From association to intervention: Rethinking CD24’s causal role in hepatocellular carcinogenesis

Table 1 Multi-omics approaches to validate cluster of differentiation 24 causality in hepatocellular carcinoma

Experimental strategy	Technical platform	Expected outcome	Clinical translation
Genetic knockout	CRISPR-Cas9 hydrodynamic transfection (Fah-/- mice)	Reduced tumor incidence, restored anti-PD-1 sensitivity	Patient selection for combination therapy
Single-cell profiling	scRNA-seq + CITE-seq + scATAC-seq	CD24+ subset resolution, regulatory network mapping	Biomarker discovery for liquid biopsy
Spatial mapping	10 × Visium + IMC (43-marker panel)	Zonal CD24 distribution, immune ecosystem architecture	Image-guided therapeutic targeting
Functional validation	In vivo CRISPR screening (AAV-sgRNA library)	Genetic interaction map, synthetic lethal targets	Rational combination design
Isoform quantification	Isoform-specific ELISA, mass spectrometry	sCD24 vs mCD24 dynamics, ADAM10/17 activity	Monitoring therapeutic response

AAV: Adeno-associated virus; ADAM10: A disintegrin and metalloproteinase domain-containing protein 10; ADAM17: A disintegrin and metalloproteinase domain-containing protein 17; CITE-seq: Cellular indexing of transcriptomes and epitopes by sequencing; CRISPR: Clustered regularly interspaced short palindromic repeats; CRISPR-Cas9: Clustered regularly interspaced short palindromic repeats-associated protein 9; ELISA: Enzyme-linked immunosorbent assay; IMC: Imaging mass cytometry; mCD24: Membrane-bound CD24 (or membrane CD24); PD-1: Programmed cell death protein 1; scATAC-seq: Single-cell assay for transposase-accessible chromatin using sequencing; sCD24: Soluble CD24; scRNA-seq: Single-cell RNA sequencing; sgRNA: Single-guide RNA.