Clonal lineage tracing and parallel multiomics profiling reveal transcriptional diversification induced by ARID1A deficiency

Phenotypic heterogeneity among genetically identical cancer cells underpins tumor progression and therapy resistance. However, how epigenetic dysregulation drives such divergence remains unclear. Here, we introduce IMPACH, a scalable platform that integrates lineage tracing, genetic perturbation, and multimodal single-cell analysis. Applying IMPACH, we demonstrate that loss of epigenetic regulators—particularly ARID1A—enhances transcriptomic and epigenetic heterogeneity under controlled conditions. ARID1A deficiency promotes stochastic chromatin opening and induces an atypical gene expression program associated with poor clinical outcomes. Despite increased epigenetic randomness, chromatin changes remain localized to biologically meaningful regulatory elements, partially linked to SMARCA4 binding sites. These findings reveal that chromatin remodeling defects promote clonal diversification through both stochastic and constrained mechanisms. Given its scalability and versatility, IMPACH provides a powerful framework for dissecting how epigenetic perturbations shape cellular heterogeneity in cancer. shPseuMO-Tag constructs targeting ARID1A (two independent shRNAs: ARI3# and ARI4#) or a nontargeting control (SCR#) were introduced into BxPC3 cells. Following single-cell cloning and approximately 20 cell divisions, 28 clonal subpopulations were established (eight controls and 20 ARID1A knockdowns). Subsequently, all 28 clones were pooled into a single cellular mixture, and scRNA-seq and scATAC-seq were performed to minimize the batch effect while evaluating transcriptional variations within and across clones.

基因背景一致的癌细胞之间的表型异质性，是肿瘤进展与治疗耐药性的核心支撑因素。然而，表观遗传失调如何驱动这类表型分化的具体机制仍不明确。本研究中，我们推出了可扩展研究平台IMPACH，该平台整合了谱系示踪、遗传扰动与多模态单细胞分析技术。通过应用IMPACH平台，我们证实：在可控实验条件下，表观遗传调控因子的缺失（尤其是ARID1A）会加剧转录组与表观基因组的异质性。ARID1A缺失会促进染色质的随机开放，并诱导出与不良临床预后相关的非典型基因表达程序。尽管表观遗传随机性有所升高，染色质的改变仍局限于具有生物学意义的调控区域，且这些区域部分与SMARCA4结合位点相关。上述研究结果表明，染色质重塑缺陷可通过随机与受限两种机制推动肿瘤细胞的克隆分化。鉴于IMPACH平台具备可扩展性与多功能性，其为解析表观遗传扰动如何塑造癌症细胞异质性提供了强有力的研究框架。将靶向ARID1A的shPseuMO-Tag构建体（包含两条独立的短发夹RNA：ARI3#与ARI4#）或非靶向对照序列（SCR#）导入BxPC3细胞中。经过单细胞克隆与约20次细胞增殖后，我们成功构建了28个克隆亚群（其中8个为对照组，20个为ARID1A敲低组）。随后，将全部28个克隆混合为单一细胞体系，通过单细胞RNA测序（scRNA-seq）与单细胞转座酶可及性测序（scATAC-seq）进行检测，在评估克隆内部与克隆间的转录组差异的同时，尽可能减少批次效应的影响。