Supplementary Materials

Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice

Jean Albrengues, Mario A. Shields, David Ng, Chun Gwon Park, Alexandra Ambrico, Morgan E. Poindexter, Priya Upadhyay, Dale L. Uyeminami, Arnaud Pommier, Victoria Küttner, Emilis Bružas, Laura Maiorino, Carmelita Bautista, Ellese M. Carmona, Phyllis A. Gimotty, Douglas T. Fearon, Kenneth Chang, Scott K. Lyons, Kent E. Pinkerton, Lloyd C. Trotman, Michael S. Goldberg, Johannes T.-H. Yeh, Mikala Egeblad

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Materials and Methods
  • Figs. S1 to S15
  • Captions for Movies S1 to S8
  • References

Images, Video, and Other Media

Movie S1
Neutrophil infiltration and neutrophil elastase activity is low in the lungs of control animals. Lung intravital imaging of a LysM-EGFP BALB/c mouse treated with PBS and injected with the NE 680 FAST probe was performed to analyze the presence of neutrophils and neutrophil elastase activity in the lungs. Neutrophils express EGFP (green), NE activity is represented in red, and DNA (blue) is labeled by injection of DAPI. Time indicated is time (h:mm:ss) after imaging was initiated, 18 hours after PBS instillation. Representative of three mice.
Movie S2
Intranasal instillation of LPS induces high neutrophil recruitment and neutrophil elastase activity in the lungs. Lung intravital imaging of a LysM-EGFP BALB/c mouse treated with LPS and injected with the NE 680 FAST probe was performed to analyze neutrophil recruitment and neutrophil elastase activity in the lungs. Neutrophils express EGFP (green), NE activity is represented in red, and DNA (blue) is labeled by injection of DAPI. Time indicated is time (h:mm:ss) after imaging was initiated, 18 hours after LPS instillation. Representative of three mice.
Movie S3
D2.0R cells are dormant in the lungs of control animals immediately after PBS instillation. A BALB/c mouse injected intravenously with FUCCI D2.0R cells at day 0 was subjected to lung intravital imaging at day 8, after PBS instillation on day 7. NE 680 FAST probe was used to detect neutrophil elastase activity. D2.0R cells express the FUCCI reporter (red, green, or yellow as represented in fig. S2D), NE activity is represented in purple (no signal was detected), and DNA (blue) is labeled by injection of DAPI. Time indicated is time (h:mm:ss) after imaging was initiated. Representative of three mice.
Movie S4
D2.0R cells remain dormant in the lungs of control animals after two PBS instillations. A BALB/c mouse injected intravenously with FUCCI D2.0R cells at day 0 was subjected to lung intravital imaging at day 11, after PBS instillation on days 7 and 10. NE 680 FAST probe was used to detect neutrophil elastase activity. D2.0R cells express the FUCCI reporter (red, green, or yellow as represented in fig. S2D), NE activity is represented in purple (no signal was detected), and DNA (blue) is labeled by injection of DAPI. Time indicated is time (h:mm:ss) after imaging was initiated. Representative of two mice.
Movie S5
D2.0R cells remain dormant in the lungs of control animals 21 days after injection. A BALB/c mouse injected intravenously with FUCCI D2.0R cells at day 0 was subjected to lung intravital imaging at day 21, after PBS instillation on days 7, 10 and 13. NE 680 FAST probe was used to detect neutrophil elastase activity. D2.0R cells express the FUCCI reporter (red, green, or yellow as represented in Fig. S2D), NE activity is represented in purple (no signal was detected), and DNA (blue) is labeled by injection of DAPI. Time indicated is time (h:mm:ss) after imaging was initiated. Representative of three mice.
Movie S6
D2.0R cells enter the G1/S transition of the cell cycle 4 days after the first LPS intranasal instillation. A BALB/c mouse injected intravenously with FUCCI D2.0R cells at day 0 was subjected to lung intravital imaging at day 11, after LPS instillation on days 7 and 10. NE680 FAST probe was used to detect neutrophil elastase activity. D2.0R cells express the FUCCI reporter (red, green, or yellow as represented in fig. S2D), NE activity is represented in purple, and DNA (blue) is labeled by injection of DAPI. Time indicated is time (h:mm:ss) after imaging was initiated; there is a gap in timeframes between 22 min and 1h 44 min due to inability to obtain good focus during this time period. Representative of three mice.
Movie S7
Clusters of dividing D2.0R cells are present in the lungs of mice 7 days after the first LPS intranasal instillation. BALB/c mice injected intravenously with FUCCI D2.0R cells at day 0 was subjected to lung intravital imaging at day 14, after LPS instillation on days 7, 10, and 13. NE680 FAST probe was used to detect neutrophil elastase activity. D2.0R cells express the FUCCI reporter (red, green, or yellow as represented in fig. S2D), NE activity is represented in purple, and DNA (blue) is labeled by injection of DAPI. Time indicated is time (h:mm:ss) after imaging was initiated. Representative of three mice.
Movie S8
Established proliferative metastasis of D2.0R cells are present in the lungs of mice 14 days after the first LPS intranasal instillation. BALB/c mice injected intravenously with FUCCI D2.0R cells at day 0 was subjected to lung intravital imaging at day 21, after LPS instillation on days 7, 10, and 13. NE 680 FAST probe was used to detect neutrophil elastase activity. D2.0R cells express the FUCCI reporter (red, green, or yellow as represented in fig. S2D), NE activity is represented in purple, and DNA (blue) is labeled by injection of DAPI. Time indicated is time (h:mm:ss) after imaging was initiated. Representative of three mice.
 
Correction (13 December 2018): In the Research Article "Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice," figure panels S6C, S6D, S7H, S8D, and S8E have been replaced. A systematic error in the transfer of raw image files into image analysis software and subsequently Adobe Illustrator led to errors in the assembly of figure panels S6C, S6D, S7H, and S8E. In addition, due to human error, an incorrect part of the gel was used for the condition "C5a" during assembly of fig. S8D. None of these changes affect the conclusions of the paper.
The original version is accessible here.