Expression, Clinical Significance, and Role in Immunotherapy Efficacy Evaluation of the Co-stimulatory Molecule LLT1 in Gastric Cancer

Summary

This research investigates LLT1 as a predictive immunotherapy biomarker in gastric cancer through multi-omics and clinical analyses. The study evaluates LLT1's role in immune evasion and tumor microenvironment modulation to facilitate precision immunotherapy approaches for enhanced treatment outcomes.

Abstract

LLT1 is a novel immune checkpoint molecule and potential therapeutic target. Immunotherapy has improved outcomes in advanced gastric cancer; however, many patients do not respond or develop resistance, and predictive biomarkers are lacking. The role of LLT1 in gastric cancer immune evasion and its effect on immunotherapy response remain unclear. To assess this, LLT1 expression was analyzed in the Cancer Genome Atlas (TCGA) gastric adenocarcinoma dataset using multiple algorithms (TIMER, CIBERSORT, and ESTIMATE) to quantify immune cell infiltration. Protein expression was validated by immunohistochemistry (IHC) in 268 gastric adenocarcinoma samples and by multiplex immunofluorescence (MIF) in 115 post-immunotherapy tissues. Integrated transcriptomic and proteomic data were analyzed for associations with tumor microenvironment features and clinical immunotherapy outcomes. The results showed that high LLT1 protein expression was significantly associated with less advanced tumor characteristics (lower invasion depth and nodal metastasis; P < 0.05) and longer overall survival (P = 0.012). LLT1 mRNA and protein levels correlated with greater CD8+ T-cell and M1 macrophage infiltration and elevated PD-L1 expression in the tumor microenvironment. Among the first cohort of 198 patients with available prognostic data, patients with high LLT1 expression (n=117) demonstrated significantly longer overall survival compared to those with low expression (n=81). In the second cohort of patients treated with combined immunotherapy and chemotherapy, tumors with high LLT1 expression (n=48) had longer progression-free survival (P = 0.014) than those with low LLT1 expression (n=67). A significant increase (P < 0.05) in PD-1+CD161+ immune cell populations was observed in Immunotherapy-effective patients (n=62) relative to treatment-resistant patients (n=53). Conclusively, LLT1 expression is a promising biomarker of a favorable prognosis in gastric cancer. To our knowledge, this is the first study to demonstrate that LLT1 expression predicts improved response to combined chemotherapy and immunotherapy in gastric cancer, supporting its potential in guiding immunotherapeutic strategies.

Introduction

Gastroesophageal cancer, particularly gastric cancer (GC), holds the distinction of being the 5^th most common form of cancer worldwide and the 5^th most deadly. Global statistics reveal that roughly 1 million new cases are identified each year, with East Asia shouldering a disproportionate share of the disease, contributing to more than half of the global incidence¹. For those diagnosed with early-stage GC, the prospect of survival is significantly favorable, with over 90% achieving 5-year survival rates following endoscopic submucosal dissection (ESD) or radical surgical intervention². However, the ....

Protocol

The research involving human subjects was reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Soochow University (Approval No. 2019026). The ethical review board of the First Affiliated Hospital of Soochow University has approved or granted an exemption for the ethical review of this data usage. Additionally, informed consent was obtained from all participants.

Bioinformatics data analysis
After a thorough Pan-cancer expression analysis examination of the TCGA and Tumor IMmune Estimation Resource (TIMER) databases, the behavior of LLT1 was investigated across various cancer types, with a ....

Results

Association between CLEC2D mRNA expression and gastric cancer subclassification/clinical parameters
An initial comprehensive cancer examination utilizing the TCGA dataset, facilitated by the TIMER database, revealed a notable elevation in CLEC2D transcript levels across various cancerous tissues, as depicted in Figure 1A. This observation implies that CLEC2D may serve as a general oncogenic factor implicated in.......

Discussion

Our study showed higher CLEC2D mRNA expression in tumor tissues than in normal tissues. Immunohistochemical results of gastric cancer revealed that high LLT1 protein expression in tumor tissues was associated with better prognosis and positively correlated with immune cell infiltration in the tumor microenvironment. Analysis of tissues from gastric cancer patients receiving immunotherapy indicated that high LLT1 expression predicts better immunotherapeutic response. Our research explored the expression and clini.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5% BSA Blocking Buffer	Beijing Solarbio Science & Technology Co., Ltd.,China	SW3015
anti-CD11c	ZSGB-BIO,China	5D11
Anti-CD161	Abcam,Cambridge, UK	ab302564
anti-CD19	ZSGB-BIO,China	EPl69
anti-CD3	ZSGB-BIO,China	LNl0
anti-CD31	ZSGB-BIO,China	UMAB30
anti-CD4	ZSGB-BIO,China	UMAB64
anti-CD56	ZSGB-BIO,China	UMAB83
anti-CD68	ZSGB-BIO,China	KP1
anti-CD8	ZSGB-BIO,China	EP334
Anti-CLEC2D	Abcam,Cambridge, UK	ab197341
anti-Foxp3	R&D Systems, USA	1054C
anti-IFN-γ	Santa Cruz Biotechnology	sc-74108
anti-IL-17	R&D Systems, USA	MAB317
anti-Ki67	ZSGB-BIO,China	8H5
Anti-pan Cytokeratin	Abcam,Cambridge, UK	ab7753
Anti-PD-1	Abcam,Cambridge, UK	ab137132
Anti-PD-L1	DAKO,Denmark	22C3
automated tissue array equipment	Estigen OU, Tartu, Estonia	Beecher ATA 27	This instrument is primarily used for the construction of tissue microarrays.
Citrate Antigen Retrieval Solution	Aladdin Biochemical Technology Co., Ltd , China	C752119
DAB chromogen kit	AiFang Biological,China	AFIHC004
Digital pathology scanner	Ningbo Jiangfeng Bio-Information Technology Co., Ltd,China	KF-FL-020	This instrument is primarily used for scanning immunohistochemistry and multiplex immunohistochemistry chips.
GraphPad Prism 10	GraphPad Software,USA	v10.0.3	This software is for Plotting Kaplan-Meier curves (with log-rank statistical analysis),Scientific data visualization, and Figure Preparation.
PBS	Aladdin Biochemical Technology Co., Ltd , China	P743267
peroxidase blocking solution	AiFang Biological,China	AFIHC012
Polymer-HRP conjugated goat anti-mouse/rabbit secondary antibody	AiFang Biological,China	AFIHC001
Six-color seven-marker multiplex immunofluorescence staining kit	AiFang Biological,China	AFIHC037	This reagent kit is designed for multiplex immunohistochemistry (mIHC)
SPSS	IBM Corporation,USA	IBM SPSS Statistics 27	The software was utilized for statistical analysis of the data.
The R Project	R Core Team	V4.4.1	All R packages, related code, and original files have been uploaded to the public database.
VISIOPHARM software	Visiopharm,Denmark	2025.02.2.18022 x64	This software is primarily designed for the quantitative analysis of pathological images.