E-spatial

Mapping out the coarse-grained connectivity structures of complex manifolds Biological systems often change over time, as old cells die and new cells are created through differentiation from progenitor cells. This means that at any given time, not all cells will be at the same stage of development. In this sense, a single-cell sample could contain cells at different stages of differentiation. By analyzing the data, we can identify which cells are at which stages and build a model for their biological transitions. By quantifying the connectivity of partitions (groups, clusters) of the single-cell graph, partition-based graph abstraction (PAGA) generates a much simpler abstracted graph (PAGA graph) of partitions, in which edge weights represent confidence in the presence of connections. In this notebook, we will introduce the concept of single-cell Trajectory Analysis using PAGA (Partition-based graph abstraction) in the context of hematopoietic differentiation.

The development of immune checkpoint-based immunotherapies has been a major advancement in the treatment of cancer, with a subset of patients exhibiting durable clinical responses. A predictive biomarker for immunotherapy response is the pre-existing T-cell infiltration in the tumor immune microenvironment (TIME). Bulk transcriptomics-based approaches can quantify the degree of T-cell infiltration using deconvolution methods and identify additional markers of inflamed/cold cancers at the bulk level. However, bulk techniques are unable to identify biomarkers of individual cell types. Although single-cell RNA sequencing (scRNAseq) assays are now being used to profile the TIME, to our knowledge there is no method of identifying patients with a T-cell inflamed TIME from scRNAseq data. Here, we describe a method, iBRIDGE, which integrates reference bulk RNAseq data with the malignant subset of scRNAseq datasets to identify patients with a T-cell inflamed TIME. Utilizing two datasets with matched bulk data, we show iBRIDGE results correlated highly with bulk assessments (0.85 and 0.9 correlation coefficients). Using iBRIDGE, we identified markers of inflamed phenotypes in malignant cells, myeloid cells, and fibroblasts, establishing type I and type II interferon pathways as dominant signals, especially in malignant and myeloid cells, and finding the TGFβ-driven mesenchymal phenotype not only in fibroblasts but also in malignant cells. Besides relative classification, per-patient average iBRIDGE scores and independent RNAScope quantifications were utilized for threshold-based absolute classification. Moreover, iBRIDGE can be applied to in vitro grown cancer cell lines and can identify the cell lines that are adapted from inflamed/cold patient tumors.

Spatial transcriptomics (ST) technology has allowed to capture of topographical gene expression profiling of tumor tissues, but single-cell resolution is potentially lost. Identifying cell identities in ST datasets from tumors or other samples remains challenging for existing cell-type deconvolution methods. Spatial Cellular Estimator for Tumors (SpaCET) is an R package for analyzing cancer ST datasets to estimate cell lineages and intercellular interactions in the tumor microenvironment. Generally, SpaCET infers the malignant cell fraction through a gene pattern dictionary, then calibrates local cell densities and determines immune and stromal cell lineage fractions using a constrained regression model. Finally, the method can reveal putative cell-cell interactions in the tumor microenvironment. In this notebook, we will illustrate an example workflow for cell type deconvolution and interaction analysis on breast cancer ST data from 10X Visium. The notebook is inspired by SpaCET's vignettes and modified to demonstrate how the tool works on BioTuring's platform.

Build single-cell trajectories with the software that introduced **pseudotime**. Find out about cell fate decisions and the genes regulated as they're made. Group and classify your cells based on gene expression. Identify new cell types and states and the genes that distinguish them. Find genes that vary between cell types and states, over trajectories, or in response to perturbations using statistically robust, flexible differential analysis. In development, disease, and throughout life, cells transition from one state to another. Monocle introduced the concept of **pseudotime**, which is a measure of how far a cell has moved through biological progress. Many researchers are using single-cell RNA-Seq to discover new cell types. Monocle 3 can help you purify them or characterize them further by identifying key marker genes that you can use in follow-up experiments such as immunofluorescence or flow sorting. **Single-cell trajectory analysis** shows how cells choose between one of several possible end states. The new reconstruction algorithms introduced in Monocle 3 can robustly reveal branching trajectories, along with the genes that cells use to navigate these decisions.