ABSTRACT
Cholangiocarcinoma (CCA) is a heterogenous malignancy that arises from the biliary epithelium and has a poor clinical prognosis. The Hippo/yes-associated protein (YAP) pathway has been reported to affect various aspects of tumorigenesis, with high expression of YAP1 being negatively associated with survival in CCA patients. Thus, we investigated the antitumoral effect of verteporfin, a YAP1 pathway inhibitor, in YAP1/AKT hydrodynamic tail vein injected murine models. We also used flow cytometry and single-cell RNA sequencing (scRNA-seq) to analyze the change in the immune cell profile and malignant cell stemness following verteporfin treatment. Our results demonstrated reduced liver weight and tumor formation in verteporfin-treated groups compared to that of a vehicle-treated group. Immune cell profiling through flow cytometry showed that relative to the vehicle, verteporfin induced a higher ratio of tumor-associated macrophage (TAM) M1/M2 and increased the percentage of activated CD8 T cell population (CD8+CD25+ and CD8+CD69+). scRNA-seq analysis showed significantly increased TAM M1 populations following verteporfin treatment and decreased proportions of stem-like cells within the malignant cell population. In summary, this study indicates that in CCA YAP/AKT murine models, verteporfin reduces tumorigenesis by polarizing anti-tumoral TAM and activating CD8 T cells and decreasing stem-like malignant cell proportions in the tumor microenvironment.
ABSTRACT
RNA from circulating blood reticulocytes was utilized to provide a robust description of genes transcribed at the final stages of erythroblast maturation. After depletion of leukocytes and platelets, Affymetrix HG-U133 arrays were hybridized with probe generated from the reticulocyte total RNA (blood obtained from 14 umbilical cords and 14 healthy adult humans). Among the cord and adult reticulocyte profiles, 698 probe sets (488 named genes) were detected in each of the 28 samples. Among the highly expressed genes, promoter analyses revealed a subset of transcription factor binding motifs encoded at higher than expected frequencies including the hypoxia-related arylhydrocarbon receptor repressor family. Over 100 probe sets demonstrated differential expression between the cord and adult reticulocyte samples. For verification, the array expression patterns for 21 genes were confirmed by real-time PCR (correlation coefficient 0.98). Only four transcripts (MAP17, FLJ32009, ARRB2, and FLJ27365) were identified as being upregulated in the adult blood transcriptome. Further analysis revealed that the lipid-regulating protein MAP17 was present in the membrane fraction of adult erythrocytes, but not detected in cord blood erythrocytes. Combined with other clinical and experimental data, these reticulocyte transcriptome profiles should be useful to better understand the molecular bases of terminal erythroid differentiation, hemoglobin switching, iron metabolism and malarial pathogenesis.
