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Introduction

Recent research has revealed critical insights into why tumors with mutations in the STK11/LKB1 gene (LKB1) show resistance to immunotherapy. Mutations in the LKB1 gene are present in approximately 20% of lung adenocarcinomas and 2-19% of squamous cell carcinomas. Despite the transformative impact of immune checkpoint blockade (ICB) on cancer treatment, tumors co-mutated with KRAS and LKB1 (KL tumors) demonstrate significant resistance to these therapies compared to LKB1 wild-type non-small cell lung cancer (NSCLC). The precise mechanisms underlying this resistance have remained elusive until now.

Key Findings

A study led by Qian et al. has delved into the effects of LKB1 loss on tumor immune infiltration and metabolism, utilizing advanced techniques such as single-cell RNA sequencing, metabolic analysis, and gene knockout experiments. The research was conducted on both mouse and human lung adenocarcinoma models, providing a comprehensive understanding of the issue.

Single-Cell RNA Sequencing Analysis

Researchers performed single-cell RNA sequencing on mouse lung adenocarcinoma models (LKR13K, LKR13KL, and LKR13KL MCT4-KO cells). In the LKR13KL model, LKB1 loss significantly upregulated glycolysis-related genes including ENO1, LDHA, and SLC16A3 (MCT4). This gene expression shift was accompanied by a notable increase in the proportions of M2 macrophages and inhibitory T cells within the tumor microenvironment of LKB1-deficient tumors.

Metabolic Analysis

Using the Seahorse XF system, the study measured the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). LKB1-deficient cells exhibited higher ECAR and lower OCR, indicating a shift towards glycolysis for energy production. Notably, knockout of the MCT4 gene in these cells significantly reduced ECAR, highlighting MCT4's crucial role in lactate secretion. CRISPR/Cas9 technology was employed for MCT4 knockout, and Western blot analysis confirmed a substantial reduction in lactate secretion.

Tumor Growth and Immune Infiltration

The researchers injected mice with various genetically modified tumor cells (LKR10KL, LKR13KL, and 344sqKL) and treated them with either anti-PD-1 antibodies or isotype controls. In the LKR13KL mouse model, anti-PD-1 antibody treatment significantly slowed tumor growth when MCT4 was knocked out. This intervention also led to a significant increase in CD8+ T cell infiltration and a reduction in M2 macrophages within the tumor microenvironment.

Lactate Levels and Immune Response

In LKB1-deficient mouse models, serum and tumor lactate levels were significantly elevated compared to controls. Following MCT4 knockout, lactate levels were markedly reduced. Flow cytometry analysis showed a significant decrease in M2 macrophages and an increase in CD8+ T cells in LKB1-deficient tumors after MCT4 knockout.

Technological Innovations

The study's innovative methodologies, such as single-cell RNA sequencing, metabolic analysis (ECAR and OCR), and CRISPR/Cas9 technology, enabled a precise examination of gene expression changes and metabolic shifts in different cell types within the tumor microenvironment. These approaches provided detailed insights into the relationship between metabolism and immune suppression, highlighting the lactate-MCT4 pathway as a potential therapeutic target.

Study Limitations and Future Directions

Despite its groundbreaking findings, the study has several limitations. It primarily focused on lactate's role in immune suppression, without extensively exploring its effects on tumor cell proliferation, migration, and invasion. Further research into the molecular mechanisms of GPR81 is needed to develop more effective receptor blockers. Future studies should aim to validate these findings with larger sample sizes, more comprehensive research methods, and human clinical trials. It is also crucial to consider potential long-term efficacy and resistance issues and to explore integrated therapeutic strategies for more significant breakthroughs in cancer treatment.

Conclusion

This study provides new theoretical insights into the mechanisms of LKB1 mutation-driven resistance to immunotherapy and demonstrates the potential of targeting the lactate-MCT4 pathway. These findings have significant implications for future research and clinical applications, paving the way for more effective cancer treatments.

Reference：

Qian, Yu, et al. "MCT4-dependent lactate secretion suppresses antitumor immunity in LKB1-deficient lung adenocarcinoma." Cancer cell 41.7 (2023): 1363-1380.