Thomas Tapmeier

Recruitment of a myeloid cell subset (CD11b/Gr1mid) via CCL2/CCR2 promotes the development of colorectal cancer liver metastasis

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. (HEPATOLOGY 2013)

Pivotal role of CD4+ T cells in renal fibrosis following ureteric obstruction

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.

Homeostatic Proliferation of Lymphocytes Results in Augmented Memory-Like Function and Accelerated Allograft Rejection

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 differentially induce nucleosome remodelling at the IL‐12p40 promoter

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 TNFα. 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.

Reimplantation of the ureter after unilateral ureteral obstruction provides a model that allows functional evaluation

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.

G-CSF rescues tumor growth and neo-angiogenesis during liver metastasis under host angiopoietin-2 deficiency

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.

The pH low insertion peptide pHLIP Variant 3 as a novel marker of acidic malignant lesions

Significance Acidic pH may distinguish aggressive from more indolent cancers. The limitation on testing this hypothesis to date has been the difficulty of measuring acidic pH in cancers. Here we show that retention of the pH low insertion peptide (pHLIP) Variant 3 (Var3) reflects acidic pH. Using pHLIP Var3, we show its ability to detect cancer with a low false-positive rate in a genetically engineered model of murine breast cancer, paving the way for testing this probe in clinical situations. 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 31P 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.

Recruitment of myeloid cells to the tumor microenvironment supports liver metastasis.

Tumor-infiltrating immune cells play important roles in metastasis. We have recently revealed the recruitment of a specific myeloid cell subset (CD11b/Gr1mid) 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.

The miRNA Mirage: How Close Are We to Finding a Non-Invasive Diagnostic Biomarker in Endometriosis? A Systematic Review

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.

Abstract 402: Plasticity of tumor associated macrophages in a metastatic melanoma model in the mouse

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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