking0.0

0.2

0.4

0.6

0.8

ns

Ly6G/Ly6C ratio

Control Smoking

0

2

4

6

8

10ns

Ly6G/Ly6C ratio

Control Smoking0

20

40

60

80

P = 0.03

A

B

Figure 4. The proportion ofmyeloid cells in the peripheral blood ofmice exposed to cigarette smoke. FVB/Nmicewere exposed to cigarette smoke for 1month(A) and3months (B). The proportions of the indicated population ofmyeloid cellswere evaluated. Eachgroup included 3mice.P valueswere calculated in two-tailed Student t test. ns, P > 0.05.

Control Smoking

0

5

10

15Gr-1–F4/80+CD 11b+ cells

% o

f C

D45

+ c

ells

ns

Gr-1+CD11b+ cells

% o

f cells

Control Smoking50

60

70

80

P = 0.03

Gr-1–F4/80+CD11b+ cells

% o

f cells

Control Smoking0

2

4

6

ns

Bone marrow

Spleen

Lung

Control Smoking0

5

10

15Gr-1+CD11b+ cells

% o

f cells

P = 0.001

Control Smoking0

2

4

6

8

10

Gr-1–F4/80+CD11b+ cells

% o

f cells

ns

CD11c+ cells

% o

f cells

Control Smoking0.0

0.5

1.0

1.5

2.0 ns

Ly6G/Ly6C ratio

Control Smoking0

10

20

30

40

50

P = 0.015

Ly6G/Ly6C ratio

Control Smoking0

20

40

60

80

ns

CD11c+ cells%

of cells

Control Smoking0.0

0.2

0.4

0.6

0.8ns

Gr-1+C D 11b+ cells

% of C

D45

+ c

ells

Control Smoking

0

5

10

15

P = 0.04

CD 11c+ cells

% o

f C

D45

+ c

ells

Non-smoking Smoking

0

5

10

15

20

25

P = 0.03

Control Smoking

0.0

0.2

0.4

0.6

0.8Ly6G/Ly6C ratio

ns

Figure 5. The proportion of myeloid cells in different organs of mice exposed to cigarette smoke. Mice were exposed to cigarette smoke for 4 months, and theproportions of the indicated populations ofmyeloid cells were evaluated in different organs. Each group included 3 to 6mice. The proportions of cells isolatedfrom the lungs were calculated among CD45þ hematopoietic cells. P values were calculated in two-tailed Student t test. ns, P > 0.05.

Ortiz et al.





leukocytes (data not shown). However, 1-month exposure tocigarette smoke caused a significant (P¼ 0.01) increase in thepresence of Gr-1þCD11bþ cells in peripheral blood. Granulo-cytic and monocytic subsets were equally increased and theirratio remained unchanged (Fig. 4A). Three-month exposure ofmice to cigarette smoke did not further increase the presenceof Gr-1þCD11bþ cells in peripheral blood. It remained 2-fold(P ¼ 0.003) higher than in control. However, the dramaticredistribution of myeloid cells toward granulocytic cells wasevident (Fig. 4B). Despite some trend to an increase in miceexposed to cigarette smoke, the presence of M� and dendriticcells was not significantly different from that in nontreatedmice (Fig. 4B).Myeloid populations in the bone marrow, spleens, and

lungs were evaluated after 4 months of cigarette smokeexposure, which caused a significant (P < 0.05) increase inthe presence of Gr-1þCD11bþ cells in all tested organs. Inthe spleens, this increase was associated with a preferentialaccumulation of granulocytic cells (Fig. 5). The population ofM� did not change in any tested organ, whereas the pro-portion of dendritic cells was significantly decreased in thelungs (Fig. 5).Thus, cigarette smoke caused a substantial increase in the

presence of Gr-1þCD11bþ cells, which could be representedby MDSC. We evaluated the immunosuppressive activity ofthese cells. Gr-1þCD11bþ cells were isolated from thespleens, lung, or bone marrow and tested in different assays.These cells did not affect the CD3/CD28–inducible T-cellproliferation (Fig. 6A) or T-cell proliferation in allogeneicmixed leukocyte reaction (Fig. 6B). These results indicatethat cigarette smoke by itself caused a substantial accumu-lation of IMC with granulocytic phenotype. However, thesecells lacked immunosuppressive activity and thus were notbona fide MDSC.

MDSC are critically important for tumor progressionTo assess the possible roles of MDSC in the development

and progression of lung tumors, WT and S100A9KO FVB/Nmice were treated with a single injection of urethane and 4-month exposure to cigarette smoke. This treatment resultedin the appearance of a small number of tumor lesions (Fig.7A). No significant differences in the number of lesions werefound between WT and S100A9KO mice at that time (Fig.7B). However, in contrast with Gr-1þCD11bþ cells isolatedfrom spleens of mice exposed to cigarette smoke alone, cellswith the same phenotype isolated from mice treated withurethane and cigarette smoke and bearing lung tumors hadpotent immunosuppressive activity (Fig. 7C). After 4 monthsof treatment, WT mice had a significant increase in thepresence of MDSC, but not M�, in their spleens and lungs(Fig. 7D). The number of MDSC in S100A9KO mice wassignificantly lower, similar to the level seen in untreatedmice (Fig. 7D). The number of M� in S100A9KO mice waslower in the spleens but not in the lungs (Fig. 7D). MDSCisolated from the spleens of S100A9KO mice had significant-ly lower T-cell suppressive activity than MDSC from thespleens of WT mice (Fig. 7C).

In a separate set of experiments, we evaluated whetherthe lack of S100A9 had an effect on the survival of micetreated with urethane and cigarette smoke. The fate of themice was drastically different. All WT mice succumbed tothe disease within 5 months after the start of the treatment.In contrast, all S100A9KO mice were still alive at that timepoint (P ¼ 0.005; Fig. 7E). Thus, cigarette smoke, by itself,caused the expansion of IMC lacking immunosuppressiveactivity. Bona fide MDSC accumulated only after the appear-ance of tumor lesions in the lung. These cells, however,played a major role in tumor progression and survival of themice.

No

IMC

0

5,000

10,000

15,000

20,000

25,000

30,000

35,000

Control

Smoking

Lung IMCSpleen IMC

1:4 1:8 1:8 1:16

cpm

cpm

Uns

timulat

ed

+CD3/

CD28

0

20,000

40,000

60,000

80,000

100,000

ControlSmoking

1:1 1:2 1:4

BA

Figure 6. Gr-1þCD11bþmyeloid cells frommice exposed to cigarette smoke do not suppress T-cell proliferation. A, splenocytes from naïve FVB/Nmice werestimulated with CD3- and CD28-specific antibodies in the presence of Gr-1þCD11bþ IMC purified from control mice and mice exposed to cigarettesmoke at the indicated IMC:splenocyte ratios. T-cell proliferation was measured in triplicate by 3H-thymidine incorporation. Each group included 3 mice.B, purified IMC from the spleens or lungs of smoking or control FVB/N mice were added at the indicated ratios to a mixed lymphocyte reaction, whichwas set up using splenocytes from BALB/C mice as responders and irradiated splenocytes from FVB/N mice as stimulators mixed at 1:1 ratio. T-cellproliferation was measured in triplicate by 3H-thymidine incorporation. Mean and SEM from three experiments are shown.






DiscussionIn this study, we investigated the possible contribution of

MDSC to the development of lung cancer. Although ampleevidence has linked inflammation and cancer, the nature ofthe immune cells directly involved in the process of tumordevelopment remained unclear. This is especially true for lungcancer. Cigarette smoke is the major cause of lung cancer, andinflammation is closely associated with smoking. BecauseMDSC are shown to be an inherent part of chronic inflamma-tion, it was tempting to speculate that MDSC may be directlyinvolved in the early stages of the development of lung tumorassociatedwith chronic inflammation. To address this question,we used two contrasting models of lung cancer. Lung cancer in

CC10Tg mice is driven by the SV40Tag oncogene and is notassociated with inflammation (refs. 13, 26; data not shown).Lung tumor lesions started as focal hyperplasia and advancedrapidly until a considerable portion of the lung parenchymawasrepresented by tumor masses. In this model, we observed theaccumulation of cells with a typical MDSC phenotype andsuppressive activity only in mice at 4 months of age when thelungs were already full of tumor lesions. Gr-1þCD11bþ cellsisolated from the spleens ofmice at 3months of age did not haveT-cell suppressive activity. The appearanceof lung tumor lesionswas associated with the accumulation of Gr-1�F4/80þCD11bþ

macrophages in the lungs. This observation is consistent with aprevious report describing the differentiation of MDSC to

Figure 7. The effect of S100A9 deletion on tumor progression inmice exposed to urethane and cigarette smoke.WT and S100A9KO FVB/Nmice were treatedwith a single injection of urethane (1.5 g/kg) and 4 months of cigarette smoke. A, typical example of lung tissue stained with H&E. Bars, 1 mm. B, thenumber of tumor lesions per lung. The differences were not significant (P > 0.1). C, immunosuppressive activity of Gr-1þCD11bþ cells isolated from thespleens of mice assessed in allogeneic mixed leukocyte reaction. Each group included 3 mice. Mean and SEM are shown. The differences between groups:��, P < 0.01; ��, P < 0.001. The differences from "No MDSC" control were significant (P < 0.05) in all groups except at 1:4 ratio. D, the number ofindicated myeloid populations in the spleens and lungs of mice. #, P < 0.05 between WT and untreated naive mice; �, P < 0.05 between WT and S100A9KOmice. Each group included 4 mice. E, survival of mice of WT (n ¼ 6) and S100A9KO (n ¼ 7) mice treated with urethane and cigarette smoke. KO, knockout.

Ortiz et al.





macrophages in tumor tissues (25). These data indicate that inCC10Tg mice MDSC were likely the consequence of tumordevelopment. These results were predictable. We expected thatthe situation would be different in the model of lung cancerassociated with the inflammation–cigarette smoke model.Indeed, even a 1-month exposure of mice to cigarette smokeled to an increase in the presence of Gr-1þCD11bþ cells in theperipheral blood, and 4 months of cigarette smoke caused asignificant increase of these cells in the lungs and spleens. Thesecells were largely represented by the granulocytic subset. How-ever, contrary to our expectation, these cells lacked immuno-suppressive activity, indicating that they were not bona fideMDSC. Immunosuppressive MDSC accumulated in these miceonly after the development of tumor lesions. These data suggestthat in this model, MDSC accumulation was also the result oftumor formation. Our results are largely consistentwith those ofprevious reports demonstrating the association of lung tumorformation caused by urethane injection with the accumulationof Gr-1þCD11bþ cells. However, whether those cells had immu-nosuppressive activity was not determined (27).These data indicate that cigarette smoke, despite causing

the expansion of IMC, was not sufficient to convert them intoMDSC. These results are consistent with the two-signal con-cept of MDSC accumulation in cancer (28), suggesting that theexpansion of IMC cells and their conversion to MDSC pheno-type is governed by different factors and signaling pathways.Some factors involved in the expansion of IMC are wellcharacterized [i.e., granulocyte macrophage colony–stimulat-ing factor (GM-CSF), macrophage colony–stimulating factor(M-CSF)]. However, the mechanism responsible for MDSC-conversion is largely unclear and needs to be determined.Although bona fide MDSC are not present in mice until theappearance of tumor lesions, these cells are critically impor-tant for the survival of the mice. We addressed the role ofMDSC in thenatural history of lung tumor progression byusingS100A9KO mice. The S100A9/S100A8 heterodimers (S100A8/A9) are expressed predominantly in myeloid cells. The differ-entiation of myelocytes and granulocytes is associated with anincrease of S100A8/A9 expression, whereas the differentiationof M� and dendritic cells is associated with the loss of theseproteins (29–32). We, and others, have shown that the accu-mulation of MDSC and the inhibition of dendritic cell differ-entiation in cancer were closely correlated with the upregula-tion of S100A8/A9 (19–21). The expansion of MDSC wassignificantly reduced in S100A9-deficient tumor-bearing miceand mice treated with CFA (21). The S100A8/A9 proteins wereimplicated in lung cancer progression. In an early study,S100A8 and S100A9 were shown to be important in attractingCD11bþ cells to premetastatic lung. Neutralizing anti-S100A8and -S100A9 antibodies blocked the morphologic changes andmigration of tumor cells and CD11bþ myeloid cells (33). In amore recent report, upregulation of S100A8/A9 in the lungadenocarcinoma was found to correlate with the stage of thedisease (34). Patients with lung cancer overexpressing S100A9had a significantly lower 5-year overall survival rate thanpatients with low or no expression of S100A9. Overexpressionof S100A9was an independent factor predictive of poor diseaseoutcome (35). Consistent with these data, another study also

found an association of the expression of S100A8/A9 in stromalcells with negative outcome in patients with lung cancer (36).Another recent report described a direct link of S100A9 andMDSC by demonstrating an accumulation of immunosuppres-sive CD14þS100A9þ monocytic MDSC in patients with non–small lung cancer. The amount of these inflammatory mono-cytes was associated with poor response to chemotherapy (37).An upregulation of the S100A8 and S100A9 genes in thebronchial airway epithelial cell brushings was found to beassociated with smoking (38).

These results suggested that a lack of S100A9 could decreasethe accumulation of MDSC and thus open the opportunity todirectly test the role of these cells in tumor progression. In ourexperiments, mice lacking S100A9 had reduced presence ofMDSC in both experimental models. In the urethane/cigarettesmoke model, it did not significantly reduce the number ofinitial tumor lesions. However, in both lung cancer models, theS100A9KO mice had much better survival than their WTcounterparts. In a recent study onmultiplemyeloma, improvedsurvival of S100A9KO tumor-bearing mice was associated withan enhanced tumor-specific response of CD8þ T cells, whichcould be reversed by the administration of MDSC (22).

Taken together, these data suggest that early stages of tumordevelopment in cigarette smoke–related lung cancer are asso-ciatedwith the accumulation of nonsuppressive IMC.Bona fideMDSC appear only after the development of tumors andcontribute greatly to tumor progression regardless of whethertumor development was associated with inflammation or not.This necessitates the search for a better way to therapeuticallytarget these cells in patients with cancer. IMC, for their part,may play an important role in tumor development viamechan-isms not involving immune suppression. We recently havefound that IMC could promote the development of skin cancerby recruiting proinflammatory CD4þ T cells (data not shown).These results indicate that IMC and MDSC may be majorfactors in the regulation of various stages of tumor develop-ment and progression and their precise role needs to be furtherelucidated.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M.L. Ortiz, L. Lu, D.I. GabrilovichDevelopment of methodology: M.L. Ortiz, L. Lu, D.I. GabrilovichAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Lu, I. RamachandranAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.L. Ortiz, L. LuWriting, review, and/or revision of themanuscript: L. Lu, I. Ramachandran,D.I. GabrilovichAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M.L. Ortiz, L. LuStudy supervision: M.L. Ortiz, L. Lu, D.I. Gabrilovich

AcknowledgmentsThe authors thank Dr. F. DeMayo (Baylor College of Medicine) for providing

them with CC10Tg mice.

Grant SupportThis study was supported by NIH grant CA 100062 to D.I. Gabrilovich and by

NIHmentor/sponsorminority supplement grant NIH3R01CA084488-11S2 toD.I.Gabrilovich and M.L. Ortiz.






The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 21, 2013; revised September 26, 2013; accepted October 4,2013; published OnlineFirst October 18, 2013.

References1. Gabrilovich DI, Nagaraj S. Myeloid-derived suppressor cells as reg-

ulators of the immune system. Nat Rev Immunol 2009;9:162–74.2. Peranzoni E, Zilio S, Marigo I, Dolcetti L, Zanovello P, Mandruzzato S,

et al. Myeloid-derived suppressor cell heterogeneity and subset def-inition. Curr Opin Immunol 2010;22:238–44.

3. Talmadge JE, Gabrilovich DI. History of myeloid derived suppressorcells (MDSCs) in themacro- andmicro-environment of tumour-bearinghosts. Nat Rev Cancer 2013;13:739–52.

4. Gabrilovich DI, Ostrand-Rosenberg S, Bronte V. Coordinated regula-tion of myeloid cells by tumours. Nat Rev Immunol 2012;12:253–68.

5. WangL,ChangEW,WongSC,OngSM,ChongDQ, LingKL. Increasedmyeloid-derived suppressor cells in gastric cancer correlate withcancer stage and plasma S100A8/A9 proinflammatory proteins.J Immunol 2013;190:794–804.

6. Porembka MR, Mitchem JB, Belt BA, Hsieh CS, Lee HM, Herndon J,et al. Pancreatic adenocarcinoma induces bone marrow mobilizationof myeloid-derived suppressor cells which promote primary tumorgrowth. Cancer Immunol Immunother 2012;61:1373–85.

7. Yuan XK, Zhao XK, Xia YC, Zhu X, Xiao P. Increased circulatingimmunosuppressive CD14(þ)HLA-DR(�/low) cells correlate with clin-ical cancer stage and pathological grade in patients with bladdercarcinoma. J Int Med Res 2011;39:1381–91.

8. Daud AI, Mirza N, Lenox B, Andrews S, Urbas P, Gao GX, et al.Phenotypic and functional analysis of dendritic cells and clinicaloutcome in patients with high-risk melanoma treated with adjuvantgranulocyte macrophage colony-stimulating factor. J Clin Oncol2008;26:3235–41.

9. Diaz-MonteroCM, SalemML,NishimuraMI, Garrett-Mayer E,ColeDJ,Montero AJ. Increased circulating myeloid-derived suppressor cellscorrelate with clinical cancer stage, metastatic tumor burden, anddoxorubicin-cyclophosphamide chemotherapy. Cancer ImmunolImmunother 2009;58:49–59.

10. Ostrand-Rosenberg S, Sinha P. Myeloid-derived suppressor cells:linking inflammation and cancer. J Immunol 2009;182:4499–506.

11. Nagaraj S, Youn J, Gabrilovich D. Reciprocal relationship betweenmyeloid-derived suppressor cells and T cells. J Immunol 2013;191:17–23.

12. Coussens LM, Zitvogel L, Palucka AK. Neutralizing tumor-promotingchronic inflammation: a magic bullet? Science 2013;339:286–91.

13. Magdaleno SM, Wang G, Mireles VL, Ray MK, Finegold MJ, DeMayoFJ. Cyclin-dependent kinase inhibitor expression in pulmonary Claracells transformed with SV40 large T antigen in transgenic mice. CellGrowth Differ 1997;8:145–55.

14. Rubin H. Synergistic mechanisms in carcinogenesis of polycyclicaromatic hydrocarbons and by tobacco smoke: a bio-historical per-spective with updates. Carcinogenesis 2001;22:1903–30.

15. Rutgers SR, Postma DS, ten Hacken NH, Kaufman HF, van Der MarkTW, Koster GH, et al. Ongoing airway inflammation in patients withCOPD who do not currently smoke. Chest 2000;117:262S.

16. Hecht SS. Carcinogenicity studies of inhaled cigarette smoke inlaboratory animals: old and new. Carcinogenesis 2005;26:1488–92.

17. Kisley LR,Barrett BS,BauerAK,Dwyer-Nield LD, Barthel B,Meyer AM,et al. Genetic ablation of inducible nitric oxide synthase decreasesmouse lung tumorigenesis. Cancer Res 2002;62:6850–6.

18. Stathopoulos GT, Sherrill TP, Cheng DS, Scoggins RM, Han W,Polosukhin VV, et al. Epithelial NF-kappaB activation promotes ure-thane-induced lung carcinogenesis. Proc Natl Acad Sci U S A2007;104:18514–9.

19. Ichikawa M, Williams R, Wang L, Vogl T, Srikrishna G. S100A8/A9activate key genes and pathways in colon tumor progression. MolCancer Res 2011;9:133–48.

20. Sinha P, Okoro C, Foell D, Freeze HH, Ostrand-Rosenberg S, Srik-rishna G. Proinflammatory s100 proteins regulate the accumulation ofmyeloid-derived suppressor cells. J Immunol 2008;181:4666–75.

21. Cheng P, Corzo CA, Luetteke N, Yu B, Nagaraj S, Bui MM, et al.Inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 protein.J Exp Med 2008;205:2235–49.

22. Ramachandran IR,MartnerA,PisklakovaA,CondamineT,ChaseT,VoglT, et al. Myeloid-derived suppressor cells regulate growth of multiplemyeloma by inhibiting T cells in bone marrow. J Immunol 2013;190:3815–23.

23. ShimizuK, LibbyP,RochaVZ, FolcoEJ, Shubiki R,GrabieN, et al. Lossofmyeloid related protein-8/14 exacerbates cardiac allograft rejection.Circulation 2011;124:2920–32.

24. Manitz MP, Horst B, Seeliger S, Strey A, Skryabin BV, Gunzer M, et al.Loss of S100A9 (MRP14) results in reduced interleukin-8-inducedCD11b surface expression, a polarized microfilament system, anddiminished responsiveness to chemoattractants in vitro. Mol Cell Biol2003;23:1034–43.

25. Corzo CA, Condamine T, Lu L, Cotter MJ, Youn JI, Cheng P, et al. HIF-1alpha regulates function and differentiation of myeloid-derived sup-pressor cells in the tumor microenvironment. J Exp Med 2010;207:2439–53.

26. Hicks SM, Vassallo JD, DieterMZ, Lewis CL,Whiteley LO, Fix AS, et al.Immunohistochemical analysis of Clara cell secretory protein expres-sion in a transgenic model of mouse lung carcinogenesis. Toxicology2003;187:217–28.

27. Rosin FC, Pedregosa JF, de Almeida JS, Bueno V. Identification ofmyeloid-derived suppressor cells and T regulatory cells in lung micro-environment after Urethane-induced lung tumor. Int Immunopharma-col 2011;11:873–8.

28. Condamine T, Gabrilovich DI. Molecular mechanisms regulating mye-loid-derived suppressor cell differentiation and function. Trends Immu-nol 2011;32:19–25.

29. Markowitz J, Carson WE III. Review of S100A9 biology and its role incancer. Biochim Biophys Acta 2013;1835:100–9.

30. SrivastavaMK, Andersson A, Zhu L, Harris-White M, Lee JM, DubinettS, et al. Myeloid suppressor cells and immune modulation in lungcancer. Immunotherapy 2012;4:291–304.

31. Ehrchen JM, Sunderkotter C, Foell D, Vogl T, Roth J. The endogenousToll-like receptor 4 agonist S100A8/S100A9 (calprotectin) as innateamplifier of infection, autoimmunity, and cancer. J Leukoc Biol2009;86:557–66.

32. Leder A, Kuo A, Cardiff RD, Sinn E, Leder P. v-Ha-ras transgeneabrogates the initiation step inmouse skin tumorigenesis: effects of ph-orbolestersandretinoicacid.ProcNatlAcadSciUSA1990;87:9178–82.

33. Hiratsuka S, Watanabe A, Aburatani H, Maru Y. Tumour-mediatedupregulation of chemoattractants and recruitment of myeloid cellspredetermines lung metastasis. Nat Cell Biol 2006;8:1369–75.

34. Su YJ, Xu F, Yu JP, Yue DS, Ren XB, Wang CL. Up-regulation of theexpression of S100A8 and S100A9 in lung adenocarcinoma and itscorrelation with inflammation and other clinical features. Chin Med J2010;123:2215–20.

35. Kawai H, Minamiya Y, Takahashi N. Prognostic impact of S100A9overexpression in non–small cell lung cancer. Tumour Biol 2011;32:641–6.

36. Ohri CM, Shikotra A, Green RH, Waller DA, Bradding P. The tissuemicrolocalisation and cellular expression of CD163, VEGF, HLA-DR,iNOS, andMRP 8/14 is correlated to clinical outcome in NSCLC. PLoSONE 2011;6:e21874.

37. Feng PH, Lee KY, Chang YL, Chan YF, Kuo LW, Lin TY, et al. CD14(þ)S100A9(þ) monocytic myeloid-derived suppressor cells and theirclinical relevance in non–small cell lung cancer. Am J Respir Crit CareMed 2012;186:1025–36.

38. Beane J, Vick J, Schembri F, Anderlind C, Gower A, Campbell J, et al.Characterizing the impact of smoking and lung cancer on the airwaytranscriptome using RNA-Seq. Cancer Prev Res 2011;4:803–17.

Ortiz et al.





2014;2:50-58. Published OnlineFirst October 18, 2013.Cancer Immunol Res Myrna L. Ortiz, Lily Lu, Indu Ramachandran, et al. CancerMyeloid-Derived Suppressor Cells in the Development of Lung

Updated version

10.1158/2326-6066.CIR-13-0129doi:

Access the most recent version of this article at:

Cited articles

http://cancerimmunolres.aacrjournals.org/content/2/1/50.full#ref-list-1

This article cites 38 articles, 17 of which you can access for free at:

Citing articles

http://cancerimmunolres.aacrjournals.org/content/2/1/50.full#related-urls

This article has been cited by 9 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerimmunolres.aacrjournals.org/content/2/1/50To request permission to re-use all or part of this article, use this link



http://cancerimmunolres.aacrjournals.org/lookup/doi/10.1158/2326-6066.CIR-13-0129

http://cancerimmunolres.aacrjournals.org/content/2/1/50.full#ref-list-1

http://cancerimmunolres.aacrjournals.org/content/2/1/50.full#related-urls

http://cancerimmunolres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerimmunolres.aacrjournals.org/content/2/1/50


Documents

Myeloid-Derived Suppressor Cells in the Development of ...€¦ · the development of lung cancer using two experimental models: a lung cancer model in CC10 transgenic mice (CC10Tg)