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.

Table 2 Comparative analysis of “don’t eat me” signals in hepatocellular carcinoma immunotherapy

Checkpoint	Ligand/receptor	Expression pattern	Clinical development	Advantages	Limitations
CD24	Siglec-10	CSCs, activated immune cells, hepatocytes	Preclinical (SWA11 mAb, CAR-T)	Low normal tissue expression, dual innate/adaptive function	Heterogeneous expression, shedding-mediated decoy effects
CD47	SIRPα	Ubiquitous (high on RBCs)	Phase I/II (Hu5F9-G4, magrolimab)	Well-established biology, broad tumor coverage	On-target anemia, dosing limitations
PD-L1	PD-1	Inducible on tumor and immune cells	Approved (atezolizumab, durvalumab)	Proven efficacy, established biomarkers	Primary resistance, immune-related adverse events
MHC-I	LILRB1/2	Variable across HCC subtypes	Early preclinical	Alternative escape mechanism	Complex regulation, technical challenges

CAR-T: Chimeric antigen receptor T cell; CSCs: Cancer stem cells; HCC: Hepatocellular carcinoma; Hu5F9-G4: Humanized anti-CD47 monoclonal antibody (5F9-G4; magrolimab); LILRB1/2: Leukocyte immunoglobulin-like receptor subfamily B member 1/2; MHC-I: Major histocompatibility complex class I; PD-1: Programmed cell death protein 1; PD-L1: Programmed death-ligand 1; RBCs: Red blood cells; Siglec-10: Sialic acid-binding Ig-like lectin 10; SIRPα: Signal regulatory protein alpha; SWA11: Anti-CD24 monoclonal antibody (clone SWA11).