Reversing immunosuppression in the tumor microenvironment of fibrolamellar carcinoma via PD-1 and IL-10 blockade.

Fibrolamellar carcinoma (FLC) is a rare liver tumor driven by the DNAJ-PKAc fusion protein that affects healthy young patients. Little is known about the immune response to FLC, limiting rational design of immunotherapy. Multiplex immunohistochemistry and gene expression profiling were performed to characterize the FLC tumor immune microenvironment and adjacent non-tumor liver (NTL). Flow cytometry and T cell receptor (TCR) sequencing were performed to determine the phenotype of tumor-infiltrating immune cells and the extent of T cell clonal expansion. Fresh human FLC tumor slice cultures (TSCs) were treated with antibodies blocking programmed cell death protein-1 (PD-1) and interleukin-10 (IL-10), with results measured by cleaved caspase-3 immunohistochemistry. Immune cells were concentrated in fibrous stromal bands, rather than in the carcinoma cell compartment. In FLC, T cells demonstrated decreased activation and regulatory T cells in FLC had more frequent expression of PD-1 and CTLA-4 than in NTL. Furthermore, T cells had relatively low levels of clonal expansion despite high TCR conservation across individuals. Combination PD-1 and IL-10 blockade signficantly increased cell death in human FLC TSCs. Immunosuppresion in the FLC tumor microenvironment is characterized by T cell exclusion and exhaustion, which may be reversible with combination immunotherapy.

Isolation of multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages.

Little is known about the differentiation capabilities of nonhematopoietic cells of the human fetal liver. We report the isolation and characterization of a human fetal liver multipotent progenitor cell (hFLMPC) population capable of differentiating into liver and mesenchymal cell lineages. Human fetal livers (74-108 days of gestation) were dissociated and maintained in culture. We treated the colonies with geneticin and mechanically isolated hFLMPCs, which were kept in an undifferentiated state by culturing on feeder layers. We derived daughter colonies by serial dilution, verifying monoclonality using the Humara assay. hFLMPCs, which have been maintained in culture for up to 100 population doublings, have a high self-renewal capability with a doubling time of 46 h. The immunophenotype is: CD34+, CD90+, c-kit+, EPCAM+, c-met+, SSEA-4+, CK18+, CK19+, albumin-, alpha-fetoprotein-, CD44h+, and vimentin+. Passage 1 (P1) and P10 cells have identical morphology, immunophenotype, telomere length, and differentiation capacity. Placed in appropriate media, hFLMPCs differentiate into hepatocytes and bile duct cells, as well as into fat, bone, cartilage, and endothelial cells. Our results suggest that hFLMPCs are mesenchymal-epithelial transitional cells, probably derived from mesendoderm. hFLMPCs survive and differentiate into functional hepatocytes in vivo when transplanted into animal models of liver disease. hFLMPCs are a valuable tool for the study of human liver development, liver injury, and hepatic repopulation.