Multiplexed Transcriptomics for Screening Drug Combinations and Defining the Mechanism of Action of HCC 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.