Integrin conformation-dependent neutrophil slowing obstructs the capillaries of the pre-metastatic lung in a model of breast cancer

Frédéric Fercoq, Gemma S. Cairns, Marco De Donatis, John B. G. Mackey, Alessia Floerchinger, Amanda McFarlane, Ximena L. Raffo-Iraolagoitia, Declan Whyte, Lindsey W. G. Arnott, Colin Nixon, Robert Wiesheu, Anna Kilbey, Leah Brown, Sarwah Al-Khalidi, Jim C. Norman, Edward W. Roberts, Karen Blyth, Seth B. Coffelt, Leo M. Carlin

Posted on: 7 October 2024 , updated on: 8 October 2024

Preprint posted on 2 August 2024

Pre-metastatic neutrophilia and lung-metastatic breast cancer: a one-two punch to the lungs

Selected by Simon Cleary

Categories: cancer biology, immunology, pathology

Background

The pulmonary vasculature traps bloodborne cells that are too large, too stiff or too adhesive to squeeze through narrow alveolar capillaries. This filtering feature of the lungs allows them to capture activated neutrophils, a process thought to contribute to remote lung injury secondary to inflammation affecting other body parts. The lungs can also sequester cancer cells that have entered the bloodstream, allowing cancer metastases to colonise the lungs.

Lung sequestration of neutrophils and metastatic cancer cells has been linked by the discovery that neutrophils help to form ‘pre-metastatic niches’ that promote metastasis of breast cancers to the lungs. So far, it has not been clear which cytokines and adhesion molecules enable neutrophils to promote pulmonary metastasis of breast cancer. In this preprint, the authors studied the molecular mediators and functional consequences of neutrophil adhesion in the lungs in a mouse model of pre-metastatic breast cancer.

Key findings

Pre-metastatic breast cancer results in pulmonary sequestration of so many neutrophils (Figure 1) that blood is less able to flow through some regions of the lung microvasculature.

**Figure 1**: Neutrophil sequestration in the lungs in pre-metastatic cancer. Adapted, with permission, from Fercoq, Cairns et al. (2024), bioRxiv, © the authors: https://doi.org/10.1101/2024.03.19.585724

Granulocyte colony stimulating factor (G-CSF), a growth factor that increases neutrophil production, also increases the number and adhesiveness of lung neutrophils.
Pulmonary neutrophil adhesion and impairment of pulmonary blood flow in pre-metastatic cancer can be reversed with an antibody (M18/2) that is thought to activate Integrin-β2 (Itgb2, CD18) (Figure 2).

**Figure 2:** Effect of the M18/2 Itgb2 antibody on distance travelled by neutrophils in ex vivo precision cut lung sections from mice bearing pre-metastatic K14-Cre:Trp53fl/fl (KP) mammary tumours. Adapted, with permission, from Fercoq, Cairns et al. (2024), bioRxiv © the authors: https://doi.org/10.1101/2024.03.19.585724

Why this work is interesting

Excessive pulmonary neutrophil sequestration resulting in lung alveoli receiving ventilation but not perfusion (alveolar dead space, or ‘wasted ventilation’) has been implicated as a cause of hypoxaemia in acute lung injury secondary to septic peritonitis. The idea that this process might be happening in pre-metastatic breast cancer is new to me.
The methods developed for use in this study are impressive, including a simplified model of p53-deficient breast cancer exhibiting deterministic, spontaneous metastasis to the lungs, and several novel imaging approaches (example in Figure 2).

Questions I would like to ask the authors about their work

Do you think the lung perfusion deficits in the pre-metastatic phase are severe enough to either directly limit gas exchange or contribute to immune checkpoint inhibitor-related pneumonitis or radiation-induced lung injury?
Does G-CSF treatment also reduce lung microvascular perfusion? I was wondering whether the effect of G-CSF treatment on pulmonary neutrophil adhesion in mice might be related to pulmonary complications that develop in some people given G-CSF as a treatment.
Do you plan to test whether the M18/2 antibody or agents blocking G-CSF signalling can limit metastasis?
How do you think the M18/2 antibody acts in vivo and would it be expected to cause immunodeficiency similar to leukocyte adhesion deficiency in people with ITGB2 mutations?

Tags: breast, cancer, metastasis, neutrophils

doi: https://doi.org/10.1242/prelights.38572

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Author's response

Leo Carlin shared

Thanks, this is a great question and adds a perspective we hadn’t considered for this work as we had primarily been concerned with the potentially pro-metastatic effects of the neutrophil behaviour. Side effects like these are driven by multiple factors, so it is difficult to disentangle this, but a pre-metastatic lung loaded with neutrophils, either via reduced perfusion or the neutrophilia itself, could react to inflammatory therapy very differently to a naïve one. Considering tissue-specific cancer-driven immune behaviour could be important to help improve patient care. This is something we had been thinking deeply about for our other work on primary lung cancer macrophage pro-fibrotic responses (https://doi.org/10.1101/2023.09.14.557344).In the pre-print under discussion here, we used enumeration of intravenously injected fluorescent beads in precision cut lung slices as an indirect assessment of pulmonary perfusion. We are now following these experiments up with approaches where we aim to be able to pinpoint the areas of low perfusion more precisely and see what is happening in those areas, this could allow us to address these questions more directly in future when combined with immunotherapy and/or radiation.
This again is a great question, and we are currently performing experiments to check. The way that G-CSF is used in the clinic to oppose neutropenic sepsis in some chemotherapy recipients as well as its ability to phenocopy the neutrophil behaviour we observed in the mammary tumour bearing mice highlighted the relevance of looking deeper into this for us.
We are currently working on this too. There are some complications in that M18/2 binds and potentially activates all β2 integrin (a widely expressed adhesion molecule on immune cells) not just that on neutrophils. This means that if the readout is directly observable in neutrophil behaviour the Ab is a great tool, but not so much for processes that might include the balance between neutrophil effects and adaptive immunity. Likewise, G-CSF regulates both the neutrophilia and the behaviour. For these reasons, we are currently focussing on the seeding phase of lung metastasis in these experiments to mitigate some of the potential confounders of long-term effects.
This is again a very interesting point outside of the cancer outcomes per se and also links to our answer to our previous question. In future, we want to try to uncover more about the way that the pathway is regulated with the aim to be able to target the slowing neutrophil behaviour specifically and mitigate the potential off target effects of long-term treatment with the Ab. This is also why just targeting the neutrophils in bulk is unlikely to be a good therapeutic avenue due to the consequences to anti-microbial defence.

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