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Protocol for profiling in vitro intratumor heterogeneity using spatially annotated single-cell sequencing.

ABSTRACT: Here, we present a protocol for spatially annotated single-cell sequencing, a technique for spatially profiling intratumor heterogeneity with deep single-cell RNA sequencing and single-cell resolution. By combining live-cell imaging and photopatterned illumination, we describe steps to identify regions of interest in an in vitro tumor model, label the selected cells with photoactivatable dyes, and isolate and subject them to scRNAseq. This protocol can be applied to a range of cell lines and could be expanded to tissue sections. For complete details on the use and execution of this protocol, please refer to Smit et al. (2022).¹.

SUBMITTER: Smit MM

PROVIDER: S-EPMC10384656 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Protocol for profiling in vitro intratumor heterogeneity using spatially annotated single-cell sequencing.

Smit Myrthe M MM Feller Kate J KJ You Li L Chien Miao-Ping MP

STAR protocols 20230714 3

Here, we present a protocol for spatially annotated single-cell sequencing, a technique for spatially profiling intratumor heterogeneity with deep single-cell RNA sequencing and single-cell resolution. By combining live-cell imaging and photopatterned illumination, we describe steps to identify regions of interest in an in vitro tumor model, label the selected cells with photoactivatable dyes, and isolate and subject them to scRNAseq. This protocol can be applied to a range of cell lines and cou ...[more]

PMID: 37453069

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Project description:BackgroundCurrent bulk transcriptomic classification systems for bladder cancer do not consider the level of intratumor subtype heterogeneity.ObjectiveTo investigate the extent and possible clinical impact of intratumor subtype heterogeneity across early and more advanced stages of bladder cancer.Design setting and participantsWe performed single-nucleus RNA sequencing (RNA-seq) of 48 bladder tumors and additional spatial transcriptomics for four of these tumors. Total bulk RNA-seq and spatial proteomics data were available from the same tumors for comparison, along with detailed clinical follow-up of the patients.Outcome measurements and statistical analysisThe primary outcome was progression-free survival for non-muscle-invasive bladder cancer. Cox regression analysis, log-rank tests, Wilcoxon rank-sum tests, Spearman correlation, and Pearson correlation were used for statistical analysis.Results and limitationsWe found that the tumors exhibited varying levels of intratumor subtype heterogeneity and that the level of subtype heterogeneity can be estimated from both single-nucleus and bulk RNA-seq data, with high concordance between the two. We found that a higher class 2a weight estimated from bulk RNA-seq data is associated with worse outcome for patients with molecular high-risk class 2a tumors. The sparsity of the data generated using the DroNc-seq sequencing protocol is a limitation.ConclusionsOur results indicate that discrete subtype assignments from bulk RNA-seq data may lack biological granularity and that continuous class scores may improve clinical risk stratification of patients with bladder cancer.Patient summaryWe found that several molecular subtypes can exist within a single bladder tumor and that continuous subtype scores can be used to identify a subgroup of patients with poor outcomes. Use of these subtype scores may improve risk stratification for patients with bladder cancer, which can help in making decisions on treatment.

Dataset Information

Protocol for profiling in vitro intratumor heterogeneity using spatially annotated single-cell sequencing.

Publications

Protocol for profiling in vitro intratumor heterogeneity using spatially annotated single-cell sequencing.

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