Mechanism of Stromal Fibroblasts in Breast Cancer Lung Metastasis

Background Introduction

Breast cancer is one of the most common cancers among women worldwide, and metastasis is the leading cause of death in breast cancer patients. The lung is one of the most common sites of breast cancer metastasis, yet the mechanisms underlying this process remain incompletely understood. The process of tumor metastasis involves not only the characteristics of tumor cells themselves but also their interaction with the surrounding microenvironment. In recent years, increasing evidence has shown that stromal cells in the tumor microenvironment, particularly fibroblasts, play a crucial role in tumor metastasis. However, the specific roles and heterogeneity of these fibroblasts in lung metastasis remain largely unexplored.

This study aims to reveal the heterogeneity of stromal fibroblasts during breast cancer lung metastasis and explore their specific roles in the formation of lung metastases. Using techniques such as single-cell RNA sequencing (scRNA-seq), the research team identified a specific subset of stromal fibroblasts that promote lung metastasis through a mechanism mediated by the tryptophan metabolite kynurenine (Kyn). These fibroblasts help metastatic tumor cells resist ferroptosis and suppress T cell function, thereby facilitating the formation of lung metastases.

Source of the Study

This research was conducted by a collaborative team from multiple institutions, including Chongqing Medical University, Emory University School of Medicine, and Cedars-Sinai Medical Center. The study was led by Professor Manran Liu, with Yongcan Liu and Shanchun Chen as the primary authors. The paper was published on August 21, 2024, in the journal Cancer Communications, with the DOI 10.1002/cac2.12608.

Research Process and Results

1. Research Process

a) Establishment of Breast Cancer Lung Metastasis Models

The researchers established a mouse model of breast cancer lung metastasis by repeatedly injecting metastatic cells purified from the lungs. Using luciferase-labeled 4T1 and E0771 murine breast cancer cell lines, they obtained highly lung-metastatic cell lines (4T1-lm3 and E0771-lm3) through in vivo selection. These cell lines were used for subsequent metastasis experiments.

b) Single-Cell RNA Sequencing (scRNA-seq) Analysis

To investigate the heterogeneity of fibroblasts in the lung metastasis microenvironment, the researchers isolated fibroblasts from mouse lung tissues at different metastatic stages (normal, pre-metastatic, micro-metastatic, and macro-metastatic) and performed single-cell RNA sequencing. Using the 10x Genomics platform, they obtained transcriptomic data from 42,348 high-quality cells, which were analyzed using the Seurat software.

c) Identification and Functional Analysis of Fibroblast Subsets

Through scRNA-seq, the researchers identified three main subsets of fibroblasts: matrix fibroblasts (MFs), myofibroblasts-1 (Myof1), and myofibroblasts-2 (Myof2). Among these, MFs were the predominant subset, and their transcriptional profiles dynamically changed as metastasis progressed.

d) Role of MFs in Lung Metastasis

To validate the specific role of MFs in lung metastasis, the researchers used a conditional knockout mouse model to specifically delete tryptophan 2,3-dioxygenase (TDO2) in MFs. Through in vivo experiments, they found that the loss of TDO2 significantly reduced the burden of lung metastasis and prolonged the survival of mice.

2. Key Findings

a) Dynamic Changes in Fibroblast Subsets

The study found that MFs dominated at all stages of lung metastasis, and their transcriptional profiles dynamically changed as metastasis progressed. In particular, MFs exhibited strong inflammatory responses and chemokine signaling pathway activation during the pre-metastatic stage, which were further enhanced during the macro-metastatic stage.

b) MFs Promote Lung Metastasis via the TDO2-Kyn Axis

The researchers discovered that MFs highly express TDO2 and generate Kyn through TDO2-catalyzed tryptophan metabolism. Kyn not only helps metastatic tumor cells resist ferroptosis but also promotes immune evasion by suppressing T cell function. By conditionally knocking out TDO2, the researchers confirmed the critical role of the TDO2-Kyn axis in lung metastasis.

c) Impact of the TDO2-Kyn Axis on Ferroptosis

Further studies revealed that TDO2high MFs upregulate the expression of ferritin heavy chain 1 (FTH1) through Kyn, thereby enhancing the resistance of metastatic tumor cells to ferroptosis. This finding provides new insights into the survival mechanisms of tumor cells in the lung metastasis microenvironment.

Conclusions and Significance

This study reveals the critical role of TDO2-positive stromal fibroblasts in breast cancer lung metastasis. Through the TDO2-Kyn axis, these fibroblasts not only help metastatic tumor cells resist ferroptosis but also promote immune evasion by suppressing T cell function. This discovery provides an important theoretical basis for developing novel therapeutic strategies targeting lung metastasis.

Research Highlights

1. Revealing Fibroblast Heterogeneity: Through single-cell RNA sequencing, the researchers systematically revealed the heterogeneity and dynamic changes of fibroblasts in the lung metastasis microenvironment for the first time.
2. Discovery of the TDO2-Kyn Axis: The study is the first to identify that TDO2-positive stromal fibroblasts promote metastasis by helping tumor cells resist ferroptosis and suppress T cell function through Kyn-mediated mechanisms.
3. Potential Therapeutic Targets: The discovery of the TDO2-Kyn axis provides potential therapeutic targets for developing new strategies against lung metastasis, with significant clinical implications.

Additional Valuable Information

The study also found that TDO2 expression is negatively correlated with lung metastasis survival in breast cancer patients, suggesting that TDO2 may serve as a biomarker for predicting the risk of lung metastasis. Additionally, the conditional knockout mouse model developed by the research team provides a powerful tool for future studies on the specific roles of fibroblasts in tumor metastasis.

This study not only deepens our understanding of the mechanisms underlying breast cancer lung metastasis but also provides important theoretical foundations and experimental tools for developing new therapeutic strategies.