Overcoming CXCR4-mediated T cell exclusion potentiates antitumor cytotoxicity in fibrolamellar carcinoma

Fibrolamellar carcinoma (FLC) is a rare liver cancer affecting young adults without underlying liver disease and is characterized by the DNAJB1-PRKACA fusion oncogene. Despite its immunogenicity, FLC shows limited response to immunotherapy. To investigate the mechanisms of immune resistance, we performed single-nucleus RNA sequencing on human FLC and matched adjacent non-tumor liver samples. Our analyses revealed dysregulated stromal-immune interactions, including CXCL12+ myofibroblast signaling that restricted T cell infiltration. Using complementary approaches, we found that CXCR4 inhibition enabled T cell entry into tumors, while PD-1 blockade enhanced T cell effector function. Combination treatment synergistically increased tumor cell death in a human tumor slice culture system. These results define mechanisms of immune evasion in FLC and provide preclinical support for combined CXCR4 and PD-1 blockade as a potential therapeutic strategy. Single-nucleus RNA-seq was performed on human fibrolamellar carcinoma (primary and metastatic) and matched adjacent liver. Nuclei were processed with Parse Evercode Mega WT v3, generating pooled sublibraries via combinatorial barcoding. Pooled libraries were sequenced (paired-end) on an Illumina NovaSeq 6000; raw multiplexed reads are deposited in SRA, and processed count matrices and cell-level annotations are provided here in GEO.