PU.1-driven Th9 Cells Promote Colorectal Cancer in Experimental Colitis Models Through Il-6 Effects in Intestinal Epithelial Cells.

BACKGROUND AND AIMS: Colorectal cancer [CRC] is one of the most frequent malignancies, but the molecular mechanisms driving cancer growth are incompletely understood. We characterised the roles of the cytokine IL-9 and Th9 cells in regulating CRC development. METHODS: CRC patient samples and samples from AOM/DSS treated mice were analysed for expression of IL-9, CD3, and PU.1 by FACS analysis and immunohistochemistry. IL-9 citrine reporter mice, IL-9 knockout mice, and PU.1 and GATA3 CD4-Cre conditional knockout mice were studied in the AOM/DSS model. DNA minicircles or hyper-IL-6 were used for overexpression of cytokines in vivo. Effects of IL-6 and IL-9 were determined in organoid and T cell cultures. Claudin2/3 expression was studied by western blotting and bacterial translocation by FISH. RESULTS: We uncovered a significant expansion of IL-9- and PU.1-expressing mucosal Th9 cells in CRC patients, with particularly high levels in patients with colitis-associated neoplasias. PU.1+ Th9 cells accumulated in experimental colorectal neoplasias. Deficiency of IL-9 or inactivation of PU.1 in T cells led to impaired tumour growth in vivo, suggesting a protumoral role of Th9 cells. In contrast, GATA3 inactivation did not affect Th9-mediated tumour growth. Mechanistically, IL-9 controls claudin2/3 expression and T cell-derived IL-6 production in colorectal tumours. IL-6 abrogated the anti-proliferative effects of IL-9 in epithelial organoids in vivo. IL-9-producing Th9 cells expand in CRC and control IL-6 production by T cells. CONCLUSIONS: IL-9 is a crucial regulator of tumour growth in colitis-associated neoplasias and emerges as potential target for therapy.

Non-Invasive Characterization of Experimental Bone Metastasis in Obesity Using Multiparametric MRI and PET/CT.

The growth of primary tumors and metastases is associated with excess body fat. In bone metastasis formation, the bone marrow microenvironment, and particularly adipocytes, play a pivotal role as growth mediators of disseminated tumor cells in the bone marrow. The aim of the present study is to non-invasively characterize the pathophysiologic processes in experimental bone metastasis resulting from accelerated tumor progression within adipocyte-rich bone marrow using multimodal imaging from magnetic resonance imaging (MRI) and positron emission tomography/computed tomography (PET/CT). To achieve this, we have employed small animal models after the administration of MDA-MB 231 breast cancer and B16F10 melanoma cells into the bone of nude rats or C57BL/6 mice, respectively. After tumor cell inoculation, ultra-high field MRI and µPET/CT were used to assess functional and metabolic parameters in the bone marrow of control animals (normal diet, ND), following a high-fat diet (HFD), and/or treated with the peroxisome proliferator-activated receptor-gamma (PPARγ) antagonist bisphenol-A-diglycidylether (BADGE), respectively. In the bone marrow of nude rats, dynamic contrast-enhanced MRI (DCE-MRI) and diffusion-weighted imaging (DWI), as well as [18F]fluorodeoxyglucose-PET/CT([18F]FDG-PET/CT), was performed 10, 20, and 30 days after tumor cell inoculation, followed by immunohistochemistry. DCE-MRI parameters associated with blood volume, such as area under the curve (AUC), were significantly increased in bone metastases in the HFD group 30 days after tumor cell inoculation as compared to controls (p < 0.05), while the DWI parameter apparent diffusion coefficient (ADC) was not significantly different between the groups. [18F]FDG-PET/CT showed an enhanced glucose metabolism due to increased standardized uptake value (SUV) at day 30 after tumor cell inoculation in animals that received HFD (p < 0.05). BADGE treatment resulted in the inversion of quantitative DCE-MRI and [18F]FDG-PET/CT data, namely a significant decrease in AUC and SUV in HFD-fed animals as compared to ND-fed controls (p < 0.05). Finally, immunohistochemistry and qPCR confirmed the HFD-induced stimulation in vascularization and glucose activity in murine bone metastases. In conclusion, multimodal and multiparametric MRI and [18F]FDG-PET/CT were able to derive quantitative parameters in bone metastases, revealing an increase in vascularization and glucose metabolism following HFD. Thus, non-invasive imaging may serve as a biomarker for assessing the pathophysiology of bone metastasis in obesity, opening novel options for therapy and treatment monitoring by MRI and [18F]FDG-PET/CT.

Impact of Endothelial Progenitor Cells in the Vascularization of Osteogenic Scaffolds.

The microvascular endothelial network plays an important role in osteogenesis, bone regeneration and bone tissue engineering. Endothelial progenitor cells (EPCs) display a high angiogenic and vasculogenic potential. The endothelialization of scaffolds with endothelial progenitor cells supports vascularization and tissue formation. In addition, EPCs enhance the osteogenic differentiation and bone formation of mesenchymal stem cells (MSCs). This study aimed to investigate the impact of EPCs on vascularization and bone formation of a hydroxyapatite (HA) and beta-tricalcium phosphate (ß-TCP)-fibrin scaffold. Three groups were designed: a scaffold-only group (A), a scaffold and EPC group (B), and a scaffold and EPC/MSC group (C). The HA/ß-TCP-fibrin scaffolds were placed in a porous titanium chamber permitting extrinsic vascularization from the surrounding tissue. Additionally, intrinsic vascularization was achieved by means of an arteriovenous loop (AV loop). After 12 weeks, the specimens were explanted and investigated by histology and CT. We were able to prove a strong scaffold vascularization in all groups. No differences regarding the vessel number and density were detected between the groups. Moreover, we were able to prove bone formation in the coimplantation group. Taken together, the AV loop is a powerful tool for vascularization which is independent from scaffold cellularization with endothelial progenitor cells' prior implantation.

Systemic PPARγ Antagonism Reduces Metastatic Tumor Progression in Adipocyte-Rich Bone in Excess Weight Male Rodents.

Primary tumors are widely associated with an excess in body fat. The role of adipose tissue on tumor cell homing to bone is yet poorly defined. In this study, we aimed to assess whether bone colonization by tumor cells is favored by an adipocyte-rich bone marrow. We delineated the accompanying alterations of the bone microenvironment and established a treatment approach that interferes with high fat diet (HFD)-induced bone metastasis formation. We were able to show that adipocytes affect skeletal tumor growth in a metastatic model of breast cancer in male rats and melanoma in male mice as well as in human breast cancer bone biopsies. Indeed, HFD-induced bone marrow adiposity was accompanied by accelerated tumor progression and increased osteolytic lesions. In human bone metastases, bone marrow adiposity correlated with tumor cell proliferation. By antagonization of the adipocyte differentiation and storage pathway linked to the peroxisome proliferator-activated receptor gamma (PPARγ) with bisphenol-A-diglycidylether (BADGE), we were able to decelerate tumor progression and subsequent osteolytic damage in the bones of two distinct metastatic animal models exposed to HFD. Overall these data show that adipose tissue is a critical factor in bone metastases and cancer-induced bone loss. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Morphological, functional, and molecular assessment of breast cancer bone metastases by experimental ultrasound techniques compared with magnetic resonance imaging and histological analysis.

BACKGROUND: The imaging of bone metastases, which is regularly performed by cross-sectional modalities, is clinically vital when characterizing and staging osseous lesions. In this paper, we aimed to establish a novel methodology using experimental ultrasound (US) techniques to assess the morphological, functional, and molecular features of breast cancer bone metastases in an animal model, compared with magnetic resonance imaging (MRI) and histological analysis. MATERIALS AND METHODS: Nude rats were implanted intra-arterially with MDA-MB-231 breast cancer cells to induce osteolytic metastasis in their right hind legs. Once tumors had developed, an experimental US technique using automatic 3D scanning and MRI were performed. For assessment of perfusion, functional imaging techniques included contrast-enhanced US (CEUS) and dynamic contrast-enhanced MRI (DCE-MRI). For molecular ultrasound, anti-VEGFR2 conjugated microbubbles were applied and correlated with immunostaining for VEGFR2 expression. RESULTS: 3D US enabled the automatic assessment of osteolytic lesions, including the largest tumor diameters along the x-, y- and z-axes as well as the segmented tumor volumes, without significant differences between US and MRI (p > 0.18). The CEUS and DCE-MRI of osseous lesions showed corresponding results for the parameters peak enhancement, wash-in area under the curve (both, r > 0.5) and wash-in perfusion index (r > 0.3) when differentiating between tumor, necrotic tissue and healthy muscle tissue (all, p < 0.01). Finally, molecular US allowed the non-invasive assessment of increased VEGFR2 expression in skeletal lesions compared with surrounding muscle tissue (p = 0.03), while a control antibody could not discriminate between these tissues (p = 0.44)-a factor which was confirmed by histological analysis. CONCLUSION: To the best of our knowledge, this is the first report on an imaging protocol for breast cancer bone metastasis using an experimental US scanner. Therefore, we present a novel methodology to characterize these osseous lesions on the morphological, functional, and molecular level in correlation with MRI and histological analysis.

Siglec-15 on Osteoclasts Is Crucial for Bone Erosion in Serum-Transfer Arthritis.

Siglec-15 is a conserved sialic acid-binding Ig-like lectin, which is expressed on osteoclasts. Deficiency of Siglec-15 leads to an impaired osteoclast development, resulting in a mild osteopetrotic phenotype. The role of Siglec-15 in arthritis is still largely unclear. To address this, we generated Siglec-15 knockout mice and analyzed them in a mouse arthritis model. We could show that Siglec-15 is directly involved in pathologic bone erosion in the K/BxN serum-transfer arthritis model. Histological analyses of joint destruction provided evidence for a significant reduction in bone erosion area and osteoclast numbers in Siglec-15-/- mice, whereas the inflammation area and cartilage destruction was comparable to wild-type mice. Thus, Siglec-15 on osteoclasts has a crucial function for bone erosion during arthritis. In addition, we generated a new monoclonal anti-Siglec-15 Ab to clarify its expression pattern on immune cells. Whereas this Ab demonstrated an almost exclusive Siglec-15 expression on murine osteoclasts and hardly any other expression on various other immune cell types, human Siglec-15 was more broadly expressed on human myeloid cells, including human osteoclasts. Taken together, our findings show a role of Siglec-15 as a regulator of pathologic bone resorption in arthritis and highlight its potential as a target for future therapies, as Siglec-15 blocking Abs are available.

Machine Learning Algorithms for Early Detection of Bone Metastases in an Experimental Rat Model.

Machine learning (ML) algorithms permit the integration of different features into a model to perform classification or regression tasks with an accuracy exceeding its constituents. This protocol describes the development of an ML algorithm to predict the growth of breast cancer bone macrometastases in a rat model before any abnormalities are observable with standard imaging methods. Such an algorithm can facilitate the detection of early metastatic disease (i.e., micrometastasis) that is regularly missed during staging examinations. The applied metastasis model is site-specific, meaning that the rats develop metastases exclusively in their right hind leg. The model's tumor-take rate is 60%-80%, with macrometastases becoming visible in magnetic resonance imaging (MRI) and positron emission tomography/computed tomography (PET/CT) in a subset of animals 30 days after induction, whereas a second subset of animals exhibit no tumor growth. Starting from image examinations acquired at an earlier time point, this protocol describes the extraction of features that indicate tissue vascularization detected by MRI, glucose metabolism by PET/CT, and the subsequent determination of the most relevant features for the prediction of macrometastatic disease. These features are then fed into a model-averaged neural network (avNNet) to classify the animals into one of two groups: one that will develop metastases and the other that will not develop any tumors. The protocol also describes the calculation of standard diagnostic parameters, such as overall accuracy, sensitivity, specificity, negative/positive predictive values, likelihood ratios, and the development of a receiver operating characteristic. An advantage of the proposed protocol is its flexibility, as it can be easily adapted to train a plethora of different ML algorithms with adjustable combinations of an unlimited number of features. Moreover, it can be used to analyze different problems in oncology, infection, and inflammation.

The JAK1/3 inhibitor tofacitinib suppresses T cell homing and activation in chronic intestinal inflammation.

BACKGROUND & AIMS: The molecular mechanism of action of the Janus kinase (JAK) inhibitor tofacitinib is poorly understood. METHODS: Here, we analysed the inhibitory effect of tofacitinib on mucosal and blood T cells from patients with ulcerative colitis (UC). Furthermore tofacitinib treatment was analysed in experimental colitis models and wound healing. Additionally, tofacitinib effects were analysed in bioassays. RESULTS: Tofacitinib significantly reduced T cell derived inflammatory cytokine production (Th2, Th9, Th17) in patients with active UC. Additionally, impaired expression of the homing receptors alpha4/beta1 and alpha4/beta7 as well as reduced gut homing capacity of T cells in a humanized mouse model of colitis were observed. Tofacitinib suppressed acute and chronic oxazolone colitis compared to untreated wild-type mice associated with downregulation of cytokines produced by Th2, Th9 and Th17 cells. Functionally, tofacitinib induced apoptosis of intestinal epithelial cells and prevented mucosal wound healing in vivo at higher concentration. Thus, our findings suggest that tofacitinib is quite effective in protecting from colitis by inhibition of a bundle of T cell derived cytokines like IL-5, IL-6, IL-9, IL-13 and IL-17A. CONCLUSION: Application of tofacitinib emerges as an attractive concept for treatment of chronic intestinal inflammation at lower concentrations, whereas higher concentrations require attention due to prolonged wound healing.

Alternative Splice Forms of CYLD Mediate Ubiquitination of SMAD7 to Prevent TGFB Signaling and Promote Colitis.

BACKGROUND & AIMS: The CYLD lysine 63 deubiquitinase gene (CYLD) encodes tumor suppressor protein that is mutated in familial cylindromatosus, and variants have been associated with Crohn disease (CD). Splice forms of CYLD that lack exons 7 and 8 regulate transcription factors and functions of immune cells. We examined the expression of splice forms of CYLD in colon tissues from patients with CD and their effects in mice. METHODS: We performed immunohistochemical analyses of colon tissues from patients with untreated CD and patients without inflammatory bowel diseases (controls). We obtained mice that expressed splice forms of CYLD (sCYLD mice) without or with SMAD7 (sCYLD/SMAD7 mice) from transgenes and CYLD-knockout mice (with or without transgenic expression of SMAD7) and performed endoscopic analyses. Colitis was induced in Rag1-/- mice by transfer of CD4+ CD62L+ T cells from C57/Bl6 or transgenic mice. T cells were isolated from mice and analyzed by flow cytometry and quantitative real-time polymerase chain reaction and intestinal tissues were analyzed by histology and immunohistochemistry. CYLD forms were expressed in mouse embryonic fibroblasts, primary T cells, and HEK293T cells, which were analyzed by immunoblot, mobility shift, and immunoprecipitation assays. RESULTS: The colonic lamina propria from patients with CD was infiltrated by T cells and had higher levels of sCYLD (but not full-length CYLD) and SMAD7 than tissues from controls. Incubation of mouse embryonic fibroblasts and T cells with transforming growth factor ß increased their production of sCYLD and decreased full-length CYLD. Transgenic expression of sCYLD and SMAD7 in T cells prevented the differentiation of regulatory T cells and T-helper type 17 cells and increased the differentiation of T-helper type 1 cells. The same effects were observed in colon tissues from sCYLD/SMAD7 mice but not in those from CYLD-knockout SMAD7 mice. The sCYLD mice had significant increases in the numbers of T-helper type 1 cells and CD44high CD62Llow memory-effector CD4+ T cells in the spleen and mesenteric lymph nodes compared with wild-type mice; sCYLD/SMAD7 mice had even larger increases. The sCYLD/SMAD7 mice spontaneously developed severe colitis, with infiltration of the colon by dendritic cells, neutrophils, macrophages, and CD4+ T cells and increased levels of Ifng, Il6, Il12a, Il23a, and Tnf mRNAs. Co-transfer of regulatory T cells from wild-type, but not from sCYLD/SMAD7, mice prevented the induction of colitis in Rag1-/- mice by CD4+ T cells. We found increased levels of poly-ubiquitinated SMAD7 in sCYLD CD4+ T cells. CYLD formed a nuclear complex with SMAD3, whereas sCYLD recruited SMAD7 to the nucleus, which inhibited the expression of genes regulated by SMAD3 and SMAD4. We found that sCYLD mediated lysine 63-linked ubiquitination of SMAD7. The sCYLD-SMAD7 complex inhibited transforming growth factor ß signaling in CD4+ T cells. CONCLUSIONS: Levels of the spliced form of CYLD are increased in colon tissues from patients with CD. sCYLD mediates ubiquitination and nuclear translocation of SMAD7 and thereby decreases transforming growth factor ß signaling in T cells. This prevents immune regulatory mechanisms and leads to colitis in mice.

Tumor immunoevasion via acidosis-dependent induction of regulatory tumor-associated macrophages.

Many tumors evolve sophisticated strategies to evade the immune system, and these represent major obstacles for efficient antitumor immune responses. Here we explored a molecular mechanism of metabolic communication deployed by highly glycolytic tumors for immunoevasion. In contrast to colon adenocarcinomas, melanomas showed comparatively high glycolytic activity, which resulted in high acidification of the tumor microenvironment. This tumor acidosis induced Gprotein-coupled receptor-dependent expression of the transcriptional repressor ICER in tumor-associated macrophages that led to their functional polarization toward a non-inflammatory phenotype and promoted tumor growth. Collectively, our findings identify a molecular mechanism of metabolic communication between non-lymphoid tissue and the immune system that was exploited by high-glycolytic-rate tumors for evasion of the immune system.

A Stat6/Pten Axis Links Regulatory T Cells with Adipose Tissue Function.

Obesity and type 2 diabetes are associated with metabolic defects and adipose tissue inflammation. Foxp3+ regulatory T cells (Tregs) control tissue homeostasis by counteracting local inflammation. However, if and how T cells interlink environmental influences with adipocyte function remains unknown. Here, we report that enhancing sympathetic tone by cold exposure, beta3-adrenergic receptor (ADRB3) stimulation or a short-term high-calorie diet enhances Treg induction in vitro and in vivo. CD4+ T cell proteomes revealed higher expression of Foxp3 regulatory networks in response to cold or ADRB3 stimulation in vivo reflecting Treg induction. Specifically, Ragulator-interacting protein C17orf59, which limits mTORC1 activity, was upregulated in CD4+ T cells by either ADRB3 stimulation or cold exposure, suggesting contribution to Treg induction. By loss- and gain-of-function studies, including Treg depletion and transfers in vivo, we demonstrated that a T cell-specific Stat6/Pten axis links cold exposure or ADRB3 stimulation with Foxp3+ Treg induction and adipose tissue function. Our findings offer a new mechanistic model in which tissue-specific Tregs maintain adipose tissue function.

Chemically induced mouse models of acute and chronic intestinal inflammation.

Inflammatory bowel diseases (IBDs) result in diarrhea and abdominal pain with further potential complications such as tissue fibrosis and stenosis. Animal models help in understanding the immunopathogenesis of IBDs and in the design of novel therapeutic concepts. Here we present an updated version of a protocol we published in 2007 for key models of acute and chronic forms of colitis induced by 2,4,6-trinitro-benzene sulfonic acid (TNBS), oxazolone and dextran sulfate sodium (DSS). This protocol update describes an adaptation of the existing protocol that modifies the technique. This protocol has been used to generate improved mouse models that better reflect the nature of IBDs in humans. In TNBS and oxazolone colitis models, topical administration of hapten reagents results in T-cell-mediated immunity against haptenized proteins and luminal antigens. By contrast, to generate DSS colitis models, mice orally receive DSS, causing death of epithelial cells, compromising barrier function and causing subsequent inflammation. The analysis of the acute colitis models can be performed within 1-2 weeks, whereas that of the chronic models may take 2-4 months. The strengths of the acute models are that they are based on the analysis of short-lasting barrier alterations, innate immune effects and flares. The advantages of the chronic models are that they may offer better insight into adaptive immunity and complications such as neoplasia and tissue fibrosis. The protocol requires basic skills in laboratory animal research.

Rectal Delivery of a DNAzyme That Specifically Blocks the Transcription Factor GATA3 and Reduces Colitis in Mice.

BACKGROUND & AIMS: GATA3 is a transcription factor that regulates T-cell production of cytokines. We investigated the role of GATA3 in development of colitis in mice. METHODS: We performed quantitative polymerase chain reaction and immunofluorescence analyses of colon tissues from patients with Crohn's disease (n = 61) or ulcerative colitis (UC, n = 74) or from patients without inflammatory bowel diseases (n = 22), to measure levels of GATA3. Colitis was induced by administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid to control mice, mice with T-cell-specific deletion of GATA3, and mice with deletion of tumor necrosis factor receptor (TNFR) 1 and TNFR2 (TNFR double knockouts); some mice were given a GATA3-specific DNAzyme (hgd40) or a control DNAzyme via intrarectal administration, or systemic injections of an antibody to TNF before or during sensitization and challenge phase of colitis induction. Colon tissues were collected and immunofluorescence and histochemical analyses were performed. Lamina propria mononuclear cells and T cells were isolated and analyzed by flow cytometry or cytokine assays. Colonic distribution of labeled DNAzyme and inflammation were monitored by in vivo imaging (endoscopy) of mice. RESULTS: Levels of GATA3 messenger RNA were higher in colon tissues from patients with UC, but not ileal Crohn's disease, than control tissues; levels of GATA3 correlated with levels of inflammatory cytokines (interleukin [IL] 9, IL17A, IL6, IL5, IL4, IL13, and TNF). We observed increased expression of GATA3 by lamina propria T cells from mice with colitis compared with controls. Mice with T-cell-specific deletion of GATA3 did not develop colitis and their colonic tissues did not produce inflammatory cytokines (IL6, IL9, or IL13). The DNAzyme hgd40 inhibited expression of GATA3 messenger RNA by unstimulated and stimulated T cells, and distributed throughout the inflamed colons of mice with colitis. Colon tissues from mice given hgd40 had reduced expression of GATA3 messenger RNA, compared with mice given a control DNAzyme. Mice given hgd40 did not develop colitis after administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid; lamina propria cells from these mice expressed lower levels of IL6, IL9, and IL13 than cells from mice given the control DNAzyme. Mini-endoscopic images revealed that hgd40 and anti-TNF reduced colon inflammation over 3 days; hgd40 reduced colitis in TNFR double-knockout mice. CONCLUSIONS: Levels of GATA3 are increased in patients with UC and correlate with production of inflammatory cytokines in mice and humans. A DNAzyme that prevents expression of GATA3 reduces colitis in mice, independently of TNF, and reduces levels of cytokines in the colon. This DNAzyme might be developed for treatment of patients with UC.