Spatially-Resolved scRNA-Seq for Intratumoral Heterogeneity
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Solution Overview
Problem
Current methods for molecular profiling of immune cells are limited in understanding intratumoral heterogeneity and immune evasion mechanisms, as they fail to accurately measure gene expression changes in single cells and mask variations within cell subpopulations, especially in primary immune cells which are difficult to perturb using traditional transfection methods.
Innovation Solution
A spatially-resolved single-cell RNA sequencing (scRNA-Seq) approach that spatially tags cells in tumor sections with unique oligonucleotides to link neighboring tumor and immune cell behaviors, identifying novel immune evasion genes and validating their effects on tumor immunity using over-expression and knockout methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional bulk RNA sequencing is used to profile immune cells, then the overall gene expression profile can be obtained, but intratumoral heterogeneity and variations within cell subpopulations are masked
Solution Approach 1:
The patent segments the bulk tissue sample into individual single cells, allowing each cell to be profiled separately. This segmentation enables the detection of gene expression variations between individual immune cells and tumor cells, preserving intratumoral heterogeneity information that would be lost in bulk sequencing where all cells are averaged together.
Solution Approach 2:
The patent adds a spatial dimension to single-cell RNA sequencing by using in situ sequencing. This spatial context allows cells to be analyzed in their native tissue architecture, providing both single-cell resolution and spatial information about cell locations and relationships within the tumor microenvironment, thereby preserving heterogeneity information.
2Ease of manufacture
If traditional transfection methods are used to perturb immune cells, then gene over-expression or knockout can be achieved, but primary immune cells are difficult to perturb due to their biological properties
Solution Approach 1:
The patent replaces traditional mechanical transfection methods (electroporation, microinjection) with a chemical approach using lentiviral vectors. The lentiviral system delivers genetic material to primary immune cells through a biological mechanism (viral infection) rather than mechanical force, significantly improving transfection efficiency and reliability for cells that are difficult to perturb.
Solution Approach 2:
The patent uses lentiviral vectors as an intermediary to deliver genetic material into primary immune cells. The viral vector acts as a mediator that naturally infects immune cells and delivers the transgene, bypassing the need for direct mechanical transfection methods that are inefficient for these cells.
3Loss of information
If spatially-resolved single-cell sequencing is implemented, then neighboring cell states and microenvironmental regulators can be identified, but the device complexity and method complexity increase
Solution Approach 1:
The patent extracts the sequencing reaction from complex live-cell imaging systems and performs it directly on fixed tissue sections using in situ sequencing. This extraction simplifies the system by eliminating the need for complex live-cell manipulation and real-time imaging equipment, while still achieving spatially-resolved single-cell sequencing data.
Solution Approach 2:
The patent uses oligonucleotide barcodes as molecular copies to capture spatial information. Each cell is assigned a unique spatial barcode that copies its location information, allowing spatial reconstruction from sequencing data without requiring complex imaging equipment. The barcode acts as a simplified informational copy of the cell's spatial position.
Data Source
AI summary
The present invention provides tools and methods for the systematic analysis of genetic interactions between cells. The present invention provides tools and methods for modulating cell phenotypes and compositions, combinatorial probing of cellular circuits, for dissecting cellular circuitry, for delineating molecular pathways, and/or for identifying relevant targets for therapeutics development.