Intratumour Oxidative Hotspots Provide a Niche for Cancer Cell Dissemination

Immunology Cell Biology Oncology Life Sciences Medicine
Intratumour heterogeneity Oxidative stress Cancer cell dissemination Microenvironment p38-Myc axis

Tumor heterogeneity is a critical issue in cancer research, encompassing genetic, phenotypic, and microenvironmental heterogeneity. Although single-cell sequencing has shed light on genetic and phenotypic heterogeneity, the study of non-genetic factors, such as microenvironmental heterogeneity, remains insufficient. Oxidative stress is a hallmark of cancer, as tumor cells face various pressures when detached from their natural microenvironment, including metabolic abnormalities, detachment from the extracellular matrix (ECM), hypoxia, and immune cell attacks, all of which lead to the accumulation of reactive oxygen species (ROS). However, the heterogeneity of intratumoral ROS and its impact on tumor behavior remain unclear, primarily due to the lack of technology to detect intratumoral ROS at the single-cell level.

To address this issue, researchers developed a tumor-targeted probe called T-AP1 to track extracellular hydrogen peroxide (H2O2), thereby visualizing and characterizing tumor cells exposed to oxidative stress. This study aims to uncover the mechanisms underlying the formation of intratumoral oxidative hotspots and explore their role in cancer cell dissemination.

Source of the Paper

This paper was co-authored by Yoshifumi Ueda, Shigeki Kiyonaka, Laura M. Selfors, and others, with the research team hailing from several renowned institutions, including Nagoya University and Kyoto University in Japan. The paper was published in Nature Cell Biology in March 2025, titled “Intratumour oxidative hotspots provide a niche for cancer cell dissemination”.

Research Process

1. Development and Validation of the T-AP1 Probe

The researchers developed a tumor-targeted probe called T-AP1, which combines HER2 antibody, Alexa Fluor 647 (AF647) (a fluorescent dye unaffected by H2O2), and Peroxy Green 1 (PG1) (a chemical probe sensitive to H2O2). T-AP1 is designed to bind to HER2-positive tumor cells and increase the fluorescence intensity ratio of PG1 to AF647 (PG1/AF647 ratio) upon extracellular H2O2 exposure, enabling the detection of H2O2 in the tumor microenvironment.

In extracellular experiments, T-AP1 demonstrated high selectivity for H2O2, and its fluorescence signal remained stable within a pH range of 6.4–7.9. Subsequently, the researchers conducted live-cell imaging experiments in multiple HER2-positive cancer cell lines, validating the specificity and sensitivity of T-AP1.

2. Discovery of Intratumoral Oxidative Hotspots

Using the T-AP1 probe, the researchers identified H2O2-rich microenvironments, termed H2O2 hotspots, in HER2-positive tumor xenograft models. These hotspots were primarily located in actively budding regions, where single cells or small cell clusters detached from the main tumor mass. Through three-dimensional imaging and quantitative analysis, the researchers confirmed the close relationship between these hotspots and tumor budding.

Additionally, the researchers validated the existence of H2O2 hotspots in various HER2-positive and HER2-negative tumor models, indicating that this phenomenon is prevalent across multiple tumor types.

3. H2O2 Hotspots and Tumor Cell Phenotypic Transition

To investigate the impact of H2O2 hotspots on tumor cell behavior, the researchers isolated tumor cells exposed to high and low H2O2 levels using cell sorting and performed RNA sequencing analysis. The results showed that tumor cells exposed to high H2O2 exhibited upregulation of MYC signaling pathway and epithelial-mesenchymal transition (EMT)-related genes, indicating partial EMT in these cells.

Further experiments revealed that H2O2 induced a mesenchymal phenotype in tumor cells by activating the p38-MYC axis, promoting cell migration and invasion. Moreover, the researchers found that tumor cells migrated away from H2O2-rich environments, suggesting an escape mechanism.

4. Role of Neutrophils in H2O2 Hotspot Formation

The researchers discovered that neutrophils play a key role in establishing intratumoral H2O2 hotspots. Through immunohistochemical analysis and neutrophil depletion experiments, the researchers confirmed the critical role of neutrophils in H2O2 hotspot formation. Additionally, the researchers developed a neutrophil-targeted H2O2 probe, L-AP1, further validating the ability of neutrophils to produce H2O2 in the tumor microenvironment.

5. Impact of NRF2 Hyperactivation on Tumor Budding

The researchers found that NRF2 (an antioxidant transcription factor) hyperactivation inhibits tumor budding in various cancer cell lines, including lung, stomach, and breast cancers. By using CRISPR-Cas9 to knock out KEAP1 (a negative regulator of NRF2), the researchers observed that NRF2-hyperactivated tumor cells exhibited significantly reduced budding despite H2O2 exposure. This suggests that NRF2 hyperactivation enables tumor cells to survive in oxidative stress environments by enhancing antioxidant programs and suppressing tumor budding signaling pathways.

Research Findings

  1. The T-AP1 probe successfully achieved high-resolution imaging of H2O2 in the tumor microenvironment, revealing the existence of intratumoral H2O2 hotspots.
  2. H2O2 hotspots are primarily located in actively budding regions of tumors, indicating a key role for H2O2 in cancer cell dissemination.
  3. Tumor cells exposed to H2O2 underwent partial EMT through p38-MYC axis activation and exhibited enhanced migration and invasion capabilities.
  4. Neutrophils are the primary source of intratumoral H2O2 hotspots, and their depletion significantly reduced tumor budding and H2O2 hotspot formation.
  5. NRF2 hyperactivation enables tumor cells to survive in oxidative stress environments by enhancing antioxidant programs and suppressing tumor budding signaling pathways.

Conclusions and Significance

This study is the first to uncover the mechanisms underlying the formation of intratumoral H2O2 hotspots and their critical role in cancer cell dissemination. By developing the T-AP1 probe, the researchers achieved high-resolution imaging of H2O2 in the tumor microenvironment, providing a new tool for studying tumor heterogeneity. The findings demonstrate that H2O2 promotes tumor dissemination by inducing partial EMT and migration in tumor cells. Additionally, the key role of neutrophils in H2O2 hotspot formation offers new insights into tumor microenvironment research.

The scientific value of this study lies in revealing how tumor cells cope with oxidative stress through physical escape mechanisms, providing new insights into the early steps of tumor metastasis. Furthermore, the inhibitory effect of NRF2 hyperactivation on tumor budding offers potential targets for developing therapeutic strategies against tumor metastasis.

Research Highlights

  1. The development of the T-AP1 probe provides a high-resolution imaging tool for detecting H2O2 in the tumor microenvironment.
  2. The first to reveal the critical role of H2O2 hotspots in cancer cell dissemination.
  3. Identified the central role of neutrophils in the formation of H2O2 hotspots.
  4. Uncovered the mechanism by which NRF2 hyperactivation enables tumor cells to survive in oxidative stress environments by enhancing antioxidant programs and suppressing tumor budding signaling pathways.

This study not only provides new perspectives for research on tumor heterogeneity and metastasis mechanisms but also offers potential targets for developing therapeutic strategies targeting the tumor microenvironment.