Tumor Cells “Refuse Entry” and Actively Package for Export: Professor Zhuowei Liu’s Team Uncovers a Novel Mechanism of Resistance in Urothelial Carcinoma

On May 21, 2026, a research team led by Professor Zhuowei Liu, Dr. Zikun Ma, and Associate Research Fellow Xiaoyu Liang from Sun Yat-sen University Cancer Center published a study in Cancer Cell entitled “Endocytic Escape Drives Tumor Resistance to Antibody–Drug Conjugate Therapy.” The study systematically characterized the adaptive remodeling processes underlying resistance to antibody-drug conjugates (ADCs) using humanized mouse models, single-cell sequencing, and spatial transcriptomics.

The investigators identified distinct tumor cell subpopulations associated with different resistance mechanisms and proposed potential therapeutic strategies to overcome resistance. These findings provide important mechanistic insights that may help optimize ADC-based treatment approaches in urothelial carcinoma and other solid tumors.

ADC Therapy Has Transformed Urothelial Carcinoma Treatment—But Resistance Remains a Major Challenge

Antibody-drug conjugates (ADCs) represent a new generation of precision cancer therapeutics. By selectively recognizing tumor-associated surface antigens, ADCs deliver highly potent cytotoxic agents directly into cancer cells while minimizing systemic exposure.

In urothelial carcinoma, Nectin-4-targeting ADCs such as enfortumab vedotin have demonstrated significant clinical benefits across multiple treatment settings, ranging from advanced disease to neoadjuvant therapy. Combination regimens pairing ADCs with anti–PD-1 immune checkpoint inhibitors have already been incorporated into clinical practice guidelines.

However, the efficacy of ADCs depends on a complex sequence of cellular events, including:

Target binding
Internalization
Intracellular trafficking
Payload release
Tumor-cell killing

Despite the remarkable progress achieved with Nectin-4 ADCs combined with PD-1 blockade, approximately half of treated patients still fail to achieve complete tumor remission.

These observations suggest that tumor cells may undergo adaptive remodeling during treatment, resulting in acquired resistance. Until now, the underlying mechanisms have remained largely unclear.

Two Distinct Resistant Tumor Cell Populations Identified

The research team first discovered that resistant tumors exhibit heterogeneous resistance phenotypes characterized by two major tumor-cell populations:

NECTIN4Low Tumor Cells

These cells demonstrated:

Downregulation of the ADC target Nectin-4
Lysosomal dysfunction
Enhanced stemness-associated features
Increased drug efflux activity
Upregulation of cellular detoxification pathways

Together, these adaptations reduce intracellular payload delivery and diminish ADC efficacy.

NECTIN4High Tumor Cells

In contrast, this population displayed:

Further upregulation of Nectin-4 expression
Markedly impaired endocytic function
Active elimination of membrane-bound ADCs through extracellular vesicle secretion

These findings were particularly striking because high target expression is traditionally viewed as a favorable predictor of ADC response.

The study revealed that high target expression alone does not guarantee effective intracellular drug delivery.

Tumor Cells Evade ADC Uptake Through Endocytic Escape

Mechanistic investigations demonstrated that uptake of Nectin-4-targeting ADCs primarily depends on the AP2M1-mediated clathrin-dependent endocytosis pathway.

However, in NECTIN4High resistant tumor cells, the enzyme AKR1C1 was found to be aberrantly overexpressed.

AKR1C1 contributes to resistance through two complementary mechanisms:

Blocking ADC Internalization

AKR1C1 binds directly to Nectin-4 and interferes with AP2M1 recognition of the receptor, thereby preventing efficient endocytosis of the ADC.

Promoting ADC Export

AKR1C1 recruits the E3 ubiquitin ligase WWP2, which stimulates extracellular vesicle formation.

As a result, ADC molecules bound to the tumor-cell surface are packaged into extracellular vesicles and expelled from the cell before they can be internalized.

This process dramatically reduces intracellular drug accumulation and ultimately drives therapeutic resistance.

The investigators further identified the transcription factor ELF3 as a key upstream regulator responsible for maintaining both the formation of this resistant tumor-cell population and its resistance-associated phenotype.

Clinical Validation in Patients with Urothelial Carcinoma

To determine whether these findings were clinically relevant, the research team collected paired tumor samples from patients with urothelial carcinoma before and after treatment with Nectin-4 ADC plus anti–PD-1 therapy.

Spatial transcriptomic analysis revealed the presence of NECTIN4High resistant tumor-cell populations within patient tumors.

Importantly, these cells exhibited molecular signatures highly consistent with those identified in preclinical models, strongly supporting the clinical relevance of the endocytic escape mechanism.

These findings suggest that the resistance pathway observed experimentally is also active in human disease.

Beyond Target Expression: A New Framework for Predicting ADC Response

One of the study’s most important conclusions is that target expression alone may be insufficient to predict response to ADC therapy.

Current clinical practice often relies heavily on immunohistochemical assessment of target abundance. However, the investigators demonstrate that ADC efficacy is influenced by multiple additional factors, including:

Endocytic efficiency
Intracellular trafficking capacity
Spatial distribution of target molecules
Adaptive resistance remodeling within tumor cells

Consequently, two tumors with similar levels of target expression may exhibit very different responses to ADC treatment.

Potential Strategies to Overcome ADC Resistance

The study also highlights several potential approaches for enhancing ADC efficacy:

Dual-Target ADCs with Alternative Internalization Mechanisms

Combining targets that utilize different endocytic pathways may reduce the likelihood of resistance arising through endocytic blockade.

Dual-Payload ADCs

ADCs carrying payloads with distinct mechanisms of action may help overcome resistance associated with a single cytotoxic agent.

Targeting Endocytic Escape Pathways

Therapeutic inhibition of resistance pathways involving AKR1C1, WWP2, or related regulators may restore ADC internalization and improve intracellular drug delivery.

These strategies provide a conceptual framework for future ADC development and combination therapies.

Significance of the Study

This work offers one of the most comprehensive analyses to date of adaptive resistance mechanisms during ADC therapy.

By demonstrating that tumor cells can effectively “refuse entry” to ADCs and actively package them into extracellular vesicles for export, the study reveals a previously underappreciated mechanism of therapeutic escape.

The findings not only deepen our understanding of ADC resistance in urothelial carcinoma but also provide important guidance for the development of next-generation ADCs and rational combination strategies aimed at improving patient outcomes.

Corresponding Authors