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Atlanta University Center DigitalCommons@Robert W. Woodruff Library, Atlanta University Center Electronic Theses & Dissertations Collection for Atlanta University & Clark Atlanta University Clark Atlanta University Spring 5-16-2016 The Contribution of Inflammatory Cells to the Progression of Prostate Cancer Kia J. Jones Clark Atlanta University, [email protected] Follow this and additional works at: http://digitalcommons.auctr.edu/cauetds Part of the Biology Commons, and the Laboratory and Basic Science Research Commons Recommended Citation Jones, Kia J., "The Contribution of Inflammatory Cells to the Progression of Prostate Cancer" (2016). Electronic Theses & Dissertations Collection for Atlanta University & Clark Atlanta University. Paper 26. This Dissertation is brought to you for free and open access by the Clark Atlanta University at DigitalCommons@Robert W. Woodruff Library, Atlanta University Center. It has been accepted for inclusion in Electronic Theses & Dissertations Collection for Atlanta University & Clark Atlanta University by an authorized administrator of DigitalCommons@Robert W. Woodruff Library, Atlanta University Center. For more information, please contact [email protected]. ABSTRACT BIOLOGICAL SCIENCES JONES, KIA JAVONE B.S., ELIZABETH CITY STATE UNIVERSITY, 2007 THE CONTRIBUTION OF INFLAMMATORY CELLS TO THE PROGRESSION OF PROSTATE CANCER Committee Chair: Cimona V. Hinton, Ph.D. Dissertation dated May 2016 In recent years, the causal relationship between inflammation and cancer has gained wider acknowledgement and acceptance. While various types of immune cells are involved in the process of inflammation, macrophages represent the major inflammatory component of many tumors. Derived from circulating monocytes, these cells migrate to tumor sites in response to molecular cues present within the tumor microenvironment. Once there, interactions with neoplastic cells shape the differentiation and functional orientation of macrophages into two phenotypically distinct subsets: the “classically” activated M1 macrophages and the “alternatively” activated M2 macrophages. The preeminent paradigm in macrophage-related cancer research is that within the tumor stoma, macrophages acquire an M2 phenotype characterized by production of proangiogenic factors, ECM degrading enzymes and up-regulation of anti-inflammatory responses, thereby promoting tumor progression. M1 macrophages, on the other hand are i thought to exert anti-tumorigenic effects due to their production of pro-inflammatory cytokines, and reactive oxygen species (ROS). While the generation of ROS during immune responses is an important aspect of immune regulation and host defense, excessive ROS production has been implicated in the pathogenesis of various degenerative diseases, including cancer. Yet, despite the wellestablished role of M1 macrophages in generating high levels of ROS via NADPH oxidase (NOX), M1 macrophages are still largely viewed as anti-tumorigenic. Hence, this study reevaluates the complex interaction between prostate cancer (PCa) cells and tumorassociated macrophages (TAMs), and operates on the premise that PCa cells promote a pro-tumor microenvironment, denoted by increased inflammation and oxidative stress, in part, through M1 macrophage-mediated, NOX-derived ROS production. Accordingly, immunofluorescent analysis of prostate tissue microarrays demonstrated an influx of M1 macrophages in prostate carcinoma. Immature monocytes co-cultured with the poorly tumorigenic prostate cell line, LNCaP, demonstrated changes in morphology and protein expression consistent with M1 macrophage polarization. PCa cells co-cultured with M1 macrophages displayed significantly higher intracellular ROS levels. Furthermore, M1mediated ROS generation through NOXs increased prostate cell invasiveness and anchorage-independent growth. Taken together, results from this study suggest a potentially novel pro-tumorigenic function of M1 macrophages in early PCa progression, and aid in understanding the complex role of inflammation in cancer. ii THE CONTRIBUTION OF INFLAMMATORY CELLS TO THE PROGRESSION OF PROSTATE CANCER A DISSERTATION SUBMITTED TO THE FACULTY OF CLARK ATLANTA UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY KIA JAVONE JONES DEPARTMENT OF BIOLOGICAL SCIENCES ATLANTA, GEORGIA MAY 2016 © 2016 KIA JAVONE JONES All Rights Reserved ACKNOWLEDGEMENTS This dissertation was made possible due to the masterly guidance of my advisor/mentor, Dr. Cimona V. Hinton. Her patience, motivation, and immense knowledge were essential in me completing my dissertation. I could not have imagined having a better advisor for my Ph.D. study. I also owe my deepest gratitude to my dissertation advisory committee members: Drs. Jaideep Chaudhary, Valerie OderoMarah, Shafiq A. Khan and Michelle Dawson for their insightful comments and encouragement, but most importantly for the hard questions they posed which not only challenged me to think critically but also enhanced my research project tremendously. It is also without question that many thanks are due to my colleagues with whom I entered into this program with. Those late night study sessions and mock presentations helped mold me into the researcher that I am today. Also, it has been an honor to work alongside both past and present members of the “Hinton lab”. Thank you all for your assistance, inspiration and stimulating discussions. I would also like to thank Clark Atlanta University, the chair and the staff of the Department of Biological Sciences and Center for Cancer Research and Therapeutic Development for the financial, academic and technical support provided. Finally, and most importantly, I would like to thank my beloved family, of which foremost recognition is given to my wonderful and supportive parents. This research was supported by the National Institute of General Medical Sciences (1R01GM106020 and 3R01GM106020); and RCMI (5G12MD007590). iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF FIGURES ........................................................................................................... vi LIST OF TABLES ............................................................................................................ vii LIST OF ABBREVIATIONS ........................................................................................... vii CHAPTER I. INTRODUCTION ............................................................................................ 1 II. LITERATURE REVIEW ................................................................................. 5 Hallmarks of Cancer ......................................................................................... 5 Inflammation: Acute vs. Chronic ...................................................................... 6 Prostate Cancer and Inflammation .................................................................... 8 Prevalence of Prostatic Inflammation ............................................................... 9 Causes of Prostatic Inflammation ................................................................... 11 Inflammatory Cells of the Prostate Tumor Stroma ......................................... 13 Tumor-Associated Macrophages .................................................................... 14 Pro-inflammatory Macrophages and Oxidative Stress ................................... 16 NADPH oxidase expression in Macrophages ................................................. 17 ROS and Prostate Cancer ................................................................................ 18 III. METHODOLOGY ......................................................................................... 21 Chemicals and Reagents ................................................................................. 21 Human Prostate Epithelial Cell Lines ............................................................. 22 Monocyte Culture and Macrophage Differentiation ....................................... 22 iv CHAPTER Monocyte/Prostate Cell Culture ...................................................................... 23 Macrophage Conditioned Media Collection ....................................................23 TMA Immunohistochemistry...........................................................................24 TMA Immunofluorescence ..............................................................................25 Transwell Migration Assay ..............................................................................26 Western Blot Analysis .....................................................................................27 ROS Microplate Assay ....................................................................................27 Invasion Assay .................................................................................................28 Anchorage-Independent Colony Formation Assay......................................... 29 Statistical Analysis .......................................................................................... 30 IV. RESULTS ....................................................................................................... 32 iNOS detection positively correlated with human PCa tissue samples .......... 32 Prostate cancer cells recruited human monocytes .......................................... 34 Classical stimuli-mediated monocyte differentiation and polarization .......... 36 Macrophage co-culture drives SC differentiation ........................................... 36 Exposure to M1s increases ROS in normal and cancerous prostate models .. 38 M1 macrophages promote prostate tumorigenesis in vitro ............................. 39 V. DISCUSSION ................................................................................................. 42 VI. CONCLUSION ............................................................................................... 46 REFERENCES ..................................................................................................................47 v LIST OF FIGURES Figure 1. The Hallmarks of Cancer ........................................................................................ 1 2. Monocyte to Macrophage Differentiation and Polarization ................................... 2 3. CXCR4 induces ROS through NOX enzymes........................................................ 3 4. Prostatitis................................................................................................................. 9 5. The role of inflammatory cells in the progression of prostate cancer................... 15 6. Macrophage Engulfing Bacterium into Phagosome….….. .................................. 20 7. Cell Transformation Assay Principle. ................................................................... 31 8. IHC analysis detected iNOS in human prostate tissue microarrays ..................... 33 9. IF analysis of iNOS expression in prostate cancer tissue ..................................... 33 10. PCa cells recruited human monocytes .................................................................. 35 11. Classical monocyte-to-macrophage differentiation .............................................. 37 12. Co-culture drives SC differentiation and polarization .......................................... 38 13. Exposure to inflammatory macrophages increases ROS in PCa cells .................. 39 14. M1s promote prostate cancer cell anchorage-independent growth....................... 40 15. M1 macrophages promote prostate cell invasiveness ........................................... 41 vi LIST OF TABLES Table 1. Human Prostate Cancer Cell Lines ....................................................................... 34 vii LIST OF ABBREVIATIONS ANOVA ...............................................................................................Analysis of Variance APO...................................................................................................................... Apocyanin BPH ....................................................................................... .Benign Prostatic Hyperplasia BSA ................................................................................................. Bovine Serum Albumin CM ......................................................................................................... Conditioned Media COX ............................................................................................................ Cyclooxygenase CXCR4................................................................................... Cysteine (C)-X-C Receptor 4 DAPI .................................................................................... 4'-6-diamidino-2-phenylindole DCF .............................................................................................. 2', 7' –dichlorofluorescein DCFDA ............................................................... 2',7'-dichlorodihydrofluorescein diacetate DNA ................................................................................................. Deoxyribonucleic Acid EGF .............................................................................................. Epidermal Growth Factor FBS ....................................................................................................... Fetal Bovine Serum H2O2 ....................................................................................................... Hydrogen Peroxide HMS ..................................................................................... Hexose Monophosphate Shunt H&E .................................................................................................Hematoxylin and Eosin IF ..........................................................................................................Immunofluorescence IFNγ .........................................................................................................Interferon Gamma IHC ................................................................................................... Immunohistochemistry viii IL-4 .................................................................................................................. Interleukin-4 IMDM .................................................................... Iscove’s Modified Dulbecco’s Medium iNOS ................................................................................................. Inducible Nitric Oxide KSFM ............................................................................. Keratinocyte Serum-Free Medium LPS ......................................................................................................... Lipopolysaccharide MCSF .....................................................................Macrophage Colony Stimulating Factor MMP ................................................................................................Matrix Metalloprotease NAC ........................................................................................................ N-Acetyl Cysteine NGS....................................................................................................... Normal Goat Serum NO ..................................................................................................................... Nitric Oxide NOX ........................................................................................................... NADPH oxidase Nrf2 ................................................................ Nuclear Factor Erythroid 2–Related Factor 2 O2 ........................................................................................................................ Superoxide O2-° ........................................................................................................... Superoxide Anion PBS ............................................................................................ Phosphate-Buffered Saline PCa ............................................................................................................... Prostate Cancer PIN ................................................................................... Prostate Intraepithelial Neoplasia PMA ............................................................................................ Phorbol Myristate Acetate PSA .............................................................................................. Prostate Specific Antigen PVDF ................................................................................................... Polyvinyl Difluoride Pyo ....................................................................................................................... Pyocyanin RFU ............................................................................................ Relative Fluorescence Unit RNA .......................................................................................................... Ribonucleic Acid RNOS ............................................................................... Reactive Nitrogen Oxide Species ix ROS .............................................................................................. Reactive Oxygen Species RPMI ................................................................................. Roswell Park Memorial Institute RT .......................................................................................................... Room Temperature SDF1α ............................................................................... Stromal-Derived Factor 1 Alpha siRNA ............................................................................................... Short Interfering RNA TAM .................................................................................. Tumor-Associated Macrophages TMA......................................................................................................... Tissue Microarray x CHAPTER I INTRODUCTION An association between chronic inflammation and the development of cancer has long-been appreciated. Over 150 years ago, Rudolf Virchow noted that tumors often occurred at sites of chronic inflammation.4 Today, the link between inflammation and cancer is well established (Figure 1); however, many of the molecular and cellular mechanisms mediating this relationship remain unresolved.5 Figure 1: The Hallmarks of Cancer. The original proposed model to define the six properties that a tumor acquires included unlimited replicative potential, ability to develop blood vessels (angiogenesis), evasion of programmed cell death (apoptosis), self-sufficiency in growth signals, insensitivity to inhibitors of growth, and tissue invasion and metastasis. A combination of recent studies, however, indicate that this model should be revised to include cancer-related inflammation as an additional hallmark.1 1 2 Figure 2: Monocyte to Macrophage Differentiation and Polarization. This schematic depicts the differential activation of macrophages and their effect on tumor growth. A key regulator of the link between inflammation and cancer are tumor-associated macrophages (TAMs).6 Derived primarily from circulating peripheral blood monocytes, TAMs form the major leukocytic infiltrate within the microenvironment of many tumor types. 7 The interaction of monocytes with cancer cells, shapes the maturation and differentiation of macrophages into two distinct subsets: the “classically” activated M1 macrophages and the “alternatively” activated M2 macrophages.6 These phenotypic states are classified by their distinct expression of cell surface markers, secreted signaling molecules, transcription and epigenetic pathways.8 Traditionally, macrophages within the tumor microenvironment are thought to acquire an M2 phenotype, characterized by their secretion of proangiogenic molecules and growth factors that promote tumor progression.9 In contrast, M1 macrophages are regarded as anti-tumorigenic largely due 3 to their production of a myriad of pro-inflammatory mediators, such as nitrogen oxide (NO) and reactive oxygen species (ROS), as part of an antimicrobial arsenal (Figure 2). While the generation of ROS during immune responses is an important aspect of host defense, excessive ROS production is also implicated as the cause of various degenerative diseases, including cancer. Consistent with this notion, our lab observed increased ROS generation in prostate cancer (PCa) cells stimulated with the inflammatory cytokine, stromal-derived factor 1 alpha (SDF1α), and demonstrated that the primary source of ROS was the membrane-bound enzyme complex, NADPH oxidase (NOX) 2 (Figure 3).10 We also established that NOX2-mediated ROS production was critical for the malignant state of prostate cancer cells.10 Interestingly, NOXs are also major sources of ROS in immune cells.11 Moreover, the isoform, NOX2, has been specifically identified as the predominant isoform responsible for superoxide generation Figure 3: CXCR4 induced ROS generation through NOX enzymes. A) C42 cells were pretreated with NAC or AMD3100 for 1 h, followed by SDF-1α treatment for 3 h. B) C42 cells were pretreated with apocynin or rotenone for 1 h, followed by SDF-1α treatment for 3 h. Fluorescence measure at 498/522 nm. 4 in macrophages.12 However, despite the well-established role of M1 macrophages in generating high levels of ROS and other tumor promoting factors, they are still largely viewed as anti-cancerous. In sharp contrast to this idea, the paradoxical concept of M1 macrophages supporting cancer is slowly emerging. Very few studies have reported observations supporting this claim. Sebens et al. observed that exposure of normal colon epithelial cells to inflammatory M1 macrophages induced activity of the anti-oxidative transcription factor, Nrf2, resulting in proteasome activation and protection from apoptosis.13 Similarly, Comito et al. inadvertently demonstrated that M1 macrophages, although to a lesser extent than their M2-polarized counterparts, increased invasiveness of PCa cells and transformed normal human prostate fibroblasts to tumor-associated fibroblasts.14 Fascinatingly, Fang et al. recently reported that persistent co-culturing of immortalized normal prostate epithelial cells with macrophages, in the absence of carcinogens, induced prostate tumorigenesis.15 Therefore, we hypothesized that prostate cancer cells promote a pro-tumor microenvironment, characterized by increased inflammation and oxidative stress, in part, through inflammatory M1 macrophagemediated ROS production. This hypothesis was explored through the investigation of the following specific aims: Aim 1: To investigate whether pro-inflammatory M1 macrophages infiltrate and mediate oxidative stress in prostate cancer. AIM 2: To determine whether M1-mediated ROS generation through NOX progresses prostate cancer in vitro. CHAPTER II LITERATURE REVIEW Hallmarks of Cancer Cancer is a complex disease in which cells in a specific tissue are no longer fully responsive to the signals that regulate cellular differentiation, survival, proliferation and death. As a result, cells continue to grow and divide in an uncontrolled and indefinite manner, causing local damage.16 Normal cells have a wide number of intrinsic defenses to protect against becoming cancerous. Moreover, several injuries (mutations) are needed to disrupt normal cell growth and survival to form cancer.17 In January 2000 a landmark paper outlining how cells acquire a cancer-like phenotype was published and detailed 6 key changes or features that make a cancer a cancer. These features are now regarded as the “hallmarks of cancer”, and represent the fundamental basis of malignancy.18 These principles are thought to govern all malignancies, simplifying and unifying the huge variety of diseases that come under the umbrella term ’cancer’. The traits (hallmarks) that the authors highlight in the paper are: (i) cancer cells stimulate their own growth (selfsufficiency in growth signals); (ii) resist inhibitory signals that might otherwise stop their growth (insensitivity to anti-growth signals); (iii) resist their programmed cell death (evading apoptosis); (iv) can multiply indefinitely (limitless replicative potential) (v) 5 6 stimulate the growth of blood vessels to supply nutrients to tumors (sustained angiogenesis); (vi) invade local tissue and spread to distant sites (tissue invasion and metastasis).17 However, since the publication of this seminal article the hallmarks of cancer have been revised to include an additional malignant trait: cancer-enabling inflammation.19 Inflammation: Acute vs. Chronic Inflammation can be classified as either acute, occurring over short periods of time (seconds, minutes, hours, and days), or chronic inflammation, which persists for extended periods of time.20 The inflammatory response is part of the body’s innate immune system. It is the body’s normal, biological response when something harmful or irritating affects a part of the body.21 Although acute inflammation is a healthy physiological response indicative of wound healing, chronic inflammation has been directly implicated in a wide range of degenerative human health disorders encompassing almost all present day diseases.22 In the book, Chronic Inflammation: Molecular Pathophysiology, Nutritional and Therapeutic Interventions, the authors cover several pathologies associated with inflammation, including aging, allergies, autoimmune disorders, atherosclerosis, cancer, chronic wounds, metabolic syndrome, and obesity.23 For many years, chronic and acute inflammatory processes were thought to be driven by different causes, through the activity of different cells and inflammation mediators, and to result in quite different outcomes. However, a more modern view suggests that these processes are interlinked. Moreover, in the setting of acute inflammation, well-regulated tissue healing can go awry and drive a chronic 7 inflammation process intertwined with fibrosis and related processes. In hepatic, pancreatic, and gastrointestinal issues, among others, this pro inflammatory pro-fibrotic environment can stimulate carcinogenesis which in turn, can lead to an altered immune/inflammatory milieu.23 Acute inflammation is the early stage of inflammation and presents with immediate signs and symptoms such as pain, heat, redness, swelling, and loss of function. These symptoms are due to the increased blood flow and increased permeability of the vasculature which is responsible for bringing neutrophils to the affected site as rapidly as possible. This protects the injured areas from further harm while the body goes into overdrive to fend off bacteria, viruses, pathogens, damaged cells, or other irritants. It not only kills off the invaders, but damaged tissue as well, until your body wins the battle. Without inflammation, wounds and infections would never heal.23 Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation and is largely characterized by an influx of macrophages. These cells, along with other cellular mediators, contribute to the simultaneous destruction and healing of the tissue during the inflammatory process. It can result from failure to eliminate an acute inflammation stimulus, an autoimmune response, and/or persistence of a chronic irritant of low intensity.23 Unfortunately, chronic inflammation will not typically produce symptoms until actual loss of function occurs. This is because chronic inflammation is low-grade and systemic, often silently damaging your tissues over an extended period of time. This process can go on for years undetected, until a disease suddenly manifests.23 8 About 20% of all human cancers are caused by chronic infection or chronic inflammatory states.24 In regards to prostate cancer, it has been proposed that exposure to environmental factors such as infectious agents and dietary carcinogens, and hormonal imbalances lead to injury of the prostate and to the development of chronic inflammation and regenerative ‘risk factor’ lesions, referred to as proliferative inflammatory atrophy (PIA).24 The development of new experimental animal models coupled with classical epidemiological studies, genetic epidemiological studies and molecular pathological approaches, has shed more light on the role of inflammation in prostate cancer, and has contributed to the advent of new strategies to prevent the disease. Prostate Cancer and Inflammation The prostate is a walnut-sized gland located between the bladder and the penis, anterior to the rectum. The urethra runs through the center of the prostate, from the bladder to the penis, allowing the outflow of urine from the body. The primary function of the prostate is to secrete fluids that nourish and protect sperm. During ejaculation, the prostate squeezes this fluid into the urethra, whereby it is then expelled with sperm as semen.25 Inflammation is very common within the normal adult prostate,26 and researchers now appreciate a potential link between chronic prostatic inflammation and prostate carcinoma (Figure 4). Histological studies have reported unexplained acute and chronic inflammation, and inflammation-associated lesions, in biopsy specimens of prostate cancer tissues.24 Moreover, analysis of surgically resected tissues of patients with benign prostate hyperplasia (BPH) or prostate cancer revealed significant variations in the expression of several signaling components involved in the inflammatory cascade, 9 including chemical signaling cytokines and their cognate chemokine receptors, suggesting a connection between inflammatory signaling and prostate carcinoma.27 Prevalence of Prostatic inflammation There are multiple different lines of evidence suggesting that inflammation is very common within the adult prostate. Prostatitis is a heterogeneous and complex entity which the National Institutes of Health (NIH) consensus classification refers to as chronic Figure 4: Prostatitis. Reprinted from Herballove.com., Retrieved October 28, 2015, from http://www.herballove.com/guide/your-guide-prostatitis. Copyright © Herballove. All Rights Reserved. prostatitis /chronic pelvic pain syndrome (CPPS).28 CPPS is divided into the following four categories, the first three of which relate to men with symptoms of disease: (i) acute bacterial prostatitis; (ii) chronic bacterial prostatitis; (iii) chronic prostatitis ⁄ CPPS; and (iv) asymptomatic inflammatory prostatitis.28 Bacterial prostatitis accounts for only an estimated 5–10% of prostatitis cases, with the most commonly implicated microorganisms being Escherichia coli (E coli) and Enterococcus spp.29, 30 In terms of 10 symptomatic prostatitis, it is estimated that up to 16% of men in the US population are afflicted at some time in their life.29, 31 The prevalence of asymptomatic prostatic inflammation (i.e. histological prostatitis) appears to be in fact much higher, as evidenced by studies examining men who undergo biopsy for prostate cancer due to elevated prostate-specific antigen (PSA) levels and test negative for cancer,32,33, 34, 35 autopsy studies36 and findings from transurethral resections for benign prostatic hyperplasia (BPH).37 A recent example of this stems from results published from the baseline data of the REDUCE (REduction by DUtasteride of prostate Cancer Events) trial, where 80% of patient biopsies were found to have some degree of Inflammation.38 Similarly, results from a prospective randomized controlled trial of 328 men with PSA levels between 2.5 and 10 ng/ml and normal digital rectal examination (DRE) indicated that more than 45% of the patients had leucocytes in expressed prostatic secretions (EPS).34 Finally, histological specimens of prostate cancer tissue frequently exhibit unexplained acute and chronic inflammation and inflammationassociated lesions.24 Evidence suggests there is also a racial and geographical difference in the prevalence of prostatic inflammation in adult men, which falls in line with the geographic distribution difference in prostate cancer incidence. For example, studies have reported an increased incidence of inflammation in biopsy specimens39 and increased expression of immune-related genes in tumor tissues 40 from African American men compared to European American men. Also, recent findings from an autopsy study done by Joshu et 11 al. revealed less inflammation in the prostates of Asian men as opposed to European American men.26 Causes of Prostatic Inflammation Multiple bacterial species are known to infect the human prostate and induce inflammation; many of which have been identified from studying patients with bacterial prostatitis.26 Interestingly, in the Ugurlu et al. study previously mentioned ,34 the patients with leucocyte-positive EPS were randomized into antibiotic (levofloxacin), antiinflammatory (naproxen sodium) and control treatment groups. Only the antibiotictreated patients exhibited a significant decrease in PSA levels, suggesting a potential contribution from an unrecognized prostatic infection.26 In addition to the most commonly implicated microorganisms in bacterial prostatitis (E. coli and Enterococcus spp), organisms such as Pseudomonas spp., Proteus mirabilis, Klebsiella spp. and Serratia spp. have also been identified.29, 30 Several sexually transmitted organisms have also been implicated in bacterial prostatitis and/or prostatic inflammation including Chlamydia trachomatis, Gonococcal organisms, Trichomonas vaginalis and Treponema pallidum.41 Mycoplasma spp. have also been implicated in chronic prostatitis.42 43 Studies attempting to define a potential correlation between prostatitis and prostate cancer risk have reported both positive and negative results.44 A study in 2010 performed in a large, multiracial and ethnic cohort as part of the California Men’s Health Study (CMHS) found an increase in risk for prostate cancer in men with a history of prostatitis and long duration of prostatitis symptoms.45 This study also found that a self-reported history of sexually transmitted disease (STD) was not associated with 12 overall prostate cancer risk; however, Latinos reporting a history of STDs had an increased risk of prostate cancer compared to Latinos with no STD history.45 Furthermore, non-US-born Latinos were found to have a greater risk of prostate cancer associated with STD history than US-born Latinos.45 Although the authors report that this study could have potentially been confounded by detection bias (e.g. men with symptomatic prostatitis may seek medical attention, which may in turn lead to a greater chance for testing and incidental detection of prostate cancer), the association between prostatitis and prostate cancer risk certainly remains an important area for further research. There are also several lines of evidence that support a potential role for asymptomatic (i.e. subclinical) prostatic inflammation caused by infectious microorganisms and prostate cancer development.26 An organism of particular interest in this respect is E. coli. Apart from being one of the most frequently isolated microorganisms from patients with bacterial prostatitis, E. coli has also been identified in both BPH and prostate cancer tissues using both culture-dependent and cultureindependent molecular techniques.46, 47 In mouse models, infection of the prostate with uropathogenic strains of E. coli (UPEC) has been reported to induce epithelial proliferation and reactive hyperplasia,48 dysplasia and oxidative DNA damage49 and a marked reduction of the haploinsufficient prostate cancer tumor suppressor, NKX3.1.50 A recent study in Wistar rats described prostatic epithelial hypertrophy and atrophy in response to UPEC E. coli infection, with a transient upregulation of ErbB2 [human epidermal growth factor receptor 2 (HER2 ⁄ neu)].51 Various strains of UPEC E. coli 13 produce a number of virulence factors, such as cytotoxic necrotizing factor 1 (CNF1), which has been shown to promote tissue damage in a rat model of prostatitis.52 Intriguingly, in a recent study examining E. coli isolates from patients presenting with acute bacterial prostatitis, more than 70% of isolates were found to express colibactin and one strain carried the cyto-lethal distending toxin (CDT) gene cluster.53 The authors of this study postulate that the production of genotoxic toxins by E. coli implicated in acute bacterial prostatitis could contribute to subsequent carcinogenesis and potentially explain the epidemiological data suggesting an increased risk for prostate cancer with previous history of prostatitis.53 Additional microorganisms (both bacterial and viral) have been implicated in stimulating prostatic inflammation that may contribute to the development of prostate carcinogenesis. Moreover, it is believed that recurrent microbial infections of the prostate resulting in the formation of lesions are due to the persistent secretion of free radicals and inflammatory cytokines by infiltrating leukocytes. As a result, leukocytic infiltrates have been identified as key players in prostate cancer development.49 Inflammatory Cells of the Prostate Tumor Stroma The inflammation identified histologically in prostate cancer tissues is most commonly chronic, being chiefly comprised of lymphocytes as well as macrophages (Figure 5), and less frequently of plasma cells and eosinophils.26 Acute inflammation is present to a lesser extent and is comprised primarily of neutrophils.26 Just as the stimuli for prostatic inflammation are largely yet to be defined, our understanding of prostate immunobiology is still relatively poor. Over the past few years, however, there have been 14 several recent advances in the characterization of the inflammatory cell types infiltrating the prostate.26 The normal prostate contains a heterogeneous inflammatory cell population of stromal and intraepithelial T and B lymphocytes, mast cells and macrophages,54 which are also present in chronically inflamed prostate tumors.26 Tumor-associated macrophages (TAMs) represent the major inflammatory components of the tumor stroma and direct multiple aspects of neoplastic growth.55 Derived from monocytic precursors in the blood and bone marrow, TAMs are mononuclear phagocytes, which once localized in tissues, acquire specialized functions depending on the requirements of the tissue. For instance, Comito et al. demonstrated that secretion of the chemokine CCL2 from PCa cells facilitated monocyte recruitment, TAM differentiation and polarization to a specific phenotype.14 56 Tumor-Associated Macrophages TAMs are traditionally sub-classified as M1 (classically-activated macrophages), or M2 alternatively activated macrophages. These phenotypic states are characterized by their distinct expression of cell surface markers, secreted signaling molecules and transcription and epigenetic pathways.8 TAMs in murine and human tumors are thought to typically display an M2-like phenotype,57 which promotes tumor growth, tissue remodeling, debris scavenging, angiogenesis and suppression of adaptive immunity. M2 macrophages are generally distinguished by their low production of pro-inflammatory cytokines, and high production of anti-inflammatory cytokines.58 M2 macrophages are 15 diverse, but are all generally involved in T helper 2 (Th2) responses, have immune regulatory function, orchestrate encapsulation and containment of parasites, and promote tissue repair, remodeling, and tumor progression;59 all of which favor tumor development and progression. Figure 5: The role of inflammatory cells in the progression of prostate cancer. Recruitment of inflammatory cells to the prostate can occur under a variety of physiological and pathophysiological conditions, such as wound, infection, prostatitis, and cancer. Genetically altered prostate epithelial cells, under the influence of resident fibroblasts, inflammatory cells (such as macrophages and lymphocytes), and endothelial cells progress further through additional genetic changes triggered by reactive oxygen species (ROS) or reactive nitrogen species (RNOS). The genetically unstable prostate cell clusters can form PIA and then proceed to PIN before becoming prostate cancer cells with increased malignant potential.2 16 M1 macrophages are referred to as “killer” macrophages due to their potent microbicidal and tumoricidal activity.60 These specialized phagocytic cells attack foreign substances, infectious microbes and cancer cells through destruction and ingestion. During the initial leukocyte migration phase, M1 macrophages produce a myriad of proinflammatory mediators, such as reactive oxygen species (ROS), as part of an antimicrobial arsenal. M1s also secrete matrix metalloproteinases (MMPs) that degrade the extracellular matrix, allowing infiltration of additional inflammatory cells to sites of tissue injury.60 Should tissue-damaging irritants persist, activated M1s further exacerbate the inflammatory response by recruiting various adaptive immune cells, thereby generating substantial tissue damage.60 Once the inflammatory stimulus is eliminated, M1-mediated inflammatory activation diminishes. On the contrary, a protective role in tumorigenesis has also been described for M1 macrophages, whereby they antagonize the suppressive activities of M2 macrophages.60 Pro-inflammatory Macrophages and Oxidative Stress During acute inflammatory responses, ROS molecules are produced in large quantities by M1 macrophages and are vital to the destruction of invading pathogens and foreign materials. These highly reactive oxygen intermediates include superoxide, hydrogen peroxide, singlet oxygen and nitric oxide.60, 61 Macrophage influx in target tissues during an immune response generates copious amounts of ROS, and as a result, leads to tissue damage.62 For instance, in rats administered large doses of vitamin A, which activated macrophages in the liver to produce ROS, tissue injury was significantly increased.63 17 The notion that oxidative stress is important for driving prostate cancer progression is bolstered by studies demonstrating that the consumption of certain types of dietary antioxidants is associated with reduced prostate cancer risk.24 Excessive, unregulated generation of ROS leads to oxidative stress. Biomolecules such as lipids, proteins, and deoxyribonucleic acid (DNA) are all targets for modification by ROS with diverse pathologic consequences, ranging from cell death (necrosis, apoptosis), to cell survival, (proliferation, invasion and migration). Accordingly, oxidative stress is implicated in the pathology of several human diseases, including cardiovascular disease, rheumatoid arthritis and cancer. We have demonstrated differential pathological responses to the ROS molecule hydrogen peroxide (H2O2), where treatment of low grade prostate cancer cells with H2O2 resulted in rapid apoptosis,64 and treatment of high grade prostate cancer tumors with H2O2 rapidly increased expression of the chemokine Gprotein coupled receptor, CXCR4, resulting in rapid cell migration and invasion through bone endothelial cells,65 independent of its ligand, SDF1α.65 We’ve also demonstrated a reciprocal relationship between CXCR4 and ROS, where treatment with SDF1α increased ROS generation specifically from the membrane associated enzyme complex NOX2 in prostate cancer cells, which led to enhanced CXCR4-mediated migration.10 Moreover, other groups support our observations and have identified NOX enzymes as the primary source of ROS in prostate tumors.10, 66 NADPH Oxidase Expression in Macrophages ROS produced in tissues are derived from two major sources: (i) the mitochondrial electron transport chain and/or (ii) NADPH oxidase enzymes (NOXs).67 18 While mitochondria generate ROS indirectly as a by-product of cellular metabolism, NOX enzymes produce ROS directly; to date ROS production is the only known biological function of NOX enzymes.11 Initially observed in leukocytes, the NOX family of enzymes is comprised of 7 known members: NOX isoforms 1-5, DUOX1 and DUOX2.11 All members of the NOX family consist of 6 transmembrane domains, a NADPH-binding domain and a FAD-binding domain. Despite their structural similarities, NOX isoforms display distinct biochemical characteristics and subcellular localizations.68 Activation of NOX enzymes require translocation of the cytosolic regulatory proteins to the plasma membrane-bound flavocytochrome b558, which, once collectively assembled, generates superoxide by transferring an electron from NADPH to oxygen, resulting in ROS generation (Figure 6) and diverse biological functions ranging from apoptosis to mitogenic signaling.11 Macrophages generate ROS via NOX enzymes, and NOX2 is the predominant form expressed in macrophages of all types.11 ROS and Prostate Cancer Prostate cancer is commonly associated with a shift in the antioxidant-prooxidant balance towards increased oxidative stress. Previous studies highlighted the altered prooxidant-antioxidant status in prostatic tissue of man, rat and also in cell lines, where the imbalance between these antagonist played a major role in the initiation of prostate carcinogenesis.69 However, there is very little idea about the cause of this imbalance. Androgens are considered to be the most powerful candidates that regulate ROS balance in the prostate, though the mechanistic relation between androgen status and redox homeostasis in the prostate is not proven.70 Tam et al.71 in this context indicated that 19 replacement of androgens reduced the oxidative stress level by down-regulating NOX expression, thereby bringing the antioxidant level to normalcy. Besides androgen, the transcription factor erythroid 2p45 (NF-E2)-related factor 2 (Nrf2) mediates the expression of key protective enzymes through the antioxidant-response element (ARE) in prostate cancer.72,73 Recent studies suggested that Nrf2 and several of its target genes are significantly down-regulated in human prostate cancer and as a result, cells were continually exposed to increased oxidative stress and may have resulted in their progression to metastatic disease.74 Another major component involved in the maintenance of redox balance in the cell is the Glutathione oxidation-reduction system. Somatic mutations, causing inactivation of the Glutathione S-Transferase gene (GSTP1) have been identified in almost all the prostate cancer cases examined by Nelson and colleagues.75 Therefore, the sensitive balance between the oxidant and anti-oxidant components of the cells and their regulatory mechanisms seem to play a major role in developing a malignant state in prostate tissue. 20 Figure 6: Macrophage engulfing a bacterium into a nascent phagosome. NADPH oxidase assembles and begins to function before the phagocytic cup has sealed. In eosinophils, macrophages, and neutrophils stimulated with soluble agonists, most NADPH oxidase assembles at the plasma membrane. The entire system is driven by NADPH oxidase activity. Electrons from cytoplasmic NADPH are translocated across a redox chain to reduce O2 to superoxide anion (O2−•) inside the phagosome or extracellularly. For each electron removed from the cell, approximately one proton is left behind. Thus NADPH oxidase activity tends to depolarize the membrane. NADPH is regenerated continuously by the hexose monophosphate shunt (HMS) during the respiratory burst.3 CHAPTER III METHODOLOGY Chemicals and Reagents The following reagents and human antibodies were from Thermo Fisher Scientific: Alexa Fluor 488 goat anti-mouse (A11001), Alexa Fluor 488 goat anti-rabbit (A-11008), Alexa Fluor 594 donkey anti-goat (A-11058), Alexa Fluor 594 goat antirabbit (A-11037) and Keratinocyte-SFM 1X (17005-042). Human recombinant SDFlα (300-28A), IFNγ and IL-4 were purchased from PeproTech. Human antibodies against COX-2 (sc-795), PSA (sc-7638) and DAPI (sc-3598) were from Santa Cruz. Apocynin (sc-203321) was also purchased from Santa Cruz Biotechnology, Inc.The following reagents and human antibodies were from Sigma Aldrich: Triton X-100 (T8532); antiαTubulin (T5168); thymidine (T1895-5G); hypoxanthine (9636-5G); 2 mercaptoethanol (M3148-25ml); LPS (L5293) and PMA (P1585-1MG). iNOS (GTX31048) and CD163 [EDHu-1] (GTX42364) were purchased from Genetex, Inc. Hemacolor rapid staining solution (111661) was purchased from EMD Millipore. COX1 human antibody (ab695) and cellular ROS/superoxide detection assay kit (ab139476) were from abcam. Sudan Black B (0593-25G) was purchased from AMRESCO. RPMI 1640 (10-040CV) was from Corning. CytoSelect 96-Well Cell Transformation Assay, Standard Soft Agar (CBA-130) kit was purchased from Cell BioLabs, Inc. 21 22 Human Prostate Epithelial Cell Lines The normal prostate epithelial cell line, RWPE1 (CRL-11609), and prostate cancer cell lines LNCaP (CRL-1740), DU145 (HTB-81) and PC3 (CRL-1435) were all obtained from American Type Culture Collection (ATCC) and maintained per manufacturer’s instructions. In brief, RWPE1 cells were cultured in complete Keratinocyte Serum-Free Medium (KSFM; supplemented with 0.05 mg/ml bovine pituitary extract (BPE) and 5 ng/ml epidermal growth factor (EGF). Prostate cancer cell lines were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640; supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids, 1% antibioticantimycotic and 1% L-glutamine). All cells were maintained in a 37 °C, 5% CO2 incubator. Serum starvation conditions consisted of culturing in reduced serum medium (unsupplemented KSFM or phenol-free RPMI containing 1% L-glutamine and 0.2% FBS) for 24 hours. Monocyte Culture and Macrophage Differentiation The human peripheral blood mononuclear cell line, SC (CRL-9855), was obtained from ATCC and maintained at a concentration between 2 x 105 and 1 x 106 cells/mL in Iscove's Modified Dulbecco's Media (IMDM) supplemented with 10% FBS, 0.05 mM 2mercaptoethanol, 0.1 mM hypoxanthine and 0.016 mM thymidine. To generate resting macrophages (Mϴ), SC cells were cultured in 60ng/mL phorbol myristate acetate (PMA) for 6 hours. Subsequent M1 or M2 polarization was achieved via treatment for an additional 62 hours with lipopolysaccharide (LPS; 0.1μg/ml) and interferon gamma (IFNγ; 20ng/ml) or interleukin 4 (IL-4; 20ng/ml), respectively. 23 Monocyte/Prostate Cell Co-culture For monocyte/prostate cell co-culture, seed 1 x 106 (1,000,000) prostate epithelial cells (RWPE1, LNCaP, DU145 and PC3) each into accompanying 0.4-µm pore transwell inserts (Corning Cat No. 3412) in 2 mL of RPMI (supplemented with 0.2% FBS) or KSFM (supplemented with 0.2% BPE; RWPE1) and incubate for 24 hours in 37°C/5% CO2 incubator. After 24 hours, without removing inserts, seed 1 x 106 SC cells into the bottom well of each well in 1 mL of serum-free IMDM. Maintain and monitor cocultures for an additional 48 hours. Macrophage Conditioned Media Collection SC cells were seeded at a concentration of 4 x 105 (400,000) cells/well in complete IMDM in a 6-well plate format. Macrophage polarization was achieved by adding 60ng/ml PMA to each well followed by incubation at 37°C in 5% CO2 incubator for 6 hrs. After 6hrs, plates were retrieved from incubator and (without removing existing well contents) M1 or M2 polarizing components were added (LPS; 0.1μg/ml and IFNγ; 20ng/ml or IL-4; 20ng/ml, respectively) and incubated for an additional 66 hours. Next, cells were pretreated for 1 hour with desired experimental conditions (apocyanin; 300µM), immediately followed by aspiration of all well contents. Serum-free IMDM was added to each well and returned to incubator for 48 hrs allowing medium conditioning to take place. After 48 hrs plates were retrieved from incubator and conditioned media collected and transferred to prelabeled/precooled 15ml conical tubes on ice. To clarify CM (remove cells and debris) samples were centrifuged at 2,000 x g at 4˚C for 15 min. After centrifugation, CM was removed being sure not to disturb any sediment, and 24 transferred to newly labeled 15ml conical tubes. Centrifugation step was repeated and CM was placed on ice for same day use. Alternatively, if CM was not needed for same day use, it was stored at -20˚C for up to 48 hours. TMA Immunohistochemistry Immunohistochemical (IHC) analysis was performed on a prostate disease spectrum tissue array (PR8011; Biomax) ranging from normal to high grade metastatic tissues. The array consisted of 80 total tissue cores including adenocarcinoma, metastatic, hyperplasia, chronic inflammation, adjacent normal tissue and normal tissue. Each individual core had a diameter of 1.5 mm and a thickness of 0.5 um. Briefly, formalinfixed, paraffin-embedded specimens were retrieved in a down-graded series of xylene and ethanol incubations. Antigen retrieval was carried out using the Biocare Digital Decloaking Chamber and Reveal Decloaker RTU per manufacturer’s instructions. Native peroxidase activity was neutralized via incubation in 0.3% hydrogen peroxide for 15 min at room temperature (RT), followed by a 1X PBS wash in a humidified chamber. Specimens were then blocked with blocking solution (5% normal goat serum/Tris-buffered saline/Tween-20; TBST) for 30 min. Inducible nitric oxide (iNOS) was detected with a rabbit anti-human iNOS polyclonal antibody (Genetex; 1:1000) in blocking solution overnight at 4°C, followed by a biotinylated affinity purified goat antirabbit IgG (H+L) secondary antibody (Vector .Laboratories; 1:1000), in blocking solution for 30 min at RT. Specimens were washed thoroughly between incubations, and avidin + biotin reagent (ABC solution) was added at a 1:200 dilution for 30 minutes at room temperature. Next, tissues were developed in diaminobenzidine (DAB) (Vector 25 Laboratories) for 3 min at RT, and nuclei counterstained with Meyer's hematoxylin for 13 min. Following counterstaining, specimens were rinsed in deionized water for 5 min and subjected to dehydration in graded-up series of ethanol followed by incubation in xylene, prior to mounting on slides. A negative control tissue sample was prepared by incubating in biotinylated affinity purified goat anti-rabbit IgG (H+L) antibody, only, as described above. The specimens were analyzed and photographed using the LSM 700 (Zeiss) confocal microscope. TMA Immunofluorescence Prostate tissue microarray’s (TMAs) (PRC481; US Biomax, Inc.) representative of normal, benign prostate hyperplasia (BPH), chronic prostatitis, low, moderate and high grade carcinoma lesions were analyzed via immunofluorescence (IF) using iNOS rabbit polyclonal antibody (1:100 dilution; Genetex), CD163 mouse monoclonal antibody (1:100 dilution; Genetex) and prostate specific antigen (PSA) goat polyclonal antibody (Santa Cruz; 1:100) all prepared in 0.1% Triton-X/PBS. In brief, TMAs were baked for 30 minutes at 60°C prior to deparaffinization/dehydration in a series of (2) 100% xylene, (1) 100% EtOH, (1) 95% EtOH, and (1) 70% EtOH immersions. Antigens were then retrieved using Dako PT Link per manufacturer’s instructions. Next, TMAs were warmed in 30°C oven for 20 minutes in preparation for staining. Using the Dako Autostainer instrument slides were first blocked with 10% normal goat serum (NGS)/0.4% Triton X/1X PBS for 20 minutes, followed by incubation with primary antibodies for 1hr at room temperature. TMAs were then washed and incubated with appropriate secondary antibodies at a concentration of 1:100 (Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 26 488 goat anti-rabbit IgG, Alexa Fluor 594 donkey anti-goat IgG and/or Alexa Fluor 594 goat anti-rabbit IgG; Invitrogen) for 1 hour and counterstained with DAPI (1:1000) for 1 minute. To reduce native tissue fluorescence TMA were then incubated with 0.1% Sudan Black B (SBB)/70% ethanol for 20 minutes at room temperature followed by three, 8 minute washes. Finally coverslips were mounted onto slides with Vectashield mounting media for fluorescence (H-1000; Vector Laboratories). Analysis of macrophage marker distribution was carried out using the LSM 700 confocal microscope (Zeiss) at 40x oil magnification. Transwell Migration Assay At roughly 80% confluency, RWPE1, LNCaP, DU145 and PC3 cells were washed in PBS and serum starved in their respective starvation media (RPMI-1640 or KSFM) for 48 hours in 37°C and 5% CO2. For control conditions serum-free IMDM, KSFM and RPMI-1640 medium was also incubated at 37°C in 5% CO2 for 48 hours. After 48 hour serum starvation, CM was removed from prostate cells and controls and transferred into labeled 15ml conical tubes on ice. CM was then clarified via centrifugation at 2000 x g at 4˚C for 15 min. After centrifugation, CM was transferred to newly labeled 15ml conical tubes and placed on ice. Using 24-well plate/5-µm-pore size polyvinylpyrrolidone-free polycarbonate transwell filters (Corning), 600µl of clarified CM was added to the lower chamber of the transwell migration plate, and 5 x 104 SC peripheral blood monocytes in 100µl were seeded onto polycarbonate filter inserts and incubated at 37°C/5% CO2. Monocytes were allowed to migrate toward CM for 24 hours, after which inserts were removed and stained using Hemacolor staining solution (EMD 27 Millipore). Stained (migrated) cells on underside of transwell insert were manually counted using a light microscope. Western Blot Analysis Monocytes, PCa cells or macrophages derived from our experimental conditions were collected and lysed for 5 minutes in 50µl of 1% Triton X-100, 2mM EGTA, 1mM phosphatase/protease inhibitor cocktail, and 1X PBS). To ensure complete cell lysis, cell lysates were then flash frozen/lysed in liquid nitrogen 3 times for 5 minutes each. Lysates were then centrifuged, protein quantified using Bradford assay, and samples were prepared containing twenty to forty micrograms of total proteins in loading buffer. Just before samples were loaded on an SDS–PAGE, they were boiled at 100°C for 5 minutes. Samples were then separated on SDS-PAGE gel and transferred onto polyvinyl difluoride (PVDF). The immunoblots were incubated in 3% bovine serum albumin (BSA), 1X TrisBuffered Saline-Tween 20 (TBS-T) for 1 hour at room temperature, incubated with primary antibodies overnight, followed by incubation with appropriate horseradish peroxidase (HRP) conjugated secondary antibodies for 1 hour at room temperate. Immunoblots were developed using enhanced chemiluminescence (ECL) method and scientific imaging film. ROS Microplate Assay Monocytes (1 x 104 cells/well) were plated, differentiated and polarized to M1macrophages (as previously described) on high-throughput screening (HTS) 96-well transwell inserts (0.4µM pore size) 3 days prior to experiment. One day prior to experiment, normal (RWPE1) and cancerous (LNCaP, DU145 and PC3) prostate 28 epithelial cells (2 x104 cells/well) were seeded in the bottom wells of transwell 96-well black plate and incubated in 37°C/5% CO2 overnight to ensure between 70-80% confluency on the day of the experiment. Several wells were left empty for the background fluorescence control measurements. On the following day, supernatant was removed and prostate cells were carefully washed with 1X Wash Buffer. Transwell inserts containing M1 macrophages were then inserted into 96 well plate containing prostate cells and allowed to co-culture for 3 hours. Positive control samples were treated with ROS Inducer (Pyocyanin) 30 minute before endpoint analysis, and negative control samples were treatment with the ROS inhibitor, N-acetyl-L-cysteine (NAC), 1hr prior to experimental end. M1 macrophages and positive/negative controls were removed and ROS/superoxide detection solution was added (100µl/well) to each well and incubated for 60 minutes at 37°C in the dark. Finally, plates were read (bottom reading), without removing the detection mix, using a fluorescence microplate reader and standard fluorescein (Ex=488nm, Em=520nm) and rhodamine (Ex=550nm, Em=610nm) filter sets. Invasion Assay Prostate epithelial cell invasion was assayed using the BioCoat Matrigel invasion chambers with 8.0μm PET membrane in 24-well plate format (Corning; 354480). To prepare plate, 500μl of warm (37°C) bicarbonate based culture medium (IMDM or RPMI) was added to the inserts and to the bottom wells. Plates were allowed to rehydrate for 2 hours in humidified tissue culture incubator (37°C; 5% CO2 atmosphere). After rehydration, media was carefully removed without disturbing the layer of Matrigel matrix 29 on the membrane. PCa cells (5 x 104) were then resuspended in 500μl of serum-free IMDM (or various experimental conditions being tested). 750μl of desired chemoattractant (SDF1α + phenol-free/serum free RPMI) or serum-free medium (phenol free RPMI) was added to the bottom well. Using sterile forceps, empty inserts were transferred to the wells containing the chemoattractant or serum-free media. Previously prepared cell suspensions (500ul) was then added to the inserts and incubated for 22hrs in a humidified tissue culture hood, at 37°C, 5%CO2 atmosphere. To remove non-invading cells, a cotton tipped swab was used to gently swabbing the membrane surface. Scrubbing was repeated with a second swab moistened with medium to ensure all noninvaded cells were removed. Prior to staining cells, they were fixed by adding 500μl of 100% methanol to the appropriate number of wells. Invaded cells were then stained using (Hemacolor staining solution (EMD millipore) following manufacturer’s instructions. Finally, inserts were allowed to dry upside, overnight. Chemotaxis was evaluated by manually counting the cells that migrated to the lower surfaces of the polycarbonate filters. Anchorage-Independent Colony Formation Assay Using the CytoSelect 96-Well Cell Transformation Assay (Soft Agar Colony Formation) kit (Cell Biolabs; CBA-130), agar Solution (1.2%) and 2X DMEM/20% FBS mixture was prepared and then added to the wells of a black polystyrene flat bottom 96 well plate (with micro-clear bottom) suitable for fluorescence measurement. The plate was then transferred to 4°C for 30 minutes to allow the base agar layer to solidify. Prostate epithelial cells were then harvested and resuspended in complete medium at 4 x 30 105 cells/ml. Equal volumes of 1.2% Agar Solution, 2X DMEM/20% FBS media, and cell suspension (1:1:1) was mixed and transferred to each well of the 96-well flat-bottom microplate already containing the solidified base agar layer (25 µL of cell suspension containing 1,000-10,000 cells/well will be seeded). Plate was then placed in 4°C for 15 minutes to allow the cell agar layer to solidify. After cell agar layer solidified, co-culture conditioned medium (CM) or control CM was added to each well and the plate was incubated 6-10 days at 37°C and 5% CO2. Cell colony formation was monitored and examined under a light microscope. Finally, culture medium was removed and agar solubilization solution was added to each well of the 96-well plate. Plate was incubated for 1 hr at 37°C. Cells were then lysed and the plate was incubated at room temperature for 15 minutes. Cell lysate (10 µL) was then transferred to another black polystyrene flat bottom 96 well plate (with micro-clear bottom) suitable for fluorescence measurement and CyQuant solution was added to each well. Plates were incubated for 10 minutes at room temperature and read using a microplate reader (fluorescence 485/520 nm filter set) Statistical Analysis Data are presented as the mean ±SE of at least three independent experiments. The data were analyzed for two-way ANOVA or Student t -test. All statistical analyses were done, and all graphs were generated using GraphPad Prism 5.0 software (GraphPad). P values less than 0.05 were considered significant. 31 Figure 7: Cell Transformation Assay Principle. Reprinted from Cell Biolabs, Inc. Retrieved October 28, 2015, from http://www.cellbiolabs.com/sites/default/files/CBA-130-cell-transformation-assay.pdf. Copyright © 2004-2012: Cell Biolabs, Inc. CHAPTER IV RESULTS iNOS detection positively correlated with human prostate cancer tissue samples To determine whether inflammatory macrophages are associated with prostate malignancies, we conducted immunohistochemical (IHC) and immunofluorescent (IF) analysis of human prostate tissue microarrays (TMAs). Tissues representative of normal, benign hyperplasia, chronic inflammation, and a range of malignant prostatic lesions were analyzed for distribution of the M1 macrophage marker, inducible nitric oxide synthase (iNOS or NOS2) and cluster of differentiation 163 (CD163; M2 marker). As indicated in Figure 8, brown (positive) staining, iNOS was detected in malignant tissues where positive iNOS detection was more prominent in the earlier stage of malignancy (Figure 8B-C). iNOS was also present in advance disease specimens, but to a lesser extent. As expected, there was little to no iNOS detected in normal adjacent tissues (Figure 8A). Confocal images of tissues stained with immunofluorescent (IF) iNOS (red) and CD163 (green) reveal a similar pattern (Figure 9). iNOS-positive macrophages were detected in BPH, chronic inflammation, and localized prostate cancer samples. CD163 detection, on the other hand, was more abundant in the advance metastatic tissue specimens. Again, little to no detection of M1 or M2 markers was observed in normal prostate tissues (Figure 9). 32 33 Figure 8: IHC analysis detected iNOS in human prostate tissue microarrays. A) Normal adjacent tissue. B) Stage II prostate carcinoma. C) Stage IV prostate carcinoma. Positive iNOS expression is indicated by brown staining. Hematoxylin (blue) was used as a counterstain. Scale bar=20μm. Arrows indicate positive iNOS staining. Figure 9: IF analysis of iNOS expression in prostate cancer tissue. IF analysis detected M1 and M2 macrophage expression in human prostate tissue microarrays. Confocal images of IF staining of iNOS (red) CD163 (green). Nuclei are revealed by DAPI staining (blue). 20X magnification. 34 Prostate cancer cells recruited human monocytes LNCaP, DU145 and PC3 cells, each exhibiting varying degrees of aggressiveness, along with normal prostate epithelial cells (RWPE-1) were utilized to model the in vivo interaction between prostate epithelial cells and macrophages at different stages of cancer. RWPE-1 cells were derived from the peripheral zone of a histologically normal adult human prostate, while human LNCaP cells originated from supraclavicular lymph node metastasis and are poorly aggressive. DU145 cells, obtained from brain metastasis of a male with grade II prostate cancer, exhibit moderate metastatic potential. The highly metastatic line, PC3, was isolated from metastases in the lumbar vertebra of a patient with grade IV prostatic adenocarcinoma and is useful in investigating the biochemical changes in advanced, malignant prostate cancer (Table 1). Table 1: Human Prostate Cancer Cell Lines Using this array of prostate epithelial cells, we assessed the ability of PCa cell lines of vary aggressiveness, to recruit macrophage precursors (monocytes), via a transwell migration assay. As detailed in the methodology section, conditioned media (CM) collected from each prostate cell line was used as chemo-attractants in the bottom 35 chamber, in which human monocytes (SC) seeded in the upper chamber of the transwell insert, were allowed to migrate towards. We observed that CM from LNCaP, DU145 and PC3 cells were able to efficiently recruit circulating monocytes, as demonstrated by their ability to migrate towards CM in the bottom chamber (Figure 10). Monocytes seeded in control, unconditioned media (IMDM or RPMI) demonstrated insignificant migration. Likewise, CM from RWPE-1 failed to induce significant chemotaxis of monocytes (Figure 10). Figure 10: PCa cells recruited human monocytes. Monocytes were serum starved and allowed to migrate for 24h toward CM from RWPE-1, LNCaP, DU145 and PC3 cells. Negative control conditions included: IMDM (monocyte culturing media), KSFM (RWPE1 culture media) and RPMI (LNCaP, DU145 and PC3 culturing media). Exposure to the chemokine SDF1α was established as a positive control. Graphical representation of results also included. 36 Classical stimuli-mediated monocyte differentiation and polarization For the classical induction of monocytes to a terminal macrophage differentiation state, SC cells were cultured in the presence of 97 nM (60ng/ml) phorbol myristate acetate (PMA) for 6 h, followed by treatment with interferon gamma (IFN-γ) and lipopolysaccharide (LPS) or IL-4, generating M1 and M2 macrophages, respectively. After differentiation/polarization, the media was removed and replaced with complete media for 24 h. As shown in Figure 11, monocytes cultured 72 hours with PMA and/or M1/M2 components caused the once non-adherent monocytes to adhere to the culture dish and displayed evident morphological changes (filopodial extensions), characteristic of mature macrophages (Figure 11). Subsequent to classical differentiation, SC cells were collected and analyzed via immunoblot analysis. As expected, SCs treated with IFNγ and LPS had higher expression of the M1 marker, COX2. Cells treated with IL-4 displayed a higher expression of COX1, a well-established marker of M2 differentiation. Results from this experiment confirmed our method of M1 and M2 differentiation was effective and therefore subsequent experiments conducted beyond this point involving differentiated macrophages were done so using these experimental parameters. Macrophage co-culture drives SC differentiation In order to analyze if exposure to tumor microenvironment features can affect monocyte differentiation, we incubated human peripheral blood monocytes with normal (RWPE1) and cancerous prostate epithelial cells (LNCaP, DU145, PC3) of varying aggressiveness. The results show that LNCaP, DU145 and PC3’s, but not RWPE1s, are able to induce evident morphological changes of monocytes that became larger, with 37 Figure 11: Classical monocyte to macrophage differentiation. A) Phase-contrast microscopy demonstrating morphological changes in monocytes in response to PMA (60ng/ml) and subsequent M1 or M2 polarization with LPS (0.1μg/ml) and IFNγ (20ng/ml) or IL-4 (20ng/ml), respectively. B) Protein expression of M1 markers (iNOS and COX-2) and M2 markers (COX-1 and TNFα) assessed via western blot analysis. ruffling membrane typical of macrophages (Figure 12A). In order to analyze the phenotype of the co-culture differentiated macrophages, we assessed the expression of inducible nitric oxide (iNOS) and COX-1. iNOS was highly expressed by macrophages cultured with the lowly aggressive cell line, LNCaP. In contrast, macrophages cocultured with the moderately aggressive, DU145 and highly metastatic PC3 cells displayed little to iNOS expression. Instead, COX-1, an M2 marker, was highly expressed in DU145 and PC3 co- cultured monocytes (Figure 12B). Normal, RWPE1 cells were unable to induce morphological or phenotypic changes in monocytes. 38 Figure 12: Prostate cancer cell/monocyte co-culture drives macrophage differentiation. A) Brightfield microscopy demonstrating morphological changes of monocytes in response to co-culture with normal prostate cells (RWPE-1) and prostate cancer cell lines of varying aggressiveness ( LNCaP, DU145, and PC3). B) Western blot analysis of co-cultured SC cells probing for M1 and M 2 markers iNOS (M1) and COX1 (M2). Exposure to M1s increases ROS in normal and cancerous prostate models To determine whether co-culture with inflammatory macrophages (M1) increases oxidative stress in normal (RWPE1) and malignant prostate epithelial cells (LNCaP, DU145 and PC3), we performed ROS microplate assays. In doing so, cells were stained with the cellular ROS indicator, c-H2DCF-DA, which upon oxidation by ROS forms the highly fluorescent compound, 2’, 7’ –dichlorofluorescein (DCF), which can be detected by fluorescence spectroscopy. Normal and cancerous cell lines were incubated with cH2DCF-DA in phenol-free RPMI or phenol-free RPMI 1640 alone (control) for 20 39 min at 37°C. Cells were then mono- or co-cultured with M1 macrophage CM for 1, 3, and 6 h. Fluorescence, in response to intracellular oxidation of c-H2DCF-DA, was measured with a microplate reader at 485nm (excitation) and 535nm (emission) wavelengths per manufacturer's instructions. As shown in Figure 13, compared to monocultured cells, M1 co-cultured cells, exhibited significantly greater fluorescence indicating higher oxidation levels. This trend was observed across all cell lines suggesting exposure to M1 macrophages alters the redox state of prostate cells (Figure 13). Figure 13: Exposure to inflammatory macrophages increases ROS in PCa cells. Prostate epithelial cells were either mono or co-cultured with M1 macrophages for the 3h. Cells were then labeled with 10µM cH2DCF-DA for 60 min at 37°C. Fluorescence was measured using a microplate reader (Excitation:488nm /Emission:520 nm). * p<0.05. PYO-Pyocyanin (ROS inducer); APO-Apocynin (NOX inhibitor). M1 macrophages promote prostate tumorigenesis in vitro To confirm that RWPE-1 cells were indeed undergoing a tumorigenic process after co-culture, RWPE-1 cells in M1 conditioned media, were plated on soft-agar to determine their anchorage independent growth. As expected, non-tumorigenic RWPE1cells were 40 able to form colonies on soft-agar after co-culture with M1 macrophage conditioned medium (Figure 14). Figure 14: M1 macrophages promote PCa cell anchorage-independent growth. Colony formation in soft agar assay of RWPE-1 cells (alone) and RWPE-1 cells co-cultured with THP-1cells. A bar graph, representative of six randomly chosen fields is shown. To evaluate the effects of exposure to inflammatory macrophages in prostate epithelial cells, we analyzed cell invasion. We found that post-M1 conditioned media exposure, all cell lines assay demonstrated an increased in invasiveness through the basement membrane-like material, matrigel. Interestingly, M1 CM exposure elicited a strong invasive spur in LNCaP cells; second only to PC3’s which are inherently highly invasive (Figure 15). 41 RWPE1 LNCaP DU145 PC3 Figure 15: M1 macrophages increase invasiveness in PCa cells. Monocytes were differentiated for 3 days and then serum starved for 48 h to obtain CM. PCa cells were incubated with collected CM for 24 h, (or serum free medium as a control), and then were allowed to invade toward medium containing SDF1α, serving as chemoattractant, for an additional 24 h. Invading cells were counted and a bar graph, representative of six randomly chosen fields, is shown. CHAPTER V DISCUSSION In recent years, evidence has mounted supporting the idea that inflammation within the normal adult prostate is not uncommon.26 In most cases, the cause of prostatic inflammation is unclear; however, various potential causative factors exist, including direct bacterial and viral infection, urine reflux, dietary factors, and hormonal changes.26 More recently, this area of research has gained more attention as researchers continue to investigate a potential link between chronic prostatitis and prostate cancer. For example, in 2010, an epidemiological study found an increased risk for prostate cancer in men with a history of prostatitis.45 Another study revealed that recurrent E. coli infections in the murine prostate induced the formation of PIN lesions, due to the persistent secretion of free radicals and inflammatory cytokines by infiltrating leukocytes.49 Interestingly, among the leukocytic infiltrates, macrophages are generally regarded as the key players supporting cancer progression within the tumor milieu.26 It is generally recognized within the scientific community that tumor-associated macrophages display an M2 phenotype, promoting pro-tumoral functions such as cell survival, proliferation, and angiogenesis. These functions are regulated through production of important mitogens, growth factors and enzymes.76 M1 macrophage 42 43 activation, on the other hand, is characterized by a high capacity to present antigen type I response, and increased production of toxic intermediates (nitric oxide (NO) and reactive oxygen species. Based on this, M1 macrophages are generally considered to be potent effector cells that kill micro-organisms and tumor cells, and produce copious amounts of pro-inflammatory cytokines.76 In general, high levels of TAMs are often, although not always, correlated with a bad prognosis, and recent studies have also highlighted a link between their abundance and the process of metastasis.77 Increasing studies have also shown that TAMs can either enhance or antagonize the antitumor efficacy of cytotoxic chemotherapy, cancer-cell targeting antibodies, and immunotherapeutic agents. Therefore, their role in mediating resistance to cancer therapies makes them an attractive target in drug development. A number of studies have demonstrated that through secretion of various chemo-protective factors such as MMP-9 and cathepsins, TAMs are able to reduce chemotherapy-induced antitumor responses, thereby promoting cancer.78 Overwhelmingly, the models used in such TAM-related studies are reported to possess characteristics of M2-like macrophages with protumor and immunosuppressive activity already prevalent in the tumor microenvironment.79 It should also be noted that the predominant detection of M2 macrophages reflects the late stage of tumor progression, while the presence, type and role of macrophages in lower staged neoplasias is only marginally studied. This observation raises the question of: (i) whether the preexisting proinflammatory environment in glandular tissues such as the prostate, before tumorigenesis, would favor M1 or M2 polarization of macrophages; and (ii) the role of such macrophages in initiating malignant transformation. 44 In the present study, we sought to explore this question and believe to have discovered a potentially novel function of M1 macrophages in promoting a pro-tumor microenvironment whereby oxidative stress and inflammation drive malignant transformation of the prostate. It is important to note, that our observations are not ignorant of the firm role of M2s in fueling cancer, but instead, aim to compliment the well-established complexity of macrophages and the tumor microenvironment as a whole. In contrast to the preeminent paradigm regarding macrophage infiltration in solid tumors, herein we demonstrate the involvement of M1 macrophages in tumor initiation, and to a lesser extent, tumor progression. In support of this notion, many studies have reported the role of inflammation and oxidative stress in promoting disease. Likewise, the ROS-generating activity of M1 macrophages is well-established. Therefore, it is conceivable that M1s may inadvertently drive cancer progression. While this concept is still only emerging, a few studies have made small inferences. Sebens et al. observed that exposure of normal colon epithelial cells to inflammatory macrophages induced activity of the anti-oxidative transcription factor, Nrf2, resulting in proteasome activation and protection from apoptosis. 13 Similarly, Comito et al. inadvertently demonstrated that M1 macrophages, although to a lesser extent than their M2-polarized counterparts, increased invasiveness of PCa cells and transformed normal human prostate fibroblasts to tumorassociated fibroblasts. 14 Fascinatingly, Fang et al. recently reported that persistent coculturing of immortalized normal prostate epithelial cells with macrophages, in the absence of carcinogens, induced prostate tumorigenesis.15 Our findings underscore the 45 potential role of M1 macrophages in prostate cancer with regard to their production of ROS. CHAPTER VI CONCLUSION TAMs are key orchestrators of the smoldering inflammation present in the tumor microenvironment. In the majority of experimental and clinical studies, TAMs have been associated with cancer progression. Their production of various growth factors for epithelial and endothelial cells, as well as inflammatory cytokines and chemokines contribute to tumor survival, proliferation and invasion. In addition, immunosuppressive mediators released by local inflammatory or tumor cells extinguish host-mediated antitumor responses and facilitate tumor progression. Thus TAMs appear as attractive candidate of novel therapeutic strategies. Three major aspects of TAM, potentially amenable of therapeutic interventions are: (i) inhibition of their recruitment and/or of their survival at the tumor site; (ii) inhibition of their positive effects on angiogenesis and tissue remodeling; (iii) reversal of their immune-suppression and restoration of antitumor cytotoxicity. 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