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Department of Pathology, University of California, San Francisco, San Francisco, California, USAComprehensive Cancer Center, University of California, San Francisco, San Francisco, California, USA
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 (
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 (
). 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 (
). 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 (
). This genetic model of squamous carcinogenesis has proven useful as a tool with which to assess functional significance of specific infiltrating immune cell types (
) 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 (
). In contrast, CD11b/MAC-1 is a cell surface beta(2)-integrin selectively expressed on neutrophils, monocytes, natural killer cells, and some lymphocytes (
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.
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.
). Quantitative comparisons of leukocyte infiltrations in wt skin versus hyperplastic and dysplastic skin and carcinomas in HPV16 mice are shown in Figure 1c.
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 2CD45 immunostaining during neoplastic progression. (a–f) 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 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 4Distinct characteristics of F4/80+versus CSF-1R+ macrophages during neoplastic progression. Macrophages were visualized by immunoreactivity to either F4/80 (a–c, g) or CSF-1-R (d–f, h) in (-)LM and pre-malignant skin and carcinomas of HPV16-transgenic mice. Higher magnification images are shown in panels i–l 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 (
) 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 (
). 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 (
). 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.
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.
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 (
). 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 (
). 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 (
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 (
). 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 (
) and experimental findings in mouse models where attenuating innate immune cell infiltration of pre-malignant tissue reduces angiogenesis and limits tumor development (
) 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) (
). 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 (
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 (
), 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 (
). 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 (
), 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 (
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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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.
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.
A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse.
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.
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