Advertisement

Analysis of Immune Cell Infiltrates during Squamous Carcinoma Development

  • Simon R. Junankar
    Affiliations
    Cancer Research Institute, University of California, San Francisco, San Francisco, California, USA
    Search for articles by this author
  • Alexandra Eichten
    Affiliations
    Cancer Research Institute, University of California, San Francisco, San Francisco, California, USA
    Search for articles by this author
  • Annegret Kramer
    Affiliations
    Cancer Research Institute, University of California, San Francisco, San Francisco, California, USA
    Search for articles by this author
  • Author Footnotes
    4 Current address: Department of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
    Karin E. de Visser
    Footnotes
    4 Current address: Department of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
    Affiliations
    Cancer Research Institute, University of California, San Francisco, San Francisco, California, USA
    Search for articles by this author
  • Lisa M. Coussens
    Correspondence
    Cancer Research Institute, University of California, San Francisco, 2340 Sutter Street, N-221, San Francisco, California 94143, USA
    Affiliations
    Department of Pathology, University of California, San Francisco, San Francisco, California, USA

    Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California, USA
    Search for articles by this author
  • Author Footnotes
    4 Current address: Department of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
      Infiltration of leukocytes into tissue is a common feature of many physiological and pathological conditions. Histopathologically, the diversity of leukocytes that infiltrate a tissue associated with a pathophysiologic response cannot be appreciated and/or examined unless highly selective immunologic detection methods are utilized. Specific populations of infiltrating leukocytes into squamous tissues harboring pre-malignant and/or malignant keratinocytes have recently been demonstrated to play a functionally significant role in the pathogenesis of squamous carcinomas. To evaluate immune cell types and quantify changes in their relative presence and localization during multi-stage neoplastic progression, we performed flow cytometry and histochemical detection using lineage-selective markers. Herein, we provide detailed methodology facilitating these analyses.
      CSF-1R
      colony-stimulating factor-1 receptor
      HPV16
      human papillomavirus type 16
      IHC
      immunohistochemical
      (-)LM
      negative littermate
      PBS
      phosphate-buffered saline
      SCC
      squamous-cell carcinoma
      wt
      wild type

      Introduction

      The immune system can be divided into two subsets based on antigen specificity and timing of activation, for example, the innate and the adaptive immune systems. In order to provide optimal protection against invading pathogens, both subsets of the immune system are intimately linked. The innate immune system, also referred to as the first line of immune defense against infection, is composed of dendritic cells, macrophages, neutrophils, basophils, eosinophils, mast cells, natural killer cells, and soluble complement components, and is relatively nonspecific and not intrinsically affected by prior contact with infectious agents. Some innate immune cells, for example, dendritic cells, macrophages, and mast cells, serve as sentinel cells, that is, they are pre-stationed in tissues and continuously monitor their microenvironment for signs of distress. When tissue homeostasis is perturbed, sentinel macrophages and mast cells instantly release soluble mediators, for example, cytokines, chemokines, matrix remodeling proteases, reactive oxygen species, and bioactive mediators such as histamine, that induce mobilization and infiltration of additional leukocytes into damaged tissue (inflammation) as well as activate vascular and fibroblast responses in order to orchestrate elimination of invading organisms and initiate local tissue repair. Dendritic cells, on the other hand, take up foreign antigens and migrate to lymphoid organs where they present their antigens to adaptive immune cells, thus representing key players in the interface between innate and adaptive immunity.
      The adaptive immune system (also called the acquired immune system) distinguishes itself from the innate immune system by its antigen specificity and memory formation. Adaptive immune cells, for example, B lymphocytes, CD4+ (helper) T lymphocytes, and CD8+ (cytotoxic) T lymphocytes, express somatically generated, diverse antigen-specific receptors, formed as a consequence of random gene rearrangements. As individual B and T lymphocytes are antigenically committed to a specific unique antigen, clonal expansion upon recognition of foreign antigens is required to obtain sufficient antigen-specific T and/or B lymphocytes to counteract infection. Together, acute activation of these distinct immune response pathways results in removal of invading organisms, “damaged” cells, and extracellular matrix, and enables subsequent normalization of cell proliferation and cell death pathways, allowing re-establishment of tissue integrity and homeostasis. Owing to their enormous plasticity, immune cells exert multiple effector functions that are continuously fine-tuned as tissue microenvironments are altered; thus, the immune system is integrally involved in maintaining tissue homeostasis as well as being implicated in the pathogenesis of many chronic diseases including arthritis, heart disease, Alzheimer's disease, and cancer (
      • Finch C.E.
      • Crimmins E.M.
      Inflammatory exposure and historical changes in human life-spans.
      ;
      • de Visser K.E.
      • Eichten A.
      • Coussens L.M.
      Paradoxical roles of the immune system during cancer development.
      ).
      Because a diverse assortment of immune cell types infiltrate neoplastic tissues, we have investigated their unique profiles during multi-stage neoplastic progression utilizing a transgenic mouse model of epithelial carcinogenesis, for example, K14-HPV16 (human papillomavirus type 16)-transgenic mice (
      • Coussens L.M.
      • Hanahan D.
      • Arbeit J.M.
      Genetic predisposition and parameters of malignant progression in K14-HPV16 transgenic mice.
      ). HPV16 mice express the early region genes of HPV16 as transgenes under control of the human keratin 14 promotor/enhancer (
      • Arbeit J.M.
      • Munger K.
      • Howley P.M.
      • Hanahan D.
      Progressive squamous epithelial neoplasia in K14-human papillomavirus type 16 transgenic mice.
      ). By 1 month of age, HPV16 mice develop epidermal hyperplasias with 100% penetrance characterized by a terminally differentiating hyperproliferative epidermis. Hyperplastic lesions advance focally into angiogenic dysplasias between 3 and 6 months of age and are distinct from hyperplasias based upon the prominent hyperproliferative epidermis that fails to undergo terminal differentiation and a dermis where intense CD45+ leukocyte infiltration occurs proximal to dilated and enlarged angiogenic vasculature (
      • Coussens L.M.
      • Raymond W.W.
      • Bergers G.
      • Laig-Webster M.
      • Behrendtsen O.
      • Werb Z.
      • et al.
      Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis.
      ;
      • van Kempen L.C.L.
      • Rhee J.S.
      • Dehne K.
      • Lee J.
      • Edwards D.R.
      • Coussens L.M.
      Epithelial carcinogenesis: dynamic interplay between neoplastic cells and their microenvironment.
      ). By 1 year of age, 60% of HPV16 mice develop malignant skin carcinomas, 50% of which are squamous-cell carcinomas (SCCs) that metastasize to regional lymph nodes with a ∼30% frequency, and ∼10% of which represent non-metastatic microcystic adnexal carcinomas (
      • van Kempen L.C.L.
      • Rhee J.S.
      • Dehne K.
      • Lee J.
      • Edwards D.R.
      • Coussens L.M.
      Epithelial carcinogenesis: dynamic interplay between neoplastic cells and their microenvironment.
      ;
      • Rhee J.S.
      • Diaz R.
      • Korets L.
      • Hodgson J.G.
      • Coussens L.M.
      TIMP-1 alters susceptibility to carcinogenesis.
      ). This genetic model of squamous carcinogenesis has proven useful as a tool with which to assess functional significance of specific infiltrating immune cell types (
      • Coussens L.M.
      • Raymond W.W.
      • Bergers G.
      • Laig-Webster M.
      • Behrendtsen O.
      • Werb Z.
      • et al.
      Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis.
      ,
      • Coussens L.M.
      • Tinkle C.L.
      • Hanahan D.
      • Werb Z.
      MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis.
      ;
      • Daniel D.
      • Meyer-Morse N.
      • Bergsland E.K.
      • Dehne K.
      • Coussens L.M.
      • Hanahan D.
      Immune enhancement of skin carcinogenesis by CD4+ T cells.
      ,
      • Daniel D.
      • Chiu C.
      • Giraud E.
      • Inoue M.
      • Mizzen L.A.
      • Chu N.R.
      • et al.
      CD4+ T cell-mediated antigen-specific immunotherapy in a mouse model for cervical cancer.
      ;
      • Giraudo E.
      • Inoue M.
      • Hanahan D.
      An amino-bisphosphonate targets MMP-9-expressing macrophages and angiogenesis to impair cervical carcinogenesis.
      ;
      • de Visser K.E.
      • Korets L.V.
      • Coussens L.M.
      De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent.
      ) that regulate de novo epithelial carcinogenesis.

      Results

      Experimental analysis of distinct leukocyte populations in tissues where cellular heterogeneity progressively changes owing to physiologic or pathologic assault/damage is aided by examining immunoreactivity of lineage-selective epitopes in either single-cell suspensions or alternatively in tissue sections. Here, we used a transgenic mouse model of epithelial carcinogenesis and qualitatively and quantitatively examined changes in the relative presence and localization of various innate and adaptive leukocytes, for example, mast cells, granulocytes (neutrophils), monocytes, macrophages, and B and T lymphocytes, at each stage of neoplastic progression. Single-cell suspensions from wild-type (wt) and neoplastic skin were analyzed by flow cytometry using antibodies for lineage-selective epitopes to assess immune cell populations expressing CD45, CD11-b, GR-1, F4/80, CD4, CD8, and CD19/B220. To complement these studies, paraffin-embedded tissue sections were used to discern changes in localization and/or abundance of CD45+ leukocytes, GR-1+ neutrophils, F4/80+ or colony stimulating factor-1 receptor (CSF-1R)+ macrophages, and serine esterase-positive mast cells.
      Figure 1 shows representative histograms representing flow cytometry analyses assessing leukocyte presence in single-cell suspensions of ear skin removed from wt mice versus dysplastic ear skin from HPV16 mice, for example, CD45+, CD11-b+, GR-1+, F4/80+, CD4+, CD8+, and CD19/B220+. CD45 (leukocyte common antigen) is expressed by all hematopoietic lineages (
      • Trowbridge I.S.
      • Thomas M.L.
      CD45: an emerging role as a protein tyrosine phosphatase required for lymphocyte activation and development.
      ), and is also expressed in fibrocytes from peripheral blood (
      • Abe R.
      • Donnelly S.C.
      • Peng T.
      • Bucala R.
      • Metz C.N.
      Peripheral blood fibrocytes: differentiation pathway and migration to wound sites.
      ). In contrast, CD11b/MAC-1 is a cell surface beta(2)-integrin selectively expressed on neutrophils, monocytes, natural killer cells, and some lymphocytes (
      • Myones B.L.
      • Dalzell J.G.
      • Hogg N.
      • Ross G.D.
      Neutrophil and monocyte cell surface p150,95 has iC3b-receptor (CR4) activity resembling CR3.
      ). GR-1 is expressed on myeloid cells but not on lymphoid or erythroid cells and increases with granulocyte maturation (
      • Fleming T.J.
      • Fleming M.L.
      • Malek T.R.
      Selective expression of Ly-6G on myeloid lineage cells in mouse bone marrow. RB6-8C5 mAb to granulocyte-differentiation antigen (Gr-1) detects members of the Ly-6 family.
      ). F4/80 is well known as a highly restricted macrophage molecule in mice (
      • Austyn J.
      • Gordon S.
      F4/80, a monoclonal antibody directed specifically against the mouse macrophage.
      ). T lymphocytes were identified by their expression of the CD4 or CD8 co-receptors (
      • Mathieson B.J.
      • Fowlkes B.J.
      Cell surface antigen expression on thymocytes: development and phenotypic differentiation of intrathymic subsets.
      ;
      • Scollay R.
      • Bartlett P.
      • Shortman K.
      T cell development in the adult murine thymus: changes in the expression of the surface antigens Ly2, L3T4 and B2A2 during development from early precursor cells to emigrants.
      ), whereas B lymphocytes were identified by their dual immunoreactivity for CD19 and B220 (
      • Li Y.S.
      • Wasserman R.
      • Hayakawa K.
      • Hardy R.R.
      Identification of the earliest B lineage stage in mouse bone marrow.
      ). Quantitative comparisons of leukocyte infiltrations in wt skin versus hyperplastic and dysplastic skin and carcinomas in HPV16 mice are shown in Figure 1c.
      Figure thumbnail gr1
      Figure 1Flow cytometric analysis of leukocytes during neoplastic progression in HPV16 mice. (a) Single-cell suspensions derived from ear-tissue from negative littermate (-)LM wt and ear-tissue from HPV16 mice were analyzed by flow cytometry to determine the percentage of positively stained cells as a percentage of total viable cells after gating on forward and side scatter and subsequent gating on live cells by exclusion of 7AAD-positive cells. (b, c) Representative histograms for CD45, CD11b, GR-1, and F4/80, and dot blots for CD4-, CD8-, and CD19/B220-positive cells are shown reflecting (-)LM versus dysplastic HPV16 skin. (d) Quantitative analysis reflecting presence of CD45-, CD11b-, GR-1-, F4/80-, CD4-, CD8-, and CD19/B220-positive cells in single-cell suspensions derived from the skin of (-)LMs and pre-malignant (hyperplastic, dysplastic) skin and carcinomas from HPV16 mice was determined as a percentage of total viable cells. Results shown are mean percentages (n=3–7 mice). Error bars represent SEM.
      Immunohistochemical (IHC) detection of leukocytes was employed to ascertain their spatial–temporal distributions in wt and neoplastic tissue from HPV16 mice (Figures 2, 3 and 4). CD45+ cells were predominately located within dermal stroma below the epithelial basement membrane in wt and pre-malignant tissue, with only occasional CD45+ cells present within the epidermis (Figure 2a–c). In carcinomas, a uniform presence of CD45+ cells was observed throughout tumor stroma (Figure 1).
      Figure thumbnail gr2
      Figure 2CD45 immunostaining during neoplastic progression. (af) Immunodetection of CD45-positive leukocytes (brown staining) in paraffin-embedded sections of staged neoplastic tissue reveals incremental increases in the number of CD45-positive leukocytes in the successive neoplastic stages. e, Epidermis; d, dermis; c, cartilage; f, hair follicle. Bar=50 μm.
      Figure thumbnail gr3
      Figure 3Infiltration of neoplastic skin by mast cells and granulocytes. (a) Mast cells (blue staining) in (-)LM and pre-malignant skin and carcinomas of HPV16-transgenic mice. (b) Immunostaining for GR-1+ granulocytes (neutrophils) in (-)LM and pre-malignant skin and carcinomas of HPV16-transgenic mice. Graphical representation of mast cell and GR-1+ neutrophil staining averaged from five high-power fields per mouse and five mice per category. Error bars represent SEM and asterisk (*) indicates statistically significant differences between HPV16 and wt (P<0.05 Mann–Whitney). e, epidermis; d, dermis; c, cartilage; f, hair follicle; SCC (C), central region of carcinoma; SCC(LE), leading edge of carcinoma. Bar=50 μm.
      Figure thumbnail gr4
      Figure 4Distinct characteristics of F4/80+ versus CSF-1R+ macrophages during neoplastic progression. Macrophages were visualized by immunoreactivity to either F4/80 (ac, g) or CSF-1-R (df, h) in (-)LM and pre-malignant skin and carcinomas of HPV16-transgenic mice. Higher magnification images are shown in panels il representing F4/80, CSF-1-R, GR-1, or mast cell staining at the leading edge of the carcinomas shown in g and h. e, epidermis; d, dermis; c, cartilage; f, hair follicle. Bar=50 μm.
      Chloroacetate esterase histochemistry is a useful enzymatic histochemical approach to evaluate presence of serine esterases in mast cells and neutrophils (
      • Leder L.D.
      The chloroacetate esterase reaction. A useful means of histological diagnosis of hematological disorders from paraffin sections of skin.
      ). The difficulty with this approach is that when both mast cells and neutrophils are present within a tissue, traditional chloroacetate esterase (
      • Leder L.D.
      The chloroacetate esterase reaction. A useful means of histological diagnosis of hematological disorders from paraffin sections of skin.
      ) will not accurately delineate between the serine esterase activities in mast cells (chymase) as opposed to that found in neutrophils (neutrophil elastase). To accomplish this and to visualize chymase activity present in mast cells more selectively, we employed a modification of the chloroacetate esterase method on paraffin-embedded tissue sections (blue staining; Figure 3a) as compared to IHC detection of granulocytes with a neutrophil-specific antibody (brown staining; Figure 3b). Individual mast cells were identified in wt dermal stroma, whereas GR-1+ neutrophils were absent. In pre-malignant tissue, similar localization of mast cells and GR-1+ neutrophils was revealed. Whereas infiltrates of Gr-1+ neutrophils were prominent in carcinomas at the leading edges (SCC-LE) as well as tumor centers (SCC-C), mast cells were only modestly associated with invasive fronts and almost completely absent in tumor centers. Whereas flow cytometric analysis of analogous single-cell suspensions generated from wt and/or neoplastic tissue revealed that GR-1+ neutrophils were predominately found in dysplastic and carcinoma tissue (Figure 1d), individual counting of positively stained cells in micrographs by contrast revealed a significant increase in GR-1+ cell as early as the hyperplastic stage (Figure 3b). This disparity reflects one benefit of micrograph quantitation where distinct cell types can be examined independently of other cellular populations as opposed to reflecting their percentage of the total number of cells counted in the suspension.
      Definitive identification of macrophages during neoplastic progression in HPV16 mice has proven difficult. Several well-characterized macrophage-selective markers have been identified and used for flow cytometry and/or IHC detection of macrophages in other organ contexts, for example, Mac-1, Mac-2 (galectin-3), CD68 (microsialin), and F4/80 (
      • Lin E.Y.
      • Nguyen A.V.
      • Russell R.G.
      • Pollard J.W.
      Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy.
      ;
      • Giraudo E.
      • Inoue M.
      • Hanahan D.
      An amino-bisphosphonate targets MMP-9-expressing macrophages and angiogenesis to impair cervical carcinogenesis.
      ;
      • Inoue T.
      • Plieth D.
      • Venkov C.D.
      • Xu C.
      • Neilson E.G.
      Antibodies against macrophages that overlap in specificity with fibroblasts.
      ). F4/80 is well known as a highly restricted macrophage molecule in mice (
      • Austyn J.
      • Gordon S.
      F4/80, a monoclonal antibody directed specifically against the mouse macrophage.
      ). Moreover, several lines of evidence indicate that the macrophage growth factor CSF-1 is required for macrophage differentiation acting through the CSF1-R expressed on macrophages (
      • Lin E.Y.
      • Gouon-Evans V.
      • Nguyen A.V.
      • Pollard J.W.
      The macrophage growth factor CSF-1 in mammary gland development and tumor progression.
      ). Using F4/80 and CSF1-R antibodies to visualize macrophage presence in wt and neoplastic tissue sections, we found that both F4/80+ and CSF1-R+ cells increased in presence during neoplastic progression as evidenced by IHC detection; however, their respective staining patterns were distinct. Moreover, histochemical analysis indicates a prominent F4/80+ cellular population that increases in presence during neoplastic progression, a population that is not identified by flow cytometry.
      • Sasmono R.T.
      • Oceandy D.
      • Pollard J.W.
      • Tong W.
      • Pavli P.
      • Wainwright B.J.
      • et al.
      A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse.
      have reported that all CSF-1R+ cells are also F4/80+ in unchallenged mice. If this is correct, this would indicate that during skin carcinogenesis in HV16 mice, disregulation of these markers occurs. It is interesting to note that F4/80+ and neutrophil markers in the skin demonstrate similar staining patterns, as assessed by IHC.
      • Huang B.
      • Pan P.Y.
      • Li Q.
      • Sato A.I.
      • Levy D.E.
      • Bromberg J.
      • et al.
      Gr-1+CD115+ immature myeloid suppressor cells mediate the development of tumor-induced T regulatory cells and T-cell anergy in tumor-bearing host.
      recently reported that a subset of Gr-1+ cells located in the spleen of tumor-bearing mice also expressed the macrophage markers F4/80 and CSF-1R, and also defined these cells as a more potent subset of myeloid suppressor cells. The biological implications of this cell type and more definitive analysis of their presence in HPV16 skin require further investigation.

      Discussion

      A functional link between inflammation and cancer has been suspected for many years (
      • Balkwill F.
      • Mantovani A.
      Inflammation and cancer: back to Virchow?.
      ;
      • Coussens L.M.
      • Werb Z.
      Inflammation and cancer.
      ). Initially, it was believed that leukocytic infiltrates in and around developing neoplasms represented an attempt of the host to eradicate neoplastic cells. Indeed, extensive infiltration of natural killer cells in human gastric or colorectal carcionoma is associated with a favorable prognosis (
      • Coca S.
      • Perez-Piqueras J.
      • Martinez D.
      • Colmenarejo A.
      • Saez M.A.
      • Vallejo C.
      • et al.
      The prognostic significance of intratumoral natural killer cells in patients with colorectal carcinoma.
      ;
      • Ishigami S.
      • Natsugoe S.
      • Tokuda K.
      • Nakajo A.
      • Che X.
      • Iwashige H.
      • et al.
      Prognostic value of intratumoral natural killer cells in gastric carcinoma.
      ). On the other hand, malignant tissues containing infiltrates of other innate immune cell types such as macrophages in human breast carcinoma and mast cells in human lung adenocarcionoma and melanoma tend to correlate with an unfavorable clinical prognosis (
      • Leek R.D.
      • Lewis C.E.
      • Whitehouse R.
      • Greenall M.
      • Clarke J.
      • Harris A.L.
      Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma.
      ,
      • Leek R.D.
      • Landers R.J.
      • Harris A.L.
      • Lewis C.E.
      Necrosis correlates with high vascular density and focal macrophage infiltration in invasive carcinoma of the breast.
      ;
      • Imada A.
      • Shijubo N.
      • Kojima H.
      • Abe S.
      Mast cells correlate with angiogenesis and poor outcome in stage I lung adenocarcinoma.
      ;
      • Ribatti D.
      • Ennas M.G.
      • Vacca A.
      • Ferreli F.
      • Nico B.
      • Orru S.
      • et al.
      Tumor vascularity and tryptase-positive mast cells correlate with a poor prognosis in melanoma.
      ). Moreover, population-based studies reveal that individuals prone to chronic inflammatory diseases exhibit enhanced risk for cancer development (
      • Balkwill F.
      • Charles K.A.
      • Mantovani A.
      Smoldering and polarized inflammation in the initiation and promotion of malignant disease.
      ), whereas long-term use of non-steroidal anti-inflammatory drugs reduces the risk of several cancer types (
      • Balkwill F.
      • Mantovani A.
      Inflammation and cancer: back to Virchow?.
      ;
      • Coussens L.M.
      • Werb Z.
      Inflammation and cancer.
      ;
      • Balkwill F.
      • Charles K.A.
      • Mantovani A.
      Smoldering and polarized inflammation in the initiation and promotion of malignant disease.
      ).
      How then do chronically activated innate immune cells participate in cancer development? Which mechanisms and which inflammatory cell-derived mediators are relevant for specific human malignancies, are these organ-, tumour stage, or etiology-dependent? Many of these questions remain unanswered; however, experimental models are beginning to elucidate molecular mechanisms by which innate immune cells regulate cancer processes (
      • de Visser K.E.
      • Eichten A.
      • Coussens L.M.
      Paradoxical roles of the immune system during cancer development.
      ). Because of their enormous plasticity and capacity to produce a myriad of cytokines, chemokines, metallo-serine and cystein proteases, reactive oxygen species, histamine, and other bioactive mediators, chronically activated innate immune cells are key modulators of cell survival (proliferation and cell death) as well as regulators of extracellular matrix metabolism. Thus, several physiological processes necessary for tumor development, such as enhanced cell survival, tissue remodeling, angiogenesis, and suppression of antitumor adaptive immune responses, are regulated by leukocytic infiltrates in neoplastic environments. This is exemplified by positive correlations between numbers of innate immune cells (macrophages, mast cells, and granulocytes) infiltrating human tumors with the number of blood vessels (
      • Yano H.
      • Kinuta M.
      • Tateishi H.
      • Nakano Y.
      • Matsui S.
      • Monden T.
      • et al.
      Mast cell infiltration around gastric cancer cells correlates with tumor angiogenesis and metastasis.
      ;
      • Esposito I.
      • Menicagli M.
      • Funel N.
      • Bergmann F.
      • Boggi U.
      • Mosca F.
      • et al.
      Inflammatory cells contribute to the generation of an angiogenic phenotype in pancreatic ductal adenocarcinoma.
      ) and experimental findings in mouse models where attenuating innate immune cell infiltration of pre-malignant tissue reduces angiogenesis and limits tumor development (
      • Coussens L.M.
      • Raymond W.W.
      • Bergers G.
      • Laig-Webster M.
      • Behrendtsen O.
      • Werb Z.
      • et al.
      Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis.
      ;
      • Sparmann A.
      • Bar-Sagi D.
      Ras-induced interleukin-8 expression plays a critical role in tumor growth and angiogenesis.
      ;
      • de Visser K.E.
      • Korets L.V.
      • Coussens L.M.
      De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent.
      ).
      We and others have utilized the K14-HPV16 mouse model of epithelial carcinogenesis (
      • Arbeit J.
      • Howley P.
      • Hanahan D.
      Chronic estrogen-induced cervical and vaginal squamous carcinogenesis in HPV16 transgenic mice.
      ;
      • Coussens L.M.
      • Hanahan D.
      • Arbeit J.M.
      Genetic predisposition and parameters of malignant progression in K14-HPV16 transgenic mice.
      ) to evaluate profiles of immune cells present during neoplastic progression in skin and cervix via IHC detection methods in tissue sections and/or by flow cytometry (Figures 1, 2, 3 and 4) (
      • Coussens L.M.
      • Raymond W.W.
      • Bergers G.
      • Laig-Webster M.
      • Behrendtsen O.
      • Werb Z.
      • et al.
      Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis.
      ,
      • Coussens L.M.
      • Tinkle C.L.
      • Hanahan D.
      • Werb Z.
      MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis.
      ;
      • van Kempen L.C.L.
      • Rhee J.S.
      • Dehne K.
      • Lee J.
      • Edwards D.R.
      • Coussens L.M.
      Epithelial carcinogenesis: dynamic interplay between neoplastic cells and their microenvironment.
      ;
      • Giraudo E.
      • Inoue M.
      • Hanahan D.
      An amino-bisphosphonate targets MMP-9-expressing macrophages and angiogenesis to impair cervical carcinogenesis.
      ;
      • de Visser K.E.
      • Korets L.V.
      • Coussens L.M.
      De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent.
      ). Interestingly, the profile of infiltrating inflammatory cells in skin is distinct as compared to cervix – pre-malignant skin lesions contain infiltrating mast cells, CD11b+, GR-1+, and F4/80+ cells predominantly (
      • Coussens L.M.
      • Raymond W.W.
      • Bergers G.
      • Laig-Webster M.
      • Behrendtsen O.
      • Werb Z.
      • et al.
      Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis.
      ;
      • de Visser K.E.
      • Korets L.V.
      • Coussens L.M.
      De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent.
      ), whereas pre-malignant cervical lesions are characterized by infiltrating F4/80+ macrophages (
      • Giraudo E.
      • Inoue M.
      • Hanahan D.
      An amino-bisphosphonate targets MMP-9-expressing macrophages and angiogenesis to impair cervical carcinogenesis.
      ), and neither are characterized by significant infiltration of T or B lymphocytes (
      • de Visser K.E.
      • Korets L.V.
      • Coussens L.M.
      De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent.
      ).
      With the aid of the methodologies described in this report allowing for accurate characterization of immune cell presence in neoplastic tissue, we hypothesized that innate immune cells were potentiating cancer development. To address this hypothesis, we generated mast cell-deficient/HPV16 mice and found attenuated neoplastic development largely owing to reduced activation of angiogenic vasculature and a failure of keratinocytes to achieve hyperproliferative growth characteristics (
      • Coussens L.M.
      • Raymond W.W.
      • Bergers G.
      • Laig-Webster M.
      • Behrendtsen O.
      • Werb Z.
      • et al.
      Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis.
      ), indicating that activation and/or degranulation of immune cells in neoplastic tissue shifts a critical balance that promotes cancer development. More significantly, studies such as these indicate that limiting or altering presence of pro-tumor innate immune cells in pre-malignant tissue minimizes oncogene-induced primary cancer development. More recently, we found that genetic deletion of the complete adaptive immune system in HPV16 mice resulted in a failure to initiate chronic inflammation during pre-malignancy, resulting in attenuated pre-malignant progression and reduced carcinoma incidence (
      • de Visser K.E.
      • Korets L.V.
      • Coussens L.M.
      De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent.
      ). Transfer of B lymphocytes or serum isolated from K14-HPV16 mice into adaptive immune-deficient/K14-HPV16 mice restored chronic inflammation in neoplastic skin as well as hallmarks of pre-malignant progression (
      • de Visser K.E.
      • Korets L.V.
      • Coussens L.M.
      De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent.
      ), indicating that B lymphocytes play a crucial role in the onset of chronic inflammation associated with pre-malignant progression, thus potentiating neoplastic cascades downstream of oncogene expression.
      Tumor-promoting roles for innate immune cells downstream of oncogene expression have also been described in other experimental tumor models (
      • Lin E.Y.
      • Nguyen A.V.
      • Russell R.G.
      • Pollard J.W.
      Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy.
      ;
      • Sparmann A.
      • Bar-Sagi D.
      Ras-induced interleukin-8 expression plays a critical role in tumor growth and angiogenesis.
      ).
      • Lin E.Y.
      • Nguyen A.V.
      • Russell R.G.
      • Pollard J.W.
      Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy.
      studied the role of CSF-1 during mammary carcinoma development by comparing transgenic mice susceptible to de novo development of mammary carcinomas (PyMT mice) with CSF-1-deficient PyMT mice (PyMT/Csf1op/op). Whereas absence of CSF-1 during early neoplastic development was without apparent consequence, development of late-stage invasive carcinomas and pulmonary metastases was significantly attenuated in PyMT/Csf1op/op mice, and correlated with a failure to recruit mature macrophages into neoplastic tissue in the absence of CSF-1. Macrophage recruitment was restored by transgenic CSF-1 expression in mammary epithelium in PyMT/Csf1op/op mice, as was characteristic primary and metastatic tumor development (
      • Lin E.Y.
      • Nguyen A.V.
      • Russell R.G.
      • Pollard J.W.
      Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy.
      ). Together, these studies indicate that the biological effect of tumor-infiltrating innate immune cells depends on the local levels of proinflammatory cytokines and numbers of innate immune cells in the neoplastic microenvironment (
      • Nesbit M.
      • Schaider H.
      • Miller T.H.
      • Herlyn M.
      Low-level monocyte chemoattractant protein-1 stimulation of monocytes leads to tumor formation in nontumorigenic melanoma cells.
      ). Immature myeloid cells or myeloid suppressor cells are a heterogeneous cell population characterized as CD11b/Gr-1 double-positive cells that typically differentiate into mature macrophages, neutrophils, or dendritic cells (
      • Kusmartsev S.
      • Gabrilovich D.I.
      Role of immature myeloid cells in mechanisms of immune evasion in cancer.
      ;
      • Serafini P.
      • Borrello I.
      • Bronte V.
      Myeloid suppressor cells in cancer: recruitment, phenotype, properties, and mechanisms of immune suppression.
      ) that can inhibit antitumor T-cell function and T-lymphocyte proliferation and promote development of regulatory T cells (
      • Huang B.
      • Pan P.Y.
      • Li Q.
      • Sato A.I.
      • Levy D.E.
      • Bromberg J.
      • et al.
      Gr-1+CD115+ immature myeloid suppressor cells mediate the development of tumor-induced T regulatory cells and T-cell anergy in tumor-bearing host.
      ). They have also been shown to have a direct pro-tumor role by secreting molecules like matrix metalloproteinase-9, an important regulator of extracellular matrix remodeling and vascular endothelial growth factor bioavailability (
      • Yang L.
      • DeBusk L.M.
      • Fukuda K.
      • Fingleton B.
      • Green-Jarvis B.
      • Shyr Y.
      • et al.
      Expansion of myeloid immune suppressor Gr+CD11b+ cells in tumor-bearing host directly promotes tumor angiogenesis.
      ). The initiation of myeloid suppressor cell differentiation cascades and/or their differential presence within neoplastic tissue could be an important feature of chronic inflammation that potentiates tumor development as opposed to chronic inflammatory scenarios such as exists in psoriatic lesions where cancer development is not a favored outcome.
      Mouse cancer models like K14-HPV16 mice provide invaluable insights into the diverse role immune cells play during cancer development. Utilizing the power of IHC detection of specific immune cell types in tissue sections or as quantified in single-cell suspensions by flow cytometry, routine access to staged pre-malignant and malignant tissues provides a unique opportunity to evaluate variances in immune cell infiltration and to define functionally significant roles for their presence in that tissue. Identifying and characterizing novel and robust markers for distinct immune cell types that infiltrate neoplastic microenvironments will enhance future investigations regarding the exact profile of immune cell types that are present in assessed tissue. Using such markers to visualize and quantify distinct immune cell populations during skin carcinogenesis has provided an insight into the role chronic inflammation plays during cancer development.

      Materials And Methods

      Transgenic mice

      Generation and characterization of HPV16-transgenic mice and neoplastic staging based on keratin intermediate filament expression has been described previously (
      • Arbeit J.M.
      • Munger K.
      • Howley P.M.
      • Hanahan D.
      Progressive squamous epithelial neoplasia in K14-human papillomavirus type 16 transgenic mice.
      ;
      • Coussens L.M.
      • Hanahan D.
      • Arbeit J.M.
      Genetic predisposition and parameters of malignant progression in K14-HPV16 transgenic mice.
      ). All mice were maintained within the UCSF Laboratory for Animal Care facility according to the IACUC procedures.

      Flow cytometry

      Leukocytic infiltrates in neoplastic tissues were analyzed by flow cytometry. Single-cell suspensions were prepared from ears of negative littermate ((-)LM) wt FVB/n mice or ears and/or carcinomas from HPV16 mice at specific ages reflecting distinct stages of neoplastic progression as reported previously (
      • Coussens L.M.
      • Hanahan D.
      • Arbeit J.M.
      Genetic predisposition and parameters of malignant progression in K14-HPV16 transgenic mice.
      ,
      • Coussens L.M.
      • Raymond W.W.
      • Bergers G.
      • Laig-Webster M.
      • Behrendtsen O.
      • Werb Z.
      • et al.
      Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis.
      ,
      • Coussens L.M.
      • Tinkle C.L.
      • Hanahan D.
      • Werb Z.
      MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis.
      ;
      • de Visser K.E.
      • Korets L.V.
      • Coussens L.M.
      Early neoplastic progression is complement independent.
      ,
      • de Visser K.E.
      • Korets L.V.
      • Coussens L.M.
      De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent.
      ). Single-cell suspensions were prepared by mincing tissue with scissors, followed by a 13-minute enzymatic digestion with 2.5 mg/ml collagenase Type II (Worthington, Lakewood, NJ), 2.5 mg/ml collagenase Type IV (Gibco, Carlsbad, CA), and 0.5 mg/ml DNase (Sigma, St Louis, MO) in phosphate-buffered saline (PBS) containing 1% BSA (Sigma) (PBS/BSA) at 37°C under continuous stirring conditions. Digests were quenched by adding DMEM (Gibco) containing 10% fetal bovine serum (Gibco) and subsequently filtered through a 70-μm nylon filter (Falcon, BD Biosciences, San Jose, CA). Single-cell suspensions were treated with PharM Lyse ammonium chloride lysing reagent (BD Biosciences, San Diego, CA) for 5 minutes to remove erythrocytes. Cells were washed with DMEM containing 10% fetal bovine serum, followed by washing with PBS/BSA. Cells were incubated for 10 minutes at 4°C with rat anti-mouse CD16/CD32mAb (clone 2.4G2, BD Biosciences) at a 1:200 dilution in PBS/BSA to prevent nonspecific antibody binding. Subsequently, cells were washed and incubated for 20 minutes with 50 μl of 1:200 dilution of allophycocyanin-conjugated anti-mouse CD45 (clone 30-F11, eBioscience, San Diego, CA), FITC-conjugated anti-mouse GR-1 (Ly-6G, clone RB6-8C5, eBioscience), phycoerythrin-conjugated anti-mouse CD31 (clone MEC13.3, BD Biosciences), FITC-conjugated anti-mouse CD4 (L3T4, clone GK1.5, eBioscience), phycoerythrin-conjugated anti-mouse CD8a (L-2, clone 53-6.7, eBioscience), allophycocyanin-conjugated anti-mouse CD19 (clone MB19.1, eBioscience), FITC-conjugated anti-mouse B220 (clone RA3-6B2, eBioscience), phycoerythrin-conjugated anti-mouse F4/80 (clone CI:A3-1, Caltag Laboratories, Burlingame, CA), and phycoerythrin-conjugated anti-mouse CD11b (clone M1/70, BD Biosciences Pharmingen, San Jose, CA). Cells were washed twice with PBS/BSA and 7-AAD (BD Biosciences) was added at a dilution of 1:10 to discriminate between viable and dead cells. Data acquisition was performed on a FACSCalibur using CellQuestPro software (BD Biosciences) and data analysis was performed using FlowJo software (Tree Star Inc.). Gates were set using negative controls and positive populations were corrected by subtraction of background and nonspecific binding of the antibody. Data shown as bar graphs as the mean±SEM reflecting a minimum of five mice per respective histopathologic stage.

      Tissue immunostaining and enzyme histochemistry

      Age-matched tissue samples from transgenic and control animals were immersion-fixed in 10% neutral-buffered formalin followed by dehydration through graded alcohols and xylene and then embedded in paraffin. Paraffin sections (5-μm-thick) were cut using a Leica 2135 microtome. Sections were deparaffinized and subjected to enzyme and IHC detection.
      Modified chloroacetate esterase histochemistry was performed to visualize chymase-like activity in mast cells. A 1 mg portion of naphthol AS-D chloroacetate (Sigma) was dissolved in 20 μl of N, N-dimethyl formamide (Sigma) and 1 ml buffer (8% N,N-dimethyl formamide, 20% ethylene glycol monoethylether in 80 mM Tris-maleate pH 7.5). Subsequently, 1 mg Fast Blue BB salt (Fluka, Buchs, Switzerland) was added, the solution filtered using a 0.45 μm filter, and then applied to deparaffinized paraffin sections for 5 minutes. Sections were rinsed in PBS, dehydrated through graded alcohols, and mounted in glycerol.
      IHC on paraffin sections was performed to visualize cells of hematopoietic origin using rat anti-mouse CD45 (Ly-5, clone 30-F11, BD Biosciences Pharmingen), macrophages using rat anti-mouse F4/80 (clone CI:A3-1, Serotec, Raleigh, NC) or rabbit anti-mouse CSF-1R (catalog no. 06-174, Upstate, Charlottesville, VA), and granulocytes using a rat anti-mouse neutrophil-specific primary antibody (clone 7/4, Cedarlane Labs, Hornby, Ontario, Canada). Antigens were retrieved by proteinase K (DAKO, Carpinteria, CA) treatment for 3 minutes for the macrophage and neutrophil stainings. Sections were blocked in blocking buffer (5% goat serum/2.5% BSA/PBS) for 30 minutes. Primary antibodies were diluted in 0.5 × blocking buffer at 1:5,000 F4/80, and 1:500 for the CD45-, CSF-1R-, and the neutrophil-specific antibody. Sections were incubated with primary antibody for 1–4 hours at room temperature, followed by PBS washing, brief (5 minutes) incubation in blocking buffer and subsequent incubation with biotinylated secondary antibody (rabbit anti-rat IgG 1:200, Vector, Burlingame, CA; except for CSF-1R, which used goat anti-rabbit IgG 1:200, Pierce, Rockford, IL) for 45 minutes at room temperature. Sections were then washed in PBS and endogenous peroxidase activity blocked by incubation in 0.6% H2O2 in methanol for 20 minutes. For the CSF-1R, CD45, and neutrophil stainings after PBS washing, Vectastain Elite ABC reagent (Vector) was applied for 30 minutes. For the F4/80 stainings after the peroxide treatment and PBS washing, streptavidin-horseradish peroxidase (Perkin Elemer, Wellesley, MA) was applied for 30 minutes, biotinyl tyramide (Perkin Elmer) was then applied for 7 minutes, followed by streptavidin-horseradish peroxidase for a further 30 minutes. Antibodies were visualized by treatment with Fast 3,3′-diaminobenzidine (Sigma), counterstained with methyl green (1% in H2O), dehydrated in isobutanol, cleared in xylene, and mounted in Cytoseal 60 (Richard-Allan Scientific, Kalamazoo, MI).
      All immunolocalization experiments were repeated on multiple tissue sections and included negative controls for determination of background staining, which was negligible. Data shown are representative of results obtained following examination of tissues removed from a minimum of five different mice per distinctive stage (or age) of neoplastic progression. Quantitative analysis of labeled cells was performed by counting cells in five random high-power fields (-40) per age-matched tissue section from five mice per group. Data presented reflect the mean total cell count per field from the ventral ear leaflet for wt and pre-malignant tissues, carcinoma centers, or carcinoma leading edge.

      Conflict of Interest

      The authors state no conflict of interest.

      ACKNOWLEDGMENTS

      We acknowledge Lidiya Korets and Aleksandr Rudik for animal husbandry assistance and the UCSF Laboratory for Cell Analysis for assistance with flow cytometry. This study was supported by NIH Grants CA72006, CA94168, CA098075, Sandler Program in Basic Sciences, National Technology Center for Networks and Pathways (U54 RR020843), a Department of Defense Breast Cancer Center of Excellence Grant (DAMD-17-02-0693), the Serono Foundation for the Advancement of Medical Science (AE), and the Dutch Cancer Society (KEdV).

      REFERENCES

        • Abe R.
        • Donnelly S.C.
        • Peng T.
        • Bucala R.
        • Metz C.N.
        Peripheral blood fibrocytes: differentiation pathway and migration to wound sites.
        J Immunol. 2001; 166: 7556-7562
        • Arbeit J.M.
        • Munger K.
        • Howley P.M.
        • Hanahan D.
        Progressive squamous epithelial neoplasia in K14-human papillomavirus type 16 transgenic mice.
        J Virol. 1994; 68: 4358-4368
        • Arbeit J.
        • Howley P.
        • Hanahan D.
        Chronic estrogen-induced cervical and vaginal squamous carcinogenesis in HPV16 transgenic mice.
        Proc Natl Acad Sci USA. 1996; 93: 2930-2935
        • Austyn J.
        • Gordon S.
        F4/80, a monoclonal antibody directed specifically against the mouse macrophage.
        Eur J Immunol. 1981; 11: 805-815
        • Balkwill F.
        • Mantovani A.
        Inflammation and cancer: back to Virchow?.
        Lancet. 2001; 357: 539-545
        • Balkwill F.
        • Charles K.A.
        • Mantovani A.
        Smoldering and polarized inflammation in the initiation and promotion of malignant disease.
        Cancer Cell. 2005; 7: 211-217
        • Coca S.
        • Perez-Piqueras J.
        • Martinez D.
        • Colmenarejo A.
        • Saez M.A.
        • Vallejo C.
        • et al.
        The prognostic significance of intratumoral natural killer cells in patients with colorectal carcinoma.
        Cancer. 1997; 79: 2320-2328
        • Coussens L.M.
        • Hanahan D.
        • Arbeit J.M.
        Genetic predisposition and parameters of malignant progression in K14-HPV16 transgenic mice.
        Am J Pathol. 1996; 149: 1899-1917
        • Coussens L.M.
        • Werb Z.
        Inflammation and cancer.
        Nature. 2002; 420: 860-867
        • Coussens L.M.
        • Raymond W.W.
        • Bergers G.
        • Laig-Webster M.
        • Behrendtsen O.
        • Werb Z.
        • et al.
        Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis.
        Genes Dev. 1999; 13: 1382-1397
        • Coussens L.M.
        • Tinkle C.L.
        • Hanahan D.
        • Werb Z.
        MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis.
        Cell. 2000; 103: 481-490
        • Daniel D.
        • Chiu C.
        • Giraud E.
        • Inoue M.
        • Mizzen L.A.
        • Chu N.R.
        • et al.
        CD4+ T cell-mediated antigen-specific immunotherapy in a mouse model for cervical cancer.
        Cancer Res. 2005; 65: 2018-2025
        • Daniel D.
        • Meyer-Morse N.
        • Bergsland E.K.
        • Dehne K.
        • Coussens L.M.
        • Hanahan D.
        Immune enhancement of skin carcinogenesis by CD4+ T cells.
        J Exp Med. 2003; 197: 1017-1028
        • de Visser K.E.
        • Eichten A.
        • Coussens L.M.
        Paradoxical roles of the immune system during cancer development.
        Nat Rev Cancer. 2006; 6: 24-37
        • de Visser K.E.
        • Korets L.V.
        • Coussens L.M.
        Early neoplastic progression is complement independent.
        Neoplasia. 2004; 6: 768-776
        • de Visser K.E.
        • Korets L.V.
        • Coussens L.M.
        De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent.
        Cancer Cell. 2005; 7: 411-423
        • Esposito I.
        • Menicagli M.
        • Funel N.
        • Bergmann F.
        • Boggi U.
        • Mosca F.
        • et al.
        Inflammatory cells contribute to the generation of an angiogenic phenotype in pancreatic ductal adenocarcinoma.
        J Clin Pathol. 2004; 57: 630-636
        • Finch C.E.
        • Crimmins E.M.
        Inflammatory exposure and historical changes in human life-spans.
        Science. 2004; 305: 1736-1739
        • Fleming T.J.
        • Fleming M.L.
        • Malek T.R.
        Selective expression of Ly-6G on myeloid lineage cells in mouse bone marrow. RB6-8C5 mAb to granulocyte-differentiation antigen (Gr-1) detects members of the Ly-6 family.
        J Immunol. 1993; 151: 2399-2408
        • Giraudo E.
        • Inoue M.
        • Hanahan D.
        An amino-bisphosphonate targets MMP-9-expressing macrophages and angiogenesis to impair cervical carcinogenesis.
        J Clin Invest. 2004; 114: 623-633
        • Huang B.
        • Pan P.Y.
        • Li Q.
        • Sato A.I.
        • Levy D.E.
        • Bromberg J.
        • et al.
        Gr-1+CD115+ immature myeloid suppressor cells mediate the development of tumor-induced T regulatory cells and T-cell anergy in tumor-bearing host.
        Cancer Res. 2006; 66: 1123-1131
        • Imada A.
        • Shijubo N.
        • Kojima H.
        • Abe S.
        Mast cells correlate with angiogenesis and poor outcome in stage I lung adenocarcinoma.
        Eur Respir J. 2000; 15: 1087-1093
        • Inoue T.
        • Plieth D.
        • Venkov C.D.
        • Xu C.
        • Neilson E.G.
        Antibodies against macrophages that overlap in specificity with fibroblasts.
        Kidney Int. 2005; 67: 2488-2493
        • Ishigami S.
        • Natsugoe S.
        • Tokuda K.
        • Nakajo A.
        • Che X.
        • Iwashige H.
        • et al.
        Prognostic value of intratumoral natural killer cells in gastric carcinoma.
        Cancer. 2000; 88: 577-583
        • Kusmartsev S.
        • Gabrilovich D.I.
        Role of immature myeloid cells in mechanisms of immune evasion in cancer.
        Cancer Immunol Immunother. 2006; 55: 237-245
        • Leder L.D.
        The chloroacetate esterase reaction. A useful means of histological diagnosis of hematological disorders from paraffin sections of skin.
        Am J Dermatopathol. 1979; 1: 39-42
        • Leek R.D.
        • Landers R.J.
        • Harris A.L.
        • Lewis C.E.
        Necrosis correlates with high vascular density and focal macrophage infiltration in invasive carcinoma of the breast.
        Br J Cancer. 1999; 79: 991-995
        • Leek R.D.
        • Lewis C.E.
        • Whitehouse R.
        • Greenall M.
        • Clarke J.
        • Harris A.L.
        Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma.
        Cancer Res. 1996; 56: 4625-4629
        • Li Y.S.
        • Wasserman R.
        • Hayakawa K.
        • Hardy R.R.
        Identification of the earliest B lineage stage in mouse bone marrow.
        Immunity. 1996; 5: 527-535
        • Lin E.Y.
        • Gouon-Evans V.
        • Nguyen A.V.
        • Pollard J.W.
        The macrophage growth factor CSF-1 in mammary gland development and tumor progression.
        J Mammary Gland Biol Neoplasia. 2002; 7: 147-162
        • Lin E.Y.
        • Nguyen A.V.
        • Russell R.G.
        • Pollard J.W.
        Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy.
        J Exp Med. 2001; 193: 727-740
        • Mathieson B.J.
        • Fowlkes B.J.
        Cell surface antigen expression on thymocytes: development and phenotypic differentiation of intrathymic subsets.
        Immunol Rev. 1984; 82: 141-173
        • Myones B.L.
        • Dalzell J.G.
        • Hogg N.
        • Ross G.D.
        Neutrophil and monocyte cell surface p150,95 has iC3b-receptor (CR4) activity resembling CR3.
        J Clin Invest. 1988; 82: 640-651
        • Nesbit M.
        • Schaider H.
        • Miller T.H.
        • Herlyn M.
        Low-level monocyte chemoattractant protein-1 stimulation of monocytes leads to tumor formation in nontumorigenic melanoma cells.
        J Immunol. 2001; 166: 6483-6490
        • Rhee J.S.
        • Diaz R.
        • Korets L.
        • Hodgson J.G.
        • Coussens L.M.
        TIMP-1 alters susceptibility to carcinogenesis.
        Cancer Res. 2004; 64: 952-961
        • Ribatti D.
        • Ennas M.G.
        • Vacca A.
        • Ferreli F.
        • Nico B.
        • Orru S.
        • et al.
        Tumor vascularity and tryptase-positive mast cells correlate with a poor prognosis in melanoma.
        Eur J Clin Invest. 2003; 33: 420-425
        • Sasmono R.T.
        • Oceandy D.
        • Pollard J.W.
        • Tong W.
        • Pavli P.
        • Wainwright B.J.
        • et al.
        A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse.
        Blood. 2003; 101: 1155-1163
        • Scollay R.
        • Bartlett P.
        • Shortman K.
        T cell development in the adult murine thymus: changes in the expression of the surface antigens Ly2, L3T4 and B2A2 during development from early precursor cells to emigrants.
        Immunol Rev. 1984; 82: 79-103
        • Serafini P.
        • Borrello I.
        • Bronte V.
        Myeloid suppressor cells in cancer: recruitment, phenotype, properties, and mechanisms of immune suppression.
        Semin Cancer Biol. 2006; 16: 53-65
        • Sparmann A.
        • Bar-Sagi D.
        Ras-induced interleukin-8 expression plays a critical role in tumor growth and angiogenesis.
        Cancer Cell. 2004; 6: 447-458
        • Trowbridge I.S.
        • Thomas M.L.
        CD45: an emerging role as a protein tyrosine phosphatase required for lymphocyte activation and development.
        Annu Rev Immunol. 1994; 12: 85-116
        • van Kempen L.C.L.
        • Rhee J.S.
        • Dehne K.
        • Lee J.
        • Edwards D.R.
        • Coussens L.M.
        Epithelial carcinogenesis: dynamic interplay between neoplastic cells and their microenvironment.
        Differentiation. 2002; 70: 501-623
        • Yang L.
        • DeBusk L.M.
        • Fukuda K.
        • Fingleton B.
        • Green-Jarvis B.
        • Shyr Y.
        • et al.
        Expansion of myeloid immune suppressor Gr+CD11b+ cells in tumor-bearing host directly promotes tumor angiogenesis.
        Cancer Cell. 2004; 6: 409-421
        • Yano H.
        • Kinuta M.
        • Tateishi H.
        • Nakano Y.
        • Matsui S.
        • Monden T.
        • et al.
        Mast cell infiltration around gastric cancer cells correlates with tumor angiogenesis and metastasis.
        Gastric Cancer. 1999; 2: 26-32