Spatially multiplexed RNA in situ hybridization to reveal tumor heterogeneity

Abstract

Multiplexed RNA in situ hybridization for the analysis of gene expression patterns plays an important role in investigating development and disease. Here, we present a method for multiplexed RNA-ISH to detect spatial tumor heterogeneity in tissue sections. We made use of a microfluidic chip to deliver ISH-probes locally to regions of a few hundred micrometers over time periods of tens of minutes. This spatial multiplexing method can be combined with ISH-approaches based on signal amplification, with bright field detection and with the commonly used format of formalin-fixed paraffin-embedded tissue sections. By using this method, we analyzed the expression of HER2 with internal positive and negative controls (ActB, dapB) as well as predictive biomarker panels (ER, PgR, HER2) in a spatially multiplexed manner on single mammary carcinoma sections. We further demonstrated the applicability of the technique for subtype differentiation in breast cancer. Local analysis of HER2 revealed medium to high spatial heterogeneity of gene expression (Cohen effect size r = 0.4) in equivocally tested tumor tissues. Thereby, we exemplify the importance of using such a complementary approach for the analysis of spatial heterogeneity, in particular for equivocally tested tumor samples. As the method is compatible with a range of ISH approaches and tissue samples, it has the potential to find broad applicability in the context of molecular analysis of human diseases.

Single-Cell RNA Sequencing Reveals Endothelial Cell Transcriptome Heterogeneity under Homeostatic Laminar Flow

“…DNA methylation heterogeneity across cells was linked to splicing variability during differentiation of induced pluripotent stem cells in another study 18 . Multiplexed RNA in situ hybridization revealed high spatial heterogeneity of biomarker gene expression in tumor tissues, suggesting the potential application of spatial heterogeneity as a complementary approach for breast cancer subtype differentiation 19 . Thus intercellular/ interregional gene expression heterogeneity has functional consequences and clinical applications, underscoring its importance.…”

Transcriptional Spatial Profiling of Cancer Tissues in the Era of Immunotherapy: The Potential and Promise

“…Zhou et al demonstrated that seqFISH is a powerful tool to study and gain a snapshot of the regulatory gene expression dynamics in T-cell maturation [ 83 ]. More recently, Voith von Voithenberg et al combined microfluidics technology with multiplexed smFISH to study tumour heterogeneity in breast cancer; they demonstrated that multiplexed smFISH can be further optimized from different angles [ 84 ].…”

Loss of CDKN1A mRNA and Protein Expression Are Independent Predictors of Poor Outcome in Chromophobe Renal Cell Carcinoma Patients

“…In contrast to genes and proteins that are highly differentially expressed in cancer, the validation of low abundance genes as diagnostic and prognostic tools in tumor pathology is a big challenge. Branched probe-based or enzymatic amplification RNA-ISH methods for the detection and quantification of transcripts in FFPE tissues [55] may be ideally suited to evaluate cancer biomarker candidates on the mRNA level. Given the huge amount of survival-related gene expression data in the TCGA database, systematic and comprehensive gene expression profiling of such candidate genes are necessary to better understand the complex regulatory network along tumor progression, which may lead to new therapeutic strategies to treat aggressive chRCC.…”