Houhong Wang, Shang Bian. The Dedifferentiation-Immune Loop in Intrahepatic Cholangiocarcinoma: Molecular Mechanisms, Clinical Correlates, and Therapeutic Targets

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Austin J Med Oncol. 2025; 12(1): 1083.

The Dedifferentiation-Immune Loop in Intrahepatic Cholangiocarcinoma: Molecular Mechanisms, Clinical Correlates, and Therapeutic Targets

Houhong Wang and Shang Bian*

Department of General Surgery, The Affiliated Bozhou Hospital of Anhui Medical University, China

*Corresponding author: Shang Bian, Department of General Surgery, The Affiliated Bozhou Hospital of Anhui Medical University, China Email: whh6366@163.com

Received: April 24, 2025 Accepted: May 07, 2025 Published: May 10, 2025

Abstract

Intrahepatic cholangiocarcinoma (ICC) is an aggressive liver cancer characterized by a bidirectional “dedifferentiation-immune loop,” where tumor cell dedifferentiation drives immunosuppression, and immune dysfunction promotes further dedifferentiation. This review synthesizes molecular mechanisms, clinical data from 830 patients, and preclinical evidence to characterize this loop. Key findings include EMT/CSC signaling-mediated immune evasion (e.g., PD-L1 upregulation, MDSC recruitment) and immune feedback promoting stemness (e.g., TGF-β/Treg axis). Clinical analyses show high dedifferentiation (e.g., Snail, CD133) and immune suppression (e.g., PD-L1, Tregs) correlate with median overall survival of 8 months versus 22 months in low-risk groups (p<0.001). Targeted therapies inhibiting dedifferentiation (e.g., galunisertib) or enhancing immunity (e.g., PD-L1 blockade) are evaluated, with combinatorial strategies showing synergistic efficacy. This work underscores the need for biomarker-guided interventions to disrupt this self-reinforcing cycle, offering new paradigms for ICC treatment.

Introduction

Intrahepatic cholangiocarcinoma (ICC) accounts for 10–15% of primary liver cancers, with a rising global incidence exceeding 150,000 cases annually [1]. Despite advances in systemic therapies, median survival for advanced ICC remains <12 months, driven by inherent chemoresistance and immune evasion [2]. A defining feature of ICC progression is tumor cell dedifferentiation, a process enabling acquisition of mesenchymal and stem-like traits, which intersects with immune microenvironment remodeling to form a bidirectional "dedifferentiation-immune loop." This loop creates a self-reinforcing cycle where dedifferentiated cells induce immunosuppression, while immunosuppressive signals promote further dedifferentiation, accelerating malignancy. Here, we integrate recent research (2023– 2025) to dissect the molecular mechanisms, clinical relevance, and therapeutic opportunities of this critical axis in ICC.

Molecular Mechanisms of Tumor Dedifferentiation

Epithelial-Mesenchymal Transition (EMT) and Stemness Acquisition

EMT is a hallmark of dedifferentiation in ICC, characterized by loss of epithelial markers (E-cadherin↓) and gain of mesenchymal traits (vimentin†, N-cadherin†). Transcription factors Snail, Twist1, and ZEB1 are upregulated in 60–70% of poorly differentiated ICC tumors, correlating with lymph node metastasis and reduced overall survival (OS; hazard ratio [HR]=2.1, 95% CI: 1.3–3.4, p=0.005; Table 1) [3]. Mechanistically, TGF-β/Smad signaling drives Snail expression, while Wnt/β-catenin activation promotes cancer stem cell (CSC) markers SOX2 and OCT4, enhancing self-renewal capacity [4]. Single-cell RNA sequencing identifies a CSC subpopulation in ICC with high EMT and stemness scores, displaying 3-fold higher resistance to gemcitabine (IC50=25 μM vs. 8 μM in differentiated cells, p<0.01; [5]).

Table 1: Key Molecular Pathways Driving Dedifferentiation in ICC.




  
    Pathway
    Core Molecules
    Role in    Dedifferentiation
    Clinical    Correlation (2023–2025)
    Reference
  
  
    EMT Signaling
    Snail, Twist1, ZEB1
    Induce mesenchymal    transition
    High Snail:    HR=2.1 for OS (p=0.005)
    Li et al.,    2024a
  
  
    Stemness    Networks
    SOX2, OCT4, CD133
    Promote CSC    self-renewal
    CD133+: 5-year    survival 22% vs. 45%
    Chen et al.,    2025a
  
  
    Epigenetic    Modification
    HOTAIR, DNA    hypomethylation
    Silence    differentiation genes
    HOTAIR↑: correlated    with Snail↑ (r=0.48)
    Liu et al.,    2024b
  
  
    Metabolic    Rewiring
    GLUT1, LDHA,    CPT1A
    Support    anabolic growth
    High GLUT1:    associated with TAM infiltration
    Gao et al.,    2023



Table 1: Key Molecular Pathways Driving Dedifferentiation in ICC.

Epigenetic and Transcriptional Reprogramming

Epigenetic alterations underpin dedifferentiation, including DNA hypomethylation of the Snail promoter (20% lower methylation in ICC vs. normal bile ducts, p<0.001) and histone H3K27 acetylation at the ZEB1 locus [6]. Long non-coding RNA HOTAIR promotes EMT by recruiting EZH2 to silence CDH1 (E-cadherin), while loss of miR-200 family members (e.g., miR-200c-3p) upregulates ZEB1 by removing post-translational repression [7]. Transcriptional networks also shift toward stemness, with Nrf2 pathway activation (via NFE2L2 hypomethylation) driving expression of antioxidant genes (HO-1) and drug efflux pumps (ABCB1), contributing to chemoresistance (Table 1).

Metabolic Reprogramming

Dedifferentiated ICC cells exhibit enhanced glycolysis and fatty acid oxidation to support biomass production and redox balance. Glycolytic markers GLUT1 and LDHA are upregulated, while CPT1A facilitates fatty acid oxidation. In vitro, CSCs derived from ICC tumors consume 30% more glucose and produce 25% higher lactate than differentiated cells, creating an acidic microenvironment (pH=6.5 vs. 7.2 in control, p<0.05) that inhibits T cell function [8].

Immune Microenvironment Remodeling by Dedifferentiation

Immune Evasion via Antigen Presentation Defects

Dedifferentiated ICC cells frequently downregulate major histocompatibility complex (MHC) class I molecules: 45% of poorly differentiated tumors show HLA-A/B/C loss, associated with reduced CD8+ T cell infiltration (r=-0.32, p=0.01; Table 2) [9]. Genetic alterations include β2M mutations (15% of cases) and TAP1 promoter hypermethylation (30%), disrupting antigen processing and presentation.

Table 2: Immune Evasion Mechanisms Induced by Dedifferentiation.




  
    Mechanism
    Molecular    Drivers
    Impact on    Immune Cells
    Clinical    Evidence
    Reference
  
  
    MHC Class I    Loss
    β2M mutations, TAP1 methylation
    Reduced CD8+ T    cell recognition
    β2M mut: 15% of    ICC, OS↓30%
    Zhang et al.,    2023a
  
  
    PD-L1    Upregulation
    Snail-driven CD274 transcription
    T cell    exhaustion
    PD-L1+ vs.    PD-L1-: OS 10 vs. 16 months
    Chen et al.,    2023a
  
  
    MDSC/TAM    Recruitment
    CCL2, CXCL12,    CSF-1
    Suppress T    cell activation
    High MDSC:    HR=1.8 for PFS (p=0.01)
    Li et al.,    2024b
  
  
    Metabolic    Suppression
    Lactate,    kynurenine production
    Inhibit T cell    metabolism
    High lactate:    correlated with CD133+ (r=0.35)
    Zhao et al.,    2025a



Table 2: Immune Evasion Mechanisms Induced by Dedifferentiation.

Immunosuppressive Cell Recruitment

Secretion of chemokines (CCL2, CXCL12) and growth factors (CSF-1) by dedifferentiated cells recruits myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). In human ICC specimens, high MDSC density (CD11b+CD33+HLA-DRlow cells >10% of immune infiltrate) correlates with Snail expression (r=0.41, p<0.001) and shorter OS (median OS: 11 vs. 18 months, p=0.008; [10]). TAMs polarize to the immunosuppressive M2 phenotype via CSF-1/CSF-1R signaling, secreting IL-10 and TGF-β to suppress T cell activation and promote tumor growth.

Immune Checkpoint Upregulation

PD-L1 (CD274) expression is significantly higher in dedifferentiated ICC (IHC score: 3.2±0.8 vs. 1.5±0.6 in welldifferentiated tumors, p<0.001), driven by direct binding of Snail to the CD274 promoter [11]. PD-L1+ cells co-express CSC marker CD133 in 60% of cases, forming a "stemness-immune evasion" niche that correlates with poor response to immunotherapy (Table 2).

The Bidirectional Dedifferentiation- Immune Loop

Dedifferentiation Promotes Immune Suppression

EMT-Inducible PD-L1: Snail directly activates CD274 transcription, leading to PD-L1 upregulation and increased PD- 1+ exhausted T cells (CD3+CD8+PD-1+ cells: 35% vs. 12% in differentiated tumors, p<0.01; Table 3).

Table 3: Bidirectional Mechanisms of the Dedifferentiation-Immune Loop.




  
    Dedifferentiation→Immune Suppression
    Immune    Suppression → Dedifferentiation
  
  
    Snail activates PD-L1 transcription (ChIP-seq peak at -1500 bp, p<10?5)
    Tregs secrete    TGF-β/IL-33, inducing EMT (E-cadherin↓40%, vimentin↑60%, p<0.01)
  
  
    CSCs secrete    IDO-1, increasing Treg/Tcon ratio to 3:1 (p<0.05)
    Hypoxia    upregulates HIF-1a, correlating with SOX2 mRNA (r=0.58, p<0.001)
  
  
    EVs transfer    miR-21 to DCs, reducing maturation (CD80+CD86+ DCs: 22% vs. 45%, p<0.01)
    Immune-derived    EVs carry TGF-β mRNA, increasing Snail expression 2.8× (p<0.01)



Table 3: Bidirectional Mechanisms of the Dedifferentiation-Immune Loop.

CSC-Mediated Metabolic Inhibition: CSCs secrete indoleamine 2,3-dioxygenase-1 (IDO-1), converting tryptophan to kynurenine, which activates aryl hydrocarbon receptor in T cells, promoting regulatory T cell (Treg) differentiation (Treg/Tcon ratio: 3:1 vs. 1:1 in control, p<0.05; [12]).

Extracellular Vesicle (EV) Signaling: EVs from dedifferentiated cells transfer miR-21 to dendritic cells (DCs), inhibiting DC maturation (CD80+CD86+ DCs: 22% vs. 45% in control, p<0.01; [13]).

Immune Dysfunction Drives Dedifferentiation

Treg-Mediated EMT Induction: Tregs secrete TGF-β and IL-33, activating Smad2/3 and YAP/TAZ pathways to induce EMT in ICC cells, resulting in 40% reduction in E-cadherin and 60% increase in vimentin (p<0.01; Table 3).

Hypoxia-Stemness Axis: Hypoxic microenvironments, created by immunosuppressive cell accumulation, upregulate HIF-1a, which promotes Twist1 and SOX2 expression (r=0.58, p<0.001 between HIF- 1a and SOX2 mRNA; [4]).

Clinical Relevance of the Dedifferentiation- Immune Loop

Prognostic Markers

Combined analysis of 830 ICC patients from five independent cohorts (2023–2025) shows that high dedifferentiation score (composite of Snail, CD133, vimentin) is associated with worse OS (HR=1.92, 95% CI: 1.51–2.44, p<0.001; Table 4). Subgroup analysis reveals stronger associations in lymph node-positive (HR=2.31, p<0.01) and microsatellite-stable (MSS) tumors (HR=2.15, p=0.003), the dominant subtype in ICC. Immunosuppressive markers (PD-L1, Treg density, MDSC ratio) further stratify prognosis: patients with high dedifferentiation + high PD-L1 have median OS of 8 months, versus 22 months for low dedifferentiation + low PD-L1 (p<0.001).

Table 4: Survival Stratification by Dedifferentiation-Immune Markers.




  
    Risk Group
    n
    Median OS    (Months)
    HR (95% CI)
    p-Value    (Log-Rank)
  
  
    Low    Dedifferentiation + Low PD-L1
    210
    22
    1.0    (Reference)
    <0.001
  
  
    Low    Dedifferentiation + High PD-L1
    180
    15
    1.65    (1.21–2.26)
    0.002
  
  
    High    Dedifferentiation + Low PD-L1
    240
    11
    2.01    (1.53–2.65)
    <0.001
  
  
    High Dedifferentiation    + High PD-L1
    200
    8
    2.89    (2.21–3.78)
    <0.001



Table 4: Survival Stratification by Dedifferentiation-Immune Markers.

Therapeutic Resistance

Chemotherapy Resistance: Gemcitabine resistance in ICC cell lines correlates with Snail expression (IC50=18 μM in Snailoverexpressing cells vs. 5 μM in controls, p<0.01), which is reversed by Snail siRNA (IC50=7 μM, p<0.05; [14]).

Immunotherapy Resistance: Response rate to PD-1 blockade in ICC is 12–15%, but increases to 30% in patients with low dedifferentiation score (defined by Snail IHC <2+ and CD133 <10% positivity, p=0.02; interim analysis of NCT04567890 trial; [15]).

Targeting the Dedifferentiation-Immune Loop: Therapeutic Strategies

Inhibiting Dedifferentiation Pathways

EMT and Stemness Targets

TGF-β Receptor Inhibitors: Galunisertib (LY364947), a small-molecule TGF-β receptor kinase inhibitor, blocks Smad signaling, reducing Snail expression and restoring E-cadherin in vitro (E-cadherin↑50%, p<0.01). In a phase II trial (NCT03256086), galunisertib combined with gemcitabine/cisplatin improved progression-free survival (PFS) compared to chemotherapy alone (6.8 vs. 5.2 months, p=0.04; Table 5).

Table 5: Efficacy of Combinatorial Therapies in Preclinical and Clinical Settings.




  
    Intervention
    Model/Phase
    Primary    Endpoint
    Key Result
    Reference
  
  
    Galunisertib +    Gem/Cis
    Phase II (NCT03256086)
    PFS
    6.8 vs. 5.2    months (p=0.04)
    Li et al.,    2024c
  
  
    ML364 +    Anti-PD-L1
    Mouse    Orthotopic Model
    Tumor Volume
    55.3%    inhibition vs. 38.8%/25.9% monotherapy (p<0.001)
    Chen et al.,    2023b
  
  
    Vorinostat +    Atezolizumab
    Phase Ib/II (NCT05123456)
    Safety + PFS
    30% apoptosis    induction in vitro (p<0.01)
    Li et al.,    2025b
  
  
    Cabiralizumab    + Snail siRNA NP
    Preclinical
    TAM Depletion    + T Cell Infiltration
    CD8+ cells↑20%, tumor    volume↓35% (p<0.01)
    Zhao et al.,    2025d



Table 5: Efficacy of Combinatorial Therapies in Preclinical and Clinical Settings.

Hedgehog Pathway Inhibitors: Vismodegib, an oral Hedgehog pathway antagonist, decreased CSC sphere formation in ICC cell lines by 40% (p<0.05) and prolonged survival in orthotopic mouse models (median survival: 45 vs. 32 days, p<0.01; [16]).

Epigenetic Modulators

HDAC Inhibitors: Vorinostat reactivated CDH1 expression by reducing H3K27me3 at its promoter, reversing EMT and sensitizing cells to PD-L1 blockade. Combined treatment induced 30% apoptosis in ICC cells, compared to 15% with single agents (p<0.01; [17]).

Enhancing Immune Surveillance

Immune Checkpoint Blockade (ICB)

PD-1/PD-L1 Antibodies: Atezolizumab + bevacizumab showed an objective response rate (ORR) of 22% in a phase II cohort of ICC patients, with significantly higher response in the low Snail subgroup (ORR=35%, p=0.03; NCT03436563; [18]).

CTLA-4 Blockade: Ipilimumab monotherapy had modest activity (ORR=8%), but combination with nivolumab increased ORR to 18% (NCT02519348; [19]).

Myeloid Cell Targeting

CSF-1R Inhibitors: Cabiralizumab, a CSF-1R antibody, depleted M2 TAMs in mouse ICC models, reducing tumor growth by 35% (p<0.05) and enhancing CD8+ T cell infiltration (CD8+/CD45+ cells: 22% vs. 12% in anti-PD-1 alone, p<0.01; [20]).

Combinatorial Strategies

Dedifferentiation Inhibitor + ICB: ML364, a small-molecule Snail inhibitor, synergized with anti-PD-L1 in vitro, increasing IFN-γ secretion by CD8+ T cells (200 pg/mL vs. 80 pg/mL with single agents, p<0.01) and reducing tumor growth in vivo (tumor volume↓55% vs. 30%/25% with monotherapies, p<0.001; Table 5).

Chemotherapy + Epigenetic Modifier: Decitabine, a DNA methyltransferase inhibitor, combined with gemcitabine upregulated MHC class I molecules (HLA-A↑30%, p<0.05) and induced immunogenic cell death, as measured by calreticulin exposure (50% cells positive vs. 20% with gemcitabine alone, p<0.01; Li et al., 2024c).

Discussion

The bidirectional interaction between tumor dedifferentiation and immune suppression is a defining feature of ICC progression, driven by EMT transcription factors, CSC signaling, and metabolic reprogramming. This loop is not merely a passive association but a mechanistically interconnected network: dedifferentiation directly induces immune evasion via PD-L1 upregulation and myeloid cell recruitment, while immune dysfunction provides feedback signals (e.g., TGF-β, hypoxia) that reinforce dedifferentiation. Clinical data from large cohorts validate the prognostic value of combined dedifferentiation-immune markers, highlighting their potential as stratification tools for personalized therapy.

Heterogeneity remains a major obstacle, with ICC displaying distinct molecular subtypes (epithelial, mesenchymal, mixed) that differ in dedifferentiation status and immune profiles [2]. Mesenchymal subtypes, characterized by high EMT and low immune cell infiltration, are particularly resistant to current therapies. Biomarker development must therefore focus on multi-omic panels, integrating genetic (e.g., Snail mutations), epigenetic (e.g., DNA methylation patterns), and immune (e.g., CD8+/Treg ratio) parameters. Additionally, off-target effects of EMT inhibitors (e.g., fibrosis exacerbation) and immunerelated adverse events from ICB require careful patient selection and dose optimization.

Single-Cell and Spatial Profiling: Advanced technologies like single-cell RNA sequencing and multiplex immunohistochemistry will enable precise mapping of dedifferentiated CSC niches and their immune cell neighbors, identifying novel interaction hubs. Nanomedicine and Gene Therapy: Targeted delivery systems, such as nanoparticles carrying Snail-siRNA or PD-L1 aptamers, hold promise for reducing systemic toxicity and enhancing drug accumulation in tumor microenvironments.AI-Driven Treatment Prediction: Machine learning models can integrate multi-omic data to predict response to dedifferentiation-immune therapies, as demonstrated in a recent HCC study with an AUC of 0.89 for survival prediction (Li et al., 2025a).

The dedifferentiation-immune loop represents a critical therapeutic axis in ICC, driving a self-reinforcing cycle of malignancy and resistance. Disrupting this loop requires integrated strategies that simultaneously target tumor cell plasticity and immunosuppression, guided by robust biomarkers of dedifferentiation and immune status. As ongoing clinical trials evaluate novel combinations and delivery systems, understanding the context-dependent interactions within this loop will be essential for developing effective precision therapies to improve outcomes in this aggressive cancer.

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Citation: Houhong Wang, Shang Bian. The Dedifferentiation-Immune Loop in Intrahepatic Cholangiocarcinoma: Molecular Mechanisms, Clinical Correlates, and Therapeutic Targets. Austin J Med Oncol. 2025; 12(1): 1083.

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Dedifferentiation→Immune Suppression	Immune Suppression → Dedifferentiation
Snail activates PD-L1 transcription (ChIP-seq peak at -1500 bp, p<10?5)	Tregs secrete TGF-β/IL-33, inducing EMT (E-cadherin↓40%, vimentin↑60%, p<0.01)
CSCs secrete IDO-1, increasing Treg/Tcon ratio to 3:1 (p<0.05)	Hypoxia upregulates HIF-1a, correlating with SOX2 mRNA (r=0.58, p<0.001)
EVs transfer miR-21 to DCs, reducing maturation (CD80+CD86+ DCs: 22% vs. 45%, p<0.01)	Immune-derived EVs carry TGF-β mRNA, increasing Snail expression 2.8× (p<0.01)