Probabilistic embedding, clustering, and alignment for integrating spatial transcriptomics data with PRECAST

Dimension reduction and (spatial) clustering is usually performed sequentially; however, the low-dimensional embeddings estimated in the dimension-reduction step may not be relevant to the class labels inferred in the clustering step. We therefore developed a computation method, Dimension-Reduction Spatial-Clustering (DR-SC), that can simultaneously perform dimension reduction and (spatial) clustering within a unified framework. Joint analysis by DR-SC produces accurate (spatial) clustering results and ensures the effective extraction of biologically informative low-dimensional features. DR-SC is applicable to spatial clustering in spatial transcriptomics that characterizes the spatial organization of the tissue by segregating it into multiple tissue structures. Here, DR-SC relies on a latent hidden Markov random field model to encourage the spatial smoothness of the detected spatial cluster boundaries. Underlying DR-SC is an efficient expectation-maximization algorithm based on an iterative conditional mode. As such, DR-SC is scalable to large sample sizes and can optimize the spatial smoothness parameter in a data-driven manner. With comprehensive simulations and real data applications, we show that DR-SC outperforms existing clustering and spatial clustering methods: it extracts more biologically relevant features than conventional dimension reduction methods, improves clustering performance, and offers improved trajectory inference and visualization for downstream trajectory inference analyses.

Spatial transcriptomics is an emerging technology that allows for analysis of cellular and molecular heterogeneity at spatial resolution. The accurate identification of pathological regions in spatial transcriptomics data is essential for understanding tissue heterogeneity and disease progression. We introduce UPSST, a comprehensive framework that integrates tissue morphology, imputes gene expression, and clusters spatial regions using a graph attention neural network (GAT). UPSST was evaluated across multiple spatial transcriptomics datasets, achieving high performance, such as achieving an Adjusted Rand Index (ARI) of 0.737 and a Fowlkes-Mallows Index (FMI) of 0.818 on slice 151671 of the LIBD human dorsolateral prefrontal cortex (DLPFC) dataset. These results highlight the robustness and precision of our approach in identifying pathology domains. Additionally, UPSST facilitates downstream analyses such as differential and enrichment analysis, which are crucial for deriving biological insights. In conclusion, UPSST offers a powerful and reliable tool for spatial transcriptomics analysis, advancing the identification of pathological regions with high accuracy.

Spatial transcriptomics is a powerful technology for high-resolution mapping of gene expression in tissue samples, enabling a molecular level understanding of tissue architecture. The acquisition entails dissecting and profiling micron-thick tissue slices, with multiple slices often needed for a comprehensive study. However, the lack of a common coordinate framework (CCF) among slices, due to slicing and displacement variations, can hinder data analysis, making data comparison and integration challenging, and potentially compromising analysis accuracy. Here we present a deep learning algorithm STaCker that unifies the coordinates of transcriptomic slices via an image registration process. STaCker derives a composite image representation by integrating tissue image and gene expression that are transformed to be resilient to noise and batch effects. STaCker overcomes the training data scarcity by training exclusively on diverse synthetic data. Its performance on various benchmarking datasets shows a significant increase in spatial concordance in aligned slices, surpassing existing methods. STaCker also successfully harmonizes multiple real spatial transcriptome datasets acquired from various platforms. These results indicate that STaCker is a valuable computational tool for constructing a CCF with spatial transcriptome data.

My talk will present three computational frameworks we developed to study cytokine signaling activities and cell-cell communications during the antitumor immune response, using tumor single-cell and spatial transcriptomics. The basic immunology tool to study cytokine signaling mostly measures cytokine release, which is transient and does not represent downstream target activities. Therefore, we first developed the CytoSig platform, providing a database of target genes modulated by cytokines and a predictive model of cytokine signaling cascades from transcriptomic profiles. We collected 20,591 transcriptome profiles for human cytokine, chemokine, and growth factor responses. This atlas of transcriptional patterns induced by cytokines enabled the reliable prediction of signaling activities in distinct cell populations in infectious diseases, chronic inflammation, and cancer using bulk and single-cell transcriptomic data. CytoSig revealed previously unidentified roles of many cytokines, such as BMP6 as an anti-inflammatory factor. Then, based on CytoSig, we developed Tres, a computational model utilizing single-cell transcriptomic data to identify signatures of T cells that are resilient to immunosuppressive signals, such as TGF-β1, TRAIL, and prostaglandin E2. Tres reliably predicts clinical responses to immunotherapy in multiple cancer types using bulk T cell transcriptomic data from pre-treatment patient tumors or infusion/pre-manufacture samples for cellular immunotherapies. Further, Tres identified FIBP as a candidate immunotherapy target to potentiate adoptive cell therapy efficacies. FIBP knockout in T cells enhanced adoptive cell therapy by down-regulating T cells' cholesterol metabolism. Last, I will briefly show our SpaCET framework for deconvolving cell fractions and identifying cell-cell interactions in tumor spatial transcriptomics data. SpaCET resolved several challenges in spatial transcriptomics analysis that previous methods did not address sufficiently. Through coupling cell fractions with ligand-receptor co-expression analysis, SpaCET reveals that intercellular interactions tend to be located at the tumor-immune boundaries. Citation Format: Peng Jiang. Inference of intercellular signaling activities in tumor spatial and single-cell transcriptomics, with applications in identifying cancer immunotherapy targets [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr IA002.

Spatial transcriptomics (ST) technologies enable the measurement of mRNA expression while simultaneously capturing spot locations. By integrating ST data, the 3D structure of a tissue can be reconstructed, yielding a comprehensive understanding of the tissue's intricacies. Nevertheless, a computational challenge persists: how to remove batch effects while preserving genuine biological structure variations across ST data. To address this, we introduce Graspot, a graph attention network designed for spatial transcriptomics data integration with unbalanced optimal transport. Graspot adeptly harnesses both gene expression and spatial information to align common structures across multiple ST datasets. It embeds multiple ST datasets into a unified latent space, facilitating the partial alignment of spots from different slices. Demonstrating superior performance compared to existing methods on four real ST datasets, Graspot excels in ST data integration, including tasks that require partial alignment. In particular, Graspot efficiently integrates multiple ST slices and guides coordinate alignment. In addition, Graspot accurately aligns the spatio-temporal transcriptomics data to reconstruct human heart developmental processes. Graspot software is available at https://github.com/zhan009/Graspot.

Spatial transcriptomics (ST) technologies enable dissecting the tissue architecture in spatial context. To perceive the global contextual information of gene expression patterns in tissue, the spatial dependence of cells must be fully considered by integrating both local and non-local features by means of spatial-context-aware. However, the current ST integration algorithm ignores for ST dropouts, which impedes the spatial-aware of ST features, resulting in challenges in the accuracy and robustness of microenvironmental heterogeneity detecting, spatial domain clustering, and batch-effects correction. Here, we developed an STMGraph, a universal dual-view dynamic deep learning framework that combines dual-remask (MASK-REMASK) with dynamic graph attention model (DGAT) to exploit ST data outperforming pre-existing tools. The dual-remask mechanism masks the embeddings before encoding and decoding, establishing dual-decoding-view to share features mutually. DGAT leverages self-supervision to update graph linkage relationships from two distinct perspectives, thereby generating a comprehensive representation for each node. Systematic benchmarking against 10 state-of-the-art tools revealed that the STMGraph has the optimal performance with high accuracy and robustness on spatial domain clustering for the datasets of diverse ST platforms from multi- to sub-cellular resolutions. Furthermore, STMGraph aggregates ST information cross regions by dual-remask to realize the batch-effects correction implicitly, allowing for spatial domain clustering of ST multi-slices. STMGraph is platform independent and superior in spatial-context-aware to achieve microenvironmental heterogeneity detection, spatial domain clustering, batch-effects correction, and new biological discovery, and is therefore a desirable novel tool for diverse ST studies

Although it has been established that the principal cause of death in COVID-19-infected patients is pneumonia and respiratory failure, little is currently known about the effects of COVID-19 on the lungs. Herein, we performed a single-nucleus RNA sequencing analysis of COVID-19 cases and controls using the human lung tissue data. Spatial transcriptomics was combined with single-nucleus RNA sequencing of human lung tissue to identify the specific cell subpopulations in COVID-19 cases and controls. We mapped ligand-receptor networks to specific cell types by combining single-cell and spatial data. Mapping the results revealed that fibroblasts are at the center of intercellular communication. These findings characterize the lung subpopulations, including fibroblasts and epithelial cells, the spatial niches in which they interact and the COVID-19 gene networks involved.

Pancreatic Cancer Ductal Adenocarcinoma (PDAC) is one of the deadliest cancers with 5-year survival of 11%. Understanding the intratumor heterogeneity is a pivotal piece to unravel the complexity of PDAC. While single cell RNASeq identifies the heterogeneous cell populations within the tumor tissue, spatially characterizing the transcriptomic profile of neoplastic and pre-neoplastic populations within the tissue remains a challenge, as the spatial dimension is usually lost upon tissue dissociation. We identified an approach to integrate spatial transcriptomics data with single cell RNASeq data. To characterize the cell populations within the tissue we performed single cell RNASeq on disease pathology-free pancreas tissue and primary PDAC samples. We profiled the transcriptomic profile of Acinar, Ductal, Acinar-to-Ductal (ADM), and Pancreatic Intraepithelial Neoplasia (PanINs) regions of interest (ROIs) across the tissue using the Nanostring GeoMx platform. Differential gene expression analysis using linear mixed-effect models of the cell-type specific ROIs defined pan-marker gene sets for each cell type, which were mapped to UMAP projections of single cell RNA sequencing data using AUCell scoring. As expected, the acinar pan-markers gene set derived from the spatial transcriptomics mapped to the manually annotated acinar population in the single cell data. On the other hand, Ductal, ADM, and PanIN pan-marker gene sets were mapped to distinct clusters that previously were not well-defined by single cell sequencing. The analysis coupled with orthogonal validation using RNAScope revealed gene signatures uniquely specific to ADM lesions and PanINs, respectively. Interestingly, our list included known markers as well as novel findings, supporting the validity of the findings. Furthermore, RNA velocity analysis using scVelo revealed a trajectory of cell evolution originating from acinar cells passing through the newly-defined ADM population and ending towards the ductal population derived from tumor samples. Overall, this integration approach of spatial and single cell transcriptomics can further define the characteristics that differentiate neoplastic and pre-neoplastic populations, as well as the potential drivers for tumorigenesis that could be therapeutically targeted. Citation Format: Ahmed M. Elhossiny, Eileen Carpenter, Padma Kadiyala, Yaqing Zhang, Filip Bednar, Arvind Rao, Timothy Frankel, Marina Pasca Di Magliano. Integrating single cell and spatial transcriptomics define gene signature for pancreatic ductal adenocarcinoma pre-neoplastic lesion [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A006.

Recent advances in spatial transcriptomics technologies have led to a growing number of diverse datasets, offering unprecedented opportunities to explore tissue organizations and functions within spatial contexts. However, it remains a significant challenge to effectively integrate and interpret these data, often originating from different samples, technologies, and developmental stages. In this paper, we present INSPIRE, a deep learning method for integrative analyses of multiple spatial transcriptomics datasets to address this challenge. With designs of graph neural networks and an adversarial learning mechanism, INSPIRE enables spatially informed and adaptable integration of data from varying sources. By incorporating non-negative matrix factorization, INSPIRE uncovers interpretable spatial factors with corresponding gene programs, revealing tissue architectures, cell type distributions and biological processes. We demonstrate the capabilities of INSPIRE by applying it to human cortex slices from different samples, mouse brain slices with complementary views, mouse hippocampus and embryo slices generated through different technologies, and spatiotemporal organogenesis atlases containing half a million spatial spots. INSPIRE shows superior performance in identifying detailed biological signals, effectively borrowing information across distinct profiling technologies, and elucidating dynamical changes during embryonic development. Furthermore, we utilize INSPIRE to build 3D models of tissues and whole organisms from multiple slices, demonstrating its power and versatility.

Rapid advances in spatial transcriptomics (ST) have revolutionized the interrogation of spatial heterogeneity and increase the demand for comprehensive methods to effectively characterize spatial domains. As a prerequisite for ST data analysis, spatial domain characterization is a crucial step for downstream analyses and biological implications. Here we propose a prior-based self-attention framework for spatial transcriptomics (PAST), a variational graph convolutional autoencoder for ST, which effectively integrates prior information via a Bayesian neural network, captures spatial patterns via a self-attention mechanism, and enables scalable application via a ripple walk sampler strategy. Through comprehensive experiments on data sets generated by different technologies, we show that PAST can effectively characterize spatial domains and facilitate various downstream analyses, including ST visualization, spatial trajectory inference and pseudotime analysis. Also, we highlight the advantages of PAST for multislice joint embedding and automatic annotation of spatial domains in newly sequenced ST data. Compared with existing methods, PAST is the first ST method that integrates reference data to analyze ST data. We anticipate that PAST will open up new avenues for researchers to decipher ST data with customized reference data, which expands the applicability of ST technology.

Since spatial transcriptomics can locate and distinguish the gene expression of functional genes in special regions and tissue, it is important for us to investigate the brain development, the development mechanism of brain diseases, and the relationship between brain structure and function in Neuroscience (or Brain science). While previous studies have introduced the crucial spatial transcriptomic techniques and data analysis methods, there are few studies to comprehensively overview the key methods, data resources, and technological applications of spatial transcriptomics in Neuroscience. For these reasons, we first investigate several common spatial transcriptomic data analysis approaches and data resources. Second, we introduce the applications of the spatial transcriptomic data analysis approaches in Neuroscience. Third, we summarize the integrating spatial transcriptomics with other technologies in Neuroscience. Finally, we discuss the challenges and future research directions of spatial transcriptomics in Neuroscience.

Spatial transcriptomics (ST) enables joint profiling of gene expression and spatial positions, thereby revealing spatially resolved biological functions. However, many existing ST analysis methods often fail to explicitly quantify the belief and uncertainty in decisions caused by noisy ST data, making it difficult to handle spots of varying quality in a fine-grained manner. In addition, domain identification is a fundamental and critical task in ST, but commonly used models that separate expression learning and clustering often struggle to learn cluster-friendly latent representations effectively. To address these issues, we propose PREST, a prototype-based evidence-aware integration framework for ST data. PREST performs multi-scale representation learning with fine-grained attention fusion and introduces learnable class prototypes to quantify belief and uncertainty in model decisions. We aim to align overall belief scores with latent semantic information to enhance uncertainty quantification and prototype learning, thereby promoting the learning of clustering-friendly representations. PREST further integrates an uncertainty-aware reconstruction module and spatial regularization to reduce overfitting to unreliable spots and promote denoised, discriminative representations. Extensive experiments on several benchmark datasets validate the effectiveness and superiority of our proposed PREST across various downstream tasks.

In recent years, the application of single-cell transcriptomics and spatial transcriptomics analysis techniques has become increasingly widespread. Whether dealing with single-cell transcriptomic or spatial transcriptomic data, dimensionality reduction and clustering are indispensable. Both single-cell and spatial transcriptomic data are often high-dimensional, making the analysis and visualization of such data challenging. Through dimensionality reduction, it becomes possible to visualize the data in a lower-dimensional space, allowing for the observation of relationships and differences between cell subpopulations. Clustering enables the grouping of similar cells into the same cluster, aiding in the identification of distinct cell subpopulations and revealing cellular diversity, providing guidance for downstream analyses. In this review, we systematically summarized the most widely recognized algorithms employed for the dimensionality reduction and clustering analysis of single-cell transcriptomic and spatial transcriptomic data. This endeavor provides valuable insights and ideas that can contribute to the development of novel tools in this rapidly evolving field.

Integrating single-cell and spatial multi-omics data is essential for resolving cellular identity, regulatory programs and tissue organization, yet remains challenging under realistic experimental designs. In practice, data are often incomplete, unpaired and partially overlapping across batches and platforms, resulting in mosaic settings where missingness, batch effects and limited correspondence are tightly coupled. Existing methods typically rely on shared cells, explicit anchors or post hoc mapping, and can become unstable as overlap diminishes or spatial structure is not explicitly modeled. Here we present GAMMI (Graph-guided Adversarial Mosaic Multi-omics Integration), a unified graph learning framework for mosaic integration of heterogeneous single-cell and spatial multi-omics data. Rather than performing direct cell or sample alignment, GAMMI learns biologically meaningful relational structure by jointly embedding cells and molecular features in a shared latent space using heterogeneous graphs that encode cell-feature, feature-feature and spatial adjacency relationships. An edge-based contrastive objective with missingness-aware negative sampling avoids false-negative supervision under unobserved modalities, while adversarial domain adaptation suppresses batch-associated variation at the embedding level. Spatial data are incorporated as structurally informative constraints during learning, enabling systematic enrichment of molecular representations across spatial locations. Across diverse mosaic single-cell benchmarks and spatial datasets, GAMMI consistently outperforms state-of-the-art methods in biological conservation, batch correction and spatial reconstruction, including in low-overlap and fully unpaired regimes.

Spatial transcriptomics (ST) measures gene expression while preserving spatial context within tissues. One of the key tasks in ST analysis is spatial domain detection, which remains challenging due to the complex structure of ST data and the varying performance of individual clustering methods. To address this, we propose SpatialESD, a Spatial EnSemble Domain detection method that integrates results from different spatial domain detection methods to improve spatial domain detection. SpatialESD captures both direct cooccurrence patterns and multiscale indirect relationships between clusters, improving the robustness and accuracy of spatial domain detection. We evaluated SpatialESD on simulated datasets and multiple 10x Visium spatial transcriptomics datasets, including human brain, breast cancer, and ovarian cancer samples. The results show that SpatialESD consistently outperforms individual methods and the existing EnSDD ensemble method in terms of clustering accuracy and stability. Based on the identified domains, we further detected domain-specific differentially expressed genes and performed trajectory and cell-cell interaction analyses. These results reveal spatial patterns of gene expression and cellular communication, offering insights into tissue organization and disease mechanisms. Overall, SpatialESD provides a reliable and effective solution for spatial domain detection in ST data and facilitates downstream biological discovery.