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Advancing novel immunotherapy strategies requires refined tools in preclinical research to thoroughly assess drug targets, biodistribution, safety, and efficacy. Light sheet fluorescence microscopy (LSFM) offers unprecedented fast volumetric ex vivo imaging of large tissue samples in high resolution. Yet, to date laborious and unstandardized tissue processing procedures have limited throughput and broader applications in immunological research. Therefore, we have developed a simple and harmonized protocol for processing, clearing and imaging of all mouse organs and even entire mouse bodies. Applying this Rapid Optical Clearing Kit for Enhanced Tissue Scanning (ROCKETS) in combination with LSFM allowed us to comprehensively study the in vivo biodistribution of an antibody targeting Epithelial Cell Adhesion Molecule (EpCAM) in 3D. Quantitative high-resolution scans of whole organs did not only reveal known EpCAM expression patterns but, importantly, uncovered several new EpCAM-binding sites. We identified choroid plexi in the brain and duodenal papillae as unexpected locations of high EpCAM-expression. These tissue locations may be considered as particularly sensitive sites due their importance for liquor production or as critical junctions draining bile and digestive pancreatic enzymes into the small bowel, respectively. These newly gained insights appear highly relevant for clinical translation of EpCAM-addressing immunotherapies. Thus, ROCKETS in combination with LSFM may help to set new standards for preclinical evaluation of immunotherapeutic strategies. Conclusively, we propose ROCKETS as an ideal platform for a broader application of LSFM in immunological research optimally suited for quantitative co-localization studies of immunotherapeutic drugs and defined cell populations in the microanatomical context of organs or even whole mice.

Here, we leveraged a large neoadjuvant PD-1 blockade trial in patients with hepatocellular carcinoma (HCC) to search for correlates of response to immune checkpoint blockade (ICB) within T cell-rich tumors. We show that ICB response correlated with the clonal expansion of intratumoral CXCL13+ CH25H+ IL-21+ PD-1+ CD4 T helper cells (CXCL13+ Th) and Granzyme K+ PD-1+ effector-like CD8 T cells, whereas terminally exhausted CD39hi TOXhi PD-1hi CD8 T cells dominated in non-responders. Strikingly, most T cell receptor (TCR) clones that expanded post-treatment were found in pre-treatment biopsies. Notably, PD-1+ TCF-1+ progenitor-like CD8 T cells were present in tumors of responders and non-responders and shared clones mainly with effector-like cells in responders or terminally differentiated cells in non-responders, suggesting that local CD8 T cell differentiation occurs upon ICB. We found that these progenitor CD8 T cells interact with CXCL13+ Th cells within cellular triads around dendritic cells enriched in maturation and regulatory molecules, or “mregDC”. Receptor-ligand analysis revealed unique interactions within these triads that may promote the differentiation of progenitor CD8 T cells into effector-like cells upon ICB. These results suggest that discrete intratumoral niches that include mregDC and CXCL13+ Th cells control the differentiation of tumor-specific progenitor CD8 T cell clones in patients treated with ICB.

Crohn’s disease is an inflammatory bowel disease (IBD) which most often presents with patchy lesions in the terminal ileum and colon and requires complex clinical care. Recent advances in the targeting of cytokines and leukocyte migration have greatly advanced treatment options, but most patients still relapse and inevitably progress. Although single-cell approaches are transforming our ability to understand the barrier tissue biology of inflammatory disease, comprehensive single-cell RNA-sequencing (scRNA-seq) atlases of IBD to date have largely sampled pre-treated patients with established disease. This has limited our understanding of which cell types, subsets, and states at diagnosis are predictive of disease severity and response to treatment. Here, through a combined clinical, flow cytometric, and scRNA-seq study, we profile diagnostic human biopsies from the terminal ileum of treatment-naive pediatric patients with Crohn’s disease (pediCD; n=14) and from non-inflamed pediatric controls with functional gastrointestinal disorders (FGID; n=13). To fully resolve and annotate epithelial, stromal, and immune cell states among the 201,883 single-cell transcriptomes, we develop and deploy a principled and unbiased tiered clustering approach, ARBOL, yielding 138 FGID and 305 pediCD end cell clusters. Notably, through both flow cytometry and scRNA-seq, we observe that at the level of broad cell types, treatment-naive pediCD is not readily distinguishable from FGID in cellular composition. However, by integrating high-resolution scRNA-seq analysis, we identify significant differences in cell states that arise during pediCD relative to FGID. Furthermore, by closely linking our scRNA-seq analysis with clinical meta-data, we resolve a vector of lymphoid, myeloid, and epithelial cell states in treatment-naive samples which can distinguish patients with less severe disease (those not on anti-TNF therapies (NOA)), from those with more severe disease at presentation who require anti-TNF therapies. Moreover, this vector was also able to distinguish those patients that achieve a full response (FR) to anti-TNF blockade from those more treatment-resistant patients who only achieve a partial response (PR). Our study jointly leverages a treatment-naive cohort, high-resolution principled scRNA-seq data analysis, and clinical outcomes to understand which baseline cell states may predict inflammatory disease trajectory.

Negative circumferential resection margins (CRM) are the cornerstone for the curative treatment of locally advanced rectal cancer (LARC). However, in up to 18.6% of patients, tumor-positive resection margins are detected on histopathology. In this proof-of-concept study, we investigated the feasibility of optical molecular imaging as a tool for evaluating the CRM directly after surgical resection to improve tumor-negative CRM rates. Methods: LARC patients treated with neoadjuvant chemoradiotherapy received an intravenous bolus injection of 4.5 mg of bevacizumab-800CW, a fluorescent tracer targeting vascular endothelial growth factor A, 2-3 d before surgery (ClinicalTrials.gov identifier: NCT01972373). First, for evaluation of the CRM status, back-table fluorescence guided imaging (FGI) of the fresh surgical resection specimens (n = 8) was performed. These results were correlated with histopathology results. Second, for determination of the sensitivity and specificity of bevacizumab-800CW for tumor detection, a mean fluorescence intensity cutoff value was determined from the formalin-fixed tissue slices (n = 42; 17 patients). Local bevacizumab-800CW accumulation was evaluated by fluorescence microscopy. Results: Back-table FGI correctly identified a tumor-positive CRM by high fluorescence intensities in 1 of 2 patients (50%) with a tumor-positive CRM. For the other patient, low fluorescence intensities were shown, although (sub)millimeter tumor deposits were present less than 1 mm from the CRM. FGI correctly identified 5 of 6 tumor-negative CRM (83%). The 1 patient with false-positive findings had a marginal negative CRM of only 1.4 mm. Receiver operating characteristic curve analysis of the fluorescence intensities of formalin-fixed tissue slices yielded an optimal mean fluorescence intensity cutoff value for tumor detection of 5,775 (sensitivity of 96.19% and specificity of 80.39%). Bevacizumab-800CW enabled a clear differentiation between tumor and normal tissue up to a microscopic level, with a tumor-to-background ratio of 4.7 +/- 2.5 (mean SD). Conclusion: In this proof-of-concept study, we showed the potential of back-table FGI for evaluating the CRM status in LARC patients. Optimization of this technique with adaptation of standard operating procedures could change perioperative decision making with regard to extending resections or applying intraoperative radiation therapy in the case of positive CRM.

This review delves into the rapidly evolving field of deep tissue imaging at cellular resolution, reviewing popular tissue clearing and staining methods in combination with light-sheet fluorescence microscopy (LSFM) including quantification and three-dimensional visualization tools, the field of applications and perspective, particularly with the focus on preclinical cancer research and drug development. The LSFM technique presented here allows an extremely fast optical sectioning for three-dimensional reconstruction of centimeter-sized tissue samples at cellular resolution. However, optical clearing methods are required to receive optical transparent tissue. Application of either tissue autofluorescence, in vivo fluorescence labeling, endogenous fluorescence or ex vivo whole-mount immunolabeling enables three-dimensional in situ visualization of morphological and functional features of unsectioned whole-mount tissue samples. This powerful and innovative imaging technique opens up a new dimension of tissue analysis providing detailed and comprehensive insights into biology. It enables the investigation of normal and pathological tissue features and disease progression and allows precise monitoring of potential therapeutic interventions in intact biological tissue on a cellular level.

This study aimed at assessing the effects of an anti-angiogenic treatment, which neutralises vascular endothelial growth factor (VEGF), on tumour heterogeneity. Murine glioma cells have been inoculated into the right brain frontal lobe of 16 mice. Anti-VEGF antibody was administered to a first group (n = 8), while a second group (n = 8) received a placebo. Magnetic resonance acquisitions, performed at days 10, 12, 15 and 23 following the implantation, allowed the derivation of a three-dimensional features dataset characterising tumour heterogeneity. Three-dimensional ultramicroscopy and standard histochemistry analysis have been performed to verify in vivo results. Placebo-treated mice displayed a highly-vascularised area at the tumour periphery, a monolithic necrotic core and a chaotic dense vasculature across the entire tumour. In contrast, the B20-treated group did not show any highly vascularised regions and presents a fragmented necrotic core. A significant reduction of the number of vessel segments smaller than 17 μm has been observed. There was no difference in overall tumour volume and growth rate between the two groups. Region-specific analysis revealed that VEGF inhibition affects only: (1) highly angiogenic compartments expressing high levels of VEGF and characterised by small capillaries, and also (2) the formation and structure of necrotic regions. These effects appear to be transient and limited in time. • VEGF inhibition affects only the highly angiogenic region and small capillaries network • VEGF inhibition is transient in time • Tumour volume is not affected by anti-angiogenic treatment • VEGF inhibition also influences the architecture of necrotic regions

The role of tissue architecture in mediating nanoparticle transport, targeting, and biological effects is unknown due to the lack of tools for imaging nanomaterials in whole organs. Here, we developed a rapid optical mapping technique to image nanomaterials in intact organs ex vivo and in three-dimensions (3D). We engineered a high-throughput electrophoretic flow device to simultaneously transform up to 48 tissues into optically transparent structures, allowing subcellular imaging of nanomaterials more than 1 mm deep into tissues which is 25-fold greater than current techniques. A key finding is that nanomaterials can be retained in the processed tissue by chemical cross-linking of surface adsorbed serum proteins to the tissue matrix, which enables nanomaterials to be imaged with respect to cells, blood vessels, and other structures. We developed a computational algorithm to analyze and quantitatively map nanomaterial distribution. This method can be universally applied to visualize the distribution and interactions of materials in whole tissues and animals including such applications as the imaging of nanomaterials, tissue engineered constructs, and biosensors within their intact biological environment.

OBJECTIVES Dynamic contrast-enhanced (DCE) micro-computed tomography (micro-CT) has emerged as a valuable imaging tool to noninvasively obtain quantitative physiological biomarkers of drug effect in preclinical studies of antiangiogenic compounds. In this study, we explored the ability of DCE micro-CT to assess the antiangiogenic treatment response in breast cancer xenografts and correlated the results to the structural vessel response obtained from 3-dimensional (3D) fluorescence ultramicroscopy (UM). MATERIAL AND METHODS Two groups of tumor-bearing mice (KPL-4) underwent DCE micro-CT imaging using a fast preclinical dual-source micro-CT system (TomoScope Synergy Twin, CT Imaging GmbH, Erlangen, Germany). Mice were treated with either a monoclonal antibody against the vascular endothelial growth factor or an unspecific control antibody. Changes in vascular physiology were assessed measuring the mean value of the relative blood volume (rBV) and the permeability-surface area product (PS) in different tumor regions of interest (tumor center, tumor periphery, and total tumor tissue). Parametric maps of rBV were calculated of the tumor volume to assess the intratumoral vascular heterogeneity. Isotropic 3D UM vessel scans were performed from excised tumor tissue, and automated 3D segmentation algorithms were used to determine the microvessel density (MVD), relative vessel volume, and vessel diameters. In addition, the accumulation of coinjected fluorescence-labeled trastuzumab was quantified in the UM tissue scans to obtain an indirect measure of vessel permeability. Results of the DCE micro-CT were compared with corresponding results obtained by ex vivo UM. For validation, DCE micro-CT and UM parameters were compared with conventional histology and tumor volume. RESULTS Examination of the parametric rBV maps revealed significantly different patterns of intratumoral blood supply between treated and control tumors. Whereas control tumors showed a characteristic vascular rim pattern with considerably elevated rBV values in the tumor periphery, treated tumors showed a widely homogeneous blood supply. Compared with UM, the physiological rBV maps showed excellent agreement with the spatial morphology of the intratumoral vascular architecture. Regional assessment of mean physiological values exhibited a significant decrease in rBV (P < 0.01) and PS (P < 0.05) in the tumor periphery after anti-vascular endothelial growth factor treatment. Structural validation with UM showed a significant reduction in reduction of relative vessel volume (rVV) (P < 0.01) and MVD (P < 0.01) in the corresponding tumor region. The reduction in rBV correlated well with the rVV (R = 0.73 for single values and R = 0.95 for mean values). Spatial maps of antibody penetration showed a significantly reduced antibody accumulation (P < 0.01) in the tumor tissue after treatment and agreed well with the physiological change of PS. Examination of vessel diameters revealed a size-dependent antiangiogenic treatment effect, which showed a significant reduction in MVD (P < 0.001) for vessels with diameters smaller than 25 μm. No treatment effect was observed by tumor volume. CONCLUSIONS Noninvasive DCE micro-CT provides valuable physiological information of antiangiogenic drug effect in the intact animal and correlates with ex vivo structural analysis of 3D UM. The combined use of DCE micro-CT with UM constitutes a complementary imaging toolset that can help to enhance our understanding of antiangiogenic drug mechanisms of action in preclinical drug research.

Purpose: For generating preclinical pharmacokinetics (PKs) of compounds, blood is drawn at different time points and levels are quantified by different analytical methods. In order to receive statistically meaningful data, 3 to 5 animals are used for each time point to get serum peak-level and half-life of the compound. Both characteristics are determined by data interpolation, which may influence the accuracy of these values. We provide a method that allows continuous monitoring of blood levels noninvasively by measuring the fluorescence intensity of labeled compounds in the eye and other body regions of anesthetized mice. Procedures: The method evaluation was performed with four different fluorescent compounds: (i) indocyanine green, a nontargeting dye; (ii) OsteoSense750, a bone targeting agent; (iii) tumor targeting Trastuzumab-Alexa750; and (iv) its F(ab ′ ) 2 -alxea750 fragment. The latter was used for a direct comparison between fluorescence imaging and classical blood analysis using enzyme-linked immunosorbent assay (ELISA). Results: We found an excellent correlation between blood levels measured by noninvasive eye imaging with the results generated by classical methods. A strong correlation between eye imaging and ELISA was demonstrated for the F(ab ′ ) 2 fragment. Whole body imaging revealed a compound accumulation in the expected regions (e.g., liver, bone). Conclusions: The combination of eye and whole body fluorescence imaging enables the simultaneous measurement of blood PKs and biodistribution of fluorescent-labeled compounds.

Fluorescent agents with specificity to cellular and subcellular moieties present promise for enhancing diagnostics and theranostics, yet challenges associated with regulatory approvals of experimental agents stifle the clinical translation. As a result, targeted fluorescent agents have remained predominantly as preclinical imaging tools. We discuss the potential of using optically labeled drugs to accelerate the clinical acceptance of optical and optoacoustic agents, in analogy to nuclear medicine approaches. This strategy, corroborated with microdosing studies, outlines a promising approach for overcoming bottlenecks and advancing photonic clinical imaging.

Die Bewertung der Entkeimungsleistung keimarm oder steril arbeitender Abfullanlagen im Lebensmittel- und Pharmabereich erfolgt praktisch durch mikrobiologische Validierungsverfahren. Grundlage bei der Validierung sind Bioindikatoren mit ausgewahlten Testkeimen, deren Resistenzen je nach Anzucht variieren und masgeblich die Abtotungseffizienz beeinflussen konnen. Fur eine reelle Aussage uber das Wirkungsspektrum von Entkeimungsvorrichtungen sind deshalb definierte Resistenzen der Testkeime unerlasslich, die im Rahmen standardisierter Untersuchungsmethoden eingesetzt werden. In Abhangigkeit der verwendeten Testmethodik sind dabei neben der erreichbaren Keimreduktion auch Aussagen uber die Praxistauglichkeit und den Hygienestatus des gesamten Abfullprozesses moglich.

Aim: Here, we evaluate the ability of dynamic contrast-enhanced micro-computed tomography (DCE micro-CT) in combination with 3D fluorescence ultramicroscopy (UM) to assess the early physiological and morphological treatment response of orthotopic breast tumors to anti-angiogenic drug therapy. Methods: Non-invasive DCE micro-CT was performed on breast cancer xenografts (KPL-4) that were treated twice with an antibody against the vascular endothelial growth factor (VEGF) or a control antibody. Tumor physiology was assessed in different tumor regions of interests measuring the relative blood volume (rBV) and the permeability-surface area product (PS). Parametric maps were calculated on a voxel-by-voxel basis to evaluate intra-tumor vascular heterogeneity. Mean physiological measurements were compared with morphological measures of the tumor vascular architecture (microvessel density (MVD) and relative vessel volume (rVV)) as obtained by 3-dimensional fluorescence UM. Additionally, vessel leakage was assessed by UM quantifying the penetration strength of a co-injected fluorescence-labeled therapeutic antibody into the tumor tissue. Results: Examination of the parametric maps revealed significantly different spatial patterns of intra-tumoral blood supply (rBV) between anti-VEGF-treated tumors and control. While a characteristic rim vascularization (high rBV values in the tumor periphery) was observed in the control tumors, treated tumors showed a widely homogeneous blood supply. Compared with high-resolution UM, these physiological rBV maps showed excellent agreement with the morphological appearance of the 3D tumor vascular architecture. Assessment of the mean physiology showed a significant decrease of rBV (p Conclusions: DCE micro-CT used along with UM provides comprehensive and complementary information of the physiological and morphological anti-angiogenic treatment response in breast cancer xenografts. This technology may help to improve current standard methods in the assessment of anti-angiogenic drug efficacy in preclinical drug development. Citation Format: Thomas Pöschinger, Anja Renner, Fabian Eisa, Michael Dobosz, Robert Brauweiler, Willi A. Kalender, Werner Scheuer. Anti-VEGF treatment in orthotopic breast cancer xenografts: Dynamic contrast-enhanced micro-CT correlates with 3D multispectral fluorescence histology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2071A. doi:10.1158/1538-7445.AM2014-2071A

Classic histology still represents the gold standard in tumor tissue analytics. However, two-dimensional analysis of single tissue slides does not provide a representative overview of the inhomogeneous tumor physiology, and a detailed analysis of complex three-dimensional structures is not feasible with this technique. To overcome this problem, we applied multispectral fluorescence ultramicroscopy (UM) to the field of tumor analysis. Optical sectioning of cleared tumor specimen provides the possibility to three-dimensionally acquire relevant tumor parameters on a cellular resolution. To analyze the virtual UM tumor data sets, we created a novel set of algorithms enabling the fully automatic segmentation and quantification of multiple tumor parameters. This new postmortem imaging technique was applied to determine the therapeutic treatment effect of bevacizumab on the vessel architecture of orthotopic KPLA breast cancer xenografts at different time points. A significant reduction of the vessel volume, number of vessel segments, and branching points in the tumor periphery was already detectable 1 day after initiation of treatment. These parameters remained virtually unchanged in the center of the tumor. Furthermore, bevacizumab-induced vessel normalization and reduction in vascular permeability diminished the penetration behavior of trastuzumab-Alexa 750 into tumor tissue. Our results demonstrated that this new imaging method enables the three-dimensional visualization and fully automatic quantification of multiple tumor parameters and drug penetration on a cellular level. Therefore, UM is a valuable tool for cancer research and drug development. It bridges the gap between common macroscopic and microscopic imaging modalities and opens up new three-dimensional (3D) insights in tumor biology.