Myeloid-Derived Cells in Tumors: Effects of Radiation

Myeloid-Derived Cells in Tumors: Effects of Radiation Ralph E. Vatner, MD, PhD and Silvia C. Formenti, MD The discrepancy between the in vitro and in vivo response to radiation is readily explained by the fact that tumors do not exist independently of the host organism; cancer cells grow in the context of a complex microenvironment composed of stromal cells, vasculature, and elements of the immune system. As the antitumor effect of radiotherapy depends in part on the immune system, and myeloid-derived cells in the tumor microenvironment modulate the immune response to tumors, it follows that understanding the effect of radiation on myeloid cells in the tumor is likely to be essential for comprehending the antitumor effects of radiotherapy. In this review, we describe the phenotype and function of these myeloid-derived cells, and stress the complexity of studying this important cell compartment owing to its intrinsic plasticity. With regard to the response to radiation of myeloid cells in the tumor, evidence has emerged demonstrating that it is both model and dose dependent. Deciphering the effects of myeloidderived cells in tumors, particularly in irradiated tumors, is key for attempting to pharmacologically modulate their actions in the clinic as part of cancer therapy. Semin Radiat Oncol 25:18-27 C 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Introduction

T

umors are not merely a collection of cancer cells, rather they function more like an organ comprising a complex and organized scaffolding of cellular and noncellular stroma and recruited cells.1,2 The primary elements of the tumor stroma are fibroblasts and the noncellular collagen-rich extracellular matrix they produce, the endothelial cells and pericytes of the tumor vasculature, and resident leukocytes such as macrophages driving progression. Tumors also recruit inflammatory immune cells, including tumor-infiltrating lymphocytes (TILs, both CD4þ and CD8þ), natural killer (NK) cells, NK T-cells, B cells, and an array of myeloid-derived cells.3 The various components of the microenvironment interact to influence the growth and function of each of the other elements, including the cancer cells themselves, mediated

Department of Radiation Oncology, New York University School of Medicine, New York, NY. S.F. is supported by grants from the National Cancer Institute, NIH, USA (R01CA161891-01), Department of Defense Breast Cancer Research Program (BC100481 and W81XWH-11-1-0530), and Breast Cancer Research Foundation, USA (13-A0-00-001870-01). The authors declare no conflict of interest. Address reprint requests to Silvia C. Formenti, MD, Department of Radiation Oncology, New York University School of Medicine, New York, NY 10016. E-mail: [email protected]

18

through direct cell-cell contact and secreted cytokines and chemokines. Myeloid-derived cells are an important part of the microenvironment, both numerically and functionally, and play a central role in regulating the antitumor immune response.3-5 Arising from the common myeloid progenitor cell, these include tumor-associated macrophages (TAMs), dendritic cells (DCs), polymorphonuclear neutrophils (PMNs), and myeloidderived suppressor cells (MDSCs). Although each of these is usually considered distinct populations, the functional distinctions in the tumor microenvironment can be somewhat artificial.6 Rather, myeloid cells in tumors exist along a spectrum of maturation and a plastic immunomodulatory phenotype that can be influenced by ionizing radiation and other exogenous agents.7-10 Functionally, myeloid cells are both friend and foe to the antitumor immune response. They are essential for facilitating antitumor immunity, but they take on an immunosuppressive phenotype in established tumors, helping to promote immune evasion.11 For example, DCs are necessary for cross-priming of cytotoxic CD8þ T lymphocytes against tumor-specific antigens and are required for immune-mediated rejection in murine tumor models.12-14 However, tumor-resident DCs often have a regulatory phenotype and express low levels of costimulatory molecules and proinflammatory cytokines, leading to T-cell anergy and tolerance.1,2,15-18 Similarly,

http://dx.doi.org/10.1016/j.semradonc.2014.07.008 1053-4296//& 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Myeloid-Derived Cells in Tumors

19 immune response in tissues and tumors.7-10,26,27 Most tumor macrophages develop from monocytes recruited from the blood. They have tremendous tissue-specific phenotypic variability, and although there is no single set of macrophagespecific surface markers, useful markers include the general myeloid-lineage marker CD11b and F4/80 (in mice) and CD14 and CD68 (in humans). Functional classification of macrophages as classically activated (M1) vs alternatively activated (M2) is a useful and commonly used paradigm to describe the extremes of a spectrum of macrophage phenotype based on patterns of gene expression and cytokine production8-11,28,29 (Fig. 1). M1 polarization develops in response to inflammatory stimuli, such as interferon (IFN)-γ and lipopolysaccharide, and is characterized by higher surface expression of major histocompatibility complex (MHC) Class II and CD86. These M1 cells generate nitric oxide (NO) catalyzed from arginine by inducible NO synthase (iNOS), signal through STAT3 and STAT1, can kill tumor cells directly, and stimulate antitumor T-cells by secreting the proinflammatory cytokines IL-12 and IL-6. Conversely, in the presence of IL-4 and IL-13 macrophages develops an M2 phenotype, which suppress antitumor immunity by producing arginase and the cytokines transforming growth factor (TGF)-β and IL-10. M2 cells can be further identified by STAT6 phosphorylation and surface expression of mannose receptor (CD206), programmed death ligand 2 (PD-L2), and restin-like molecule-a.

macrophages can promote immune effector function, yet TAMs usually have an immunosuppressive phenotype that promotes tumor growth, invasion, and metastasis.3,19-22 The fact that myeloid-derived cells contribute to an immunosuppressive tumor microenvironment is no accident. Most tumors evolve in the context of a competent immune system that recognizes neoplastic cells by the expression of tumor-specific neoantigens and embryonic antigens normally expressed exclusively during development. Tumors can only grow after selection of immune escape variants by immunoediting, and clinically apparent tumors have undergone this process, developing the means to escape immune recognition.3-5,23-25 Myeloid-derived cells are at the center of immune escape. They are actively recruited by tumor-secreted factors and maintained in an immunosuppressive phenotype that allows for tumor growth in the context of an otherwise competent immune system.5,6,8

Phenotype and Function of Myeloid-Derived Cells in the Tumor Microenvironment Macrophages Macrophages are large mononuclear phagocytes that ingest cellular debris, promote wound healing, and modify the local

Type 1

Type 2

Immune Suppression

Monocyte RegDC

DC

ARG1 M1 Macrophage

M2 Macrophage

Maturaon

H2O2 ONOOMonocyte

Granulocyte

N1 PMN

Effector T-Cell

IL-10 TGF-β M-MDSC

Myeloid Progenitor Cell

L-Arginine

PD-L1/2

CCL22

NK Cell

DC G-MDSC

H2O2 ONOO-

RegDC

Treg

N2 PMN

Figure 1 Myeloid-derived cells in tumors and mechanisms of immune suppression. Tumor-associated myeloid cells originate from a myeloid progenitor cell in the bone marrow. These produce the granulocytes (primarily PMNs) and granulocytic MDSCs (g-MDSC), and the monocytic cells including monocytes, macrophages, dendritic cells (DCs), and monocytic MDSCs (m-MDSC). Influenced by their environment, myeloid cells can acquire a phenotype ranging from proinflammatory (type 1) to immunosuppressive (type 2). Type 2 myeloid cells suppress antitumor immunity through the following 4 general mechanisms: (1) They produce arginase (ARG-1), which depletes L-arginine and inhibits T-cell function. (2) They produce reactive oxygen species such as hydrogen peroxide (H2O2) and reactive nitrogen intermediates such as peroxynitrite (ONOO), which modify receptors for antigens and chemokines on T-cells and impairing their function. (3) They produce cytokines such as IL-10 and TGF-β that impair effector T-cells and NK cells and convert DCs into regulatory DCs, which can further impair T-cell function. (4) They produce chemokines such as CCL22, which selectively attract Tregs, and TGF-β also directly stimulates Tregs to suppress antitumor effector T-cells. (Color version of figure is available online.)

R.E. Vatner and S.C. Formenti

20 Macrophages in established tumors are usually M2 polarized, suppressing antitumor immunity and directly promoting tumor growth, invasion, and metastasis. TAMs are driven to adopt this phenotype by factors secreted by cancer cells and TILs, including tumor necrosis factor (TNF), prostaglandins, and IL-10.11,22,30 Various mechanisms to explain this immunosuppression have been proposed, most of which are shared by MDSCs. Gabrilovich et al30 neatly parses these into 4 categories: (1) nutrient depletion, (2) oxidative stress, (3) impairment of lymphocyte trafficking and viability, and (4) promotion of Treg numbers and function. T-cells depend on certain nutrients such as the amino acid L-arginine for normal growth and function, and arginase production by TAMs can suppress antitumor immunity by modifying the metabolic milieu of the tumor microenvironment. Arginase depletes L-arginine and results in suppression of the effector activity of TILs through downregulation of the expression of zeta chain of the T-cell receptor (TCR) and progression through the cell cycle.12-18,31-35 Another immunosuppressive mechanism used by both TAMs (and MDSCs) is the generation of reactive oxygen species (ROS).36-39 These ROS can modify the structure of both the MHC Class I expressed on tumor cells and the TCR of TILs, which interferes with the specific recognition and killing of tumor cells.23-25,40 Reactive nitrogen intermediates can also modify chemokines such as CCL2, preventing the recruitment of tumor-specific lymphocytes.41 TAMs interfere with T-cell function in other ways as well. Although more associated with M1 TAMs and MDSCs, NO is another agent produced by TAMs that can directly interfere with T-cell activation and proliferation by reversibly blocking signaling through the IL-2 receptor.19-22,42 In addition to inhibiting effector TILs, M2 TAMs can directly inhibit and even kill effector TILs. A potential mechanism is through direct cytotoxicity of effector TILs by engagement of the PD-1 receptor with its ligands PL-L1 and PD-L2, which are expressed by TAMs. M2 TAMs can inhibit the cytotoxic antitumor effector functions of TILs through production of the anti-inflammatory cytokines IL-10 and TGF-β. These cytokines also promote the development of Tregs and regulatory DCs (regDCs), both of which suppress antitumor immunity through their own array of mechanisms. Furthermore, TAMs selectively recruit immunosuppressive Tregs to the tumor microenvironment through the secretion of chemokines such as CCL22.12-14,43 Although TAMs in most tumors tend to have an immunosuppressive M2 phenotype, it is worth noting that TAMs have the potential to adopt M1 polarization, which are generally thought to have an antitumor effect.28 M1 macrophages do produce the proinflammatory cytokines IL-1β, TNF, IL-6, and IL-12, which promote antitumor T-helper type 1 immune responses and effector function of cytotoxic T lymphocytes (CTLs) and NK cells. M1 TAMs also recruit T-helper type 1 cells through secretion of chemokines such as CXCL9 and CXCL10.28 Furthermore, M1-polarized TAMs can kill tumor cells directly through antibody-dependent cellular cytotoxicity as well as TNF production.44,45 In fact, the antitumor effect of M1 TAMs was initially proposed to explain a correlation

between tumor regression and elevated NO production by macrophages in murine tumors.46,47 This association holds true in some human tumors in which elevated M1-M2 TAM ratios correlate with improved clinical outcomes including survival.48-50 However, not all evidence supports an antitumor effect of M1 TAMs. NO synthase, one of the quintessential M1 gene products, depletes arginine, which suppresses T-cell function, and generates NO and reactive nitrogen species, which can further inhibit antitumor immunity and promote tumor progression.51-53

Dendritic Cells DCs are monocytic myeloid-derived antigen-presenting cells (APCs) in the tumor microenvironment that mediate both priming and tolerizing antitumor T-cells.12-14 Like macrophages, DCs are a heterogeneous cell type with at least 5 subtypes, each functionally and sometimes anatomically distinct.54,55 Although there are experimental conditions that provide evidence for the importance of just about every subtype of DC in T-cell priming, the most important players seem to be the CD11bþ CD11cþCD4CD8αþ subset, the CD11bþCD11cþCD4CD8 monocyte-derived population, and the plasmacytoid DCs.56 The major population in tumors are monocyte-derived DCs, which endocytose tumor antigens and transport them to the draining lymph nodes where they prime antigen-specific CD4þ and CD8þ T-cells in concert with CD8αþ DCs. Plasmacytoid DCs also have an important role in cross-priming antitumor CD8þ T-cell responses. These cells are factories for type I INF, which has been implicated as a vital part of the immune response to tumors. As early as 1983, it was observed that depletion of type I INF could enhance tumor growth in mice.57 Type I INF was later identified as a mediator of cancer immunosurveillance in mice and is essential for radiation-induced cross-priming of tumor-specific CTLs by its action on CD8αþ DCs.24,58-63 Analogous to the M1-M2 paradigm for macrophages, DCs can adopt a regulatory phenotype that promotes T-cell anergy and tolerance.15,64 These regDCs can be identified by their surface markers CD11bhighCD11clowGR1, but in practice, differentiating between TAMs and DCs can be difficult, as histologic appearance and surface markers can give divergent or ambiguous results. Functionally, regDCs mediate T-cell anergy by presenting antigen in the context of decreased surface expression of costimulatory molecules and production of the anti-inflammatory cytokines IL-4, IL-10, and IL-13. In the Lewis lung carcinoma and B16 melanoma tumor models, regDCs could suppress T-cell activation and proliferation in response to stimulation with the mitogen concanavalin A in vitro, and adoptive transfer of regDCs promoted the formation of tumor metastases.65

Polymorphonuclear Neutrophils Polymorphonuclear granulocytes, or PMNs, are the most abundant leukocytes and important mediators of acute inflammation and protection against bacterial infection. The M1-M2 dichotomy has been extended to PMNs, which can

Myeloid-Derived Cells in Tumors assume a spectrum of phenotypes anywhere from tumoricidal (N1) to suppressive of antitumor immunity (N2).36,38,39 However, the data supporting this distinction in tumorassociated neutrophils (TANs) are still inconclusive. Furthermore, TANs are often defined as CD11bþLy6Gþ cells, which conflates them with the immunosuppressive granulocytic MDSCs (g-MDSCs), now considered a distinct population from mature PMNs.66 Gene expression data support the concept of a spectrum of maturation and expression level of a similar gene profile, with N1 TANs representing activated PMNs and N2 TANs resembling g-MDSCs that can mature into N1 TANs with the appropriate stimulus.67 In essence, the distinction between N1 and N2 is functional, with N1 defined as proinflammatory and N2 as immunosuppressive. The biological significance of TANs is still under investigation, and unlike MDSCs and tumor macrophages, it is not clear whether PMNs are more proinflammatory or immunosuppressive in tumors.36 In some human cancers such as clear cell renal carcinoma, esophageal carcinoma, gastric carcinoma, and squamous cell carcinoma of the head and neck, TAN number and density are prognostic factors that correlate with poor clinical outcome.68-71 This finding is consistent with the concept that TANs, macrophages, and MDSCs all share similar tools for immune suppression, including ROS such as hydrogen peroxide and expression of iNOS for the depletion of 72 L-arginine and production of NO. Somewhat paradoxically, in studies of PMNs in vitro, iNOS and ROS mediate tumor cytotoxicity, which is in stark contrast to g-MDSC that use the same means to suppress antitumor immunity, as described later. These contrasting ideas have not been resolved experimentally, but it may be a result of the anatomical location of the cells and immune mediators.

Myeloid-Derived Suppressor Cells MDSC is a collective term to describe a heterogeneous population of immature myeloid cells that have immunosuppressive properites.10,53,73-75 They are found in the blood, lymphoid organs, and tumor microenvironment in animals and patients with cancer. Although they are defined functionally by their ability to suppress immune responses, they also can be distinguished by their expression of surface markers. There are 2 general populations of MDSCs that reflect the 2 lineages of myeloid cells: monocytic and granulocytic.76,77 In mice, monocytic MDSCs (m-MDSC) are CD11bþLy6ChighLy6G, in contrast to g-MDSC, which are CD11bþLy6ClowLy6Gþ. Humans do not express a marker homologous to Ly6C/G (Gr1). Instead, human MDSCs are defined as CD11bþCD14CD33þ, making it impossible to distinguish between m-MDSCs and g-MDSCs based on surface markers.78 Both populations of MDSCs are prevalent in the lymphoid organs, peripheral blood, and tumors of tumor-bearing animals, with m-MDSCs more prevalent in the tumor microenvironment. MDSCs in the serum and tumor correlate with poor clinical outcomes in patients and mediate suppression of the antitumor immune response in mice both by antigen-specific and

21 nonspecific mechanisms.74,79 A mechanism shared between TAMs and m-MDSCs—the predominant MDSC population in tumors—is the expression of iNOS and arginase. In addition to inhibiting T-cell function by depleting L-arginine, iNOS generates NO, which inhibits signaling through the IL-2 receptor, interfering with T-cell activation and proliferation. The fact that M1 TAMs, which overall support antitumor immunity, also express iNOS is somewhat of a paradox, but perhaps the immunosuppressive effect of iNOS in these cells is overwhelmed by their other proinflammatory and antitumor mediators. In addition to arginine depletion and NO generation, m-MDSCs generate ROS and reactive nitrogen species such as peroxynitrite mediated by iNOS and arginase.80 Much like the ROS generated by TAMs, peroxynitrite can modify the TCR of antitumor T-cells making them unable to bind to their cognate MHC-peptide antigen when presented by tumors and other APCs in the tumor.81 These reactive nitrogen species can also modify chemokines making them inactive and preventing the recruitment of antitumor immune cells. Myeloid MDSCs can also suppress antitumor immunity indirectly. Like M2 TAMs, MDSCs generate the cytokines IL-10 and TGF-β that can suppress antitumor TILs, generate Tregs in the tumor, and convert DCs into a regulatory phenotype.82 MDSCs can also recruit Tregs to tumors in a TGF-β–independent pathway.83 The g-MDSCs have many of the same immunosuppressive mechanisms as m-MDSCs, but they also generate ROS that, like reactive nitrogen species, can modify the TCR of TILs through direct cell-to-cell contact.84

Effect of Ionizing Radiation on Tumor Myeloid Cells Ionizing radiation profoundly affects the tumor microenvironment, including the myeloid cells in tumors. Data supporting this have been reported from many different tumor models using a variety of dose and fractionation regimens and are summarized in the Table. The data are so disparate, dependent on radiation dose and tumor model, that upon first glance it seems difficult to draw any conclusions about a consistent effect of radiotherapy (RT) on tumor myeloid cells. However, close examination reveals some patterns arising from the noise of variability.85 The data supporting an effect of RT on tumor myeloid cells can be characterized broadly as causing the 5 following effects: recruitment, removal, reorganization, repolarization, and representation, each of which will be explained in more detail later.

Recruitment Recruitment of myeloid-derived cells to tumors is one of the more consistent findings associated with RT. The recruited cells are primarily M2 TAMs and MDSCs and usually promote tumor growth and immune evasion. There is some human data from patients who underwent enucleation for recurrent or persistent uveal melanoma with a history of radiation delivered by brachytherapy. The mean dose delivered to the tumor apex

R.E. Vatner and S.C. Formenti

22 Table Studies Demonstrating and Effect of Radiation on Tumor Myeloid Cells Ref

Model

Dose or Fx

86

Human uveal melanoma

87

Human glioma

88

Mouse Panc02 pancreas

89

Mouse TRAMP-C1 prostate, ALTS1C1, and GL261 astrocytoma

25 Gy  1

98

Mouse TRAMP-C1 prostate Mouse TRAMP-C1 prostate

25 Gy  1 4 Gy  15 25 Gy  1 4 Gy  15

Mouse RM-1, RM-9, and Myc-CaP prostate

3 Gy  5 (mice)

Human prostate

Standard definitive Tx (humans) 2 Gy  5 15 Gy  1

100

92

93

U251 human glioblastoma xenograft

95

Human 54A lung xenograft Mouse MCa8 mammary

Brachytherapy median 86 Gy Brachytherapy minimum 60 Gy 20 Gy  3

20 Gy  1 15 Gy þ 6 Gy tumor RT 15 Gy þ 6 Gy Total Body RT 12 Gy  1

96

Mouse TUBO mammary Mouse MC38 colon

106

Mouse MC57G fibrosarcoma

10 Gy  1

90

Human FaDu hypopharynx xenograft Mouse LLC lung, MC38 colon, and SCCVII Mouse MT1A2 mammary Mouse RIF fibrosarcoma

12 Gy x 1

107

15 Gy  1 20 Gy  1 20 Gy  1

TAM

Recruitment, 50% increase on day 7. M2 polarized. Tumor control improved in NF-κB/ mice. TAM, PMN, Reorganization, CD11blow/CD68þ TAMs in hypoxic regions, and G-MDSC CD11blow/F4/80þ TAMs between hypoxic and necrotic regions. CD11bþGr1þ PMN/G-MDSC in necrotic areas. Increase in CD11bþGr1þ cells (20%), no TAM increase. Tumor model and tissue effects. TAM Recruitment, CD68þ TAMs to hypoxic areas.

TAM

TAM and MDSC

Repolarization, M2 genes (Arg-1 and COX2) up within days, lasting weeks. Adoptive transfer of these TAMs shows a trend toward faster tumor growth. Recruitment, RT - ABL1 nuclear translocation - CSF-1 transcription and translation - MDSC and TAM recruitment. Patients treated for prostate cancer have an increase in CSF1 after RT.

Recruitment, RT - vasculature damage - hypoxia - HIF-1 - SDF-1, binds to CXCR4, and recruits TAM/MDSC. Blocking HIF-1 or SDF-1/CXCR4 prevents myeloid cell recruitment and tumor. TAM, MDSC, Recruitment, RT induces SDF-1, recruits CD11bþF4/80þ and CD11bþ TAMs and CD11bþF4/80Gr1 myeloid cells. Blocking CXCR4 abrogates recruitment and improves tumor control myeloid cells after RT.

TAM and MDSC

TAM, MDSC, and PMN

Removal, no increase in MDSC 3 and 10 d. RT increased PDL1 on tumor and myeloid cells. RT þ PD-L1 antagonist controlled tumor and induced systemic antitumor immunity. TAM Re-presentation, RT - tumor Ag cross-presentation by TAMs, required for tumor elimination by RT þ CTL adoptive transfer. CD11bþ Recruitment, RT recruits CD11bþ myeloid cells. Inhibiting CD11b or CD18 inhibits RT-mediated myeloid cell myeloid cells recruitment and improves tumor response to RT.

DC

Mouse B16-OVA 15 Gy  1 melanoma 3 Gy  5 Mouse B16-D5 melanoma 8.5 Gy  5

108

Recruitment, o1 mo and persistent for 41 y.

15 Gy  3

91

Mouse B16-SIY melanoma Mouse B16 melanoma

TAM and microglia

20-25 Gy  1

Mouse RT5 insulinoma 0.5-6 Gy  1 Human MeWo melanoma xenograft Human pancreas

104

Effect of RT Recruitment, up to 5 y after RT.

CD11bþ Recruitment, model specific, primarily MMP-9þ. myeloid cells MMP-9þ myeloid cells are important for tumor vasculogenesis and growth after RT. TAM Repolarization, M2 - M1 by lower dose RT (r2 Gy) or adoptive transfer of irradiated macrophages - iNOS recruits endogenous or adoptive transferred T-cells. Doses 42 Gy deplete TILs. RT 0.5-5 Gy to human pancreas cancer increases TILs. DC, TAM, and Recruitment, DC, TAM, and MDSC. MDSC Re-presentation and DC, and possible macrophage, in TDLN. DC Re-presentation and exogenous DC cross-present host tumor antigen. RT does not affect maturation. DC Re-presentation, DC, and possible macrophage, in TDLN

101

105

Myeloid Cell TAM

Re-presentation and DC required for RT-induced crosspriming

Abbreviations: MMP, matrix metalloproteinase; Ref, reference; SDF, stromal-derived factor; TDLN, tumor-draining lymph node.

Myeloid-Derived Cells in Tumors in these patients was 86 Gy, which resulted in an influx of macrophages into the tumor and surrounding irradiated sclera, an effect that persisted for years after RT.86 Similar long-lived macrophage recruitment was observed in the brain in patients treated with brachytherapy to a minimum dose of 60 Gy for glioma.87 Histopathologic analysis was performed at autopsy, ranging from 3 weeks to 5 years after treatment, and at all time points CD68þ macrophages and microglia were observed in the treated tissues and tumors around areas of necrosis, which occurred inside the 72-Gy isodose line. In both of these studies, parts of the tumors were treated with much higher doses of RT owing to the dose distribution properties of brachytherapy. This recruitment of TAMs in response to RT is seen in a variety of murine tumor models as well. Lewis lung carcinoma implanted subcutaneously and treated with 5 daily fractions of 6 Gy develops an influx of Arg-1þ F4/80þ macrophages at all time points tested, from 3-14 days after treatment (Fig. 2). Crittenden et al88 observed an influx of TAM and MDSCs in a transplantable model of pancreatic adenocarcinoma (Panc02) and metastatic breast cancer (4T1) after treatment with 3 fractions of 20 Gy. Gene array analysis of the TAMs revealed an increase in transcription of M2-related messenger RNAs, which was dependent on nuclear factor-κB (NF-κB), with indirect evidence that M2 TAMs increase radiation resistance of tumors, as Panc02 tumors in NF-κB/ mice responded better to RT than tumors grown in wild-type mice. In the TRAMP-C1

Figure 2 Radiation therapy increases tumor-infiltrating myeloid cells: subcutaneous tumors of Lewis lung carcinoma were irradiated with 6 Gy on 5 consecutive days and harvested at 3, 7, and 14 days after treatment completion. Tumor-infiltrating CD11bþ/Arg-1þ myeloid cells (A, sham) were increased in irradiated (B) at all time points. (Courtesy of Dr Barcellos-Hoff.)

23 model of prostate cancer, two-thirds of all stromal cells in untreated tumors are TAMs and MDSCs, which increases to 85% after a single dose of 25 Gy. TAMs were the most prominent myeloid cell type, but the recruited cells in response to RT were primarily CD11bþGr1þ MDSCs or PMNs.89 Using a xenograft model with human FaDu nasopharyngeal cancer cells implanted into mice, Ahn et al90 observed a 2-4-fold increase in CD11bþ myeloid cells 1 day and 7 days after treatment with a single fraction of 20 Gy. Administering a monoclonal antibody against CD11b abrogated this myeloid cell recruitment and improved the tumor response to a single fraction of 12 or 20 Gy of RT, an effect also seen in an immunocompetent murine model of head and neck squamous cell carcinoma, SCCVII. A similar improvement in tumor control after RT (15 Gy) was seen using the Lewis lung carcinoma and MC38 colon carcinoma models in a murine host deficient in CD11 and CD18 that has decreased recruitment of myeloid cells to tumors. Lugade et al91 observed an increase in tumor-infiltrating DCs in addition to TAMs after treatment of B16-OVA tumors with either 5 daily fractions of 3 Gy or a single fraction of 15 Gy. Two different mechanisms have been proposed to explain myeloid cell recruitment to tumors in response to RT. Using the RM-1, RM-9, and Myc-CaP murine models of prostate cancer, Xu et al92 observed a 3-fold increase in TAMs and MDSCs one week after treatment with 5 daily fractions of 3 Gy, which correlated with increased serum concentrations of the macrophage chemokine and growth factor colony stimulating factor 1 (CSF-1). Blocking the CSF-1 receptor with a small molecule inhibitor prevented the RTinduced recruitment of myeloid cells, suggesting an essential role for CSF-1 in the myeloid response to RT. Investigating the mechanism further, they found that a single fraction of 3 Gy induced CSF-1 gene transcription in vitro, which was enhanced by nuclear translocation of the ABL1 kinase where it binds to the CSF-1 promoter, an effect that was abolished by depleting the cells of ABL1 using small interfering RNA. These data suggest that RT induces translocation of ABL1 into the nucleus where it binds to the CSF-1 promoter, inducing gene expression and production of CSF-1 by tumor cells, which is responsible for the influx of MDSCs and TAMs in response to RT. A separate mechanism for RT-induced TAM recruitment was elucidated by the work of the Brown group.93 Using the U251 xenograft model in athymic mice, they observed that a single 15-Gy fraction of RT disrupts the tumor vasculature inducing tumor hypoxia, which results in expression of the hypoxia-inducible factor-1 (HIF-1) as determined by an in vivo luciferase reporter. HIF-1 is known to stimulate secretion of stromal-derived factor-1, which recruits myeloid-derived cells by binding CXCR4.94 Blocking the activity of HIF-1 with a small molecule inhibitor or by genetic knockdown, or inhibiting the stromal-derived factor-1-CXCR4 interaction on myelomonocytic cells abrogates the recruitment of macrophages to the tumor microenvironment. Consistent with the findings described earlier, preventing recruitment of TAMs improves the tumor response to RT. These findings were supported by a separate group studying the 54A human lung cancer xenograft

R.E. Vatner and S.C. Formenti

24 and MCa8 mouse mammary tumors treated with 20-21 Gy in 1-2 fractions.95

Removal Although the preponderance of data supports a recruitment effect of RT on tumor myeloid cells, there is a report of TAM and MDSC depletion in response to RT. Deng et al96 recently demonstrated a decrease in number of MDSCs in the TUBO mammary carcinoma and MC38 colon carcinoma models 3 days after treatment with a single fraction of 12 Gy when combined with anti-PD-L1 immunotherapy, an effect that was not observed when tumors were treated with RT alone. Depletion of MDSCs after combined treatment was dependent on CD8þ T lymphocytes, and the number of MDSCs increased when CD8þ T-cells were depleted. Although Crittenden et al88,97 observed an increase in TAMs and MDSCs in 4T1 and Panc02 tumors after treatment with 3 fractions of 20 Gy, they observed a profound decrease in peripheral MDSCs after treatment. This finding seemingly conflicts with the results of Xu et al92 albeit in a different tumor model (prostate) and with a substantially lower dose of RT (3 Gy  5 fractions) and may simply reflect the systemic response to improved tumor control after treatment with such a large dose of RT.

Reorganization In addition to affecting the numbers of myeloid cells in tumors, RT causes a reorganization of macrophages in tumors. This work has mostly been performed by the Hong group, who demonstrated that a single fraction of 25 Gy induces regions of hypoxia and necrosis in multiple tumor models, including 2 glioma models and a prostate cancer model.89,98 They found that CD11blow/CD68þ TAMs clustered in hypoxic regions, CD11blow/F4/80þ TAMs clustered in between hypoxic and necrotic areas, and CD11bþGr1þ PMNs/MDSCs centered in necrotic regions of the tumor after RT. This migration and segregation of TAMs was model dependent. Myeloid cells in TRAMP-C1 prostate tumors and GL261 gliomas assumed this clustering even in the absence of RT, whereas this reorganization only occurred after RT in the ALTS1C1 glioma model. Furthermore, RT-induced reorganization of TAMs was dependent on tissue location in the ALTS1C1 glioma model, with clustering of TAMs in intracranial tumors dependent on RT, but independent of RT in tumors grown intramuscularly. An explanation for these findings is that reorganization of tumor myeloid cells is dependent on necrosis and hypoxia. Reorganization may occur independently of RT in tumor models that have more spontaneous necrosis and hypoxia, whereas tumors with improved oxygenation may only develop hypoxia and subsequent myeloid cell reorganization after RT.

Repolarization Macrophages and other myeloid cells have a plastic phenotype that is exquisitely sensitive to environmental stimuli, and there are many reports that radiation can repolarize TAMs, usually

toward an M2 phenotype.28 TAMs and other myeloid cells in tumors have a phenotype that promotes tumor growth and suppresses antitumor immunity, partially owing to factors secreted by the cancer cells themselves.99 For example, in the TRAMP-C1 prostate cancer model, 15 fractions of 4 Gy or a single fraction of 25 Gy induced the M2 genes ARG-1 and COX2 within days of RT, and the effect lasted for many weeks.100 This effect was also reported in the Panc02 model after 3 fractions of 20 Gy, which induced M2 genes and proteins in an NF-κB dependent manner.88 Interestingly, in the RT5 insulinoma pancreatic cancer model, and in MeWo human melanoma xenografts, Klug et al101 recently reported that lower doses of RT, from 0.5-2 Gy, polarized TAMs toward an M1 phenotype with increased production of iNOS. This repolarization by lower doses of RT facilitated both endogenous and adoptively transferred lymphocyte recruitment to tumors and resulted in an improved tumor control when a single dose of RT was combined with adoptive transfer of tumor-specific T-cells. Although this is in contrast to findings using higher doses of RT, it is consistent with data from experiments using these lower doses of RT on peritoneal macrophages in vitro.102

Re-presentation As professional APCs, macrophages and DCs can ingest tumor antigens and re-present them on the cell surface in the context of MHC Class I and Class II after proteolytic processing.103 This is essential for priming tumor-specific CD4þ helper and CD8þ cytotoxic T-cells, and the evidence suggests that RT induces re-presentation of tumor antigens by myeloid cells. A number of studies demonstrate increased re-presentation of tumor antigens by DCs in the tumor-draining lymph nodes after RT. In the B16-SIY and B16-OVA melanoma models, RT doses from 15-25 Gy, or 5 fractions of 3 Gy all increase the number of APCs cross-presenting tumor-specific antigens in the tumor-draining lymph nodes, which correlated with increased priming of antitumor T-cell responses.91,104 When exogenous DCs are therapeutically introduced into poorly immunogenic B16-D5 tumors, RT (8.5 Gy  5 fractions) also increases tumor antigen uptake and presentation as well as cross-priming.105 Among myeloid cells in tumors, DCs are generally considered the heroes of the antitumor immune response, essential for T-cell priming, liberated by RT from the suppressive and tolerogenic tumor microenvironment. Macrophages, by contrast, are presented as villains (as the data suggest), but the Schreiber group has reported that TAMs are actually essential for effective tumor rejection by the immune system.106 Using a murine sarcoma model, a single fraction of 10 Gy induces TAMs to cross-present tumor antigens on MHC Class I, a transient effect that peaks 2 days after RT and is undetectable 4 days after treatment. It is not clear whether this crosspresentation by TAMs is stimulating the tumor-specific T-cells, making them more effective at killing tumor cells, or if TAMs must be eliminated and cross-presentation makes them better targets for CTL killing. In either case, this is an interesting finding and may help explain why combinations of

Myeloid-Derived Cells in Tumors immunotherapy and RT are less effective when the 2 treatments are not given concurrently.

Conclusion Myeloid cells in the tumor microenvironment are important modulators of the immune response to tumors. As described elsewhere in this issue, RT can promote both local and systemic antitumor immunity; however, myeloid-derived cells in the tumor often counteract development of an effective immune response. Depending on the dose, fractionation, and tumor model system, RT can have either a protumor or antitumor effect by recruiting, removing, repolarizing, and reorganizing, and inducing antigen re-presentation using myeloid-derived cells in tumors. Most effects of RT reported in the literature involve the recruitment and repolarization of tumor-promoting immunosuppressive cells, making depletion of TAMs, MDSCs, and other myeloid cells in the tumor microenvironment an attractive therapeutic approach to combine with RT. However, caution is warranted as these treatments may remove DCs and M1-polarized TAMs that are essential for effective antitumor immunity. Clearly there is more work to be done, and as we move forward there are some questions that must be answered to improve the clinical and therapeutic relevance of manipulating these cells: What markers can specifically differentiate one subset of TAM or DC or MDSC from another and are these subsets biologically meaningful? What is the effect of radiation dose and fractionation on tumor myeloid cells? Are the experimentally convenient single high-dose treatments relevant and is the radiobiology of low doses (o2 Gy) completely different? If so, what is the dose-dependent trigger that affects this distinction? Many of these questions will be more readily addressed after testing whether the efficacy of small molecule inhibitors of TAM and MDSC recruitment affects the systemic immune response generated by RT. However, the main question is how the field will move forward with an ever-expanding appreciation of the complexity of the tumor microenvironment, or even within a class of cells as reviewed herein. One anticipates the need for an integrated analysis of how radiation affects the tumor as a system. Each cell type of the immune system interacts with all the others and often changes in response to the evolving signals of a dynamic tumor. A better understanding is necessary of which cells are critical and when they are most amenable to therapeutic intervention in conjunction with radiation and thus achieve long-term benefit by reigniting tumor immunity.

Acknowledgment We would like to thank Mary Helen Barcellos-Hoff for her critical reading of the manuscript.

References 1. Balkwill FR, Capasso M, Hagemann T: The tumor microenvironment at a glance. J Cell Sci 125:5591-5596, 2012

25 2. Pietras K, Ostman A: Hallmarks of cancer: Interactions with the tumor stroma. Exp Cell Res 316:1324-1331, 2010 3. Gajewski TF, Schreiber H, Fu Y-X: Innate and adaptive immune cells in the tumor microenvironment. Nat Immunol 14:1014-1022, 2013 4. Hanahan D, Coussens LM: Accessories to the crime: Functions of cells recruited to the tumor microenvironment. Cancer Cell 21:309-322, 2012 5. Gajewski TF, Meng Y, Blank C, et al: Immune resistance orchestrated by the tumor microenvironment. Immunol Rev 213:131-145, 2006 6. Geissmann F, Gordon S, Hume DA, et al: Unravelling mononuclear phagocyte heterogeneity. Nat Rev Immunol 10:453-460, 2010 7. Solito S, Pinton L, Damuzzo V, et al: Highlights on molecular mechanisms of MDSC-mediated immune suppression: Paving the way for new working hypotheses. Immunol Invest 41:722-737, 2014 8. Sica A, Porta C, Morlacchi S, et al: Origin and functions of tumorassociated myeloid cells (TAMCs). Cancer Microenviron 5:133-149, 2012 9. Becker JC: Tumor-educated myeloid cells: Impact the micro- and macroenvironment. Exp Dermatol 23:157-158, 2014 10. Ostrand-Rosenberg S, Sinha P, Beury DW, et al: Cross-talk between myeloid-derived suppressor cells (MDSC), macrophages, and dendritic cells enhances tumor-induced immune suppression. Semin Cancer Biol 22:275-281, 2012 11. Schouppe E, Mommer C, Movahedi K, et al: Tumor-induced myeloidderived suppressor cell subsets exert either inhibitory or stimulatory effects on distinct CD8þ T-cell activation events. Eur J Immunol 43:2930-2942, 2013 12. McDonnell AM, Robinson BWS, Currie AJ: Tumor antigen crosspresentation and the dendritic cell: Where it all begins? Clin Dev Immunol 2010:539519, 2010 13. Huang AY, Golumbek P, Ahmadzadeh M, et al: Role of bone marrowderived cells in presenting MHC class I-restricted tumor antigens. Science 264:961-965, 1994 14. Jung S, Unutmaz D, Wong P, et al: In vivo depletion of CD11cþ dendritic cells abrogates priming of CD8þ T cells by exogenous cellassociated antigens. Immunity 17:211-220, 2002 15. Gabrilovich D: Mechanisms and functional significance of tumourinduced dendritic-cell defects. Nat Rev Immunol 4:941-952, 2004 16. Cuenca A, Cheng F, Wang H, et al: Extra-lymphatic solid tumor growth is not immunologically ignored and results in early induction of antigenspecific T-cell anergy: Dominant role of cross-tolerance to tumor antigens. Cancer Res 63:9007-9015, 2003 17. Staveley-O’Carroll K, Sotomayor E, Montgomery J, et al: Induction of antigen-specific T cell anergy: An early event in the course of tumor progression. Proc Natl Acad Sci U S A 95:1178-1183, 1998 18. Chaput N, Conforti R, Viaud S, et al: The Janus face of dendritic cells in cancer. Oncogene 27:5920-5931, 2008 19. Qian B-Z, Pollard JW: Macrophage diversity enhances tumor progression and metastasis. Cell 141:39-51, 2010 20. Condeelis J, Pollard JW: Macrophages: Obligate partners for tumor cell migration, invasion, and metastasis. Cell 124:263-266, 2006 21. Pollard JW: Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer 4:71-78, 2004 22. Mantovani A, Sica A: Macrophages, innate immunity and cancer: Balance, tolerance, and diversity. Curr Opin Immunol 22:231-237, 2010 23. Smyth MJ, Godfrey DI, Trapani JA: A fresh look at tumor immunosurveillance and immunotherapy. Nat Immunol 2:293-299, 2001 24. Dunn GP, Koebel CM, Schreiber RD: Interferons, immunity and cancer immunoediting. Nat Rev Immunol 6:836-848, 2006 25. Prestwich RJ, Errington F, Hatfield P, et al: The immune system—Is it relevant to cancer development, progression and treatment? Clin Oncol (R Coll Radiol) 20:101-112, 2008 26. Murray PJ, Wynn TA: Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 11:723-737, 2011 27. Allavena P, Mantovani A: Immunology in the clinic review series; focus on cancer: Tumour-associated macrophages: Undisputed stars of the inflammatory tumour microenvironment. Clin Exp Immunol 167:195-205, 2012

26 28. Sica A, Mantovani A: Macrophage plasticity and polarization: In vivo veritas. J Clin Invest 122:787-795, 2012 29. Van Overmeire E, Laoui D, Keirsse J, et al: Mechanisms driving macrophage diversity and specialization in distinct tumor microenvironments and parallelisms with other tissues. Front Immunol 5:127, 2014 30. Gabrilovich DI, Ostrand-Rosenberg S, Bronte V: Coordinated regulation of myeloid cells by tumours. Nat Rev Immunol 12:253-268, 2012 31. Rodríguez PC, Quiceno DG, Zabaleta J , et al: Arginase I production in the tumor microenvironment by mature myeloid cells inhibits T-cell receptor expression and antigen-specific T-cell responses. Cancer Res 64:5839-5849, 2004 32. Rodríguez PC, Quiceno DG, Ochoa AC: L-Arginine availability regulates T-lymphocyte cell-cycle progression. Blood 109:1568-1573, 2007 33. Doedens AL, Stockmann C, Rubinstein MP, et al: Macrophage expression of hypoxia-inducible factor-1 alpha suppresses T-cell function and promotes tumor progression. Cancer Res 70:7465-7475, 2010 34. Movahedi K, Laoui D, Gysemans C, et al: Different tumor microenvironments contain functionally distinct subsets of macrophages derived from Ly6C(high) monocytes. Cancer Re 70:5728-5739, 2010 35. Aoe T, Okamoto Y, Saito T: Activated macrophages induce structural abnormalities of the T cell receptor-CD3 complex. J Exp Med 181:1881-1886, 1995 36. Fridlender ZG, Albelda SM: Tumor-associated neutrophils: Friend or foe? Carcinogenesis 33:949-955, 2012 37. Otsuji M, Kimura Y, Aoe T, et al: Oxidative stress by tumor-derived macrophages suppresses the expression of CD3 zeta chain of T-cell receptor complex and antigen-specific T-cell responses. Proc Natl Acad Sci U S A 93:13119-13124, 1996 38. Mishalian I, Bayuh R, Levy L, et al: Tumor-associated neutrophils (TAN) develop pro-tumorigenic properties during tumor progression. Cancer Immunol Immunother 62:1745-1756, 2013 39. Fridlender ZG, Sun J, Kim S, et al: Polarization of tumor-associated neutrophil phenotype by TGF-beta: “N1” versus “N2” TAN. Cancer Cell 16:183-194, 2009 40. Lu T, Ramakrishnan R, Altiok S, et al: Tumor-infiltrating myeloid cells induce tumor cell resistance to cytotoxic T cells in mice. J Clin Invest 121:4015-4029, 2011 41. Molon B, Ugel S, Del Pozzo F, et al: Chemokine nitration prevents intratumoral infiltration of antigen-specific T cells. J Exp Med 208:1949-1962, 2011 42. Bingisser RM, Tilbrook PA, Holt PG, et al: Macrophage-derived nitric oxide regulates T cell activation via reversible disruption of the Jak3/ STAT5 signaling pathway. J Immunol 160:5729-5734, 1998 43. Curiel TJ, Coukos G, Zou L, et al: Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 10:942-949, 2004 44. Fidler IJ, Schroit AJ: Recognition and destruction of neoplastic cells by activated macrophages: Discrimination of altered self. Biochim Biophys Acta 948:151-173, 1988 45. Mantovani A, Caprioli V, Gritti P, et al: Human mature macrophages mediate antibody-dependent cellular cytotoxicity on tumour cells. Transplantation 24:291-293, 1977 46. Mills CD, Shearer J, Evans R, et al: Macrophage arginine metabolism and the inhibition or stimulation of cancer. J Immunol 149:2709-2714, 1992 47. Mills CD, Kincaid K, Alt JM, et al: M-1/M-2 macrophages and the Th1/ Th2 paradigm. J Immunol 164:6166-6173, 2000 48. Edin S, Wikberg ML, Dahlin AM, et al: The distribution of macrophages with a M1 or M2 phenotype in relation to prognosis and the molecular characteristics of colorectal cancer. PLoS One 7:e47045, 2012 49. Zhang M, He Y, Sun X, et al: A high M1/M2 ratio of tumor-associated macrophages is associated with extended survival in ovarian cancer patients. J Ovarian Res 7:19, 2014 50. Ma J, Liu L, Che G, et al: The M1 form of tumor-associated macrophages in non-small cell lung cancer is positively associated with survival time. BMC Cancer 10:112, 2010 51. Thomsen LL, Miles DW: Role of nitric oxide in tumour progression: Lessons from human tumours. Cancer Metastasis Rev 17:107-118, 1998 52. Lala PK, Orucevic A: Role of nitric oxide in tumor progression: Lessons from experimental tumors. Cancer Metastasis Rev 17:91-106, 1998

R.E. Vatner and S.C. Formenti 53. Gabrilovich DI, Nagaraj S: Myeloid-derived suppressor cells as regulators of the immune system (Available at:). Nat Rev Immunol 9:162-174, 2009http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi? dbfrom=pubmed&id=19197294&retmode=ref&cmd=prlinks 54. Dudziak D, Kamphorst AO, Heidkamp GF, et al: Differential antigen processing by dendritic cell subsets in vivo. Science 315:107-111, 2007 55. Villadangos JA, Schnorrer P: Intrinsic and cooperative antigenpresenting functions of dendritic-cell subsets in vivo. Nat Rev Immunol 7:543-555, 2007 56. Shortman K, Liu Y-J: Mouse and human dendritic cell subtypes. Nat Rev Immunol 2:151-161, 2002 57. Gresser I, Belardelli F, Maury C, et al: Injection of mice with antibody to interferon enhances the growth of transplantable murine tumors. J Exp Med 158:2095-2107, 1983 58. Picaud S, Bardot B, De Maeyer E, et al: Enhanced tumor development in mice lacking a functional type I interferon receptor. J Interferon Cytokine Res 22:457-462, 2002 59. Dunn GP, Bruce AT, Sheehan KCF, et al: A critical function for type I interferons in cancer immunoediting. Nat Immunol 6:722-729, 2005 60. Diamond MS, Kinder M, Matsushita H, et al: Type I interferon is selectively required by dendritic cells for immune rejection of tumors. J Exp Med 208:1989-2003, 2011 61. Fuertes MB, Kacha AK, Kline J, et al: Host type I IFN signals are required for antitumor CD8þ T cell responses through CD8{alpha}þ dendritic cells. J Exp Med 208:2005-2016, 2011 62. Honda K, Yanai H, Negishi H, et al: IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature 434:772-777, 2005 63. Takagi H, Fukaya T, Eizumi K, et al: Plasmacytoid dendritic cells are crucial for the initiation of inflammation and T cell immunity in vivo. Immunity 35:958-971, 2011 64. Seliger B, Massa C: The dark side of dendritic cells: Development and exploitation of tolerogenic activity that favor tumor outgrowth and immune escape. Front Immunol 4:419, 2013 65. Zhong H, Gutkin DW, Han B, et al: Origin and pharmacological modulation of tumor-associated regulatory dendritic cells. Int J Cancer 134:2633-2645, 2014 66. Brandau S, Moses K, Lang S : The kinship of neutrophils and granulocytic myeloid-derived suppressor cells in cancer: Cousins, siblings or twins? Semin Cancer Biol 23:171-182, 2013 67. Fridlender ZG, Sun J, Mishalian I, et al: Transcriptomic analysis comparing tumor-associated neutrophils with granulocytic myeloid-derived suppressor cells and normal neutrophils. PLoS One 7:e31524, 2012 68. Ohno Y, Nakashima J, Ohori M, et al: Pretreatment neutrophil-tolymphocyte ratio as an independent predictor of recurrence in patients with nonmetastatic renal cell carcinoma. J Urol 184:873-878, 2010 69. Sharaiha RZ, Halazun KJ, Mirza F, et al: Elevated preoperative neutrophil:lymphocyte ratio as a predictor of postoperative disease recurrence in esophageal cancer. Ann Surg Oncol 18:3362-3369, 2011 70. Perisanidis C, Kornek G, Pöschl PW, et al: High neutrophil-tolymphocyte ratio is an independent marker of poor disease-specific survival in patients with oral cancer. Med Oncol 30:334, 2013 71. Jiang N, Deng J-Y, Liu Y, et al: The role of preoperative neutrophillymphocyte and platelet-lymphocyte ratio in patients after radical resection for gastric cancer. Biomarkers 19(6):444-451, 2014 72. Schmielau J, Finn OJ: Activated granulocytes and granulocyte-derived hydrogen peroxide are the underlying mechanism of suppression of T-cell function in advanced cancer patients. Cancer Res 61:4756-4760, 2001 73. Peranzoni E, Zilio S, Marigo I, et al: Myeloid-derived suppressor cell heterogeneity and subset definition. Curr Opin Immunol 22:238-244, 2010 74. Montero AJ, Diaz-Montero CM, Kyriakopoulos CE, et al: Myeloidderived suppressor cells in cancer patients: A clinical perspective. J Immunother 35:107-115, 2012 75. Nagaraj S, Gabrilovich DI: Myeloid-derived suppressor cells in human cancer. Cancer J 16:348-353, 2010 76. Dolcetti L, Peranzoni E, Ugel S, et al: Hierarchy of immunosuppressive strength among myeloid-derived suppressor cell subsets is determined by GM-CSF. Eur J Immunol 40:22-35, 2010

Myeloid-Derived Cells in Tumors 77. Youn J-I, Nagaraj S, Collazo M, et al: Subsets of myeloid-derived suppressor cells in tumor-bearing mice. J Immunol 181:5791-5802, 2008 78. Ochoa AC, Zea AH, Hernandez C, et al: Arginase, prostaglandins, and myeloid-derived suppressor cells in renal cell carcinoma. Clin Cancer Res 13:721s-726s, 2007 79. Meyer C, Cagnon L, Costa-Nunes CM, et al: Frequencies of circulating MDSC correlate with clinical outcome of melanoma patients treated with ipilimumab. Cancer Immunol Immunother 63:247-257, 2014 80. Bronte V, Serafini P, De Santo C, et al: IL-4-induced arginase 1 suppresses alloreactive T cells in tumor-bearing mice. J Immunol 170:270-278, 2003 81. Nagaraj S, Gupta K, Pisarev V, et al: Altered recognition of antigen is a mechanism of CD8þ T cell tolerance in cancer. Nat Med 13:828-835, 2007 82. Yang R, Cai Z, Zhang Y, et al: CD80 in immune suppression by mouse ovarian carcinoma-associated Gr-1þCD11bþ myeloid cells. Cancer Res 66:6807-6815, 2006 83. Serafini P, Mgebroff S, Noonan K, et al: Myeloid-derived suppressor cells promote cross-tolerance in B-cell lymphoma by expanding regulatory T cells. Cancer Res 68:5439-5449, 2008 84. Movahedi K, Guilliams M, Van den Bossche J, et al: Identification of discrete tumor-induced myeloid-derived suppressor cell subpopulations with distinct T cell-suppressive activity. Blood 111:4233-4244, 2008 85. Gough MJ, Young K, Crittenden M: The impact of the myeloid response to radiation therapy. Clin Dev Immunol 2013:281958, 2013 86. Toivonen P, Kivelä T: Infiltrating macrophages in extratumoural tissues after brachytherapy of uveal melanoma. Acta Ophthalmol 90:341-349, 2012 87. Julow J, Szeifert GT, Bálint K, et al: The role of microglia/macrophage system in the tissue response to I-125 interstitial brachytherapy of cerebral gliomas. Neurol Res 29:233-238, 2007 88. Crittenden MR, Cottam B, Savage T, et al: Expression of NF-κB p50 in tumor stroma limits the control of tumors by radiation therapy. PLoS One 7:e39295, 2012 89. Chiang C-S, Fu SY, Wang S-C, et al: Irradiation promotes an m2 macrophage phenotype in tumor hypoxia. Front Oncol 2:89, 2012 90. Ahn G-O, Tseng D, Liao C-H, et al: Inhibition of Mac-1 (CD11b/CD18) enhances tumor response to radiation by reducing myeloid cell recruitment. Proc Natl Acad Sci U S A 107:8363-8368, 2010 91. Lugade AA, Moran JP, Gerber SA, et al: Local radiation therapy of B16 melanoma tumors increases the generation of tumor antigen-specific effector cells that traffic to the tumor. J Immunol 174:7516-7523, 2005 92. Xu J, Escamilla J, Mok S, et al: CSF1R signaling blockade stanches tumor-infiltrating myeloid cells and improves the efficacy of radiotherapy in prostate cancer. Cancer Res 73:2782-2794, 2013 93. Kioi M, Vogel H, Schultz G, et al: Inhibition of vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after irradiation in mice. J Clin Invest 120:694-705, 2010

27 94. Ceradini DJ, Kulkarni AR, Callaghan MJ, et al: Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nat Med 10:858-864, 2004 95. Kozin SV, Kamoun WS, Huang Y, et al: Recruitment of myeloid but not endothelial precursor cells facilitates tumor regrowth after local irradiation. Cancer Res 70:5679-5685, 2010 96. Deng L, Liang H, Burnette B, et al: Irradiation and anti-PD-L1 treatment synergistically promote antitumor immunity in mice. J Clin Invest 124:687-695, 2014 97. Crittenden MR, Savage T, Cottam B, et al: The peripheral myeloid expansion driven by murine cancer progression is reversed by radiation therapy of the tumor. PLoS One 8:e69527, 2013 98. Chen F-H, Chiang C-S, Wang C-C, et al: Radiotherapy decreases vascular density and causes hypoxia with macrophage aggregation in TRAMP-C1 prostate tumors. Clin Cancer Res 15:1721-1729, 2009 99. Cavnar MJ, Zeng S, Kim TS, et al: KIT oncogene inhibition drives intratumoral macrophage M2 polarization. J Exp Med 210:2873-2886, 2013 100. Tsai C-S, Tsai C-S, Chen F-H, et al: Macrophages from irradiated tumors express higher levels of iNOS, arginase-I and COX-2, and promote tumor growth. Int J Radiat Oncol Biol Phys 68:499-507, 2007 101. Klug F, Prakash H, Huber PE, et al: Low-dose irradiation programs macrophage differentiation to an iNOSþ/M1 phenotype that orchestrates effective T cell immunotherapy. Cancer Cell 24:589-602, 2013 102. Wunderlich R, Ernst A, Rödel F, et al: Low and moderate dose of ionising radiation up to 2 Gy modulates transmigration and chemotaxis of activated macrophages, provokes an anti-inflammatory cytokine milieu, but does not impact on viability and phagocytic function. Clin Exp Immunol 2014. Epub ahead of print 103. Heath WR, Belz GT, Behrens GMN, et al: Cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens. Immunol Rev 199:9-26, 2004 104. Lee Y, Auh SL, Wang Y, et al: Therapeutic effects of ablative radiation on local tumor require CD8þ T cells: Changing strategies for cancer treatment. Blood 114:589-595, 2009 105. Teitz-Tennenbaum S, Li Q, Okuyama R, et al: Mechanisms involved in radiation enhancement of intratumoral dendritic cell therapy. J Immunother 31:345-358, 2008 106. Zhang B, Bowerman NA, Salama JK, et al: Induced sensitization of tumor stroma leads to eradication of established cancer by T cells. J Exp Med 204:49-55, 2007 107. Ahn G-O, Brown JM: Matrix metalloproteinase-9 is required for tumor vasculogenesis but not for angiogenesis: Role of bone marrow-derived myelomonocytic cells. Cancer Cell 13:193-205, 2008 108. Burnette BC, Liang H, LEE Y, et al: The efficacy of radiotherapy relies upon induction of type I interferon-dependent innate and adaptive immunity. Cancer Res 71:2488-2496, 2011