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Background CD68, a glycosylated scavenger receptor traditionally used as a macrophage marker, has recently been identified in tumor cells. TCGA analysis 1 revealed CD68 expression correlates with worse overall survival across multiple cancers, including hepatocellular carcinoma (HCC). Clinical samples showed CD68+ tumor regions associate with reduced CD8+ T cell infiltration and poorer outcomes following IO + chemotherapy treatment (Blando et al., 2023, unpublished).Methods HCC patient scRNAseq data 2 was analyzed to compare CD68highversus CD68low tumor cells identified by cytokeratin/CD68 co-expression. Huh-1 and SNU-423 cells were treated with EGF (0.5-50 ng/ml) for 48h to assess CD68 and SNAIL expression by Simple Western (SW). CD68 knockout (KO) clones in SNU-423 and SK-HEP-1 lines were validated by flow cytometry (FC) and SW. Transwell assays measured migration of CD3/CD28-stimulated CD8+ T cells from seven donors co-cultured with wild-type (WT) or KO cells. CD155, Galectin-3, PD-L1, and LAMP1 expression was assessed by FC and scRNAseq analysis. VEGF secretion was quantified using ProteinSimple ELLA.Results scRNAseq revealed elevated Galectin-3, CD155, VEGFA, and LAMP1 in CD68 high tumor cells. EGF treatment upregulated CD68 in SNU-423 (3.8-fold at 50 ng/ml; 1.8-fold at 0.5 ng/ml) and Huh-1 (2-fold at 50 ng/ml), with concurrent SNAIL increases. CD8+ T cell migration significantly improved with CD68 KO cells versus WT (p<0.0001). Surface expression of Galectin-3, PD-L1, CD155, and LAMP1 was higher on SNU-423 WT cells, while PD-L1 and LAMP1 surface expression was absent in KO cells despite similar intracellular levels. VEGF secretion was reduced in KO cells (6.8 ng/ml) compared to WT (21 ng/ml), aligning with scRNAseq findings.Conclusions Analysis of HCC patient scRNAseq data, CD68 knockout models, and clinical observations suggests tumor cell CD68 expression correlates with epithelial-mesenchymal transition and promotes CD8+ T cell exclusion. CD68 + tumor cells showed elevated Galectin-3, PD-L1, PVR, and VEGF expression, suggesting a role in tumor heterogeneity, metastasis, and immune evasion. These findings highlight the potential strategy of using CD68+ cells for novel bispecific immuno-oncology and combination therapies in HCC.References Zhang J, Li S, Liu F, et al. Role of CD68 in tumor immunity and prognosis prediction in pan-cancer. Sci Rep. 2022;12:7844.Ma L, Wang L, et al. Single-cell atlas of tumor cell evolution in response to therapy in hepatocellular carcinoma and intrahepatic cholangiocarcinoma. J Hepatol. 2021;75(6):1397–1408.