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Uterine Fibroids, or leiomyomata, affect millions of women world-wide, with a high incidence of 75% within women of reproductive age. In ~30% of patients, uterine fibroids cause menorrhagia, or heavy menstrual bleeding, and more than half of the patients experience symptoms such as heavy menstrual bleeding, pelvic pain, or infertility. Treatment is symptomatic with limited options including hysterectomy as the most radical solution. The genetic foundations of uterine fibroid growth have been traced to somatic driver mutations (MED12, HMGA2, FH−/−, and COL4A5-A6). These also lead to downstream expression of angiogenic factors including IGF-1 and IGF-2, as opposed to the VEGF-driven mechanism found in the angiogenesis of hypoxic tumors. The resulting vasculature supplying the fibroid with nutrients and oxygen is highly irregular. Of particular interest is the formation of a pseudocapsule around intramural fibroids, a unique structure within tumor angiogenesis. These aberrations in vascular architecture and network could explain the heavy menstrual bleeding observed. However, other theories have been proposed such as venous trunks, or venous lakes caused by the blocking of normal blood flow by uterine fibroids, or the increased local action of vasoactive growth factors. Here, we review and discuss the evidence for the various hypotheses proposed.

Uterine Fibroids (leiomyomata) and endometriosis affect millions of women world-wide. Although aetiology and natural history of the conditions are markedly different, symptoms can overlap and make differential diagnoses necessary, often using invasive methods such as laparoscopy. Considerable comorbidity exists between the two conditions and needs to be taken into account when treating fibroids and/or endometriosis. The genetic foundations of both uterine fibroids and endometriosis remain to be fully understood but recent evidence suggest common underpinnings. Here, we discuss the comorbidity of uterine fibroids and endometriosis and the implications for diagnosis, treatment and risks.

Endometriosis is a common chronic inflammatory condition causing pelvic pain and infertility in women, with limited treatment options and 50% heritability. We leveraged genetic analyses in two species with spontaneous endometriosis, humans and the rhesus macaque, to uncover treatment targets. We sequenced DNA from 32 human families contributing to a genetic linkage signal on chromosome 7p13-15 and observed significant overrepresentation of predicted deleterious low-frequency coding variants in NPSR1, the gene encoding neuropeptide S receptor 1, in cases (predominantly stage III/IV) versus controls (P = 7.8 × 10-4). Significant linkage to the region orthologous to human 7p13-15 was replicated in a pedigree of 849 rhesus macaques (P = 0.0095). Targeted association analyses in 3194 surgically confirmed, unrelated cases and 7060 controls revealed that a common insertion/deletion variant, rs142885915, was significantly associated with stage III/IV endometriosis (P = 5.2 × 10-5; odds ratio, 1.23; 95% CI, 1.09 to 1.39). Immunohistochemistry, qRT-PCR, and flow cytometry experiments demonstrated that NPSR1 was expressed in glandular epithelium from eutopic and ectopic endometrium, and on monocytes in peritoneal fluid. The NPSR1 inhibitor SHA 68R blocked NPSR1-mediated signaling, proinflammatory TNF-α release, and monocyte chemotaxis in vitro (P < 0.01), and led to a significant reduction of inflammatory cell infiltrate and abdominal pain (P < 0.05) in a mouse model of peritoneal inflammation as well as in a mouse model of endometriosis. We conclude that the NPSR1/NPS system is a genetically validated, nonhormonal target for the treatment of endometriosis with likely increased relevance to stage III/IV disease.

IntroductionMillions of women suffer from the consequences of endometriosis and uterine fibroids, with fibroids the cause for over 50% of hysterectomies in the USA, and direct costs for their treatment estimated at between US$4 and US$9 billion. Endometriosis commonly affects millions of women worldwide predominantly during reproductive age, with severe menstrual and non-menstrual pain and subfertility the main symptoms. Due to the ‘unhappy triad’ of endometriosis—lack of awareness, lack of clinically relevant biomarkers and the unspecific nature of symptoms—women wait on average for 8–12 years before the definitive endometriosis diagnosis is made. Treatment options for both conditions are not satisfactory at the moment, especially with a view to preserving fertility for the women and families affected. In the Fibroids and Endometriosis Oxford (FENOX) study, we combine the investigation of fibroids and endometriosis, and plan to collect high-quality tissue samples and medical data of participants over a time frame of 5 years after surgical intervention.Methods and analysisBiological samples such as blood, saliva, urine, fat, peritoneal fluid and—if found—endometrial tissue or fibroids as well as detailed clinical and intraoperative data will be collected from women undergoing surgery and participating in the study after informed consent. We plan to recruit up to 1200 participants per disease arm (ie, endometriosis and uterine fibroids) over 5 years. Participants will fill in detailed and validated questionnaires on their medical history and quality of life, with follow-ups for 5 years. Enrolment started on 2 April 2018, and FENOX will close on 31 March 2028. We will analyse the biological samples using state-of-the-art molecular biology methods and correlate the findings with the medical records and questionnaire data.Ethics and disseminationThe findings will be published in high-ranking journals in the field and presented at national and international conferences.Trial registration numberNCT13560263.

Endometriosis is a common gynaecological disease of women in reproductive age, and is thought to arise from retrograde menstruation and implantation of endometrial tissue, mostly into the peritoneal cavity. The condition is characterized by a chronic, unresolved inflammatory process thereby contributing to pain as cardinal symptom in endometriosis. Elevated reactive oxygen species (ROS) and oxidative stress have been postulated as factors in endometriosis pathogenesis. We here set out for a systematic study to identify novel mechanisms and pathways relating to oxidative stress in ectopic peritoneal lesions. Using combined proteomic and transcriptomic approaches, we identified novel targets including upregulated pro-oxidative enzymes, such as amine oxidase 3/vascular adhesion protein 1 (AOC3/VAP1) as well as downregulated protective factors, in particular alkenal reductase PTGR1 and methionine sulfoxide reductase. Consistent with an altered ROS landscape, we observed hemoglobin / iron overload, ROS production and lipid peroxidation in ectopic lesions. ROS-derived 4-hydroxy-2-nonenal induced interleukin IL-8 release from monocytes. Notably, AOC3 inhibitors provoked analgesic effects in inflammatory pain models in vivo, suggesting potential translational applicability.

Background Endometriosis is a gynaecological condition characterised by immune cell infiltration and distinct inflammatory signatures found in the peritoneal cavity. In this study, we aim to characterise the immune microenvironment in samples isolated from the peritoneal cavity in patients with endometriosis. Methods We applied mass cytometry (CyTOF), a recently developed multiparameter single-cell technique, in order to characterise and quantify the immune cells found in peritoneal fluid and peripheral blood from endometriosis and control patients. Results Our results demonstrate the presence of more than 40 different distinct immune cell types within the peritoneal cavity. This suggests that there is a complex and highly heterogeneous inflammatory microenvironment underpinning the pathology of endometriosis. Stratification by clinical disease stages reveals a dynamic spectrum of cell signatures suggesting that adaptations in the inflammatory system occur due to the severity of the disease. Notably, among the inflammatory microenvironment in peritoneal fluid (PF), the presence of CD69+ T cell subsets is increased in endometriosis when compared to control patient samples. On these CD69+ cells, the expression of markers associated with T cell function are reduced in PF samples compared to blood. Comparisons between CD69+ and CD69− populations reveal distinct phenotypes across peritoneal T cell lineages. Taken together, our results suggest that both the innate and the adaptive immune system play roles in endometriosis. Conclusions This study provides a systematic characterisation of the specific immune environment in the peritoneal cavity and identifies cell immune signatures associated with endometriosis. Overall, our results provide novel insights into the specific cell phenotypes governing inflammation in patients with endometriosis. This prospective study offers a useful resource for understanding disease pathology and opportunities for identifying therapeutic targets.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Objective To demonstrate the feasibility of studying exosomes directly from peritoneal fluid, we isolated exosomes from endometriosis patient samples and from controls, and characterized their cargo. Design Case-control experimental study. Setting Academic clinical center. Patient (s) Women with and without endometriosis who underwent laparoscopic surgery (n = 28 in total). Intervention (s) None. Main Outcome Measure (s) Concentration of exosomes within peritoneal fluid and protein content of the isolated exosomes. Result (s) Peritoneal fluid samples were pooled according to the cycle phase and disease stage to form six experimental groups, from which the exosomes were isolated. Exosomes were successfully isolated from peritoneal fluid in all the study groups. The concentration varied with cycle phase and disease stage. Proteomic analysis showed specific proteins in the exosomes derived from endometriosis patients that were absent in the controls. Five proteins were found exclusively in the endometriosis groups: PRDX1, H2A type 2-C, ANXA2, ITIH4, and the tubulin α-chain. Conclusion (s) Exosomes are present in peritoneal fluid. The characterization of endometriosis-specific exosomes opens up new avenues for the diagnosis and investigation of endometriosis.

Background: Endometriosis is a common disorder of the reproductive age group, characterised by the presence of ectopic endometrial tissue. The disease not only causes enormous suffering to the affected women, but also brings a tremendous medical and economic burden to bear on society. There is a long lag phase between the onset and diagnosis of the disease, mainly due to its non-specific symptoms and the lack of a non-invasive test. Endometriosis can only be diagnosed invasively by laparoscopy. A specific, non-invasive test to diagnose endometriosis is an unmet clinical need. The recent discovery of microRNAs (miRNAs) as modulators of gene expression, and their stability and specificity, make them an attractive candidate biomarker. Various studies on miRNAs in endometriosis have identified their cardinal role in the pathogenesis of the disease, and have proposed them as potential biomarkers in endometriosis. Rationale/Objectives: The aims of this review were to study the role of circulatory miRNAs in endometriosis, and bring to light whether circulatory miRNAs could be potential non-invasive biomarkers to diagnose the disease. Search methods: Three databases, PubMed, EMBASE, and BIOSIS were searched, using a combination of Mesh or Emtree headings and free-text terms, to identify literature relating to circulating miRNAs in endometriosis published from 1996 to 31 December 2017. Only peer-reviewed, full-text original research articles in English were included in the current review. The studies meeting the inclusion criteria were critically assessed and checked using the QUADAS-2 (Quality Assessment of Diagnostic Accuracy Studies) tool. The dysregulated miRNAs were assessed regarding the concordance between the various studies and their role in the disease. Outcomes: Nine studies were critically analysed, and 42 different miRNAs were found to be dysregulated in them, with only one common miRNA (miR-20a) differentially expressed in more than one study. miR-17-5p/20a, miR-200, miR-199a, miR-143, and miR-145 were explored for their pivotal role in the aetiopathogenesis of endometriosis. Wider implications: It is emerging that miRNAs play a central role in the pathogenesis of endometriosis and have the potential of being promising biomarkers. Circulating miRNAs as a non-invasive diagnostic tool may shorten the delay in the diagnosis of the disease, thus alleviating the suffering of women and reducing the burden on health care systems. However, despite numerous studies on circulating miRNAs in endometriosis, no single miRNA or any panel of them seems to meet the criteria of a diagnostic biomarker. The disagreement between the various studies upholds the demand of larger, well-controlled systematic validation studies with uniformity in the research approaches and involving diverse populations.

Genome-wide association studies in the fields of reproductive medicine and endocrinology are yielding robust genetic variants associated with disease. Integrated genomic, transcriptomic, and epigenomic molecular profiling studies are common methodologies used to understand the biologic pathways perturbed by these variants. However, molecular profiling resources do not include the tissue most relevant to many female reproductive traits, the endometrium, while the parameters influencing variability of results from its molecular profiling are unclear. We investigated the sources of DNA methylation and RNA expression profile variability in endometrium (n = 135), endometriotic disease tissue (endometriosis), and subcutaneous abdominal fat samples from 24 women, quantifying between-individual, within-tissue (cellular heterogeneity), and technical variation. DNA samples (n = 96) were analyzed using Illumina HumanMethlylation450 BeadChip arrays; RNA samples (n = 39) were analyzed using H12-expression arrays. Variance-component analyses showed that, for the top 10–50% variable DNA methylation/RNA expression sites, between-individual variation far exceeded within-tissue and technical variation. Menstrual-phase accounted for most variability in methylation/expression patterns in endometrium (Pm = 7.8 × 10−3, Pe = 8.4 × 10−5) but not in fat and endometriotic tissue; age was significantly associated with DNA methylation profile of endometrium (Pm = 9 × 10−5) and endometriotic disease tissue (Pm = 2.4 × 10−5); and smoking was significantly associated with DNA methylation in adipose tissue (Pm = 1.8 × 10−3). Hierarchical cluster analysis showed significantly different methylation signatures between endometrium and endometriotic tissue enriched for WNT signaling, angiogenesis, cadherin signaling, and gonadotropin-releasing-hormone-receptor pathways. Differential DNA methylation/expression analyses suggested detection of a limited number of sites with large fold changes (FC > 4), but power calculations accounting for different sources of variability showed that for robust detection >500 tissue samples are required. These results enable appropriate study design for large-scale expression and methylation tissue-based profiling relevant to many reproductive and endocrine traits.

Adenomyosis (AM) remains one of the unsolved conundrums of gynecology. After decades of debate and numerous studies it is now well accepted that adenomyosis does not represent a sub-entity of endometriosis, although some symptoms appear to overlap. One of the few established facts about AM is the descriptive diagnosis of endometrial glandular epithelial and stromal cells surrounded by hypertrophy and hyperplasia in the myometrial compartment of the uterus (1). Unfortunately, even the most fundamental clinical details including reliable data about the true prevalence, possible familial accumulations or risk factors are sparse and are either based on retrospective studies using hysterectomy specimen or, more recently, on ultrasound or magnetic resonance imaging (MRI) reports without internationally agreed diagnostic standards (2).

Experimental unilateral ureteral obstruction (UUO) is widely used to study renal fibrosis; however, renal injury can only be scored semiobjectively by histology. We sought to improve the UUO model by reimplanting the obstructed ureter followed by removal of the contralateral kidney, thus allowing longitudinal measurements of renal function. Mice underwent UUO for different lengths of time before ureteral reimplantation and contralateral nephrectomy. Measurement of blood urea nitrogen (BUN) allows objective evaluation of residual renal function. Seven weeks after reimplantation and contralateral nephrectomy, mean BUN levels were increased with longer duration of UUO. Interstitial expansion, fibrosis, and T-cell and macrophage infiltration were similar in kidneys harvested after 10 days of UUO or following 10 weeks of ureter reimplantation, suggesting that the inflammatory process persisted despite relief of obstruction. Urinary protein excretion after reimplantation was significantly increased compared to control animals. Our study shows that functional assessment of the formerly obstructed kidney can be made after reimplantation and may provide a useful model to test therapeutic strategies for reversing renal fibrosis and preserving or restoring renal function.

Current strategies for early detection of breast and other cancers are limited in part because some lesions identified as potentially malignant do not develop into aggressive tumors. Acid pH has been suggested as a key characteristic of aggressive tumors that might distinguish aggressive lesions from more indolent pathology. We therefore investigated the novel class of molecules, pH low insertion peptides (pHLIPs), as markers of low pH in tumor allografts and of malignant lesions in a mouse model of spontaneous breast cancer, BALB/neu-T. pHLIP Variant 3 (Var3) conjugated with fluorescent Alexa546 was shown to insert into tumor spheroids in a sequence-specific manner. Its signal reflected pH in murine tumors. It was induced by carbonic anhydrase IX (CAIX) overexpression and inhibited by acetazolamide (AZA) administration. By using (31)P magnetic resonance spectroscopy (MRS), we demonstrated that pHLIP Var3 was retained in tumors of pH equal to or less than 6.7 but not in tissues of higher pH. In BALB/neu-T mice at different stages of the disease, the fluorescent signal from pHLIP Var3 marked cancerous lesions with a very low false-positive rate. However, only ∼60% of the smallest lesions retained a pHLIP Var3 signal, suggesting heterogeneity in pH. Taken together, these results show that pHLIP can identify regions of lower pH, allowing for its development as a theranostic tool for clinical applications.

Optical microscopy imaging techniques have enabled a wide spectrum of biomedical applications. Among visualization, a quantitative analysis of tumour cell growth in lungs is of great importance. The main challenges inherently linked with such data analysis are: local contrast changes related to tissue depth, lack of clear object boundaries due to the presence of noise, and cluttering with motion artefacts due to translational shift of the specimen and non-linear lung tissue collapse. This paper aims to address these problems by introducing a novel image registration framework specifically designed to correct for motion artefacts from optical microscopy of lung tumour cells imaging. For this purpose, a previously developed modality independent neighbourhood descriptor (MIND) was adapted to cope with multiple image channels for optical microscopy data. Two versions of this novel multichannel MIND (mMIND) are here presented. The proposed registration technique estimates both rigid transformations and non-linear deformations both common in the optical microscopy volumes and time-sequences acquisition. The performance of our registration technique based on a novel multichannel image representation is demonstrated using two distinctive optical imaging data sets of lung cells: 3D volumes with translation motion artefacts only, and time-sequences with both rigid and non-linear motion artefacts. Visual inspection of the registration outcomes and reported results of the qualitative evaluation show a promising improvement compared to images without correction.

Liver metastasis from colorectal cancer is a leading cause of cancer mortality. Myeloid cells play pivotal roles in the metastatic process, but their prometastatic functions in liver metastasis remain incompletely understood. To investigate their role, we simulated liver metastasis in C57BL/6 mice through intrasplenic inoculation of MC38 colon carcinoma cells. Among the heterogeneous myeloid infiltrate, we identified a distinct population of CD11b/Gr1mid cells different from other myeloid populations previously associated with liver metastasis. These cells increased in number dramatically during establishment of liver metastases and were recruited from bone marrow by tumor-derived CCL2. Liver metastasis of Lewis lung carcinoma cells followed this pattern but this mechanism is not universal as liver colonization by B16F1 melanoma cells did not recruit similar subsets. Inhibition of CCL2 signaling and absence of its cognate receptor CCR2 reduced CD11b/Gr1mid recruitment and decreased tumor burden. Depletion of the CD11b/Gr1mid subset in a transgenic CD11b-diphtheria toxin receptor mouse model markedly reduced tumor cell proliferation. There was no evidence for involvement of an adaptive immune response in the prometastatic effects of CD11b/Gr1mid cells. Additionally, an analogous myeloid subset was found in liver metastases of some colorectal cancer patients. Conclusion: Collectively, our findings highlight the importance of myeloid cells-in this case a selective CD11b/Gr1mid subset-in sustaining development of colorectal cancer liver metastasis and identify a potential target for antimetastatic therapy. © 2012 American Association for the Study of Liver Diseases.

Suppression of neo-angiogenesis is a clinically used anti-tumor strategy with new targets such as angiopoietin-2 (Ang2) being proposed. However, the functions of Ang2 in vascular remodeling, inflammation and tumor growth are not consistent. We examined effect of depletion of host Ang2 on liver colony formation using Ang2 deficient (Ang2-/-) mice. Surprisingly, the metastatic colonies formed in Ang2-/- mice were larger than those in the wild type. These colonies had greater vascular density with more pericyte coverage than the vessels in liver colonies in the wild type. Liver VEGF concentration in both genotypes was equivalent, and thus, the differences appeared VEGF independent. However, after colony formation, the serum concentration of granulocyte-colony stimulating factor (G-CSF) and CXCL1 in Ang2-/- mice was 12 and 6 times greater than after colony formation in wild type. Increase of these two cytokines was associated with two times greater numbers of neutrophils recruited to the liver. Two times more Tie2+/CD11b+/CD31- cells were present in the tumors in Ang2-/- than in the wild type livers. These results suggest that the depletion of host Ang2 induced compensatory VEGF-independent angiogenic mechanisms and thus enhanced liver metastatic colony growth and colony vascularity. They further indicate organotypic differences in response to tumor metastasis. In contrast, Ang2 deficiency inhibited tumor growth during metastatic colony formation in the lung, consistent with the reports of decreased pulmonary seeding of tumor cells after pharmacological inhibition of Ang2. Further studies are thus required to assess the effects of pharmacological Ang2 blockade for cancer patients particularly in the liver. What's new? Suppression of neo-angiogenesis is a clinically used anti-tumor strategy, with new targets such as angiopoietin-2 (Ang2) being proposed. However, the functions of Ang2 in vascular remodeling, inflammation, and tumor growth are not consistent. This work shows that genetic depletion of host Ang2 unexpectedly enhanced liver metastatic colony vascularity and growth. The more aggressive neo-angiogenesis and tumor growth were associated with VEGF-independent angiogenic mechanisms, including G-CSF production and neutrophil infiltration. Further studies are thus required to assess the effects of pharmacological Ang2 blockade for cancer patients, particularly in the liver. Copyright © 2012 UICC.

Tumor-infiltrating immune cells play important roles in metastasis. We have recently revealed the recruitment of a specific myeloid cell subset (CD11b/Gr1(mid)) to hepatic metastases. Such a recruitment relies on CCL2/CCR2 signaling and acts to sustain metastatic growth. A similar cell subset was identified in patients bearing hepatic metastases of colorectal cancer, highlighting the potential therapeutic relevance of our findings.

Homeostatic proliferation is a normal physiological process triggered by lymphopenia to maintain a constant level of T cells. It becomes the predominant source of new T cells in adulthood after thymus regression. T cells that have undergone homeostatic proliferation acquire the memory phenotype, cause autoimmune disease, and are resistant to tolerance induction protocols. Transplantation is a rare example in which lymphopenia is deliberately induced for its immunosuppressive effect. However, it is not known whether the homeostatic proliferation that follows will have the opposite effect and accelerate rejection. We show that T cells that have undergone homeostatic proliferation acquire a memory phenotype, spontaneously skews toward the Th1 phenotype, even in the absence of antigenic stimulus. Interestingly, in contrast, the percentage of Foxp3+ regulatory T cells increased by 28-fold following homeostatic proliferation. Using a mouse life-sustaining kidney transplant model, we showed that T cells that have gone through homeostatic proliferation in lymphopenic hosts transformed chronic rejection to acute rejection of a single MHC class II-mismatched kidney allograft. T cells that have undergone homeostatic proliferation consistently cause reliable rejection even when bona fide memory T cells cannot. These functional changes are long-lasting and not restricted to the acute phase of homeostatic proliferation. Our findings have important implications for tolerance induction or graft-prolonging protocols involving leukocyte depletion such as irradiation bone marrow chimera, T cell-depleting Abs, and lymphopenia induced by infections such as CMV and HIV.

Toll-like receptors (TLRs) mediate recognition of microbial components. Despite activation of a shared set of signal transduction molecules, the biological effects of certain TLR agonists differ considerably. In macrophages and dendritic cells, stimulation by the prototypical stimuli CpG-DNA (TLR9), lipopolysaccharide (LPS; TLR4) and lipoteichoic acid (LTA; TLR2) resulted in striking differences in expression of IL-12. However, these stimuli induced similar amounts of the common proinflammatory cytokine TNFalpha. Surprisingly, an IL-12p40 promoter reporter construct was activated equally by CpG-DNA, LPS and LTA. Examinations of the chromatin structure of the endogenous IL-12p40 promoter revealed that nucleosome remodelling contributed to differential IL-12 induction. Upon stimulation, nucleosome architecture was changed to provide increased access to the IL-12p40 promoter. In dendritic cells, a differential induction of nucleosome remodelling at the IL-12p40 promoter was observed upon triggering with different TLR agonists. These results identify nucleosome remodelling as an additional restriction point in differential TLR signalling.

Metastatic colorectal cancer (CRC) is one of the most prominent causes of cancer-related mortality. However, therapeutic options for patients with metastatic CRC are limited and development of new and more effective therapeutics aimed at downstaging the disease and prolonging survival is urgently required. Primary tumor dissemination to secondary sites, predominantly the liver, is a dynamic process dependent on the interactions between tumor cells and the host tissue microenvironment. Immune cells in the tumor microenvironment have been shown to play pivotal roles in the metastatic process but their pro-metastatic functions in CRC liver metastasis remains incompletely understood. To simulate liver metastasis, MC38 colon carcinoma cells were injected into the spleen of C57BL/6 mice. A population of CD11b/Gr1mid cells, distinct from other metastasis-associated myeloid cells previously described, increased dramatically during establishment of liver metastases. Adoptive transfer of bone marrow cells confirmed that these CD11b/Gr1mid cells were derived from bone marrow progenitor cells. MC38 tumor cells expressed high levels of CCL2 and serum levels were elevated in tumor-bearing mice compared to controls. Inhibition of tumor-derived CCL2 using shRNA, and absence of its cognate receptor in CCR2 KO mice significantly diminished CD11b/Gr1mid cell infiltration into tumor-bearing livers. Importantly, inhibition of CD11b/Gr1mid cell recruitment led to a marked reduction in tumor burden. Moreover, when CD11b/Gr1mid cells were depleted in a transgenic CD11b-DTR mouse model, a striking decrease in tumor vessel density was observed, with a concomitant reduction in tumor cell proliferation. Serum CCL2 concentrations were similarly elevated in CRC patients, including those who present metastasis, compared to normal controls. Furthermore, CD11b/CCR2 myeloid cells, analogous to the CD11b/Gr1mid population described in our mouse model, were identified in liver metastases of several CRC patients. Together, our studies demonstrate the importance of a distinct CD11b/Gr1mid myeloid subset in sustaining CRC liver metastasis and implicate a CCL2/CCR2-dependent pathway in their recruitment to metastatic sites. Our findings reinforce how tumor cells utilize host systems for successful propagation and identify a potential target for therapeutic manipulation. Citation Format: Su Yin Lim, Lei Zhao, Alex Gordon-Weeks, Thomas Tapmeier, Ruth Muschel. Tumor-derived CCL2 promotes recruitment of CD11b/Gr1mid myeloid cells to sustain colorectal cancer liver metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2871. doi:10.1158/1538-7445.AM2013-2871

In a pulmonary metastasis model using B16F10 melanoma, we found monocyte/macrophage infiltration gradually increasing in the course of metastatic progression. The gene expression pattern of the infiltrating macrophages revealed an early (d3) inflammatory response that shifted to a tumor-promoting late response (d21). The pro-tumoral expression pattern was characterized by Arg1 and elevated expression of Ccl2, Vcam1, Vegfa, Stab1, Lyve1 and Ptgs2. In contrast, while enhancing their anti-inflammatory phenotype, resident lung (alveolar) macrophages also expressed pro-inflammatory genes, notably Il12b. We found at least three chemokine receptors involved in the recruitment of macrophages into the metastatic lung: CCR1, CCR2 and CCR5. The B16F10 cells secreted large quantities of RANTES which in turn activates CCR1 and CCR5, and thus contribute to macrophage chemotaxis. Infiltrating monocytes/macrophages, as well as alveolar macrophages, revealed an increased chemokine expression level in response to tumor challenge. These chemokines included the ligands for CCR1 and CCR5, Mip1α and Mip1β, thus providing a constant means of further macrophage recruitment. Interestingly, the expression of the CCR2 ligand, CCL2, was found strikingly increased by infiltrating monocytes/ macrophages, thus ensuring a positive feed-back loop for macrophage recruitment. In order to explore the involvement of CC chemokine receptors in pulmonary metastasis, we treated the mice with antagonists against CCR1, CCR5 and CCR2 after i.v. B16F10 challenge. The CCR1 and CCR5 antagonist treatments resulted in a 20% decrease in the number of lung colonies each. FACS analysis of the B16F10 colony bearing lungs revealed a distribution of macrophage subsets quite different from untreated lungs, highlighting the importance of CC-chemokine receptors in the recruitment of pro-tumoral macrophages. The effect of a CCR2 antagonist on lung colony formation will be discussed, together with its effect on the phenotype of macrophage subpopulations. Citation Format: Thomas T. Tapmeier, Annamaria Gal, Ruth J. Muschel. The response of infiltrating and resident macrophages to melanoma challenge in the mouse lung: Involvement of the CC-chemokine axis in the recruitment of pro-tumoral macrophages. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-324. doi:10.1158/1538-7445.AM2013-LB-324

Macrophages acquire different functional traits, with the pro-inflammatory, classically activated M1 and the tissue-repair, alternatively activated M2 phenotypes the best characterized to date. Tumor-associated macrophages (TAMs) have been described as alternatively activated and exert pro-tumoral activity. We investigated TAMs isolated from subcutaneous tumors and experimental lung metastases at an early (72 h) and late (3 wk) stage within a B16F10 mouse melanoma model. The macrophages were characterized according to their expression of genes, surface markers and secreted factors. Interestingly, the mRNA profile revealed both M1- and M2-associated gene expression in macrophages derived from s.c. melanoma, including Cxcl9, Cxcl11, Il-1a, Nos2, and Retnla (FIZZ-1), Arg-1 and Fn1, respectively. In early pulmonary metastases, we found that macrophages also expressed both pro-inflammatory (Il1b, Il12b, Ccl2), and anti-inflammatory (Ccl17 and Ccl22) cytokines and chemokines and other M1- and M2-associated genes, such as Cd40, Cd86, and Il27ra and Itgax (Cdd11c), respectively. Macrophages from late stage lung metastases displayed predominantly alternatively activated characteristics with Arg1, Retnla and Fn1 expression, but pro-inflammatory genes, such as Ccl2, Il1b and Cxcl10 were also induced/upregulated. We therefore suggest an intermediate phenotype of TAMs which does not fit the orthodox M1/M2 paradigm. To describe the mechanism by which macrophages are recruited to pulmonary metastases, we investigated chemokine receptor expression of macrophages infiltrating the lung. While Ccr2 was expressed at high level in macrophages associated with both the control and B16F10 metastases bearing lung, we found that Ccr1 and Ccr5 expression was induced after tumor cell challenge. The secretion profile of B16F10 cells revealed CCL5 (RANTES), a ligand for these receptors, secreted at high level, which suggests that CCL5 attracts macrophages to lung metastases via CCR1 and CCR5. The effect of anti-CCL5 antibody treatment, and s.c melanoma growth and pulmonary metastasis development in CCR1-/- and CCR5-/- mice will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 402. doi:1538-7445.AM2012-402

For growth and survival in metastasis, tumor colonies must develop new intratumoral vasculature for supply of nutrients and oxygen. Since angiopoietin-2 (Ang2) resulted in a burst of angiogenesis in the glioma model, we examined the effects of host Ang2 on colony growth and neo-angiogenesis during the liver colonization. Liver colonization was induced through the intrasplenic injection of MC38-GFP (murin colorectal carcinoma cells) into two genotype mice of the ang2−/− mice and the wild type mice. When colonies were established, the livers were isolated and analyzed using flow cytometry, immunoflorescent histochemistry and ELISA. In addition, the blood was collected from vena cava for measurement of MCP-1 at 14 days. Flow cytometry analyses of whole livers showed that the tumor cell population in the ang2−/− mice was approximately 2 times larger than in the wild type mice. Immunoflorescent histochemistry of the liver tissue using antibodies for CD31, CD34, CD146, VE-cadherin and NG2 showed that intratumoral vascular density and vascular maturity in the ang2−/− mice were approximately 2 times higher than the wild type mice. Intratumoral vessels were significantly positive for CD146 and CD34, markers more frequently associated with neo-angiogenesis, but much less for CD31. There was no significant difference in VEGF level which is known to mediate vascular response in combination with Ang2. CD11b positive cells and MCP-1 concentration in the livers were equivalent in two genotypes. Notably, there were approximately 10 times more Tie2+/CD11b+ cells in the ang2−/− mice than the wild type mice. CD11c+/CD45+ cell populations were slightly different in two genotypes. These results suggested that host Ang2 would inhibit tumor growth and neo-angiogenesis in the liver, and that the inhibition is correlated with the decreased recruitment of TEMs, one of myeloid cells, into the liver. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2847. doi:10.1158/1538-7445.AM2011-2847

Tubulointerstitial fibrosis is a common consequence of a diverse range of kidney diseases that lead to end-stage renal failure. The degree of fibrosis is related to leukocyte infiltration. Here, we determined the role of different T cell populations on renal fibrosis in the well-characterized mouse model of unilateral ureteric obstruction. Depletion of CD4 + T cells in wild-type mice with a monoclonal antibody significantly reduced the amount of interstitial expansion and collagen deposition after 2 weeks of obstruction. Reconstitution of lymphopenic RAG knockout mice with purified CD4 + but not CD8 + T cells, prior to ureteric obstruction, resulted in a significant increase in interstitial expansion and collagen deposition. Wild-type mice had significantly greater interstitial expansion and collagen deposition compared with lymphopenic RAG −/− mice, following ureteric obstruction; however, macrophage infiltration was equivalent in all groups. Thus, our results suggest that renal injury with subsequent fibrosis is likely to be a multifactorial process, with different arms of the immune system involved at different stages. In this ureteric obstruction model, we found a critical role for CD4 + T cells in kidney fibrosis. These cells could be a potential target of therapeutic intervention to prevent excessive fibrosis and loss of function due to renal injury.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.