Multiplexed Transcriptomics for Screening Drug Combinations and Defining the Mechanism of Action of <scp>HCC</scp> Therapeutics at Single‐Cell Resolution

Compared to classical drug screening, single-cell screening not only significantly enhances throughput but also provides richer transcriptional response information. In this study, we employed the high-throughput and high-sensitive single-nucleus sequencing platform, snHH-seq, to screen clinical drug combinations with anti-hepatocellular carcinoma (HCC) activity. Single-cell transcriptomics analysis revealed that the HY combination (HHT and YM155) exhibited the strongest suppression of tumour cell proliferation, a finding validated by both in vitro and in vivo functional assays. Further investigation suggested that HY triggers ferroptosis, as evidenced by rescue from cell death upon co-treatment with the ferroptosis inhibitor Fer-1. Subcluster analysis identified distinct tumour cell subclusters' responses to HY treatment. A gene regulatory network analysis highlighted JUN as a key regulator mediating proliferation inhibition, primarily active in the apoptotic cell subcluster. These findings illustrate how integrating high-throughput screening with mechanistic dissection can accelerate the discovery of targeted drug combination therapies, and offer a blueprint for precise interventions using pathway vulnerabilities and cellular heterogeneity in HCC.