Search

BackgroundThe inflammatory tumor microenvironment (TME) is formed by various immune cells, being closely associated with tumorigenesis. Especially, the interaction between tumor-infiltrating T-cells and macrophages has a crucial impact on tumor progression and metastatic spread. The purpose of this study was to investigate whether oscillating-gradient diffusion-weighted MRI (OGSE-DWI) enables a cell size-based discrimination between different cell populations of the TME.MethodsSine-shaped OGSE-DWI was combined with the Imaging Microstructural Parameters Using Limited Spectrally Edited Diffusion (IMPULSED) approach to measure microscale diffusion distances, here relating to cell sizes. The accuracy of IMPULSED-derived cell radii was evaluated using in vitro spheroid models, consisting of either pure cancer cells, macrophages, or T-cells. Subsequently, in vivo experiments aimed to assess changes within the TME and its specific immune cell composition in syngeneic murine breast cancer models with divergent degrees of malignancy (4T1, 67NR) during tumor progression, clodronate liposome-mediated depletion of macrophages, and immune checkpoint inhibitor (ICI) treatment. Ex vivo analysis of IMPULSED-derived cell radii was conducted by immunohistochemical wheat germ agglutinin staining of cell membranes, while intratumoral immune cell composition was analyzed by CD3 and F4/80 co-staining.ResultsOGSE-DWI detected mean cell radii of 8.8±1.3 µm for 4T1, 8.2±1.4 µm for 67NR, 13.0±1.7 for macrophage, and 3.8±1.8 µm for T-cell spheroids. While T-cell infiltration during progression of 4T1 tumors was observed by decreasing mean cell radii from 9.7±1.0 to 5.0±1.5 µm, increasing amount of intratumoral macrophages during progression of 67NR tumors resulted in increasing mean cell radii from 8.9±1.2 to 12.5±1.1 µm. After macrophage depletion, mean cell radii decreased from 6.3±1.7 to 4.4±0.5 µm. T-cell infiltration after ICI treatment was captured by decreasing mean cell radii in both tumor models, with more pronounced effects in the 67NR tumor model.ConclusionsOGSE-DWI provides a versatile tool for non-invasive profiling of the inflammatory TME by assessing the dominating cell type T-cells or macrophages.

The endothelin (ET) axis plays a pivotal role in cardiovascular diseases. Enhanced levels of circulating ET-1 have been correlated with an inferior clinical outcome after myocardial infarction (MI) in humans. Thus, the evaluation of endothelin-A receptor (ETAR) expression over time in the course of myocardial injury and healing may offer valuable information toward the understanding of the ET axis involvement in MI. We developed an approach to track the expression of ETAR with a customized molecular imaging probe in a murine model of MI. The small molecular probe based on the ETAR-selective antagonist 3-(1,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyl)methyl]-2(5H)-furanone (PD156707) was labeled with fluorescent dye, IRDye800cw. Mice undergoing permanent ligation of the left anterior descending artery (LAD) were investigated at day 1, 7, and 21 post surgery after receiving an intravenous injection of the ETAR probe. Cryosections of explanted hearts were analyzed by cryotome-based CCD, and fluorescence reflectance imaging (FRI) and fluorescence signal intensities (SI) were extracted. Fluorescence-mediated tomography (FMT) imaging was performed to visualize probe distribution in the target region in vivo. An enhanced fluorescence signal intensity in the infarct area was detected in cryoCCD images as early as day 1 after surgery and intensified up to 21 days post MI. FRI was capable of detecting significantly enhanced SI in infarcted regions of hearts 7 days after surgery. In vivo imaging by FMT localized enhanced SI in the apex region of infarcted mouse hearts. We verified the localization of the probe and ETAR within the infarct area by immunohistochemistry (IHC). In addition, neovascularized areas were found in the affected myocardium by CD31 staining. Our study demonstrates that the applied fluorescent probe is capable of delineating ETAR expression over time in affected murine myocardium after MI in vivo and ex vivo.

Tyrosine kinase inhibitors have significantly improved treatment of patients with different malignancies, but their utilization can be compromised by unintended toxicities. Ibrutinib is a first-in-class covalent inhibitor of the Bruton´s tyrosine kinase inhibitor that has been approved for the treatment of patients with chronic lymphocytic leukemia, mantle cell lymphoma and Waldenstrom's macroglobulinemia and is connected with toxicities, caused by high dosage. To minimize toxicities of ibrutinib we linked ibrutinib to a cell-targeted, internalizing antibody. To this end, we synthesized a poly-anionic derivate of ibrutinib, ibrutinib-Cy3.5 that retains full functionality. This anionic inhibitor is complexed by our anti-CD20-protamine targeting conjugate and free protamine and thereby spontaneously assembles into an electrostatically stabilized vesicular nanocarrier. The complexation led to an accumulation of the drug driven by the CD20 antigen internalization to the intended cells and an amplification of its physiological effectivity. In vivo , we observed a significant enrichment of the drug in xenograft lymphoma tumors in immune-compromised mice and a significantly better response to significantly lower doses compared to the original untargeted drug . This combination of a cell-determining antibody, electrostatically connected to a molecular targeted inhibitor defines a first-in-class antibody-inhibitor conjugate.

Tumor development and metastasis are dependent on tumor infiltrating immune cells which form a characteristic tumor microenvironment (TME). Activated monocytes secrete the protein heterodimer S100A8/A9 promoting TME formation. Monocyte-dependent proteases facilitate local tumor cell invasion by degradation of the extracellular matrix. We aimed for target specific in vivo imaging of S100A8 and proteases to provide differentiating biomarkers for local tumor growth and metastatic potential. Murine breast cancer cells of the 4T1 model with graduated metastatic potential (4T1 and 4T07: both hematogenous metastasis > 168FAR: lymph-node metastasis > 67NR: no metastasis) were orthotopically implanted into female BALB/c mice. At 4 mm size, tumors were investigated by injecting the protease-specific probe ProSense 750EX (PerkinElmer, 4T1 n = 7, 4T07 n = 10, 168FAR n = 16, 67NR n = 15) and anti-S100A8-Cy5.5 (n = 6 each) and performing fluorescence reflectance imaging at 0 and 24 h after injection. In vivo imaging was validated with immunohistochemistry. At 24 h, S100A8-specific signals in 4T1 and 4T07 were significantly higher (1714.05/1683.45 AU) as compared to 168FAR and 67NR (174.85/167.95 AU, p = 0.0012/p = 0.0003), reflecting the capability of hematogenous spread. Protease-specific signals were significantly higher in 4T1 and 4T07 (348.01/409.93 AU) as compared to 168FAR (214.91 AU) and 67NR (129.78 AU p

Autoimmune diseases, such as psoriasis and arthritis, show a patchy distribution of inflammation despite systemic dysregulation of adaptive immunity. Thus, additional tissue-derived signals, such as danger-associated molecular patterns (DAMPs), are indispensable for manifestation of local inflammation. S100A8/S100A9 complexes are the most abundant DAMPs in many autoimmune diseases. However, regulatory mechanisms locally restricting DAMP activities are barely understood. We now unravel for the first time, to our knowledge, a mechanism of autoinhibition in mice and humans restricting S100-DAMP activity to local sites of inflammation. Combining protease degradation, pull-down assays, mass spectrometry, and targeted mutations, we identified specific peptide sequences within the second calcium-binding EF-hands triggering TLR4/MD2-dependent inflammation. These binding sites are free when S100A8/S100A9 heterodimers are released at sites of inflammation. Subsequently, S100A8/S100A9 activities are locally restricted by calcium-induced (S100A8/S100A9)2 tetramer formation hiding the TLR4/MD2-binding site within the tetramer interphase, thus preventing undesirable systemic effects. Loss of this autoinhibitory mechanism in vivo results in TNF-α-driven fatal inflammation, as shown by lack of tetramer formation in crossing S100A9-/- mice with 2 independent TNF-α-transgene mouse strains. Since S100A8/S100A9 is the most abundant DAMP in many inflammatory diseases, specifically blocking the TLR4-binding site of active S100 dimers may represent a promising approach for local suppression of inflammatory diseases, avoiding systemic side effects.

Background To facilitate onsite decision-making during endoscopy, both accurate detection and in vivo characterization of preneoplasia are prerequisites. However, no endoscopy technique is available that meets both demands satisfactorily. We evaluated endothelin-receptor A (ETAR)-guided fluorescence endoscopy (FE) in vivo and fluorescence reflectance imaging (FRI) ex vivo for detection and characterization of early dysplastic colitis-associated colonic lesions. Methods Colorectal cancerogenesis was investigated in the inflammatory driven AOM-DSS model and spontaneous adenoma development in ApcMin mice. A Cy5.5-labeled nonpeptidic ETAR-specific imaging probe was injected intravenously to assess tumor development in vivo by white light endoscopy (WLE) and FE. Ex vivo tumors were evaluated by FRI, histological examination, and western blot analysis. In addition, tissue samples from patients with colitis-associated malignant and nonmalignant mucosal alterations were analyzed. Specificity experiments were performed using an unspecific Cy3.5-glycine tracer. Results Overall, 62 adenomas were observed. FE was able to detect and quantify ETAR expression targeting the ETAR-specific photoprobe. A significantly higher fluorescent contrast was detected in colonic adenomas compared to adjacent nonmalignant mucosa by FE (64.3 ± 7.9 vs. 56.6. ± 7.0; P < 0.001). These results were confirmed by FRI examination, immunochemistry, and western blot analysis. Additionally, ETAR expression in samples from human patients with colitis-associated cancer was highly elevated compared to nonmalignant alterations. Specificity experiments indicated a high binding-specificity of the applied ETAR photoprobe (1.4 ± 0.3 vs. 2.5 ± 0.7; P < 0.001). Conclusions We introduced ETAR guided FE in mice for successful in vivo detection and characterization of colorectal neoplasia on a molecular level.

Background Rheumatoid arthritis synovial fibroblasts (RASFs) are known to travel via the bloodstream from sites of cartilage destruction to new locations where they reinitiate the destructive processes at distant articular cartilage surfaces. In this study, we examined the role of interleukin (IL)-1-induced cartilage changes and their chemotactic effect on RASF transmigratory capacity. Methods To investigate synovial fibroblast (SF) transmigration through endothelial layers, we used a modified Boyden chamber with an endothelioma cell layer (bEnd.5) as a barrier and IL-1-treated murine cartilage explants as a chemotactic stimulus for SFs from human tumor necrosis factor–transgenic (hTNFtg) mice. We injected recombinant IL-1 or collagenase into knee joints of wild-type mice, followed by tail vein injection of fluorescence-labeled hTNFtg SFs. The distribution and intensity of transmigrating hTNFtg SFs were measured by fluorescence reflectance imaging with X-ray coregistration. Toluidine blue staining was performed to evaluate the amount of cartilage destruction. Results Histomorphometric analyses and in vivo imaging revealed a high degree of cartilage proteoglycan loss after intra-articular IL-1 and collagenase injection, accompanied by an enhanced in vivo extravasation of hTNFtg SFs into the respective knee joints, suggesting that structural cartilage damage contributes significantly to the attraction of hTNFtg SFs into these joints. In vitro results showed that degraded cartilage was directly responsible for the enhanced transmigratory capacity because stimulation with IL-1-treated cartilage, but not with IL-1 or cartilage alone, was required to increase hTNFtg SF migration. Conclusions The present data indicate that structural cartilage damage facilitates the migration of arthritic SF into affected joints. The prevention of early inflammatory cartilage damage may therefore help prevent the progression of rheumatoid arthritis and its spread to previously unaffected joints.

The urothelial carcinoma is the most frequent malignancy of the urinary bladder (UBC). The transition into invasive growth is accompanied by several histological changes including an oncofoetal reorganization of the extracellular matrix. Recently, the occurrence of oncofoetal fibronectin with an O-linked glycosylation in the IIICS region (oncf Fn) was shown to be present in urine from UBC patients and was recommended as a tumour marker. Until now there are no data available regarding the source and distribution of oncf Fn in UBC and its value for the assessment of invasiveness.oncf Fn was analysed in noninvasive and invasive UBC using immunohistochemistry and western blot. Additionally, the mRNA expression of the IIICS splicing region was evaluated by quantitative real time RT-PCR.Immunohistochemical results reveal a highly significant correlation of oncf Fn to invasiveness. Papillary tumours regularly show no positivity. In western blot, invasive UBC show a strongly increased amount of the 250 kDa oncf Fn. Additionally, several smaller bands could be shown suggesting a proteolytic processing of Fn. The mRNA of the IIICS region shows a 21.5-fold increase in invasive UBC compared with noninvasive carcinomas.In summary, immunohistochemistry of oncf Fn is a valuable histological marker for invasiveness of urothelial carcinoma of the urinary bladder. The restricted and invasion-associated tissue distribution of immunoreactivity enables to monitor the recurrence of invasive UBC by a quantitative evaluation of IIICS O-linked glycosylated Fn in urine.

Tumors recruit and reprogram immune cells to support tumor development and spread, the most prominent among them being of monocytic origin such as tumor-associated macrophages (TAM) or myeloid-derived suppressor cells (MDSC). The alarmin S100A8/A9 has been implicated in the induction of TAM and MDSC. We assessed S100A9 as a molecular imaging marker for the activity of tumor-associated immune cells in a syngeneic murine breast cancer model. S100A9 could serve as a surrogate marker for tumor immune crosstalk as a function of malignancy, providing a tool with the potential for both basic research in tumor immunology and clinical stratification of patients.BALB/c mice were inoculated with murine breast cancer cells of common origin but different metastatic capability. At different times during tumor development, optical imaging was performed using a S100A9-specific probe to visualize activated monocytes. To further explore the impact of tumor-educated monocytes, splenic myeloid cells were isolated from either healthy or tumor-bearing animals and injected into tumor-bearing mice. We analyzed the effect of the cell transfer on immune cell activity and tumor development.We could prove S100A9-driven imaging to sensitively and specifically reflect monocyte activity in primary tumor lesions. The imaging results were corroborated by histology and fluorescence-activated cell sorting analyses. In a prospective experiment, S100A9 imaging proved indicative of the individual tumor growth, with excellent correlation. Moreover, we could show that the monocyte activity as depicted by S100A9 activity in the primary tumor lesion mirrored the tumor's metastatic behavior. Treatment with tumor-primed splenic monocytes induced increased tumor growth, accompanied by an augmented infiltration of activated myeloid cells (MDSC and TAM) into the tumor. The consecutive S100A9 expression as depicted by in vivo imaging was significantly increased.S100A9 proved to be a sensitive and specific marker for the activity of tumor-associated immune cells. To our knowledge, S100A9 imaging represents a first in vivo imaging approach for the estimation of recruitment and activity of tumor-associated myeloid immune cells. We demonstrated the potential value of this imaging approach for prediction of local and systemic tumor development.

Inflammation has a key role in the pathogenesis of various human diseases. The early detection, localization and monitoring of inflammation are crucial for tailoring individual therapies. However, reliable biomarkers to detect local inflammatory activities and to predict disease outcome are still missing. Alarmins, which are locally released during cellular stress, are early amplifiers of inflammation. Here, using optical molecular imaging, we demonstrate that the alarmin S100A8/S100A9 serves as a sensitive local and systemic marker for the detection of even sub-clinical disease activity in inflammatory and immunological processes like irritative and allergic contact dermatitis. In a model of collagen-induced arthritis, we use S100A8/S100A9 imaging to predict the development of disease activity. Furthermore, S100A8/S100A9 can act as a very early and sensitive biomarker in experimental leishmaniasis for phagocyte activation linked to an effective Th1-response. In conclusion, the alarmin S100A8/S100A9 is a valuable and sensitive molecular target for novel imaging approaches to monitor clinically relevant inflammatory disorders on a molecular level., Alarmins are locally released during inflammation and are early amplifiers of inflammation. Here Vogl et al. show that the alarmin S100A8/S100A9 can be used as a sensitive marker to detect subclinical inflammation and follow disease progression in a variety of disease models.

Biomedical imaging has offered a dramatic leap forward in cancer research by enabling non-invasive assessment of tumor biology in its native environment. Multiple modalities are currently utilized for this purpose, including molecular (e.g. PET, SPECT, optical imaging), functional (e.g. Doppler ultrasound) or anatomical (e.g. ultrasound, MRI, CT) endpoints. However, integrated multi-modality imaging can be impractical due to cost or infrastructure requirements, while co-registration of sequential imaging sessions can pose technical challenges. A single modality that offers anatomical, functional and molecular contrast could therefore provide the next leap forward in cancer research. Multispectral Optoacoustic Imaging (MSOT) is an emerging modality that combines ultrasound resolution of 150 μm and acquisition times of a few microseconds with optical contrast in the near infrared (NIR) spectral region. Multispectral imaging allows the localization of injected targeted fluorophores with NIR absorbance to offer molecular contrast, while visualization of endogenous contrast such as deoxy-/oxy-hemoglobin or melanin offers both functional and anatomical information. A core imaging rate of 10 images/second allows the acquisition of a multispectral image within a second or even less. In the present work, MSOT was used to track the fate of injected monocytes, labeled with an NIR chromophore, in a model of dss-induced colitis. Monocyte distribution in lymphoid organs (e.g. spleen) and chronic sites of inflammation, often a prerequisite for tumor development, in the gut was monitored over a 48 hour period. The axial image plane enables the discrimination of signal origin throughout the entire depth of the mouse, while 3D renderings of axial image stacks allow visualization of monocyte distribution throughout the abdominal region. The functional response to an injection of a vascular disrupting agent in the HT1090 fibrosarcoma model was assessed by MSOT-mediated differentiation of oxygenated and deoxygenated hemoglobin signals. Truncated tissue factor (tTF) fused to an NGR-peptide (tTF-NGR) targets CD13, which is overexpressed in tumor endothelial cells. After binding, it is known to cause vascular disruption, blood pooling and vessel occlusions within the tumor vasculature. These processes were associated with an increase in MSOT-associated deoxyhemoblin signal in the tumor. In summary, MSOT offers an imaging modality that can provide anatomical, functional, molecular and kinetic information at high temporal and spatial resolution. When combined with (molecular) imaging agents that possess appropriate targeting properties, this modality can be leveraged for new biomarker imaging approaches in cancer research. Citation Format: Wouter H. P. Driessen, Neal Burton, Christian Schwöppe, Wolfgang E. Berd, Toni Weinhage, Georg Varga, Christiane Geyer. Multispectral optoacoustic tomography (MSOT) as a novel approach for dynamic, noninvasive assessment of tumor therapy response and chronic inflammation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5114. doi:10.1158/1538-7445.AM2015-5114

Tumour progression in oral squamous cell carcinoma (OSCC) is associated with a reorganisation of extracellular matrix. Laminin-5 (Ln-5) plays an important role for tumour migration and shows an increased expression in areas of direct tumour/stroma interactions. We have previously shown stromal spot like Ln-5/gamma2 chain deposits distant from the basement membrane region. In this study we have analysed which cell type is responsible for Ln-5/gamma2 chain synthesis in situ. Furthermore, we studied its spatial relation to TGF-beta1 as well as the Ln-5 modulating enzymes matrix metalloproteinase (MMP) 2, membrane type-1 (MT1-) MMP and bone morphogenetic protein (BMP-) 1 by different techniques including triple immunofluorescence labelling and in situ hybridisation in OSCC. We found that the stromal spot-like Ln-5 deposits occurred in the invasive front in the vicinity of mesenchymal cells and vessel structures. In particular, not only carcinoma cells but also mesenchymal cells were shown to express the Ln-5/gamma2 chain mRNA. Moreover, stromal Ln-5 deposits showed a spatial association with TGF-beta1 as well as with MT1-MMP and BMP-1. Based on these findings we suggest that mesenchymal cells contribute to the promotion of tumour cell migration as well as vessel formation in OSCC by providing and organising promigratory Ln-5 fragments.