Preclinical evaluation of EpCAM-binding designed ankyrin repeat proteins (DARPins) as targeting moieties for bimodal near-infrared fluorescence and photoacoustic imaging of cancer

PURPOSE Fluorescence-guided surgery (FGS) can play a key role in improving radical resection rates by assisting surgeons to gain adequate visualization of malignant tissue intraoperatively. Designed ankyrin repeat proteins (DARPins) possess optimal pharmacokinetic and other properties for in vivo imaging. This study aims to evaluate the preclinical potential of epithelial cell adhesion molecule (EpCAM)-binding DARPins as targeting moieties for near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging of cancer. METHODS EpCAM-binding DARPins Ac2, Ec4.1, and non-binding control DARPin Off7 were conjugated to IRDye 800CW and their binding efficacy was evaluated on EpCAM-positive HT-29 and EpCAM-negative COLO-320 human colon cancer cell lines. Thereafter, NIRF and PA imaging of all three conjugates were performed in HT-29_luc2 tumor-bearing mice. At 24 h post-injection, tumors and organs were resected and tracer biodistributions were analyzed. RESULTS Ac2-800CW and Ec4.1-800CW specifically bound to HT-29 cells, but not to COLO-320 cells. Next, 6 nmol and 24 h were established as the optimal in vivo dose and imaging time point for both DARPin tracers. At 24 h post-injection, mean tumor-to-background ratios of 2.60 ± 0.3 and 3.1 ± 0.3 were observed for Ac2-800CW and Ec4.1-800CW, respectively, allowing clear tumor delineation using the clinical Artemis NIRF imager. Biodistribution analyses in non-neoplastic tissue solely showed high fluorescence signal in the liver and kidney, which reflects the clearance of the DARPin tracers. CONCLUSION Our encouraging results show that EpCAM-binding DARPins are a promising class of targeting moieties for pan-carcinoma targeting, providing clear tumor delineation at 24 h post-injection. The work described provides the preclinical foundation for DARPin-based bimodal NIRF/PA imaging of cancer

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Introduction In November 2011 archaeologists of City of Rotterdam Archaeological Service (BOOR) conducted underwater research in the Yangtze harbour, Rotterdam Maasvlakte, The Netherlands. The research was carried out by order of Port of Rotterdam Authority and supervised by the Cultural Heritage Agency of the Netherlands. The results of geological, botanical, zoological and archaeological analyses of the retrieved material generated new information on the occupation of a relatively high river dune by prehistoric hunter-gatherers, and on the development history of the surrounding landscape ca. 9,000 years ago. Methods Rather than employing divers the underwater investigations were carried out on board a vessel using a wire-operated, horizontal closing grab. Three small trenches (total area ca. 375m²) were excavated in layers in a fairly controlled fashion. Underwater excavations cannot achieve the same level of precision as is possible on land, but the many soil core samples taken in the project’s preliminary phase allowed detailed descriptions of the geomorphological stratigraphy. The excavation resulted in 316 bulk bags of soil. All soil was sieved on land, using sieves with mesh sizes of 10 and 2mm, after which archaeologists and volunteers carefully sorted the residues, documenting a total of ca. 46,000 finds. Results Plenty of Mesolithic occupation remains were retrieved at all three grab locations, from depths ranging between 17 to 21m below modern MSL. The finds span the age range from ca. 8400 to 6500BC, when the site transformed from dryland (an inland dune) to wetland (drowned delta subsurface). At the foot of the inland dune, the depositional conditions allowed for excellent preservation of bone, charcoal and plant material as well as stone artefacts. The site provides an unusually rich and detailed body of evidence on environmental conditions and the Middle Mesolithic palaeo-economy. The landscape ecotones around the site yielded an abundance of food while gradually being transformed, due to rising sea levels, from a valley containing the rivers Rhine and Meuse into the mouth area of those rivers. At 6500 BC, the site was finally transgressed: drowned in an estuary and swallowed up by the sea. Conclusion The Rotterdam Yangtze Harbour research project demonstrates the preservation of Mesolithic sites along the river Rhine, at depths in nowadays coastal and offshore areas. Furthermore, it demonstrates the feasibility of archaeological investigation of such submerged sites, even at depths of 18 to 20 m beneath sea, lake and harbour floors. Never before had such a submerged site been excavated at such a great depth. The scientific report (in English) will appear in the autumn of 2014, providing a full description of all finds as well as their landscape context

Integrating geoarchaeological techniques to reveal the invisible stratigraphy at Umhlatuzana rockshelter, South Africa: a grid-based approach

Umhlatuzana rockshelter is an archaeological site with an occupational record spanning the last ~70,000 years, covering the Middle Stone Age (MSA), Later Stone Age (LSA), and Iron Age. The deposits bearing Pleistocene archaeological assemblages at Umhlatuzana rockshelter appear homogeneous with no macroscopically visible stratigraphic boundaries. This means the integrity of the archaeological assemblages is difficult to ascertain. Moreover, the sedimentation rate, taphonomic history, and the environmental context across the sequence are unclear. This study aims to resolve these issues by integrating different geoarchaeological techniques in order to explore fine-resolution geochemical differentiations of the sediments that are macroscopically invisible. Samples were systematically retrieved from the western profile of the site following a grid-based sampling strategy and analysed for pH, elemental composition (XRF), and Magnetic Susceptibility. These methods were chosen because they provide insight into ‘invisible’ geoarchaeological dynamics, related to sediment input (geogenic and anthropogenic), taphonomy, and environmental conditions. Additionally, the results were integrated with preliminary micromorphological observations. Our study reveals a gradual change in the geochemistry of the deposits throughout the Pleistocene, related to a combination of environmental change and occupation intensity. Furthermore, the gradual change within the geochemical data indicates that no large-scale sediment mixing took place (contrary to previous suggestions), while small-scale mixing related to bioturbation is visible in the micromorphological thin sections. Our study offers a successful multi-proxy approach to systematically sample and analyse archaeological deposits at the macro and micro scale, integrating a variety of geoarchaeological techniques. The approach provides insight into the depositional and postdepositional history of the site, and allows questions of stratigraphic integrity, anthropogenic input, preservation, and environmental change to be addressed.NWOVidi 276-60-004Human Origin

Making the invisible stratigraphy visible: a grid-based, multi-proxy geoarchaeological study of Umhlatuzana rockshelter, South Africa

Umhlatuzana rockshelter is an archaeological site with an occupational record covering the Middle Stone Age, Later Stone Age, and Iron Age. The presence of both Middle and Later Stone Age assemblages makes Umhlatuzana the ideal location for the study of the MSA-LSA transition (20-40 ka) in southern Africa. This transitional period is characterized by important modifications in stone tool technology, from prepared core technology to a toolkit based on microlith production. These changes are argued to have occurred in response to changes in climate and environment leading up to the Last Glacial Maximum. The deposits bearing the transitional assemblages at Umhlatuzana rockshelter appear homogeneous with no visible stratigraphic boundaries. This study integrates geoarchaeological techniques in order to explore fine-resolution geochemical differentiations of the sediments that are macroscopically invisible, and that will provide insight into (post-)depositional processes over time. Samples were systematically retrieved from the western profile of the site following a grid-based sampling strategy and analyzed for pH, elemental composition (XRF), and Magnetic Susceptibility. Additionally, the results were integrated with preliminary micromorphological observations. Our study reveals a steady, gradual change in the geochemistry of the deposits throughout the Pleistocene, related to a combination of environmental change and occupation intensity. We suggest that the part of the sequence reported to bear Middle to Later Stone Age transitional industries is characterized by wetter environmental conditions compared to the underlying deposits. Additionally, we support results from previous studies that excluded large scale post-depositional movement of the sedimentary sequence. Our study offers a successful multi-proxy approach to systematically sample and study archaeological deposits at the macro and micro scale, integrating a variety of geoarchaeological techniques. The approach provides insight into the depositional and post-depositional history of the site, and allows for questions of stratigraphic integrity, anthropogenic input, preservation, and environmental change to be addressed.Thrombosis and Hemostasi

Tissue level, activation and cellular localisation of TGF-β1 and association with survival in gastric cancer patients

Transforming growth factor-β1 (TGF-β1), a tumour suppressing as well as tumour-promoting cytokine, is stored as an extracellular matrix-bound latent complex. We examined TGF-β1 activation and localisation of TGF-β1 activity in gastric cancer. Gastric tumours showed increased stromal and epithelial total TGF-β1 staining by immunohistochemistry. Active TGF-β1 was present in malignant epithelial cells, but most strongly in smooth muscle actin expressing fibroblasts. Normal gastric mucosa from the same patient showed some staining for total, and little for active TGF-β1. Active TGF-β1 levels were determined by ELISA on tissue homogenates, confirming a strong increase in active TGF-β1 in tumours compared to corresponding normal mucosa. Moreover, high tumour TGF-β1 activity levels were significantly associated with clinical parameters, including worse survival of the patients. Total and active TGF-β1 levels were not correlated, suggesting a specific activation process. Of the different proteases tested, active TGF-β1 levels were only correlated with urokinase activity levels. The correlation with urokinase activity suggests a role for plasmin in TGF-β1 activation in the tumour microenvironment, resulting in transformation of resident fibroblasts to tumour promoting myofibroblasts. In conclusion we have shown localisation and clinical relevance of TGF-β1 activity levels in gastric cancer

Preclinical evaluation of EpCAM-binding designed ankyrin repeat proteins (DARPins) as targeting moieties for bimodal near-infrared fluorescence and photoacoustic imaging of cancer

PurposeFluorescence-guided surgery (FGS) can play a key role in improving radical resection rates by assisting surgeons to gain adequate visualization of malignant tissue intraoperatively. Designed ankyrin repeat proteins (DARPins) possess optimal pharmacokinetic and other properties for in vivo imaging. This study aims to evaluate the preclinical potential of epithelial cell adhesion molecule (EpCAM)-binding DARPins as targeting moieties for near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging of cancer.MethodsEpCAM-binding DARPins Ac2, Ec4.1, and non-binding control DARPin Off7 were conjugated to IRDye 800CW and their binding efficacy was evaluated on EpCAM-positive HT-29 and EpCAM-negative COLO-320 human colon cancer cell lines. Thereafter, NIRF and PA imaging of all three conjugates were performed in HT-29_luc2 tumor-bearing mice. At 24 h post-injection, tumors and organs were resected and tracer biodistributions were analyzed.ResultsAc2-800CW and Ec4.1-800CW specifically bound to HT-29 cells, but not to COLO-320 cells. Next, 6 nmol and 24 h were established as the optimal in vivo dose and imaging time point for both DARPin tracers. At 24 h post-injection, mean tumor-to-background ratios of 2.60 & PLUSMN; 0.3 and 3.1 & PLUSMN; 0.3 were observed for Ac2-800CW and Ec4.1-800CW, respectively, allowing clear tumor delineation using the clinical Artemis NIRF imager. Biodistribution analyses in non-neoplastic tissue solely showed high fluorescence signal in the liver and kidney, which reflects the clearance of the DARPin tracers.ConclusionOur encouraging results show that EpCAM-binding DARPins are a promising class of targeting moieties for pan-carcinoma targeting, providing clear tumor delineation at 24 h post-injection. The work described provides the preclinical foundation for DARPin-based bimodal NIRF/PA imaging of cancer.Vascular SurgerySurgical oncolog

Matrix metalloproteinase-2 is a consistent prognostic factor in gastric cancer

In a pioneer study, we showed 10 years ago that enhanced tissue levels of the matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in gastric cancers, as determined by zymography, were related with worse overall survival of the patients. To corroborate these observations, we now assessed MMP-2 and MMP-9 with new techniques in an expanded group of gastric cancer patients (n=81) and included for comparison MMP-7, MMP-8 and the tissue inhibitors of MMPs, TIMP-1 and -2. All MMPs and TIMP-1 were significantly increased in tumour tissue compared to normal gastric mucosa. Matrix metalloproteinase-7, -8 and -9, and the TIMPs showed some correlations with the clinicopathologic parameters TNM, WHO and Laurén classification, but their levels were not related with survival. Regardless of the determination method used, that is, enzyme-linked immunosorbent assay or bioactivity assay, an enhanced tumour MMP-2 level did not show a significant correlation with any of the clinicopathological parameters, but was confirmed to be an independent prognostic factor in gastric cancer

Implant removal associated complications in children with limb fractures due to trauma

Surger