结直肠癌中LC3与树突状细胞募集、TLS形成的相关性及其临床意义

doi:10.19401/j.cnki.1007-3639.2023.09.002

摘要/Abstract

摘要：

背景与目的：肿瘤细胞免疫逃逸是肿瘤治疗中的难点，而肿瘤细胞中自噬过度激活会引起肿瘤细胞表面抗原的增加，参与抗肿瘤免疫。本研究通过临床样本检测结直肠癌中自噬关键因子微管相关蛋白轻链3（light chain 3，LC3）表达、成熟树突状细胞（mature dendritic cell，mDC）数目及三级淋巴结构（tertiary lymphoid structure，TLS）的形成，探讨其相关性及临床意义，旨在为增强抗肿瘤免疫治疗提供新思路。方法：采用免疫组织化学EnVision法检测2016年1月—2017年6月滨州医学院附属医院收治的经手术切除的179例T₂期高危及T₃期结直肠癌患者的癌组织及癌旁组织中LC3、DC-lamp的表达和TLS的形成。采用蛋白质印迹法（Western blot）及实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）检测TLS⁺及TLS^-结直肠癌组织中LC3、NY-ESO-2、淋巴毒素β（lymphotoxin-beta，LTβ）、CC族趋化因子配体21（CC chemokine ligand 21，CCL21）、CXC族趋化因子配体13（CXC chemokine ligand 13，CXCL13）及白细胞介素17（interleukin-17，IL-17）的表达，分析其相关性及临床意义。采用log-rank检验比较组间预后差异，采用COX比例风险回归模型进行多因素生存分析。结果：结直肠癌组织中LC3、DC-lamp的表达量均高于癌旁组织（P<0.05），LC3与DC-lamp的表达呈正相关（P<0.05）。TLS⁺组中LC3、NY-ESO-2、LTβ、CXCL13及CCL21的蛋白水平及mRNA表达均高于TLS^-组（P <0.05），而IL-17的表达量则低于TLS^-组（P<0.05），LC3表达与TLS/生发中心（germinal center，GC）⁺、TLS/GC^-两亚型均呈正相关（P <0.05），且与NY-ESO-2、LTβ、CXCL13及CCL21的表达呈正相关（P<0.05）。DC-lamp在TLS/GC⁺和TLS/GC^-亚型组中的表达量均高于其余两亚组（P<0.05），且呈正相关（P<0.05）。Log-rank检验及COX比例风险回归模型显示，结直肠癌中LC3、DC-lamp、TLS及淋巴结转移与结直肠癌患者的预后密切相关，且均是结直肠癌预后的独立危险因素。结论：结直肠癌中LC3异常表达可通过增加表面抗原激活mDC，进而募集淋巴细胞，促进TLS的形成与成熟，最终影响患者的预后。

关键词: 结直肠肿瘤, 轻链3, 成熟树突状细胞, 三级淋巴结构, 临床意义

Abstract:

Background and purpose: It has been recognized as a complex problem in tumor therapy to deal with the tumor immune escape, and over-activated autophagy can cause the increase of tumor surface antigen, which participates in anti-tumor immunity. In this study, the expressions of microtubule-associated protein light chain 3 (LC3), mature dendritic cell (mDC) and the formation of tertiary lymphoid structure (TLS), an essential autophagy factor in colorectal cancer, were detected in clinical samples. The results had important clinical implications and provided new insights for enhancing anti-tumor immunity. Methods: Immunohistochemical EnVision method was used to detect the expressions of LC3, DC-lamp and the formation of TLS in cancer tissues and normal mucosal tissues of 179 patients with T₂ stage high-risk and T₃ stage colorectal cancer who underwent surgical resection at Binzhou Medical University Hospital from January 2016 to June 2017. Western blot and real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) were used to detect the expressions of LC3, NY-ESO-2, lymphotoxin-beta (LTβ), CC chemokine ligand 21 (CCL21), CXC chemokine ligand 13 (CXCL13) and interleukin-17 (IL-17) in TLS⁺ and TLS^- colorectal cancer tissues. Then the correlation and clinical significance were analyzed. Log-rank test was used to compare the prognostic differences between groups, and COX proportional risk regression model was used for multivariate survival analysis. Results: Clinical samples showed that the expressions of LC3 and DC-lamp were higher in colorectal cancer tissues than in normal mucosa tissues (P<0.05), and the expressions of LC3 and DC-lamp were positively correlated (P<0.05). The protein and mRNA expressions of LC3, NY-ESO-2, LTβ, CXCL13 and CCL21 were higher in TLS⁺ group than in TLS^- group. The expression of IL-17 was higher in the TLS^- group than in the TLS⁺ group (P<0.05). The expression of LC3 was positively correlated with TLS/germinal center (GC)⁺ and TLS/GC^- subtypes and positively correlated with the expression of NY-ESO-2, LTβ, CXCL13 and CCL21 (P<0.05). The expression of DC-lamp was higher in TLS/GC⁺ and TLS/GC^- subtype groups than in the other two subgroups (P<0.05), and there was a positive correlation. Kaplan-Meier and COX regression models showed that LC3, DC-lamp, TLS and lymph node metastasis were closely related to the prognosis of patients with colorectal cancer, and they were independent risk factors for the prognosis of colorectal cancer. Conclusion: The abnormal expression of LC3 in colorectal cancer can activate mDC to recruit lymphocytes and promote the expression and maturation of TLS, ultimately affecting the prognosis of patients.

Key words: Colorectal cancer, Light chain 3, Mature dendritic cell, Tertiary lymphoid structure, Clinical significance

中图分类号:

武寒, 徐磊, 王苗苗, 张睿哲, 许晓阳, 郭宁杰, 吴淑华. 结直肠癌中LC3与树突状细胞募集、TLS形成的相关性及其临床意义[J]. 中国癌症杂志, 2023, 33(9): 818-828.

WU Han, XU Lei, WANG Miaomiao, ZHANG Ruizhe, XU Xiaoyang, GUO Ningjie, WU Shuhua. Correlation of LC3 and the recruitment of dendritic cell and the formation of TLS in colorectal cancer and its clinical significance[J]. China Oncology, 2023, 33(9): 818-828.

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Gene	Sequence (3’-5’)
Gene	Forward primer	Reverse primer
LC3	GGCGCTTACAGCTCAATGCT	CTCCTGGGAGGCATAGACCA
NY-ESO-2	CTTCTGCGCAGGATGGAAG	ATCAACAGGGAAAGCTGCTG
CXCL13	TCTCTGCTTCTCATGCTGCT	TTGTGTAATAGACCTCCAGAACAC
LTB	CCAGCTGCCCACCTCATA	GAAACGCCTGTTCCTTCGT
CCL21	GAACCAAGCTTAGGCTGCTC	CTTTGGGTCTGCACATAGCTC
IL-17	TGGGAAGACCTCATTGGTGT	GGATTTCGTGGGATTGTGAT
GAPDH	GGCTCTCCAGAACATCATCCCTGC	GGGTGTCGCTGTTGAAGTCAGAGG

Item	Case n	LC3		Pvalue	DC-lamp		P value
Item	Case n	+	-	Pvalue	+	-	P value
Para-cancerous tissues	179	25 (13.9)	154 (86.1)	0.001	48 (26.8)	131 (73.2)	0.001
Cancer tissues	179	103 (57.6)	76 (42.4)	0.001	93 (51.9)	86 (49.1)	0.001

Item	Case n	DC-lamp		rvalue	Pvalue
Item	Case n	+	-	rvalue	Pvalue
LC3				0.192	0.010
+	103	62	41
-	76	31	45

TLS⁺										TLS^-
Item	Case n	GC⁺		rvalue	Pvalue	GC^-		rvalue	Pvalue	DLI		rvalue	Pvalue	SLI		rvalue	Pvalue
Item	Case n	+	-	rvalue	Pvalue	+	-	rvalue	Pvalue	+	-	rvalue	Pvalue	+	-	rvalue	Pvalue
LC3				0.270	0.001			0.230	0.009			-0.175	0.031			-0.443	0.001
+	103	35	86			52	51			13	90			3	100
-	76	5	71			23	53			20	56			28	48

TLS⁺										TLS^-
Item	Case n	GC⁺		rvalue	Pvalue	GC^-		rvalue	Pvalue	DLI		rvalue	Pvalue	SLI		rvalue	Pvalue
Item	Case n	+	-	rvalue	Pvalue	+	-	rvalue	Pvalue	+	-	rvalue	Pvalue	+	-	rvalue	Pvalue
DC-lamp				0.194	0.012			0.227	0.003			-0.148	0.047			-0.358	0.001
+	93	28	65			49	44			12	81			4	89
-	86	12	74			26	60			21	65			27	59