Spatial Sequence Research Articles

Guidelines of inclusive architecture design for autism spectrum disorder: What is new?

IntroductionAutism spectrum disorder (ASD) is a complex neuro-developmental condition. According to the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), restricted interests and repetitive behaviors and difficulties with social communication and interaction characterize ASD. Different ways of learning, moving, or paying attention are related to the degree of impairments. By reducing environmental and social obstacles in school, work, and other areas of life, architecture could play a pivotal role in helping people on the spectrum become more independent and acquire more abilities.ObjectivesThe aim of this study was to outline the recommendations and guidelines of the inclusive architecture design for ASD.MethodsWe conducted a comprehensive review of the scientific literature using the following keywords: inclusive design, architecture, autism or ADS.ResultsOur research found that the Autism ASPECTSS design index reported in 2013 by Magda Mostafa from Canada, which was based on the sensory design theory, is the world’s first set of evidence-based design guidelines for managing built environments to serve ADS individuals interaction, particularly in schools and workspaces. ASPECTSS conceptual framework delineate seven design concepts: acoustics, spatial sequencing, escape space, compartmentalization, transition spaces, sensory zoning, and safety. In 2023, the same author published an autism friendly design guide for the world’s first autism-friendly university. This guide is characterized by a better understanding of human-centered design and advocates beyond the mere inclusion, aspiring to a state where the boundaries between ‘normal’ and ‘special’ are blurred in order to treat all users as human beings with equal rights, thus calling for equal opportunities beyond the ADS spectrum.ConclusionsWith such well-established conceptual framework, it is nowadays imperative to expand our buildings in cities, schools, workplaces, hospitals, and public areas using the guidelines of autism-friendly environments. These buildings will enhance our individual and social well-being.Disclosure of InterestNone Declared

Spatial co-transcriptomics reveals discrete stages of the arbuscular mycorrhizal symbiosis

The symbiotic interaction of plants with arbuscular mycorrhizal (AM) fungi is ancient and widespread. Plants provide AM fungi with carbon in exchange for nutrients and water, making this interaction a prime target for crop improvement. However, plant–fungal interactions are restricted to a small subset of root cells, precluding the application of most conventional functional genomic techniques to study the molecular bases of these interactions. Here we used single-nucleus and spatial RNA sequencing to explore both Medicago truncatula and Rhizophagus irregularis transcriptomes in AM symbiosis at cellular and spatial resolution. Integrated, spatially registered single-cell maps revealed infected and uninfected plant root cell types. We observed that cortex cells exhibit distinct transcriptome profiles during different stages of colonization by AM fungi, indicating dynamic interplay between both organisms during establishment of the cellular interface enabling successful symbiosis. Our study provides insight into a symbiotic relationship of major agricultural and environmental importance and demonstrates a paradigm combining single-cell and spatial transcriptomics for the analysis of complex organismal interactions.

Sequence prediction of missing data of spatio-temporal series of marine environmental elements based on ST-ResNet model

Spatial data of marine environmental elements are of great significance to the marine industry and marine safety, and they are also affected by external factors. This results in a new method for spatial and temporal sequence prediction of marine environmental elements based on the ST-ResNet model. Specifically, the temporal distance, period, and trend of marine regions are modelled by residual networks, and different residual sub-networks are used for each characteristic, in each sub-network, different weights are assigned to different branches and regions by simulating the changes of marine data, and the output is finally fitted to obtain the output. The ST-ResNet model was validated on the ERA5 dataset as an example, and compared with other prediction models, the ST-ResNet model achieved better results, which verified the effectiveness of the method in predicting the temporal and spatial sequences of marine environmental elements.

Single-Nucleus Multiomic Analyses Identifies Gene Regulatory Dynamics of Phenotypic Modulation in Human Aneurysmal Aortic Root.

Aortic root aneurysm is a potentially life-threatening condition that may lead to aortic rupture and is often associated with genetic syndromes, such as Marfan syndrome (MFS). Although studies with MFS animal models have provided valuable insights into the pathogenesis of aortic root aneurysms, this understanding of the transcriptomic and epigenomic landscape in human aortic root tissue remains incomplete. This knowledge gap has impeded the development of effective targeted therapies. Here, this study performs the first integrative analysis of single-nucleus multiomic (gene expression and chromatin accessibility) and spatial transcriptomic sequencing data of human aortic root tissue under healthy and MFS conditions. Cell-type-specific transcriptomic and cis-regulatory profiles in the human aortic root are identified. Regulatory and spatial dynamics during phenotypic modulation of vascular smooth muscle cells (VSMCs), the cardinal cell type, are delineated. Moreover, candidate key regulators driving the phenotypic modulation of VSMC, such as FOXN3, TEAD1, BACH2, and BACH1, are identified. In vitro experiments demonstrate that FOXN3 functions as a novel key regulator for maintaining the contractile phenotype of human aortic VSMCs through targeting ACTA2. These findings provide novel insights into the regulatory and spatial dynamics during phenotypic modulation in the aneurysmal aortic root of humans.

Role of Tumor-Associated Macrophages in Cervical Cancer: Integrating Classical Perspectives with Recent Technological Advances.

Tumor-associated macrophages (TAMs) play a pivotal role in the tumor microenvironment, influencing cancer progression and contributing to poor prognosis. However, in cervical cancer (CC), their significance and involvement are relatively less studied than in other gynecological cancers such as ovarian and endometrial cancer. This review aims to provide an overview of TAMs, covering their origins and phenotypes and their impact on CC progression, along with major TAM-targeted therapeutic approaches. Furthermore, we advocate for the integration of cutting-edge research methodologies, such as single-cell RNA sequencing and spatial RNA sequencing, to enable in-depth and comprehensive investigations into TAMs in CC, which would be beneficial in leading to more personalized and effective immunotherapy strategies for patients with CC.

Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights.

Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset.

Semantic network analysis of spatial gene sequence in Dabaodao neighbourhood

ABSTRACT Dabaodao neighbourhood was a central gathering place of Chinese in the period of German colonial rule in Qingdao (north-eastern China), so the style and form of it express the integration of Chinese and Western cultures. With the help of the semantic network method, this paper proposes a gene analysis method suitable for historic neighbourhoods and analyses the genetic heritability of the Dabaodao neighbourhood. Regarding specific content, the spatial genes of the Dabaodao neighbourhood are identified as the primary research object, and two genetic research samples are proposed. Next, the spatial texture of the neighbourhoods is analysed to construct a semantic network model. Finally, with the help of the topological quantification method, a specific gene sequence analysis of the Dabaodao neighbourhood is conducted to discuss the degree of genetic influence expressed by it. On this basis, the effectiveness of gene analysis in neighbourhood renewal is discussed in the context of the renewal in the Dabaodao neighbourhood. Semantic network gene analysis not only provides an indicative strategy for the renewal of the Dabaodao neighbourhood but is also a formal analysis method different from the traditional quantitative analysis.

Abstract 6169: High throughput in-situ spatial sequencing of proteins and RNA in FFPE tissue

Abstract We have developed an high throughput high resolution in situ multi-omic platform to enable studies in the rapidly evolving landscape of oncology and immunology research. Despite the potential of existing tools, the speed and throughput of current methodologies pose significant limitations. Here, we introduce a novel spatial sequencing platform employing a rapid 4-color SBS chemistry with sub-micron resolution imaging and an ultra-high throughput capacity. The platform simultaneously profiles RNA transcripts and proteins within formalin-fixed paraffin-embedded (FFPE) tissues. With up to 40 cm^2 of tissue area imaging within a 24-hour timeframe, the platform can power extensive, multi-omic studies in cancer research. To evaluate the performance of the platform, we prepared 5 um serial sections from healthy and tumor kidney FFPE block and used a novel tool to precisely position multiple tissue sections on a slide. Following probe binding and amplification, we used SBS readout to profile 105 transcripts and 6 proteins (via DNA-conjugated antibodies). Images were analyzed using a custom pipeline for feature detection and decoding, and cell segmentation using Cellpose. Results showed strong correlation to single-cell transcription profiles and cell type signatures defined by scRNA-Seq. Additionally, we see good agreement between single cell transcript and protein profiles. Existing spatial biology platforms are known to struggle with degraded and challenging clinical sample types (e.g. decalcified bone marrow), due to RNA fragmentation. We successfully profiled immune cells in FFPE-embedded bone marrow biopsies, measuring 143 transcript targets and 8 proteins. We believe this represents a significant step forward for in-situ analysis of bone marrow samples, and could be valuable for studying the pathology and response to treatment in blood cancers such as AML and CML. In-situ sequencing of antibody genes and T-cell receptors provides a deeper view of cellular immune response. As a step towards this goal, we demonstrated in-situ sequencing of antibody heavy and light chains within a B cell line, resulting in accurate readout of 28 bases within the CDR3 regions. In conclusion, we present an SBS-based approach for in-situ detection of targeted gene transcripts and proteins at an unprecedented throughput, and its application to the analysis of human kidney and bone marrow FFPE tissue samples. The capability to deeply profile tissues at high throughput could enable larger translational studies, and assembly of 3D maps of gene and protein expression at sub-cellular resolution. Citation Format: Michael Lawson, Yuji Ishitsuka, Andrew Pawlowski, Zhenmin Hong, Jake Koh, Kenneth Gouin, Nathan Ing, Richard Que, Yeoan Youn, Sara Ding, Tony Facchini, Ryan Costello, Katelyn Nelson, Jamie Stover, Howon Lee, Sarthak Duggal, Taylor Plaziak, Kaitlin Cameron, Mimi Abdu, Daan Witters, Martin Fabani, Eli Glezer. High throughput in-situ spatial sequencing of proteins and RNA in FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6169.

Abstract 4312: KRAS oncogenic activation in Gramd2 + AT1 cells form lung adenocarcinoma with distinct transcriptomic and tumor microenvironment signatures

Abstract Lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer and presents with a high degree of biological heterogeneity and distinct clinical outcomes. The current paradigm of LUAD etiology recognizes alveolar epithelial type II (AT2) cells as the primary cell of origin, while the role of AT1 cells in LUAD oncogenesis remains relatively unknown. Here, we examined transformation via activation of the oncogenic KRASG12D mutation in a Gram-domain containing 2 (Gramd2)+ AT1 cell lineage tracing model system, which resulted in multifocal LUAD, primarily of papillary histology. Spatial transcriptomic sequencing revealed significant differences of transcriptomic signatures and signaling activities between Gramd2+ AT1 cell-derived and Sftpc+ AT2 cell-derived LUAD. Single-cell RNA sequencing of macrodissected LUAD lesions demonstrated heterogeneous cell composition of the tumor microenvironment (TME), with Gramd2+ AT1 cell-derived LUAD fostering a proinflammatory, immunoreactive TME and Sftpc+ AT2-derived LUAD presenting with an immunosuppressive TME. Collectively, our study suggests that cell of origin for LUAD is associated with histologic subtype, transcriptomic aberrations, and immune cell composition within LUAD that may influence response to therapy. Citation Format: Minxiao Yang, Jonathan Castillo, Edgar Gonzalez, Chunli Yan, Evanthia Roussos Torres, Zea Borok, Crystal Marconett. KRAS oncogenic activation in Gramd2 + AT1 cells form lung adenocarcinoma with distinct transcriptomic and tumor microenvironment signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4312.

Abstract 5389: Development of T-cell exclusion in melanoma

Abstract Immune checkpoint inhibitors (ICIs), which prevent immune cell exhaustion and boost an antitumor response, have shown remarkable treatment efficacy in patients with advanced melanoma. However, its efficacy is limited by the emergence of resistance associated with an immunosuppressive tumor microenvironment (TME). The contribution of abnormal tumor vasculature to limited immune cell infiltration into the tumor core has been reported but the mechanisms of its development are still elusive. We analyzed how tumor angiogenesis affects the immune cell exclusion at different time points of primary melanoma using syngeneic murine melanomas. The spatial distribution of various immune cell populations and vascular integrity were analyzed in whole tumor cross-sections via multiplex immunofluorescence staining. We found that the exclusion pattern of CD8+ T cells was less evident with a balanced distribution across the whole tumor regions at the early stage. However, as the tumor grows, there is a pronounced accumulation of CD8+ T cells at the tumor periphery with a conspicuous absence in the core. Tumor blood vessels were structurally uniform and functionally stable in the early developmental tumor but became unstable and leaky as the tumor progressed, which was associated with the development of CD8+ T-cell exclusion. To understand the mechanism underlying vascular regulation of immune exclusion during tumor progression, we will investigate the expression of genes related to tumor angiogenesis using spatial transcriptomic analysis and RNA sequencing of endothelial cells (CD45+CD31-) sorted from the murine melanomas. These results highlight the contribution of spatial tumor vascular destabilization to the T-cell exclusion development, implying the potential of targeting tumor vasculature to enhance immune activation and the therapeutic efficacy of ICI in melanoma. Citation Format: Ha-Ram Park, Hee Won Yang, Minah Kim. Development of T-cell exclusion in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5389.

Abstract 4407: Simultaneous spatial epigenomic and transcriptomic analysis of gastric adenocarcinoma reveals regulatory patterns governing tumor and microenvironment architecture at the cellular level

Abstract Recent advances in spatial transcriptomics and spatial proteomics have enabled increasingly complex questions on the nature of gene regulation and expression in cellular subtypes in tumor tissue and the tumor microenvironment. However, most spatial omics techniques do not profile the epigenomic landscape responsible for downstream gene expression. Furthermore, current spatial technologies have yet to profile the epigenome and transcriptome simultaneously, and thus it remains a challenge to correlate multi-omics data across sections of extremely heterogenous tumor tissue. Recently, co-profiling of spatial epigenomics and transcriptomics using principles of Deterministic Barcoding in Tissue for spatial omics sequencing (DBiT-seq) has been demonstrated on normal brain tissue. Joint spatial profiling of chromatin states and whole transcriptome in tissue allows for parallel characterization of gene regulation programs across all cell types, while preserving the tissue architecture for greater understanding of the cellular environment. Here we present the first application of spatial ATAC-seq and spatial transcriptomics on the same tissue section to characterize the tumor microenvironment of an invasive gastric adenocarcinoma (GAC) and adjacent normal tissue. GAC is the fifth most common cancer and commonly exhibits mutations in epigenetic modifiers, including ARID1A and MLL1-4. Distinct spatial clusters representing different cell subtypes were identified via both spatial chromatin accessibility and spatial transcriptomics. Spatial ATAC-seq profiling of accessible regulatory elements correlated well with RNA expression of target genes. Spatial patterns of transcription factor motif accessibility also correlated well with the observed transcriptional program of tumor tissue. When compared to adjacent normal tissue, spatial co-profiling of chromatin accessibility and the transcriptome revealed that the epigenetic landscape is significantly altered in tumorigenesis of GAC. Future work will focus on development of co-profiling of histone modifications and the transcriptome to enable the study of another layer of the epigenomic landscape, especially as targeting epigenetic modifiers such as EZH2 has been identified as a potential therapeutic strategy in GACs. Overall, we present a solution to profile multiple layers of gene regulation and expression with spatial context, which can be applied to most tumor types for better understanding of tumorigenesis and the consequences of new targeted therapies. Citation Format: Katelyn J. Noronha, Jennifer M. Garbarino, Daniel Massucci, Abigail R. Tyree, Colin Ng. Simultaneous spatial epigenomic and transcriptomic analysis of gastric adenocarcinoma reveals regulatory patterns governing tumor and microenvironment architecture at the cellular level [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4407.

Abstract 1910: Advancing therapeutic interventions for NUT carcinoma: Orchestrating the precision of CRISPR/Cas9-mediated NUTM1 suppression and synergistic ADC strategies

Abstract Purpose: NUT carcinoma (NC) is aggressive carcinoma defined by translocations involving the NUTM1 and frequent squamous differentiation. NUTM1 gene encodes a testicular nuclear protein (NUT), exclusively expressed in the testis. The fusion of NUTM1 leads to abnormal cell growth, resulting in the development of NC. This study aims to discover advanced approaches for inducing the death of cancer cells, specifically targeting those expressing NUT proteins. Methods: Spatial Transcriptome analysis and RNA sequencing were applied to formalin-fixed paraffin-embedded NUTM1 fusion-positive cancer tissues. Virus-like particles were used to introduce CRISPR/Cas9 into cells expressing the NUTM1 fusion oncoprotein. The inhibitory effect was confirmed through multiple in vitro assays. The effectiveness of Anti-Trophoblastic cell-surface antigen (TROP-2) Antibody-Drug Conjugate (ADC) was demonstrated in vivo and in vitro in cells with increased TROP2 expression subsequent to NUT suppression. Results: NCs revealed a relatively uniform pattern of gene expression, forming six unique groups. Notably, the NUT protein predominantly located in the inner regions of the tumor tissues. We employed CRISPR/Cas9 technology to specifically target cells harboring the NUTM1 fusion gene. The precise genetic editing was remarkably effective, achieving up to 90% success in the targeted area. This led to a notable decrease in both RNA and protein levels of NUT expression, effectively disrupting associated downstream pathways. The cell growth rate was reduced in several NC cell lines, and differentiation, accompanied by morphological changes, was observed in most edited cells. RNA sequencing analysis of these edited cells uncovered a substantial increase in expression of cytosolic molecules, with a marked increase in TACSTD2 expression. Interestingly, when we combined this genetic editing approach with a targeted therapy - the TROP2-ADC loaded with interferon-beta - the results were even more striking. Cells treated with this combination showed almost double the rate of growth inhibition compared to those receiving only a single form of treatment. Conclusion: This study provides valuable insights into identifying novel therapeutic targets and elucidating potential mechanisms by analyzing NUT cancer tissues using various assays. Our results support that CRISPR/Cas9-mediated targeting of NUTM1 could be a useful approach for NUT oncoprotein ablation, especially when used in combination with other therapies such as ADCs. Further studies are needed to investigate the efficacy of NUT inhibition in preclinical and clinical settings. the elimination of tumor cells facilitated by CRISPR/Cas9, along with additional tumor markers targeted by ADCs, holds potential for developing treatment strategies against incurable cancers driven by genetic alterations. Citation Format: Juyoung Choi, Hwa Lin Yi, Hee Geon Park, Misook Lee, Daesik Kim, Sung Youl Hong, Young Kee Shin, Yoon-la Choi. Advancing therapeutic interventions for NUT carcinoma: Orchestrating the precision of CRISPR/Cas9-mediated NUTM1 suppression and synergistic ADC strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1910.

Abstract 1620: Multiregion spatial sequencing of early-stage head and neck squamous cell carcinoma revealed theranostic intratumor heterogeneity

Abstract Introduction: Head and neck squamous cell carcinoma (HNSCC) is characterized by substantial intratumor heterogeneity (ITH) at the molecular level that may foster tumor evolution, adaptation, and therapeutic failure. Personalized-medicine strategies mostly rely on single tumor-biopsy samples which might not capture the mutational burden of heterogeneous tumors. We investigated genetic ITH portrayed from multiregion spatial sequencing in early-stage HNSCC patients. Patients and Method: Seventy-eight early-stage HNSCC patients treated with upfront curative-intent surgery in the prospective biobanking SCANDARE study (NCT03017573) were included. We developed a pathological measure of ITH, based on the relative contribution of multiple poorly and well-differentiated intratumor regions (n = 199) in resected tumor samples. To characterize genetic ITH, we performed targeted next-generation sequencing on multiple spatially separated intratumor regions by using a custom panel of 571 genes (named DRAGON), in combination with 3’-Tag RNA sequencing and pathway enrichment analysis. Results: Fifty-nine patients displaying a minimum of two sequenced intratumor regions (total, n = 180) were analyzed. We observed genetic ITH in 22/59 patients (37%), of whom 6/22 (27%) based on theranostic molecular alterations. Three of the six patients exhibited molecular alterations in PIK3CA (n = 2) and TSC2 (n = 1) that have supporting clinical evidence of actionability in some cancer types (OncoKB level 1) and three patients exhibited actionable molecular alterations in PTEN (n = 2) and CDKN2A/B (n = 1) supported by substantial research evidence (OncoKB level 4). Two patients exhibited molecular alterations in SMARCA4 that is likely oncogenic, albeit not yet annotated by OncoKB. The pathological measure of ITH accounted for a significant source of variation in gene expression. Notably, we identified 346 differentially expressed genes imposing p-values ≤ 0.05 and |log fold-change| ≥ 1 involved in several Reactome’s signaling pathways, including the cell junction organization (R-HSA-446728) and the tight junction interaction (R-HSA-420029) pathways. Conclusions: We reported clinically relevant genetic ITH in early-stage HNSCC, which can lead to underestimation of the mutational landscape portrayed from single tumor biopsy and may present major challenges to precision medicine. Our findings uncovered the biology behind ITH and may have translational implications for biomarkers discovery in HNSCC. Citation Format: Grégoire Marret, Sophie Vacher, Constance Lamy, Ladidi Ahmanache, Laura Courtois, Choumouss Kamoun, Maral Halladjian, Zakhia El Beaino, Jerzy Klijanienko, Charlotte Martinat, Emmanuelle Jeannot, Joey Martin, Guillaume Rougier, Nathalie Badois, Maria Lesnik, Antoine Dubray-Vautrin, Olivier Choussy, Wahib Ghanem, Rabah Taouachi, Julien Masliah Planchon, Nicolas Servant, Maud Kamal, Ivan Bièche, Christophe Le Tourneau. Multiregion spatial sequencing of early-stage head and neck squamous cell carcinoma revealed theranostic intratumor heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1620.

Abstract 2860: High-resolution mapping of oncogenic structural changes in osteosarcoma

Abstract We have leveraged long-read and spatial sequencing modalities to identify cancer-specific oncogenic alterations for treatment of osteosarcomas. Osteosarcoma is archetypal of cancers driven by aneuploidy and structural rearrangement rather than point mutations. The functional consequences of many SVs stem from their effect on multidimensional genome organization. SVs can enable enhancer hijacking, alter boundaries of topologically associated domains (TADs), and move gene loci to different regulatory compartments to affect gene expression. SVs can also lead to the formation of extrachromosomal DNA amplicons (ecDNA), which can be megabases in size and can incorporate both genes and regulatory elements to result in substantial increases in gene transcription and intratumor heterogeneity. Complex SVs may have profound implications for prognosis and treatment in OS and other cancers yet they remain poorly understood. By incorporating long-read optical genome mapping (OGM) and chromatin conformation capture sequencing (HiC), we studied SVs in osteosarcoma with the goal of elucidating their contribution to cancer development. We used a unique panel of 11 OS patient-derived xenograft (PDX) cell lines which have been previously characterized regarding response to targeted therapies as well as genomically via whole genome (WGS), RNA (RNA-seq), and chromatin accessibility (ATAC-seq) sequencing. To understand the complex genomic reorganization that occurs in osteosarcoma, we integrated OGM and WGS data for all 11 cell lines to describe detailed genome-wide SVs including ecDNA formation. PDX lines assayed using OGM exhibited between 106 and 704 translocations each (versus zero detected in germline controls) and we identified dozens of potential ecDNA amplicons, including one amplicon which may explain exceptionally high expression of YAP1 and BIRC3 transcripts. Further, we used HiC to describe the topological environment resulting from these SVs and observed potential enhancer hijacking events affecting dozens of known oncogenes including MYC and CDK4. HiC interaction maps among six cell lines reveal genomic compartments and TAD boundaries in detail, with 4,167 unique TAD boundaries identified among six cell lines using 50kb genomic bins. Despite the observed complexity of these genomes, we note that 71% of TAD boundaries are consistent in at least four of the six HiC assayed cell lines, and 44% are present in all six lines. Changing TAD boundaries are associated with numerous SVs and/or copy number discrepancies, and are associated with genomic regions that contain genes with dysregulated RNA transcript levels. Overall, OGM and HiC modalities add considerably to our ability to detect oncogenic SVs and the genes they affect. Citation Format: Andrew Scott Clugston, Stan Leung, Eunice Fuentes, Leanne Sayles, Megan Ostrowski, Coco Wu, Christina Curtis, Zhicheng Ma, Yanding Zhao, Georgi K. Marinov, Vijay Ramani, Marcus Breese, Alejandro Sweet-Cordero. High-resolution mapping of oncogenic structural changes in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2860.

Multi-slice spatial transcriptome domain analysis with SpaDo

With the rapid advancements in spatial transcriptome sequencing, multiple tissue slices are now available, enabling the integration and interpretation of spatial cellular landscapes. Herein, we introduce SpaDo, a tool for multi-slice spatial domain analysis, including modules for multi-slice spatial domain detection, reference-based annotation, and multiple slice clustering at both single-cell and spot resolutions. We demonstrate SpaDo’s effectiveness with over 40 multi-slice spatial transcriptome datasets from 7 sequencing platforms. Our findings highlight SpaDo’s potential to reveal novel biological insights in multi-slice spatial transcriptomes.

Integrated single-cell sequencing, spatial transcriptome sequencing and bulk RNA sequencing highlights the molecular characteristics of parthanatos in gastric cancer.

Parthanatos is a novel programmatic form of cell death based on DNA damage and PARP-1 dependency. Nevertheless, its specific role in the context of gastric cancer (GC) remains uncertain. In this study, we integrated multi-omics algorithms to investigate the molecular characteristics of parthanatos in GC. A series of bioinformatics algorithms were utilized to explore clinical heterogeneity of GC and further predict the clinical outcomes. Firstly, we conducted a comprehensive analysis of the omics features of parthanatos in various human tumors, including genomic mutations, transcriptome expression, and prognostic relevance. We successfully identified 7 cell types within the GC microenvironment: myeloid cell, epithelial cell, T cell, stromal cell, proliferative cell, B cell, and NK cell. When compared to adjacent non-tumor tissues, single-cell sequencing results from GC tissues revealed elevated scores for the parthanatos pathway across multiple cell types. Spatial transcriptomics, for the first time, unveiled the spatial distribution characteristics of parthanatos signaling. GC patients with different parthanatos signals often exhibited distinct immune microenvironment and metabolic reprogramming features, leading to different clinical outcomes. The integration of parthanatos signaling and clinical indicators enabled the creation of novel survival curves that accurately assess patients' survival times and statuses. In this study, the molecular characteristics of parthanatos' unicellular and spatial transcriptomics in GC were revealed for the first time. Our model based on parthanatos signals can be used to distinguish individual heterogeneity and predict clinical outcomes in patients with GC.

Sequence stratigraphy of the paleozoic successions in Ghadames Basin, southern Tunisia: Insights from integrated interpretation of well logs and seismic data.

The Ghadames Basin is considered as huge intracratonic structure, which preserved over 4000 m of Paleozoic sedimentary succession deposited over the Precambrian Basement. The present work consists of an integrated interpretation of the Paleozoic interval in the Ghadames basin of southern Tunisia using well logs and calibrated seismic data. Thus, the adopted sequence stratigraphic approach allowed the identification of a total of 3 s-order sequences, bounded by Taconic, Caledonian, and Hercynian unconformities recognized as major tectonic discontinuities in the study area. The determined second-order sequences have been further subdivided into fourteen third-order sequences with their associated systems tracts, which are limited by sequence boundaries (SB), and presenting transgressive surfaces (TS), and maximum flooding surfaces (mfs). Each sequence is characterized by a unique stacking pattern and distinctive facies, thus reflecting the lithology and the corresponding depositional environment. These sequences are composed of claystone and sandstone facies deposited in varying depositional environments, ranging from a shallow marine setting during the Ordovician period to a deep marine environment during the Early Silurian. A complex deltaic environment was evidenced during the Late Silurian involving to a fluvial environment during the Devonian period. The integration of well logs and seismic data proved to be an efficient approach in the delineation of sequence stratigraphy, revealing in detail the spatial distribution and sequence organizations within the study area. These later are controlled by the relative sea level variations, with eustasy is being the dominant factor. The obtained results are mainly correlatable with the sequence stratigraphy of North African basins at a regional scale and Snedden and Lee chart (2010) at a global scale.

Precursor exhausted CD8+T cells in colorectal cancer tissues associated with patient's survival and immunotherapy responsiveness.

Exhausted CD8+T cells represent a distinct cellular lineage that emerges during both chronic infections and cancers. Recent studies have shown that persistent antigen exposure can drive the differentiation of precursor exhausted CD8+T cells, termed Tpex cells, which are characterized as TCF-1+PD-1+CD8+T cells. Elevated Tpex cell frequencies in the tumor microenvironment (TME) are associated with improved overall survival (OS) in cancer patients and heightened responsiveness to anti-PD-1 therapy. In our present study, we utilized multi-color immunohistochemistry (mIHC) to determine the localization and clinical implications of tumor-infiltrating Tpex cells within the TME of human colorectal cancer (CRC) tissues. We also conducted a multi-omics integrative analysis using single-cell RNA sequencing (scRNA-seq) data derived from both the murine MC38 tumor model and human CRC tissues. This analysis helped delineate the transcriptional and functional attributes of Tpex cells within the CRC TME. Furthermore, we employed spatial transcriptome sequencing data from CRC patients to investigate the interactions between Tpex cells and other immune cell subsets within the TME. In conclusion, our study not only established a method for Tpex cell detection using mIHC technology but also confirmed that assessing Tpex cells within the CRC TME could be indicative of patients' survival. We further uncovered the transcriptional and functional characteristics of Tpex cells in the TME and ascertained their pivotal role in the efficacy of immunotherapy against CRC.

Domain generalization enables general cancer cell annotation in single-cell and spatial transcriptomics

Single-cell and spatial transcriptome sequencing, two recently optimized transcriptome sequencing methods, are increasingly used to study cancer and related diseases. Cell annotation, particularly for malignant cell annotation, is essential and crucial for in-depth analyses in these studies. However, current algorithms lack accuracy and generalization, making it difficult to consistently and rapidly infer malignant cells from pan-cancer data. To address this issue, we present Cancer-Finder, a domain generalization-based deep-learning algorithm that can rapidly identify malignant cells in single-cell data with an average accuracy of 95.16%. More importantly, by replacing the single-cell training data with spatial transcriptomic datasets, Cancer-Finder can accurately identify malignant spots on spatial slides. Applying Cancer-Finder to 5 clear cell renal cell carcinoma spatial transcriptomic samples, Cancer-Finder demonstrates a good ability to identify malignant spots and identifies a gene signature consisting of 10 genes that are significantly co-localized and enriched at the tumor-normal interface and have a strong correlation with the prognosis of clear cell renal cell carcinoma patients. In conclusion, Cancer-Finder is an efficient and extensible tool for malignant cell annotation.

Comparative Roles of the Caudate and Putamen in the Serial Order of Behavior: Effects of Striatal Glutamate Receptor Blockade on Variable versus Fixed Spatial Self-Ordered Sequencing in Marmosets.

Self-ordered sequencing is an important executive function involving planning and executing a series of steps to achieve goal-directed outcomes. The lateral frontal cortex is implicated in this behavior, but downstream striatal outputs remain relatively unexplored. We trained marmosets on a three-stimulus self-ordered spatial sequencing task using a touch-sensitive screen to explore the role of the caudate nucleus and putamen in random and fixed response arrays. By transiently blocking glutamatergic inputs to these regions, using intrastriatal CNQX microinfusions, we demonstrate that the caudate and putamen are both required for, but contribute differently to, flexible and fixed sequencing. CNQX into either the caudate or putamen impaired variable array accuracy, and infusions into both simultaneously elicited greater impairment. We demonstrated that continuous perseverative errors in variable array were caused by putamen infusions, likely due to interference with the putamen's established role in monitoring motor feedback. Caudate infusions, however, did not affect continuous errors, but did cause an upward trend in recurrent perseveration, possibly reflecting interference with the caudate's established role in spatial working memory and goal-directed planning. In contrast to variable array performance, while both caudate and putamen infusions impaired fixed array responding, the combined effects were not additive, suggesting possible competing roles. Infusions into either region individually, but not simultaneously, led to continuous perseveration. Recurrent perseveration in fixed arrays was caused by putamen, but not caudate, infusions. These results are consistent overall with a role of caudate in planning and flexible responding and the putamen in more rigid habitual or automatic responding.