The Spatial Biology Week™ 2023

Integrating the spatial omics approach with other multimodal techniques can enable a more comprehensive view of cell to cell interaction, tissue architecture, and pathological conditions. This session covers innovative research projects and their related methods that integrate multiple technologies from single cell to spatial multiomics and multimodal imaging. These approaches will enhance understanding of biological mechanisms underlying disease development, progression, and therapeutic resistance.

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Why does spatial organization of cells matter? Can these data help us to better understand disease progression? We need to explore the niche or neighborhood in the multicellular system we want to study. Since proximity speaks to cellular activity, spatial organization of cells will certainly play a role in modulating clinical outcomes.  However, this type of complex study cannot be performed due to the shortage of tools to analyze single cell with spatial context in the multicellular system such as cancer.  A tumor cell clearly recognizes the importance of its neighborhood and restructures the neighborhood to meet its needs for its own benefit. What if we compare this interaction to a model system developed by our own behaviours? How do we decide where we want to live in a city?

With higher-plexed imaging, we can now characterize spatially resolved mRNA (spatial transcriptomics). and protein (spatial proteomics) simultaneously.  An analogy to this is that we can tell whether an individual has enough money to buy food to live in a particular neighborhood of a city. These points bring us back to another aspect, tissue heterogeneity. To avoid redundancy, we need to analyze a single tissue section using as many omics platforms as we can so that one unique and reliable city map can be generated

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Glioblastoma (GBM) is the most common primary adult brain tumor, with standard-of-care therapy inevitably leading to therapy resistance in 96% of patients, who relapse with incurable disease. This talk will review the biological profile of treatment-refractory glioblastoma, and discuss the application of an integrative multi-omics target discovery platform for human GBM which incorporates DNA cellular barcoding, glycocapture profiling of the cell surface proteome, RNA sequencing, single-cell sequencing and genome-wide CRISPR screening of primary and recurrent GBM. We will introduce a scientific program designed to address our gap in knowledge of the spatial heterogeneity of GBM, which is built together with patients and families who chose to participate in an innovative rapid autopsy GBM donor program. 

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Whilst the peripheral immune response to COVID-19 infection is well characterised, less is known about the tissue response in the lungs of affected patients. We conducted a multi-omic investigation in post-mortem lung tissue from fatal COVID-19 infection comprising bulk Quantseq RNA sequencing, spatial transcriptomic profiling on the Nanostring GeoMx, multiplex immunohistochemistry on the Lunaphore COMET™ and in-situ transcriptomcics with RNAscope. We found the SARS-CoV-2 virus was progressively cleared in more temporally advanced disease. Furthermore, collagen VI was found to be transcriptionally up-regulated by bulk sequencing and within the interstitium on spatial transcriptomics. These findings were supported by multiplex immunohistochemistry at a proteomic level which showed fibrillar deposition of collagen VI in the interstitum. To validate these findings clinically, we looked at blood markers for collagen VI synthesis and degradation by ELISA in a cohort of severe and mildly infected COVID-19 patients. Blood markers of collagen VI synthesis (PRO-C6) was significantly predictive for mortality in hospitalised (p = 0.0065) and intensive care (p = 0.028) COVID-19 patients. Overall, blood PRO-C6 levels, based on increase interstitial lung deposition, could help identify high-risk of patients enabling better treatment stratification. The study also demonstrates the power of post-mortem investigation and spatial profiling in elucidating clinically relevant insights into complex disease phenomena.

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RNAscope in situ hybridization (ISH) technology is powerful method for detecting RNA expression with single molecule sensitivity in spatial and morphological context. Backed by over 8,000 peer reviewed publications, RNAscope technology enables visualization of the widest range of diverse RNAs commercially available. 

In this presentation, Michaeline Bunting, PhD. from the Spatial Biology Division at BioTechne will discuss RNAscope™ multiomic spatial applications and how they can be used to uncover new insights into health and disease and advance therapeutic development. Learn more about how RNAscope works and future directions to automate RNAscope on the Lunaphore COMET™ system to develop the first fully automated same-slide spatial multiomics solution.  

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The brain is a complex organ that exhibits cellular and molecular heterogeneity within and between brain regions. This session features cutting edge research projects where spatial biology has been used to interrogate the brain and gather previously unknown insights. The discussions emphasize the field’s transformative impact on advancing knowledge of brain function and its diseases.

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To increase the throughput of spatial profiling of the human central nervous system (CNS), a CNS-specific library of 100+ markers with an associated analysis platform was developed and validated using the Lunaphore COMET™. Optimizing antibody concentrations, cycle order, and standard operating procedures, while managing subsequent image analysis with background and autofluorescence subtraction, have allowed for the visualization of molecular signaling at the subcellular level. Our approach is project-driven, from onboarding projects to interrogation sessions with investigators, with the associated panels customized to each specific scientific question. This strategy can be used to validate data such as single-cell sequencing or for novel discovery and hypothesis generation.

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Over the past decade, emerging technologies have enabled investigators to begin to bridge the gap between cancer and neuroscience research to unveil the interactive roles of nerves in cancer. We are now beginning to define how the nervous system contributes to cancer initiation, growth, spread, recurrence, and even resistance to oncologic therapeutic strategies. Indeed, neuromodulation with both genetic and pharmacological approaches has been shown to affect not only tumor growth but also antitumor immune response. Collectively, studies have demonstrated a significant and pressing need to recognize cancer neuroscience interplay as a hallmark of cancer to 1) establish the neoneurogenic process as a highly relevant therapeutic target for both the prevention and treatment of cancer, 2) foster interdisciplinary cross-talk among experts in cancer biology and neuroscience, which have traditionally progressed along parallel paths, and 3) integrate human variables such as biological sex, age, race, and gender as underlying factors that can impact cancer neuroscience.  

This session introduces the current viewpoint of burgeoning cancer neuroscience field and discusses the most seminal papers over the last 10 years as well as promising up-and-coming directions of the field towards its establishment as a hallmark of cancer and implement it as a therapeutic approach in clinical oncology.

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Talk abstract N/A.

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Intratumor heterogeneity underlies cancer evolution and treatment resistance, but targetable mechanisms driving intratumor heterogeneity are poorly understood. Meningiomas are the most common primary intracranial tumors and are resistant to all current medical therapies. High-grade meningiomas cause significant neurological morbidity and mortality and are distinguished from low-grade meningiomas by increased intratumor heterogeneity arising from clonal evolution and divergence. Here we integrate spatial transcriptomic and spatial protein profiling approaches across high-grade meningiomas to identify genomic, biochemical, and cellular mechanisms linking intratumor heterogeneity to molecular, temporal, and spatial evolution. We show divergent intratumor gene and protein expression programs distinguish high-grade meningiomas that are otherwise grouped together by current clinical classification systems. Analyses of matched pairs of primary and recurrent meningiomas reveal spatial expansion of sub–clonal copy number variants underlies treatment resistance. Multiplexed sequential immunofluorescence (seqIF) and spatial deconvolution of meningioma single-cell RNA sequencing show decreased immune infiltration, decreased MAPK signaling, increased PI3K-AKT signaling, and increased cell proliferation drive meningioma recurrence. To translate these findings to clinical practice, we use epigenetic editing and lineage tracing approaches in meningioma organoid models to identify new molecular therapy combinations that target intratumor heterogeneity and block tumor growth. Our results establish a foundation for personalized medical therapy to treat patients with high-grade meningiomas and provide a framework for understanding therapeutic vulnerabilities driving intratumor heterogeneity and tumor evolution. 

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Spatial biology techniques have become highly valuable in biomarker development, where bulk analyses have reached a bottleneck. Researchers are pursuing novel biomarker signatures to better understand diseases and stratify patients. This session showcases real world examples of spatial biology studies to identify clinically relevant biomarkers. These case studies demonstrate how the identification and characterization of spatial biomarkers are fundamental for personalized medical and diagnostic approaches.

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With the advent of complement therapeutics, it becomes critical to understand whether and how complement contributes to the pathophysiology of diseases affecting the kidney to provide personalized treatments. During the talk, we will illustrate how we developed an ultra-high in situ Complementomics approach using the COMET™ Imager. We combined the staining for complement proteins, receptors and activation fragments with tissue structure markers and markers for phenotyping of the immune infiltrate.  Through this innovative approach, we successfully untangled the intricacies of complement activation within diverse kidney diseases and kidney cancers, as well as its interconnectedness with the infiltration of immune cells. Ultimately, this method sheds light on the role of complement in various disorders, opening doors to the discovery of novel biomarkers and therapeutic strategies.

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High dimensional morphological profiling allows the generation of unbiased fingerprints indicative of cell states, and allows identification of genes lying on common pathways or drugs acting via common mechanisms of actions. We have developed a system for pooled genetic screening of iPSC-derived neurons which combines peptide barcoding of short guide RNAs for CRISPR perturbation; a panel of antibodies for morphological and cell state characterization via highly multiplexed immunostaining; and subsequent feature extraction pipelines to reduce the data dimensionality into useful feature vectors. Our pilot experiments validate computationally predicted interactions between genes related to Huntington’s disease.

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Intraductal papillary mucinous neoplasms (IPMN) of the pancreas are bona fide precursor lesions of pancreatic ductal adenocarcinoma (PDAC). The most common subtype of IPMNs harbors a gastric foveolar-type epithelium, and these low-grade mucinous neoplasms are harbingers of IPMNs with high-grade dysplasia and cancer. To explore the molecular underpinning of gastric differentiation in IPMNs, we conducted spatial transcriptomics on a cohort of IPMNs, followed by hyper-plex cyclic immunofluorescence staining using the COMET™ platform and cross-species validation studies. Our study identifies NKX6-2 as a key transcription factor driving indolent gastric differentiation in IPMN pathogenesis. 

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Clinical studies with immune checkpoint inhibitors (ICI) have shown limited response rates, yet several case reports have shown encouraging responses and outcomes. Novel, combined modality treatments, including immunotherapies, have the potential to be effective in chordoma, a disease where conventional cytotoxic chemotherapy and radiotherapy to date have failed to provide meaningful long-term control. To optimize an immunotherapy intervention, we need a comprehensive understanding of the immune composition, the dynamic communication between cells, and the expression of immune checkpoint molecules of chordomas. Here, we comprehensively characterized the immune landscape of chordomas by employing high throughput methods such as single-cell transcriptomics (scRNA-seq), and single-T–cell/B–cell receptor sequence. Our data show that chordomas induce tumor-reactive B- and T-cell immunity inside and outside (blood) the tumor. Spatial transcriptomics and proteomics revealed that most T cells localize in the peritumoral areas, with a broad expression of PD-1 and CTLA-4, and exhausted phenotype. Investigating the cell-cell interactions driving tumor-mediated T-cell exhaustion, we identified M2 tumor-associated macrophages and a plasma cell phenotype-like subset upregulating TGFB and IL10 expression, referred to as regulatory B cells (Bregs). Thus, immunosuppression governs the chordoma microenvironment. However, the existence of tumor-reactive T cells in the circulation that are not subject to the tumor immunosuppressive suggests that a neoadjuvant ICI intervention is preferred over an adjuvant intervention. Using DNA methylome, we established a fast and easy way to define the immune composition of the tumor from biopsies. Thus, the DNA methylome of biopsies might be an effective way to stratify and select patients is a high probability to respond to neoadjuvant ICI.

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As the number of biomarkers detected by spatial biology techniques increases, the complexity of images escalates, and their interpretation becomes more challenging. Quantitative data must be extracted from images to infer cellular interactions and dependencies. Speakers showcased novel workflows to transform hyperplex images into biologically relevant datasets that can address basic and translation research questions.

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Highly-multiplexed imaging technologies enable the acquisition of dozens of markers generating large amounts of data in the process. The streamlined processing of such complex data through steps like image preprocessing, single cell segmentation, quantification of marker intensities and cell phenotyping poses a major challenge. In this talk, I will highlight how we utilized MCMICRO to process terabytes of Lunaphore COMET™ data with a focus on myocardial infarction.

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Tumor buds are clusters of up to 4 tumor cells that detached themselves from the main tumor. They are a powerful prognostic factor in colorectal cancer among others. However, they are also notoriously cumbersome to annotate and investigate, as there currently exist no phenotypic marker that differentiates them from other tumor cells.

 
We’ve built a pipeline to automatically detect tumor buds on COMET™ images as well as assess their expression patterns. I will discuss problems and lessons learned when working with sequential immunofluorescence images, and how it is important to take the biological context of the research question into account. 

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Spatial biology enables the interrogation of tissue architecture on a single-cell level. Transforming the rich image output into meaningful quantitative data is the next significant hurdle on the path to fully comprehend and interpret the intricate imaging data. HORIZON™ is an image analysis software tailored to COMET™ hyperplex immunofluorescence images. It seamlessly extends the COMET™ workflow, smoothly handling the large data sets. With its straightforward and user-friendly approach, this analysis tool supports cell-based analysis and provides researchers with a toolset for early and high-level assessment of data in a hyperplex immunofluorescent image. We will walk through image analysis workflow on HORIZON™ from a COMET™ image to biological insights.

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The tumor microenvironment (TME) is the seat of multiple cell interactions, including tumor cells, immune cells, stromal cells, and others. The identification of the markers expressed, and their spatial distribution can help not only to establish a prognosis of the disease, but also to direct therapeutic selection.

In this presentation, Mr. Pedro Espinosa Gonzalez presents how the HALO® and HALO AI platform can be used to analyze images of immuno-oncology panels encompassing a highly multiplexed biomarker panel from the COMET™ platform.

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Understanding the immune system in solid tissues requires highplex staining, imaging, and analysis for multi-marker cellular phenotypes. We present a comprehensive workflow for highplex images, covering tissue and cell segmentation and cellular phenotyping in a single software package. Visualization templates for marker subsets and pre-trained nuclear segmentation algorithms are reusable and paint-to-train tissue segmentation is easy to perform. Spatial biodistribution metrics, heatmaps and interactive partitioned t-SNE plots can be generated for each tissue type with a minimum of work. This workflow for highplex image analysis enables biologists and immunologists by circumventing the need for expert programming for each specific application. 

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Spatial biology is the new frontier in life sciences. However, despite the great potential of spatial techniques, their adoption remains challenging for many laboratories In this session, attendees will discover how spatial biology enables success. From basic research laboratory to translational research group, success stories stemming from Lunaphore’s Access Lab projects featured their journey in quickly adopting spatial biology and integrating a customized workflow in the field of immunology, immuno oncology, and beyond.

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Cancer (PCa) is a predominant male cancer, with rising incidence and progression rates emphasizing the need for effective treatments. Chronic inflammation plays a pivotal role in PCa progression, influencing genetic changes and the tumor environment. This is exacerbated by reactive oxygen species leading to DNA damage, increasing mutation risks, and altering the tumor environment to support growth. Contrary to traditional beliefs, our study of 825 samples revealed significant inflammation in prostate cancer. Using Hyperplex IF techniques, we discerned distinct CD3+ cell patterns in castration-resistant prostate cancer (CRPC), correlating with patient survival. Notably, nodular inflammation patterns hinted at tertiary lymphoid structures (TLSs) fostering immunosuppression. Further analyses differentiated between “hot” and “cold” tumors, and highlighted associations with mature and immature TLSs, potentially indicative of different immune responses. Our findings provide a nuanced understanding of the CRPC immune context, presenting avenues for enhanced immunotherapy and improved patient prognosis in prostate cancer treatment. 

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The immune microenvironment of pediatric gliomas is relatively understudied despite being a leading cause of childhood mortality. Pilocytic astrocytomas (PA) are the most common pediatric glioma and surgical resection is often curative. However, PAs are not always amenable to surgery and can have significant long-term morbidity and mortality. To identify PA patients that may be responsive to immune checkpoint inhibitors and to guide prioritization of available immunotherapeutic treatments, orthogonal immune profiling strategies were used to identify patients that may benefit from the next generation of immune therapeutics. 

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Non-Hodgkin lymphomas are a very heterogenous group of tumors arising from lymphocytes at different stages of differentiation. In order to reliably anticipate responses to treatment, lymphoma models must preserve the spatial organization and the functional interdependency between malignant and non-malignant cells. However, modeling lymphoma ex vivo is challenging due its highly heterogeneous nature and its intrinsic cellular composition. To address these challenges, we have established a tissue explant culture system for murine and human lymphoma that can potentially be used to assess therapy response in lymphoma patients (personalized medicine) and can help uncover novel aspects of lymphoma biology. 

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