“HA糖外衣”调控ER+乳腺癌细胞干性在内分泌治疗耐药中的作用

doi:10.3969/j.issn.1674-8115.2025.10.005

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (10): 1298-1307.doi: 10.3969/j.issn.1674-8115.2025.10.005

“HA糖外衣”调控ER⁺乳腺癌细胞干性在内分泌治疗耐药中的作用

吴诗怡¹, 陈思², 刘泊含², 刘宇婷¹, 刘鷖雯², 何怡青², 杜艳², 张国良², 郭倩², 高锋², 杨翠霞¹^,²()

^1.上海交通大学医学院附属第六人民医院检验科，上海 200233
^2.上海交通大学医学院附属第六人民医院中心实验室，上海 200233

收稿日期:2024-12-30 接受日期:2025-06-23 出版日期:2025-10-28 发布日期:2025-10-28
通讯作者: 杨翠霞，主任技师，博士；电子信箱：dr.steven@163.com。
基金资助:
国家自然科学基金(82273462);国家自然科学基金(82503431)

Role of "HA coat" in modulating stemness and endocrine resistance in ER⁺ breast cancer

WU Shiyi¹, CHEN Si², LIU Bohan², LIU Yuting¹, LIU Yiwen², HE Yiqing², DU Yan², ZHANG Guoliang², GUO Qian², GAO Feng², YANG Cuixia¹^,²()

^1.Department of Clinical Laboratory, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200233, China
^2.Department of Molecular Biology, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200233, China

Received:2024-12-30 Accepted:2025-06-23 Online:2025-10-28 Published:2025-10-28
Contact: YANG Cuixia, E-mail:dr.steven@163.com.
Supported by:
National Natural Science Foundation of China(82273462)

摘要/Abstract

摘要：

目的·探究雌激素受体阳性（estrogen receptor-positive，ER⁺）乳腺癌的内分泌治疗耐药微环境中透明质酸（hyaluronan，HA）的表达情况及其对耐药性的影响。方法·借助化学免疫发光法检测氟维司群（fulvestrant）耐药乳腺癌细胞培养上清中的HA含量；通过免疫荧光法（immunofluorescence，IF）检测耐药细胞系（MCF7/FulR）细胞表面CD44与HA的表达及共定位情况；利用前期研究构建的内分泌治疗耐药乳腺癌小鼠模型，采用免疫组织化学法（immunohistochemistry，IHC）检测耐药的ER⁺乳腺癌组织中HA的表达情况；通过单细胞转录组测序（scRNA-seq）、RNA测序（RNA-seq）方法分析fulvestrant耐药ER⁺乳腺癌细胞的转录组特征及HA相关基因的表达情况；借助流式细胞术（flow cytometry，FCM）分析MCF7/FulR细胞中CD44⁺CD24^-细胞的占比；利用GEO数据库分析ER⁺乳腺癌中HA合成基因集与细胞干性的相关性；利用IF观察透明质酸酶（hyaluronidase，HAase）清除MCF7/FulR细胞表面HA的情况；通过荧光定量聚合酶链反应（reverse transcription quantitative real-time PCR，RT-qPCR）和免疫印迹法分析经HAase处理后MCF7/FulR细胞中干性相关分子的表达变化；利用CCK-8实验分析HAase处理后MCF7/FulR细胞对fulvestrant的敏感性变化；利用FCM分析HAase处理后细胞周期的变化；利用Hoechst 33258染色分析HAase处理后凋亡细胞比例的变化。结果·IF结果显示，与MCF7细胞相比，MCF7/FulR细胞表面的“HA糖外衣”显著增厚；IHC结果显示，HA在耐药小鼠乳腺癌组织中的潴留显著增加；scRNA-seq和RNA-seq结果显示，fulvestrant耐药乳腺癌细胞的干性基因表达增加，HA合成相关基因表达增加；相关性分析结果显示，在ER⁺乳腺癌中，HA合成与细胞干性呈正相关；IF和RT-qPCR结果显示，清除MCF7/FulR表面“HA糖外衣”后，其干性相关分子的表达显著下降；CCK-8、FCM和Hoechst 33258染色结果显示，去除“HA糖外衣”后MCF7/FulR对fulvestrant耐受能力减弱，凋亡细胞增多。结论·内分泌治疗耐药乳腺癌细胞表面的“HA糖外衣”显著增厚，fulvestrant耐药肿瘤细胞的干性增强；清除“HA糖外衣”可以显著抑制肿瘤细胞干性，通过诱导耐药癌细胞凋亡，恢复其对fulvestrant的敏感性。

关键词: 乳腺癌, 内分泌治疗耐药, 透明质酸糖外衣

Abstract:

Objective ·To determine hyaluronan (HA) expression in the endocrine-resistant microenvironment of estrogen receptor-positive (ER⁺) breast cancer and elucidate its impact on the acquired resistance. Methods ·Chemiluminescent immunoassay was used to quantify HA levels in the culture supernatants of fulvestrant-resistant breast cancer cells. An immunofluorescence (IF) assay was performed to visualize the colocalization of CD44 and HA in MCF7/FulR cells. Using an established adaptive endocrine-resistant breast cancer mouse model, HA expression in resistant breast cancer tissues was assessed by immunohistochemistry (IHC) assay. Single-cell RNA sequencing (scRNA-seq) and RNA sequencing (RNA-seq) were conducted to examine transcriptomic profiles and alterations in HA-related genes in resistant breast cancer cells. Flow cytometry (FCM) was utilized to measure the proportion of CD44⁺CD24^- cells in MCF7/FulR. The correlation between HA synthesis genes and cell stemness was investigated in clinical ER⁺ breast cancers from GEO data sets. Hyaluronidase (HAase) treatment was applied to remove the "HA coat", and RT-qPCR and Western blotting analysis were carried out to monitor changes in stemness-related molecules. CCK-8 assays, flow cytometry (FCM), and Hoechst 33258 staining were performed to determine changes in apoptosis and fulvestrant efficiency after HAase treatment. Results ·IF results revealed that compared with MCF7 cells, the "HA coat" on the surface of MCF7/FulR cells was significantly thickened. IHC demonstrated markedly increased HA retention in fulvestrant-resistant mouse breast cancer tissues. ScRNA-seq and RNA-seq analyses indicated elevated expression of stemness-related genes and HA synthesis-associated genes in fulvestrant-resistant breast cancer cells. Correlation analysis revealed a positive association between HA synthesis and cancer stemness in ER⁺ breast cancer. IF and RT-qPCR results demonstrated that removing the HA coating from the surface of MCF7/FulR cells led to a significant reduction in the expression of stemness-related molecules; concurrently, CCK-8 assays, FCM analysis, and Hoechst 33258 staining revealed that "HA coat" clearance reduced MCF7/FulR' tolerance to fulvestrant and increased apoptosis. Conclusion ·Endocrine-resistant breast cancer cells develop an enriched "HA coat", which promotes stemness in fulvestrant-resistant tumors. Disruption of this HA coat through HAase treatment effectively reduces cell stemness, induces apoptosis, and re-sensitizes breast cancer cells to fulvestrant.

Key words: breast cancer, endocrine therapy-resistant tumor, hyaluronan coat

中图分类号:

R737.9

吴诗怡, 陈思, 刘泊含, 刘宇婷, 刘鷖雯, 何怡青, 杜艳, 张国良, 郭倩, 高锋, 杨翠霞. “HA糖外衣”调控ER⁺乳腺癌细胞干性在内分泌治疗耐药中的作用[J]. 上海交通大学学报（医学版）, 2025, 45(10): 1298-1307.

WU Shiyi, CHEN Si, LIU Bohan, LIU Yuting, LIU Yiwen, HE Yiqing, DU Yan, ZHANG Guoliang, GUO Qian, GAO Feng, YANG Cuixia. Role of "HA coat" in modulating stemness and endocrine resistance in ER⁺ breast cancer[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(10): 1298-1307.

图/表 5

表1 引物序列

Tab 1 Primer sequences

Gene	Primer sequence
KLF4	Forward	5'-CATCTCAAGGCACACCTGCGAA-3'
	Reverse	5'-TCGGTCGCATTTTTGGCACTGG-3'
SNAIL	Forward	5'-TGCCCTCAAGATGCACATCCGA-3'
	Reverse	5'-GGGACAGGAGAAGGGCTTCTC-3'
SOX2	Forward	5'-GCTACAGCATGATGCAGGACCA-3'
	Reverse	5'-TCTGCGAGCTGGTCATGGAGTT-3'
SOX9	Forward	5'-AGGAAGCTCGCGGACCAGTAC-3'
	Reverse	5'-GGTGGTCCTTCTTGTGCTGCAC-3'
TWIST	Forward	5'-GCCAGGTACATCGACTTCCTCT-3'
	Reverse	5'-TCCATCCTCCAGACCGAGAAGG-3'
VIM	Forward	5'-AGGCAAAGCAGGAGTCCACTGA-3'
	Reverse	5'-ATCTGGCGTTCCAGGGACTCAT-3'
ZEB1	Forward	5'-GGCATACACCTACTCAACTACGG-3'
	Reverse	5'-TGGGCGGTGTAGAATCAGAGTC-3'
NANOG	Forward	5'-CTCCAACATCCTGAACCTCAGC-3'
	Reverse	5'-CGTCACACCATTGCTATTCTTCG-3'
ACTB	Forward	5'-CCCGAGCTGATTGACTACGG-3'
	Reverse	5'-GCAGTTCCAACGATGTCTTCG-3'

图1 耐药乳腺癌细胞表面“HA糖外衣”的潴留情况Note： A. Comparative quantification of HA secretion levels in MCF7/FulR and T47D/FulR versus their parental counterparts. B. IF analysis showing colocalization of HA and CD44 on the membrane of MCF7/FulR cells. C. IHC staining of HA in matched pairs of fulvestrant-sensitive and -resistant breast tumors (n=3). D. Quantitative analysis of HA-positive areas using ImageJ IHC Toolbox software (n=9). ①P=0.001, ②P<0.001.

Fig 1 Enrichment of the HA glycocalyx on the surface of fulvestrant-resistant breast cancer cells

图2 “HA糖外衣”与内分泌治疗耐药乳腺癌细胞干性维持的关系Note： A. UMAP presenting nine distinct cellular clusters within the endocrine-sensitive and -resistant breast cancers. B. GSVA revealing pathway enrichment profiles of differentially expressed genes from scRNA-seq. Red indicates the characteristics significantly enriched in fulvestrant-resistant cancer cells, while blue represents sensitive cancer cells. C. Expression of stemness-associated genes (Abcg2, Sox9) through UMAP and violin plots from scRNA-seq. D. Heatmap of stemness-related genes from RNA-seq. Color gradient reflects Z-score normalized expression levels. E. Heatmap of hyaluronan metabolism-associated genes from RNA-seq. F/G. FCM analysis (F) and Western blotting (G) of CD44+/CD24- cell proportion and its expression in MCF7/FulR cells. H. Correlation matrix heatmap illustrating associations between stemness-related pathways and HA biosynthesis genes in clinical ER+ breast cancers (GEO: GSE17705). ①P=0.003, ②P<0.001.

Fig 2 Association between the HA glycocalyx and stemness maintenance in endocrine therapy-resistant breast cancer

图3 清除“HA糖外衣”对耐药乳腺癌细胞干性的影响Note：A. IF analysis of HA in MCF7/FulR cells treated with 50 μg·mL-1 HAase for 24 h. Quantitative analysis of mean fluorescence intensity was performed using ImageJ software. B/C. Expression of stemness-related transcriptional regulators after HAase-mediated glycocalyx removal, analyzed by RT-qPCR (B) and immunoblotting (C) in MCF7/FulR cells. ①P<0.001, ②P=0.001, ③P=0.014.

Fig 3 Impact of HA glycocalyx removal on stem-like characteristics in fulvestrant-resistant cancer cells

图4 清除“HA糖外衣”对耐药细胞药物敏感性的影响Note： A. CCK-8 proliferation assays of MCF7/FulR cells treated with HAase and fulvestrant for 24 h at increasing doses. B‒D. Propidium iodide-based FCM analysis (B) and Hoechst 33258 nuclear staining (C/D) of MCF7/FulR cells following HAase-mediated glycocalyx removal. ①P<0.001, compared with HAase group; ②P<0.001, ③P=0.002.

Fig 4 Impact of removal of HA glycocalyx on drug sensitivity in drug-resistant cells

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“HA糖外衣”调控ER⁺乳腺癌细胞干性在内分泌治疗耐药中的作用

Role of "HA coat" in modulating stemness and endocrine resistance in ER⁺ breast cancer

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“HA糖外衣”调控ER+乳腺癌细胞干性在内分泌治疗耐药中的作用

Role of "HA coat" in modulating stemness and endocrine resistance in ER+ breast cancer

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“HA糖外衣”调控ER⁺乳腺癌细胞干性在内分泌治疗耐药中的作用

Role of "HA coat" in modulating stemness and endocrine resistance in ER⁺ breast cancer