Correlation of LC3 and the recruitment of dendritic cell and the formation of TLS in colorectal cancer and its clinical significance

doi:10.19401/j.cnki.1007-3639.2023.09.002

Abstract

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

CLC Number:

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