ScRNA-seq is particularly powerful for investigating mechanisms of action as it resolves which cells respond to a therapy, how they respond at the pathway level, and why responses differ across subpopulations.
Specific cellular pathways activated in response to therapies
Therapeutic failure in cancer often stems from the emergence of drug resistance, driven by dynamic adaptations in both cancer cells and the TME. Therapy exposure can remodel cellular plasticity, differentiation states, and immune–stromal interactions, fostering a resistant tumor phenotype. Using single-cell RNA-seq with combinatorial barcoding, investigators of a seminal PDAC study found therapy-induced remodeling of the TME—such as enhanced IL-6 family signaling (CLCF1–CNTFR, LIF–IL6ST) between cancer-associated fibroblasts and neural-like cancer stem cells—driving STAT3 activation and resistance through altered ligand–receptor communication.
Similar studies leverage CITE-seq, scATAC-seq, and spatial transcriptomics to profile on-target effects and adaptive rewiring in situ, combined with computational tools (GSVA, RNA velocity, NicheNet) to map pathway activity, predict functional interactions, and identify biomarkers for combination therapy.
Longitudinal single cell profiling, like before, during, and post-treatment, can link specific pathway activation to resistant clones, informing strategies such as co-targeting IL-6/JAK-STAT signaling with chemotherapy or blocking ABC transporter activity alongside PI3K inhibition
How different cancer cell states respond to therapeutics, and what mechanisms could underpin these responses
Over 90% of cancer mortality is attributed to the development of multidrug resistance (MDR).
MDR rises from mainly two fronts: CSCs and the protection conferred to the tumor by the TME.
Cancer stem cells (CSCs), are often quiescent (G0 phase), and highly proficient in DNA repair, thanks to the ATM/ATR–CHK1/CHK2 pathways that render them resistant to DNA-damaging and proliferation-targeted therapies such as radiotherapy or cisplatin or cyclophosphamide.
CSCs resist therapy by expressing high levels of aldehyde dehydrogenases that detoxify drug-induced aldehydes and by upregulation of genes like BCL2 family which protects them from radiation, and chemotherapies like cisplatin, or doxorubicin.
Their stemness and capacity for self-renewal, migration, and colonization are maintained through Notch, Wnt/β-catenin, and PI3K/Akt/mTOR signaling. The tumor microenvironment (TME) further enhances resistance: CAFs and TAMs release cytokines that activate pro-survival pathways ( STAT3, NF-κB), while endothelial cells supply oxygen and growth factors that buffer oxidative stress. Single cell analysis of cancer and TME can reveal these pathways and mutation-driven responses, accelerating the discovery of new targets.
What are the off-target effects of novel chemotherapeutics
Novel and known agents can have unwanted effects when they bind or attack unintended, healthy cells. These therapies damage the bone marrow, the gastrointestinal and cardiovascular systems; kidneys and liver functions are also impaired.
The underlying mechanisms include DNA damage, impaired DNA and RNA synthesis, mitochondrial dysfunction with increased production of ROS resulting in cellular toxicity and death.
ScRNA-seq is being applied to investigate the off-target effects of novel therapeutics. In a study on medulloblastoma (MB) scRNA-seq with combinatorial barcoding was used to investigate OLIG2 expression across MB subgroups.
OLIG2 is a transcription factor that in the developing brain promotes differentiation in the oligodendrocyte lineage, but also maintains neural stem and progenitor cells in an undifferentiated, stem-like state that drives tumor growth. The authors assessed the therapeutic potential of OLIG2 inhibition using a novel small molecule, CT-179. The CT-179 treatment significantly decreased OLI2 expression, demonstrating that CT-179 specifically blocked OLIG2-driven transcription, making it a candidate novel therapeutic.
Another study explores the power of combining two therapeutics in combating GB by targeting the CSCs, recognized as a major barrier to effective cancer therapy, persisting after conventional treatments that eliminate bulk tumor cells. However, in a study on glioblastoma, scRNA-seq demonstrated that a combined treatment with transient radiation and adenylcyclase activator forskolin led to loss of glioma stem cells and prolonged median survival in mouse models of glioblastoma, opening a path to effective therapy by forcing CSCs into differentiation.
Ask a Single Cell
Investigating Mechanisms of Action with Single Cell Sequencing
Understanding how therapies function at the cellular level requires resolving how different cell types respond immediately and directly to treatment. Tumors comprise diverse cancer and non-malignant cell populations whose transcriptional programs and signaling pathways are differentially modulated by therapeutic exposure. Single cell RNA sequencing enables systematic interrogation of these responses, linking drug treatment to pathway modulation, cellular state changes, and shifts in cell-cell communication across the tumor ecosystem.
Characterizing cellular responses to therapy
- How do cancer cells and non-malignant cells respond transcriptionally to therapeutic intervention?
- Which signaling pathways and gene programs are modulated following treatment?
- Are therapeutic responses uniform across cells, or do they vary by cell type or state?
Mapping pathway engagement and functional impact
- Which molecular pathways are directly affected by treatment in different cellular compartments?
- How does pathway modulation manifest across transcriptional programs and cellular functions?
- Are observed transcriptional changes consistent with the intended mechanism of action?
Characterizing treatment-induced changes in cellular state
- Does therapy alter differentiation, activation, or functional states within cancer or stromal populations?
- How do cell state distributions shift following treatment exposure?
- Which cellular programs are altered as an immediate consequence of therapy?
Assessing system-level effects and off-target responses
- How does treatment influence communication between cancer, immune, and stromal cells?
- Which non-malignant cell types exhibit transcriptional responses to therapy?
- Are stress, damage, or compensatory programs induced outside the intended target population?
TLDR: The impact of a therapy is impacted by the cellular responses it triggers across a heterogeneous tumor. Even when a drug engages its intended target, downstream signaling and transcriptional effects can vary widely across cancer, immune, and stromal cells. These differences shape efficacy, adaptation, and off-target effects. Single cell RNA sequencing reveals how therapies act within individual cells, exposing pathway engagement, state transitions, and changes in cell to cell communication that are obscured in aggregate measurements. This chapter examines how resolving mechanisms of action at cellular resolution clarifies therapeutic effects and guides rational treatment design.
