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Comprehensive molecular profiling of combined hepatocellular carcinoma and cholangiocarcinoma reveals distinct Notch signaling subgroups with prognostic significance

  • 양찬모 (아주대학교 일반대학원)

초록/요약

Combined hepatocellular–cholangiocarcinoma (cHCC-CCA) is an uncommon primary liver malignancy that integrates hepatocytic and cholangiocytic differentiation programs. Although Notch signaling governs hepatic lineage specification and stemness, its precise contribution to cHCC-CCA pathogenesis remains unresolved. Resection specimens collected at Ajou University Medical Center (2017–2023) comprised cHCC-CCA (n = 35), hepatocellular carcinoma (HCC; n = 38), cholangiocarcinoma (CCA; n = 32), and normal liver tissue (n = 3). Lineage and Notch-pathway proteins (NOTCH1, HES5) were analyzed by immunohistochemistry with H-score quantification, and Notch receptors plus their canonical targets were profiled by qRT-PCR. Using predefined cutoffs, cHCC-CCA cases were divided into three Notch-based subgroups. QuantSeq 3′ RNA-seq combined with gene-ontology and gene-set enrichment analyses benchmarked each subgroup against HCC and CCA molecular signatures. To explore signaling dynamics, an optogenetic, light-switchable construct (optoNOTCH) was introduced into doxorubicin-enriched HepG2 cancer stem-like cells, enabling transient, continuous, and repetitive activation patterns. Spatial transcriptomics (Xenium In Situ 5K) performed on a custom tissue microarray delineated microenvironmental influences. Overall, cHCC-CCA displayed heightened Notch activity relative to HCC or CCA yet showed pronounced intertumoral variability. Stratification by NOTCH1/HES5 identified three molecular classes; Group 2 (NOTCH1 high/HES5 high) exhibited the most aggressive histopathologic features and significantly poorer progression-free survival, paralleling CCA-like biology. Transcriptomic analysis confirmed enrichment of NOTCH1-upregulated programs and invasion/survival pathways in Group 2. Optogenetic experiments demonstrated that repetitive Notch stimulation elicited stronger downstream responses than brief or sustained inputs— indicating that activation duration encodes distinct functional outputs. Spatial mapping further revealed POSTN expression in peritumoral fibroblasts juxtaposed with CD44-positive tumor cells, implicating a stromal POSTN—NOTCH1—CD44 axis in maintaining high Notch activity. Collectively, cHCC-CCA comprises biologically distinct, Notch-defined subgroups with prognostic relevance. Repetitive Notch activation—potentially driven by fibroblast-derived POSTN and reinforced by nuclear export mechanisms—characterizes an aggressive, CCA-like subset. These findings highlight the POSTN–NOTCH1 interface and temporal regulation of Notch signaling as promising therapeutic targets in cHCC-CCA. Keywords: Combined hepatocellular carcinoma and cholangiocarcinoma, Notch signaling pathway, Optogenetics, Spatial Transcriptomics, Prognostic prediction

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목차

Ⅰ. Introduction 1
Ⅱ. Materials and Methods. 6
1. Patient characteristics. 6
2. Immunohistochemical staining and analysis 6
3. Quantitative real-time polymerase chain reaction (qRT-PCR). 7
4. RNA sequencing analysis. 8
5. Optogenetically activatable Notch engineering 9
6. Generation of hepatic cancer stem -like cells, cell culture, and transfection 10
7. Lentivirus production and in vitro transduction 10
8. Live cell imaging and light stimulation. 11
9. Light-Emitting Diode (LED) Stimulation. 11
10. Immunocytochemistry. 12
11. Wound healing assay 12
12. Tissue microarray construction. 13
13. Spatial Transcriptomic Assay (Xenium In Situ). 14
14. Stat i s t i ca l ana lys i s. 14
Ⅲ. Results 16
1. Analysis of Notch pathway components in primary liver cancers. 16
2. Subgrouping of combined hepatocellular carcinoma and cholangiocarcinoma by Notch signaling activation. 40
3. Transcriptomic profi l ing of cHCC-CCA subgroups, HCC, and CCA. 49
4. Survival outcomes among cHCC-CCA subgroups. 65
5. Optogenetic analysis of Notch signaling activation. 68
6. Spatial transcriptomic analysis of combined hepatocellular carcinoma and cholangiocarc inoma 87
Ⅳ. Discussion. 116
Reference 124
국문요약 135

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