Multiomics identifies a cholesterol–TFEB–PLD3–TLR9 axis driving immunosuppressive tumor-associated macrophage polarization in esophageal squamous cell carcinoma

Licheng Tan; Hongyu Zhou; Baifeng Zhang; Dora Lai Wan Kwong; Yongxu Jia; Jiayi Huang; Kin To Hugo Siu; Shuang Zhang; Jiao Huang; Jie Luo; Yuma Yang; Qin Liu; Yingchen Lyu; Liuxian Ban; Ziyang Qi; Nanzhou Yu; Chaohui He; Weiguang Zhang; Yanru Qin; Beilei Liu; Xinyuan Guana

doi:10.1073/pnas.2520427123

Multiomics identifies a cholesterol–TFEB–PLD3–TLR9 axis driving immunosuppressive tumor-associated macrophage polarization in esophageal squamous cell carcinoma

Licheng Tan (Co-first Author), Hongyu Zhou (Co-first Author), Baifeng Zhang (Co-first Author), Dora Lai Wan Kwong, Yongxu Jia, Jiayi Huang, Kin To Hugo Siu, Shuang Zhang, Jiao Huang, Jie Luo, Yuma Yang, Qin Liu, Yingchen Lyu, Liuxian Ban, Ziyang Qi, Nanzhou Yu, Chaohui He, Weiguang Zhang, Yanru Qin, Beilei Liu^*Xinyuan Guana^*

^*Corresponding author for this work

Research output: Journal Publications and Reviews › RGC 21 - Publication in refereed journal › peer-review

Abstract

Tumor-associated macrophages (TAMs) reshape the tumor immune microenvironment and promote tumor progression, yet the underlying mechanisms remain largely unclear. Through integration of single-cell RNA (scRNA) sequencing datasets from esophageal squamous cell carcinoma (ESCC), we identified a distinct protumoral macrophage population with elevated expression of phospholipase D3 (PLD3). Multiomics investigations revealed that high infiltration of these PLD3-high macrophages was associated with poor clinical outcomes in ESCC patients. Mechanistically, tumor cells secreted cholesterol to modulate the microenvironment. Upon the uptake by TAMs, cholesterol triggered the nuclear translocation of transcription factor EB (TFEB), which directly bound to the PLD3 promoter region and activated its transcription. The overexpressed PLD3 localized to lysosomes, enzymatically degrading single-stranded nucleic acids, thereby suppressing the activation of the toll-like receptor 9 (TLR9) pathway. This cascade ultimately impaired effector T cell function and sustained an immunosuppressive tumor microenvironment (TME). Notably, therapeutic intervention using ODN2216-siPLD3 in murine models enhanced CD8 T cell infiltration and significantly inhibited tumor growth. Our findings highlight PLD3-high macrophages as a promising diagnostic biomarker and a therapeutic target for ESCC, paving the way for potential clinical translation. Copyright © 2026 the Author(s).

Original language	English
Article number	e2520427123
Number of pages	11
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	123
Issue number	3
Online published	12 Jan 2026
DOIs	https://doi.org/10.1073/pnas.2520427123
Publication status	Published - 20 Jan 2026

Funding

We thank the Centre for PanorOmic Sciences, The University of Hong Kong, for the support. Graphic figures were drawn via Biorender(https://www.biorender.com/).This work is supported by grants from the Hong Kong Research Grant Council grants including Collaborative Research Funds (C7065-18GF, C7026-18GF, and C4039-19GF), Research Impact Fund (R4017-18, R1020-18F, and R7022-20), General Research Fund (17119322), Theme-based Research Scheme Fund (T12-703/22-R), the National Natural Science Foundation of China(82072738, 82273483, 82403295), Shenzhen Key Laboratory for cancer metastasis and personalized therapy (ZDSYS20210623091811035), Shenzhen Science and Technology Program (JCYJ20220818103014030, KQTD20180411185028798,JCYJ20220818103012025, and JCYJ20240813113012017), Guangdong Science and Technology Department (2020B1212030004 and 2024A1515220039), and the Program for Guangdong Introducing Innovative and Entrepreneurial Team (2019BT02Y198). X.Y.G. is the Sophie Y. M. Chan Professor in Cancer Research.

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

Publisher's Copyright Statement

COPYRIGHT TERMS OF DEPOSITED FINAL PUBLISHED VERSION FILE: This full text is made available under CC-BY-NC-ND 4.0. https://creativecommons.org/licenses/by-nc-nd/4.0/

RGC Funding Information

RGC-funded

Access Output

10.1073/pnas.2520427123

Other Access Options

Check CityUHK Library

Permanent Link

Right click to copy link

Cite this

Tan, L., Zhou, H., Zhang, B., Wan Kwong, D. L., Jia, Y., Huang, J., Hugo Siu, K. T., Zhang, S., Huang, J., Luo, J., Yang, Y., Liu, Q., Lyu, Y., Ban, L., Qi, Z., Yu, N., He, C., Zhang, W., Qin, Y., ... Guana, X. (2026). Multiomics identifies a cholesterol–TFEB–PLD3–TLR9 axis driving immunosuppressive tumor-associated macrophage polarization in esophageal squamous cell carcinoma. Proceedings of the National Academy of Sciences of the United States of America, 123(3), Article e2520427123. https://doi.org/10.1073/pnas.2520427123

@article{4d3f2617f1b4425ab64814c6be41edaf,

title = "Multiomics identifies a cholesterol–TFEB–PLD3–TLR9 axis driving immunosuppressive tumor-associated macrophage polarization in esophageal squamous cell carcinoma",

abstract = "Tumor-associated macrophages (TAMs) reshape the tumor immune microenvironment and promote tumor progression, yet the underlying mechanisms remain largely unclear. Through integration of single-cell RNA (scRNA) sequencing datasets from esophageal squamous cell carcinoma (ESCC), we identified a distinct protumoral macrophage population with elevated expression of phospholipase D3 (PLD3). Multiomics investigations revealed that high infiltration of these PLD3-high macrophages was associated with poor clinical outcomes in ESCC patients. Mechanistically, tumor cells secreted cholesterol to modulate the microenvironment. Upon the uptake by TAMs, cholesterol triggered the nuclear translocation of transcription factor EB (TFEB), which directly bound to the PLD3 promoter region and activated its transcription. The overexpressed PLD3 localized to lysosomes, enzymatically degrading single-stranded nucleic acids, thereby suppressing the activation of the toll-like receptor 9 (TLR9) pathway. This cascade ultimately impaired effector T cell function and sustained an immunosuppressive tumor microenvironment (TME). Notably, therapeutic intervention using ODN2216-siPLD3 in murine models enhanced CD8 T cell infiltration and significantly inhibited tumor growth. Our findings highlight PLD3-high macrophages as a promising diagnostic biomarker and a therapeutic target for ESCC, paving the way for potential clinical translation. Copyright {\textcopyright} 2026 the Author(s).",

author = "Licheng Tan and Hongyu Zhou and Baifeng Zhang and {Wan Kwong}, {Dora Lai} and Yongxu Jia and Jiayi Huang and {Hugo Siu}, {Kin To} and Shuang Zhang and Jiao Huang and Jie Luo and Yuma Yang and Qin Liu and Yingchen Lyu and Liuxian Ban and Ziyang Qi and Nanzhou Yu and Chaohui He and Weiguang Zhang and Yanru Qin and Beilei Liu and Xinyuan Guana",

year = "2026",

month = jan,

day = "20",

doi = "10.1073/pnas.2520427123",

language = "English",

volume = "123",

journal = "Proceedings of the National Academy of Sciences of the United States of America",

issn = "0027-8424",

publisher = "National Academy of Sciences",

number = "3",

}

Tan, L, Zhou, H, Zhang, B, Wan Kwong, DL, Jia, Y, Huang, J, Hugo Siu, KT, Zhang, S, Huang, J, Luo, J, Yang, Y, Liu, Q, Lyu, Y, Ban, L, Qi, Z, Yu, N, He, C, Zhang, W, Qin, Y, Liu, B & Guana, X 2026, 'Multiomics identifies a cholesterol–TFEB–PLD3–TLR9 axis driving immunosuppressive tumor-associated macrophage polarization in esophageal squamous cell carcinoma', Proceedings of the National Academy of Sciences of the United States of America, vol. 123, no. 3, e2520427123. https://doi.org/10.1073/pnas.2520427123

Multiomics identifies a cholesterol–TFEB–PLD3–TLR9 axis driving immunosuppressive tumor-associated macrophage polarization in esophageal squamous cell carcinoma. / Tan, Licheng (Co-first Author); Zhou, Hongyu (Co-first Author); Zhang, Baifeng (Co-first Author) et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 123, No. 3, e2520427123, 20.01.2026.

Research output: Journal Publications and Reviews › RGC 21 - Publication in refereed journal › peer-review

TY - JOUR

T1 - Multiomics identifies a cholesterol–TFEB–PLD3–TLR9 axis driving immunosuppressive tumor-associated macrophage polarization in esophageal squamous cell carcinoma

AU - Tan, Licheng

AU - Zhou, Hongyu

AU - Zhang, Baifeng

AU - Wan Kwong, Dora Lai

AU - Jia, Yongxu

AU - Huang, Jiayi

AU - Hugo Siu, Kin To

AU - Zhang, Shuang

AU - Huang, Jiao

AU - Luo, Jie

AU - Yang, Yuma

AU - Liu, Qin

AU - Lyu, Yingchen

AU - Ban, Liuxian

AU - Qi, Ziyang

AU - Yu, Nanzhou

AU - He, Chaohui

AU - Zhang, Weiguang

AU - Qin, Yanru

AU - Liu, Beilei

AU - Guana, Xinyuan

PY - 2026/1/20

Y1 - 2026/1/20

N2 - Tumor-associated macrophages (TAMs) reshape the tumor immune microenvironment and promote tumor progression, yet the underlying mechanisms remain largely unclear. Through integration of single-cell RNA (scRNA) sequencing datasets from esophageal squamous cell carcinoma (ESCC), we identified a distinct protumoral macrophage population with elevated expression of phospholipase D3 (PLD3). Multiomics investigations revealed that high infiltration of these PLD3-high macrophages was associated with poor clinical outcomes in ESCC patients. Mechanistically, tumor cells secreted cholesterol to modulate the microenvironment. Upon the uptake by TAMs, cholesterol triggered the nuclear translocation of transcription factor EB (TFEB), which directly bound to the PLD3 promoter region and activated its transcription. The overexpressed PLD3 localized to lysosomes, enzymatically degrading single-stranded nucleic acids, thereby suppressing the activation of the toll-like receptor 9 (TLR9) pathway. This cascade ultimately impaired effector T cell function and sustained an immunosuppressive tumor microenvironment (TME). Notably, therapeutic intervention using ODN2216-siPLD3 in murine models enhanced CD8 T cell infiltration and significantly inhibited tumor growth. Our findings highlight PLD3-high macrophages as a promising diagnostic biomarker and a therapeutic target for ESCC, paving the way for potential clinical translation. Copyright © 2026 the Author(s).

AB - Tumor-associated macrophages (TAMs) reshape the tumor immune microenvironment and promote tumor progression, yet the underlying mechanisms remain largely unclear. Through integration of single-cell RNA (scRNA) sequencing datasets from esophageal squamous cell carcinoma (ESCC), we identified a distinct protumoral macrophage population with elevated expression of phospholipase D3 (PLD3). Multiomics investigations revealed that high infiltration of these PLD3-high macrophages was associated with poor clinical outcomes in ESCC patients. Mechanistically, tumor cells secreted cholesterol to modulate the microenvironment. Upon the uptake by TAMs, cholesterol triggered the nuclear translocation of transcription factor EB (TFEB), which directly bound to the PLD3 promoter region and activated its transcription. The overexpressed PLD3 localized to lysosomes, enzymatically degrading single-stranded nucleic acids, thereby suppressing the activation of the toll-like receptor 9 (TLR9) pathway. This cascade ultimately impaired effector T cell function and sustained an immunosuppressive tumor microenvironment (TME). Notably, therapeutic intervention using ODN2216-siPLD3 in murine models enhanced CD8 T cell infiltration and significantly inhibited tumor growth. Our findings highlight PLD3-high macrophages as a promising diagnostic biomarker and a therapeutic target for ESCC, paving the way for potential clinical translation. Copyright © 2026 the Author(s).

UR - http://www.scopus.com/inward/record.url?scp=105027108198&partnerID=8YFLogxK

UR - https://www.scopus.com/record/pubmetrics.uri?eid=2-s2.0-105027108198&origin=recordpage

U2 - 10.1073/pnas.2520427123

DO - 10.1073/pnas.2520427123

M3 - RGC 21 - Publication in refereed journal

C2 - 41525484

SN - 0027-8424

VL - 123

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 3

M1 - e2520427123

ER -

Tan L, Zhou H, Zhang B, Wan Kwong DL, Jia Y, Huang J et al. Multiomics identifies a cholesterol–TFEB–PLD3–TLR9 axis driving immunosuppressive tumor-associated macrophage polarization in esophageal squamous cell carcinoma. Proceedings of the National Academy of Sciences of the United States of America. 2026 Jan 20;123(3):e2520427123. Epub 2026 Jan 12. doi: 10.1073/pnas.2520427123

Multiomics identifies a cholesterol–TFEB–PLD3–TLR9 axis driving immunosuppressive tumor-associated macrophage polarization in esophageal squamous cell carcinoma

Abstract

Funding

UN SDGs

Publisher's Copyright Statement

RGC Funding Information

Access Output

Other Access Options

Permanent Link

Other Files and Links

Embargoed Document

Fingerprint

RIF-ExtU-Lead: Patient-Derived Preclinical Models for Translational Cancer Research: a Hong Kong-based Biotechnology Centre for Genomic Medicine

RIF: Development of an Integrated Microfluidics System for Multi-level High Content Screening of Anti-tumor Drugs

Cite this

Multiomics identifies a cholesterol–TFEB–PLD3–TLR9 axis driving immunosuppressive tumor-associated macrophage polarization in esophageal squamous cell carcinoma

Abstract

Funding

UN SDGs

Publisher's Copyright Statement

RGC Funding Information

Access Output

Other Access Options

Permanent Link

Other Files and Links

Embargoed Document

Fingerprint

Projects

RIF-ExtU-Lead: Patient-Derived Preclinical Models for Translational Cancer Research: a Hong Kong-based Biotechnology Centre for Genomic Medicine

RIF: Development of an Integrated Microfluidics System for Multi-level High Content Screening of Anti-tumor Drugs

Cite this