Three-dimensional analysis of human pancreatic cancer specimens by phase-contrast based X-ray tomography - the next dimension of diagnosis.

BACKGROUND: The worldwide increase of pancreatic ductal adenocarcinoma (PDAC), which still has one of the lowest survival rates, requires novel imaging tools to improve early detection and to refine diagnosis. Therefore, the aim of this study was to assess the feasibility of propagation-based phase-contrast X-ray computed tomography of already paraffin-embedded and unlabeled human pancreatic tumor tissue to achieve a detailed three-dimensional (3D) view of the tumor sample in its entirety. METHODS: Punch biopsies of areas of particular interest were taken from paraffin blocks after initial histological analysis of hematoxylin and eosin stained tumor sections. To cover the entire 3.5 mm diameter of the punch biopsy, nine individual tomograms with overlapping regions were acquired in a synchrotron parallel beam configuration and stitched together after data reconstruction. Due to the intrinsic contrast based on electron density differences of tissue components and a voxel size of 1.3 µm achieved PDAC and its precursors were clearly identified. RESULTS: Characteristic tissue structures for PDAC and its precursors, such as dilated pancreatic ducts, altered ductal epithelium, diffuse immune cell infiltrations, increased occurrence of tumor stroma and perineural invasion were clearly identified. Certain structures of interest were visualized in three dimensions throughout the tissue punch. Pancreatic duct ectasia of different caliber and atypical shape as well as perineural infiltration could be contiguously traced by viewing serial tomographic slices and by applying semi-automatic segmentation. Histological validation of corresponding sections confirmed the former identified PDAC features. CONCLUSION: In conclusion, virtual 3D histology via phase-contrast X-ray tomography visualizes diagnostically relevant tissue structures of PDAC in their entirety, preserving tissue integrity in label-free, paraffin embedded tissue biopsies. In the future, this will not only enable a more comprehensive diagnosis but also a possible identification of new 3D imaging tumor markers.

The murine male reproductive organ at a glance: Three-dimensional insights and virtual histology using label-free light sheet microcopy.

BACKGROUND: The unique anatomy of the male reproductive organ reflects its complex function from sperm maturation to their storage for months until emission. Since light microscopy in two dimensions (2d) cannot sufficiently demonstrate its complex morphology, a comprehensive visualization is required to identify pathologic alterations in its entire anatomical context. OBJECTIVES: Aim of this study was to use three-dimensional (3d) light sheet fluorescence microscopy (LSFM) to visualize entire murine testes in 3d, label-free and at subcellular resolution, and to assign local autofluorescence to testicular and deferent structures. MATERIALS AND METHODS: Murine testes were fixed with four different fixatives and subsequently cleared with benzoic acid/benzyl benzoate. Hereafter, complete murine testes were scanned with LSFM with different fluorescence filter sets and subsequently embedded in paraffin for further conventional planar histology. RESULTS: Autofluorescence signals of the murine reproductive organ allowed the unambiguous identification of the testicular anatomy from the seminiferous tubules to the vas deferens with their specific stratification independent of the used fixative. Blood vessels were visualized from the pampiniform plexus to the small capillaries of single tubules. Moreover, due to the specific intrinsic fluorescence properties of the efferent ducts and the epididymis, luminal caliber, the epithelial stratification and retronuclear cytoplasmic inclusions gave a unique insight into the interface of both morphological structures. Subsequent 2d histology confirmed the identified morphological structures. DISCUSSION: LSFM analysis of the murine reproductive organ allows due to its intrinsic fluorescence a simple, label-free 3d assessment of its entire duct morphology, the epithelial composition, and the associated blood supply in its anatomical relation. CONCLUSION: LSFM provides the technical basis for comprehensive analyses of pathologically altered murine testes in its entirety by depicting specific autofluorescence. Thereby it facilitates mouse studies of testicular disease or their drug-related alterations in more detail potentially for clinical translation assessing human testicular biopsies.

Identification of CD318, TSPAN8 and CD66c as target candidates for CAR T cell based immunotherapy of pancreatic adenocarcinoma.

A major roadblock prohibiting effective cellular immunotherapy of pancreatic ductal adenocarcinoma (PDAC) is the lack of suitable tumor-specific antigens. To address this challenge, here we combine flow cytometry screenings, bioinformatic expression analyses and a cyclic immunofluorescence platform. We identify CLA, CD66c, CD318 and TSPAN8 as target candidates among 371 antigens and generate 32 CARs specific for these molecules. CAR T cell activity is evaluated in vitro based on target cell lysis, T cell activation and cytokine release. Promising constructs are evaluated in vivo. CAR T cells specific for CD66c, CD318 and TSPAN8 demonstrate efficacies ranging from stabilized disease to complete tumor eradication with CD318 followed by TSPAN8 being the most promising candidates for clinical translation based on functionality and predicted safety profiles. This study reveals potential target candidates for CAR T cell based immunotherapy of PDAC together with a functional set of CAR constructs specific for these molecules.

3D virtual histology of human pancreatic tissue by multiscale phase-contrast X-ray tomography.

A multiscale three-dimensional (3D) virtual histology approach is presented, based on two configurations of propagation phase-contrast X-ray tomography, which have been implemented in close proximity at the GINIX endstation at the beamline P10/PETRA III (DESY, Hamburg, Germany). This enables the 3D reconstruction of characteristic morphological features of human pancreatic normal and tumor tissue, as obtained from cancer surgery, first in the form of a large-scale overview by parallel-beam illumination, followed by a zoom into a region-of-interest based on zoom tomography using a Kirkpatrick-Baez mirror with additional waveguide optics. To this end 1 mm punch biopsies of the tissue were taken. In the parallel tomography, a volumetric throughput on the order of 0.01 mm3 s-1 was achieved, while maintaining the ability to segment isolated cells. With a continuous rotation during the scan, a total acquisition time of less than 2 min was required for a full tomographic scan. Using the combination of both setups, islets of Langerhans, a three-dimensional cluster of cells in the endocrine part of the pancreas, could be located. Cells in such an islet were segmented and visualized in 3D. Further, morphological alterations of tumorous tissue of the pancreas were characterized. To this end, the anisotropy parameter Ω, based on intensity gradients, was used in order to quantify the presence of collagen fibers within the entire biopsy specimen. This proof-of-concept experiment of the multiscale approach on human pancreatic tissue paves the way for future 3D virtual pathology.

3D virtual histology of murine kidneys -high resolution visualization of pathological alterations by micro computed tomography.

The increasing number of patients with end stage chronic kidney disease not only calls for novel therapeutics but also for pioneering research using convincing preclinical disease models and innovative analytical techniques. The aim of this study was to introduce a virtual histology approach using micro computed tomography (µCT) for the entire murine kidney in order to close the gap between single slice planar histology and a 3D high resolution dataset. An ex vivo staining protocol based on phosphotungstic acid diffusion was adapted to enhance renal soft tissue x-ray attenuation. Subsequent CT scans allowed (i) the detection of the renal cortex, medulla and pelvis in greater detail, (ii) the analysis of morphological alterations, (iii) the quantification of the volume as well as the radio-opacity of these portions and (iv) the quantification of renal fibrotic remodeling based on altered radio-opacity using the unilateral ureteral obstruction model. Thus, virtual histology based on PTA contrast enhanced CT will in future help to refine the outcome of preclinical research on kidney associated murine disease models.

µCT of ex-vivo stained mouse hearts and embryos enables a precise match between 3D virtual histology, classical histology and immunochemistry.

The small size of the adult and developing mouse heart poses a great challenge for imaging in preclinical research. The aim of the study was to establish a phosphotungstic acid (PTA) ex-vivo staining approach that efficiently enhances the x-ray attenuation of soft-tissue to allow high resolution 3D visualization of mouse hearts by synchrotron radiation based µCT (SRµCT) and classical µCT. We demonstrate that SRµCT of PTA stained mouse hearts ex-vivo allows imaging of the cardiac atrium, ventricles, myocardium especially its fibre structure and vessel walls in great detail and furthermore enables the depiction of growth and anatomical changes during distinct developmental stages of hearts in mouse embryos. Our x-ray based virtual histology approach is not limited to SRµCT as it does not require monochromatic and/or coherent x-ray sources and even more importantly can be combined with conventional histological procedures. Furthermore, it permits volumetric measurements as we show for the assessment of the plaque volumes in the aortic valve region of mice from an ApoE-/- mouse model. Subsequent, Masson-Goldner trichrome staining of paraffin sections of PTA stained samples revealed intact collagen and muscle fibres and positive staining of CD31 on endothelial cells by immunohistochemistry illustrates that our approach does not prevent immunochemistry analysis. The feasibility to scan hearts already embedded in paraffin ensured a 100% correlation between virtual cut sections of the CT data sets and histological heart sections of the same sample and may allow in future guiding the cutting process to specific regions of interest. In summary, since our CT based virtual histology approach is a powerful tool for the 3D depiction of morphological alterations in hearts and embryos in high resolution and can be combined with classical histological analysis it may be used in preclinical research to unravel structural alterations of various heart diseases.