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Radiation exposure elicits a neutrophil-driven response in healthy lung tissue that enhances metastatic colonization

Nature Cancer volume 3, pages 173–187 (2022)Cite this article

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An Author Correction to this article was published on 04 April 2022

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Abstract

Radiotherapy is one of the most effective approaches to achieve tumor control in cancer patients, although healthy tissue injury due to off-target radiation exposure can occur. In this study, we used a model of acute radiation injury to the lung, in the context of cancer metastasis, to understand the biological link between tissue damage and cancer progression. We exposed healthy mouse lung tissue to radiation before the induction of metastasis and observed a strong enhancement of cancer cell growth. We found that locally activated neutrophils were key drivers of the tumor-supportive preconditioning of the lung microenvironment, governed by enhanced regenerative Notch signaling. Importantly, these tissue perturbations endowed arriving cancer cells with an augmented stemness phenotype. By preventing neutrophil-dependent Notch activation, via blocking degranulation, we were able to significantly offset the radiation-enhanced metastases. This work highlights a pro-tumorigenic activity of neutrophils, which is likely linked to their tissue regenerative functions.

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Fig. 1: Radiation exposure in healthy lung tissue enhances metastasis.
Fig. 2: Radiation exposure induces the infiltration and local activation of lung neutrophils.
Fig. 3: Primed neutrophils support metastatic colonization in irradiated lungs.
Fig. 4: Radiation-primed neutrophils perturb the lung tissue environment.
Fig. 5: Notch is activated in the lung epithelium and enhances spontaneous metastasis.
Fig. 6: Radiation exposure boosts Notch signaling and stemness in metastatic cells.
Fig. 7: Radiation-primed neutrophils boost lung metastasis via their degranulation activity.
Fig. 8: Inhibition of Notch signaling attenuates the radiation-driven enhancement of metastatic growth in vivo.

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Data availability

The bulk RNA sequencing datasets (GSE180823) and the single-cell RNA sequencing datasets (GSE181306) are deposited in the Gene Expression Omnibus (GEO, NCBI) repository. The proteomic datasets are deposited in PRoteomics IDEntifications (PRIDE) repository (PXD027628). Source data are provided with this paper.

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Acknowledgments

We are grateful to N. Osborne from the Safety, Health and Sustainability Team at the Francis Crick Institute for his invaluable support during this work. We thank E. Nye from the Experimental Histopathology Unit at the Crick Institute for histological processing and advice and D. Barry from the Advanced Light Microscopy Facility at the Crick Institute for image analysis advice. We are grateful to P. Chakravarty from the Bioinformatics & Biostatistics Facility at the Crick Institute for bioinformatics support. We thank B. Snijders from the Proteomics Facility at the Crick Institute and R. Goldstone and A. Edwards from the Advanced Sequencing Facility at the Crick Institute for their technical support. We are also grateful for support from the Flow Cytometry Unit, the Cell Services Unit and the Biological Resources Unit at the Francis Crick Institute, and in particular we thank D. Poniskaitiene and T. Zverev specifically for their extended contributions to welfare monitoring of experimental mice and for performing oral gavages. We are grateful to S. Quezada for his support for the image-guided focused irradiations. We thank A. Wilkins, Clinician Scientist at the ICR and Honorary Consultant at the Royal Marsden, London, for providing critical input on the manuscript sections discussing the clinical relevance of the study. We thank R. Ferreira for critical reading of the manuscript. This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (grant no. FC001112), the UK Medical Research Council (grant no. FC001112), and the Wellcome Trust (grant no. FC001112) and the European Research Council (grant no. ERC CoG-H2020-725492). The work at UCL was supported by the Radiation Research Unit at the Cancer Research UK City of London Centre Award (no. C7893/A28990).

Author information

Author notes

  1. Peter Faull

    Present address: Center for Biomedical Research Support Biological Mass Spectrometry Facility, The University of Texas at Austin, Austin, TX, USA

  2. These authors contributed equally: Luigi Ombrato, Adam Karoutas, Nicolas Rabas.

Authors and Affiliations

  1. Tumour Host Interaction laboratory, The Francis Crick Institute, London, UK

    Emma Nolan, Victoria Louise Bridgeman, Luigi Ombrato, Adam Karoutas, Nicolas Rabas, Celine Angeli Natascha Sewnath, Felipe Silva Rodrigues & Ilaria Malanchi

  2. Barts Cancer Institute, Queen Mary University of London, London, UK

    Luigi Ombrato

  3. Cancer Immunology Unit, UCL Cancer Institute, University College London, London, UK

    Marcos Vasquez

  4. Bioinformatics & Biostatistics Unit, The Francis Crick Institute, London, UK

    Stuart Horswell

  5. Proteomics Unit, The Francis Crick Institute, London, UK

    Peter Faull

  6. Preclinical Radiotherapy TTP, CRUK-City of London Centre, UCL Cancer Institute, University College London, London, UK

    Rebecca Carter

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Contributions

E.N. designed and performed most of the experiments, analyzed and interpreted the data. V.L.B. managed colony breeding and generated experimental mice, supported the animal work and provided technical support and discussion. L.O. developed the sLP-Cherry labeling tool and provided valuable discussion. A.K and N.R. performed the single-cell RNA analysis. C.A.N.S. performed the γ-H2AX immunofluorescence and SA-β-gal staining. M.V. performed the injections and harvested mice for the image-guided partial-lung irradiation. S.H. performed bioinformatic analysis. F.S.R. provided technical support. P.F. performed the neutrophil proteomics. R.C. planned and performed the image-guided, focused partial-lung radiation. L.O., A.K., N.R. and V.L.B. critically reviewed the manuscript. I.M. designed and supervised the study and interpreted the data. I.M and E.N. wrote the manuscript.

Corresponding author

Correspondence to Ilaria Malanchi.

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Nature Cancer thanks Marc Vooijs and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Radiation exposure in healthy lung tissue enhances metastasis.

a, Representative H&E images of metastatic lungs from control (UT) and irradiated BALB/c mice orthotopically injected with 4T1 breast cancer cells to generate a primary tumour. The metastatic area is depicted with a dashed line (n = 4 mice per group, 2 independent experiments). Scale bar, 250 μm. b,c, FACS quantification of GFP+ tumour cells in the metastatic lung (b) and primary tumour volume (c) from control and irradiated mice (n = 4 mice per group, one experiment). d,e, Representative H&E images (d) and FACS quantification of GFP+ tumour cells (e) from metastatic lungs from control (UT) or irradiated FVB mice intravenously injected with GFP+ MMTV-PyMT primary mammary tumour cells at day 7 (n = 5 per group, one experiment). Scale bar, 100 μm. f, Table and representative immunostaining of human-specific Lamin A/C to detect human lung cancer cells growing in BALB/c Nude mice (n = 4 mice per group for each cell line tested). Mice were intravenously injected with H460 or A549 human NSCLC cells 7 days following targeted lung irradiation (or sham-irradiation for control mice) and lungs harvested 3 weeks later. The table depicts the number of mice in which h-Lamin A/C+ cancer cells were present in the lungs. No foci were detected in control mice from either experimental group. The representative images show an example of a Lamin A/C+ metastatic foci within an irradiated lung, for each cell line. The H460 metastatic lesion (left) is indicated with the arrow. Scale bar, 250 μm. g, Violin plot showing the number of metastatic foci in the non-irradiated left lung lobe from mice that received image-guided targeted radiation to the right lung (n = 5 for 8 Gy mice; n = 7 for 12 Gy mice), compared with control mice (n = 6). The violin plot displays the median, 25th and 75th percentiles as well as the density of data points. Dots represent individual mice. All data represented as mean ± s.e.m. Statistical analysis by non-parametric two-tailed Mann-Whitney test for (b), (c) and (e) and a one-way ANOVA with multiple comparisons for (g). UT, untreated. IR, irradiated. Gating strategies for FACS analysis provided in Extended Data Fig. 2. Histology quantification process outlined in Supplementary File 1.

Source data

Extended Data Fig. 2 FACS gating strategy.

a-c, Example of FACS gating strategy to determine the frequency of (a) GFP+ cancer cells or (b) Ly6G+ neutrophils in the lung tissue of control/irradiated mice, or (c) Lineage–/EpCAM+ epithelial cells in the irradiated metastatic niche (Cherry-labelled, left), or unlabelled distant lung (Cherry-negative, right). All samples were gated to exclude debris and doublets, followed by live cell discrimination by DAPI staining. All gates were set based on fluorescence-minus-one (FMO) controls, containing all antibodies minus the one of interest, to determine the background signal. Importantly, lung tissue displays a high level of autofluorescence, which needs to be considered when excluding dead cells (an FMO-DAPI is critical for this).

Extended Data Fig. 3 Radiation exposure induces lung perturbations including neutrophil infiltration and activation.

a,b, Representative immunofluorescent images (a) and quantification (b) of phospho-Histone H2A.X (Ser139) (green) and DAPI (blue) stained lungs from control/irradiated mice, 7 days post-irradiation (n = 3 mice per group, one experiment). Scale bar, 10 μm. 6 fields of view were quantified per mouse. c,d, Representative images of senescence-associated β-galactosidase (SA-β-gal) staining on lung cryosections (c) and quantification (d) from control/irradiated mice, 7 days post-irradiation (n = 3 mice per group, one experiment). Scale bar, 25 μm. e, Quantification of immunostaining for S100A9+ neutrophils in metastases from control and irradiated lungs at day 14 (7 days post-i.v., Fig. 1d) (n = 6 mice per group, 2 independent experiments). The number of neutrophils within the metastatic area was normalised to tumour area. f, FACS quantification of GFP+ cancer cells in control/irradiated lungs from RAG1-ko mice at day 14 (7 days post-IV). n = 9 mice per group, two independent experiments, grey dots C57BL/6J and white dots FVB background. g,h, Volcano plots showing protein expression from irradiated versus control lung (g) and bone marrow (h) neutrophils. A selection of differentially expressed proteins in the lung are depicted in red, with the same proteins shown in bone marrow samples (n = 3 mice per group). i, Table of granule proteins within primary, secondary or tertiary granules with their fold change in irradiated lung neutrophils (IR) vs untreated control (UT). Nd = not detected. Highly enriched proteins are highlighted in red. j, Mice received an intraperitoneal EdU injection (25 mg/kg) 1 h prior to lung irradiation. Lungs/bone marrow were harvested 1 h or 7 days later, and EdU incorporation assessed by FACS. k, EdU incorporation in Ly6G+ neutrophils from bone marrow (left) and lungs (right) from control (UT) and irradiated (IR) mice (n = 2 control/irradiated mice at 2 h, n = 3 control/irradiated mice at day 7). A representative of two independent experiments is shown (total for both experiments n = 5 control/irradiated at 2 h,, n = 6 control/irradiated mice at day 7). All data represented as mean ± s.e.m. Statistical analysis by two-tailed t-test with Welch’s correction for (d) and (e), two-tailed non-parametric Mann-Whitney test for (f) and (k) and one-sample t-test (for value different from 2) for (b). UT, untreated; IR, irradiated. FACS gating strategies provided in Extended Data Fig. 2.

Source data

Extended Data Fig. 4 Radiation-primed neutrophil fuel metastatic growth independently of NETosis and extravasation.

a, Experimental setup. Irradiated mice were given daily injections of an anti-Ly6G neutrophil depletion antibody or IgG control, beginning the day before an IV injection of 4T1-GFP+ cancer cells. Mice were collected 72 h post-i.v. b,c, Frequency of Ly6G+ neutrophils (b) and GFP+ cancer cells (c) among live cells by FACS (n = 7 mice per group, 2 independent experiments). d, Representative immunofluorescent images of myeloperoxidase (MPO, green), citrullinated histone-H3 (Cit-H3, red) and DAPI (blue) stained lungs at 2 h, 24 h and 7 days post-irradiation (n = 3 mice per group, each timepoint). The positive control represents lung tissue from a mouse infected intratracheally with C.albicans and harvested 24 h later. Scale bar, 100 μm. e, Schematic of 3D co-cultures. GFP+MMTV-PyMT cancer cells were seeded in AlvetexTM Scaffold 96-well plates with MACS-sorted Ly6G+ neutrophils from control or irradiated mice, harvested 7 days after irradiation. f, GFP signal quantification. Cancer cell growth on the scaffold is shown as the fold change compared to cancer cells alone (n = 3 mice, each with at least 3 technical replicates, see methods). g, Schematic of GFP+MMTV-PyMT cancer cell proliferation in 2D co-culture with MACS-sorted Ly6G+ lung neutrophils from control (UT) or irradiated mice, harvested 7 days after irradiation (n = 6 mice per group). Cells were treated with EdU (20 μM) and incorporation was assessed by FACS 6 h later. h, Quantification of EdU+ cells among GFP+ cancer cells. PyMT cancer cells co-cultured with neutrophils isolated from control (UT) or irradiated (IR) lungs (n = 6 mice per group) were compared to PyMT cells cultured alone (n = 2 replicates). All data represented as mean ± s.e.m. Statistical analysis by one-way ANOVA for (b) and (c), two-way ANOVA for (f) and an unpaired two-tailed t-test for (h). UT, untreated; IR, irradiated; NT, neutrophils. Gating strategies for FACS analysis provided in Extended Data Fig. 2.

Source data

Extended Data Fig. 5 Recombinant G-CSF treatment permits controlled neutrophil recruitment.

a, Experimental setup for rG-CSF time course. Mice were given a subcutaneous injection of rGCSF every second day for a total of 4 doses, beginning the day before irradiations. On day 0, 2, 4, 6, 7 and 8, a blood sample was taken from each mouse to quantify Ly6G+ neutrophils. Mice were harvested at day 8. b, FACS analysis of Ly6G+CD11b+ neutrophils among CD45+ cells in the blood (n = 6 mice per group). The day of rGCSF injections is depicted in blue and indicated by an arrow. The day of irradiation (day 0), the day of cancer cell injection (day 7) and the day of lung seeding (day 8) are indicated. Each dot represents an individual mouse, treated with either rGCSF (blue dots) or PBS (black dots). IV, intravenous; rG-CSF, recombinant G-CSF; ko, knock-out.

Source data

Extended Data Fig. 6 Primed neutrophils influence the lung epithelial cell response to radiation-induced injury.

a, Principle Component Analysis (PCA) of Lin-EpCAM−mesenchymal cell signatures following RNA-seq analysis of control, irradiated and neutrophil-depleted irradiated lungs. Each dot represents an individual mouse, ovals enclose samples from each group to highlight their similarity in the PCA plot (n = 4 mice per group). b,c, 3D co-culture of GFP+MMTV-PyMT+ cancer cells on AlvetexTM Scaffolds with EpCAM+ lung epithelial cells isolated from control (UT), irradiated (IR) and neutrophil-depleted irradiated (IR + α-Ly6G) mice, 7 days after irradiation with (b) showing GFP signal quantification at day 4, normalised to cancer cell growth alone and (c) displaying representative images of GFP intensity at day 4 (n = 9 mice total per group, 3 independent experiments). Each dot in (b) represents the average of n = 3 mice for an independent experiment, with at least 3 technical replicates quantified per mouse in each experiment. Scale bar, 400 μm. d,e, 3D culture of GFP+EpCAM+ epithelial cells to assess survival on the scaffold, with (d) showing representative images of GFP intensity on the scaffold at day 4 (n = 3 mice per group, at least 3 technical replicates per mouse) and (e) showing GFP signal quantification (n = 3 mice per group, at least 3 technical replicates per mouse). Each dot represents an individual mouse. GFP+EpCAM+ cells were sorted from the lungs of control, irradiated and neutrophil-depleted irradiated actin-GFP mice 7 days after irradiation, and seeded in AlvetexTM Scaffolds. f, Heatmap of Lin-EpCAM−mesenchymal cells from control, irradiated and neutrophil-depleted irradiated lungs (hierarchically clustered samples in columns and genes in rows) (n = 4 mice per group). All data represented as mean ± s.e.m. Statistical analysis by one-way ANOVA for (b). UT, untreated; IR, irradiated; Ep, epithelial.

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Extended Data Fig. 7 Radiation-induced Notch signalling in the lung environment is boosted by the presence of neutrophils.

a, Representative images and quantification of immunofluorescent staining for the Notch Intracellular Domain (NCID) in irradiated EpCAM+ lung epithelial cells that were MACS-sorted and seeded on coverslips. Cells were harvested at day 7 from irradiated mice that were treated daily with an anti-Ly6G neutrophil depletion antibody or an IgG control antibody. Cells were quantified using CellProfiler, each dot represents the intensity of nuclear NCID staining in an individual cell (n = 130 IgG cells, n = 205 α-Ly6G cells, see methods). n = 3 mice per group, 3 technical replicates quantified per mouse. Scale bar, 5 μm. b, Quantitative RT-PCR validation of differentially-expressed genes identified by RNA-sequencing in sorted Lin-EpCAM+ lung epithelial cells. Lungs were harvested from an independent cohort of control, irradiated and neutrophil-depleted irradiated mice (n = 3 mice per group). c, Quantitative RT-PCR expression of Notch genes in sorted CD31+ lung endothelial cells from control, irradiated and neutrophil-depleted irradiated mice (n = 4 mice per group). Gapdh was used as a housekeeper gene for normalisation in (b) and (c). All data represented as mean ± s.e.m. Statistical analysis by unpaired two-tailed t-test for (a) and one-way ANOVA for (b) and (c). UT, untreated; IR, irradiated; Ep, epithelial.

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Extended Data Fig.8 Radiation-exposure boosts Notch-signalling and stemness in metastatic cells.

a, Quantitative RT-PCR analysis of Notch1 and target gene Hes1 in lineage-traced lung alveolar type 2 cells from SPC-Cre-ERT2 control mice and SPC-Cre-ERT2/Rosa26NICD-IRES-GFP Notch-activated mice. Mice were administered tamoxifen by oral gavage (40 mg/kg) over three consecutive days. Lungs were harvested 14 days after the last tamoxifen dose and GFP+ cells were sorted by flow cytometry (n = 3 control mice, n = 4 Notch mice). b, Primary tumour weight from PyMT/Control (n = 6) and PyMT/Notch mice (n = 8) harvested two weeks post-tamoxifen induction. c, Quantification and representative images of Hes1 immunostaining in lung metastases from PyMT/Control (n = 5) and PyMT/Notch mice (n = 7) mice (metastatic lungs harvested over n = 5 independent tamoxifen administrations, immunostaining quantification in methods). The enlarged inset shows nuclear localisation. Scale bar, 100 μm (main image), 10 μm (enlarged inset). d, Combined Uniform Manifold Approximation and Projection (UMAP) plot of cells from the mCherry+ niche and mCherry- distant lung from irradiated mice (n = 10 mice, pooled). The expression level of EpCAM (distinguishing epithelial cells), Pdgfrα and Pdgfrβ (fibroblasts) and Pecam1(CD31) (endothelial cells) is indicated in blue. e, Representative immunostaining for RBPJ (top panel) and Hes1 (lower panel) in metastatic lungs from control, irradiated, and neutrophil-depleted irradiated mice harvested at day 14 (n = 7 mice per group, 2 independent experiments). Quantification shown in Fig. 6d,e. The enlarged inset shows nuclear localisation within tumour cells. Hes1+ cells are indicated by arrows. Scale bar, 100 μm (main image), 10 μm (enlarged inset). f, Representative immunostaining and quantification of RBPJ staining intensity in metastatic lungs from irradiated FVB mice pre-treated with rGCSF or PBS prior to injection of MMTV-PyMT cancer cells (Fig. 3e, n = 5 mice per group). Staining intensity within the metastatic area was measured using ImageJ (see methods). Scale bar, 100 μm. All data represented as mean ± s.e.m. Statistical analysis by non-parametric two-tailed Mann-Whitney test for (a), a two-tailed unpaired t-test for (b), (c) and (f). UT, untreated; IR, irradiated. The violin plot displays the median, 25th and 75th percentiles as well as the density of data points. Dots represent individual mice. Gating strategies for FACS sorting for (d) provided in Extended Data Fig. 2.

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Supplementary File 1 (Metastasis quantification strategy) and Tables 1 and 2.

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Nolan, E., Bridgeman, V.L., Ombrato, L. et al. Radiation exposure elicits a neutrophil-driven response in healthy lung tissue that enhances metastatic colonization. Nat Cancer 3, 173–187 (2022). https://doi.org/10.1038/s43018-022-00336-7

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