Abcam (United Kingdom)

In support of the Pathfinder Off-Plane Grating Rocket Experiment (OGRE-P), Penn State researchers and engineers needed to create a large collimated light source to test alignment of the Joint European Telescope for X-ray astronomy (JET-X) flight module. This was done by using a hobbyist 18-inch Dobsonian telescope in reverse in order to generate a collimated light source large enough to cover the entire JET-X module. Here we present technical details of our test bed execution and discuss pitfalls we encountered so that others will be able to follow a similar reverse-telescope test setup for in-lab testing.

The Pathfinder Off-plane Grating Rocket Experiment (OGRE-P) plans to use Electron-Multiplying CCDs as the primary detectors for the mission. Electron-multiplying CCDs incorporate an additional gain register that amplifies the photoelectron signal before readout, increasing sensitivity by reducing the impact of readout noise, making them ideal for low-light and high-speed X-ray imaging applications. With the additional gain, EM-CCDs amplify the signal to increase signal to noise for each detected photon which is important for photon counting X-ray observations. In addition, the EM-CCDs used have high spatial resolution and moderate energy resolution which works well with the high quantum efficiency in the soft-X-ray band. The EM-CCDs being used are being developed by XCAM in cooperation with the Open University. The mission consists of four EM-CCDs; three of which are located on a circle around the focal axis to be used for spectral data collection and the fourth directly in the center of the focal plane. We present promising results on the use of EM-CCDs for X-ray detection and spectral analysis for the mission as well as an update on camera calibration and operation.

Antibody-based research applications are critical for biological discovery. Yet there are no industry standards for comparing the performance of antibodies in various applications. We describe a knockout cell line-based antibody characterization platform, developed and approved jointly by industry and academic researchers, that enables the systematic comparison of antibody performance in western blot, immunoprecipitation and immunofluorescence. The scalable protocols, which require minimal technological resources, consist of (1) the identification of appropriate cell lines for antibody characterization studies, (2) development/contribution of isogenic knockout controls, and (3) a series of antibody characterization procedures focused on the most common applications of antibodies in research. We provide examples of expected outcomes to guide antibody users in evaluating antibody performance. Central to our approach is advocating for transparent and open data sharing, enabling a community effort to identify specific antibodies for all human proteins. Mid-level graduate students with training in biochemistry and prior experience in cell culture and microscopy can complete the protocols for a specific protein within 1 month while working part-time on this effort. Antibody characterization is needed to meet standards for resource validation and data reproducibility, which are increasingly required by journals and funding agencies.

BackgroundThe advancement of spatially resolved, multiplex proteomic and transcriptomic technologies has revolutionized and redefined the approaches to complex biological questions pertaining to tissue heterogeneity, tumor microenvironments, cellular interactions, cellular diversity, and therapeutic response. While spatial transcriptomics has traditionally led the way in plex, multiple studies have demonstrated a poor correlation between RNA expression and protein abundance, owing to transcriptional and translational regulation, target turnover, and most critically, post-translational protein modifications. Therefore, a more holistic proteomic atlas approach becomes critical to discovery biology. Previously, lack of successful detection of antibody-based probes well into the 100s served as a barrier to proteome-based interrogation of tissue while maintaining spatial context.The GeoMx® Digital Spatial Profiler (DSP) platform is uniquely suited to support high-plex proteomics, allowing for the simultaneous analyses of proteins from discrete regions of interest (ROIs) in FFPE tissue sections while preserving spatial context. The assay relies upon abcam antibodies coupled to photocleavable DNA barcodes read out with NGS sequencing, allowing for theoretically unlimited plex. Here we present the Human GeoMx Immuno-Oncology Proteome Atlas (IPA), a >500-plex antibody-based proteomic discovery panel, compatible with immunohistochemistry on FFPE tissues with NGS readout. The panel content focuses on key areas of immuno-oncology, oncology, immunology, epigenetics, metabolism, cell death, and specific signaling pathway regulation.MethodsWe validated the specificity and sensitivity of the IPA on the GeoMx across >90 cell types and >50 human tissue types, normal and cancerous, representing FDA guidelines for antibody cross-reactivity testing. Using the validated IPA, we evaluated the proteomic landscape of various diseased colon tissue including adenocarcinoma, hyperplasia, and chronic inflammation (ulcerative colitis, Crohn’s disease).ResultsEach of the individual antibodies in the IPA passed the specificity and sensitivity requirements which include exhibiting a maximum positive signal divided by the limit of detection, plus two standard deviations (SD) that is >5 in both cell pellet arrays and tissue microarrays; such a threshold gives a false positive rate of less than 10%. In addition, we compared the colonic diseased tissue to normal tissue and observed an upregulation of specific pathways associated with tumorigenesis and/or inflammation. Furthermore, we observed distinct differences in protein expression between several of the colonic diseases.ConclusionsWe demonstrate the power of the combination of the GeoMx DSP and curated Human GeoMx Immuno-Oncology Proteome Atlas to enable discovery biology by rapidly screening large numbers of tissues across critical potential therapeutic targets.FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

Abstract The glioblastoma multiforme (GBM) microenvironment is highly immunosuppressive, leading to poor clinical outcomes and median survival of less than 15 months. Information about the nature of immunosuppressive and immunostimulatory cell types in GBM is fragmented, and a better understanding is urgently needed. We have developed a spatial phenotyping application that permits comprehensive characterization of the human GBM immune microenvironment. The Phenocycler-Fusion is a fast spatial biology solution that affords ultrahigh-plex single-cell spatial readouts. We used this solution for deep immune phenotyping of >50 proteins, comprising immune cell lineages, activation states and checkpoints, as well as markers for tumor, vascular and neuronal landscapes of GBM. Via single cell spatial phenotyping, we then isolated spatial signatures within the GBM tissue immune microenvironment. We focused on large numbers of glioma-associated macrophages that were identified via the combinatorial expression of key markers CD68, CD163, STAT1, STAT6, Iba-1 and SIRPα. The macrophages and their biomarker expression profiles displayed significant inter- and intra-tumoral heterogeneity, indicative of the complex biology of GBM. In addition, we profiled other immunosuppressive elements, including Tregs and exhausted T cells. Our data provide a comprehensive account of diverse immune cell populations in GBM and they may uncover systematic differences among GBM patient samples. Our application has enormous potential to further our understanding of the GBM immune microenvironment and is ready to be deployed by other laboratories.

Abstract Background: The tumor microenvironment (TME) consists of malignant cells and supporting non-malignant cellular and non-cellular components that form the tumor stroma. The tumor stroma plays an important role in tumor progression and has emerged as a modulator of anti-tumor immunity (Salmon et al., Nat Rev Cancer 2019) and responses to therapy (Hirata and Sahai, Cold Spring Harb Perspect Med 2017). As such, several therapeutic approaches have recently been developed to target stromal cells as anti-cancer treatments (Valkenburg et al., Nat Rev Clin Oncol 2018, Bejarano et al., Cancer Discov 2021). In addition, the composition of the TME has been recognized as a prognostic factor for survival in cancer patients (Pagès et al., Oncogene 2011). Current protein-based approaches to characterize and better understand the cell composition of the tumor stroma face many limitations such as reagent availability and lengthy protocols. In this study, we identified a list of 22 markers to characterize non-tumoral immune cells, fibroblasts and endothelial cells in the TME, in a single tissue slide. We propose an approach that overcomes reagent incompatibility and opens new avenues of research of tumor stroma. Method: Multiorgan Tumor Microarray (TMA) was interrogated with a sequential immunofluorescence (seqIF™) panel encompassing protein markers enabling characterization of TME. A 22-plex panel was created based on expanding an already established 13-plex panel (CD3, CD4, CD8, CD11c, CD20, CD45, CD56, CD68, aSMA, FoxP3, Ki67, PD1, PD-L1) by adding 9 additional antibodies (CD11b, CD14, CD31, CD47, CK, FAP, LaminB1, SIRPα, Vimentin). Hyperplex immunofluorescent staining was performed using automated staining-imaging COMET™ platform generating ome-tiff images containing 25 layers: DAPI, 2 autofluorescent and 22 marker channels. Postprocessing of images was done with HORIZON™ image analysis software. Results: We established a panel of 22 markers that can be analyzed simultaneously on a single tissue slide despite limited variability in primary antibody species. Using seqIF™ protocol allowed the study of colocalization and co-expression of markers not compatible to study simultaneously in the traditional immunofluorescence approach. Our hyperplex data revealed distinct composition of the stromal compartment between different tumor types, highlighting high heterogeneity in the tissue composition and stromal architecture. Conclusion: COMET™ platform enabled studying in detail TME components and highlighted heterogeneity of tumor stroma across different tissue types. SeqIF™ protocol lifted the limitation imposed by same species antibodies and allowed simultaneous interrogation of markers’ expression preserving their spatial relationship. Citation Format: François Rivest, Victor de Gautard, Vytautas Navikas, Nadine Nelson, Bastian Nicolai, Joanna Kowal, Saska Brajkovic. Automated multiplex immunofluorescence enables single cell analysis of tumor stroma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4616.

BackgroundThe spatial interactions between the immune system and tumor cells greatly influence antitumoral immunity, patient prognosis, and therapeutic efficacy. However, few methods exist to query large numbers of immune biomarkers at subcellular spatial resolution. Launched earlier this year, the CosMx™ Spatial Molecular Imager (SMI) platform captures high-plex single cell and subcellular detection of proteins from FFPE tissues. To detect these key drivers of cancer progression and immune cell activation states and functions, we designed and validated a high-plex protein panel for the CosMx SMI platform. This panel contains 4 markers for cell segmentation and pre-experimental imaging and 64 barcoded antibodies emphasizing immuno-oncology focused targets.MethodsThe CosMx protein assay uses antibodies conjugated with oligonucleotides, which are detected using universal, multi-analyte CosMx readout reagents. The fully automated CosMx instrument carries a widefield water immersion objective with 1.1 NA. The CosMx Human Immuno-oncology panel was optimized to comprehensively phenotype lymphoid and stromal lineages within the tumor microenvironment. The CosMx protein assay reagents were validated on multi-organ FFPE tissue microarrays covering prevalent solid tumor types and matched controls, and 52 human FFPE cell lines, including overexpression lines for key targets such as GITR, CD278, PD-L1, and PD-1.ResultsWe achieved 86% sensitivity and 90% specificity across well-characterized human cell lines compared to GeoMx spatial profiling, and further benchmarked to multiple orthogonal datasets (e.g., the Human Protein Atlas, low-plex IHC). Within the tissue sample profiled, we captured immune cell localization across and within the tumor, key signaling markers related to lymphoid and myeloid activation such as checkpoint engagement, and myeloid cell polarization and antigen cross-presentation markers. We made the data from the study profiling cancer tissue freely available online. This includes the protein images, AI-based cell segmentation, and the per-cell protein abundance profiles.ConclusionsCosMx SMI is a high-plex spatial multiomics platform that enables detection of more than 64 proteins at subcellular resolution in real-world FFPE tissues. Our 64-plex human immuno-oncology protein panel enables in-depth study of the tumor microenvironment, including markers covering cell typing and lineage, immune activation, and checkpoints. This new platform will enable researchers to collect high-plex protein immunophenotyping data and understand immune molecular mechanisms with full spatial context.FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

Passive immunization using monoclonal antibodies will play a vital role in the fight against COVID-19. The recent emergence of viral variants with reduced sensitivity to some current antibodies and vaccines highlights the importance of broad cross-reactivity. This study describes deep-mining of the antibody repertoires of hospitalized COVID-19 patients using phage display technology and B cell receptor (BCR) repertoire sequencing to isolate neutralizing antibodies and gain insights into the early antibody response. This comprehensive discovery approach has yielded a panel of potent neutralizing antibodies which bind distinct viral epitopes including epitopes conserved in SARS-CoV-1. Structural determination of a non-ACE2 receptor blocking antibody reveals a previously undescribed binding epitope, which is unlikely to be affected by the mutations in any of the recently reported major viral variants including B.1.1.7 (from the UK), B.1.351 (from South Africa) and B.1.1.28 (from Brazil). Finally, by combining sequences of the RBD binding and neutralizing antibodies with the B cell receptor repertoire sequencing, we also describe a highly convergent early antibody response. Similar IgM-derived sequences occur within this study group and also within patient responses described by multiple independent studies published previously.

The <i>Journal of Astronomical Telescopes, Instruments, and Systems</i> (JATIS) publishes peer-reviewed papers reporting on original research in the development, testing, and application of telescopes, instrumentation, techniques, and systems for ground- and space-based astronomy.