Behavior-coupled single-cell transcriptomics reveals trafficking protein-dependent regulation of tumor cell migration
- 1Erasmus University Medical Center
Abstract
Aggressive cellular migration underlies cancer invasion and metastatic dissemination, yet the molecular programs defining highly migratory tumor cell states remain poorly characterized. In head and neck squamous cell carcinoma (HNSCC), metastatic progression is associated with poor clinical outcome, motivating method developments that link migratory behavior to underlying mechanisms. Here, we present behavior-coupled single-cell sequencing (BC-seq), a live-cell imaging-guided transcriptomic platform that enables real-time identification, isolation, and profiling of individual cells based on migratory behavior. Applying BC-seq to 10 HPV-negative oral squamous cell carcinoma (OSCC), the most common HNSCC subtype, we distinguished fast-migrating cells (FMCs) from slow-migrating cells (SMCs) and identified transcriptional programs associated with aggressive migration. Mechanistically, BC-seq uncovered cholesterol-regulated signaling pathways and associated trafficking proteins involving SREBP2, STARD4, Cavin1/3, and VEGFR2 that promotes actin cytoskeletal remodeling and cellular motility. Disruption of STARD4-mediated cholesterol trafficking or Cavin1/3-dependent VEGFR2 nuclear translocation significantly impaired cell migration, linking cholesterol metabolism to migratory machinery. We next examined whether molecular programs associated with aggressive migration are reflected in clinical outcomes. A 15-gene expression signature derived from FMCs predicted distant metastasis with 83.6% accuracy in an independent OSCC cohort, outperforming conventional clinicopathological risk stratification. Together, our study establishes BC-seq as an approach for integrating dynamic cellular behavior with single-cell transcriptomics to uncover mechanisms of aggressive migration and clinically relevant molecular signatures.
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BibTeX
@misc{chien2026behaviorcoupled,
title = {{Behavior-coupled single-cell transcriptomics reveals trafficking protein-dependent regulation of tumor cell migration}},
author = {M.-P. Chien and R. Ghadiri Rad and J. Huang and M. T. Lopez-Cascales and L. You and J. J. H. Traets and D. Bellomo and T.-C. Chou and W. Bennik and J. Singh and Y. Begce and J. A. U. Hardillo and D. Vadgama and T. van den Bosch and V. van Dis},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.15.759353},
url = {https://www.biorxiv.org/content/10.64898/2026.10.15.759353v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Behavior-coupled single-cell transcriptomics reveals trafficking protein-dependent regulation of tumor cell migration
AU - M.-P. Chien
AU - R. Ghadiri Rad
AU - J. Huang
AU - M. T. Lopez-Cascales
AU - L. You
AU - J. J. H. Traets
AU - D. Bellomo
AU - T.-C. Chou
AU - W. Bennik
AU - J. Singh
AU - Y. Begce
AU - J. A. U. Hardillo
AU - D. Vadgama
AU - T. van den Bosch
AU - V. van Dis
PY - 2026
DA - 2026/10/15
PB - bioRxiv
DO - 10.64898/2026.10.15.759353
UR - https://www.biorxiv.org/content/10.64898/2026.10.15.759353v1
AB - Aggressive cellular migration underlies cancer invasion and metastatic dissemination, yet the molecular programs defining highly migratory tumor cell states remain poorly characterized. In head and neck squamous cell carcinoma (HNSCC), metastatic progression is associated with poor clinical outcome, motivating method developments that link migratory behavior to underlying mechanisms. Here, we present behavior-coupled single-cell sequencing (BC-seq), a live-cell imaging-guided transcriptomic platform that enables real-time identification, isolation, and profiling of individual cells based on migratory behavior. Applying BC-seq to 10 HPV-negative oral squamous cell carcinoma (OSCC), the most common HNSCC subtype, we distinguished fast-migrating cells (FMCs) from slow-migrating cells (SMCs) and identified transcriptional programs associated with aggressive migration. Mechanistically, BC-seq uncovered cholesterol-regulated signaling pathways and associated trafficking proteins involving SREBP2, STARD4, Cavin1/3, and VEGFR2 that promotes actin cytoskeletal remodeling and cellular motility. Disruption of STARD4-mediated cholesterol trafficking or Cavin1/3-dependent VEGFR2 nuclear translocation significantly impaired cell migration, linking cholesterol metabolism to migratory machinery. We next examined whether molecular programs associated with aggressive migration are reflected in clinical outcomes. A 15-gene expression signature derived from FMCs predicted distant metastasis with 83.6% accuracy in an independent OSCC cohort, outperforming conventional clinicopathological risk stratification. Together, our study establishes BC-seq as an approach for integrating dynamic cellular behavior with single-cell transcriptomics to uncover mechanisms of aggressive migration and clinically relevant molecular signatures.
ER -