MMP14在胰腺癌中的表达及其与肿瘤免疫微环境特征的相关性研究

doi:10.3969/j.issn.1674-8115.2022.03.008

摘要/Abstract

摘要：

目的·探究基质金属蛋白酶14（matrix metalloproteinase 14，MMP14）在胰腺癌组织中的表达及其与临床特征的相关性，并分析其与胰腺癌肿瘤免疫微环境特征的相关性。方法·通过GEPIA（Gene Expression Profiling Interactive Analysis）数据平台分析MMP家族成员在30种常见肿瘤组织中的表达情况。通过R语言整合GTEx（Genotype-Tissue Expression）数据库中167例胰腺正常组织和TCGA（The Cancer Genome Atlas）数据库中178例胰腺癌组织及4例配对癌旁组织的转录组数据，比较MMP14在胰腺癌与正常组织及癌旁组织中的表达差异。使用GEPIA数据平台对不同MMP14表达水平胰腺癌患者进行生存分析。从TCGA数据库中下载178例胰腺癌患者的相关临床信息，应用Perl和R语言分析MMP14的表达与临床特征的相关性。应用R语言和GSEA v3.0进行京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）通路富集分析和基因本体论（Gene Ontology，GO）功能富集分析。采用CIBERSORT反卷积算法计算胰腺癌免疫微环境中各类免疫细胞亚群比例，分析MMP14的表达与各免疫细胞亚群比例的相关性。收集6例新鲜胰腺癌组织和配对癌旁组织，流式细胞术检测各类免疫细胞亚群；通过t-SNE降维与统计学分析，描述胰腺癌免疫微环境的特征。利用胰腺癌组织芯片进行多色荧光免疫组织化学实验，检测MMP14的表达与免疫细胞亚群的比例，验证生物信息学分析结果。结果·在生物信息学数据库中，与MMP家族其他成员相比，MMP14在多种肿瘤中高表达，且胰腺癌中MMP14的表达水平高于其他肿瘤；MMP14在胰腺癌中表达水平显著高于胰腺正常组织和癌旁组织（P=0.000），TNM分期Ⅱ期肿瘤MMP14表达水平显著高于Ⅰ期（P=0.012），病理组织学分级G2和G3期肿瘤MMP14显著高于G1期（均P=0.000）。MMP14低表达组患者生存预后显著优于高表达组（P=0.033）。KEGG和GO分析结果显示MMP14主要富集在胰腺癌和免疫相关通路。MMP14的表达水平与CD8⁺ T细胞、单核细胞的比例呈负相关（均P<0.05），与巨噬细胞的比例呈正相关（P=0.000）。流式细胞实验结果提示胰腺癌肿瘤微环境呈现免疫抑制性。多色荧光免疫组织化学实验结果显示，MMP14在肿瘤组织中表达水平显著高于癌旁组织（P=0.000），TNM分期较晚及病理组织学分级较高的肿瘤MMP14的表达水平也相对较高（均P<0.05）。在MMP14高表达的肿瘤组织中，CD8⁺ T细胞比例降低（P=0.001），巨噬细胞比例升高（P=0.000），与生物信息学分析结果一致。结论·与正常胰腺组织和癌旁组织相比，MMP14在胰腺癌中的表达显著升高且与患者不良预后相关；MMP14高表达的胰腺癌组织中CD8⁺ T细胞比例降低，巨噬细胞比例升高；高表达MMP14的胰腺癌肿瘤微环境呈现高度免疫抑制性。

关键词: 胰腺癌, 基质金属蛋白酶14, 肿瘤免疫微环境, 生物信息学分析, 流式细胞术, 多色荧光免疫组织化学法

Abstract:

Objective·To explore the expression of matrix metalloproteinase 14 (MMP14) in pancreatic cancer tissues and its correlation with clinical characteristics, and analyze its correlation with the characteristics of tumor immune microenvironment in pancreatic cancer.

Methods·GEPIA (Gene Expression Profiling Interactive Analysis) platform was used to analyze the MMPs expression in 30 common tumor tissues. R software was used to integrate transcriptomic data of 167 normal pancreatic tissues from GTEx (Genotype-Tissue Expression) database with 178 pancreatic cancer tissues and 4 para-tumor tissues from TCGA (The Cancer Genome Atlas) database to compare the differences of MMP14 expression. GEPIA platform was used to perform survival analysis in the patients with different MMP14 expression levels. Perl and R project software were used to analyze the correlation of MMP14 expression and clinical characteristics. R project software and GSEA v3.0 software were used for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and Gene Ontology (GO) functional enrichment analysis. CIBERSORT analysis was used to calculate the proportions of immune cell subsets in the tumor immune microenvironment, and analyze correlation of MMP14 expression and the proportion of immune cell subsets. Six fresh surgical samples of pancreatic cancer tissues and paired para-tumor tissues were collected to detect various immune cell subsets by flow cytometry. t-SNE dimensionality reduction and statistical analysis were used to explore the characteristics of tumor immune microenvironment in pancreatic cancer. A tissue microarray was used to conduct multiplex immunohistochemistry (mIHC) to detect MMP14 expression and the proportions of immune cell subsets to verify bioinformatics analysis results.

Results·In the bioinformatics database, compared with the other members of MMPs, MMP14 was highly expressed in a variety of tumors, and MMP14 expression in pancreatic cancer was higher than that in the other cancers. The expression of MMP14 in pancreatic cancer tissues was significantly higher than that in normal pancreatic tissues and para-tumor tissues (P=0.000). The expression of MMP14 in the TNM stage II tumor was higher than that in the stage I tumor (P=0.012). The expressions of MMP14 in the histological grade 2 (G2) and grade 3 (G3) tumors were higher than those in the grade 1 (G1) tumor (P=0.000). The survival prognosis of the patients in MMP14-low expression group was significantly better than that in high expression group (P=0.033). KEGG and GO analysis results showed that MMP14 was enriched in the pancreatic cancer pathway and immune-related pathways. The expression level of MMP14 was negatively correlated with the percentages of CD8⁺ T cells and monocytes (P<0.05) , and positively correlated with the percentage of macrophages (P=0.000). Flow cytometry analysis suggested that tumor microenvironment of pancreatic cancer was immunosuppressive. The results of mIHC showed that MMP14 expression in the tumor tissues was higher than that in the para-tumor tissues (P=0.000). Tumor tissues with higher TNM stage and histological grade expressed relatively higher MMP14 levels (P<0.05). In the tumor tissues with high MMP14 expression level, the percentage of CD8⁺ T cells decreased (P=0.001) and the percentage of macrophages increased (P=0.000), which were consistent with bioinformatics analysis results.

Conclusion·Compared with normal pancreatic tissues and para-tumor tissues, the expression of MMP14 significantly increases in pancreatic tumor tissues and the high expression is associated with poor prognosis of the patients; in the pancreatic tumor tissues with high MMP14 expression, the percentage of CD8⁺ T cells decreases and the percentage of macrophages increases; with high MMP14 expression, the tumor microenvironment of pancreatic cancer is highly immunosuppressive.

Key words: pancreatic cancer, matrix metalloproteinase 14 (MMP14), tumor immune microenvironment, bioinformatics analysis, flow cytometry, multiplex immunohistochemistry (mIHC)

中图分类号:

R735.9

许静轩, 杜少倩, 曹源, 王红霞, 黄伟翼. MMP14在胰腺癌中的表达及其与肿瘤免疫微环境特征的相关性研究[J]. 上海交通大学学报（医学版）, 2022, 42(3): 312-322.

XU Jingxuan, DU Shaoqian, CAO Yuan, WANG Hongxia, HUANG Weiyi. MMP14 expression in pancreatic cancer and its correlation with characteristics of tumor immune microenvironment[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(3): 312-322.

图/表 6

表1 抗体信息

Tab 1 Information of antibodies

Antibody	Source	Identifier	Usage
Anti-CD45	BD Biosciences	Cat# 564585	Flow cytometry
Anti-CD3	BD Biosciences	Cat# 566517	Flow cytometry
Anti-CD4	BD Biosciences	Cat# 560909	Flow cytometry
Anti-CD8	BD Biosciences	Cat# 612943	Flow cytometry
Anti-TCRγδ	BD Biosciences	Cat# 562511	Flow cytometry
Anti-CD19	BD Biosciences	Cat# 612917	Flow cytometry
Anti-CD56	BD Biosciences	Cat# 557919	Flow cytometry
Anti-CD16	BD Biosciences	Cat# 748850	Flow cytometry
Anti-CD11b	BD Biosciences	Cat# 747357	Flow cytometry
Anti-CD14	BD Biosciences	Cat# 566465	Flow cytometry
Anti-CD66b	BD Biosciences	Cat# 561645	Flow cytometry
Anti-CD11c	BD Biosciences	Cat# 612968	Flow cytometry
Anti-CD123	BD Biosciences	Cat# 563072	Flow cytometry
Anti-HLA-DR	BD Biosciences	Cat# 561358	Flow cytometry
Anti-CD206	BD Biosciences	Cat# 741860	Flow cytometry
Anti-CD45	Santa Cruz Biotechnology	Cat# sc-19597	mIHC
Anti-CD8	Abcam	Cat# ab237709	mIHC
Anti-CD14	Abcam	Cat# ab133335	mIHC
Anti-CD68	Abcam	Cat# ab955	mIHC
Anti-MMP14	Abcam	Cat# ab51074	mIHC

图1 生物信息学数据库中 MMP14 在胰腺癌组织中的表达情况及其与患者临床特征的相关性分析Note： A. Expression of MMPs in 30 common tumor tissues. B. MMP14 expression in the pancreatic cancer tissues and the normal tissues. C. Comparison of MMP14 expression among different TNM stages of the patients with pancreatic adenocarcinoma. D. Comparison of MMP14 expression among different grades of the patients with pancreatic adenocarcinoma. E. Overall survival analysis of MMP14-high expression group and MMP14-low expression group. ①P=0.000, ②P=0.012. ACC—adrenocortical carcinoma; BLCA—bladder urothelial carcinoma; BRCA—breast invasive carcinoma; CESC—cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL—cholangiolar carcinoma; COAD—colon adenocarcinoma; DLBC—lymphoid neoplasm diffuse large B-cell lymphoma; ESCA—esophageal carcinoma; GBM—glioblastoma multiforme; HNSC—head and neck squamous cell carcinoma; KICH—kidney chromophobe; KIRC—kidney renal clear cell carcinoma; KIRP—kidney renal papillary cell carcinoma; LAML—acute myeloid leukemia; LGG—brain lower grade glioma; LIHC—liver hepatocellular carcinoma; LUAD—lung adenocarcinoma; LUSC—lung squamous cell carcinoma; MESO—mesothelioma; OV—ovarian serous cystadenocarcinoma; PAAD—pancreatic adenocarcinoma; PCPG—pheochromocytoma and paraganglioma; PRAD—prostate adenocarcinoma; READ—rectum adenocarcinoma; STAD—stomach adenocarcinoma; TGCT—testicular germ cell tumor; THCA—thyroid carcinoma; THYM—thymoma; UCEC—uterine corpus endometrial carcinoma; UCS—uterine carcinosarcoma.

Fig 1 Bioinformatics analysis of MMP14 expression in the pancreatic cancer tissues and its correlation with the patients’ clinical characteristics

图2 TCGA数据库中MMP14信号通路和功能富集分析Note： A. KEGG pathway enrichment analysis. B. GO enrichment analysis.

Fig 2 KEGG analysis and GO analysis of MMP14 in TCGA database

图3 TCGA数据库中胰腺癌 MMP14 不同表达水平组肿瘤浸润免疫细胞比例差异及其与 MMP14 表达的相关性Note： A. Percentage differences of immune cell subsets in MMP14-high expression group (blue) and MMP14-low expression group (yellow). B. Correlation analysis of MMP14 expression and percentage of three immune cell subsets. ①P=0.009, ②P=0.000.

Fig 3 Distribution differences of tumor-infiltrating immune cells in the pancreatic cancer with different MMP14 expression levels and the correlation between the percentage of immune cell subsets and MMP14 expression in TCGA database

图4 胰腺癌和配对癌旁组织的免疫微环境流式细胞分析Note： A. t-SNE analysis of CD45+ immune cells from different tissues and colored by cell clusters. B. t-SNE plots of 24 000 CD45+ immune cells from a pair of tissues colored by relative expression of CD3, CD4, CD8, TCR γδ, CD19, CD14, CD206, CD56, HLA-DR, CD11c, CD11b and CD66b. C. Frequency analysis of all immune cell types for six pairs of pancreatic cancer and para-tumor tissues. ①P=0.025, ②P=0.021, ③P=0.037.

Fig 4 Flow cytometry analysis of immune microenvironment in pancreatic cancer and paired para-tumor tissues

图5 MMP14在胰腺癌组织中的表达及其与免疫微环境相关性的分析Note： A. IHC analysis of MMP14 expression in the pancreatic cancer and paired para-tumor tissues of a representative patient. Scale bar=200 μm. B. IHC scores of MMP14 expression in pancreatic cancer and paired para-tumor tissues. C. Comparison of MMP14 expression among different TNM stages of patients. D. Comparison of MMP14 expression among different grades of patients. E. mIHC images of two representative patients with different MMP14 expression using MMP14 (green), CD45 (purple), CD8 (red), CD14 (orange), CD68 (yellow) and DNA (blue) markers. The white arrows show CD8+ T cells. The yellow arrows show CD68+ macrophages. Scale bar=200 μm (upper). Scale bar=20 μm (lower). F. Frequency analysis of CD8+ T cells, CD68+ macrophages and CD14+ monocytes in MMP14-high expression group and MMP14-low expression group. ①P=0.000, ②P=0.017, ③P=0.006, ④P=0.022, ⑤P=0.030, ⑥P=0.018,⑦P=0.001.

Fig 5 Analysis of MMP14 expression in the pancreatic cancer tissues and its correlation with immune microenvironment

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