Spatially Annotated Single Cell Sequencing for Unraveling Intratumor Heterogeneity.

Myrthe M Smit,Cecile Beerens,Yasin Begce,Jelle Storteboom,Kate J Feller,Li You,Miao-Ping Chien

doi:10.3389/fbioe.2022.829509

Abstract

Intratumor heterogeneity is a major obstacle to effective cancer treatment. Current methods to study intratumor heterogeneity using single-cell RNA sequencing (scRNA-seq) lack information on the spatial organization of cells. While state-of-the art spatial transcriptomics methods capture the spatial distribution, they either lack single cell resolution or have relatively low transcript counts. Here, we introduce spatially annotated single cell sequencing, based on the previously developed functional single cell sequencing (FUNseq) technique, to spatially profile tumor cells with deep scRNA-seq and single cell resolution. Using our approach, we profiled cells located at different distances from the center of a 2D epithelial cell mass. By profiling the cell patch in concentric bands of varying width, we showed that cells at the outermost edge of the patch responded strongest to their local microenvironment, behaved most invasively, and activated the process of epithelial-to-mesenchymal transition (EMT) to migrate to low-confluence areas. We inferred cell-cell communication networks and demonstrated that cells in the outermost ∼10 cell wide band, which we termed the invasive edge, induced similar phenotypic plasticity in neighboring regions. Applying FUNseq to spatially annotate and profile tumor cells enables deep characterization of tumor subpopulations, thereby unraveling the mechanistic basis for intratumor heterogeneity.

Highlights

Intratumor heterogeneity, both at the genetic and transcriptomic level, is commonly observed in various cancer types and complicates diagnosis and treatment (Gerlinger et al, 2012; Patel et al, 2014; Morrissy et al, 2017; Puram et al, 2017; Berglund et al, 2018)
We focused on the epithelial-tomesenchymal transition (EMT), as this is an important source for intratumor heterogeneity (Nieto et al, 2016)
Cells were first incubated with photoactivatable Janelia Fluor 646 (JF646) dye, after which we phototagged the outer one-third of the patch (Figure 1B; cells emit red fluorescence after photoactivation)

Summary

Introduction

Intratumor heterogeneity, both at the genetic and transcriptomic level, is commonly observed in various cancer types and complicates diagnosis and treatment (Gerlinger et al, 2012; Patel et al, 2014; Morrissy et al, 2017; Puram et al, 2017; Berglund et al, 2018). We have recently developed a functionally annotated transcriptomic profiling technique, called functional single cell sequencing (FUNseq), to study heterogeneous populations of tumor cells based on functional features (You et al, 2021). This technology uses live-cell imaging to identify cells with a phenotype of interest (e.g., cell migration or morphology), which can be Spatially Annotated Single Cell Sequencing phototagged (via a photopatterned device) with a photoactivatable dye, isolated and subjected to single-cell RNA sequencing (scRNA-seq). FUNseq links phenotypic traits to gene expression profiles of rare subpopulations of tumor cells, thereby identifying the underlying mechanisms of intratumor heterogeneity. Cells are currently labeled using a single dye, making it impossible to discern cells based on their spatial organization

Methods

Results

Conclusion