Galectin-9阳性肿瘤相关巨噬细胞在肌层浸润性膀胱癌中的表型、功能及临床治疗意义

doi:10.3969/j.issn.1674-8115.2022.12.003

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (12): 1666-1676.doi: 10.3969/j.issn.1674-8115.2022.12.003

• 论著 · 基础研究 • 上一篇

Galectin-9阳性肿瘤相关巨噬细胞在肌层浸润性膀胱癌中的表型、功能及临床治疗意义

戚炀炀¹(), 熊鹰²()

^1.上海交通大学基础医学院免疫学与微生物学系，上海市免疫学研究所，上海 200025
^2.复旦大学附属中山医院泌尿外科，上海 200032

收稿日期:2022-09-19 接受日期:2022-11-18 出版日期:2022-12-28 发布日期:2022-12-28
通讯作者: 熊鹰 E-mail:qiyangyang@shsmu.edu.cn;xiong.ying@zs-hospital.sh.cn
作者简介:戚炀炀（1995—），女，助理实验师，硕士；电子信箱：qiyangyang@shsmu.edu.cn。
基金资助:
国家自然科学基金青年科学基金项目(81902563)

Phenotype, function and clinical significance of galectin-9 positive tumor-associated macrophages in muscle-invasive bladder cancer

QI Yangyang¹(), XIONG Ying²()

^1.Department of Immunology and Microbiology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai Institute of Immunology, Shanghai 200025, China
^2.Department of Urology, Zhongshan Hospital, Fudan University, Shanghai 200032, China

Received:2022-09-19 Accepted:2022-11-18 Online:2022-12-28 Published:2022-12-28
Contact: XIONG Ying E-mail:qiyangyang@shsmu.edu.cn;xiong.ying@zs-hospital.sh.cn
Supported by:
National Natural Science Foundation of China—Youth Fund Project(81902563)

摘要/Abstract

摘要：

目的·分析半乳糖凝集素9阳性肿瘤相关巨噬细胞（galectin-9⁺ tumor-associated macrophages， galectin-9⁺TAMs）在肌层浸润性膀胱癌（muscle-invasive bladder cancer，MIBC）组织中的表型特征，探究MIBC微环境对galectin-9⁺TAMs的调控作用，galectin-9⁺TAMs抑制CD8⁺T细胞反应的机制及其临床治疗意义。方法·通过流式细胞术检测MIBC与癌旁组织中galectin-9⁺TAMs的表型特征。利用TCGA（The Cancer Genome Atlas）数据库筛选与LGALS9和LGALS9巨噬细胞基因集显著相关的细胞因子。用人重组细胞因子体外刺激巨噬细胞，分为人重组巨噬细胞集落刺激因子（recombinant human macrophage-stimulating factor，rhM-CSF）刺激组、人重组白介素-16（recombinant human interleukin-16，rhIL-16）刺激组和人重组干扰素γ（recombinant human interferon-γ，rhIFN-γ）刺激组，并通过流式细胞术检测3组galectin-9表达情况。流式细胞术检测加入rhM-CSF中和性抗体后巨噬细胞表达galectin-9的情况。Anti-galectin-9抗体处理MIBC单细胞悬液后，通过流式细胞术检测TAMs的效应功能变化。分选癌与癌旁组织中galectin-9⁺TAMs和人外周血CD8⁺T细胞并进行体外共培养，通过流式细胞术检测CD8⁺T细胞效应功能变化。采用anti-galectin-9抗体和程序性死亡受体1（programmed cell death protein 1，PD-1）抗体单独及协同处理体外培养的肿瘤组织，通过流式细胞术检测肿瘤细胞凋亡及CD8⁺T细胞效应功能变化。结果·Galectin-9⁺TAMs高表达人类白细胞DR抗原（human leukocyte antigen DR，HLA-DR）、CD86、CD206和细胞程序性死亡-配体1（programmed cell death-ligand 1，PD-L1），分泌IL-10和转化生长因子-β（transforming growth factor-β，TGF-β）增加，分泌肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）减少。TCGA数据库筛选结果显示，M-CSF、IL-16和IFN-γ与LGALS9和LGALS9巨噬细胞基因集的相关性最显著。用rhM-CSF、rhIL-16和rhIFN-γ体外刺激巨噬细胞，rhM-CSF刺激组中galectin-9的表达显著升高，加入中和性抗体后表达显著下调。阻断galectin-9后，TAMs表型从高表达抑制性分子向促炎症分子转换，其表面表达PD-L1显著下降。体外共培养galectin-9⁺TAMs和CD8⁺T细胞后，galectin-9⁺TAMs能够抑制CD8⁺T细胞的效应功能，该作用部分依赖于galectin-9。联合阻断galectin-9和PD-1后，肿瘤细胞凋亡比例、CD8⁺T细胞的增殖能力和效应分子的分泌与单独阻断PD-1相比，均显著增多或增强。结论·Galectin-9⁺TAMs具有免疫抑制表型和功能。肿瘤来源M-CSF诱导TAMs高表达galectin-9。Galectin-9⁺TAMs抑制CD8⁺T细胞功能从而促进MIBC免疫逃逸。联合阻断galectin-9和PD-1能够更有效地重激活CD8⁺T细胞功能。

关键词: 半乳糖凝集素-9, galectin-9阳性肿瘤相关巨噬细胞, 肌层浸润性膀胱癌, CD8⁺T细胞, 免疫逃逸

Abstract:

Objective ·To explore the phenotype of galectin-9⁺ tumor-associated macrophages (galectin-9⁺TAMs) in muscle-invasive bladder cancer (MIBC). To clarify the regulation of galectin-9⁺TAMs in MIBC microenvironment, and elucidate the mechanism of galectin-9⁺TAMs inhibiting the effector function of CD8⁺T cells and its clinical therapeutic significance in MIBC. Methods ·Phenotype of galectin-9⁺TAMs from MIBC peritumor and tumor tissues was detected by flow cytometry. TCGA (The Cancer Genome Atlas) database was used to sort out the cytokines most relevant to LGALS9 and LGALS9 macrophage gene set. Macrophages were stimulated by recombinant human cytokines in vitro, divided into recombinant human macrophage stimulating factor (rhM-CSF) stimulation group, recombinant human interleukin-16 (rhIL-16) stimulation group and recombinant human interferon-γ (rhIFN-γ) stimulation group. Expression level of galectin-9 among the three groups was verified by flow cytometry. Expression level of galectin-9 on macrophages was detected by flow cytometry after adding M-CSF neutralizing antibody. Effector functions of TAMs were detected by flow cytometry after treating MIBC single cell suspension with anti-galectin-9 antibody. Galectin-9⁺TAMs from peritumor and tumor tissues, and human peripheral blood CD8⁺T cells were sorted and co-cultured in vitro. Effector functions of CD8⁺T cells were detected by flow cytometry. Tumor tissues cultured in vitro were treated with anti-galectin-9 antibody and programmed cell death protein 1 (PD-1) antibody alone or in combination. Tumor cell apoptosis and effector function of CD8⁺T cells were detected by flow cytometry. Results ·Galectin-9⁺TAMs exhibited the phenotype with high expression of human leukocyte antigen DR (HLA-DR), CD86, CD206 and programmed cell death-ligand 1 (PD-L1). Increased IL-10 and transforming growth factor-β (TGF-β) and decreased tumor necrosis factor-α (TNF-α) were secreted by itself. M-CSF, IL-16 and IFN-γ showed the significant difference with LGALS9 and LGALS9 macrophage gene set in TCGA database. Galectin-9 on macrophages increased significantly in rhM-CSF stimulated group and its expression decreased by adding neutralizing antibody. Galectin-9 inhibition switched the activation of TAMs from an immunosuppressive phenotype to a more inflammatory state and PD-L1 on its surface significantly decreased. After cultured in vitro, galectin-9⁺TAMs inhibited the effect of CD8⁺T cells in a partly galectin-9-dependent manner. Compared with applying PD-1 inhibitor alone, percentage of tumor cell apoptosis, the proliferation of CD8⁺T cells and its effector molecules were significantly enhanced or increased in both galectin-9 and PD-1 blockade. Conclusion ·Galectin-9⁺TAMs exhibit an immunosuppressive phenotype and function. Tumor-derived M-CSF induced TAMs to express galectin-9. Galectin-9⁺TAMs inhibit the function of CD8⁺T cells to promote the immune escape of MIBC. Galectin-9 and PD-1 blockade can reactivate the function of CD8⁺T cells more effectively and synergistically.

Key words: galectin-9, galectin-9⁺tumor-associated macrophages (galectin-9⁺TAMs), muscle-invasive bladder cancer (MIBC), CD8⁺T cells, immune evasion

中图分类号:

R392.12

戚炀炀, 熊鹰. Galectin-9阳性肿瘤相关巨噬细胞在肌层浸润性膀胱癌中的表型、功能及临床治疗意义[J]. 上海交通大学学报（医学版）, 2022, 42(12): 1666-1676.

QI Yangyang, XIONG Ying. Phenotype, function and clinical significance of galectin-9 positive tumor-associated macrophages in muscle-invasive bladder cancer[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(12): 1666-1676.

图/表 6

图1 Galectin-9+TAMs在MIBC患者中的表型特征Note： A. Representative flow cytometric plots of galectin-9+TAMs (galectin-9+CD45+CD68+cells) from peritumor and tumor tissues. B. Expression of HLA-DR, CD86, CD80 and CD206 on peritumoral and intratumoral galectin-9+TAMs. C. Immunosuppressive cytokines (IL-10 and TGF-β) and pro-inflammatory cytokines (TNF-α and IL-12) secreted by peritumoral and intratumoral galectin-9+TAMs. D. Expression of immune checkpoint molecules (PD-L1, PD-1 and CTLA-4) on peritumoral and intratumoral galectin-9+TAMs. Flow cytometry charts and statistical analysis are included in B?D (n=10).

Fig 1 Phenotype of galectin-9+TAMs in MIBC patients

图2 TCGA数据库筛选与galectin-9显著相关的细胞因子Note： A. GSEA analysis uncovered an enrichment of cytokine-cytokine receptor interaction involved in galectin-9 high tumors. NES, non-enrichment score. B. Volcano plots of the top three upregulated cytokines genes related to LGALS9 high and LGALS9 macrophage gene set high group. Log2 fold change and -Log10 adjusted P value were written in sequence. LGALS9 (high vs low): CSF-1:1.001,6.821; IL-16:1.181,6.859; IFNG:2.112,6.934. LGALS9 macrophage gene set (high vs low): CSF-1:1.652,16.192; IL-16: 1.795,13.691; IFNG: 3.131,11.069.

Fig 2 Screening for cytokines significantly related to galectin-9 in TCGA database

图3 IFN-γ、M-CSF和IL-16对巨噬细胞表达galectin-9的影响Note： A. Galectin-9 expression on macrophages induced from patient PBMCs after stimulation with rhIFN-γ, rhM-CSF and rhIL-16 by flow cytometry (n=3).B. M-CSF concentration between tumor and peritumor tissues or between PTS and TTS (n=10). C. Correlation between M-CSF concentration and percentage of galectin-9+TAMs in MIBC tumors (n=20). D. Galectin-9 expression on macrophages induced from patient PBMCs exposed to TTS with anti-M-CSF for 24 h by flow cytometry(n=3).

Fig 3 Effect of IFN-γ, M-CSF and IL-16 on expression of galectin-9 in macrophages

图4 阻断galectin-9对TAMs的表型及功能影响Note： A/B. Flow cytomtery images (A) and quantification of CD206 and HLA-DR (B) within TAMs (CD45+CD68+cells), n=10. C. Quantification of IL-10, TGF-β, TNF-α, IL-12 and PD-L1 within TAMs, n=10.

Fig 4 Effects of galectin-9 blockade on phenotype and function of TAMs

图5 膀胱癌组织中galectin-9+TAMs 对CD8+T细胞增殖和功能的影响Note： A.Flow cytometry charts. B. Statistical analysis (n=3).

Fig 5 Effect of galectin-9+TAMs on proliferation and function of CD8+T cells in MIBC tumor tissues

图6 Galectin-9与PD-1的协同效应Note： A.Flow cytometry charts of CD8+T cells and tumor cells (Epcam+CD45-). B. Quantification of Annexin V within tumor cells. C?E. Quantification of Ki67 (C), IFN-γ (D) and GZMB (E) within CD8+T cells.Tumor cell suspensions were treated with isotype group, anti-galectin-9 antibody group, pembrolizumab group and anti-galectin-9+pembrolizumab group for 12 h (n=8?10).

Fig 6 Synergistic effect of galectin-9 and PD-1

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Galectin-9阳性肿瘤相关巨噬细胞在肌层浸润性膀胱癌中的表型、功能及临床治疗意义

Phenotype, function and clinical significance of galectin-9 positive tumor-associated macrophages in muscle-invasive bladder cancer

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