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Updated: Aug 11, 2026
Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models
Published on: December 9, 2016
Spike-in normalization for single-cell RNA-seq reveals dynamic global transcriptional activity mediating anticancer
Xin Wang1, Jane Frederick2, Hongbin Wang1
1Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
Abstract:
The transcriptional plasticity of cancer cells promotes intercellular heterogeneity in response to anticancer drugs and facilitates the generation of subpopulation surviving cells. Characterizing single-cell transcriptional heterogeneity after drug treatments can provide mechanistic insights into drug efficacy. Here, we used single-cell RNA-seq to examine transcriptomic profiles of cancer cells treated with paclitaxel, celecoxib and the combination of the two drugs. By normalizing the expression of endogenous genes to spike-in molecules, we found that cellular mRNA abundance shows dynamic regulation after drug treatment. Using a random forest model, we identified gene signatures classifying single cells into three states: transcriptional repression, amplification and control-like. Treatment with paclitaxel or celecoxib alone generally repressed gene transcription across single cells. Interestingly, the drug combination resulted in transcriptional amplification and hyperactivation of mitochondrial oxidative phosphorylation pathway linking to enhanced cell killing efficiency. Finally, we identified a regulatory module enriched with metabolism and inflammation-related genes activated in a subpopulation of paclitaxel-treated cells, the expression of which predicted paclitaxel efficacy across cancer cell lines and in vivo patient samples. Our study highlights the dynamic global transcriptional activity driving single-cell heterogeneity during drug response and emphasizes the importance of adding spike-in molecules to study gene expression regulation using single-cell RNA-seq.
Insights
Cancer cells exhibit transcriptional plasticity, leading to survival heterogeneity. This study reveals how drug combinations alter gene expression, impacting cancer cell survival and predicting treatment efficacy.
Area of Science:
- Cancer Biology
- Molecular Oncology
- Genomics
Background:
- Cancer cells display transcriptional plasticity, contributing to heterogeneity and survival under drug treatment.
- Understanding single-cell transcriptional changes during drug response is crucial for improving cancer therapy efficacy.
Purpose of the Study:
- To investigate single-cell transcriptomic heterogeneity in cancer cells treated with paclitaxel, celecoxib, or their combination.
- To identify gene signatures associated with distinct cellular states and predict drug efficacy.
Main Methods:
- Single-cell RNA sequencing (scRNA-seq) was employed to analyze transcriptomic profiles.
- Random forest modeling was used to classify cells into transcriptional states.
- Gene expression was normalized using spike-in molecules.
Main Results:
- Paclitaxel and celecoxib alone generally repressed gene transcription.
- The drug combination induced transcriptional amplification and activated mitochondrial oxidative phosphorylation, enhancing cell killing.
- A specific gene module (metabolism and inflammation-related) predicted paclitaxel efficacy in cell lines and patient samples.
Conclusions:
- Global transcriptional activity dynamically regulates single-cell heterogeneity during drug response.
- Spike-in molecules are essential for accurate gene expression studies in scRNA-seq.
- Identifying predictive gene signatures can guide personalized cancer treatment strategies.
