Visualization of immediate immune responses to pioneer metastatic cells in the lung

Abstract

Lung metastasis is the lethal determinant in many cancers1,2 and a number of lines of evidence point to monocytes and macrophages having key roles in its development3,4,5. Yet little is known about the immediate fate of incoming tumour cells as they colonize this tissue, and even less known about how they make first contact with the immune system. Primary tumours liberate circulating tumour cells (CTCs) into the blood and we have developed a stable intravital two-photon lung imaging model in mice6 for direct observation of the arrival of CTCs and subsequent host interaction. Here we show dynamic generation of tumour microparticles in shear flow in the capillaries within minutes of CTC entry. Rather than dispersing under flow, many of these microparticles remain attached to the lung vasculature or independently migrate along the inner walls of vessels. Using fluorescent lineage reporters and flow cytometry, we observed ‘waves’ of distinct myeloid cell subsets that load differentially and sequentially with this CTC-derived material. Many of these tumour-ingesting myeloid cells collectively accumulated in the lung interstitium along with the successful metastatic cells and, as previously understood, promote the development of successful metastases from surviving tumour cells3. Although the numbers of these cells rise globally in the lung with metastatic exposure and ingesting myeloid cells undergo phenotypic changes associated with microparticle ingestion, a consistently sparse population of resident conventional dendritic cells, among the last cells to interact with CTCs, confer anti-metastatic protection. This work reveals that CTC fragmentation generates immune-interacting intermediates, and defines a competitive relationship between phagocyte populations for tumour loading during metastatic cell seeding.

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Figure 1: Intravital imaging of the first hours of lung seeding by B16 melanoma.
Figure 2: Encounter and uptake of tumour-derived cytoplasts by lung myeloid cells.
Figure 3: Discrete waves of cytoplast loaded myeloid cells define the early metastatic niche.
Figure 4: Lung-resident dendritic cells inhibit metastasis of B16 melanoma.

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Acknowledgements

We thank D. Hume for providing MacBlue mice; Z. Werb for providing MDA-MB231 GFP cells; J. Massegue and S. Abrams for providing PyMT-B cells; J. Cyster for providing us with CCR2-knockout mice. We would also like to thank E. Thornton for her initial work in developing lung intravital imaging. M. Werner for early technical assistance in this project. All members of the Krummel laboratory, BIDC, and M Koch for discussion, support, and guidance while developing this work. This work was supported in part by a Department of Defense post-doctoral fellowship to M.B.H. (W81XWH-13-1-0009) and NIH grants U54 CA163123, P01 HL024136 and R21CA167601.

Author information

Affiliations

Authors

Contributions

M.B.H. designed and conducted most of the experiments, data analysis, and drafted the manuscript; A.B. developed the Intercostal Imaging Window; A.N. generated B16ZsGreen cell line and discussed data; E.W.R. generated mTmG mouse embryonic fibroblasts and provided editorial support on manuscript; M.L.R. participated in development and troubleshooting of LIVM method, discussed data and project direction; A.G. participated in intravital LN experiments and provided editorial support on manuscript. M.F.K. designed experiments, interpreted data, and with other authors, developed the completed manuscript.

Corresponding author

Correspondence to Matthew F. Krummel.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Loading of lung immune cells by prospective metastatic B16 melanoma.

a, Schema for assessing immune cell loading by i.v.-injected B16ZsGreen cells. b, Representative plots of ZsGreen+ populations in the lungs of mice injected with B16ZsGreen over the first 24 h following injection. Data are gated on the basis of expression of the immune cell marker CD45. c, Quantification of b with n = 6 per group, error bars are s.d. d, Confocal imaging of CD45+ZsGreen+ cells sorted from a lung digest from a ubiquitous membrane-bound TdTomato fluorescent mouse 24 h after i.v. injection with B16ZsGreen.

Extended Data Figure 2 Intercostal insertion window for lung intravital microscopy.

a, Top, side, and bottom views of the intercostal insertion window. The window accommodates an 8 mm coverslip and allows for visualization of a 4 mm field of the left lung lobe. be, Images detailing surgical insertion of the intercostal window. b, Mouse is intubated, attached to ventilator, and placed in right lateral decubitis position and surgical field is shaved. c, An ~6 mm incision is made immediately above ribs 4 and 5 over the anterior surface of left lung lobe. d, The intercostal window is slipped between ribs 4 and 5, and attached to a rigid support. e, Around 20 mm Hg of vacuum suction is applied to the window to secure a small portion of lung to the coverglass. f, Schema showing approach for two-photon intravital microscopy of lung seeding by B16ZsGreen cells.

Extended Data Figure 3 Assessment of tumour cell activity and cytoplast characterization.

a, Binarized maximum intensity projections of representative cells at 2 or 24 h after injection. Cells were time-projected over a 30 min window to assess overall cellular activity during the interval. Images show the beginning time point (0 min), the ending time point (30 min), the time projection, and the overlay. White-filled space in the overlay represents the region of the cell that was stable during the analysed interval. These data are associated with Supplementary Video 3. These data are representative from imaging performed in 3 mice. b, Quantification of the cell activity index (defined as the area calculated from the projection of all positions over time as a ratio of the common area over all time (for example, area of overlap)) from analysis in d (n = 10 cells per group, horizontal bars are mean value). c, Flow cytometric quantification of FSC for tumour cells isolated from lung via digestion at 15 min, 2 h, 4 h, 6 h, and 24 h after injection. *P < 0.05, one-way ANOVA with Bonferroni post-hoc test, error bars are s.d. d, Representative data from flow cytometric discrimination of nucleated karyoplasts and anucleate cytoplasts derived from B16ZsGreen tumour cells in the lung in vivo over a 24 h time course, full data set quantified in Fig. 1e. e, confocal analysis of Hoechst and Mitotracker-labelled B16ZsGreen karyoplasts and cytoplasts sorted from in vitro culture of B16ZsGreen cells.

Extended Data Figure 4 Autonomous motility of cytoplasts in vivo.

a, Flow cytometric quantification of cytoplasts in vitro following 24 h treatment with Z-VAD at indicated concentrations. b, Flow cytometric quantification of cytoplasts in vivo from lung digests of mice treated with 10 μg Z-VAD i.v at the time of injection with 2.5 × 105 B15ZsGreen cells (a and b, n = 4 per group; no significant difference detected between groups, unpaired t-test, error bars are s.d.). c, Image series for B16ZsGreen cytoplast migrating autonomously through the lung microvasculature of a MacBlue host. Arrows represent the direction of the trajectory of the cytoplast at indicated time point. These data are associated with Supplementary Video 6. These data are representative of imaging collected from at least 12 mice. d, Representative tracking of a cytoplast from Supplementary Video 6 (and Extended Data Fig. 4c). e, A superposition image of 23 consecutive time points of a cytoplast migrating through lung microvasculature. Image shows the change in position in the y axis of direction as defined in d at each subsequent timepoint as the cytoplast migrates up and down the vessel.

Extended Data Figure 5 Characterization of tumour-interacting myeloid cell waves.

a, Representative gating for total lung myeloid populations. b, Schema detailing method for discrimination of intravascular versus extravascular localization of lung myeloid populations. c, d, Representative histograms of intravascular CD45 staining used to discriminate between intravascular and extravascular localization of lung myeloid cells at 4 and 24 h after injection with B16ZsGreen. Data quantified in Fig. 3d. In the leftmost panels, alveolar macrophages at 24 h after tumour injection are shown as a known control for extravascular staining. c, Total lung myeloids. d, ZsGreen+ myeloid cells. e, Quantification ZsGreen+ myeloid populations by flow cytometry in lungs of mice bearing 2-week subdermal B16ZsGreen tumours (n = 4 per group). Error bars are s.d.

Extended Data Figure 6 Stimulatory capacity of CD103+ DCs in lung-draining lymph node.

a, CD69 vs Nur77-GFP expression 24 h after culture from ex vivo coculture of OT-I TCR transgenic CD8+ T cells with sorted APCs from mLNs, where the latter were isolated 72 h post-injection with B16ZsGreenSL8. b, Quantification of a. c, Dilution of SE670 as an index of proliferation 72 h after culture with indicated APC populations. d, Quantification of c. n = 6 (b) or 6–12 (d) per group from 2 experiments; *P < 0.05, one-way ANOVA with Bonferroni post-hoc test, horizontal bars are mean value.

Extended Data Figure 7 cDCs confer anti-metastatic activity in the presence of a primary tumour.

a, Experimental schema for evaluation of role of cDCs in lung metastasis in the presence of a primary tumour. b, Representative images of lungs from Zbtb46-DTR bone marrow chimaeras treated with PBS or DT after implantation of a primary subdermal tumour (1 × 105 B16F10 in matrigel) and i.v. metastases (1.5 × 105 B16ZsGreen). Metastases were assessed one week after i.v. injection. c, Quantification of total number of visible ZsGreen+ lung metastases in PBS- or DT-treated Zbtb46-DTR bone marrow chimaeras. n = 4–5 per group, representative of 2 experiments; *P < 0.05, unpaired t-test, horizontal bars are mean value.

Supplementary information

Intravital imaging of initial arrival of B16F10 Melanoma in the lung Vasculature

Intravital imaging of pulmonary vasculature of mTmG mice (red) during injection of Hoechst-labeled B16-ZsGreen cells (Green cells with blue nuclei). Evans Blue was coinjected at time of i.v. injection with tumor cells to label vascular flow. Z volumes were collected every 20 seconds and Video represents 2 minutes and 20 seconds of real time. White arrows indicate production and release of cytoplasts microparticles. This Video corresponds to data in Figure 1D. (MOV 6277 kb)

Intravital imaging of the first 8 hrs of B16F10 Melanoma following arrest in the lung vasculature

Intravital imaging of pulmonary vasculature of an Actin-CFP mouse (blue) from 15 minutes until 8 hrs and 38minutes post-IV injection with Hoechst-labeled B16-ZsGreen tumor cells (green cells with blue nuclei). Z volumes were collected every 30 seconds for the first 1hr15 minutes and every 60 seconds thereafter. White arrows and text highlight various features of imaging throughout. This Video corresponds to Figure 1E. (MOV 27324 kb)

Comparitive imaging of tumor karyoplasts 2hrs and 24hrs post- lung entry demonstrates a progressive reduction in cell activity over the first 24hrs

Intravital imaging of pulmonary vasculature of an Actin-CFP mouse (blue). Tumor cells were injected either 24 hrs (B16-DsRed, red cells) or 2 hrs (B16-ZsGreen, green cells) prior to intravital imaging. Z volumes were collected every 2 min and Video represents 30 minutes of real time. This Video corresponds to Figure S3A-B. (MOV 1213 kb)

Spontaneous lysis of a melanoma tumor cell in lung vasculature

Intravital imaging of pulmonary vasculature of mTmG mice (red) 1 hr post-injection with Hoechst- labeled B16-ZsGreen cells. Evans Blue was co-injected at time of i.v. injection with tumor cells to label vascular flow. White arrows and text highlight the moment of lysis of a B16-ZsGreen melanoma cell (loss of green fluorescence signal), the nucleus remains intact during this event and is swept away by vascular flow. Z volumes were collected every 30 seconds and Video represents ˜12 minutes of real time. (MOV 947 kb)

Spontaneous arrest and release of circulating cytoplast microparticles in lung

Intravital imaging of pulmonary vasculature of mTmG mice (red) 2 hrs post-injection with Hoechst- labeled B16-ZsGreen cells. Evans Blue was co-injected at time of I.V. injection with tumor cells to label vascular flow. Imaging shows a region of the lung devoid of any arrested tumor karyoplasts. Over time multiple cytoplast microparticles (indicated by white arrows, green blebs lacking a Hoechst+ nucleus) circulate through this vascular region and in some case spontaneously arrest (these specific events are indicated by red arrows) and then release back into circulation. Z volumes were collected every 20 seconds and Video represents ˜60 minutes of real time. (MOV 11738 kb)

Intravital imaging of a cytoplast migrating through lung vasculature

Intravital imaging of pulmonary vasculature of an Actin-CFP mouse (blue) 1hr post-IV injection with B16-ZsGreen tumor cells (green). Z volumes were collected every 3 minutes. White arrow highlights the release of a cytoplast microparticle and subsequent migration and directional changes within the lung vasculature. This Video corresponds to Figure S4. (MOV 2177 kb)

Ingestion of tumor cytoplast by myeloid cells in lung vasculature

Intravital imaging of pulmonary vasculature of a MacBlue mouse (blue cells are myeloid cells expressing CFP driven by a CSF1R-promoter reporter) 3 hrs and 15 minutes post- IV injection with B16-ZsGreen tumor cells (green). Green blebs shown are cytoplast microparticles. Z volumes were collected every 1min 25 seconds and Video represents ˜41 minutes of real time. White arrow highlights the moment of ingestion of a microparticle by a migrating MacBlue+ myeloid cell. This Video corresponds to Figure 2E. (MOV 2167 kb)

Targeting of recently released cytoplast by myeloid cells in lung vasculature

Intravital imaging of pulmonary vasculature of a MacBlue mouse (blue cells are myeloid cells expressing CFP driven by a CSF1R-promoter reporter) from 15 minutes until 6 hrs post-IV injection with B16-ZsGreen tumor cells (green cell). Z volumes were collected every 30 seconds and Video represents ˜5 hrs and 45 min of real time. White arrows and text highlight the formation and release of a cytoplast microparticle from a parental Karyoplast. Following this event migration of the cytoplast up a vascular branch can be observed and subsequent recognition and interaction of the cytoplast by lung myeloid cells. This Video corresponds to Figure 2F. (MOV 28635 kb)

Tumor antigen bearing macrophages stably interact with 24hr surviving metastatic melanoma in lung

Intravital imaging of pulmonary vasculature of a MacBlue mouse x CD11c-mCherry where coexpression of CFP and mCherry label monocyte-derived Macrophages. Imaging was performed 24hrs post-injection of B16-ZsGreen tumor cells IV (green cells). Z volumes were collected every 2mins and Video represents ˜57 min of real time. Video clearly shows the presence of CFP and CFP+ CD11c+ macrophages with ZsGreen+ inclusions surrounding this surviving metastasis. This Video corresponds to Figure 4A-D. (MOV 2018 kb)

Tumor antigen loaded Dendritic cells interact with activated antigen specific T cells in the mediastinal LN

Live explant imaging of a mediastinal LN from a CD11c-mCherry reporter mouse, taken 72 hrs post-IV injection of with B16-ZsGreenOVA. GFP+ cells are OT-1 T cells specific to the ovalbumin model tumor antigen. Arrows highlight CD11c+ dendritic cells bearing tumor antigen (yellow) engaged in interaction with a scrum of OT-1 T cells. Z volumes were taken every 20 seconds and Video represents 30 minutes of real time. This Video corresponds to Figure 4H. This was updated on 7 April 2016 to correct the video legend (MOV 2863 kb)

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Headley, M., Bins, A., Nip, A. et al. Visualization of immediate immune responses to pioneer metastatic cells in the lung. Nature 531, 513–517 (2016). https://doi.org/10.1038/nature16985

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